25101287
Astrocytic Activation and Delayed Infarct Expansion after Permanent Focal Ischemia in Rats. Part I: Enhanced Astrocytic Synthesis of S-100β in the Periinfarct Area Precedes Delayed Infarct Expansion
An astrocytic protein S-100β enhances the expression of inducible nitric oxide synthase in cultured astrocytes at micromolar concentrations, leading to nitric oxide-mediated death of cocultured neurons. The present study examined whether S-100β production by reactive astrocytes accumulating within the periinfarct area was related to delayed expansion of infarct volume after permanent middle cerebral artery occlusion in the rat. After rapid increases during the initial 24 hours, the increase of infarct volume then decelerated while maintaining the increasing tendency until 168 hours in this model, attaining a significant difference compared with that at 24 hours. In the periinfarct area, the number of reactive astrocytes expressing both S-100 and glial fibrillary acidic protein, the tissue level of S-100β as measured by the sandwich enzyme-linked immunosolvent assay method using anti-S-100β monoclonal antibody, and the number of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling-positive cells were significantly increased preceding the delayed expansion of infarct volume. The CSF concentration of S-100β showed a biphasic increase, presumably reflecting the immediate release from astrocytes within the ischemic core and the subsequent production in reactive astrocytes within the periinfarct area. These results show for the first time that the enhanced synthesis of S-100β by reactive astrocytes participates in the inflammatory responses within the periinfarct area, which may be related to the occurrence of delayed infarct expansion as a major component of the cytokine network.
31623720
Apoptosis and Necrosis after Reversible Focal Ischemia: An in Situ DNA Fragmentation Analysis
Apoptosis is one of the two forms of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain (Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385–388). We now show that a significant number of striatal and cortical neurons exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.
40726384
Temporal Profile of in situ DNA Fragmentation after Transient Middle Cerebral Artery Occlusion in the Rat
We measured the temporal profile and anatomic distribution of cells exhibiting DNA fragmentation at various durations of reperfusion after middle cerebral artery (MCA) occlusion in the rat. Focal cerebral ischemia was induced in male Wistar rats (n = 62) using an intraluminal monofilament blockade of the MCA. After 2 h of MCA occlusion, the animals were killed at different durations of reperfusion (0.5, 3, 6, 9, and 12 h and 1, 2, 4, 7, 14, 21, and 28 days, n = 4 per time point). Sham-operated rats (n = 4) and normal rats not subjected to any surgical procedure (n = 4) were used as controls. Coronal brain sections (5 μm) were analyzed, using an in situ ApopTag kit, hematoxylin and eosin, and immunohistochemical double-staining methods. Six rats subjected to 2 h of MCA occlusion were killed at 24 h for measurement of DNA fragmentation by gel electrophoresis. Our data indicate that within a coronal section, DNA fragmentation was present in zero to three cells in each hemisphere of normal and sham-operated rats as well as in the contralateral hemisphere of ischemic rats. The number of cells exhibiting DNA fragmentation increased as early as 0.5 h (8 ± 6), peaked at 24–48 h (213 ± 59), and persisted for 4 weeks (10 ± 2) after onset of reperfusion (p < 0.01). Groups of cells exhibiting DNA fragmentation (>95% neurons) were located primarily in the inner boundary zone of the infarct. With use of gel electrophoresis, purified DNA obtained from the ischemic tissue exhibited the characteristic nucleosome ladder pattern associated with apoptosis. The presence and anatomical location of cells exhibiting DNA fragmentation after transient MCA occlusion suggest that apoptosis contributes to the development of ischemic infarct. In addition, the prolonged presence of DNA fragmentation after the onset of ischemia suggests that apoptotic ischemic brain damage is a dynamic ongoing process.
