28989238
Pathogenesis of Brain Edema and Investigation into Anti-Edema Drugs
Brain edema is a potentially fatal pathological state that occurs after brain injuries such as stroke and head trauma. In the edematous brain, excess accumulation of extracellular fluid results in elevation of intracranial pressure, leading to impaired nerve function. Despite the seriousness of brain edema, only symptomatic treatments to remove edema fluid are currently available. Thus, the development of novel anti-edema drugs is required. The pathogenesis of brain edema is classified as vasogenic or cytotoxic edema. Vasogenic edema is defined as extracellular accumulation of fluid resulting from disruption of the blood-brain barrier (BBB) and extravasations of serum proteins, while cytotoxic edema is characterized by cell swelling caused by intracellular accumulation of fluid. Various experimental animal models are often used to investigate mechanisms underlying brain edema. Many soluble factors and functional molecules have been confirmed to induce BBB disruption or cell swelling and drugs targeted to these factors are expected to have anti-edema effects. In this review, we discuss the mechanisms and involvement of factors that induce brain edema formation, and the possibility of anti-edema drugs targeting them.
23530928
Pathology and new players in the pathogenesis of brain edema
Brain edema continues to be a major cause of mortality after diverse types of brain pathologies such as major cerebral infarcts, hemorrhages, trauma, infections and tumors. The classification of edema into vasogenic, cytotoxic, hydrocephalic and osmotic has stood the test of time although it is recognized that in most clinical situations there is a combination of different types of edema during the course of the disease. Basic information about the types of edema is provided for better understanding of the expression pattern of some of the newer molecules implicated in the pathogenesis of brain edema. These molecules include the aquaporins, matrix metalloproteinases and growth factors such as vascular endothelial growth factors A and B and the angiopoietins. The potential of these agents in the treatment of edema is discussed. Since many molecules are involved in the pathogenesis of brain edema, effective treatment cannot be achieved by a single agent but will require the administration of a “magic bullet” containing a variety of agents released at different times during the course of edema in order to be successful.
12117827
Brain edema in acute liver failure and chronic liver disease: Similarities and differences
Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that typically develops as a result of acute liver failure or chronic liver disease. Brain edema is a common feature associated with HE. In acute liver failure, brain edema contributes to an increase in intracranial pressure, which can fatally lead to brain stem herniation. In chronic liver disease, intracranial hypertension is rarely observed, even though brain edema may be present. This discrepancy in the development of intracranial hypertension in acute liver failure versus chronic liver disease suggests that brain edema plays a different role in relation to the onset of HE. Furthermore, the pathophysiological mechanisms involved in the development of brain edema in acute liver failure and chronic liver disease are dissimilar. This review explores the types of brain edema, the cells, and pathogenic factors involved in its development, while emphasizing the differences in acute liver failure versus chronic liver disease. The implications of brain edema developing as a neuropathological consequence of HE, or as a cause of HE, are also discussed.
12817374
Intracranial Pressure Monitoring: Invasive versus Non-Invasive Methods—A Review
Monitoring of intracranial pressure (ICP) has been used for decades in the fields of neurosurgery and neurology. There are multiple techniques: invasive as well as noninvasive. This paper aims to provide an overview of the advantages and disadvantages of the most common and well-known methods as well as assess whether noninvasive techniques (transcranial Doppler, tympanic membrane displacement, optic nerve sheath diameter, CT scan/MRI and fundoscopy) can be used as reliable alternatives to the invasive techniques (ventriculostomy and microtransducers). Ventriculostomy is considered the gold standard in terms of accurate measurement of pressure, although microtransducers generally are just as accurate. Both invasive techniques are associated with a minor risk of complications such as hemorrhage and infection. Furthermore, zero drift is a problem with selected microtransducers. The non-invasive techniques are without the invasive methods' risk of complication, but fail to measure ICP accurately enough to be used as routine alternatives to invasive measurement. We conclude that invasive measurement is currently the only option for accurate measurement of ICP.
46650798
Edema and brain trauma
Brain edema leading to an expansion of brain volume has a crucial impact on morbidity and mortality following traumatic brain injury (TBI) as it increases intracranial pressure, impairs cerebral perfusion and oxygenation, and contributes to additional ischemic injuries. Classically, two major types of traumatic brain edema exist: "vasogenic" due to blood-brain barrier (BBB) disruption resulting in extracellular water accumulation and "cytotoxic/cellular" due to sustained intracellular water collection. A third type, "osmotic" brain edema is caused by osmotic imbalances between blood and tissue. Rarely after TBI do we encounter a "hydrocephalic edema/interstitial" brain edema related to an obstruction of cerebrospinal fluid outflow. Following TBI, various mediators are released which enhance vasogenic and/or cytotoxic brain edema. These include glutamate, lactate, H(+), K(+), Ca(2+), nitric oxide, arachidonic acid and its metabolites, free oxygen radicals, histamine, and kinins. Thus, avoiding cerebral anaerobic metabolism and acidosis is beneficial to control lactate and H(+), but no compound inhibiting mediators/mediator channels showed beneficial results in conducted clinical trials, despite successful experimental studies. Hence, anti-edematous therapy in TBI patients is still symptomatic and rather non-specific (e.g. mannitol infusion, controlled hyperventilation). For many years, vasogenic brain edema was accepted as the prevalent edema type following TBI. The development of mechanical TBI models ("weight drop," "fluid percussion injury," and "controlled cortical impact injury") and the use of magnetic resonance imaging, however, revealed that "cytotoxic" edema is of decisive pathophysiological importance following TBI as it develops early and persists while BBB integrity is gradually restored. These findings suggest that cytotoxic and vasogenic brain edema are two entities which can be targeted simultaneously or according to their temporal prevalence.
30443325
Brain oedema in focal ischaemia: molecular pathophysiology and theoretical implications
Focal cerebral ischaemia and post-ischaemic reperfusion cause cerebral capillary dysfunction, resulting in oedema formation and haemorrhagic conversion. There are substantial gaps in understanding the pathophysiology, especially regarding early molecular participants. Here, we review physiological and molecular mechanisms involved. We reaffirm the central role of Starling's principle, which states that oedema formation is determined by the driving force and the capillary "permeability pore". We emphasise that the movement of fluids is largely driven without new expenditure of energy by the ischaemic brain. We organise the progressive changes in osmotic and hydrostatic conductivity of abnormal capillaries into three phases: formation of ionic oedema, formation of vasogenic oedema, and catastrophic failure with haemorrhagic conversion. We suggest a new theory suggesting that ischaemia-induced capillary dysfunction can be attributed to de novo synthesis of a specific ensemble of proteins that determine osmotic and hydraulic conductivity in Starling's equation, and whose expression is driven by a distinct transcriptional program.
26851965
Advances in the management of intracerebral hemorrhage
Intracerebral hemorrhage (ICH) is one of the most detrimental sub-types of stroke and accounts for 10–15 % of all strokes Qureshi et al. (Lancet 373(9675):1632–1644, 2009). ICH has an incidence of 10–30 cases per 100,000 people/year which is increasing and expected to double by the year 2050 Qureshi et al. (N Engl J Med 344 (19):1450–1460, 2001). Mortality rates still remain poor (30–50 %) and functional dependency after ICH is high (~75 %) van Asch et al. (Lancet Neurol 9 (2):167–176, 2010). Up to now, all randomized controlled trials investigating treatment approaches in ICH have failed to document improvements on clinical endpoints Mayer et al. (N Engl J Med 358 (20):2127–2137, 2008); Brouwers and Goldstein (Neurotherapeutics 9 (1):87–98, 2012). Only a specialized treatment of severely injured patients at dedicated neuro intensive care units [NICU] has been shown to be beneficial Qureshi et al. (Lancet 373(9675):1632–1644, 2009); Suarez et al. (Crit Care Med 32 (11):2311–2317, 2004). Currently, ongoing trials are investigating aggressive blood pressure lowering, hemostatic therapies, different operative strategies, intraventricular thrombolysis as well as neuroprotective approaches, and brain edema therapies. This review will summarize advanced treatment strategies and novel approaches which are currently under investigation.
3186870
The brain in acute on chronic liver failure
Acute-on-chronic liver failure (ACLF) is a newly defined clinical entity with significant morbidity and mortality (~40–90 % at 1 year dependent on need for organ support at presentation). It defines a presentation with acute severe liver injury, often with multiorgan dysfunction, on a background of previously known or unknown cirrhosis. In its severest form, it is almost indistinguishable from acute liver failure, as similarly in around 5 % may rapidly progress to intracranial hypertension and cerebral oedema culminating in coma and/or death. Our understanding of such cerebral sequelae is currently limited to clinical observation, though our knowledge base is rapidly expanding since recent consensus clinical definition and guidance. Moreover, there are now animal models of ACLF and imaging modalities to better characterize events in the brain that occur with ACLF. However, as yet there has been little in the way of interventional study of this condition which are much needed. In this review we dissect existing clinical and experimental data to better characterise the manifestations of ACLF on the brain and allow for the development of targeted therapy as currently the plethora of existing interventions were designed to treat either the effects of cirrhosis or acute liver injury independently.
10207985
Treatment of Cerebral Edema
Background:Cerebral edema is a potentially devastating complication of various acute neurologic disorders. Its successful treatment may save lives and preserve neurologic function. Review Summary:Different pathophysiological mechanisms are responsible for the formation of cytotoxic and vasogenic edema. Yet, these 2 types of edema often coexist and their treatment tends to overlap, with the exception of corticosteroids, which should be only used to ameliorate vasogenic edema. Currently available to control brain swelling include osmotic agents (with emphasis on mannitol and hypertonic saline solutions), corticosteroids, hyperventilation, sedation (propofol, barbiturates), neuromuscular paralysis, hypothermia, and surgical interventions. This article discusses the indications, advantages, and limitations of each treatment modality following an evidence-based approach. Conclusions:The therapy for brain edema remains largely empirical. More research aimed at enhancing our understanding of the pathophysiology of cerebral edema is needed to identify new and more effective forms of treatment.
23365177
Medical management of cerebral edema.
Cerebral edema is frequently encountered in clinical practice in critically ill patients with acute brain injury from diverse origins and is a major cause of increased morbidity and death in this subset of patients. The consequences of cerebral edema can be lethal and include cerebral ischemia from compromised regional or global cerebral blood flow (CBF) and intracranial compartmental shifts due to intracranial pressure gradients that result in compression of vital brain structures. The overall goal of medical management of cerebral edema is to maintain regional and global CBF to meet the metabolic requirements of the brain and prevent secondary neuronal injury from cerebral ischemia. Medical management of cerebral edema involves using a systematic and algorithmic approach, from general measures (optimal head and neck positioning for facilitating intracranial venous outflow, avoidance of dehydration and systemic hypotension, and maintenance of normothermia) to specific therapeutic interventions (controlled hyperventilation, administration of corticosteroids and diuretics, osmotherapy, and pharmacological cerebral metabolic suppression). This article reviews and highlights the medical management of cerebral edema based on pathophysiological principles in acute brain injury.
1576435
Novel Treatment Targets for Cerebral Edema
Cerebral edema is a common finding in a variety of neurological conditions, including ischemic stroke, traumatic brain injury, ruptured cerebral aneurysm, and neoplasia. With the possible exception of neoplasia, most pathological processes leading to edema seem to share similar molecular mechanisms of edema formation. Challenges to brain-cell volume homeostasis can have dramatic consequences, given the fixed volume of the rigid skull and the effect of swelling on secondary neuronal injury. With even small changes in cellular and extracellular volume, cerebral edema can compromise regional or global cerebral blood flow and metabolism or result in compression of vital brain structures. Osmotherapy has been the mainstay of pharmacologic therapy and is typically administered as part of an escalating medical treatment algorithm that can include corticosteroids, diuretics, and pharmacological cerebral metabolic suppression. Novel treatment targets for cerebral edema include the Na(+)-K(+)-2Cl(−) co-transporter (NKCC1) and the SUR1-regulated NCCa-ATP (SUR1/TRPM4) channel. These two ion channels have been demonstrated to be critical mediators of edema formation in brain-injured states. Their specific inhibitors, bumetanide and glibenclamide, respectively, are well-characterized Food and Drug Administration-approved drugs with excellent safety profiles. Directed inhibition of these ion transporters has the potential to reduce the development of cerebral edema and is currently being investigated in human clinical trials. Another class of treatment agents for cerebral edema is vasopressin receptor antagonists. Euvolemic hyponatremia is present in a myriad of neurological conditions resulting in cerebral edema. A specific antagonist of the vasopressin V1A- and V2-receptor, conivaptan, promotes water excretion while sparing electrolytes through a process known as aquaresis.
30038585
Drowning stars: reassessing the role of astrocytes in brain edema
Edema formation frequently complicates brain infarction, tumors, and trauma. Despite the significant mortality of this condition, current treatment options are often ineffective or incompletely understood. Recent studies have revealed the existence of a brain-wide paravascular pathway for cerebrospinal (CSF) and interstitial fluid (ISF) exchange. The current review critically examines the contribution of this 'glymphatic' system to the main types of brain edema. We propose that in cytotoxic edema, energy depletion enhances glymphatic CSF influx, whilst suppressing ISF efflux. We also argue that paravascular inflammation or 'paravasculitis' plays a critical role in vasogenic edema. Finally, recent advances in diagnostic imaging of glymphatic function may hold the key to defining the edema profile of individual patients, and thus enable more targeted therapy.
205500476
Astrocyte–endothelial interactions at the blood–brain barrier
The blood–brain barrier, which is formed by the endothelial cells that line cerebral microvessels, has an important role in maintaining a precisely regulated microenvironment for reliable neuronal signalling. At present, there is great interest in the association of brain microvessels, astrocytes and neurons to form functional 'neurovascular units', and recent studies have highlighted the importance of brain endothelial cells in this modular organization. Here, we explore specific interactions between the brain endothelium, astrocytes and neurons that may regulate blood–brain barrier function. An understanding of how these interactions are disturbed in pathological conditions could lead to the development of new protective and restorative therapies.
23399070
Brain endothelial cells and the glio-vascular complex
We present and discuss the role of endothelial and astroglial cells in managing the blood-brain barrier (BBB) and aspects of pathological alterations in the BBB. The impact of astrocytes, pericytes, and perivascular cells on the induction and maintenance of the gliovascular unit is largely unidentified so far. An understanding of the signaling pathways that lie between these cell types and the endothelium and that possibly are mediated by components of the basal lamina is just beginning to emerge. The metabolism for the maintenance of the endothelial barrier is intimately linked to and dependent on the microenvironment of the brain parenchyma. We report the structure and function of the endothelial cells of brain capillaries by describing structures involved in the regulation of permeability, including transporter systems, caveolae, and tight junctions. There is increasing evidence that caveolae are not only vehicles for endo- and transcytosis, but also important regulators of tight-junction-based permeability. Tight junctions separate the luminal from the abluminal membrane domains of the endothelial cell (“fence function”) and control the paracellular pathway (“gate function”) thus representing the most significant structure of the BBB. In addition, the extracellular matrix between astrocytes/pericytes and endothelial cells contains numerous molecules with inherent signaling properties that have to be considered if we are to improve our knowledge of the complex and closely regulated BBB.
2202060
The blood–brain barrier: an overview Structure, regulation, and clinical implications
The blood-brain barrier (BBB) is a diffusion barrier, which impedes influx of most compounds from blood to brain. Three cellular elements of the brain microvasculature compose the BBB-endothelial cells, astrocyte end-feet, and pericytes (PCs). Tight junctions (TJs), present between the cerebral endothelial cells, form a diffusion barrier, which selectively excludes most blood-borne substances from entering the brain. Astrocytic end-feet tightly ensheath the vessel wall and appear to be critical for the induction and maintenance of the TJ barrier, but astrocytes are not believed to have a barrier function in the mammalian brain. Dysfunction of the BBB, for example, impairment of the TJ seal, complicates a number of neurologic diseases including stroke and neuroinflammatory disorders. We review here the recent developments in our understanding of the BBB and the role of the BBB dysfunction in CNS disease. We have focused on intraventricular hemorrhage (IVH) in premature infants, which may involve dysfunction of the TJ seal as well as immaturity of the BBB in the germinal matrix (GM). A paucity of TJs or PCs, coupled with incomplete coverage of blood vessels by astrocyte end-feet, may account for the fragility of blood vessels in the GM of premature infants. Finally, this review describes the pathogenesis of increased BBB permeability in hypoxia-ischemia and inflammatory mechanisms involving the BBB in septic encephalopathy, HIV-induced dementia, multiple sclerosis, and Alzheimer disease.
1495418
Glutamate excitotoxicity in transient global cerebral ischemia.
The glutamate excitotoxicity hypothesis of ischemic cell damage holds that cell damage caused by transient cerebral ischemia is triggered by glutamate, released during ischemia from the intracellular compartment into the synaptic cleft: high extracellular glutamate levels activate ionotropic glutamate receptors, thus inducing an overflow of calcium ions into the neurones and a calcium-induced activation of catabolic processes. However, several arguments (and much of the evidence) in favour of this hypothesis do not bear closer examination. On the other hand, evidence is accumulating that, after transient ischemia, calcium fluxes through ionotropic glutamate receptors of the non-NMDA type may play a major role in the manifestation of ischemic cell damage. Calcium fluxes through non-NMDA receptors are determined by mRNA editing of non-NMDA receptor subunits: calcium fluxes are blocked in the presence of an edited subunit. A possible role of mRNA editing in the development of ischemic cell damage is discussed.
24511407
Mitochondria in traumatic brain injury and mitochondrial‐targeted multipotential therapeutic strategies
Traumatic brain injury (TBI) is a major health and socioeconomic problem throughout the world. It is a complicated pathological process that consists of primary insults and a secondary insult characterized by a set of biochemical cascades. The imbalance between a higher energy demand for repair of cell damage and decreased energy production led by mitochondrial dysfunction aggravates cell damage. At the cellular level, the main cause of the secondary deleterious cascades is cell damage that is centred in the mitochondria. Excitotoxicity, Ca2+ overload, reactive oxygen species (ROS), Bcl‐2 family, caspases and apoptosis inducing factor (AIF) are the main participants in mitochondria‐centred cell damage following TBI. Some preclinical and clinical results of mitochondria‐targeted therapy show promise. Mitochondria‐ targeted multipotential therapeutic strategies offer new hope for the successful treatment of TBI and other acute brain injuries.
1317241
Molecular mechanisms of cognitive dysfunction following traumatic brain injury
Traumatic brain injury (TBI) results in significant disability due to cognitive deficits particularly in attention, learning and memory, and higher-order executive functions. The role of TBI in chronic neurodegeneration and the development of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and most recently chronic traumatic encephalopathy (CTE) is of particular importance. However, despite significant effort very few therapeutic options exist to prevent or reverse cognitive impairment following TBI. In this review, we present experimental evidence of the known secondary injury mechanisms which contribute to neuronal cell loss, axonal injury, and synaptic dysfunction and hence cognitive impairment both acutely and chronically following TBI. In particular we focus on the mechanisms linking TBI to the development of two forms of dementia: AD and CTE. We provide evidence of potential molecular mechanisms involved in modulating Aβ and Tau following TBI and provide evidence of the role of these mechanisms in AD pathology. Additionally we propose a mechanism by which Aβ generated as a direct result of TBI is capable of exacerbating secondary injury mechanisms thereby establishing a neurotoxic cascade that leads to chronic neurodegeneration.
205839528
The blood-brain barrier in brain homeostasis and neurological diseases.
Brain endothelial cells are unique among endothelial cells in that they express apical junctional complexes, including tight junctions, which quite resemble epithelial tight junctions both structurally and functionally. They form the blood-brain-barrier (BBB) which strictly controls the exchanges between the blood and the brain compartments by limiting passive diffusion of blood-borne solutes while actively transporting nutrients to the brain. Accumulating experimental and clinical evidence indicate that BBB dysfunctions are associated with a number of serious CNS diseases with important social impacts, such as multiple sclerosis, stroke, brain tumors, epilepsy or Alzheimer's disease. This review will focus on the implication of brain endothelial tight junctions in BBB architecture and physiology, will discuss the consequences of BBB dysfunction in these CNS diseases and will present some therapeutic strategies for drug delivery to the brain across the BBB.
36798835
Role of glial cells in cerebral ischemia
Despite intense efforts at the bench and at the bedside, few therapeutic strategies exist to combat the consequences of cerebral ischemia. Traditionally, a “neurocentric” view has dominated research in this field. Evidence is now accumulating that glial cells, in particular astrocytes, play an active and important role in the pathophysiology of cerebral ischemia. Brain energetics, water and ion homeostasis, inflammation, trophic factor production, vascular regulation, neuroneogenesis, and vasculogenesis, among others, are all under the control of glial cells. As a consequence, glial cells have been identified as promising targets for novel therapeutic approaches in brain protection. This review aims at dissecting possible protective as well as destructive roles of astrocytes (and other glial cells) in cerebral ischemia. By emphasizing open issues in this field, we hope to stimulate further research into this relatively unexplored aspect of brain pathophysiology. © 2005 Wiley‐Liss, Inc.
14037633
Absence of the Chemokine Receptor CCR2 Protects Against Cerebral Ischemia/Reperfusion Injury in Mice
Background and Purpose— The chemokine, monocyte chemoattractant protein-1 (CCL2), is a major factor driving leukocyte infiltration into the brain parenchyma in a variety of neuropathologic conditions associated with inflammation, including stroke. In addition, recent studies indicate that CCL2 and its receptor (CCR2) could have an important role in regulating blood-brain barrier (BBB) permeability. This study evaluated the role of the CCL2/CCR2 axis in regulating postischemic inflammation, BBB breakdown, and vasogenic edema formation. Methods— CCR2−/− and CCR2+/+ mice were subjected to focal transient cerebral ischemia. BBB permeability and brain edema formation were observed at days 1 and 5 of reperfusion by evaluating the product surface area for fluorescein isothiocyanate–albumin and measuring water and electrolyte contents. Immunohistochemistry was used to assess leukocyte infiltration. cDNA gene and protein arrays for inflammatory cytokines were used to assess inflammatory profiles in CCR2+/+ and CCR2−/− mice. Results— CCR2−/− mice had reduced infarct sizes and significantly reduced BBB permeability and brain edema formation in the affected ischemic hemisphere compared with CCR2+/+ mice. This reduction in injury was closely associated with reduced infiltration of not only monocytes but also neutrophils (7- and 4-fold decreases, respectively). In addition, CCR2−/− mice had reduced expression/production of inflammatory cytokines during reperfusion. Conclusions— These data suggest that inhibiting the CCL2/CCR2 axis affects brain reperfusion outcome by reducing brain edema, leukocyte infiltration, and inflammatory mediator expression.
10653781
VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown
Breakdown of the blood-brain barrier (BBB) is an early and significant event in CNS inflammation. Astrocyte-derived VEGF-A has been implicated in this response, but the underlying mechanisms remain unresolved. Here, we identify the endothelial transmembrane tight junction proteins claudin-5 (CLN-5) and occludin (OCLN) as targets of VEGF-A action. Down-regulation of CLN-5 and OCLN accompanied up-regulation of VEGF-A and correlated with BBB breakdown in experimental autoimmune encephalomyelitis, an animal model of CNS inflammatory disease. In cultures of brain microvascular endothelial cells, VEGF-A specifically down-regulated CLN-5 and OCLN protein and mRNA. In mouse cerebral cortex, microinjection of VEGF-A disrupted CLN-5 and OCLN and induced loss of barrier function. Importantly, functional studies revealed that expression of recombinant CLN-5 protected brain microvascular endothelial cell cultures from a VEGF-induced increase in paracellular permeability, whereas recombinant OCLN expressed under the same promoter was not protective. Previous studies have shown CLN-5 to be a key determinant of trans-endothelial resistance at the BBB. Our findings suggest that its down-regulation by VEGF-A constitutes a significant mechanism in BBB breakdown.
6091720
Molecular mechanisms of ischemic cerebral edema: role of electroneutral ion transport.
The brain achieves homeostasis of its intracellular and extracellular fluids by precisely regulating the transport of solute and water across its major cellular barriers: endothelia of the blood-brain barrier (BBB), choroid plexus epithelia, and neuroglial cell membranes. Cerebral edema, the pathological accumulation of fluid in the brain's intracellular and extracellular spaces, is a major cause of morbidity and mortality following stroke and other forms of ischemic brain injury. Until recently, mechanisms of cerebral edema formation have been obscure; consequently, its treatment has been empiric and suboptimal. Here, we provide a paradigm for understanding ischemic cerebral edema, showing that its molecular pathogenesis is a complex yet step-wise process that results largely from impaired astrocytic cell volume regulation and permeability alterations in the cerebral microvasculature, both of which arise from pathological changes in the activities of specific ion channels and transporters. Recent data has implicated the bumetanide-sensitive NKCC1, an electroneutral cotransporter expressed in astrocytes and the BBB, in cerebral edema formation in several different rodent models of stroke. Pharmacological inhibition or genetic deficiency of NKCC1 decreases ischemia-induced cell swelling, BBB breakdown, cerebral edema, and neurotoxicity. Combination pharmacological strategies that include NKCC1 as a target might thus prove beneficial for the treatment of ischemic, and potentially other types of, cerebral edema.
207342478
Treatment of Brain Edema in Acute Liver Failure
Opinion statementCerebral edema is very common in patients with acute liver failure and encephalopathy. In severe cases, it produces brain tissue shift and potentially fatal herniation. Brain swelling in acute liver failure is produced by a combination of cytotoxic (cellular) and vasogenic edema. Accumulation of ammonia and glutamine leads to disturbances in the regulation of cerebral osmolytes, increased free radical production and calcium-mediated mitochondrial injury, and alterations in glucose metabolism (inducing high levels of brain lactate), resulting in astrocyte swelling. Activation of inflammatory cytokines can cause increased blood-brain barrier permeability leading to vasogenic edema, although the relative contribution of vasogenic edema is probably minor compared with cellular swelling. Cerebral blood flow is disturbed and generally increased in patients with acute liver failure; persistent vasodilatation and loss of autoregulation may generate hyperemia, and the consequent augmentation in cerebral blood volume may exacerbate brain edema.Adequate management of intracranial hypertension demands continuous monitoring of intracranial pressure and cerebral perfusion pressure. Coagulation status should be assessed and bleeding diathesis should be treated prior to insertion of the intracranial pressure monitor. Standard treatment measures such as hyperventilation and osmotic agents (e.g., mannitol, hypertonic saline) remain useful first-line interventions. Although hypertonic saline may be preferred in patients with coexistent hyponatremia, the rate of correction of hyponatremia must be gradual to avoid the risk of osmotic demyelination. Barbiturate coma and intravenous indomethacin are available options in refractory cases. The most promising novel therapeutic alternative is the induction of moderate hypothermia (aiming for a core temperature of 32–34°C). However, the safety and efficacy of therapeutic hypothermia for brain swelling caused by liver failure still needs to be proven in randomized, controlled clinical trials. Management of intracranial pressure in patients with acute liver failure should be guided by well-defined treatment protocols.
28842643
Cytotoxic edema: mechanisms of pathological cell swelling.
Cerebral edema is caused by a variety of pathological conditions that affect the brain. It is associated with two separate pathophysiological processes with distinct molecular and physiological antecedents: those related to cytotoxic (cellular) edema of neurons and astrocytes, and those related to transcapillary flux of Na+ and other ions, water, and serum macromolecules. In this review, the authors focus exclusively on the first of these two processes. Cytotoxic edema results from unchecked or uncompensated influx of cations, mainly Na+, through cation channels. The authors review the different cation channels that have been implicated in the formation of cytotoxic edema of astrocytes and neurons in different pathological states. A better understanding of these molecular mechanisms holds the promise of improved treatments of cerebral edema and of the secondary injury produced by this pathological process.
38794307
Cold-induced brain edema in mice. Involvement of extracellular superoxide dismutase and nitric oxide.
The role of extracellular superoxide in the pathogenesis of vasogenic edema was studied using transgenic mice expressing a 5-fold increase in extracellular superoxide dismutase (EC-SOD) activity in their brains. Increased EC-SOD expression offered significant protection against edema development after cold-induced injury (44% less edema than nontransgenic littermates, p < 0.05). Since iron may contribute to vasogenic edema by catalyzing the production of hydroxyl radical from superoxide and hydrogen peroxide, the effects of the chelator deferoxamine were studied. Deferoxamine reduced edema formation after cold-induced injury (43% less edema than controls, p < 0.05); however, treatment with iron-saturated deferoxamine also reduced edema development in mice (32-48% less edema, p < 0.05). This suggested that the protection offered by deferoxamine was independent of its ability to chelate iron. An iron-independent mechanism by which superoxide can contribute to vasogenic edema is via reaction with nitric oxide to produce the potentially toxic peroxynitrite anion, which is also scavenged by deferoxamine. Mice treated with an inhibitor of nitric oxide synthase were protected against cold-induced edema (37% less edema, p < 0.05). EC-SOD transgenic mice received no additional protection by inhibition of nitric oxide synthesis, supporting this novel alternative mechanism of edema formation.
2130545
Cold-Injury of the Cerebral Cortex: Immunolocalization of Cellular Proteins and Blood-Brain Barrier Permeability Studies
The occurrence of blood-brain barrier (BBB) permeability alterations and neovascularization are well documented in the cerebral cortical cold-injury model. This model was used to determine whether the glucose transporter (glutl) protein was present in endothelium of cerebral vessels with breakdown of BBB to protein and when regenerating endothelial cells become immunoreactive for glutl protein. Secondly, the protein products of c-fos and c-jun were localized to determine whether these early immediate genes are activated in this model. Observations were made over a period of 12 hours to 14 days after the cold-injury. Blood-brain barrier permeability was assessed using horseradish peroxidase (HRP) as a tracer. Since HRP may not be able to enter thrombosed vessels within the cold lesion, immunohistochemistry was used to detect extravasation of endogenous serum proteins using antisera to rat serum proteins. The proteins—glutl, GFAP, c-fos and c-jun—were localized by immunohistochemistry. Endothelium of vessels which were permeable to protein, whether in the cold-injury site or in the perilesional area, all contained glutl protein; hence, the presence of glutl did not appear to correlate with an intact BBB to protein. An interesting point is that in the process of neovascularization, regenerating endothelial cells become immunoreactive for glutl at 5 days and this coincides with the presence of tight junctions in these cells. Immunoreactivity for c-fos was observed in regenerating endothelium within the lesion site, in astrocytes, and to a lesser extent in endothelial cells and neurons in the perilesional area. Few astrocytes showed immunoreactivity for c-jun at 4 and 5 days. Possibly, the growth factors generated to promote angiogenesis and repair led to activation of the c-fos gene with deposition of c-fos protein. The results suggest that during nervous system development or endothelial regeneration, the presence of glutl in cerebral endothelium coincides with the presence of an intact BBB to protein and protein tracers. However, in pathological states presence of glutl in cerebral endothelium does not appear to correlate with an intact BBB to protein. This model lends itself to the study of angiogenesis and repair processes in the cerebral cortex in an environment unaffected by ischemia and thus the findings may be relevant to traumatic injuries of the human cerebral cortex.
33113217
Cold injury in mice: a model to study mechanisms of brain edema and neuronal apoptosis
Small rodents, mice in particular, have been widely used for genetic manipulation because of the extensive knowledge in development, embryology and other molecular aspects of this species. However, the use of mice for neurobiology research in the area of brain edema and neuronal injury has not been common. Here we summarize the studies of cold injury-induced brain edema and neuronal apoptosis using mice. Blood-brain barrier (BBB) permeability, demonstrated by extravasation of a serum albumin tracer, Evans Blue, was increased immediately after the injury and returned to the control level by 24 hr. Water content was maximized at 24 hr, whereas a secondary lesion gradually progressed up to 72 hr after cold injury. The mechanism of the development of the cold injury-induced edema and the secondary lesion, involving of oxygen radicals in particular, was determined using superoxide dismutase (SOD)-1 transgenic (Tg) mice with overexpressed copper, zinc-SOD. All of the parameters, BBB permeability, water content and secondary lesion, were attenuated in the Tg mice as compared to littermate non-Tg mice. This clearly demonstrates that oxygen radicals, superoxide anion in particular, mediate cold injury. We also studied whether apoptosis contributes to brain injury following cold injury. Staining with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed the apoptotic cells widespread throughout the entire lesion while still remaining in the margin. DNA laddering was exhibited by gel electrophoresis. These studies indicate that oxidative mediates the development of cold injury-induced edema and the secondary injury, and induces apoptotic cell death. We believe that cold injury in mice provides a simple animal model to study the pathogenesis of brain edema and apoptosis in genetically altered animals.
2426708
Inhibition of Bradykinin Receptor B1 Protects Mice from Focal Brain Injury by Reducing Blood–Brain Barrier Leakage and Inflammation
Kinins are proinflammatory and vasoactive peptides that are released during tissue damage and may contribute to neuronal degeneration, inflammation, and edema formation after brain injury by acting on discrete bradykinin receptors, B1R and B2R. We studied the expression of B1R and B2R and the effect of their inhibition on lesion size, blood–brain barrier (BBB) disruption, and inflammatory processes after a focal cryolesion of the right parietal cortex in mice. B1R and B2R gene transcripts were significantly induced in the lesioned hemispheres of wild-type mice (P<0.05). The volume of the cortical lesions and neuronal damage at 24 h after injury in B1R −/− mice were significantly smaller than in wild-type controls (2.5±2.6 versus 11.5±3.9 mm3, P<0.001). Treatment with the B1R antagonist R-715 1 h after lesion induction likewise reduced lesion volume in wild-type mice (2.6±1.4 versus 12.2±6.1 mm3, P<0.001). This was accompanied by a remarkable reduction of BBB disruption and tissue inflammation. In contrast, genetic deletion or pharmacological inhibition of B2R had no significant impact on lesion formation or the development of brain edema. We conclude that B1R inhibition may offer a novel therapeutic strategy after acute brain injuries.
808926
Cyclic GMP phosphodiesterase inhibition alters the glial inflammatory response, reduces oxidative stress and cell death and increases angiogenesis following focal brain injury
J. Neurochem. (2010) 112, 807–817.
42403838
Mechanisms of cerebral edema in traumatic brain injury: therapeutic developments