2569548
Intact, injured, necrotic and apoptotic cells after focal cerebral ischemia in the rat
Middle cerebral artery occlusion (MCAo) leads to brain cell death. However, quantitation of injured brain cells and inflammatory cells after MCAo has not been determined in the rat. Transient (2 h) MCAo was therefore induced in male Wistar rats by means of an intraluminal monofilament. Immunohistochemical and histochemical procedures performed at 46 h after MCAo were used to identify specific cellular populations in ischemic and control rats (n = 11). In the ischemic core of the lesion, approximately 24.7% of cells disappeared. Forty-four point eight percent of parenchymal cells consisted of intact (13.0%) or reversibly injured swollen (7.6%) and scalloped/shrunken dark (24.2%) cells. The percentage of irreversibly damaged cells was 55.2%, and included 49.9% necrotic cells (10.5% red and 39.4% ghost) and 5.4% apoptotic cells. In the inner boundary zone of the lesion, 15.9% of cells disappeared. Viable cells constituted 62.0% of all remaining cells. Neutrophils and macrophages were localized to this area. In the outer boundary zone of the lesion, 9.0% of cells disappeared. Viable cells constituted 91.6% of all remaining cells. The ratio of apoptotic to necrotic cells was 1:9, 1:6, 1:13 in the ischemic core, inner and outer boundary zones, respectively. Our data suggest the presence of three zones within the ischemic lesion: the core, and inner and outer boundaries. At 46 h after 2 h of MCAo the ischemic lesion is highly heterogeneous containing relatively large percentages of morphologically intact cells, suggesting the possibility of an extended window of therapeutic opportunity.
10017472
Viability thresholds and the penumbra of focal ischemia
The classic concept of the viability thresholds of ischemia differentiates between two critical flow rates, the threshold of electrical failure and the threshold of membrane failure. These thresholds mark the upper and lower flow limits of the ischemic penumbra which is thought ot suffer only functional but not structural injury. Recent studies of the functional and metabolic disturbances suggest a more complex pattern of thresholds. At declining flow rates, protein synthesis is inhibited at first (at a threshold of about 0.55 ml/gm/min), followed by a stimulation of anaerobic glycolysis (at 0.35 ml/gm/min), the release of neurotransmitters and the beginning disturbance of energy metabolism (at about 0.20 ml/min), and finally the anoxic depolariztion (<0.15 ml/gm/min). The penumbra, as defined by the classic flow thresholds, does not remain viable for extended periods. Since viability of the tissue requires maintenance of energy‐dependent metabolic processes, penumbra is redefined as a region of constrained blood supply in which the energy metabolism is preserved. Imaging of the penumbra by combining autoradiographic cerebral blood flow measurements with bioluminescent images of adenosine triphosphate (ATP) demonstrates a gradual expansion of the infarct core (in which ATP is depleted) into the penumbra until, after a few hours, the penumbra has disappeared. It is suggested that the limited survival of the penumbra is due to periinfarct depolarizations, which result in repeated episodes of tissue hypoxia, because the increased metabolic workload is not coupled to an adequate increase of collateral blood supply. This explains pharmacological suppression of periinfarct depolarizations lowering the threshold of metabolic disturbances and reducing the volume of the ischemic infarct.
25328439
Brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (Wistar rat).