Purpose of reviewAlthough a number of factors contribute to the high mortality and morbidity associated with traumatic brain injury (TBI), the development of cerebral edema with brain swelling remains the most significant predictor of outcome. The present review summarizes the most recent advances in the understanding of mechanisms associated with development of posttraumatic cerebral edema, and highlights areas of therapeutic promise. Recent findingsDespite the predominance of cytotoxic (or cellular) edema in the first week after traumatic brain injury, brain swelling can only occur with addition of water to the cranial vault from the vasculature. As such, regulation of blood–brain barrier permeability has become a focus of recent research seeking to manage brain edema. Aquaporins, matrix metalloproteinases and vasoactive inflammatory agents have emerged as potential mediators of cerebral edema following traumatic brain injury. In particular, kinins (bradykinins) and tachykinins (substance P) seem to play an active physiological role in modulating blood–brain barrier permeability after trauma. Substance P neurokinin-1 receptor antagonists show particular promise as novel therapeutic agents. SummaryAttenuating blood–brain barrier permeability has become a promising approach to managing brain edema and associated swelling given that increases in cranial water content can only be derived from the vasculature. Inflammation, both classical and neurogenic, offers a number of attractive targets.
9976605
Blood–Brain Barrier Pathophysiology in Traumatic Brain Injury
The blood–brain barrier (BBB) is formed by tightly connected cerebrovascular endothelial cells, but its normal function also depends on paracrine interactions between the brain endothelium and closely located glia. There is a growing consensus that brain injury, whether it is ischemic, hemorrhagic, or traumatic, leads to dysfunction of the BBB. Changes in BBB function observed after injury are thought to contribute to the loss of neural tissue and to affect the response to neuroprotective drugs. New discoveries suggest that considering the entire gliovascular unit, rather than the BBB alone, will expand our understanding of the cellular and molecular responses to traumatic brain injury (TBI). This review will address the BBB breakdown in TBI, the role of blood-borne factors in affecting the function of the gliovascular unit, changes in BBB permeability and post-traumatic edema formation, and the major pathophysiological factors associated with TBI that may contribute to post-traumatic dysfunction of the BBB. The key role of neuroinflammation and the possible effect of injury on transport mechanisms at the BBB will also be described. Finally, the potential role of the BBB as a target for therapeutic intervention through restoration of normal BBB function after injury and/or by harnessing the cerebrovascular endothelium to produce neurotrophic growth factors will be discussed.
39244787
Review: Molecular pathogenesis of blood–brain barrier breakdown in acute brain injury
S. Nag, A. Kapadia and D. J. Stewart (2011) Neuropathology and Applied Neurobiology37, 3–23 Molecular pathogenesis of blood–brain barrier breakdown in acute brain injury
11540783
Experimental traumatic brain injury
Traumatic brain injury, a leading cause of death and disability, is a result of an outside force causing mechanical disruption of brain tissue and delayed pathogenic events which collectively exacerbate the injury. These pathogenic injury processes are poorly understood and accordingly no effective neuroprotective treatment is available so far. Experimental models are essential for further clarification of the highly complex pathology of traumatic brain injury towards the development of novel treatments. Among the rodent models of traumatic brain injury the most commonly used are the weight-drop, the fluid percussion, and the cortical contusion injury models. As the entire spectrum of events that might occur in traumatic brain injury cannot be covered by one single rodent model, the design and choice of a specific model represents a major challenge for neuroscientists. This review summarizes and evaluates the strengths and weaknesses of the currently available rodent models for traumatic brain injury.
12090797
Animal models of traumatic brain injury
Traumatic brain injury (TBI) is a leading cause of mortality and morbidity both in civilian life and on the battlefield worldwide. Survivors of TBI frequently experience long-term disabling changes in cognition, sensorimotor function and personality. Over the past three decades, animal models have been developed to replicate the various aspects of human TBI, to better understand the underlying pathophysiology and to explore potential treatments. Nevertheless, promising neuroprotective drugs that were identified as being effective in animal TBI models have all failed in Phase II or Phase III clinical trials. This failure in clinical translation of preclinical studies highlights a compelling need to revisit the current status of animal models of TBI and therapeutic strategies.
25748914
Lateral fluid percussion brain injury: a 15-year review and evaluation.
This article comprehensively reviews the lateral fluid percussion (LFP) model of traumatic brain injury (TBI) in small animal species with particular emphasis on its validity, clinical relevance and reliability. The LFP model, initially described in 1989, has become the most extensively utilized animal model of TBI (to date, 232 PubMed citations), producing both focal and diffuse (mixed) brain injury. Despite subtle variations in injury parameters between laboratories, universal findings are evident across studies, including histological, physiological, metabolic, and behavioral changes that serve to increase the reliability of the model. Moreover, demonstrable histological damage and severity-dependent behavioral deficits, which partially recover over time, validate LFP as a clinically-relevant model of human TBI. The LFP model, also has been used extensively to evaluate potential therapeutic interventions, including resuscitation, pharmacologic therapies, transplantation, and other neuroprotective and neuroregenerative strategies. Although a number of positive studies have identified promising therapies for moderate TBI, the predictive validity of the model may be compromised when findings are translated to severely injured patients. Recently, the clinical relevance of LFP has been enhanced by combining the injury with secondary insults, as well as broadening studies to incorporate issues of gender and age to better approximate the range of human TBI within study design. We conclude that the LFP brain injury model is an appropriate tool to study the cellular and mechanistic aspects of human TBI that cannot be addressed in the clinical setting, as well as for the development and characterization of novel therapeutic interventions. Continued translation of pre-clinical findings to human TBI will enhance the predictive validity of the LFP model, and allow novel neuroprotective and neuroregenerative treatment strategies developed in the laboratory to reach the appropriate TBI patients.
8966122
Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice
Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair. Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20. We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.
10639038
Multimodal MR imaging of acute and subacute experimental traumatic brain injury: Time course and correlation with cerebral energy metabolites
Background Traumatic brain injury (TBI) is one of the leading causes of death and permanent disability world-wide. The predominant cause of death after TBI is brain edema which can be quantified by non-invasive diffusion-weighted magnetic resonance imaging (DWI). Purpose To provide a better understanding of the early onset, time course, spatial development, and type of brain edema after TBI and to correlate MRI data and the cerebral energy state reflected by the metabolite adenosine triphosphate (ATP). Material and Methods The spontaneous development of lateral fluid percussion-induced TBI was investigated in the acute (6 h), subacute (48 h), and chronic (7 days) phase in rats by MRI of quantitative T2 and apparent diffusion coefficient (ADC) mapping as well as perfusion was combined with ATP-specific bioluminescence imaging and histology. Results An induced TBI led to moderate to mild brain damages, reflected by transient, pronounced development of vasogenic edema and perfusion reduction. Heterogeneous ADC patterns indicated a parallel, but mixed expression of vasogenic and cytotoxic edema. Cortical ATP levels were reduced in the acute and subacute phase by 13% and 27%, respectively, but were completely normalized at 7 days after injury. Conclusion The partial ATP reduction was interpreted to be partially caused by a loss of neurons in parallel with transient dilution of the regional ATP concentration by pronounced vasogenic edema. The normalization of energy metabolism after 7 days was likely due to infiltrating glia and not to recovery. The MRI combined with metabolite measurement further improves the understanding and evaluation of brain damages after TBI.
19237831
Regional patterns of blood-brain barrier breakdown following central and lateral fluid percussion injury in rodents.
In order to determine how fluid percussion injury (FPI) effect is distributed throughout the brain, and to assess the extent to which individual brain nuclei and regions are affected, the pattern of blood-brain barrier (BBB) breakdown was determined in groups with different injury cannula locations. Injury cannulas were placed either at midline, or 2 or 4 mm to the side. One hour following FPI, animals were given horseradish peroxidase (HRP) and the brains were stained using the TMB method. The distribution of HRP leakage varied considerably depending upon the location of the injury cannula, however, there were also common sites of leakage among these groups. Locally the cortex and hippocampus under and adjacent to the injury cannula were heavily affected, with a clear asymmetric effect in the lateral cannula groups. Common sites of leakage included the dorsal thalamus, septal area, pontine tegmentum, periaqueductal gray, substantia nigra, and narrow zones adjacent to ventricular or cisternal surfaces. The hippocampus tended to be involved at greater distances than the cerebral cortex. The cervicomedullary junction proved to be especially vulnerable to FPI with extensive HRP leakage, and petechial hemorrhage ranging from minor to fatal coalescent hemorrhage. A very narrow threshold separated these outcomes. Neurologic impairment of the animals correlated most directly with the extent of cervico-medullary junction injury. Thus FPI produces a mix of local and diffuse effects on the BBB. Injury at the cervicomedullary junction is a prominent effect and is the limiting factor in trying to establish more severe diffuse injury.
6790203
The blood-brain barrier disruption to circulating proteins in the early period after fluid percussion brain injury in rats.
Breakdown of the blood-brain barrier (BBB) immediately after traumatic brain injury is not clearly understood. In the present study we focused on the integrity of the BBB to circulating proteins within the first hour after injury. For this purpose, vascular permeability to endogenous albumin and to the exogenous protein tracer horseradish peroxidase (HRP) was examined after a lateral fluid percussion brain injury in rats. Albumin was immunolocalized in brain sections at 3 and 60 min after impact. This distribution was compared with the histochemical localization of HRP given before impact at the same time points. In a separate experiment HRP was given prior to sacrifice to determine the time course for the barrier disruption. Permeability to this protein was assessed at 13, 30, and 60 min after impact. Prominent extravasation of albumin occurred within 3 min of injury and was present in multiple foci within the injured hemisphere. At 60 min the extravasated albumin was present in the same sites, where it was widely distributed. Throughout the related brain parenchyma, little difference was found between the extravascular distribution of albumin and HRP. In the delayed administration paradigm breakdown of the BBB was noted in the impact site, hemorrhagic site in the deep cortical layer, hippocampus, thalamus, and midbrain at 13 min after injury. This injured barrier was restored in most regions by 30 min. However, the impact site and hemorrhagic site remained permeable up to 60 min postinjury. In addition, newly developed barrier disruption to HRP occurred in the parasagittal cortex at 30 and 60 min. In conclusion, widespread breakdown of the BBB to circulating proteins occurred within a few minutes after traumatic brain injury. The time course for this barrier disruption is characterized by three different patterns: (1) transient, (2) prolonged, and (3) delayed opening. This variation in the development of barrier disruption may be related to the secondary barrier failure as well as the primary opening after injury.
39613812
Selective inhibition of inducible nitric oxide synthase reduces neurological deficit but not cerebral edema following traumatic brain injury
The role of inducible nitric oxide synthase (iNOS) in cerebral edema and neurological deficit following traumatic brain injury (TBI) is not yet clear-cut. Therefore, the aim of this study was to investigate the effect of three different iNOS inhibitors on cerebral edema and functional outcome after TBI. First, the time courses of blood--brain barrier (BBB) breakdown, cerebral edema, and neurological deficit were studied in a rat model of fluid percussion-induced TBI. The permeability of BBB to Evans blue was increased from 1 h to 24 h after TBI. Consistently, a significant increase in brain water content (BWC) was observed at 6 and 24 h post-TBI. A deficit in sensorimotor neurological functions was also observed from 6 h to 7 days with a maximum 24 h after TBI. Second, a single dose of aminoguanidine (AG; 100 mg/kg, i.p.), L-N-iminoethyl-lysine (L-NIL; 20 mg/kg, i.p.), or N-[3-(aminomethyl)benzyl]acetamide (1400W; 20 mg/kg, s.c.) was administered at 6 h post-TBI. Treatment with AG reduced by 71% the increase in BWC evaluated at 24 h, while L-NIL and 1400W had no effect. In contrast, the three iNOS inhibitors reduced the neurological deficit from 30% to 40%. Third, 1400W (20 mg/kg, s.c.) was administered at 5 min, 8 and 16 h post-TBI. Although this treatment paradigm had no effect on cerebral edema evaluated at 24 h, it significantly reduced the neurological deficit and iNOS activity. In conclusion, iNOS contributes to post-TBI neurological deficit but not to cerebral edema. The beneficial effect of iNOS inhibitors is not due to their anti-edematous effect, and the reduction of cerebral edema by AG is unlikely related to iNOS inhibition. The 6 h therapeutic window of iNOS inhibitors could allow their use in the treatment of functional deficit at the acute phase of TBI.
22577293
Key role of sulfonylurea receptor 1 in progressive secondary hemorrhage after brain contusion.
An important but poorly understood feature of traumatic brain injury (TBI) is the clinically serious problem of spatiotemporal progression ("blossoming") of a hemorrhagic contusion, a phenomenon we term progressive secondary hemorrhage (PSH). Molecular mechanisms of PSH are unknown and efforts to reduce it by promoting coagulation have met with equivocal results. We hypothesized that PSH might be due to upregulation and activation of sulfonylurea receptor 1 (SUR1)-regulated NC(Ca-ATP) channels in capillary endothelial cells, predisposing to oncotic death of endothelial cells and catastrophic failure of capillary integrity. Anesthetized adult male rats underwent left parietal craniectomy for induction of a focal cortical contusion. The regulatory subunit of the channel, SUR1, was prominently upregulated in capillaries of penumbral tissues surrounding the contusion. In untreated rats, PSH was characterized by progressive enlargement of the contusion deep into the site of cortical impact, including corpus callosum, hippocampus, and thalamus, by progressive accumulation of extravasated blood, with a doubling of the volume during the first 12 h after injury, and by capillary fragmentation in penumbral tissues. Block of SUR1 using low-dose (non-hypoglycemogenic) glibenclamide largely eliminated PSH and capillary fragmentation, and was associated with a significant reduction in the size of the necrotic lesion and in preservation of neurobehavioral function. Antisense oligodeoxynucleotide against SUR1, administered after injury, reduced both SUR1 expression and PSH, consistent with a requirement for transcriptional upregulation of SUR1. Our findings provide novel insights into molecular mechanisms responsible for PSH associated with hemorrhagic contusions, and point to SUR1 as a potential therapeutic target in TBI.
23325066
Blood–brain barrier permeability is positively correlated with cerebral microvascular perfusion in the early fluid percussion-injured brain of the rat
The blood–brain barrier (BBB) opening following traumatic brain injury (TBI) provides a chance for therapeutic agents to cross the barrier, yet the reduction of the cerebral microvascular perfusion after TBI may limit the intervention. Meanwhile, optimizing the cerebral capillary perfusion by the strategies such as fluid administration may cause brain edema due to the BBB opening post trauma. To guide the TBI therapy, we characterized the relationship between the changes in the cerebral capillary perfusion and BBB permeability after TBI. First, we observed the changes of the cerebral capillary perfusion by the intracardiac perfusion of Evans Blue and the BBB disruption with magnetic resonance imaging (MRI) in the rat subjected to lateral fluid percussion (FP) brain injury. The correlation between two variables was next evaluated with the correlation analysis. Since related to BBB breakdown, matrix metalloproteinase-9 (MMP-9) activity was finally detected by gelatin zymography. We found that the ratios of the perfused microvessel numbers in the lesioned cortices were significantly reduced at 0 and 1 h post trauma compared with that in the normal cortex, which then dramatically recovered at 4 and 24 h after injury, and that the BBB permeability was greatly augmented in the ipsilateral parts at 4, 12, and 24 h, and in the contralateral area at 24 h after injury compared with that in the uninjured brain. The correlation analysis showed that the BBB permeability increase was related to the restoration of the cerebral capillary perfusion over a 24-h period post trauma. Moreover, the gelatin zymography analysis indicated that the MMP-9 activity in the injured brain increased at 4 h and significantly elevated at 12 and 24 h as compared to that at 0 or 1 h after TBI. Our findings demonstrate that the 4 h post trauma is a critical turning point during the development of TBI, and, importantly, the correlation analysis may guide us how to treat TBI.
23575946
Interleukin-10 Improves Outcome and Alters Proinflammatory Cytokine Expression after Experimental Traumatic Brain Injury
Traumatic injury to the central nervous system initiates inflammatory processes that are implicated in secondary tissue damage. These processes include the synthesis of proinflammatory cytokines, leukocyte extravasation, vasogenic edema, and blood-brain barrier breakdown. Interleukin-10 (IL-10), a cytokine with antiinflammatory properties, negatively modulates proinflammatory cascades at multiple levels. We examined the hypothesis that IL-10 treatment can improve outcome in a clinically relevant model of traumatic brain injury (TBI). IL-10 was administered via different routes and dosing schedules in a lateral fluid-percussion model of TBI in rats. Intravenous administration of IL-10 (100 micrograms) at 30 min before and 1 h after TBI improved neurological recovery and significantly reduced TNF expression in the traumatized cortex at 4 h after injury. Such treatment was associated with lower IL-1 expression in the injured hippocampus, and to a lesser extent, in the injured cortex. Subcutaneous IL-10 administration (100 micrograms) at 10 min, 1, 3, 6, 9, and 12 h after TBI also enhanced neurological recovery. In contrast, intracerebroventricular administration of IL-10 (1 or 6 micrograms) at 15 min, 2, 4, 6, and 8 h after TBI was not beneficial. These results indicate that IL-10 treatment improves outcome after TBI and suggest that this improvement may relate, in part, to reductions in proinflammatory cytokine synthesis.
23631527
Injury-related production of cysteinyl leukotrienes contributes to brain damage following experimental traumatic brain injury.
The leukotrienes belong to a family of biologically active lipids derived from arachidonate that are often involved in inflammatory responses. In the central nervous system, a group of leukotrienes, known as the cysteinyl leukotrienes, is generated in brain tissue in response to a variety of acute brain injuries. Although the exact clinical significance of this excess production remains unclear, the cysteinyl leukotrienes may contribute to injury-related disruption of the brain-blood barrier and exacerbate secondary injury processes. In the present study, the formation and role of cysteinyl leukotrienes was explored in the fluid percussion injury model of traumatic brain injury in rats. The results showed that levels of the cysteinyl leukotrienes were elevated after fluid percussion injury with a maximal formation 1 hour after the injury. Neutrophils contributed to cysteinyl leukotriene formation in the injured brain hemisphere, potentially through a transcellular biosynthetic mechanism. Furthermore, pharmacological reduction of cysteinyl leukotriene formation after the injury, using MK-886, resulted in reduction of brain lesion volumes, suggesting that the cysteinyl leukotrienes play an important role in traumatic brain injury.
20165952
Alterations in blood-brain barrier permeability to large and small molecules and leukocyte accumulation after traumatic brain injury: effects of post-traumatic hypothermia.
We investigated the temporal and regional profile of blood-brain barrier (BBB) permeability to both large and small molecules after moderate fluid percussion (FP) brain injury in rats and determined the effects of post-traumatic modest hypothermia (33 degrees C/4 h) on these vascular perturbations. The visible tracers biotin-dextrin-amine 3000 (BDA-3K, 3 kDa) and horseradish peroxidase (HRP, 44 kDa) were injected intravenously at 4 h or 3 or 7 days post-TBI. At 30 min after the tracer infusion, both small and large molecular weight tracers were detected in the contusion area as well as remote regions adjacent to the injury epicenter in both cortical and hippocampal structures. In areas adjacent to the contusion site, increased permeability to the small molecular weight tracer (BDA-3K) was evident at 4 h post-TBI and remained visible after 7 days survival. In contrast, the larger tracer molecule (HRP) appeared in these remote areas at acute permeable sites but was not detected at later post-traumatic time periods. A regionally specific relationship was documented at 3 days between the late-occurring permeability changes observed with BDA-3K and the accumulation of CD68-positive macrophages. Mild hypothermia initiated 30 min after TBI reduced permeability to both large and small tracers and the infiltration of CD68-positive cells. These results indicate that moderate brain injury produces temperature-sensitive acute, as well as more long-lasting vascular perturbations associated with secondary injury mechanisms.
44397437
Diffusion-weighted imaging of edema following traumatic brain injury in rats: effects of secondary hypoxia.
Hypoxia and edema are frequent and serious complications of traumatic brain injury (TBI). Therefore, we examined the effects of hypoxia on edema formation after moderate lateral fluid percussion (LFP) injury using NMR diffusion-weighted imaging (DWI). Adult Sprague-Dawley rats were separated into four groups: sham uninjured (S), hypoxia alone (H), trauma alone (T), and trauma and hypoxia (TH). Animals in Groups T and TH received LFP brain injury, with Groups H and TH undergoing 30 min of moderately severe hypoxia (FiO2 = 0.11) immediately after surgery or TBI (respectively). DWIs were obtained at 2, 4, and 24 h and at 1 week post injury, and apparent diffusion coefficient (ADC) maps were constructed. Animals in Groups T and TH showed an early decrease (p < 0.001) in ADC values in the cortex ipsilateral to TBI 4 hr post injury, followed by elevated ADCs 1 week later (p < 0.05). No significant differences in ADC values were seen between T and TH groups in the ipsilateral cortex. In contrast, the ipsilateral hippocampus for Group TH showed only increasing ADC values. This hyperintensity in the ADC map began at 2 h after TBI, was significant by 24 h (p < 0.05), and reached a maximum at 1 week. This hyperintensity was not observed in Group T. Histopathology seen in TBI animals corresponded well with the pathology observed with MRI. Midline shifts reflecting edema were only observed in TBI animals with little difference between normoxic (T) and hypoxic animals (TH). In sum, this study demonstrates that the development and extent of brain edema following TBI can be examined in vivo in rats using DWI technology. TBI resulted in an early decrease in ADC values indicating cytotoxic edema in the cortex that was followed at 1 week by an increase in the ADC that was associated with decreased tissue cellularity. Histopathology corresponded well to the regions of brain injury and edema visualized by T2 and DWI procedures. Overall, the addition of hypoxia to brain injury resulted in a small increase in the magnitude of edema in hippocampus and cortex over that seen with trauma alone.
3007083
Global Cerebral Edema After Subarachnoid Hemorrhage: Frequency, Predictors, and Impact on Outcome
Background and Purpose— Cerebral edema visualized by CT is often seen after subarachnoid hemorrhage (SAH). Inflammatory or circulatory mechanisms have been postulated to explain this radiographic observation after SAH. We sought to determine the frequency, causes, and impact on outcome of early and delayed global cerebral edema after SAH. Methods— We evaluated the presence of global edema on admission and follow-up CT scans in 374 SAH patients admitted within 5 days of onset to our Neurological Intensive Care Unit between July 1996 and February 2001. Using multivariate analysis, we identified predictors of global cerebral edema and evaluated the impact of global edema on outcome 3 months after onset with the modified Rankin Scale. Results— Global edema was present on admission CT scans in 8% (n=29) and developed secondarily in 12% (n=44) of the patients. Global edema on admission was predicted by loss of consciousness at ictus and increasing Hunt-Hess grade. Delayed global edema was predicted by aneurysm size >10 mm, loss of consciousness at ictus, use of vasopressors, and increased SAH sum scores. Thirty-seven percent (n=137) of the patients were dead or severely disabled (modified Rankin Scale 4 to 6) at 3 months. Death or severe disability was predicted by any global edema, aneurysm size >10 mm, loss of consciousness at ictus, increased National Institutes of Health Stroke Scale scores, and older age. Conclusions— Global edema is an independent risk factor for mortality and poor outcome after SAH. Loss of consciousness, which may reflect ictal cerebral circulatory arrest, is a risk factor for admission global edema, and vasopressor-induced hypertension is associated with the development of delayed global edema. Critical care management strategies that minimize edema formation after SAH may improve outcome.
20960331
Intracerebral haemorrhage
Intracerebral haemorrhage is an important public health problem leading to high rates of death and disability in adults. Although the number of hospital admissions for intracerebral haemorrhage has increased worldwide in the past 10 years, mortality has not fallen. Results of clinical trials and observational studies suggest that coordinated primary and specialty care is associated with lower mortality than is typical community practice. Development of treatment goals for critical care, and new sequences of care and specialty practice can improve outcome after intracerebral haemorrhage. Specific treatment approaches include early diagnosis and haemostasis, aggressive management of blood pressure, open surgical and minimally invasive surgical techniques to remove clot, techniques to remove intraventricular blood, and management of intracranial pressure. These approaches improve clinical management of patients with intracerebral haemorrhage and promise to reduce mortality and increase functional survival.
23503310
Experimental intracerebral hemorrhage: relationship between brain edema, blood flow, and blood-brain barrier permeability in rats.
There have been few investigations of brain edema formation after intracerebral hemorrhage (ICH), despite the fact that mass effect and edema are important clinical complications. The present study was designed to investigate the time course for the formation and resolution of brain edema and to determine how changes in cerebral blood flow (CBF) and blood-brain barrier (BBB) permeability are temporally related to edema formation following ICH. Anesthetized adult rats received a sterile injection of 100 microliters of autologous blood into the caudate nucleus. Water and ion contents were measured immediately, at 4 and 12 hours, and daily to Day 7 (10 time points, six rats at each time) after experimental ICH. The water content of the ipsilateral basal ganglia increased progressively (p < 0.002) over the first 24 hours, then remained constant until after Day 5, when the edema began to resolve. Edema was most severe in the tissue immediately surrounding the hemorrhage; however, it was also present in the ipsilateral cortex, the contralateral cortex, and the basal ganglia. Measurements of local CBF (using [14C]-iodoantipyrine) and BBB permeability (using [3H]-alpha-aminoisobutyric acid) were obtained in separate groups of six to eight rats at various time intervals between 1 and 48 hours after ICH. Cerebral blood flow was reduced to 50% of control at 1 hour, returned to control values by 4 hours, but then decreased to less than 50% of control between 24 and 48 hours after ICH. The BBB permeability increased significantly prior to the occurrence of significant edema in the tissue surrounding the clot. However, BBB permeability in the more distant structures remained normal despite the development of edema. These results demonstrate a time course for the formation and resolution of brain edema following ICH similar to that observed during focal ischemia. Brain edema forms in the immediate vicinity of the clot as a result of both BBB disruption and the local generation of osmotically active substances and then spreads to adjacent structures. While local ischemia, due to the mass effect of the hemorrhage, may play a role in producing cytotoxic and vasogenic edema, the release of toxic substances from the clot should also be considered. Since edema is nearly maximal by 24 hours after ICH, therapy directed at reducing edema formation must be instituted within the 1st day.
22601053
Exploring neuroprotective drug therapies for intracerebral hemorrhage.
Intracerebral hemorrhage (ICH) is a devastating neurological disorder with high mortality and poor prognosis, for which virtually no effective drug therapies are available at present. Experimental animal models, based on intrastriatal injection of collagenase or autologous blood, have enabled great advances in elucidation of cellular/molecular events contributing to brain pathogenesis associated with ICH. Many lines of evidence indicate that blood constituents, including hemoglobin-derived products as well as proteases such as thrombin, play important roles in the pathogenic events. Inflammatory reactions involving neutrophils, activated microglia, and production of proinflammatory cytokines also constitute a critical aspect of pathology leading to neurodegeneration and tissue damage. Efforts are continuing to find drugs that potentially alleviate pathological and neurological outcomes of ICH. Various drugs that possess antioxidative, anti-inflammatory or neurotrophic/neuroprotective properties have been demonstrated to produce therapeutic effects on ICH animal models. Drugs already in clinical use such as minocycline, statins, and several nuclear receptor ligands are among the list of effective drugs, but whether they also show therapeutic efficacy in human ICH patients remains unproven. Here, current knowledge of ICH pathogenesis and problems arising with respect to exploration of new drug candidates are discussed.
22611943
Blood-brain barrier function in intracerebral hemorrhage.
In this paper, we review current knowledge on blood-brain barrier (BBB) dysfunction following intracerebral hemorrhage (ICH). BBB disruption is a hallmark of ICH-induced brain injury. Such disruption contributes to edema formation, the influx of leukocytes, and the entry of potentially neuroactive agents into the perihematomal brain, all of which may contribute to brain injury. A range of factors have been implicated in inducing BBB disruption, including inflammatory mediators (e.g., cytokines and chemokines), thrombin, hemoglobin breakdown products, oxidative stress, complement, and matrix metalloproteinases. While there is interaction between some of these mediators, it is probable that prevention of ICH-induced BBB disruption will involve blocking multiple pathways or blocking a common end pathway (e.g., by stabilizing tight junction structure). While the effects of ICH on BBB passive permeability have been extensively examined, effects on other 'barrier' properties (metabolic and transport functions) have been less well-studied. However, recent data suggests that ICH can affect transport and that this may help protect the BBB and the brain. Indeed, it is possible in small bleeds that BBB disruption may be beneficial, and it is only in the presence of larger bleeds that disruption has detrimental effects.
10068658
Vascular disruption and blood–brain barrier dysfunction in intracerebral hemorrhage
This article reviews current knowledge of the mechanisms underlying the initial hemorrhage and secondary blood–brain barrier (BBB) dysfunction in primary spontaneous intracerebral hemorrhage (ICH) in adults. Multiple etiologies are associated with ICH, for example, hypertension, Alzheimer’s disease, vascular malformations and coagulopathies (genetic or drug-induced). After the initial bleed, there can be continued bleeding over the first 24 hours, so-called hematoma expansion, which is associated with adverse outcomes. A number of clinical trials are focused on trying to limit such expansion. Significant progress has been made on the causes of BBB dysfunction after ICH at the molecular and cell signaling level. Blood components (e.g. thrombin, hemoglobin, iron) and the inflammatory response to those components play a large role in ICH-induced BBB dysfunction. There are current clinical trials of minimally invasive hematoma removal and iron chelation which may limit such dysfunction. Understanding the mechanisms underlying the initial hemorrhage and secondary BBB dysfunction in ICH is vital for developing methods to prevent and treat this devastating form of stroke.
14958392
The Molecular Mechanisms that Promote Edema After Intracerebral Hemorrhage
Intracerebral hemorrhage (ICH) is a devastating type of stroke with no effective therapies. Clinical advances in ICH treatment are limited by an incomplete understanding of the molecular mechanisms responsible for secondary injury and poor outcome. Increasing evidence suggests that cerebral edema is a major contributor to secondary injury and poor outcome in ICH. ICH activates specific signaling pathways that promote edema and damage neuronal tissue. By increasing our understanding of these pathways, we may be able to target them pharmaceutically to reduce edema in ICH patients. In this review, we focus on three major signaling pathways that promote edema after ICH: (1) the coagulation cascade and thrombin, (2) the inflammatory response and matrix metalloproteinases, and (3) the complement cascade and hemoglobin toxicity. We will describe the experimental evidence that confirms these pathways promote edema in ICH, discuss potential targets for new therapies, and comment on important directions for future research.
205161637
Experimental Intracerebral Hemorrhage: Avoiding Pitfalls in Translational Research
Intracerebral hemorrhage (ICH) has the highest mortality of all stroke subtypes, yet treatments are mainly limited to supportive management, and surgery remains controversial. Despite significant advances in our understanding of ICH pathophysiology, we still lack preclinical models that accurately replicate the underlying mechanisms of injury. Current experimental ICH models (including autologous blood and collagenase injection) simulate different aspects of ICH-mediated injury but lack some features of the clinical condition. Newly developed models, notably hypertension- and oral anticoagulant therapy-associated ICH models, offer added benefits but further study is needed to fully validate them. Here, we describe and discuss current approaches to experimental ICH, with suggestions for changes in how this condition is studied in the laboratory. Although advances in imaging over the past few decades have allowed greater insight into clinical ICH, there remains an important role for experimental models in furthering our understanding of the basic pathophysiologic processes underlying ICH, provided limitations of animal models are borne in mind. Owing to differences in existing models and the failed translation of benefits in experimental ICH to clinical practice, putative neuroprotectants should be trialed in multiple models using both histological and functional outcomes until a more accurate model of ICH is developed.
7937486
Comparison of different preclinical models of intracerebral hemorrhage.
Intracerebral hemorrhage (ICH) is the most devastating type of stroke. It is characterized by spontaneous bleeding in brain parenchyma and is associated with a high rate of morbidity and mortality. Presently, there is neither an effective therapy to increase survival after intracerebral hemorrhage nor a treatment to improve the quality of life for survivors. A reproducible animal model of spontaneous ICH mimicking the development of acute and delayed brain injury after ICH is an invaluable tool for improving our understanding of the underlying mechanisms of ICH-induced brain injury and evaluating potential therapeutic interventions. A number of models have been developed. While different species have been studied, rodents have become the most popular and widely utilized animals used in ICH research. The most often used methods for experimental induction of ICH are injection of bacterial collagenase and direct injection of blood into the brain parenchyma. The "balloon" method has also been used to mimic ICH for study. In this summary, we intend to provide a comparative overview of the technical methods, aspects, and pathologic findings of these types of ICH models. We will also focus on the similarities and differences among these rodent models, achievements in technical aspects of the ICH model, and discuss important aspects in selecting relevant models for study.
8493066
The rodent endovascular puncture model of subarachnoid hemorrhage: mechanisms of brain damage and therapeutic strategies
Subarachnoid hemorrhage (SAH) represents a considerable health problem. To date, limited therapeutic options are available. In order to develop effective therapeutic strategies for SAH, the mechanisms involved in SAH brain damage should be fully explored. Here we review the mechanisms of SAH brain damage induced by the experimental endovascular puncture model. We have included a description of similarities and distinctions between experimental SAH in animals and human SAH pathology. Moreover, several novel treatment options to diminish SAH brain damage are discussed.SAH is accompanied by cerebral inflammation as demonstrated by an influx of inflammatory cells into the cerebral parenchyma, upregulation of inflammatory transcriptional pathways and increased expression of cytokines and chemokines. Additionally, various cell death pathways including cerebral apoptosis, necrosis, necroptosis and autophagy are involved in neuronal damage caused by SAH.Treatment strategies aiming at inhibition of inflammatory or cell death pathways demonstrate the importance of these mechanisms for survival after experimental SAH. Moreover, neuroregenerative therapies using stem cells are discussed as a possible strategy to repair the brain after SAH since this therapy may extend the window of treatment considerably. We propose the endovascular puncture model as a suitable animal model which resembles the human pathology of SAH and which could be applied to investigate novel therapeutic therapies to combat this debilitating insult.
20010256
Inadvertent hyponatremia leading to acute cerebral edema 
and early evidence of herniation
IntroductionFor years, the maintenance of normal or supranormal serum sodium (Na) concentrations has been believed to be beneficial in brain injuries. Recently published guidelines for cerebral trauma recommend the use of hypertonic saline to achieve hypernatremia for the management of increased intracranial pressure and these standards are generally practiced across most diseases in neurocritical care including stroke, hemorrhage and tumors. Severe hyponatremia has long been known to be detrimental, but objective evidence for the harm of mild hyponatremia as a secondary injury has been scarce.Description of CaseIn this case report, we describe a child with aneurysmal subarachnoid hemorrhage who had a sudden, inadvertent decrease in serum Na (128 meq/l) that was associated with a deterioration of her neurological examination and evidence of early transtentorial herniation on emergent brain CT scan. These findings were quickly reversed after the serum Na was corrected.DiscussionThis report emphasizes that close monitoring of serum Na and osmolarity in acute head injured children is important, and provides evidence that alterations of these parameters is a substantial risk for cerebral edema in children with evolving brain injuries and briefly reviews the literature regarding the risks of hyponatremia in children.
12306590
Aquaporin-4 and brain edema
Aquaporin-4 (AQP4) is a water-channel protein expressed strongly in the brain, predominantly in astrocyte foot processes at the borders between the brain parenchyma and major fluid compartments, including cerebrospinal fluid (CSF) and blood. This distribution suggests that AQP4 controls water fluxes into and out of the brain parenchyma. Experiments using AQP4-null mice provide strong evidence for AQP4 involvement in cerebral water balance. AQP4-null mice are protected from cellular (cytotoxic) brain edema produced by water intoxication, brain ischemia, or meningitis. However, AQP4 deletion aggravates vasogenic (fluid leak) brain edema produced by tumor, cortical freeze, intraparenchymal fluid infusion, or brain abscess. In cytotoxic edema, AQP4 deletion slows the rate of water entry into brain, whereas in vasogenic edema, AQP4 deletion reduces the rate of water outflow from brain parenchyma. AQP4 deletion also worsens obstructive hydrocephalus. Recently, AQP4 was also found to play a major role in processes unrelated to brain edema, including astrocyte migration and neuronal excitability. These findings suggest that modulation of AQP4 expression or function may be beneficial in several cerebral disorders, including hyponatremic brain edema, hydrocephalus, stroke, tumor, infection, epilepsy, and traumatic brain injury.
12675648
Cerebral blood flow of rats with water-intoxicated brain edema.
Water intoxication brain edema was produced in rats by intraperitoneal loading of excessive amounts of distilled water (DW). In 10% and 20% groups, DW in amounts of 10 or 20% of body weight was injected, respectively. Water content of brain tissue increased proportionally to the amount of injected water, as follows: 79.8% of wet weight in control, 80.5 and 82.4 in 10% and 20% DW groups, respectively. Since cerebral blood flow (CBF) values measured by laser Doppler (LD) flowmetry were found to give a good correlation with those by hydrogen clearance method in a preliminary experiment, CBF measurement were carried out using LD flowmetry thereafter. Before the injection, CBF values were around 50 ml/min/100 g. Two hours after the water loading, CBF values in 10% and 20% DW groups were 25.6 and 20.3 ml/100 g/min, respectively. CBF values under these edematous condition decreased significantly (p < 0.001 by paired t-test) in proportion to the severity of the brain edema.
19709357
Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke
Cerebral edema contributes significantly to morbidity and death associated with many common neurological disorders. However, current treatment options are limited to hyperosmolar agents and surgical decompression, therapies introduced more than 70 years ago. Here we show that mice deficient in aquaporin-4 (AQP4), a glial membrane water channel, have much better survival than wild-type mice in a model of brain edema caused by acute water intoxication. Brain tissue water content and swelling of pericapillary astrocytic foot processes in AQP4-deficient mice were significantly reduced. In another model of brain edema, focal ischemic stroke produced by middle cerebral artery occlusion, AQP4-deficient mice had improved neurological outcome. Cerebral edema, as measured by percentage of hemispheric enlargement at 24 h, was decreased by 35% in AQP4-deficient mice. These results implicate a key role for AQP4 in modulating brain water transport, and suggest that AQP4 inhibition may provide a new therapeutic option for reducing brain edema in a wide variety of cerebral disorders.
28176383
Glial Cell Aquaporin-4 Overexpression in Transgenic Mice Accelerates Cytotoxic Brain Swelling*
Aquaporin-4 (AQP4) is a water transport protein expressed in glial cell plasma membranes, including glial cell foot processes lining the blood-brain barrier. AQP4 deletion in mice reduces cytotoxic brain edema produced by different pathologies. To determine whether AQP4 is rate-limiting for brain water accumulation and whether altered AQP4 expression, as occurs in various pathologies, could have functional importance, we generated mice that overexpressed AQP4 in brain glial cells by a transgenic approach using the glial fibrillary acid protein promoter. Overexpression of AQP4 protein in brain by ∼2.3-fold did not affect mouse survival, appearance, or behavior, nor did it affect brain anatomy or intracranial pressure (ICP). However, following acute water intoxication produced by intraperitoneal water injection, AQP4-overexpressing mice had an accelerated progression of cytotoxic brain swelling, with ICP elevation of 20 ± 2 mmHg at 10 min, often producing brain herniation and death. In contrast, ICP elevation was 14 ± 2 mmHg at 10 min in control mice and 9.8 ± 2 mmHg in AQP4 knock-out mice. The deduced increase in brain water content correlated linearly with brain AQP4 protein expression. We conclude that AQP4 expression is rate-limiting for brain water accumulation, and thus, that altered AQP4 expression can be functionally significant.
19349814
Targeted Overexpression of Endothelin-1 in Astrocytes Leads to More Severe Cytotoxic Brain Edema and Higher Mortality
Transgenic mice overexpressing endothelin-1 (ET-1) in astrocytes (GET-1) displayed more severe brain edema and neurologic dysfunction after experimental ischemic stroke. However, it was not clear whether astrocytic ET-1 contributed to cytotoxic or vasogenic edema associated with stroke. In this study, the role of astrocytic ET-1 in cytotoxic edema and brain injury was investigated. Upon acute water intoxication, the GET-1 mice had a lower survival rate and more severe neurologic deficits. Such an exacerbated condition in the GET-1 mice may be a result of a significant increase in cerebral water content and increased expression of the water channel protein, aquaporin 4 (AQP-4). The GET-1 mice treated with OPC-31260, a nonpeptide arginine vasopressin V2 receptor antagonist, were alleviated from the cerebral water accumulation and neurologic deficit during the early time period after water intoxication. In addition, a significant reduction of AQP-4 expression was observed in astrocytic end-feet AQP-4 in the hippocampus of the GET-1 mice treated with OPC-31260. Therefore, ET-1-induced AQP-4 expression and cerebral water accumulation are the key factors in brain edema associated with acute water intoxication. The V2 receptor antagonist, OPC-31260, may be one of the effective drugs for the early treatment of ET-1-induced cytotoxic edema and brain injury.
2191656
Both water intoxication and osmotic BBB disruption increase brain water content in rats.
Our previous experiments revealed that water intoxication and osmotic BBB disruption in the rat allow penetration of high-molecular substances into the brain and that resulting changes in the internal environment of the CNS lead to pathological development, such as the loss of integrity of myelin. The aim of the present study was to determine whether the previously described phenomena are associated with increased water content in the brain. To answer the question following methods were used: a) water intoxication: intraperitoneal administration of distilled water, b) osmotic BBB disruption: application of mannitol (20 %) selectively into the internal carotid artery, c) brain wet weight was measured after decapitation, and subsequently (after six days in thermostat set at 86 °C) the dry weight were estimated d) in animals with 20 % and 30 % hyperhydration the degree of myelin deterioration was estimated e) animal locomotor activity was tested by continuous behavior tracking and analysis. Brain water content after water intoxication and following the administration of mannitol was higher than in the control group. Different degrees of hyperhydration led to different levels of brain water content and to different degrees of myelin impairment. Hyperhydration corresponding to 20 % of the body weight brought about lower locomotor activity. Increased water content in the brain after the BBB osmotic disruption is surprising because this method is frequently used in the clinical practice.
5669316
Neurological complications of acute liver failure: Pathophysiological basis of current management and emerging therapies
One of the major causes of mortality in patients with acute liver failure (ALF) is the development of hepatic encephalopathy (HE) which is associated with increased intracranial pressure (ICP). High ammonia levels, increased cerebral blood flow and increased inflammatory response have been identified as major contributors to the development of HE and the related brain swelling. The general principles of the management of patients with ALF are straightforward. They include identifying the insult causing hepatic injury, providing organ systems support to optimize the patient's physical condition, anticipation and prevention of development of complications. Increasing insights into the pathophysiological mechanisms of ALF are contributing to better therapies. For instance, the evident role of cerebral hyperemia in the pathogenesis of increased ICP has led to a re-evaluation of established therapies such as hyperventilation, N-acetylcysteine, thiopentone sodium and propofol. The role of systemic inflammatory response in the pathogenesis of increased ICP has also gained importance supporting the concept that antibiotics given prophylactically reduce the risk of developing sepsis during the course of illness. Moderate hypothermia has also been established as a therapy able to reduce ICP in patients with uncontrolled intracranial hypertension and to prevent increases in ICP during orthopic liver transplantation. Ornithine phenylacetate, a new drug in the treatment of liver failure, and liver replacement therapies are still being investigated both experimentally and clinically. Despite many advances in the understanding of the pathophysiological basis and the management of intracranial hypertension in ALF, more clinical trials should be conducted to determine the best therapeutic management for this difficult clinical event.
29209513
Low grade cerebral edema and the pathogenesis of hepatic encephalopathy in cirrhosis
Whereas cerebral edema is a long-known complication of fulminant liver failure,1 patients with cirrhosis and hepatic encephalopathy (HE) usually show no clinical signs of overt cerebral edema and increased intracranial pressure (ICP). However, a first indication of the presence of low grade cerebral edema in patients with cirrhosis was derived from 1HMRS studies of the human brain in vivo. These noted a reduction in the myo-inositol signal in the brain (reflecting an osmosensitive myo-inositol pool) from patients with HE, together with an increased glutamine/glutamate signal. The glial localization of these osmolytes suggested a disturbance of astrocyte volume homeostasis.2 Based on this observation and available data from animal and in vitro studies, it was hypothesized that HE in cirrhosis reflects the clinical manifestation of a low grade cerebral edema, which may worsen under the influence of precipitating factors and thereby trigger cell hydration-dependent alterations of astrocyte function.2,3 Such alterations may affect glial-neuronal communication, whose disturbance is considered to be relevant to the pathogenesis of HE.4 Meanwhile, the existence of a low grade cerebral edema in patients with cirrhosis and HE in vivo was also demonstrated in studies on magnetization transfer ratios5,6 and by quantitative cerebral water mapping based on a new magnetic resonance (MR) technique for fast quantitative mapping of T1 and water content.7 Also, patients with noncirrhotic portal vein thrombosis and minimal HE exhibit MR findings consistent with increased brain water8 and as noted in this issue of HEPATOLOGY, mild brain edema develops in a recently characterized animal model of hepatic encephalopathy in cirrhosis.9 MR findings consistent with a low grade cerebral edema are already found in patients with minimal HE and become more pronounced when HE severity increases,2,8,10 for example, after implantation of a transjugular intrahepatic portalsystemic stent2 or experimentally induced hyperammonemia.11 Conversely, these magnetic resonance spectroscopy (MRS) abnormalities can resolve after liver transplantation5 or successful medical treatment of HE.6 Thus, fluctuations of HE severity in patients with cirrhosis roughly parallel the extent of low grade cerebral edema. In vitro studies on the mechanisms underlying this low grade cerebral edema have largely focused on the astrocyte. Astrocytes are the only compartment in the brain capable of ammonia detoxification via glutamine synthesis12 and when exposed to ammonia, can undergo socalled Alzheimer type II changes as a morphological counterpart of astrocyte swelling. Intra-astrocytic accumulation of osmotically active glutamine in liver cirrhosis and other hyperammonemic states contributes to the development of a low grade brain edema, as evidenced by consistently elevated 1H-MR spectroscopic glutamine signals in the brain of patients with cirrhosis. Further, methionine sulfoximine, an inhibitor of glutamine synthetase, prevents astrocyte swelling following an ammonia load in experimental animals.13 However, not only ammonia, but also benzodiazepines, hyponatremia and inflammatory cytokines can induce astrocyte swelling in vitro. Thus, different neurotoxins, repeatedly implicated in the pathogenesis of HE, may synergistically promote astrocyte swelling as one common pathogenetic endpoint. This may provide an explanation for another key feature of HE in patients with cirrhosis,2,3 namely that the syndrome is precipitated by rather heterogeneous factors, such as bleeding, infections, sedatives or electrolyte disturbances. However, it would be an oversimplification to postulate that ammonia-induced astrocyte swelling arises solely as a consequence of an accumulation of osmotically active glutamine. In cultured astrocytes, ammonia triggers the generation of oxidative stress,14,15 also induced in response to hypoosmotic swelling, benzodiazepines16 or inflammatory cytokines such as tumor necrosis factoror interferons (for review see Schliess et al.17). This oxidative stress response is mediated by activation of N-methyl-Daspartate (NMDA) glutamate receptors,15-17 but the mechanisms underlying this activation are unclear. Possible explanations are swellingand ammonia-induced glutamate release from astrocytes17,18 and/or deinhibition of Abbreviations: HE, hepatic encephalopathy; ICP, increased intracranial pressure; MR, magnetic resonance; MRS, magnetic resonance spectroscopy; NMDA, N-methyl-D-aspartate; PBR, peripheral type benzodiazepine receptor; NOS, nitric oxide synthases; PTN, protein tyrosine nitration. Address reprint requests to: Dieter Häussinger, Heinrich-Heine-Universitat — Medizinische Universitatsklinik, Moorenstrasse 5 Düsseldorf D-40225, Germany. E-mail: haeussin@uni-duesseldorf.de. fax: (49) 211-811-8838. Copyright © 2006 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.21235 Potential conflict of interest: Nothing to report.
23253577
Brain edema in liver failure: Basic physiologic principles and management
In patients with severe liver failure, brain edema is a frequent and serious complication that may result in high intracranial pressure and brain damage. This short article focuses on basic physiologic principles that determine water flux across the blood‐brain barrier. Using the Starling equation, it is evident that both the osmotic and hydrostatic pressure gradients are imbalanced across the blood‐brain barrier in patients with acute liver failure. This combination will tend to favor cerebral capillary water influx to the brain. In contrast, the disequilibration of the Starling forces seems to be less pronounced in patients with cirrhosis because the regulation of cerebral blood flow is preserved and the arterial ammonia concentration is lower compared with that of patients with acute liver failure. Treatments that are known to reverse high intracranial pressure tend to decrease the osmotic pressure gradients across the blood‐brain barrier. Recent studies indicate that interventions that restrict cerebral blood flow, such as hyperventilation, hypothermia, and indomethacin, are also efficient in preventing edema and high intracranial pressure, probably by decreasing the transcapillary hydrostatic pressure gradient. In our opinion, it is important to recall that rational fluid therapy, adequate ventilation, and temperature control are of direct importance to controlling cerebral capillary water flux in patients with acute liver failure. These simple interventions should be secured before more advanced experimental technologies are instituted to treat these patients.
31741264
Encephalopathy and Cerebral Edema in the Setting of Acute Liver Failure: Pathogenesis and Management
Cerebral edema is a potential life-threatening complication in patients with acute liver failure who progress to grade III/IV encephalopathy. The incidence is variably reported but appears to be most prevalent in those patients with hyperacute liver failure as opposed to subacute forms of liver failure. In those patients who are deemed at risk of cerebral edema and raised intracranial pressure, insertion of an intra-cranial pressure monitoring device may be considered to optimize treatment and interventions. The pathogenesis of cerebral edema in this setting remains controversial, although recent work suggests a pivotal role for arterial ammonia, whose effects appear to be potentiated by the presence of systemic inflammation. Recent work has also suggested the import of free radical formation occurring at a mitochondrial level as being the potential mediator of cellular dysfunction as opposed to ammonia per se. Treatment of such patients requires a multi-disciplinary approach incorporating both hepatology and critical care. In a significant proportion of such cases, consideration of liver transplantation may be required. Treatment should be focused at optimizing liver function and regenerative capacity and minimizing the inflammatory milieu. Controlled studies are lacking and much of the management has been extrapolated from neurocritical care. Sustained elevation of intracranial pressure may be responsive to mannitol or hypertonic saline bolus, and in those with hyperemia indomethacin has been reported as beneficial in case series. Recently, interest has developed into the use of cooling in the management of patients with acute liver failure and raised intracranial pressure. Animal studies support this treatment option as do case series, although randomized trials are still awaited.
36182271
Pathophysiology of cerebral oedema in acute liver failure.
Cerebral oedema is a devastating consequence of acute liver failure (ALF) and may be associated with the development of intracranial hypertension and death. In ALF, some patients may develop cerebral oedema and increased intracranial pressure but progression to life-threatening intracranial hypertension is less frequent than previously described, complicating less than one third of cases who have proceeded to coma since the advent of improved clinical care. The rapid onset of encephalopathy may be dramatic with the development of asterixis, delirium, seizures and coma. Cytotoxic and vasogenic oedema mechanisms have been implicated with a preponderance of experimental data favouring a cytotoxic mechanism. Astrocyte swelling is the most consistent neuropathological finding in humans with ALF and ammonia plays a definitive role in the development of cytotoxic brain oedema. The mechanism(s) by which ammonia induces astrocyte swelling remains unclear but glutamine accumulation within astrocytes has led to the osmolyte hypothesis. Current evidence also supports an alternate 'Trojan horse' hypothesis, with glutamine as a carrier of ammonia into mitochondria, where its accumulation results in oxidative stress, energy failure and ultimately astrocyte swelling. Although a complete breakdown of the blood-brain barrier is not evident in human ALF, increased permeation to water and other small molecules such as ammonia has been demonstrated resulting from subtle alterations in the protein composition of paracellular tight junctions. At present, there is no fully efficacious therapy for cerebral oedema other than liver transplantation and this reflects our incomplete knowledge of the precise mechanisms underlying this process which remain largely unknown.
187378
Prevention and management of brain edema in patients with acute liver failure
1 Intracranial pressure is the pressure exerted by the cranial contents on the dural envelope and consists of the partial pressures of the brain, blood, and cerebrospinal fluid. 2 Severe cases of acute liver failure are frequently complicated by brain edema (due to cytotoxic edema) and an increase in cerebral blood flow while the cerebrospinal fluid volume remains constant. 3 The development of intracranial hypertension in patients with acute liver failure may be controlled by manipulation of the position, body temperature, plasma tonicity, arterial carbon dioxide tension, and arterial pressure. 4 If intracranial hypertension evolves despite these first‐tier interventions, increased sedation, induction of hypothermia (body temperature of 33°C to 34°C), and the use of anti‐inflammatory drugs may help secure brain viability.
33243087
Endotoxemia produces coma and brain swelling in bile duct ligated rats
This study explores the hypothesis that the inflammatory response induced by administration of lipopolysaccharide (LPS) exacerbates brain edema in cirrhotic rats; and if so whether this is associated with altered brain metabolism of ammonia or anatomical disturbance of the blood‐brain barrier. Adult Sprague‐Dawley rats 4 weeks after bile duct ligation (BDL)/Sham‐operation, or naïve rats fed a hyperammonemic diet (HD), were injected with LPS (0.5 mg/kg, intraperitoneally) or saline, and killed 3 hours later. LPS administration increased brain water in HD, BDL, and sham‐operated groups significantly (P < 0.05), but this was associated with progression to pre‐coma stages only in BDL rats. LPS induced cytotoxic brain swelling and maintained anatomical integrity of the blood‐brain barrier. Plasma/brain ammonia levels were higher in HD and BDL rats than in sham‐operated controls and did not change with LPS administration. Brain glutamine/myoinositol ratio was increased in the HD group but reduced in the BDL animals. There was a background pro‐inflammatory cytokine response in the brains of cirrhotic rats, and plasma/brain tumor necrosis factor alpha (TNF‐α) and IL‐6 significantly increased in LPS‐treated animals. Plasma nitrite/nitrate levels increased significantly in LPS groups compared with non‐LPS controls; however, frontal cortex nitrotyrosine levels only increased in the BDL + LPS rats (P < 0.005 versus BDL controls). Conclusion: Injection of LPS into cirrhotic rats induces pre‐coma and exacerbates cytotoxic edema because of the synergistic effect of hyperammonemia and the induced inflammatory response. Although the exact mechanism of how hyperammonemia and LPS facilitate cytotoxic edema and pre‐coma in cirrhosis is not clear, our data support an important role for the nitrosation of brain proteins. (HEPATOLOGY 2007.)
20621358
Ammonia and related amino acids in the pathogenesis of brain edema in acute ischemic liver failure in rats
The pathogenesis of brain edema in acute liver failure is poorly understood. We have previously shown that rats with ischemic acute liver failure (portacaval anastomosis followed by hepatic artery ligation) exhibit brain edema and intracranial hypertension, with swelling of cortical astrocytes as the most prominent neuropathological abnormality. Because ammonia has been shown to induce swelling of astrocytes in vivo and in vitro, we examined the relationship between brain ammonia, amino acids generated from ammonia metabolism and brain water content in this model. Four groups of animals were studied: rats subjected to two sham operations, rats subjected to portacaval anastomosis and a sham operation, rats subjected to a sham operation and hepatic artery ligation and rats subjected to portacaval anastomosis and hepatic artery ligation. The last group of animals was studied at three progressive stages of encephalopathy. Cortical gray matter water increased from 80.26% ± 0.22% (sham + sham) to 82.46% ± 0.06% (last stage of devascularization). In cerebral cortex, brain ammonia increased to a maximum of 5.4 mmol/L. Glutamine, generated in glial cells from ammonia and glutamate, increased sixfold to 24 mmol/L and remained at this level throughout all stages of encephalopathy. Alanine, which may be generated from the transamination of glutamine, increased in parallel to the increase in water (r = 0.80, n = 15). In this model of fulminant liver failure and associated brain edema, brain ammonia increases to levels associated with in vitro swelling of brain slices and glial cells. The accumulation of osmogenic aminoacids such as glutamine and alanine may contribute to the selective astrocyte swelling seen in this condition. Ammonia, by its metabolically related amino acids, may play a role in the pathogenesis of brain edema in acute liver failure. (Hepatology 1992;15:449–453).
207068000
Aquaporin-4 deletion in mice reduces encephalopathy and brain edema in experimental acute liver failure
Brain edema and associated astrocyte swelling leading to increased intracranial pressure are hallmarks of acute liver failure (ALF). Elevated blood and brain levels of ammonia have been implicated in the development of brain edema in ALF. Cultured astrocytes treated with ammonia have been shown to undergo cell swelling and such swelling was associated with an increase in the plasma membrane expression of aquaporin-4 (AQP4) protein. Further, silencing the AQP4 gene in cultured astrocytes was shown to prevent the ammonia-induced cell swelling. Here, we examined the evolution of brain edema in AQP4-null mice and their wild type counterparts (WT-mice) in different models of ALF induced by thioacetamide (TAA) or acetaminophen (APAP). Induction of ALF with TAA or APAP significantly increased brain water content in WT mice (by 1.6% ± 0.3 and 2.3 ± 0.4%, respectively). AQP4 protein was significantly increased in brain plasma membranes of WT mice with ALF induced by either TAA or APAP. In contrast to WT-mice, brain water content did not increase in AQP4-null mice. Additionally, AQP4-null mice treated with either TAA or APAP showed a remarkably lesser degree of neurological deficits as compared to WT mice; the latter displayed an inability to maintain proper gait, and demonstrated a markedly reduced exploratory behavior, with the mice remaining in one corner of the cage with its head tilted downwards. These results support a central role of AQP4 in the brain edema associated with ALF.
12015650
Glutamine in the mechanism of ammonia-induced astrocyte swelling
Brain edema and the subsequent increase in intracranial pressure are the major neurological complications in fulminant hepatic failure (FHF). Brain edema in FHF is predominantly "cytotoxic" due principally to astrocyte swelling. It is generally believed that ammonia plays a key role in this process, although the mechanism by which ammonia brings about such swelling is yet to be defined. It has been postulated that glutamine accumulation in astrocytes subsequent to ammonia detoxification results in increased osmotic forces leading to cell swelling. While the hypothesis is plausible and has gained support, it has never been critically tested. In this study, we examined whether a correlation exists between cellular glutamine levels and the degree of cell swelling in cultured astrocytes exposed to ammonia. Cultured astrocytes derived from rat brain cortices were exposed to ammonia (5 mM) for different time periods and cell swelling was measured. Cultures treated with ammonia for 1-3 days showed a progressive increase in astrocyte cell volume (59-127%). Parallel treatment of astrocyte cultures with ammonia showed a significant increase in cellular glutamine content (60-80%) only at 1-4 h, a time when swelling was absent, while glutamine levels were normal at 1-3 days, a time when peak cell swelling was observed. Thus no direct correlation between cell swelling and glutamine levels was detected. Additionally, acute increase in intracellular levels of glutamine by treatment with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) after ammonia exposure also did not result in swelling. On the contrary, DON treatment significantly blocked (66%) ammonia-induced astrocyte swelling at a later time point (24 h), suggesting that some process resulting from glutamine metabolism is responsible for astrocyte swelling. Additionally, ammonia-induced free radical production and induction of the mitochondrial permeability transition (MPT) were significantly blocked by treatment with DON, suggesting a key role of glutamine in the ammonia-induced free radical generation and the MPT. In summary, our findings indicate a lack of direct correlation between the extent of cell swelling and cellular levels of glutamine. While glutamine may not be acting as an osmolyte, we propose that glutamine-mediated oxidative stress and/or the MPT may be responsible for the astrocyte swelling by ammonia.
33787400
Glutamine in the pathogenesis of acute hepatic encephalopathy
Hepatic encephalopathy (HE) is the major neurological disorder associated with liver disease. It presents in chronic and acute forms, and astrocytes are the major neural cells involved. While the principal etiological factor in the pathogenesis of HE is increased levels of blood and brain ammonia, glutamine, a byproduct of ammonia metabolism, has also been implicated in its pathogenesis. This article reviews the current status of glutamine in the pathogenesis of HE, particularly its involvement in some of the events triggered by ammonia, including mitochondrial dysfunction, generation of oxidative stress, and alterations in signaling mechanisms, including activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB). Mechanisms by which glutamine contributes to astrocyte swelling/brain edema associated with acute liver failure (ALF) will also be described.
19092025
NF-κB in the mechanism of brain edema in acute liver failure: Studies in transgenic mice
Astrocyte swelling and brain edema are major complications of the acute form of hepatic encephalopathy (acute liver failure, ALF). While elevated brain ammonia level is a well-known etiological factor in ALF, the mechanism by which ammonia brings about astrocyte swelling is not well understood. We recently found that astrocyte cultures exposed to ammonia activated nuclear factor-κB (NF-κB), and that pharmacological inhibition of such activation led to a reduction in astrocyte swelling. Although these findings suggest the involvement of NF-κB in astrocyte swelling in vitro, it is not known whether NF-κB contributes to the development of brain edema in ALF in vivo. Furthermore, pharmacological agents used to inhibit NF-κB may have non-specific effects. Accordingly, we used transgenic (Tg) mice that have a functional inactivation of astrocytic NF-κB and examined whether these mice are resistant to ALF-associated brain edema. ALF was induced in mice by treatment with the hepatotoxin thioacetamide (TAA). Wild type (WT) mice treated with TAA showed a significant increase in brain water content (1.65%) along with prominent astrocyte swelling and spongiosis of the neuropil, consistent with the presence of cytotoxic edema. These changes were not observed in Tg mice treated with TAA. Additionally, WT mice with ALF showed an increase in inducible nitric oxide synthase (iNOS) immunoreactivity in astrocytes from WT mice treated with TAA (iNOS is known to be activated by NF-κB and to contribute to cell swelling). By contrast, Tg mice treated with TAA did not exhibit brain edema, histological changes nor an increase in iNOS immunoreactivity. We also examined astrocytes cultures derived from Tg mice to determine whether these cells exhibit a lesser degree of swelling and cytopathological changes following exposure to ammonia. Astrocyte cultures derived from Tg mice showed no cell swelling nor morphological abnormalities when exposed to ammonia for 24h. By contrast, ammonia significantly increased cell swelling (31.7%) in cultured astrocytes from WT mice and displayed cytological abnormalities. Moreover, we observed a lesser increment in iNOS and NADPH oxidase activity (the latter is also known to be activated by NF-κB and to contribute to astrocyte swelling) in astrocyte cultures from Tg mice treated with ammonia, as compared to ammonia-treated WT mice astrocytes. These findings strongly suggest that activation of NF-κB is a critical factor in the development of astrocyte swelling/brain edema in ALF.
6190987
Sulfonylurea Receptor 1 Contributes to the Astrocyte Swelling and Brain Edema in Acute Liver Failure
Astrocyte swelling (cytotoxic brain edema) is the major neurological complication of acute liver failure (ALF), a condition in which ammonia has been strongly implicated in its etiology. Ion channels and transporters are known to be involved in cell volume regulation, and a disturbance in these systems may result in cell swelling. One ion channel known to contribute to astrocyte swelling/brain edema in other neurological disorders is the ATP-dependent, nonselective cation (NCCa-ATP) channel. We therefore examined its potential role in the astrocyte swelling/brain edema associated with ALF. Cultured astrocytes treated with 5 mM ammonia showed a threefold increase in the sulfonylurea receptor type 1 (SUR1) protein expression, a marker of NCCa-ATP channel activity. Blocking SUR1 with glibenclamide significantly reduced the ammonia-induced cell swelling in cultured astrocytes. Additionally, overexpression of SUR1 in ammonia-treated cultured astrocytes was significantly reduced by cotreatment of cells with BAY 11–7082, an inhibitor of NF-κB, indicating the involvement of an NF-κB-mediated SUR1 upregulation in the mechanism of ammonia-induced astrocyte swelling. Brain SUR1 mRNA level was also found to be increased in the thioacetamide (TAA) rat model of ALF. Additionally, we found a significant increase in SUR1 protein expression in rat brain cortical astrocytes in TAA-treated rats. Treatment with glibenclamide significantly reduced the brain edema in this model of ALF. These findings strongly suggest the involvement of NCCa-ATP channel in the astrocyte swelling/brain edema in ALF and that targeting this channel may represent a useful approach for the treatment of the brain edema associated with ALF.
28258289
Brain Aquaporin-4 in Experimental Acute Liver Failure
Intracranial hypertension caused by brain edema and associated astrocyte swelling is a potentially lethal complication of acute liver failure (ALF). Mechanisms of edema formation are not well understood, but elevated levels of blood and brain ammonia and its by-product glutamine have been implicated in this process. Since aquaporin-4 (AQP4) has been implicated in brain edema in other conditions, we examined its role in a rat model of ALF induced by the hepatotoxin thioacetamide. Rats with ALF showed increased AQP4 protein in the plasma membrane (PM). Total tissue levels of AQP4 protein and mRNA levels were not altered, indicating that increased AQP4 is not transcriptionally mediated but likely reflects a more stable anchoring of AQP4 to the PM and/or interference with its degradation. An increase inAQP4 immunoreactivity in thePM was observed in perivascular astrocytes in ALF. Rats with ALF also showed increased levels of &agr;-syntrophin, a protein involved in anchoringAQP4 to perivascular astrocytic end-feet. Increased AQP4 and&agr;-syntrophin levels were inhibited by L-histidine, an inhibitor of glutamine transport into mitochondria, suggesting a role for glutamine in the increase of PM levels of AQP4. These results indicate that increased AQP4 PM levels in perivascular astrocytic end-feet are likely critical to the development of brain edema in ALF.
5209633
Brain edema in acute liver failure: inhibition by L-histidine.
Brain edema and the associated increase in intracranial pressure are potentially lethal complications of acute liver failure (ALF). Astrocyte swelling (cytotoxic edema) represents a significant component of the brain edema in ALF, and elevated blood and brain ammonia levels have been strongly implicated in its formation. We earlier showed in cultured astrocytes that oxidative stress (OS) and the mitochondrial permeability transition (mPT) play major roles in the mechanism of ammonia-induced astrocyte swelling. Glutamine, a byproduct of ammonia metabolism, has also been shown to induce OS, the mPT, and astrocyte swelling. Such effects of glutamine were suggested to be mediated by its hydrolysis in mitochondria, potentially yielding high levels of ammonia in this organelle and leading to OS and the mPT. L-histidine, an inhibitor of mitochondrial glutamine transport, was recently shown to mitigate OS, mPT, and cell swelling in cultured astrocytes treated with ammonia. The present study examined whether L-histidine similarly abolishes OS, the mPT, and brain edema in a rat model of ALF. Treatment of rats with thioacetamide caused a significant degree of brain edema, which was associated with induction of OS and the mPT. These changes were completely abolished by L-histidine, supporting a key role of mitochondrial glutamine transport and hydrolysis in the mechanism of the brain edema associated with ALF.
18748250
Brain edema in acute liver failure: mechanisms and concepts
Brain edema and associated increase in intracranial pressure continue to be lethal complications of acute liver failure (ALF). Abundant evidence suggests that the edema in ALF is largely cytotoxic brought about by swelling of astrocytes. Elevated blood and brain ammonia levels have been strongly implicated in the development of the brain edema. Additionally, inflammation and sepsis have been shown to contribute to the astrocyte swelling/brain edema in the setting of ALF. We posit that ammonia initiates a number of signaling events, including oxidative/nitrative stress (ONS), the mitochondrial permeability transition (mPT), activation of the transcription factor (NF-κB) and signaling kinases, all of which have been shown to contribute to the mechanism of astrocyte swelling. All of these factors also impact ion-transporters, including Na+, K+, Cl− cotransporter and the sulfonylurea receptor 1, as well as the water channel protein aquaporin-4 resulting in a perturbation of cellular ion and water homeostasis, ultimately resulting in astrocyte swelling/brain edema. All of these events are also potentiated by inflammation. This article reviews contemporary knowledge regarding mechanisms of astrocyte swelling/brain edema formation which hopefully will facilitate the identification of therapeutic targets capable of mitigating the brain edema associated with ALF.
2248990
Brain region-selective mechanisms contribute to the progression of cerebral alterations in acute liver failure in rats.
BACKGROUND & AIMS
Patients with acute liver failure (ALF) often die of intracranial pressure (IP) and cerebral herniation. Main contributors to increased IP are ammonia, glutamine, edema, and blood flow. The sequence of events and underlying mechanisms, as well as the temporal pattern, regional distribution, and contribution of each parameter to the progression of neurologic deterioration and IP, are unclear. We studied rats with ALF to follow the progression of changes in ammonia, glutamine, grade and type (vasogenic or cytotoxic) of edema, blood-brain barrier permeability, cerebral blood flow, and IP. We assessed whether the changes in these parameters were similar between frontal cortex and cerebellum and evaluated the presence, type, and progression of edema in 12 brain areas.