The progression from ischemic injury to pannecrosis that occurs in the rat brain several hours after occluding a large artery may be partly attributable to a worsening of the circulation through the microvessels. The objective of this study was to quantitate selected structural changes involving astrocytes and endothelial cells within an area of focal brain ischemia created by the occlusion of a middle cerebral artery. The magnitude of these structural changes was correlated with alterations in the patency to a circulating macromolecule through the microvessels (< or = 15 mu in diameter) located within the territory of the occluded artery. One hundred eighty-five adult male Wistar rats had the right middle cerebral artery occluded after threading a nylon monofilament through the external carotid artery. Experiments were terminated by either cardiovascular perfusion or decapitation and immersion fixation at intervals ranging between 30 minutes and 7 days after the arterial occlusion. Randomly selected animals from each experimental subgroup were injected intravenously with horseradish peroxidase (molecular weight 44 kd) approximately 20 minutes before death. The progressive decline in the area fraction comprised by the vessels filled with horseradish peroxidase was preceded at 30 to 60 minutes by an increase in the surface area occupied (on a cross-section of a microvessel) by endothelial cells (both nucleus and cytoplasm). This was followed by an increase of 23.7% in the mean diameter of astrocytes nuclei and a decrease of approximately 35% in lumenal surface of the microvessels. These observations suggest that the occlusion of a large cerebral artery causes prompt swelling of endothelial cells and astrocytes; both of these early biological responses may interfere with erythrocyte circulation and oxygen delivery, which (after the arterial occlusion) are entirely dependent on the circulation provided by the collateral arterial connections. Through its interference with microvascular patency and oxygen delivery, cell swelling may influence the rate at which neurons become necrotic. In this model of brain infarct the number of necrotic neurons peaks approximately 72 hours after middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 400 WORDS)
25150736
Temporal profile of ischemic tissue damage, neutrophil response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat
We investigated the temporal profile of ischemic tissue damage, neutrophil response, and vascular occlusion after permanent and transient middle cerebral artery occlusion in the rat. Focal cerebral ischemia was induced by advancing a nylon monofilament to occlude middle cerebral artery (MCA). Two groups of rats were investigated: (1) those with permanent MCA occlusion (n = 29), and (2) and those having the arterial occlusion released after 2 h (n = 34). Experiments were terminated at 6, 12, 24, 48, 72, 96 and 168 h after the onset of ischemia, and brain sections were stained with hematoxylin and eosin for histological evaluation. Initially, the cortical lesion was smaller in rats subjected to transient MCA occlusion than in rats subjected to permanent MCA occlusion (p < 0.02). The surface area of the lesion was identical in both groups at 48 h after the onset of ischemia. Neutrophil infiltration into tissue and the time of peak neutrophil infiltration occurred earlier after transient MCA occlusion than after permanent MCA occlusion (6 h, 48 h in transient; 12 h, 72 h in permanent). Within the lesions, the number of occluded vessels was significantly lower in the transient ischemia group than in the permanent ischemia group during the time interval between 12-48 h (p < 0.01). Our data suggest that the temporal evolution of the lesion, the pattern of neutrophil infiltration and the chronology of microvascular occlusion differs depending on whether the MCA occlusion is transient (2 h) or permanent; however, significant differences in the size of the brain lesion disappeared 48 h after onset of ischemia.
42950692
Enlargement of human cerebral ischemic lesion volumes measured by diffusion‐weighted magnetic resonance imaging
We aimed to determine the frequency and time course of the enlargement of ischemic cerebral lesions following human stroke and to study the effect of the state of perfusion on lesion enlargement. Acute lesion volumes were measured on diffusion‐weighted magnetic resonance images and compared with lesion volumes measured on T2‐weighted images at 7 days or later. Forty‐four measurements were performed between 2 and 53 hours after stroke onset in 28 patients. Thirteen patients also had magnetic resonance perfusion imaging performed. In 12 (43%) of 28 patients the initial lesion volume increased by 20% or more. The number of studies showing enlargement of the ischemic lesion volume ranged from 12 (43%) of 28 at or after 2 hours to 10 (38%) of 26 at or after 6 hours, 5 (33%) of 15 at or after 24 hours, and 2 (33%) of 6 at or after 48 hours. In 7 of the 10 patients in whom the hypoperfusion volume acutely exceeded the volume of the abnormality on diffusion‐weighted images, lesion volume increased by 20% or more. This study provided evidence that substantial enlargement of human cerebral ischemic lesion volumes can occur beyond the first 6, 12 or 24 hours after onset. A mismatch acutely between the region of hypoperfusion (larger) and the region of diffusion abnormality (smaller) may be predictive of ischemic lesion enlargement.