METHODS
ALF was induced by injection of galactosamine. The grade and type of edema was assessed by measuring the apparent diffusion coefficient by magnetic resonance imaging. Cerebral blood flow was measured by magnetic resonance and blood-brain barrier permeability by Evans blue-albumin extravasation.


RESULTS
Increased IP arises from an early increase of blood-brain barrier permeability in certain areas (including cerebellum but not frontal cortex) followed by vasogenic edema. Ammonia and glutamine then increase progressively, leading to cytotoxic edema in many areas. Alterations in lactate and cerebral blood flow are later events that further increase IP.


CONCLUSIONS
Different mechanisms in specific regions of the brain contribute, with different temporal patterns, to the progression of cerebral alterations and IP in ALF.
11169951
TIMP-1/MMP-9 imbalance in brain edema in rats with fulminant hepatic failure.
BACKGROUND
Fulminant hepatic failure (FHF) is a devastating disease. When coma sets in, brain edema develops, changing FHF into a lethal condition. Liver transplantation is the definitive treatment. However, a third of these patients die as the result of brain edema before a donor becomes available. Tissue inhibitor of matrix metalloproteinase (MMP), or TIMP, and MMP-9 are implicated in ischemic brain edema. We thus hypothesized that an imbalance in TIMP-1/MMP-9 relationship plays a role in the development of increased brain extravasation and edema in FHF.


MATERIALS AND METHODS
FHF was induced with a single intraperitoneal injection of D-galactosamine (250 mg/kg). Control rats received saline. GM6001, a synthetic MMP inhibitor, was administered (30 mg/kg) every 12 h for 3 doses starting at 12 h after D-galactosamine injection. MMP-9 was assayed with standard gelatin zymography. Brain extravasation, a measurement of the blood-brain barrier permeability, was determined with Evans blue. Brain edema was determined using specific gravity method.


RESULTS
The active MMP-9 in the systemic circulation was significantly increased in the comatose FHF as compared to the precoma FHF and control animals (6.5 +/- 0.7 versus 4.6 +/- 0.4 versus 2.6 +/- 0.5 pg/microg, respectively; P < 0.05). Conversely, TIMP-1 was steadily decreased in precoma and coma FHF rats by 35% and 45%, respectively. Blocking MMP-9 activity with GM6001 significantly attenuated brain extravasation and edema in rats with FHF.


CONCLUSIONS
Our study strongly supports that the perturbation of decreased TIMP-1 and increased MMP-9 contributes to the pathogenesis of brain edema in FHF. Our findings present a potential therapeutic approach to effectively increase the window of opportunity for life-saving liver transplantation.
1946772
Increased aquaporin-4 expression in ammonia-treated cultured astrocytes
Brain edema is a serious complication of hepatic encephalopathy associated with fulminant hepatic failure (FHF). Factors responsible for such swelling are not clear, but ammonia, a toxin strongly implicated in FHF, is known to induce astrocyte swelling. The mechanism(s) responsible for ammonia-induced swelling, however, are not known. Aquaporins are plasma membrane proteins that mediate transmembrane water movement. To investigate the potential role of aquaporins in astrocyte swelling, we measured aquaporin-4 (AQP-4) protein expression in cultured astrocytes exposed to 5 mM NH4Cl. AQP-4 levels significantly increased 10 h after treatment with ammonia, and displayed a progressive rise up to 48 h, which appeared to precede the onset of astrocyte swelling. AQP-4 may be involved in the astrocyte swelling associated with hyperammonemic states.
9251205
Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling*
Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH4Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation.
5920685
Brain Water Content: a Misunderstood Measurement?
Brain edema is a major contributor to poor outcome following ischemic and hemorrhagic stroke. In animal models, edema has historically been quantified as a change in brain water content expressed as a percentage of wet weight (% water content). As described in this communication, this number can be misleading, as “small” changes in percentage of brain water content actually reflect much bigger changes in brain swelling. Using either water content, expressed as grams per gram of dry weight, or a measure of brain swelling better reflect the impact of edema after stroke and brain injury.
44288756
A simple gravimetric technique for measurement of cerebral edema.
A simple method was developed for the laboratory preparation of gradient columns of specific gravity used in measurement of brain-tissue water. By this automated technique, virtually linear and repeatable density gradients were obtained from which values of tissue specific gravity could be determined. The specific gravity of both solid and fresh cortex and white matter from adult cats was measured and converted to units of percent water per gram tissue using conversion factors derived for this purpose and applicable to studies of brain edema.
33489600
Spreading of vasogenic edema and cytotoxic edema assessed by quantitative diffusion and T2 magnetic resonance imaging.
BACKGROUND AND PURPOSE
The apparent diffusion coefficient (ADC) of water should be sensitive to the cytotoxic edema triggered by energy failure during ischemia. Elevated values of T2. the nuclear MR transverse relaxation time of water, seen on T2 nuclear MR images detect vasogenic edema and infarcted areas. The temporal and spatial changes in ADC and T2 abnormalities after occlusion of the middle cerebral artery (MCAO) were therefore estimated by these two quantitative techniques.


METHODS
Permanent MCAO was performed on rats. Quantitative ADC and T2 maps of brain water were obtained, from which the ischemic volumes were calculated at various times up to 48 hours after MCAO.


RESULTS
The areas of decreased ADC represented 36 +/- 7% of the final infarct volume (24 hours) at 0.5 hours and 64 +/- 4% at 5 hours after MCAO, suggesting that there is recruitment of peripheral areas with disturbed energy metabolism and cytotoxic edema. The ADC and T2 contours closely matched at 3.5, 24, and 48 hours after MCAO.


CONCLUSIONS
T2 imaging can assess ischemic insults as well as ADC imaging, but only 3.5 hours after the onset of ischemia. Assessment of edematous swelling (approximately 24.5% of total infarcted volume) demonstrates that ADC and therefore T2 imaging detect all the tissue that will become infarcted approximately 7 hours after occlusion. The spread of ADC and T2 abnormalities would therefore stop at approximately 7 hours, and any further increase in volume observed on the images would be mainly due to edematous swelling.
19883373
Temporal and Regional Evolution of Aquaporin-4 Expression and Magnetic Resonance Imaging in a Rat Pup Model of Neonatal Stroke
Edema formation can be observed using magnetic resonance imaging (MRI) in patients with stroke. Recent studies have shown that aquaporin-4 (AQP4), a water channel, is induced early after stroke and potentially participates in the development of brain edema. We studied whether induction of AQP4 correlated with edema formation in a rat pup filament stroke model using high field (11.7-Tesla) MRI followed by immunohistochemical investigation of AQP4 protein expression. At 24 h, we observed increased T2 values and decreased apparent diffusion coefficients (ADC) within injured cortical and striatal regions that reflected the edema formation. Coincident with these MR changes were significant increases in AQP4 expression on astrocytic end-feet in the border regions of injured tissues. Striatal imaging findings were still present at 72 h with a slow normalization of AQP4 expression in the border regions. At 28 d, AQP4 expression normalized in the border while in this region ADC values increased. We show that induction of AQP4 is increased during the period of active edema formation in the border region without regional correlation with edema. Finally, induction of AQP4 on astrocyte end-feet could participate in tissue preservation after ischemia in the immature rat brain.
30002765
Lasting cytotoxic edema as an indicator of irreversible brain damage: a case of neonatal stroke.
We describe a case of neonatal stroke in the territory of the left middle cerebral artery. Although the ischemic lesion appeared rather homogeneously hyperintense on T2-weighted MR images, corresponding diffusion-weighted images clearly delineated two separate zones of different cellular swelling and, thus, different prognoses. Lasting cytotoxic edema heralded infarction. We believe that the different rates of disintegration of neurons and glial cells may have caused the change from intracellular to interstitial volume fraction during the evolution of edema.
12274082
Biology of vascular endothelial growth factors
Angiogenesis is the process by which new blood vessels are formed from existing vessels. The vascular endothelial growth factors (VEGFs) are considered as key molecules in the process of angiogenesis. The VEGF family currently includes VEGF‐A, ‐B, ‐C, ‐D, ‐E, ‐F and placenta growth factor (PlGF), that bind in a distinct pattern to three structurally related receptor tyrosine kinases, denoted VEGF receptor‐1, ‐2, and ‐3. VEGF‐C and VEGF‐D also play a crucial role in the process of lymphangiogenesis. Here, we review the biology of VEGFs and evaluate their role in pathological angiogenesis and lymphangiogenesis.
24870277
Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats.
BACKGROUND AND PURPOSE
Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and also has the potential to increase vascular permeability. Therefore, it may contribute to the recovery of brain cells from ischemic insult through potentiating neovascularization or may exacerbate brain damage by forming brain edema. However, the exact role of this protein in cerebral ischemia is not fully understood. We investigated temporal, spatial, and cellular profiles of the induction of VEGF gene expression after transient focal cerebral ischemia at both mRNA and protein levels.


METHODS
We used a transient middle cerebral artery (MCA) occlusion model. Northern blot analysis was performed to assess the chronological pattern of induction and the impact of length of ischemia on mRNA expression. Western blot analysis was performed to ensure the selective detection of immunoreactive VEGF with an antibody. Temporal, spatial, and cellular changes of immunohistochemical VEGF expression were compared with different periods of reperfusion from 1 hour to 7 days after transient MCA occlusion.


RESULTS
(1) Northern blot analysis revealed no detectable VEGF mRNA in the control brains. The mRNA became evident at 1 hour after reperfusion, peaked at 3 hours, and then decreased. The length of ischemia from 1 to 3 hours made no differences in the degree and temporal profile of the subsequent induction of VEGF mRNA. (2) Western blot analysis showed no band in the control brain, but two bands with molecular weights of 38 and 45 kD, corresponding to VEGF121 and VEGF165, were induced at 1 hour of reperfusion, peaked at 3 hours of reperfusion, and then decayed. (3) Neurons in the cerebral cortex of the MCA territory expressed VEGF at 1 hour after reperfusion with a peak at 3 hours and then diminished by 1 day. Pial cells of the MCA territory also expressed immunoreactive VEGF from 1 hour of reperfusion that was sustained until 3 to 7 days after reperfusion.


CONCLUSIONS
Rapid induction of VEGF gene expression after transient MCA occlusion was demonstrated at both mRNA and protein levels. Cortical neurons and pial cells were the source of VEGF production in this model, but the temporal profiles of the induction between these cells were different. The early but dissociative induction of VEGF between neuronal and pial cells suggests different roles of the protein in their cells after transient MCA occlusion.
21274271
Differential Expression of Vascular Endothelial Growth Factor‐A (VEGF‐A) and VEGF‐B After Brain Injury
Our previous study demonstrated that vascular endothelial growth factor (VEGF), now referred to as VEGF-A, plays a significant role in blood-brain barrier (BBB) breakdown and angiogenesis after brain injury. In this study, VEGF-A expression was compared with that of VEGF-B in the rat cortical cold injury model over a period of 6 hours to 6 days post-injury. VEGF-A and VEGF-B mRNA were detected by in situ hybridization and their protein was detected by immunohistochemistry. The presence of VEGF-A and VEGF-B proteins in endothelium of lesion vessels was related to BBB breakdown by double labeling for either of these growth factors and fibronectin, which was used as a marker of BBB breakdown. Significant induction of both VEGF-A and VEGF-B mRNA occurred at the lesion site during the period of maximal endothelial proliferation. VEGF-A mRNA levels peaked at 3 and 4 days post-injury and returned to basal expression by day 6, while VEGF-B mRNA levels remained elevated up to day 6. VEGF-B protein was constitutively expressed in endothelium of all cerebral vessels. After brain injury, there was increased immunoreactivity for VEGF-B at the lesion site, this protein being present in the endothelium and vascular smooth muscle cells of pial vessels, in inflammatory cells, and later in proliferating endothelial cells, endothelium of neovessels, and astrocytes. Lesion vessels showing BBB breakdown to fibronectin showed endothelial VEGF-A protein but not VEGF-B protein. Constitutive expression of VEGF-B in normal endothelium suggests that it may have a role in maintenance of the BBB in steady states, while its induction at both the gene and protein level post-injury indicates that it has an essential role in angiogenesis and the repair processes after brain injury.
33658679
VEGF and VEGF receptor expression after experimental brain contusion in rat.
Angiogenesis following traumatic brain injury (TBI) may be of importance not only for post-traumatic reparative processes but also for the development of secondary injuries. Vascular endothelial growth factor (VEGF) is a major regulator of endothelial cell proliferation, angiogenesis, and vascular permeability, though its possible involvement in secondary injuries after TBI is largely unknown. This study was undertaken to analyze the expression of VEGF and the VEGF receptors in experimental brain contusion in rat. Twenty-three adult female Sprague-Dawley rats were subjected to a focal cerebral contusion injury by use of a weight-drop model. Four additional rats underwent craniotomy only. The animals were sacrificed 6 h, or 1, 2, 4, 6, 8, or 16 days post-injury. Expression of VEGF and the VEGF receptors VEGFR1 (Flt-1) and VEGFR2 (Flk-1) were studied by in situ hybridization and immunohistochemistry. VEGF messenger (m)RNA and protein expression were detected in astrocytes, neutrophils, and macrophages in or adjacent to the injury from 1 day after injury, with a peak expression after 4-6 days. Flt-1 and Flk-1 mRNA and protein were detected in vessels adjacent to the lesion from 1 day after injury throughout day 6 after injury. It was also noted that Flt-1/Flk-1 and VEGF-positive vessels often were negative for SMI-71, a marker for vessels in areas with blood-brain barrier (BBB). In conclusion, we have demonstrated that TBI leads to an upregulation of VEGF, Flt-1, and Flk-1 mRNA and protein in and around the lesion. The data provide a foundation for future pharmacological intervention studies focusing on posttraumatic angiogenesis and possible injury repair effects of the VEGF system in TBI.
21791783
Vascular endothelial growth factor and its receptor, KDR, in human brain tissue after ischemic stroke.
Ischemic stroke results from a reduction in cerebral blood flow to a focal region of the brain after the occlusion of an artery, causing damage to nervous tissue. There is a region of cerebral ischemic tissue (penumbra) surrounding an acute cerebral infarct that is dysfunctional but potentially viable. Restoration of perfusion in the penumbra may ameliorate the tissue damage. The identity and the role of growth factors that control the extent of tissue damage and its repair are poorly understood. Angiogenesis has been demonstrated to occur in brain tissues of patients surviving an acute ischemic stroke. In this paper we have investigated the status of a potent angiogenesis factor, vascular endothelial growth factor (VEGF), in patients after acute ischemic brain stroke. Western blotting and immunohistochemistry were used to determine protein expression, and in situ hybridization was used to quantify and localize mRNA synthesis. The expression of VEGF protein was increased in the penumbra compared with infarcted brain and contralateral hemisphere. Neurones, endothelial cells, and astrocytes in the penumbra in all patients studied had significant up-regulation of both VEGF165 and VEGF189 mRNA (p < 0.01, Wilcoxon Matched-Pairs Signed-Ranks Test) compared with infarcted tissue and the normal looking contralateral hemisphere that was used as a control. Immunohistochemistry demonstrated that kinase insert domain receptor was present in blood vessels within the infarct/penumbra and absent from the normal contralateral hemisphere. VEGF, which is important in angiogenesis, may also influence long term neuronal survival, and possibly its modulation may prove to be of therapeutic value for patients with ischemic stroke.
12941881
Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct.
Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.
22618262
VEGF expression in human brain tissue after acute ischemic stroke.
Ischemic stroke is the third most common cause of death in humans, requiring further studies to elucidate its pathophysiological background. One potential mechanism to increase oxygen delivery to the affected tissue is induction of angiogenesis. The most potent proangiogenic factor is VEGF. For this reason, our study investigated immunohistochemically VEGF reactivity in different cellular brain compartments from 15 ischemic stroke patients, as well as from 2 age control cases. By enzymatic immunohistochemistry, we investigate VEGF expression in different brain cell compartments and then we quantified its signal intensity by assessing integrated optical densities (IOD). To establish the exact cellular brain topography of VEGF immunoreactivity we performed double fluorescent immunohistochemistry series (VEGF÷NeuN, GFAP, CD68, CD105). In control samples, VEGF reactivity was observed especially in neurons from the Brodmann cortical layers IV to VI and in protoplasmic astrocytes from the deeper layers of gray matter and in endothelial cells from normal blood vessels because of systemic hypoxia generated after death. In acute ischemic stroke samples, this reactivity was noticed in all brain cellular compartments but with different intensities. The most reactive compartment was the neurons, the intensity of VEGF reaction decreasing with the lesional age from the core infarct toward intact adjacent brain cortex. With a lower intensity, VEGF reaction was noticed in astrocytes compartments, especially in gemistocytic astrocytes adjacent to the liquefaction zone. We also noticed a weak reaction in activated non-phagocytic microglia from the periphery of liquefaction zones, and high VEGF-CD105 colocalization values at the level of microvessels that surround the infarcted brain area. In conclusion, this reactivity could suggest that VEGF might exhibit neuronal and glial protective effects and also a neoangiogenic property in acute ischemic stroke, facts that may have significant therapeutically impact on these patients.
561172
Vascular Endothelial Growth Factor Is Increased in Cerebrospinal Fluid after Traumatic Brain Injury in Infants and Children
OBJECTIVEVascular endothelial growth factor (VEGF) is an important regulator of angiogenesis, the formation of which is triggered by hypoxia, cytokines, and growth factors and is also induced by activation of the adenosine 2B receptor. VEGF is neuroprotective in several models of experimental brain injury and is increased in brain after traumatic brain injury (TBI) in humans and experimental animals. Adenosine is a neuroprotective purine metabolite that increases in cerebrospinal fluid (CSF) after clinical TBI in children. We hypothesized that VEGF levels would 1) be increased in CSF after TBI in infants and children, and 2) be preceded by increases in CSF adenosine. To test this hypothesis, we designed a case-control study to compare the CSF of infants and children after severe TBI with that of uninjured children. METHODSUsing an Institutional Review Board-approved protocol, we compared CSF concentrations of VEGF (by enzyme-linked immunosorbent assay) and adenosine (by high-performance liquid chromatography) in 73 samples from 14 infants and children with severe TBI (Glasgow Coma Scale score ≤8) with those in CSF from 5 noninjured control subjects. Patients received standard neurointensive care. RESULTSMean VEGF levels were increased after TBI versus control (39.8 ± 6.2 versus 14.9 ± 1.5 ng/dl, mean ± standard error of the mean, P = 0.0002) and peaked at 91.6 ± 26.4 ng/dl, approximately 6 times control (P = 0.001). Peak VEGF occurred at 22.4 hours after injury. There was a trend toward increased adenosine concentration after TBI versus control (18.3 ± 3.5 versus 11.5 ± 2.3 nmol/L), but this did not reach statistical significance. A multivariate regression model showed an independent, significant association between the concentrations of VEGF and adenosine. CONCLUSIONVEGF is increased in CSF after pediatric TBI, and this increase is associated with an increase in CSF adenosine. These results may imply that a component of the vascular regenerative response of the brain is initiated rapidly after TBI and continues for several days after injury. Further investigation is warranted to determine 1) whether this association is causative, 2) the role of adenosine in triggering the increase in CSF VEGF concentration, and 3) the exact role VEGF that plays after injury.
7123496
Delayed Progression of Edema Formation Around a Hematoma Expressing High Levels of VEGF and MMP-9 in a Patient With Traumatic Brain Injury: Case Report
The mechanisms accounting for the development of tissue damage following traumatic brain injury (TBI) have been studied for several decades. A variety of mediators, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), which play a crucial role in edema formation after TBI, have been identified. We experienced a case of brain edema that progressed continuously at least until 13 days after head injury. The brain edema occurred around the hemorrhage from an intracerebral contusion. The evacuated hematoma was investigated based on the inference that the unexpected expansion of edema was induced by the mediators within the hematoma itself. A 64-year-old woman was admitted to our hospital following a traffic injury. Left brain contusion was revealed by head computed tomography (CT) on admission. Three hours later, formation of an intracerebral hematoma became evident. Serial CT examination revealed that brain edema had developed progressively till 13 days after the injury. A hematoma removal operation was performed on Day 13. The hematoma was centrifuged and the supernatant was analyzed for the expression of VEGF and MMP-9. The values of both (4400 pg/ml and 920 ng/ml, respectively) were extremely high compared with values reported previously in serum and cerebrospinal fluid collected from patients with intracranial infection or injury. This case suggested that the delayed exacerbation of edema following traumatic intracranial hemorrhage was possibly induced by secretory factors such as VEGF and MMP-9 released from within and around the hematoma.
5636010
Clinical significance of plasma VEGF value in ischemic stroke - research for biomarkers in ischemic stroke (REBIOS) study
BackgroundVascular endothelial growth factor (VEGF) is a well-known molecule mediating neuronal survival and angiogenesis. However, its clinical significance in ischemic stroke is still controversial. The goal of this study was to examine the temporal profile of plasma VEGF value and its clinical significance in ischemic stroke with taking its subtypes into consideration.MethodsWe prospectively enrolled 171 patients with ischemic stroke and age- and gender-matched healthy subjects. The stroke patients were divided into 4 subtypes: atherothrombotic infarction (ATBI, n = 34), lacunar infarction (LAC, n = 45), cardioembolic infarction (CE, n = 49) and other types (OT, n = 43). Plasma VEGF values were measured as a part of multiplex immunoassay (Human MAP v1.6) and we obtained clinical information at 5 time points (days 0, 3, 7, 14 and 90) after the stroke onset.ResultsPlasma VEGF values were significantly higher in all stroke subtypes but OT than those in the controls throughout 90 days after stroke onset. There was no significant difference in the average VEGF values among ATBI, LAC, and CE. VEGF values were positively associated with neurological severity in CE patients, while a negative association was found in ATBI patients. After adjustment for possible confounding factors, plasma VEGF value was an independent predictor of poor functional outcome in CE patients.ConclusionsAlthough plasma VEGF value increases immediately after the stroke onset equally in all stroke subtypes, its significance in functional outcome may be different among the stroke subtypes.
205160264
An Analysis of Regional Microvascular Loss and Recovery following Two Grades of Fluid Percussion Trauma: A Role for Hypoxia-Inducible Factors in Traumatic Brain Injury
Secondary hypoxic/ischemic injuries, stemming from reductions in cerebral blood flow are important contributing factors in progressive neuronal dysfunction after brain trauma. A greater preclinical understanding of how brain trauma leads to secondary hypoxia/ischemia is necessary in the development of posttraumatic brain injury (TBI) therapeutics. To this end, we examined the density of microvascular coverage in the injured and contralateral cortical hemispheres using two intensities of fluid percussion trauma in rats. A silicone microangiography technique showed a significant loss in microvascular density in 2 atmosphere (atm) (16.9±3.8%) and 3 atm (15.7±1.3%) injured animals relative to sham animals (29.9±2.5%; P<0.01). RECA-1 immunohistochemistry indicated that capillary changes involved a reduction in capillary number and diameter. Reduction in microvascular density was shown to be a diffuse phenomenon occurring up to 4 mm rostral and caudal to the injury epicenter. Recovery of microvasculature occurred by 2 weeks after injury only in the 2 atm injury group. Expression of HIF1α and increased vascular endothelial growth factor expression were observed in the ipsilateral hippocampus suggesting sufficiently impaired microcirculation resulting in the expression of hypoxic-response proteins. Collectively, the results indicate diffuse and heterogeneous microvascular alterations as well as endogenous expression of neuroprotective and neovascularization pathways after TBI.
24510768
Vascular endothelial growth factor is involved in mediating increased de novo hippocampal neurogenesis in response to traumatic brain injury.
Stimulating the endogenous repair process after traumatic brain injury (TBI) can be an important approach in neuroregenerative medicine. Vascular endothelial growth factor (VEGF) is one of the molecules that can increase de novo hippocampal neurogenesis. Here, we tested whether VEGF signaling through Flk1 (VEGF receptor 2) is involved in the neurogenic process after experimental TBI. We found that Flk1 is expressed both by neuroblasts in the subgranular layer (SGL) and by maturing granule neurons in the adult dentate gyrus (DG) of the hippocampus. After lateral fluid percussion TBI (LFP-TBI) in the rat, we detected elevated VEGF levels and also increased numbers of de novo neurons in the ipsilateral DG. To test the involvement of VEGF and Flk1 in the neurogenic process directly, we delivered recombinant VEGF or SU5416, an inhibitor to Flk1, into the ipsilateral cerebral ventricle of injured animals. We found that VEGF infusion significantly increased the number of BrdU+/Prox1+ new neurons, decreased the number of TUNEL+ cells, but did not change the number of BrdU+ newborn cells per se. Infusion with SU5416 caused no significant changes. Our results suggest that (a) VEGF is a part of the molecular signaling network that mediates de novo hippocampal neurogenesis after TBI; (b) VEGF predominantly mediates survival of de novo granule neurons rather than proliferation of neuroblasts in the injured brain; and (c) additional VEGF receptor(s) and/or other molecular mechanism(s) are also involved in mediating increased neurogenesis following injury.
10770246
Cerebral angiogenesis and expression of VEGF after subarachnoid hemorrhage (SAH) in rats
Subarachnoid hemorrhage (SAH) leads to the development of vasospasm in which endothelin-1 plays a very important role. The effect of its vasoconstricting action is hypoxia of the nervous tissue, which stimulates the release of growth factors. Vascular endothelial growth factor (VEGF) released in excessive amounts from hypoxically altered cerebrovascular endothelial cells is the most potent angiogenic factor and may enhance angiogenesis after SAH. If endothelin-1 is mainly responsible for vasospasm after SAH, it is possible that early administration of endothelin converting enzyme inhibitor or endothelin receptor antagonist may protect neurons against. The aim of the study was to establish whether prolonged vasospasm and endothelial cell hypoxia stimulate VEGF expression and, in consequence, promote angiogenesis in the central nervous system after subarachnoid hemorrhage. Investigations were also performed to determine whether the administration of phosphoramidon, an endothelin-converting enzyme (ECE) inhibitor, and BQ-123, an endothelin receptor ET(A) antagonist, suppresses angiogenesis and VEGF expression. Experiments were carried out in male Wistar rats injected with phosphoramidon or BQ-123 into the cisterna magna following the induction of subarachnoid hemorrhage. The brains were removed 48 h after the hemorrhage for histopathological and immunohistochemical examinations of VEGF expression and angiogenesis in the cerebral hemispheres, brainstem, and cerebellum. Statistical analysis was performed using nonparametric Wilcoxon test (P<0.05). The results obtained have shown for the first time a close correlation between endothelial hypoxia after SAH in cerebral microvessels and enhanced angiogenesis. There is also an increase in VEGF expression in cerebral vessels and neurons within the cerebral hemispheres, brainstem, and cerebellum. The administration of phosphoramidon or BQ-123 has been found to inhibit angiogenesis. Angiogenesis in the chronic phase of SAH-induced vasospasm is the result of prolonged narrowing of vessels due to excessive secretion of endothelin by damaged endothelial cells. Present results obtained indicate that it is possible to reduce or prevent the late effects of SAH, i.e., neuronal hypoxia and cerebral edema, through the inhibition of endothelin-1 induced vasospasm.
25486203
Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression
Blood–brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier; however, they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein, we employed oxygen–glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3‐h before co‐culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8‐D1A cells). Incubation of OGD‐treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin‐5 proteins as well as increased vascular endothelial growth factor (VEGF) secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.
23144583
Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease.
In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.
17864420
Vascular Endothelial Growth Factors Enhance the Permeability of the Mouse Blood-brain Barrier
The blood-brain barrier (BBB) impedes entry of many drugs into the brain, limiting clinical efficacy. A safe and efficient method for reversibly increasing BBB permeability would greatly facilitate central nervous system (CNS) drug delivery and expand the range of possible therapeutics to include water soluble compounds, proteins, nucleotides, and other large molecules. We examined the effect of vascular endothelial growth factor (VEGF) on BBB permeability in Kunming (KM) mice. Human VEGF165 was administered to treatment groups at two concentrations (1.6 or 3.0 µg/mouse), while controls received equal-volume saline. Changes in BBB permeability were measured by parenchymal accumulation of the contrast agent Gd-DTPA as assessed by 7 T magnetic resonance imaging (MRI). Mice were then injected with Evans blue, sacrificed 0.5 h later, and perfused transcardially. Brains were removed, fixed, and sectioned for histological study. Both VEGF groups exhibited a significantly greater signal intensity from the cerebral cortex and basal ganglia than controls (P<0.001). Evans blue fluorescence intensity was higher in the parenchyma and lower in the cerebrovasculature of VEGF-treated animals compared to controls. No significant brain edema was observed by diffusion weighted MRI (DWI) or histological staining. Exogenous application of VEGF can increase the permeability of the BBB without causing brain edema. Pretreatment with VEGF may be a feasible method to facilitate drug delivery into the CNS.
1692015
Claudin-based barrier in simple and stratified cellular sheets.
For homeostasis in multicellular organisms, isolation and compartmentalisation of the internal environment are essential, and are established by various cellular sheets. For these cellular sheets to function as barriers, the intercellular route must be sealed. Recent advances reveal that claudins - major cell adhesion molecules in tight junctions - are directly involved in this intercellular sealing, not only in simple but also in stratified cellular sheets in vertebrates.
14085307
Size-selective loosening of the blood-brain barrier in claudin-5–deficient mice
Tight junctions are well-developed between adjacent endothelial cells of blood vessels in the central nervous system, and play a central role in establishing the blood-brain barrier (BBB). Claudin-5 is a major cell adhesion molecule of tight junctions in brain endothelial cells. To examine its possible involvement in the BBB, claudin-5–deficient mice were generated. In the brains of these mice, the development and morphology of blood vessels were not altered, showing no bleeding or edema. However, tracer experiments and magnetic resonance imaging revealed that in these mice, the BBB against small molecules (<800 D), but not larger molecules, was selectively affected. This unexpected finding (i.e., the size-selective loosening of the BBB) not only provides new insight into the basic molecular physiology of BBB but also opens a new way to deliver potential drugs across the BBB into the central nervous system.
428210
Occludin: a novel integral membrane protein localizing at tight junctions
Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no proteins with significant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an integral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which we designated as "occludin."
2137640
Vascular Endothelial Growth Factor Antagonist Reduces Brain Edema Formation and Venous Infarction
Background and Purpose— Cerebral venous ischemia often induces severe brain edema. Vascular endothelial growth factor (VEGF), which induces angiogenesis, is also known as vascular permeability (VP) factor. The present study was undertaken to investigate whether the inhibition of VEGF could reduce brain edema formation and cerebral venous infarction (CVI) in a rat 2-vein occlusion (2-VO) model. Methods— We used 2-VO model in which 2 adjacent cortical veins were photochemically occluded. Male Wistar rats (n=25) were divided into 2 groups: one group was treated with a VEGF antagonist (antagonist group, n=10) and the second group was treated with phosphate-buffered solution (PBS) (PBS group, n=15). VEGF antagonist or PBS was injected intraperitoneally immediately after 2-VO. The developing ischemic infarct was evaluated by magnetic resonance imaging (MRI) and histology 24 hours after occlusion. Results— VEGF expression was observed in the cytoplasm of neurons exclusively in the area of vasogenic edema that was shown as a high-intensity area in the apparent diffusion coefficient of water map. Ischemic volumes calculated from each MR images, which are related to infarction and/or vasogenic edema, respectively, were significantly smaller in the antagonist group as compared with the PBS group (P<0.05) Conclusions— Our study is the first to provide evidence that the inhibition of VEGF attenuates VP and reduces CVI in the acute stage. Although VEGF is a significant angiogenesis factor, we concluded that the inhibition of VEGF might be a new therapy for both brain edema formation and CVI.
35496460
VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain.
VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.
23426831
Effect of VEGF receptor antagonist (VGA1155) on brain edema in the rat cold injury model.
Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis and a strong vascular permeability factor. Blockade of VEGF may have a potential to treat brain edema after brain injury. In the rat cold injury model, the VEGF receptor antagonist VGA1155 significantly reduced the brain water content and the maximum effect was obtained when given at 30 minutes after injury. This effect was in dose-dependent manner and the dose of 25 mg/kg showed the maximum effect. With this dose, VGA1155 also significantly reduced vascular permeability. Histological evaluation of brain lesion showed no significant reduction of damaged area 1 week after injury by VGA1155. The data suggest that VGA1155 may have antiedematous effect in acute phase after cold injury.
22768665
Regulation of matrix biology by matrix metalloproteinases.
Matrix metalloproteinases (MMPs) are endopeptidases that contribute to growth, development and wound healing as well as to pathologies such as arthritis and cancer. Until recently, it has been thought that MMPs participate in these processes simply by degrading extracellular matrix (ECM) molecules. However, it is now clear that MMP activity is much more directed and causes the release of cryptic information from the ECM. By precisely cleaving large insoluble ECM components and ECM-associated molecules, MMPs liberate bioactive fragments and growth factors and change ECM architecture, all of which influence cellular behavior. Thus, MMPs have become a focal point for understanding matrix biology.
23663847
Role of the Matrix Metalloproteinase and Plasminogen Activator-Plasmin Systems in Angiogenesis
Abstract—Extracellular proteolysis is an absolute requirement for new blood vessel formation (angiogenesis). This review examines the role of the matrix metalloproteinase (MMP) and plasminogen activator (PA)-plasmin systems during angiogenesis. Specifically, a role for gelatinases (MMP-2, MMP-9), membrane-type 1 MMP (MMP-14), the urokinase-type PA receptor, and PA inhibitor 1 has been clearly defined in a number of model systems. The MMP and PA-plasmin systems have also been implicated in experimental vascular tumor formation, and their role during this process will be examined. Antiproteolysis, particularly in the context of angiogenesis, has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization.
7352183
Neurovascular matrix metalloproteinases and the blood-brain barrier.
Blood-brain barrier (BBB) leakage and brain edema is a critical part of stroke pathophysiology. In this mini-review, we briefly survey the potential role of matrix metalloproteinases (MMPs) in BBB dysfunction. A large body of data in both experimental models as well as clinical patient populations suggests that MMPs may disrupt BBB permeability and interfere with cell-cell signaling in the neurovascular unit. Hence, ongoing efforts are underway to validate MMPs as potential biomarkers in stroke as well as pursue MMP blockers as therapeutic opportunities. Because BBB perturbations may also occur in neurodegeneration, MMPs and associated neurovascular unit mechanisms may also be potential targets in a broader range of CNS disorders.
6356504
Matrix Metalloproteinases Increase Very Early during Experimental Focal Cerebral Ischemia
Microvascular integrity is lost during focal cerebral ischemia. The degradation of the basal lamina and extracellular matrix are, in part, responsible for the loss of vascular integrity. Matrix metalloproteinases (MMPs) may play a primary role in basal lamina degradation. By using a sensitive modification of gelatin zymography, the authors investigated the activity of MMP-2 and MMP-9 in frozen 10-µm sections of ischemic and nonischemic basal ganglia and plasma samples of 27 non-human primates after middle cerebral artery occlusion/reperfusion (MCAO/R) for various periods. The gelatinolytic activities were compared with parallel cell dUTP incorporation in the ischemic zones of adjacent sections. In the brain, the integrated density of MMP-2 increased significantly by 1 hour after MCAO and was persistently elevated thereafter. Matrix metalloproteinase-2 expression was highly correlated with the extent of neuron injury and the number of injured neurons (r = 0.9763, SE = 0.004, 2P < 0.0008). Matrix metalloproteinase-9 expression only was significantly increased in subjects with hemorrhagic transformation. In plasma, only MMP-9 increased transiently at 2 hours of MCAO. These findings highlight the early potential role of MMP-2 in the degradation of basal lamina leading to neuronal injury, and an association of MMP-9 with hemorrhagic transformation after focal cerebral ischemia.
41889891
ELEVATION OF MATRIX METALLOPROTEINASES 3 AND 9 IN CEREBROSPINAL FLUID AND BLOOD IN PATIENTS WITH SEVERE TRAUMATIC BRAIN INJURY
OBJECTIVETraumatic brain injury (TBI) causes an increase in matrix metalloproteinases (MMPs), which are associated with neuroinflammation, blood-brain barrier disruption, hemorrhage, and cell death. We hypothesized that patients with TBI have an increase in MMPs in ventricular cerebrospinal fluid (CSF) and plasma. METHODSPatients with TBI and a ventricular catheter were entered into the study. Samples of CSF and plasma were collected at the time of catheter placement and at 24 and 72 hours after admission. Seven TBI patients were entered into the study, with 6 having complete data for analysis. Only patients who had a known time of insult that fell within a 6-hour window from initial insult to ventriculostomy were accepted into the study. Control CSF came from ventricular fluid in patients undergoing shunt placement for normal pressure hydrocephalus. Both MMP-2 and MMP-9 were measured with gelatin zymography and MMP-3 with Western immunoblot. RESULTSWe found a significant elevation in the levels of the latent form of MMP-9 (92-kD) in the CSF obtained at the time of arrival (P < 0.05). Elevated levels of MMP-2 were detected in plasma at 72 hours, but not in the CSF. Using albumin from both CSF and blood, we calculated the MMP-9 index, which was significantly increased in the CSF, indicating endogenous MMP production. Western immunoblot showed elevated levels of MMP-3 in CSF at all times measured, whereas MMP-3 was not detected in the CSF of normal pressure hydrocephalus. CONCLUSIONWe show that MMPs are increased in the CSF of TBI patients. Although the number of patients was small, the results were robust and clearly demonstrated increases in MMP-3 and MMP-9 in ventricular CSF in TBI patients compared with controls. Although these preliminary results will need to be replicated, we propose that MMPs may be important in blood-brain barrier opening and hemorrhage secondary to brain injury in patients.
207445274
Matrix metalloproteinases and their tissue inhibitors in serum and cerebrospinal fluid of patients with moderate and severe traumatic brain injury.
OBJECTIVE
In this study, we investigated matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) in cerebrospinal fluid (CSF) and plasma of traumatic brain injury (TBI) patients.


PATIENTS AND METHODS
A total of 30 patients with moderate and severe TBI and 15 age-matched controls were enrolled in this study. Plasma and CSF samples were collected within 24 h (as the initial value), at 72 and 120 h post injury. CSF and plasma MMP-9, MMP-2, TIMP-1 and TIMP-2 were estimated using ELISA. Different levels of these indexes were compared in the two groups and further investigated the correlation between each other.


RESULTS
There was a significant elevation in the levels of the initial MMP-9 in the CSF (P < 0.05), which lasted for 72 h post injury. TIMP-1 kept increasing within 120 h post injury and it was different compared with TIMP-1 at 24 and 72 h post injury. Plasma levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 in TBI patients were also significantly different from those in controls. Furthermore the CSF MMP-9 in patients with severe TBI was higher than that in patients with moderate TBI. In addition, there was a positive relationship between the initial MMP-9 and TIMP-1 at 120 h post injury (r = 0.614, P < 0.01).