22815044
Longitudinal magnetic resonance imaging study of perfusion and diffusion in stroke: Evolution of lesion volume and correlation with clinical outcome
A prospective longitudinal diffusion‐weighted and perfusion‐weighted magnetic resonance imaging (DWI/PWI) study of stroke patients (n = 21) at five distinct time points was performed to evaluate lesion evolution and to assess whether DWI and PWI can accurately and objectively demonstrate the degree of ischemia‐induced deficits within hours after stroke onset. Patients were scanned first within 7 hours of symptom onset and then subsequently at 3 to 6 hours, 24 to 36 hours, 5 to 7 days, and 30 days after the initial scan. Lesion evolution was dynamic during the first month after stroke. Most patients (18 of 19, 95%) showed increased lesion volume over the first week and then decreased at 1 month relative to 1 week (12 of 14, 86%). Overall, lesion growth appeared to depend on the degree of mismatch between diffusion and perfusion at the initial scan. Abnormal volumes on the acute DWI and PWI (<7 hours) correlated well with initial National Institutes of Health (NIH) stroke scale scores, outcome NIH stroke scale scores, and final lesion volume. DWI and PWI can provide an early measure of metabolic and hemodynamic insufficiency, and thus can improve our understanding of the evolution and outcome after acute ischemic stroke.
24213258
Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat.
Focal brain ischemia induced in rats by occlusion of an intracranial artery is a widely used paradigm of human brain infarct. Details of the structural changes that develop in either the human or the rat brain at various times after occlusion of an intracranial artery are incompletely characterized. We studied, in 48 adult Wistar rats, structural alterations involving the cerebral hemisphere ipsilateral to an arterial occlusion, at intervals ranging from 30 min to 7 days. Microscopic changes developed over time in separate areas of the corresponding cerebral hemisphere in a predictable pattern, appearing as small lesions in the preoptic area (30 minutes), enlarging to involve the striatum, and finally involving the cerebral cortex. Two types of neuronal responses were noted according to the time elapsed; acute changes (up to 6 hours) included scalloping, shrinkage, and swelling, whereas delayed changes (eosinophilia and karyolysis) appeared later (> or = 12 hours). Three types of astrocytic responses were noted. 1) Cytoplasmic disintegration occurred in the preoptic area at a time and in a place where neurons appeared minimally injured. 2) Nuclear and cytoplasmic swelling were prominent responses in the caudoputamen and cerebral cortex at a time when neurons showed minimal alterations. 3) Increased astrocytic glial fibrillary acidic protein reactivity was noted at the interface between the lesion and the surrounding brain tissue after 4 to 6 hours. The gross pattern of the brain lesion and the maturation of neuronal changes typical of a brain infarct have a predictable progression. Focal brain ischemia of up to 6-hour duration does not induce coagulation necrosis.
25108539
Neuronal necrosis after middle cerebral artery occlusion in Wistar rats progresses at different time intervals in the caudoputamen and the cortex.
BACKGROUND AND PURPOSE
Most brain lesions that develop after an artery is occluded evolve from an initial stage of "ischemic injury" (probably reversible) to an infarct or an area where most neurons become necrotic. There is scant information on the time that must elapse after the arterial occlusion for neurons to undergo irreversible injury. The objective of these experiments was to chart the time course and the topographic distribution of the neuronal necrosis that follows the occlusion of a large cerebral artery.


METHODS
One hundred fifty-one adult rats (including 15 controls) were used in this study. One hundred forty-seven had the right middle cerebral artery occluded for variable periods ranging from 30 minutes to 7 days. After processing the brains for histology, a meticulous structural evaluation of each specimen, including quantitation of necrotic neurons, was followed by a detailed statistical analysis of the neuronal counts.