CONCLUSION
MMPs and TIMPs are increased in both CSF and plasma of TBI patients. TIMP-1 has a positive correlation with MMP-9 and the initial MMP-9 is associated with the neurological outcomes.
45699192
Matrix metalloproteinase-9 expression and protein levels after fluid percussion injury in rats: the effect of injury severity and brain temperature.
The temporal and regional expression profiles of matrix metalloproteinase-9 (MMP-9), after moderate or severe traumatic brain injury (TBI) were measured to investigate the effects of post-traumatic hypothermia (33 degrees C) or hyperthermia (39 degrees C). In the first phase of this study, adult male Sprague-Dawley rats were randomly assigned to groups of moderate TBI (1.8-2.2 atm), severe TBI (2.4-2.7 atm), and sham-injured control. The rats were killed at 4, 6, 12, 24, 48, and 72 h, or 1 week after TBI, for mRNA and protein analysis. In the second phase, rats underwent moderate fluid percussion brain injury, followed immediately by 4 h of post-traumatic normothermia (37 degrees C), hyperthermia (39 degrees C), or hypothermia (32 degrees C). The rats were killed at 12 and 48 h after TBI for mRNA expression analyses, or killed at 24 and 72 h after TBI for protein expression analyses. Brain samples, including the cerebral cortex and hippocampus (both ipsilateral and contralateral hemispheres of each group), were assayed using RT-PCR and Western blot techniques. MMP-9 levels in both the ipsilateral and contralateral hemispheres were significantly increased after TBI compared with those of sham injured animals (p < 0.01). Two expression peaks of MMP-9 were observed in the ipsilateral cortex and hippocampus. An increase in injury severity was associated with an increase in mRNA (12 and 48 h), and protein (24 and 72 h) levels of MMP-9. Post-traumatic hypothermia attenuated the increase in both the mRNA and protein levels of MMP-9, compared with normothermia and hyperthermia (p < 0.01). In contrast, hyperthermia had no significant effect on mRNA (at 12 h) and protein levels (at 24 h) of MMP-9, compared with normothermic values (p > 0.05), but resulted in a significant increase in the levels of MMP-9 mRNA and protein at 24 and 72 h, respectively (p < 0.01). Increases in MMP-9 mRNA and protein after TBI were proportional to injury severity in this model. The effects of post-traumatic hypothermia on the expression of MMP-9 may partially explain the observed effects of post-traumatic temperature on secondary injury after TBI.
6582947
Metalloproteinase inhibition blocks edema in intracerebral hemorrhage in the rat
Clinical worsening often occurs 1 to 2 days after an intracerebral hemorrhage. Extracellular matrix proteolysis by metalloproteinases, which attack the basal lamina and open the blood-brain barrier, may be one contributing factor. Matrix metalloproteinases and plasminogen activators are increased 16 to 24 hours after a bacterial collagenase-induced intracerebral hemorrhage, suggesting that agents that block metalloproteinases may reduce the brain swelling after hemorrhage. Therefore, we injected 0.2, 0.3, 0.4, or 0.5 units bacterial collagenase intracerebrally in rats to produce an intracerebral hemorrhage. Twenty-four hours later, brain tissue was removed for measurement of brain water and electrolytes. Proteases were assayed by zymography. Treatment with a matrix metalloproteinase inhibitor, BB-1101, was begun 6 hours after the collagenase lesion, when the hematomas were formed and the secondary edema was increasing. Bacterial collagenase caused a dose-dependent hematoma at the injection site with secondary brain edema in both posterior regions. The lower bacterial collagenase doses (0.2 and 0.3 units) mainly caused brain edema in the tissue around the injection site, whereas the higher doses (0.4 and 0.5 units) also affected the opposite hemisphere. Administration of BB-1101 significantly reduced the brain water and sodium contents in regions away from the injection site in rats with 0.4 unit lesions (p < 0.05). Zymography showed an increase in 92-kDa type IV collagenase and urokinase-type plasminogen activator at 24 hours. Inhibitors of proteolytic cascade enzymes may be useful in treatment of secondary brain edema in intracerebral hemorrhage.
23391894
Expression and Activation of Matrix Metalloproteinase-2 and -9 in Rat Brain after Transient Focal Cerebral Ischemia
Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions during development and after injury. By means of zymography, Western blot and immunohistochemistry, we studied MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in rat brain after focal cerebral ischemia. The control rat brain showed constitutive MMP-2 and, to a lesser extent, MMP-9, which were mainly present as prozymogens. MMP-2 protein was located in the cell body of neurons, glia, and endothelium, whereas MMP-9 was associated to neurons and myelinated fibre tracts. Ischemia greatly increased MMP activation in two temporal waves, in the first one, MMP-9 protein was induced from 4 h to 4 days, and also a small and short-lasting increase in MMP-2 was detected at 4 h. The second wave showed a massive increase in MMP-2 protein expression and activation by day 4, which was compatible with abundant MMP-2 in reactive microglia/macrophages. Our results are compatible with progressive induction of MMP-9 proform, likely in neurons, shortly after ischemia. For MMP-2, the results suggest a discrete production immediately after reperfusion, while a very enhanced expression and activation of MMP-2 attributable to microglia/macrophages occurs on day 4, and it might contribute to the phagocytic action of these reactive cells.
32955697
Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures
Reperfusion damages the blood-brain barrier (BBB). Matrix metalloproteinases (MMPs) are associated with the opening of the BBB, but their cellular localization and activation mechanisms are uncertain. We used immunohistochemistry to determine the cellular localization of the MMPs in reperfused rat brain, and cell cultures to study their activation. Spontaneously hypertensive rats (SHR) had a 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion for times from 3 h to 21 days. Frozen sections were immunostained with antibodies to gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). Sham-operated control rats showed MMP-2 immunostaining in astrocytic processes next to blood vessels. After 3 h of the onset of reperfusion MMP-2 immunostaining increased in astrocytes. At 24 h immunoreactivity for MMP-3 and MMP-9 appeared. MMP-3 co-localized with activated microglia (Ox-42+) and ischemic neurons (NeuN+). MMP-9 immunostaining was seen at 48 h in endothelial cells, neutrophils, and neurons. At 5 and 21 days intense MMP-2 staining was seen in reactive astrocytes around the ischemic core. Studies of activation of the MMP were done in lipopolysaccharide (LPS)-stimulated astrocyte and microglia cultures. Stimulated astrocytes produced an activated form of MMP-2. When microglia were stimulated, they activated MMP-9. Immunostaining showed MMP-3 in cultures of enriched microglial cells. The hydroxymate-type, MMP inhibitor, BB-1101, blocked the activation of MMP-2 and MMP-9 by LPS in mixed glial cultures. We propose that MMP-2 is normally present in astrocytic end feet, and that during ischemia MMP-9 and MMP-3 are produced. MMP-3 in microglia/macrophages may be activating proMMP-9. Our results show that a differential expression of MMPs by astrocytes, microglia, and endothelial cells at the blood vessels is involved in the proteolytic disruption of the BBB.
25381219
Matrix metalloproteases and their inhibitors are produced by overlapping populations of activated astrocytes.
Matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) are involved in many cell migration phenomena and produced by many cell types, including neurons and glia. To assess their possible roles in brain injury and regeneration, we investigate their production by glial cells, after brain injury and in tissue culture, and we investigate whether they are capable of digesting known axon-inhibitory proteoglycans. To determine the action of MMPs, we incubated astrocyte conditioned medium with activated MMPs, then did western blots for several chondroitin sulphate proteoglycans. MMP-3 digested all five proteoglycans tested, whereas MMP-2 digested only two and MMP-9 none. To determine whether MMPs or TIMPs are produced by astrocytes in vitro, we tested both primary cultures and astrocyte cell lines by western blotting, and compared them with Schwann cells. All cultures produced at least some MMPs and TIMPs, with no obvious correlation with the ability of axons to grow on those cells. Both MMP-9 and TIMP-3 were regulated by various cytokines. To determine which cells produce MMPs and TIMPs after brain injury, we made lesions of adult rat cortex, and did immunohistochemistry. MMP-2 was seen to be induced in activated astrocytes through the whole thickness of the cortex but not deeper, but MMP-3 was not seen in the injured brain. TIMP-2 and TIMP-3 immunoreactivities were induced in activated astrocytes in deep cortex and the underlying white matter. In situ hybridisation confirmed induction of TIMP-2 in glia as well as neurons, but showed no expression of TIMP-4. These results show that both MMPs and TIMPs are produced by some astrocytes, but TIMP production is particularly strong, especially in deep cortex and white matter which is more inhibitory for axon regeneration. Conversely the MMPs produced may not be adequate to promote migration of cells and axons within the glial scar.
23277874
Overexpression of Copper and Zinc Superoxide Dismutase in Transgenic Mice Prevents the Induction and Activation of Matrix Metalloproteinases after Cold Injury-Induced Brain Trauma
Matrix metalloproteinases (MMPs), a family of proteolytic enzymes which degrade the extracellular matrix, are implicated in blood—brain barrier disruption, which is a critical event leading to vasogenic edema. To investigate the role of reactive oxygen species (ROS) in the expression of MMPs in vasogenic edema, the authors measured gelatinase activities before and after cold injury (CI) using transgenic mice that overexpress superoxide dismutase-1. A marked induction of pro-gelatinase B (pro-MMP-9) was seen 2 hours after CI and was maximized at 12 hours in wild-type mice. The pro-MMP-9 level was significantly lower in transgenic mice 4 hours (P < 0.001) and 12 hours (P < 0.05) after CI compared to wild-type mice. The activated MMP-9 was detected from 6 to 24 hours after injury. A mild induction of pro-gelatinase A (pro-MMP-2) was seen at 6 hours and was sustained until 7 days. In contrast, the activated form of MMP-2 appeared at 24 hours, was maximized at 7 days, and was absent in transgenic mice. Western blot analysis showed that the tissue inhibitors of metalloproteinases were not modified after CI. The results suggest that ROS production after CI may contribute to the induction and/or activation of MMPs and could thereby exacerbate endothelial cell injury and the development of vasogenic edema after injury.
29142255
Matrix Metalloproteinase Inhibition Prevents Oxidative Stress-Associated Blood–Brain Barrier Disruption after Transient Focal Cerebral Ischemia
Oxidative stress generated during stroke is a critical event leading to blood–brain barrier (BBB) disruption with secondary vasogenic edema and hemorrhagic transformation of infarcted brain tissue, restricting the benefit of thrombolytic reperfusion. In this study, the authors demonstrate that ischemia-reperfusion–induced BBB disruption in mice deficient in copper/zinc-superoxide dismutase (SOD1) was reduced by 88% (P < 0.0001) and 73% (P < 0.01), respectively, after 3 and 7 hours of reperfusion occurring after 1 hour of ischemia by the inhibition of matrix metalloproteinases. Accordingly, the authors show that local metalloproteinase-generated proteolytic imbalance is more intense in ischemic regions of SOD1 mice than in wild-type litter mates. Moreover, active in situ proteolysis is, for the first time, demonstrated in ischemic leaking capillaries that produce reactive oxygen species. By showing that oxidative stress mediates BBB disruption through metalloproteinase activation in experimental ischemic stroke, this study provides a new target for future therapeutic strategies to prevent BBB disruption and potentially reperfusion-triggered intracerebral hemorrhage.
2509951
Gelatinase B and TIMP‐1 are regulated in a cell‐ and time‐dependent manner in association with neuronal death and glial reactivity after global forebrain ischemia
Matrix metalloproteinases (MMPs) belong to a large family of endopeptidases that regulate the pericellular environment through the cleavage of protein components of the extracellular matrix, membrane receptors and cytokines. MMP activity is controlled by the multifunctional tissue inhibitors of metalloproteinases (TIMPs). Proteases and their inhibitors are critically involved in developmental and pathological processes in numerous organs, including the brain. Global transient cerebral ischemia induces selective delayed neuronal death and neuroinflammation. We compared, in discrete vulnerable and resistant areas of the ischemic rat hippocampus, the kinetics and cellular distribution of gelatinase B and its principal inhibitor TIMP‐1 and we assessed by in situ zymography, the net gelatinolytic activity at the cellular level. We show that gelatinases are expressed and active in neurons, suggesting that MMPs play a role in maintaining neural homeostasis. In the ischemic rat brain, expression and activity of gelatinase B, and expression of TIMP‐1 are altered in a time‐, region‐ and cell‐dependent manner. Gelatinase B is induced first in reactive microglia and subsequently in reactive astrocytes. In situ, increases in gelatinase activity accompanied the progression of neuronal death and glial reactivity. Our results suggest that MMPs and TIMPs are involved in cell viability and tissue remodelling in the ischemic brain, and reinforces the idea that the MMP/TIMP system contributes both to neuronal demise and tissue repair in the context of glial reactivity.
9868426
Effects of Matrix Metalloproteinase-9 Gene Knock-Out on the Proteolysis of Blood–Brain Barrier and White Matter Components after Cerebral Ischemia
Deleterious processes of extracellular proteolysis may contribute to the progression of tissue damage after acute brain injury. We recently showed that matrix metalloproteinase-9 (MMP-9) knock-out mice were protected against ischemic and traumatic brain injury. In this study, we examined the mechanisms involved by focusing on relevant MMP-9 substrates in blood–brain barrier, matrix, and white matter. MMP-9 knock-out and wild-type mice were subjected to transient focal ischemia. MMP-9 levels increased after ischemia in wild-type brain, with expression primarily present in vascular endothelium. Western blots showed that the blood–brain barrier-associated protein and MMP-9 substrate zonae occludens-1 was degraded after ischemia, but this was reduced in knock-out mice. There were no detectable changes in another blood–brain barrier-associated protein, occludin. Correspondingly, blood–brain barrier disruption assessed via Evans Blue leakage was significantly attenuated in MMP-9 knock-out mice compared with wild types. In white matter, ischemic degradation of the MMP-9 substrate myelin basic protein was significantly reduced in knock-out mice compared with wild types, whereas there was no degradation of other myelin proteins that are not MMP substrates (proteolipid protein and DM20). There were no detectable changes in the ubiquitous structural protein actin or the extracellular matrix protein laminin. Finally, 24 hr lesion volumes were significantly reduced in knock-out mice compared with wild types. These data demonstrate that the protective effects of MMP-9 gene knock-out after transient focal ischemia may be mediated by reduced proteolytic degradation of critical blood–brain barrier and white matter components.
16605693
Neuroprotection by inhibition of matrix metalloproteinases in a mouse model of intracerebral haemorrhage.
Intracerebral haemorrhage (ICH) is an acute neurological disorder without effective treatment. Mechanisms of acute brain injury after ICH remain to be clarified. Although a few studies suggested a detrimental role for the gelatinase matrix metalloproteinase (MMP)-9 in ICH, the relationship between MMP-9 activity and acute brain injury after ICH is not determined. In this study, we first examined the expression of gelatinases in vivo using a collagenase-induced mouse model of ICH. Gel zymography revealed that MMP-9 was activated and upregulated after ICH. In situ zymography showed that gelatinase activity was mostly co-localized with neurons and endothelial cells of the blood vessel matrix. Inhibition with a broad-spectrum metalloproteinase inhibitor GM6001 (100 mg/kg) ameliorated dysregulated gelatinase activity, neutrophil infiltration, production of oxidative stress, brain oedema and degenerating neurons. Functional improvement and a decrease in injury volume were also observed. We provide evidence that MMP-9 may play a deleterious role in acute brain injury within the first 3 days after ICH. Blockade of MMP activity during this critical period may have efficacy as a therapeutic strategy for the treatment of acute brain injury after ICH.
15852727
Matrix metalloproteinase-9 is associated with blood-brain barrier opening and brain edema formation after cortical contusion in rats.
Matrix metalloproteinases (MMPs) are associated with blood-brain opening and may be involved in the pathophysiology of acute brain injury. Previous research demonstrated that knockout mice deficient in MMP-9 subjected to transient focal cerebral ischemia had reduced blood-brain barrier (BBB) disruption and attenuated cerebral infarction. In this study, we examined MMP-9 up-regulation, BBB disruption, and brain edema formation after cortical impact injury in rats. Cortical contusion was induced by controlled cortical impact. Animals were sacrificed at intervals after injury. MMP up-regulation was assessed by gelatin zymography, and BBB integrity was evaluated using Evans blue dye with a spectrophotometric assay. Brain water content was measured by comparing wet and dry weights of each hemisphere as an indicator of brain edema. Zymograms showed elevated MMP-9 as early as at 3 hours after injury, reaching a maximum at 18 hours. Peak levels of BBB disruption occurred 6 hours after injury. Brain edema became progressively more severe, peaking 24 hours after injury. Compared to control group, treatment with MMP-inhibitor GM6001 significantly reduced BBB disruption 6 hours and brain water content (85.9 +/- 0.5% vs. 82.6 +/- 0.3%; p < 0.05) 24 hours after injury. These findings suggest that MMP-9 may contribute to BBB disturbance and subsequent brain edema after traumatic brain injury.
2137112
Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain.
BACKGROUND AND PURPOSE
Reperfusion disrupts cerebral capillaries, causing cerebral edema and hemorrhage. Middle cerebral artery occlusion (MCAO) induces the matrix-degrading metalloproteinases, but their role in capillary injury after reperfusion is unknown. Matrix metalloproteinases (MMPs) and tissue inhibitors to metalloproteinases (TIMPs) modulate capillary permeability. Therefore, we measured blood-brain barrier (BBB) permeability, brain water and electrolytes, MMPs, and TIMPs at multiple times after reperfusion.


METHODS
Adult rats underwent MCAO for 2 hours by the suture method. Brain uptake of 14C-sucrose was measured from 3 hours to 14 days after reperfusion. Levels of MMPs and TIMPs were measured by zymography and reverse zymography, respectively, in contiguous tissues. Other rats had water and electrolytes measured at 3, 24, or 48 hours after reperfusion. Treatment with a synthetic MMP inhibitor, BB-1101, on BBB permeability and cerebral edema was studied.


RESULTS
Brain sucrose uptake increased after 3 and 48 hours of reperfusion, with maximal opening at 48 hours and return to normal by 14 days. There was a correlation between the levels of gelatinase A at 3 hours and the sucrose uptake (P<0.05). Gelatinase A (MMP-2) was maximally increased at 5 days, and TIMP-2 was highest at 5 days. Gelatinase B and TIMP-1 were maximally elevated at 48 hours. The inhibitor of gelatinase B, TIMP-1, was also increased at 48 hours. Treatment with BB-1101 reduced BBB opening at 3 hours and brain edema at 24 hours, but neither was affected at 48 hours.


CONCLUSIONS
The initial opening at 3 hours correlated with gelatinase A levels and was blocked by a synthetic MMP inhibitor. The delayed opening, which was associated with elevated levels of gelatinase B, failed to respond to the MMP inhibitor, suggesting different mechanisms of injury for the biphasic BBB injury.
29037024
Early Beneficial Effect of Matrix Metalloproteinase Inhibition on Blood—Brain Barrier Permeability as Measured by Magnetic Resonance Imaging Countered by Impaired Long-Term Recovery after Stroke in Rat Brain
Proteolytic disruption of the extracellular matrix with opening of the blood—brain barrier (BBB) because of matrix metalloproteinases (MMPs) occurs in reperfusion injury after stroke. Matrix metalloproteinase inhibition blocks the early disruption of the BBB, but the long-term consequences of short-term MMP inhibition are not known. Recently, a method to quantify BBB permeability by graphical methods was described, which provides a way to study both early disruption of the BBB and long-term effects on recovery in the same animal. We used a broad-spectrum MMP inhibitor, BB1101, to determine both the usefulness of the Magnetic resonance imaging (MRI) method for treatment studies and the long-term effects on recovery. Magnetic resonance imaging studies were performed in control (N = 6) and drug-treated (N = 8) groups on a dedicated 4.7-T MRI scanner. Adult Wistar—Kyoto underwent a 2-h middle cerebral artery occlusion followed by an MRI study after 3 h of reperfusion, which consisted of T2- and diffusion-weighted techniques. Additionally, a rapid T1 mapping protocol was also implemented to acquire one pre-gadolinium-diethylenetriaminepentaacetic acid baseline data set followed by postinjection data sets at 3-min intervals for 45 mins. The same animal was imaged again at 48 h for lesion size estimation. Data was postprocessed pixel-wise to generate apparent diffusion coefficient and permeability coefficient maps. Treatment with BB-1101 significantly reduced BBB permeability at 3 h, but failed to reduce lesion size at 48 h. Behavioral studies showed impairment in recovery in treated rats. Magnetic resonance imaging allowed for the monitoring of multiple parameters in the same animal. Our studies showed that BB-1101 was an excellent inhibitor of the BBB damage. However, results show that BB-1101 may be responsible for significant deterioration in neurologic status of treated animals. Although these preliminary results suggest that BB-1101 is useful in reducing early BBB leakage owing to reperfusion injury in stroke, further studies will be needed to determine whether the later detrimental effects can be eliminated by shorter time course of drug delivery.
10225809
Treatment of cold injury-induced brain edema with a nonspecific matrix metalloproteinase inhibitor MMI270 in rats.
Blood-brain barrier (BBB) disruption is a critical event leading to vasogenic brain edema and secondary brain damage after cold injury-induced brain trauma. Matrix metalloproteinases (MMPs), a family of proteolytic enzymes which degrade the extracellular matrix, are implicated in BBB disruption in this model. The purpose of this study was to examine the effects of MMI270 (N-hydroxy-2(R)-[(4-methoxysulfony) (3-picolyl)-amino]-3-metylbutaneamide hydrochloride monohydrate), a synthetic nonspecific MMP inhibitor, on cold injury-induced brain edema in rats. Cold injury was induced by applying a copper probe cooled with liquid nitrogen on the parietal skull for 30 sec in 38 rats. Treatment with MMI270, a bolus injection at a dose of 30 mg/kg, was started immediately after the induction of cold injury and was continued for 24 h at a dose of 40 mg/kg/day using an intraperitoneal osmotic minipump (n = 7). In the untreated control group (n = 7), rats were administered a vehicle and implanted with a vehicle-containing osmotic pump. Two percent Evans Blue (EB) in saline (1 mL/kg) was administrated intravenously immediately after the cold injury in another group of rats, six of which were untreated and six of which were treated with MMI270 at the above dose. At 24 h after the cold injury, the brain water content and the BBB permeability to EB were determined. To assess the protective effect of MMI270 on secondary brain lesion after the cold injury, the MMI270-treated rats received a bolus injection at a dose of 30 mg/kg, followed by a continuous administration of MMI270 for 7 days at a dose of 40 mg/kg/day using an osmotic minipump (n = 6). In the untreated control group (n = 6), the rats were administered the vehicle and implanted with a vehicle-containing osmotic pump. At 7 days after cold injury, the secondary brain lesion was assessed using hematoxylin and eosin (H-E) staining. Compared with the untreated control group, treatment with MMI270 significantly reduced the brain water content in the ipsilateral core and intermediate areas (p < 0.05 and p < 0.01) and protected the BBB integrity to EB in the ipsilateral core area (p < 0.05) at 24 h after the cold injury. The secondary lesion was significantly smaller in the MMI270-treated animals compared with the untreated animals (p < 0.05) a 7 days after the cold injury. O kur results indicate that treatment with MMI270 in rats exhibits protection in acute brain edema formation and secondary brain damage by attenuating the BBB permeability after cold injury.
205508671
Aquaporin water channels in the nervous system
The aquaporins (AQPs) are plasma membrane water-transporting proteins. AQP4 is the principal member of this protein family in the CNS, where it is expressed in astrocytes and is involved in water movement, cell migration and neuroexcitation. AQP1 is expressed in the choroid plexus, where it facilitates cerebrospinal fluid secretion, and in dorsal root ganglion neurons, where it tunes pain perception. The AQPs are potential drug targets for several neurological conditions. Astrocytoma cells strongly express AQP4, which may facilitate their infiltration into the brain, and the neuroinflammatory disease neuromyelitis optica is caused by AQP4-specific autoantibodies that produce complement-mediated astrocytic damage.
14751575
Aquaporin 4: a player in cerebral edema and neuroinflammation
Neuroinflammation is a common pathological event observed in many different brain diseases, frequently associated with blood brain barrier (BBB) dysfunction and followed by cerebral edema. Neuroinflammation is characterized with microglia activation and astrogliosis, which is a hypertrophy of the astrocytes. Astrocytes express aquaporin 4, the water channel protein, involved in water homeostasis and edema formation. Aside from its function in water homeostasis, recent studies started to show possible interrelations between aquaporin 4 and neuroinflammation. In this review the roles of aquaporin 4 in neuroinflammation associated with BBB disruption and cerebral edema will be discussed with recent studies in the field.
45197500
Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin‐1
Aquaporin‐1 (AQP1) is a water channel expressed strongly at the ventricular‐facing surface of choroid plexus epithelium. We developed novel methods to compare water permeability in isolated choroid plexus of wild‐type vs. AQP1 null mice, as well as intracranial pressure (ICP) and cerebrospinal fluid (CSF) production and absorption. Osmotically induced water transport was rapid in choroid plexus from wild‐type mice and reduced by fivefold by AQP1 deletion. AQP1 deletion did not affect choroid plexus size or structure. By stereotaxic puncture of the lateral ventricle with a microneedle, ICP was 9.5 ± 1.4 cm H2O in wild‐type mice and 4.2 ± 0.4 cm H2O in AQP1 null mice. CSF production, an isosmolar fluid secretion process, was measured by a dye dilution method involving fluid collections using a second microneedle introduced into the cisterna magna. CSF production in wild‐type mice was (in µl min–1) 0.37 ± 0.04 (control), 0.16 ± 0.03 (acetazolamide‐treated), and 1.14 ± 0.15 (forskolin‐treated), and reduced by ~25% in AQP1 null mice. Pressure‐dependent CSF outflow, measured from steady‐state ICP at different ventricular infusion rates, was not affected by AQP1 deletion. In a model of focal brain injury, AQP1 null mice had remarkably reduced ICP and improved survival compared with wild‐type mice. The reduced ICP and CSF production in AQP1 null mice provides direct functional evidence for the involvement of AQP1 in CSF dynamics, suggesting AQP1 inhibition as a novel option for therapy of elevated ICP.
10158561
Astrocyte-Specific Expression of Aquaporin-9 in Mouse Brain is Increased after Transient Focal Cerebral Ischemia
Aquaporin-9 (AQP9) is a new member of the aquaporin family of water-selective channels mainly expressed in liver and testis, presenting the characteristic of also being permeable to various solutes, particularly lactate. Recent data have shown the presence of AQP9 on tanycytes in the rat brain. In the current study, the authors show the expression of AQP9 in astrocytes in the mouse brain and changes in its expression after cerebral ischemia. Indeed, in control mouse, the AQP9 immunolabeling is present on astrocytic processes bordering the subarachnoid space and ventricles. The labeling also is observed on astrocytes in the white matter, hippocampus, hypothalamus, and lateral septum. After focal transient ischemia, an increase of the immunolabeling is detected on astrocytes in periinfarct areas. This AQP9 distribution study in mouse brain suggests a role of AQP9 in water homeostasis in the central nervous system. Furthermore, the overexpression of AQP9 on astrocytes surrounding an ischemic lesion suggests that AQP9 may also play a role in the regulation of postischemia edema and, in view of its permeability to monocarboxylates, in the clearance of lactate from the ischemic focus.
44597132
Increased expression of aquaporin-4 in human traumatic brain injury and brain tumors.
OBJECTIVE
To characterize the expression of aquaporin-4 (AQP4), one of the aquaporins (AQPs), in human brain specimens from patients with traumatic brain injury or brain tumors.


METHODS
Nineteen human brain specimens were obtained from the patients with traumatic brain injury, brain tumors, benign meningioma or early stage hemorrhagic stroke. MRI or CT imaging was used to assess brain edema. Hematoxylin and eosin staining were used to evaluate cell damage. Immunohistochemistry was used to detect the AQP4 expression.


RESULTS
AQP4 expression was increased from 15 h to at least 8 d after injury. AQP4 immunoreactivity was strong around astrocytomas, ganglioglioma and metastatic adenocarcinoma. However, AQP4 immunoreactivity was only found in the centers of astrocytomas and ganglioglioma, but not in metastatic adenocarcinoma derived from lung.


CONCLUSION
AQP4 expression increases in human brains after traumatic brain injury, within brain-derived tumors, and around brain tumors.
3013905
Activation of P2X7 Promotes Cerebral Edema and Neurological Injury after Traumatic Brain Injury in Mice
Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part to the absence of viable drug targets. In the present study, genetic inhibition (P2X7−/− mice) of the purinergic P2x7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-1β (IL-1β) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, brilliant blue G (BBG), a clinically non-toxic P2X7 inhibitor, inhibited IL-1β expression, limited edemic development, and improved neurobehavioral outcomes after TBI. The beneficial effects of BBG followed either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and attenuated the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation.
11460596
Brain edema after intracerebral hemorrhage in rats: the role of iron overload and aquaporin 4.
OBJECT
Brain edema formation following intracerebral hemorrhage (ICH) appears to be partly related to erythrocyte lysis and hemoglobin release. An increase of brain water content was associated with an increase of brain iron, which is an erythrocyte degradation product. Expression of AQP4 is highly modified in several brain disorders, and it can play a key role in cerebral edema formation. However, the question whether AQP4 is regulated by drugs lacks reliable evidence, and the interacting roles of iron overload and AQP4 in brain edema after ICH are unknown. The goal of this study was to clarify the relationship between iron overload and AQP4 expression and to characterize the effects of the iron chelator deferoxamine (DFO) on delayed brain edema after experimental ICH.


METHODS
A total of 144 Sprague-Dawley rats weighing between 250 and 300 g were used in this work. The animals were randomly divided into 4 groups. The ICH models (Group C) were generated by injecting 100 microl autologous blood stereotactically into the right caudate nucleus; surgical control rats (Group B) were generated in a similar fashion, by injecting 100 microl saline into the right caudate nucleus. Intervention models (Group D) were established by intraperitoneal injection of DFO into rats in the ICH group. Healthy rats (Group A) were used for normal control models. Brain water content, iron deposition, and AQP4 in perihematomal brain tissue were evaluated over the time course of the study (1, 3, 7, and 14 days) in each group.


RESULTS
Iron deposition was found in the perihematomal zone as early as the 1st day after ICH, reaching a peak after 7 days and remaining at a high level thereafter for at least 14 days following ICH. Rat brain water content around the hematoma increased progressively over the time course, reached its peak at Day 3, and still was evident at Day 7 post-ICH. Immunohistochemical analysis showed that AQP4 was richly expressed over glial cell processes surrounding microvessels in the rat brain; there was upregulation of the AQP4 expression in perihematomal brain during the observation period, and it reached maximum at 3 to 7 days after ICH. The changes of brain water content were accompanied by an alteration of AQP4. The application of the iron chelator DFO significantly reduced iron overload, brain water content, and AQP4 level in the perihematomal area compared with the control group.


CONCLUSIONS
Iron overload and AQP4 may play a critical role in the formation of brain edema after ICH. In addition, AQP4 expression was affected by iron concentration. Importantly, treatment with DFO significantly reduced brain edema in rats and inhibited the AQP4 upregulation after ICH. Deferoxamine may be a potential therapeutic agent for treating ICH.
42760195
Decreased hemispheric Aquaporin-4 is linked to evolving brain edema following controlled cortical impact injury in rats
The cerebral Aquaporin-4 (AQP4) water channel is suggested to be involved in brain edema formation aggravated by reduced cerebral blood flow early after traumatic brain injury (TBI). Therefore, the temporal profile of brain edema formation, AQP4 expression, and cortical perfusion were investigated following focal TBI in rats. Brain edema was maximal by 24 h. Concurrently, AQP4 protein expression was decreased in both hemispheres, being more pronounced in the traumatized hemisphere (-50%) 48 h after trauma. Cortical perfusion was only decreased in the ipsilateral cortex (-40%) between 4 and 8 h after trauma, reaching baseline values at 24 h. Globally reduced AQP4 expression following induction of a focal contusion coincides with edema development and seems to be independent of changes in cortical perfusion.
35302929
Chemical preconditioning-induced reactive astrocytosis contributes to the reduction of post-ischemic edema through aquaporin-4 downregulation
Aquaporins are a family of membrane proteins that promote the transmembrane diffusion of water. Aquaporin-4 (AQP4) is a predominant water channel protein in the brain and is concentrated in the end-feet of astrocytes. A critical question is what role astrocytic AQP4 plays in pathological conditions. Another matter to be elucidated is the relationship between morphological changes in astrocytes and AQP4 expression in such cases. We investigated the correlation between AQP4 expression and post-ischemic brain edema formation with astrocytic molecular markers after 3-nitropropionic acid (3NP) preconditioning. 3NP is a mitochondrial toxin, which can induce tolerance to ischemia at subtoxic levels. Rats were treated with 3NP at the tolerance-inducible and the non-tolerance-inducible stage (TS or NTS) before focal ischemia. The control group was injected with physiological saline. After ischemia, the hemispheric enlargement (HE) was volumetrically measured. Immunohistochemical and immunofluorescence analyses of AQP4, glial fibrillary acidic protein (GFAP), and glutamine synthetase (GS) were also conducted after the 3NP treatment and a vehicle was applied. HE was found to be significantly smaller in the TS group than in the vehicle group or the NTS group. The immunofluorescence analyses demonstrated that the AQP4 immunoreactivity in the cortex and striatum was significantly reduced in the TS group but not in the NTS group. In contrast, both GFAP expression and GS expression in the TS group were enhanced, with reactive astrocytosis. AQP4 downregulation in reactive astrocytosis may be one of the factors contributing to the role of 3NP preconditioning in attenuating post-ischemic edema.
25872192
Aquaporin‐4 facilitates reabsorption of excess fluid in vasogenic brain edema
Aquaporin‐4 (AQP4) is the major water channel in the brain, expressed predominantly in astroglial cell membranes. Initial studies in AQP4‐deficient mice showed reduced cellular brain edema following water intoxication and ischemic stroke. We hypothesized that AQP4 deletion would have the opposite effect (increased brain swelling) in vasogenic (noncellular) edema because of impaired removal of excess brain water through glial limitans and ependymal barriers. In support of this hypothesis, we found higher intracranial pressure (ICP, 52±6 vs. 26±3 cm H2O) and brain water content (81.2±0.1 vs. 80.4±0.1%) in AQP4‐deficient mice after continuous intraparenchymal fluid infusion. In a freeze‐injury model of vasogenic brain edema, AQP4‐deficient mice had remarkably worse clinical outcome, higher ICP (22±4 vs. 9±1 cm H2O), and greater brain water content (80.9±0.1 vs. 79.4±0.1%). In a brain tumor edema model involving stereotactic implantation of melanoma cells, tumor growth was comparable in wild‐ type and AQP4‐deficient mice. However, AQP4‐deficient mice had higher ICP (39±4 vs. 19±5 cm H2O at seven days postimplantation) and corresponding accelerated neurological deterioration. Thus, AQP4‐mediated transcellular water movement is crucial for fluid clearance in vasogenic brain edema, suggesting AQP4 activation and/or up‐regulation as a novel therapeutic option in vasogenic brain edema.
24022070
Effects of Aquaporin-4 on edema formation following intracerebral hemorrhage
OBJECTIVE
Intracerebral hemorrhage (ICH) constitutes 10% to 15% of all strokes and is associated with high morbidity and mortality. To date, little is known about the role of AQP4 (Aquaporin-4), which is abundantly expressed in pericapillary astrocyte foot processes and in edema formation after intracerebral hemorrhage. The purpose of this study was to examine the role of AQP4 in edema formation after ICH by using AQP4(-/-) mice.


METHODS
ICH was induced by microinjecting 5microl autologous whole blood into the striatum of AQP4(+/+) and AQP4(-/-) mice. We compared neurological deficits, brain edema contents of whole hemorrhagic ipsilateral hemisphere, specific gravity of brain tissue surrounding hematoma, Evans blue leakage and ultrastructure of brain microvessels between AQP4(+/+) and AQP4(-/-) mice following ICH. Histological changes were also detected with Nissl's staining and TUNEL staining.


RESULTS
Our experiments showed a significant increase of AQP4 expression following ICH in AQP4(+/+) mice. AQP4 deletion aggravated neurological deficits and brain edema contents of whole hemorrhagic ipsilateral hemisphere. Besides, it also reduced the specific gravity of brain tissue surrounding hematoma. Moreover, it enhanced Evans blue leakage and ultrastructure of brain microvessel damage. Histology also showed less Nissl's staining and more TUNEL staining in AQP4(-/-) mice following ICH.


CONCLUSIONS
These results suggest that AQP4 deletion increases ICH damage, including edema formation, blood-brain barrier damage and neuronal death/TUNEL-positive cells. Further studies on the protective role of activated AQP4 expression following ICH may provide useful therapeutic target for ICH-induced brain injury.
8428058
Pretreatment with a novel aquaporin 4 inhibitor, TGN-020, significantly reduces ischemic cerebral edema
We investigated the in vivo effects of a novel aquaporin 4 (AQP4) inhibitor 2-(nicotinamide)-1,3,4-thiadiazole, TGN-020, in a mouse model of focal cerebral ischemia using 7.0-T magnetic resonance imaging (MRI). Pretreatment with TGN-020 significantly reduced brain edema associated with brain ischemia, as reflected by percentage of brain swelling volume (%BSV), 12.1 ± 6.3% in the treated group, compared to (20.8 ± 5.9%) in the control group (p < 0.05), and in the size of cortical infarction as reflected by the percentage of hemispheric lesion volume (%HLV), 20.0 ± 7.6% in the treated group, compared to 30.0 ± 9.1% in the control group (p < 0.05). The study indicated the potential pharmacological use of AQP4 inhibition in reducing brain edema associated with focal ischemia.
4992077
Aquaporin-4 Inhibition Mediates Piroxicam-Induced Neuroprotection against Focal Cerebral Ischemia/Reperfusion Injury in Rodents
Background and Purpose Aquaporin-4(AQP4) is an abundant water channel protein in brain that regulates water transport to maintain homeostasis. Cerebral edema resulting from AQP4 over expression is considered to be one of the major determinants for progressive neuronal insult during cerebral ischemia. Although, both upregulation and downregulation of AQP4 expression is associated with brain pathology, over expression of AQP4 is one of the chief contributors of water imbalance in brain during ischemic pathology. We have found that Piroxicam binds to AQP4 with optimal binding energy value. Thus, we hypothesized that Piroxicam is neuroprotective in the rodent cerebral ischemic model by mitigating cerebral edema via AQP4 regulation. Methods Rats were treated with Piroxicam OR placebo at 30 min prior, 2 h post and 4 h post 60 minutes of MCAO followed by 24 hour reperfusion. Rats were evaluated for neurological deficits and motor function just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, biochemical analysis, RT-PCR and western blot experiments. Results Piroxicam pretreatment thirty minutes prior to ischemia and four hour post reperfusion afforded neuroprotection as evident through significant reduction in cerebral infarct volume, improvement in motor behavior, neurological deficit and reduction in brain edema. Furthermore, ischemia induced surge in levels of nitrite and malondialdehyde were also found to be significantly reduced in ischemic brain regions in treated animals. This neuroprotection was found to be associated with inhibition of acid mediated rise in intracellular calcium levels and also downregulated AQP4 expression. Conclusions Findings of the present study provide significant evidence that Piroxicam acts as a potent AQP4 regulator and renders neuroprotection in focal cerebral ischemia. Piroxicam could be clinically exploited for the treatment of brain stroke along with other anti-stroke therapeutics in future.
33538624
The Na–K–Cl Co-transporter in astrocyte swelling
Ion channels, exchangers and transporters are known to be involved in cell volume regulation. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na+–K+–Cl− cotransporters has been shown to regulate cell volume in many conditions. The Na+–K+–Cl− cotransporters (NKCC) are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. Studies have established the role of NKCC1 in astrocyte swelling/brain edema in ischemia and trauma. Our recent studies suggest that NKCC1 activation is also involved in astrocyte swelling induced by ammonia and in the brain edema in the thioacetamide model of acute liver failure. This review will focus on mechanisms of NKCC1 activation and its contribution to astrocyte swelling/brain edema in neurological disorders, with particular emphasis on ammonia neurotoxicity and acute liver failure.
23997
Na+-Dependent Chloride Transporter (NKCC1)-Null Mice Exhibit Less Gray and White Matter Damage after Focal Cerebral Ischemia
We previously demonstrated that pharmacological inhibition of Na+−K+−Cl− cotransporter isoform 1 (NKCC1) is neuroprotective in in vivo and in vitro ischemic models. In this study, we investigated whether genetic ablation of NKCC1 provides neuroprotection after ischemia. Focal ischemia was induced by 2 hours occlusion of the left middle cerebral artery (MCAO) followed by 10 or 24 hours reperfusion. Two hours MCAO and ten or twenty-four hours reperfusion caused infarction (˜85 mm3) in NKCC1 wild-type (NKCC1+/+) mice. Infarction volume in NKCC1−/− mice was reduced by ˜30% to 46%. Heterozygous mutant (NKCC1+/–) mice showed ˜28% reduction in infarction (P>0.05). Two hours MCAO and twenty-four hours reperfusion led to a significant increase in brain edema in NKCC1+/+ mice. In contrast, NKCC1+/– and NKCC1−/− mice exhibited ˜50% less edema (P<0.05). Moreover, white matter damage was assessed by immunostaining of amyloid precursor protein (APP). An increase in APP was detected in NKCC1+/+ mice after 2 hours MCAO and 10 hours reperfusion. However, NKCC1−/− mice exhibited significantly less APP accumulation (P<0.05). Oxygen-glucose deprivation (OGD) induced ˜67% cell death and a fourfold increase in Na+ accumulation in cultured NKCC1+/+ cortical neurons. OGD-mediated cell death and Na+ influx were significantly reduced in NKCC1−/− neurons (P<0.05). In addition, inhibition of NKCC1 by bumetanide resulted in similar protection in NKCC1+/+ neurons and astrocytes (P<0.05). These results imply that stimulation of NKCC1 activity is important in ischemic neuronal damage.
18088820
Bumetanide Inhibition of the Blood-Brain Barrier Na-K-Cl Cotransporter Reduces Edema Formation in the Rat Middle Cerebral Artery Occlusion Model of Stroke
Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl cotransporter is stimulated by factors present during ischemia, suggesting that the cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6–30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40–67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia.
20906697
NKCC1-mediated traumatic brain injury-induced brain edema and neuron death via Raf/MEK/MAPK cascade
Objective:Brain edema is one of the characteristic features of patients with severe traumatic brain injury. The aim of this study was to examine the effects of Na+-K+-2Cl− co-transporter on traumatic brain injury-induced brain edema and neuron damage and to elucidate the relationship between Na+-K+-2Cl− co-transporter and mitogen-activated protein kinase (MAPK) cascade. Design:Laboratory investigation. Setting:University research laboratory. Subjects:Male Wistar rats weighing 350–400 g. Interventions:Anesthetized animals were subjected to a weight-drop device (450-g weight, 1.8-m height) to induce traumatic brain injury. Measurements and Main Results:The expression of Na+-K+-2Cl− co-transporter and phosphorylation of MAPK cascade were determined by Western blot test. We also analyzed the degree of brain edema and neuronal damage in this study. We found that the messenger RNA and protein of Na+-K+-2Cl− co-transporter were up-regulated mainly in hippocampus neurons from 2 to 24 hrs after traumatic brain injury. After traumatic brain injury, animals displayed severe brain edema and neuron damage. The phosphorylation of extracellular signal-regulated kinase, MAPK kinase, and Raf also was significantly elevated after traumatic brain injury. Bumetanide (15.2 mg/kg), a specific Na+-K+-2Cl− co-transporter inhibitor, significantly attenuated the neuronal damage and brain edema after traumatic brain injury by decreasing the phosphorylation of Raf/MEK/ERK cascade proteins. Conclusions:The present study suggests that Na+-K+-2Cl− co-transporter plays an important role in TBI-induced brain edema and neuronal damage via activation of MAPK cascade.
25972155
The Na-K-Cl cotransporter in the brain edema of acute liver failure.
BACKGROUND & AIMS
Astrocyte swelling and brain edema associated with increased intracranial pressure are major complications of acute liver failure (ALF). The mechanism for such astrocyte swelling/brain edema, however, is not well understood. We recently found that ammonia, a key etiological factor in ALF, caused the activation of the Na-K-Cl cotransporter-1 (NKCC1) in cultured astrocytes, and that inhibition of such activation led to a reduction in astrocyte swelling, suggesting that NKCC1 activation may be an important factor in the mechanism of brain edema in ALF. To determine whether NKCC activation is also involved in brain edema in vivo, we examined whether NKCC activation occurs in the thioacetamide (TAA) rat model of ALF and determined whether treatment with the NKCC inhibitor bumetanide reduces the severity of brain edema in TAA-treated rats.


METHODS
Brain water content was measured using the gravimetric method. NKCC1 phosphorylation and protein expression were measured by Western blots. NKCC activity was measured in brain cortical slices.


RESULTS
NKCC activity was elevated in brain cortical slices of TAA-treated rats as compared to sham animals. Western blot analysis showed significant increases in total as well as phosphorylated (activated) NKCC1 protein expression in the cortical tissue. These findings were associated with a significant increase in brain water content which was attenuated by treatment with the NKCC inhibitor bumetanide.


CONCLUSIONS
Our studies suggest the involvement of NKCC in the development of brain edema in experimental ALF, and that targeting NKCC may represent a useful therapeutic strategy in humans with ALF.
25166987
Cell Swelling and a Nonselective Cation Channel Regulated by Internal Ca2+ and ATP in Native Reactive Astrocytes from Adult Rat Brain
Hypoxia–ischemia and ATP depletion are associated with glial swelling and blebbing, but mechanisms involved in these effects remain incompletely characterized. We examined morphological and electrophysiological responses of freshly isolated native reactive astrocytes (NRAs) after exposure to NaN3, which depletes cellular ATP. Here we report that NaN3 caused profound and sustained depolarization attributable to activation of a novel 35 pS Ca2+-activated, [ATP]i-sensitive nonselective cation (NCCa-ATP) channel, found in >90% of excised membrane patches. The channel was impermeable to Cl−, was nearly equally permeable to monovalent cations, with permeabilities relative to K+ being PCs+/PK+(1.06) ≈ PNa+/PK+(1.04) ≈ PRb+/PK+(1.02) ≈ PLi+/PK+(0.96), and was essentially impermeable to Ca2+ and Mg2+(PCa2+/PK+≈ PMg2+/PK+< 0.001), with intracellular Mg2+ (100 μm to 1 mm) causing inward rectification. Pore radius, estimated by fitting relative permeabilities of organic cations to the Renkin equation, was 0.41 nm. This channel exhibited significantly different properties compared with previously reported NCCa-ATP channels, including different sensitivity to block by various adenine nucleotides (EC50of 0.79 μm for [ATP]i, with no block by AMP or ADP), and activation by submicromolar [Ca]i. The apparent dissociation constant for Ca2+ was voltage dependent (0.12, 0.31, and 1.5 μm at −40, −80, and −120 mV, respectively), with a Hill coefficient of 1.5. Channel opening by [ATP]i depletion was accompanied by and appeared to precede blebbing of the cell membrane, suggesting participation of this channel in cation flux involved in cell swelling. We conclude that NRAs from adult rat brain express a 35 pS NCCa-ATP channel that may play an important role in the pathogenesis of brain swelling.
496684
Newly expressed SUR1-regulated NCCa-ATP channel mediates cerebral edema after ischemic stroke
Pathological conditions in the central nervous system, including stroke and trauma, are often exacerbated by cerebral edema. We recently identified a nonselective cation channel, the NCCa-ATP channel, in ischemic astrocytes that is regulated by sulfonylurea receptor 1 (SUR1), is opened by depletion of ATP and, when opened, causes cytotoxic edema. Here, we evaluated involvement of this channel in rodent models of stroke. SUR1 protein and mRNA were newly expressed in ischemic neurons, astrocytes and capillaries. Upregulation of SUR1 was linked to activation of the transcription factor Sp1 and was associated with expression of functional NCCa-ATP but not KATP channels. Block of SUR1 with low-dose glibenclamide reduced cerebral edema, infarct volume and mortality by 50%, with the reduction in infarct volume being associated with cortical sparing. Our findings indicate that the NCCa-ATP channel is crucially involved in development of cerebral edema, and that targeting SUR1 may provide a new therapeutic approach to stroke.
2220110
Sulfonylurea receptor 1 in central nervous system injury: a focused review
The sulfonylurea receptor 1 (Sur1)-regulated NCCa-ATP channel is a nonselective cation channel that is regulated by intracellular calcium and adenosine triphosphate. The channel is not constitutively expressed, but is transcriptionally upregulated de novo in all cells of the neurovascular unit, in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). The channel is linked to microvascular dysfunction that manifests as edema formation and delayed secondary hemorrhage. Also implicated in oncotic cell swelling and oncotic (necrotic) cell death, the channel is a major molecular mechanism of ‘accidental necrotic cell death’ in the CNS. In animal models of SCI, pharmacological inhibition of Sur1 by glibenclamide, as well as gene suppression of Abcc8, prevents delayed capillary fragmentation and tissue necrosis. In models of stroke and TBI, glibenclamide ameliorates edema, secondary hemorrhage, and tissue damage. In a model of SAH, glibenclamide attenuates the inflammatory response due to extravasated blood. Clinical trials of an intravenous formulation of glibenclamide in TBI and stroke underscore the importance of recent advances in understanding the role of the Sur1-regulated NCCa-ATP channel in acute ischemic, traumatic, and inflammatory injury to the CNS.
13108024
Endothelial sulfonylurea receptor 1-regulated NC Ca-ATP channels mediate progressive hemorrhagic necrosis following spinal cord injury.
Acute spinal cord injury (SCI) causes progressive hemorrhagic necrosis (PHN), a poorly understood pathological process characterized by hemorrhage and necrosis that leads to devastating loss of spinal cord tissue, cystic cavitation of the cord, and debilitating neurological dysfunction. Using a rodent model of severe cervical SCI, we tested the hypothesis that sulfonylurea receptor 1-regulated (SUR1-regulated) Ca(2+)-activated, [ATP](i)-sensitive nonspecific cation (NC(Ca-ATP)) channels are involved in PHN. In control rats, SCI caused a progressively expansive lesion with fragmentation of capillaries, hemorrhage that doubled in volume over 12 hours, tissue necrosis, and severe neurological dysfunction. SUR1 expression was upregulated in capillaries and neurons surrounding necrotic lesions. Patch clamp of cultured endothelial cells exposed to hypoxia showed that upregulation of SUR1 was associated with expression of functional SUR1-regulated NC(Ca-ATP) channels. Following SCI, block of SUR1 by glibenclamide or repaglinide or suppression of Abcc8, which encodes for SUR1 by phosphorothioated antisense oligodeoxynucleotide essentially eliminated capillary fragmentation and progressive accumulation of blood, was associated with significant sparing of white matter tracts and a 3-fold reduction in lesion volume, and resulted in marked neurobehavioral functional improvement compared with controls. We conclude that SUR1-regulated NC(Ca-ATP) channels in capillary endothelium are critical to development of PHN and constitute a major target for therapy in SCI.
13728782
Glibenclamide reduces secondary brain damage after experimental traumatic brain injury
Following traumatic brain injury (TBI) SUR1-regulated NCCa-ATP (SUR1/TRPM4) channels are transcriptionally up-regulated in ischemic astrocytes, neurons, and capillaries. ATP depletion results in depolarization and opening of the channel leading to cytotoxic edema. Glibenclamide is an inhibitor of SUR-1 and, thus, might prevent cytotoxic edema and secondary brain damage following TBI. Anesthetized adult Sprague-Dawley rats underwent parietal craniotomy and were subjected to controlled cortical impact injury (CCI). Glibenclamide was administered as a bolus injection 15min after CCI injury and continuously via osmotic pumps throughout 7days. In an acute trial (180min) mean arterial blood pressure, heart rate, intracranial pressure, encephalographic activity, and cerebral metabolism were monitored. Brain water content was assessed gravimetrically 24h after CCI injury and contusion volumes were measured by MRI scanning technique at 8h, 24h, 72h, and 7d post injury. Throughout the entire time of observation neurological function was quantified using the "beam-walking" test. Glibenclamide-treated animals showed a significant reduction in the development of brain tissue water content(80.47%±0.37% (glibenclamide) vs. 80.83%±0.44% (control); p<0.05; n=14). Contusion sizes increased continuously within 72h following CCI injury, but glibenclamide-treated animals had significantly smaller volumes at any time-points, like 172.53±38.74mm(3) (glibenclamide) vs. 299.20±64.02mm(3) (control) (p<0.01; n=10; 24h) or 211.10±41.03mm(3) (glibenclamide) vs. 309.76±19.45mm(3) (control) (p<0.05; n=10; 72h), respectively. An effect on acute parameters, however, could not be detected, most likely because of the up-regulation of the channel within 3-6h after injury. Furthermore, there was no significant effect on motor function assessed by the beam-walking test throughout 7days. In accordance to these results and the available literature, glibenclamide seems to have promising potency in the treatment of TBI.
4119214
Glibenclamide—10-h Treatment Window in a Clinically Relevant Model of Stroke
Glibenclamide improves outcomes in rat models of stroke, with treatment as late as 6 h after onset of ischemia shown to be beneficial. Because the molecular target of glibenclamide, the sulfonylurea receptor 1 (Sur1)-regulated NCCa-ATP channel, is upregulated de novo by a complex transcriptional mechanism, and the principal pathophysiological target, brain swelling, requires hours to develop, we hypothesized that the treatment window would exceed 6 h. We studied a clinically relevant rat model of stroke in which middle cerebral artery occlusion (75% < reduction in LDF signal ≤90%) was produced using an intra-arterial occluder. Recanalization was obtained 4.5 h later by removing the occluder. At that time, we administered recombinant tissue plasminogen activator (rtPA; 0.9 mg/kg IV over 30 min). Immunolabeling showed modest expression of Sur1 5 h after onset of ischemia, with expression increasing 7- to 11-fold (P < 0.01) by 24 h. Rats were administered either vehicle or glibenclamide (10 μg/kg IP loading dose plus 200 ng/h by constant subcutaneous infusion) beginning 4.5 or 10 h after onset of ischemia. In rats treated at 4.5 or 10 h, glibenclamide significantly reduced hemispheric swelling at 24 h from (mean ± SEM) 14.7 ± 1.5% to 8.1 ± 1.6% or 8.8 ± 1.1% (both P < 0.01), respectively, and significantly reduced 48-h mortality from 53% to 17% or 12% (both P < 0.01), and improved Garcia scores at 48 h from 3.8 ± 0.62 to 7.6 ± 0.70 or 8.4 ± 0.74 (both P < 0.01). We conclude that, in a clinically relevant model of stroke, the treatment window for glibenclamide extends to 10 h after onset of ischemia.
25029399
Glibenclamide in Cerebral Ischemia and Stroke
The sulfonylurea receptor 1 (Sur1)–transient receptor potential 4 (Trpm4) channel is an important molecular element in focal cerebral ischemia. The channel is upregulated in all cells of the neurovascular unit following ischemia, and is linked to microvascular dysfunction that manifests as edema formation and secondary hemorrhage, which cause brain swelling. Activation of the channel is a major molecular mechanism of cytotoxic edema and “accidental necrotic cell death.” Blockade of Sur1 using glibenclamide has been studied in different types of rat models of stroke: (i) in conventional non-lethal models (thromboembolic, 1–2 h temporary, or permanent middle cerebral artery occlusion), glibenclamide reduces brain swelling and infarct volume and improves neurological function; (ii) in lethal models of malignant cerebral edema, glibenclamide reduces edema, brain swelling, and mortality; (iii) in models with rtPA, glibenclamide reduces swelling, hemorrhagic transformation, and death. Retrospective studies of diabetic patients who present with stroke have shown that those whose diabetes is managed with a sulfonylurea drug and who are maintained on the sulfonylurea drug during hospitalization for stroke have better outcomes at discharge and are less likely to suffer hemorrhagic transformation. Here, we provide a comprehensive review of the basic science, preclinical experiments, and retrospective clinical studies on glibenclamide in focal cerebral ischemia and stroke. We also compare the preclinical work in stroke models to the updated recommendations of the Stroke Therapy Academic Industry Roundtable (STAIR). The findings reviewed here provide a strong foundation for a translational research program to study glibenclamide in patients with ischemic stroke.
23336175
Molecular pharmacology and pathophysiological significance of endothelin.
Since the discovery of the most potent vasoconstrictor peptide, endothelin, in 1988, explosive investigations have rapidly clarified much of the basic pharmacological, biochemical and molecular biological features of endothelin, including the presence and structure of isopeptides and their genes (endothelin-1, -2 and -3), regulation of gene expression, intracellular processing, specific endothelin converting enzyme (ECE), receptor subtypes (ETA and ETB), intracellular signal transduction following receptor activation, etc. ECE was recently cloned, and its structure was shown to be a single transmembrane protein with a short intracellular N-terminal and a long extracellular C-terminal that contains the catalytic domain and numerous N-glycosylation sites. In addition to acute contractile or secretory actions, endothelin has been shown to exert long-term proliferative actions on many cell types. In this case, intracellular signal transduction appears to converge to activation of mitogen-activated protein kinase. As a recent dramatic advance, a number of non-peptide and orally active receptor antagonists have been developed. They, as well as current peptide antagonists, markedly accelerated the pace of investigations into the true pathophysiological roles of endogenous endothelin-1 in mature animals; e.g., hypertension, pulmonary hypertension, acute renal failure, cerebral vasospasm, vascular thickening, cardiac hypertrophy, chronic heart failure, etc. Thus, the interference with the endothelin pathway by either ECE-inhibition or receptor blockade may provide an exciting prospect for the development of novel therapeutic drugs.
24625268
Pharmacology and physiopathology of the brain endothelin system: an overview.
The endothelin system, consisting of three peptides, two peptidases and two G-protein coupled receptors, is widely expressed in the brain cell types and brain-derived tumor cell lines. The stimulation of endothelin receptors elicits a variety of short- and long-term changes at cellular level but the effects of the pharmacological modulation of the endothelin system in brain physiology and pathophysiology are, at the present time, poorly understood. Altered expression of endothelins (ETs) in reactive astrocytes has been observed in many pathological conditions of the human brain, such as infarcts, lacunae, traumatic conditions, Alzheimer's disease and inflammatory diseases of the brain. In addition, recent studies have shown that endothelin antagonists might inhibit growth and induce cell death in human melanoma cells in vitro and in vivo, and have emphasized a possible role of endothelin peptides as autocrine or paracrine factor in the proliferation and dissemination of tumor cell lines. Given the fact that brain cell and a variety of brain tumor cell lines express functional endothelin receptors, further studies are warranted to demonstrate a possible therapeutic role of endothelin agonists and antagonist in the pharmacological treatment of brain-related diseases and brain tumors.
14743313
Endothelin-1 as a neuropeptide: neurotransmitter or neurovascular effects?
Endothelin-1 (ET-1) is an endothelium-derived peptide that also possesses potent mitogenic activity. There is also a suggestion the ET-1 is a neuropeptide, based mainly on its histological identification in both the central and peripheral nervous system in a number of species, including man. A neuropeptide role for ET-1 is supported by studies showing a variety of effects caused following its administration into different regions of the brain and by application to peripheral nerves. In addition there are studies proposing that ET-1 is implicated in a number of neural circuits where its transmitter affects range from a role in pain and temperature control to its action on the hypothalamo-neurosecretory system. While the effect of ET-1 on nerve tissue is beyond doubt, its action on nerve blood flow is often ignored. Here, we review data generated in a number of species and using a variety of experimental models. Studies range from those showing the distribution of ET-1 and its receptors in nerve tissue to those describing numerous neurally-mediated effects of ET-1.
23605915
Targeting endothelin receptors for pharmacotherapy of ischemic stroke: current scenario and future perspectives.
Increased expression of endothelin (ET) peptide and its receptors following ischemic stroke is found to regulate many critical aspects of stroke pathophysiology. Many attempts have been made to target ET receptors in various animal models of stroke, but it is very difficult to draw a definite line of conclusion, because these studies differ in many aspects, such as animal model, treatment schedule, parameters and techniques used for assessing these parameters. A meta-analysis of all studies showed a significant reduction in the lesion volume and improvement in functional outcome in focal cerebral ischemia. ET(A) receptor antagonists appear to offer an essential advantage of multiple neuroprotective mechanisms, including prevention of blood-brain barrier disruption and leukocyte infiltration.
25195264
Endothelin receptor expression in the normal and injured spinal cord: potential involvement in injury-induced ischemia and gliosis
The endothelins (ETs) are a family of peptides that exert their biological effects via two distinct receptors, the endothelin A receptor (ET(A)R) and the endothelin B receptor (ET(B)R). To more clearly define the potential actions of ETs following spinal cord injury, we used immunohistochemistry and confocal microscopy to examine the protein expression of ET(A)R and ET(B)R in the normal and injured rat spinal cord. In the normal spinal cord, ET(A)R immunoreactivity (IR) is expressed by vascular smooth muscle cells and a subpopulation of primary afferent nerve fibers. ET(B)R-IR is expressed primarily by radial glia, a small population of gray and white matter astrocytes, ependymal cells, vascular endothelial cells, and to a lesser extent in smooth muscle cells. Fourteen days following compression injury to the spinal cord, there was a significant upregulation in both the immunoexpression and number of astrocytes expressing the ET(B)R in both gray and white matter and a near disappearance of ET(B)R-IR in ependymal cells and ET(A)R-IR in primary afferent fibers. Conversely, the vascular expression of ET(A)R and ET(B)R did not appear to change. As spinal cord injury has been shown to induce an immediate increase in plasma ET levels and a sustained increase in tissue ET levels, ETs would be expected to induce an initial marked vasoconstriction via activation of vascular ET(A)R/ET(B)R and then days later a glial hypertrophy via activation of the ET(B)R expressed by astrocytes. Strategies aimed at blocking vascular ET(A)R/ET(B)R and astrocyte ET(B)Rs following spinal cord injury may reduce the resulting ischemia and astrogliosis and in doing so increase neuronal survival, regeneration, and function.
21236565
Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS
The ability of mammalian central nervous system (CNS) neurons to survive and/or regenerate following injury is influenced by surrounding glial cells. To identify the factors that control glial cell function following CNS injury, we have focused on the endothelin B receptor (ETBR), which we show is expressed by the majority of astrocytes that are immunoreactive for glial acid fibrillary protein (GFAP) in both the normal and crushed rabbit optic nerve. Optic nerve crush induces a marked increase in ETBR and GFAP immunoreactivity (IR) without inducing a significant increase in the number of GFAP‐IR astrocytes, suggesting that the crush‐induced astrogliosis is due primarily to astrocyte hypertrophy. To define the role that endothelins play in driving this astrogliosis, artificial cerebrospinal fluid (CSF), ET‐1 (an ETAR and ETBR agonist), or Bosentan (a mixed ETAR and ETBR antagonist) were infused via osmotic minipumps into noninjured and crushed optic nerves for 14 days. Infusion of ET‐1 induced a hypertrophy of ETBR/GFAP‐IR astrocytes in the normal optic nerve, with no additional hypertrophy in the crushed nerve, whereas infusion of Bosentan induced a significant decrease in the hypertrophy of ETBR/GFAP‐IR astrocytes in the crushed but not in the normal optic nerve. These data suggest that pharmacological blockade of astrocyte ETBR receptors following CNS injury modulates glial scar formation and may provide a more permissive substrate for neuronal survival and regeneration. GLIA 41:180–190, 2003. © 2003 Wiley‐Liss, Inc.
1925619
Absence of Glial Fibrillary Acidic Protein and Vimentin Prevents Hypertrophy of Astrocytic Processes and Improves Post-Traumatic Regeneration
The regenerative capacity of the CNS is extremely limited. The reason for this is unclear, but glial cell involvement has been suspected, and oligodendrocytes have been implicated as inhibitors of neuroregeneration (Chen et al., 2000, GrandPre et al., 2000; Fournier et al., 2001). The role of astrocytes in this process was proposed but remains incompletely understood (Silver and Miller, 2004). Astrocyte activation (reactive gliosis) accompanies neurotrauma, stroke, neurodegenerative diseases, or tumors. Two prominent hallmarks of reactive gliosis are hypertrophy of astrocytic processes and upregulation of intermediate filaments. Using the entorhinal cortex lesion model in mice, we found that reactive astrocytes devoid of the intermediate filament proteins glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-), and consequently lacking intermediate filaments (Colucci-Guyon et al., 1994; Pekny et al., 1995; Eliasson et al., 1999), showed only a limited hypertrophy of cell processes. Instead, many processes were shorter and not straight, albeit the volume of neuropil reached by a single astrocyte was the same as in wild-type mice. This was accompanied by remarkable synaptic regeneration in the hippocampus. On a molecular level, GFAP-/-Vim-/- reactive astrocytes could not upregulate endothelin B receptors, suggesting that the upregulation is intermediate filament dependent. These findings show a novel role for intermediate filaments in astrocytes and implicate reactive astrocytes as potent inhibitors of neuroregeneration.
2182986
Endothelins Promote the Activation of Astrocytes in Rat Neostriatum through ETB Receptors
The effects of endothelin (ET)‐3 and an ETB receptor agonist on astrocytic activation in rat caudate putamen were examined by an immunohistochemical staining of glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes. A single injection of 40 pmol ET‐3 into rat caudate putamen increased the number of GFAP positive cells compared to that in the contralateral saline‐injected side. Ala1,3,11,15‐ET‐1 (40 pmol), an ETB receptor agonist, also increased the number of striatal GFAP positive cells. The increases in GFAP positive cells were maximum (about 150% of the control side) in 1–2 weeks after injections of the ETs, and then reduced in 4 weeks. A continuous infusion of BQ788, an ETB receptor antagonist (23 nmol/day), into the lateral ventricle of the cerebrum antagonized the effect of Ala1,3,11,15‐ET‐1, while 80788 also reduced the number of GFAP positive cells in saline‐injected caudate putamen. Intrastriatal injection of 40 pmol Ala1,3,11,15‐ET‐1 did not affect the number of cells stained by B4 isolectin from Griffonia simplicifolia, which labels activated microglia/macrophages. Intraperitoneal administration of 5 mg/kg per day chloroquine and 0.2 mg/kg per day colchicine did not affect the action of Ala1,3,11,15‐ET‐1. These results suggest that activation of ETB receptors is involved in the induction of reactive astrocytes.
42277956
Intracerebroventricular administration of an endothelin ETB receptor agonist increases expressions of GDNF and BDNF in rat brain
Endothelins (ETs) are suggested to be involved in functional alterations of astrocytes after brain injury, including proliferation, hypertrophy and production of neurotrophic factors. In this study, effects of Ala1,3,11,15‐endothelin‐1 (Ala1,3,11,15‐ET‐1), an ETB receptor selective agonist, on neurotrophic factor production were examined in rat brain. A continuous intracerebroventricular administration of Ala1,3,11,15‐ET‐1 (500 pmol/day for 7 days) increased the numbers of GFAP‐ and vimentin‐positive astrocytes in the hippocampus, caudate putamen and cerebrum. Ala1,3,11,15‐ET‐1 did not induce neuronal degeneration and activation of microglia/macrophage in these brain regions. The intracerebroventricular administration of Ala1,3,11,15‐ET‐1 for 7 days caused two‐ to three‐fold increases in glial cell line‐derived neurotrophic factors (GDNF) mRNA in the hippocampus and cerebrum. The mRNA levels of brain‐derived neurotrophic factors (BDNF) in caudate putamen were increased by Ala1,3,11,15‐ET‐1. Expressions of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) mRNA in these regions were not largely affected by Ala1,3,11,15‐ET‐1, except cerebral NGF mRNA level was increased. The Ala1,3,11,15‐ET‐1‐induced increases in GDNF and BDNF mRNA levels were accompanied by increases in immunoreactive GDNF and BDNF. Immunohistochemical observations showed that GFAP‐positive astrocytes expressed GDNF and BDNF in the brain regions of Ala1,3,11,15‐ET‐1‐infused rats. In cultured rat astrocytes, Ala1,3,11,15‐ET‐1 (100 nm) increased mRNA levels of GDNF and BDNF. These results suggest that activation of brain ETB receptors induced GDNF and BDNF expression in astrocytes.
19179348
Intracerebroventricular administration of an endothelin ET(B)-receptor agonist increases expression of matrix metalloproteinase-2 and -9 in rat brain.
Matrix metalloproteinases (MMPs), a family of zinc-endopeptidases, have a critical role in the pathophysiological responses in damaged brains. MMPs are up-regulated in brain pathologies. To clarify the extracellular signals involved in brain MMP production, the effects of endothelins (ETs), a family of vasoconstricting peptides, were examined. Intracerebroventricular administration of 500 pmol/day Ala(1,3,11,15)-ET-1, an ET(B)-receptor agonist, increased the mRNAs of MMP2 and MMP9 in rat hippocampus and cerebrum. Ala(1,3,11,15)-ET-1 did not affect mRNA levels of MMP 1, 12, and 14. Administration of Ala(1,3,11,15)-ET-1 for 7 days also increased the protein content and proteolytic activities of MMP2 and MMP9 in the cerebrum. Immunohistochemical observations showed that astrocytes in the hippocampus and the cerebrum of ET-infused rats had MMP2 and MMP9 reactivities. In rat cultured astrocytes, both Ala(1,3,11,15)-ET-1 (100 nM) and ET-1 (100 nM) increased MMP2 and MMP9 mRNAs. ET-1 stimulated the protein releases and the proteolytic activities of MMP2 and MMP9 from cultured astrocytes. BQ788, an ET(B) antagonist, inhibited the effects of ET-1 on astrocytic MMP2 and MMP9. The ET-induced expression of MMP9, but not MMP2, was inhibited by pyrrolidine dithiocarbamate, proteasome inhibitor I, and MG132. These results suggest that ET stimulates astrocytic MMP2 and MMP9 production through ET(B) receptors.
46121962
I.c.v administration of an endothelin ETB receptor agonist stimulates vascular endothelial growth factor-A production and activates vascular endothelial growth factor receptors in rat brain
Vascular endothelial growth factors (VEGFs), a family of angiogenic factors, are upregulated by nerve injuries. To clarify the extracellular signals involved in VEGF production in the brain, the effects of endothelins (ETs), a family of vasoconstricting peptides, were examined. I.c.v. administration of 500 pmol/d Ala(1,3,11,15)-ET-1, an ET(B) receptor agonist, increased the level of VEGF-A mRNA in the rat cerebrum, whereas those of VEGF-B, placental growth factor (PLGF), angiopoietin (ANG)-1, and ANG-2 mRNAs were not largely affected by Ala(1,3,11,15)-ET. The ET-induced increases in cerebrum VEGF-A mRNA were reduced by coadministration of 1 nmol/d BQ788, an ET(B) antagonist. Ala(1,3,11,15)-ET-1 also stimulated the production of VEGF-A proteins in the cerebrum. Immunohistochemical observations in the cerebrum of Ala(1,3,11,15)-ET-1-infused rats showed that glial fibrillary acidic protein (GFAP)-positive astrocytes had VEGF-A immunoreactivity. Neurons, microglia, and brain capillary endothelial cells in the Ala(1,3,11,15)-ET-1-infused rats did not show VEGF-A reactivity. The i.c.v. administration of Ala(1,3,11,15)-ET-1 stimulated tyrosine phosphorylations of VEGF-R1 and R2 receptors in the rat cerebrum, whereas expression levels of total VEGF-R1 and R2 proteins were not largely changed. Immunoreactivity of tyrosine-phosphorylated VEGF-R1 was selectively shown in GFAP-positive astrocytes in the cerebrum of Ala(1,3,11,15)-ET-1-infused rats. Tyrosine-phosphorylated VEGF-R2 proteins were present in astrocytes and brain capillary endothelial cells. These findings indicate that activation of brain ET(B) receptors increases production of VEGF-A and stimulates VEGF receptor signaling in the brain.
5806445
Endothelins reciprocally regulate VEGF‐A and angiopoietin‐1 production in cultured rat astrocytes: Implications on astrocytic proliferation
Vascular endothelial growth factors (VEGFs) and angiopoietins (ANGs) are involved in pathophysiological responses in damaged nerve tissues. Astrocytes produce VEGFs and ANGs upon brain ischemia and traumatic injury. To clarify the extracellular signals regulating VEGF and ANG production, effects of endothelins (ETs), a family of endothelium‐derived peptides, were examined in cultured rat astrocytes. ET‐1 (100 nM) and Ala1,3,11,15‐ET‐1 (100 nM), an ETB receptor agonist, increased VEGF‐A mRNA levels in cultured astrocytes, while ANG‐1 mRNA was decreased by ETs. ET‐1 did not affect astrocytic VEGF‐B, placental growth factor (PLGF), and ANG‐2 mRNA levels. The effects of ET‐1 on VEGF‐A and ANG‐1 mRNAs were inhibited by BQ788, an ETB antagonist. Release of VEGF‐A proteins from cultured astrocytes was increased by ET‐1. In contrast, ET‐1 reduced release of astrocytic ANG‐1. Exogenous ET‐1 (100 nM) and VEGF165 (100 ng/mL), an isopeptide of VEGF‐A, stimulated bromodeoxyuridine (BrdU) incorporation into cultured astrocytes. Treatment with ET‐1 and VEGF165 increased the numbers of cyclin D1‐positive astrocytes. Exogenous ANG‐1 (250 ng/mL) did not stimulate the BrdU incorporation. Increases in BrdU incorporation by ET‐1 and VEGF165 were not affected by ANG‐1. In 60–70% confluent cultures, SU4312 (10 μM), a VEGF receptor tyrosine kinase inhibitor, partially reduced the effects of ET‐1 on BrdU incorporation and cyclin D1 expression. ET‐induced BrdU incorporation and cyclin D1 expression were reduced by a neutralizing antibody against VEGF‐A. Our findings suggest that ET‐1 is a factor regulating astrocytic VEGF‐A and ANG‐1, and that increased VEGF‐A production potentiates ET‐induced astrocytic proliferation by an autocrine mechanism. © 2012 Wiley Periodicals, Inc.
14389657
Amelioration of Cold Injury-Induced Cortical Brain Edema Formation by Selective Endothelin ETB Receptor Antagonists in Mice
Brain edema is a potentially fatal pathological condition that often occurs in stroke and head trauma. Following brain insults, endothelins (ETs) are increased and promote several pathophysiological responses. This study examined the effects of ETB antagonists on brain edema formation and disruption of the blood-brain barrier in a mouse cold injury model (Five- to six-week-old male ddY mice). Cold injury increased the water content of the injured cerebrum, and promoted extravasation of both Evans blue and endogenous albumin. In the injury area, expression of prepro-ET-1 mRNA and ET-1 peptide increased. Intracerebroventricular (ICV) administration of BQ788 (ETB antagonist), IRL-2500 (ETB antagonist), or FR139317 (ETA antagonist) prior to cold injury significantly attenuated the increase in brain water content. Bolus administration of BQ788, IRL-2500, or FR139317 also inhibited the cold injury-induced extravasation of Evans blue and albumin. Repeated administration of BQ788 and IRL-2500 beginning at 24 h after cold injury attenuated both the increase in brain water content and extravasation of markers. In contrast, FR139317 had no effect on edema formation when administrated after cold injury. Cold injury stimulated induction of glial fibrillary acidic protein-positive reactive astrocytes in the injured cerebrum. Induction of reactive astrocytes after cold injury was attenuated by ICV administration of BQ788 or IRL-2500. These results suggest that ETB receptor antagonists may be an effective approach to ameliorate brain edema formation following brain insults.
17576150
Status Epilepticus Induces Vasogenic Edema via Tumor Necrosis Factor-α/ Endothelin-1-Mediated Two Different Pathways
Status epilepticus (SE) induces vasogenic edema in the piriform cortex with disruptions of the blood-brain barrier (BBB). However, the mechanisms of vasogenic edema formation following SE are still unknown. Here we investigated the endothelin B (ETB) receptor-mediated pathway of SE-induced vasogenic edema. Following SE, the release of tumor necrosis factor-α (TNF-α) stimulated endothelin-1 (ET-1) release and expression in neurons and endothelial cells. In addition, TNF-α-induced ET-1 increased BBB permeability via ETB receptor-mediated endothelial nitric oxide synthase (eNOS) activation in endothelial cells. ETB receptor activation also increased intracellular reactive oxygen species by NADPH oxidase production in astrocytes. These findings suggest that SE results in BBB dysfunctions via endothelial-astroglial interactions through the TNF-α-ET-1-eNOS/NADPH oxidase pathway, and that these ETB receptor-mediated interactions may be an effective therapeutic strategy for vasogenic edema in various neurological diseases.
44060263
Neuroprotection afforded by antagonists of endothelin-1 receptors in experimental stroke
Endothelin-1 (ET-1) is involved on the development of cerebral edema in acute ischemic stroke. As edema is a therapeutic target in cerebral ischemia, our aim was to study the effect of antagonists for ET-1 receptors (Clazosentan® and BQ-788, specific antagonists for receptors A and B, respectively) on the development of edema, infarct volume and sensorial-motor deficits in rats subjected to ischemia by occlusion of the middle cerebral artery (MCAO). We used Wistar rats (280-320 g) submitted to ischemia by intraluminal transient (90 min) MCAO. After ischemia, rats were randomized into 4 groups (n = 6) treated with; 1) control group (saline), 2) Clazosentan® group (10 mg/kg iv), 3) BQ-788 group (3 mg/kg iv), and 4) combined treatment (Clazosentan® 10 mg/kg plus BQ-788 3 mg/kg iv). We observed that rats treated with Clazosentan® showed a reduction of edema, measured by MRI, at 72 h (hours) and at day 7 (both p < 0.0001), and a decrease in the serum levels of ET-1 at 72 h (p < 0.0001) and at day 7 (p = 0.009). The combined treatment also induced a reduction of edema at 24 h (p = 0.004), 72 h (p < 0.0001) and at day 7 (p < 0.0001), a reduction on infarct volume, measured by MRI, at 24 and 72 h, and at day 7 (all p < 0.01), and a better sensorimotor recovery at 24 and 72 h, and at day 7 (all p < 0.01). Moreover, Clazosentan® induced a decrease in AQP4 expression, while BQ-788 induced an increase in AQP9 expression. These results suggest that antagonists for ET-1 receptors may be a good therapeutic target for cerebral ischemia.
17642347
The management of brain edema in brain tumors
This review focuses on pathophysiology, clinical signs, and imaging of brain edema associated with intracranial tumors and its treatment. Brain edema in brain tumors is the result of leakage of plasma into the parenchyma through dysfunctional cerebral capillaries. The latter type of edema (ie, vasogenic edema) and the role of other types in brain tumors is discussed. Vascular endothelial growth factor-induced dysfunction of tight junction proteins probably plays an important role in the formation of edema. Corticosteroids are the mainstay of treatment of brain edema. When possible, corticosteroids should be used in a low dose (eg, 4 mg dexamethasone daily) to avoid serious side effects such as myopathy or diabetes. Higher doses of dexamethasone (16 mg/day or more), sometimes together with osmotherapy (mannitol, glycerol) or surgery, may be used in emergency situations. On tapering, one should be aware of the possible development of corticosteroid dependency or withdrawal effects. Novel therapies include vascular endothelial growth factor receptor inhibitors and corticotropin releasing factor, which should undergo further clinical testing before they can be recommended in practice.
6380960
Effects of dexamethasone on peritumoural oedematous brain: a DT-MRI study
Objectives: Glucocorticoids (dexamethasone) are thought to reduce peritumoural brain oedema by decreasing the permeability of neoplastic capillaries and/or enhancing the clearance of extracellular water. Diffusion tensor magnetic resonance imaging (DT-MRI) was used to measure the water diffusion parameters of oedematous and normal brain in a group of patients with intracranial tumours before and after steroid treatment. Methods: Fifteen patients with intracranial tumours (seven with high-grade glioma, four with metastatic carcinoma and four with meningioma) were examined before and 48–72 h after dexamethasone treatment (16 mg/day). The mean diffusivity () and fractional anisotropy (FA) were measured for oedematous brain and apparently normal contralateral white matter before and after steroid therapy. Results: In all three patient groups there was a significant decrease in  of oedematous brain after steroid treatment (p<0.01). There was no significant change in FA of oedematous brain after treatment in any of the three groups. There was also no significant change in either  or FA of apparently normal contralateral white matter after treatment. Conclusion: These data indicate that dexamethasone produces a localised reduction in the magnitude of extracellular water molecule mobility, and hence water content, in peritumoural oedematous brain. Furthermore, the magnitude of these changes is similar for both intra- and extra-axial tumours.
6692091
Effect of steroids on edema and sodium uptake of the brain during focal ischemia in rats.
Steroids reduce permeability of the blood-brain barrier and inhibit active sodium transport by brain capillaries in vitro. Since the rate of edema formation during the early stages of ischemia is related to the rate of sodium transport from blood to brain, this study was designed to determine whether steroids reduce ischemic edema formation by inhibiting blood-brain barrier sodium transport. Dexamethasone was compared with progesterone since the latter is a more potent inhibitor of sodium transport in isolated capillaries. Sprague-Dawley rats were treated with vehicle (n = 22) or 2 mg/kg of either dexamethasone (n = 22) or progesterone (n = 17) 1 hour before occlusion of the middle cerebral artery. After 4 hours of ischemia, brain water content and blood-brain barrier permeability to [3H] alpha-aminoisobutyric acid and sodium-22 were determined. In controls, mean +/- SEM water content of tissue in the center of the ischemic zone was 82.4 +/- 0.2%. Brain edema was significantly reduced following pretreatment with either dexamethasone (80.6 +/- 0.1%, p less than 0.001) or progesterone (81.5 +/- 0.3%, p less than 0.05). There was also a significant reduction in blood-brain barrier permeability to alpha-aminoisobutyric acid in normal brain following either treatment (e.g., 2.21 +/- 0.19 and 1.37 +/- 0.10 microliters/g/min, p less than 0.001, for control and dexamethasone treatments, respectively), but no effect on the permeability to sodium (e.g., 1.19 +/- 0.05 and 1.12 +/- 0.11 microliters/g/min for control and dexamethasone treatments, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
22810774
A novel brain trauma model in the mouse: effects of dexamethasone treatment
Abstract. We describe a novel methodological approach for inducing cold lesion in the mouse as a model of human cortical contusion trauma. To validate its reproducibility and reliability, dexamethasone (Dxm) was repeatedly applied to demonstrate possible antioedematous drug effects. Following the induction of anaesthesia with halothane, the dura was exposed via trephination. Using a micromanipulator a pre-cooled (–78°C) copper cylinder, 3 mm in diameter, was pressed down to a depth of 1 mm onto the dura for 30 s under microscopic control. The body temperature was held constant at 37°C throughout the procedure. Blood pressure (BP), measured by a modified photosensor-monitored tail-cuff method, and acid–base status were not significantly different when analysed before and after cold lesion and prior to sacrifice. However, there was a marginal mixed respiratory and metabolic acidosis. The antioedematous action of Dxm was studied in four standard pre- and post-treatment paradigms: 2×0.5 mg/kg (II), 2×12.5 mg/kg (III) and 4×6.25 mg/kg (IV: 3× pre-, 1× post-treatment; V: 1× pre-, 3× post-treatment). Physiological saline injections served as controls. High doses of Dxm (III–V) significantly attenuated the cold-lesion-induced loss of body mass. Dxm treatment also resulted in a reduction of brain water content (III; P<0.05), and brain swelling (IV; P<0.05) in the lesioned hemisphere, relative to controls. In conclusion, we have characterized a novel cold lesion model in the mouse to mimic traumatic brain injury and the beneficial effect of Dxm treatment on the extent of brain oedema.
23267722
Low doses of dexamethasone decrease brain water content of collagenase-induced cerebral hematoma.
Different doses of dexamethasone were evaluated for the treatment of cerebral trauma using a rat model of cerebral hematoma induced by intracerebral (IC) stereotaxic injections of collagenase. Control animals received an intracerebral collagenase injection followed by intraperitoneal (IP) saline injection. Sham operated animals received saline only (IC, IP). Forty-eight hours following the surgeries, the brains were removed from the euthanized animals. Cerebral hemispheres were separated and the 4 coronal sections (antero-posterior plane) were weighed. Each slice was dried for 24 h (100 degrees C) and weighed again to establish brain water content. In hematoma-induced saline treated rats, significant differences in brain water content were observed when compared to sham operated animals. Rats treated with 1 mg/kg dexamethasone had a significant brain water content decrease; however, no significant differences were observed with higher doses of dexamethasone. In conclusion, low doses of dexamethasone seem to be beneficial for the treatment of cerebral trauma.
24621037
Dexamethasone inhibits ICAM-1 and MMP-9 expression and reduces brain edema in intracerebral hemorrhagic rats
BackgroundThe molecular mechanism of hemorrhagic stroke is unclear, and the identification of therapeutic agents for attenuating post-stroke brain damage remains an unresolved challenge. Dexamethasone (DEX) is used clinically to treat spinal cord injury and brain tumor patients by reducing edema formation, but has produced conflicting results in stroke management.MethodsIn this study, intracerebral hemorrhage (ICH) was induced in rats by intracranial stereotactic injection of collagenase into the caudate nucleus. DEX was given immediately and 3 days after ICH. The expression of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), nuclear factor (NF)-κB, and IκB were analyzed by Western blotting, and perihematomal edema formation was evaluated by magnetic resonance imaging.ResultsThe results showed that ICH caused an increase of ICAM-1 and MMP-9 expression from 4 h to 7 days, which was inhibited following the administration of DEX. The perihematomal edema volume in ICH rats was high, with two peak periods at 12 h and 3 days, which was also reduced in DEX-treated groups. Furthermore, the administration of DEX not only maintained IκB in cytoplasm, but also decreased NF-κB elevation in the nucleus at 3 and 5 days in ICH rats.ConclusionsIn conclusion, these data show that DEX successfully reduced post-stroke brain edema by decreasing MMP-9 and ICAM-1 levels, partially through the IκB/NF-κB signaling pathway. The timing of DEX administration in relation to the onset of brain injury may be critical.
8706472
Inflammation and brain edema: new insights into the role of chemokines and their receptors.
Brain edema is associated with a variety of neuropathological conditions such as brain trauma, ischemic and hypoxic brain injury, central nervous system infection, acute attacks of multiple sclerosis, and brain tumors. A common finding is an inflammatory response, which may have a significant impact on brain edema formation. One critical event in the development of brain edema is blood-brain barrier (BBB) breakdown, which may be initiated and regulated by several proinflammatory mediators (oxidative mediators, adhesion molecules, cytokines, chemokines). These mediators not only regulate the magnitude of leukocyte extravasation into brain parenchyma, but also act directly on brain endothelial cells causing the loosening of junction complexes between endothelial cells, increasing brain endothelial barrier permeability, and causing vasogenic edema. Here we review junction structure at the BBB, the effects of pro-inflammatory mediators on that structure, and focus on the effects of chemokines at the BBB. New evidence indicates that chemokines (chemoattractant cytokines) do not merely direct leukocytes to areas of injury. They also have direct and indirect effects on the BBB leading to BBB disruption, facilitating entry of leukocytes into brain, and inducing vasogenic brain edema formation. Chemokine inhibition may be a new therapeutic target to reduce vasogenic brain edema.
823559
Dexamethasone coordinately regulates angiopoietin-1 and VEGF: a mechanism of glucocorticoid-induced stabilization of blood-brain barrier.
Glucocorticoids stabilize the blood-brain barrier (BBB), leading to attenuation of vasogenic brain edema. However, the action mechanism of glucocorticoids has been poorly elucidated. To elucidate the mechanism, we investigated whether dexamethasone (Dex), a synthetic glucocorticoid hormone, regulates the levels of key permeability regulating factors such as angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor (VEGF) in the three types of cells comprising BBB. Dex increased the level of angiopoietin-1 mRNA and protein and decreased VEGF mRNA and protein in brain astrocytes and pericytes, but not in endothelial cells. The mRNA and protein of angiopoietin-2 were detected only in endothelial cells and not regulated by Dex. The Dex-induced regulation of angiopoietin-1 and VEGF was inhibited by RU486, suggestive of glucocorticoid receptor mediation. The mRNA stability of angiopoietin-1 and VEGF was not changed by Dex treatment, implying that Dex increases angiopoietin-1 and decreases VEGF through transcriptional regulation. This is the first study showing the coordinate regulation of angiopoietin-1 and VEGF by glucocorticoids, suggesting a novel mechanism underlying glucocorticoids-induced stabilization of BBB.
39183989
Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells
The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. An immortalised rat brain endothelial cell line (GPNT) was used to investigate the mechanisms underlying dexamethasone-induced decrease in paracellular permeability. Following treatment with 1 microM dexamethasone there was a decrease in transmonolayer paracellular permeability mainly to sucrose, fluorescein and dextrans of up to 20 KDa. According to pore theory, these differences in permeability were consistent with a decrease in the number of pores between brain endothelial cells. This effect was accompanied by a concentration of filamentous actin and cortactin to the cell periphery. Concomitantly, the continuity of the tight junctional protein ZO-1 at the cell borders was improved and was associated with an increase in both ZO-1 and occludin expression. By contrast, the expression and distribution of adherens junctional proteins such as beta-catenin and p100/p120 remained unchanged. These observations suggest that glucocorticoids induce a more differentiated BBB phenotype in cultured brain endothelial cells through modification of tight junction structure.
17024426
Glyburide is Associated with Attenuated Vasogenic Edema in Stroke Patients
BackgroundBrain edema is a serious complication of ischemic stroke that can lead to secondary neurological deterioration and death. Glyburide is reported to prevent brain swelling in preclinical rodent models of ischemic stroke through inhibition of a non-selective channel composed of sulfonylurea receptor 1 and transient receptor potential cation channel subfamily M member 4. However, the relevance of this pathway to the development of cerebral edema in stroke patients is not known.MethodsUsing a case–control design, we retrospectively assessed neuroimaging and blood markers of cytotoxic and vasogenic edema in subjects who were enrolled in the glyburide advantage in malignant edema and stroke-pilot (GAMES-Pilot) trial. We compared serial brain magnetic resonance images (MRIs) to a cohort with similar large volume infarctions. We also compared matrix metalloproteinase-9 (MMP-9) plasma level in large hemispheric stroke.ResultsWe report that IV glyburide was associated with T2 fluid-attenuated inversion recovery signal intensity ratio on brain MRI, diminished the lesional water diffusivity between days 1 and 2 (pseudo-normalization), and reduced blood MMP-9 level.ConclusionsSeveral surrogate markers of vasogenic edema appear to be reduced in the setting of IV glyburide treatment in human stroke. Verification of these potential imaging and blood biomarkers is warranted in the context of a randomized, placebo-controlled trial.