RESULTS
Few neurons in isolated sites showed morphological signs of necrosis during the initial 4 hours; the first significant increase in the percentage of necrotic neurons (15%) was observed within the territory of the occluded artery after 6 hours (P < .05); 12 hours after the arterial occlusion most neurons (65%) had become necrotic (P < .0001). Pannecrosis involving neurons, glial cells, and blood vessels was observed at 72 to 96 hours. However, even at this time pannecrosis involved only the preoptic area and the lateral putamen; a few intact neurons remained visible in the cortex, and scattered necrotic neurons could be identified beyond the edges of the "area of pallor," which does not become clearly demarcated until 4 to 5 days after the arterial occlusion.


CONCLUSIONS
There is a predictable progression in the development of neuronal necrosis after a permanent arterial occlusion. Irreversible changes appear first in the caudoputamen and then spread to the cortex. The causes for the progression of the lesion are not known; however, therapeutic interventions that start within the first 1 to 2 hours after the arterial occlusion may alter the histopathologic responses to this form of injury. It remains to be determined whether the extent of the neurological deficit induced by an arterial occlusion correlates with the number of necrotic neurons.
33371960
Delayed Treatment with Aminoguanidine Decreases Focal Cerebral Ischemic Damage and Enhances Neurologic Recovery in Rats
Delayed treatment with aminoguanidine (AG), a relatively selective inhibitor of inducible nitric oxide synthase, ameliorates brain damage produced by occlusion of the rat's middle cerebral artery (MCA). We investigated whether the protection exerted by AG is dose-dependent and whether it is associated with improved neurologic outcome. We also studied the effect of the timing of administration of AG relative to the induction of cerebral ischemia. Halothane-anesthetized spontaneously hypertensive rats underwent permanent MCA occlusion distal to the lenticulostriate branches. Neurologic deficits were assessed daily by the postural reflex test and beam balance test. Infarct volume was determined in thionin- stained sections 96 hours after ischemia and values corrected for swelling. Treatment with AG (intraperitoneally, twice daily), starting 24 hours after MCA occlusion, decreased neocortical infarct volume in comparison to vehicle-treated rats. After correction for swelling, the decrease was 8 ± 12% at 50 mg/kg (n = 8; P > .05; analysis of variance), 25 ± 13% at 100 mg/kg (n = 7; P < .05), 30 ± 16% at 200 mg/kg (n = 7; P < .05) and 32 ± 9% at 400 mg/kg (n = 5; P < .05). Twenty-four hours after induction of ischemia neurologic deficits scores did not differ between treated and untreated rats (P > .05). However, from 48 to 96 hours after ischemia, neurologic deficits improved significantly in rats treated with AG (100 to 400 mg/kg) compared to rats in which vehicle was administered (P < .05). The decrease in neocortical infarct volume was greatest when AG (100 mg/kg; twice daily) was administered 12 (26 ± 17%; n = 9) or 24 hours (25 ± 13; n = 7) after MCA occlusion. The findings show that AG decreases ischemic brain damage dose-dependently and improves neurologic recovery. Delayed treatment with AG may be a therapeutic strategy to selectively target the evolution of ischemic damage that occurs in the post-ischemic period.
23292153
Influx of leukocytes and platelets in an evolving brain infarct (Wistar rat).
The results of several experimental studies of focal ischemia and anecdotal observations suggest that leukocytes may contribute to the injury initiated by an arterial occlusion. The timing and the nature of leukocyte responses in evolving brain infarcts (either human or experimental) are incompletely characterized. This is a study of experimental brain lesions in 96 Wistar rats that underwent occlusion of a large intracranial artery for variable intervals ranging between 30 minutes and 7 days. The experimental model, based on the occlusion of a middle cerebral artery ostium via the insertion of a nylon monofilament through the external carotid artery, does not require opening the skull; therefore, the inflammatory response is not influenced by the effects of craniotomy and changes in intracranial pressure are only those induced by the ischemic lesion. All 96 animals having the same type of arterial occlusion developed an ischemic brain lesion (limited to the territory of the corresponding artery) that evolved into an area of extensive neuronal necrosis over a period of 6 to 12 hours followed by pan-necrosis (infarct) approximately 60 hours later. In this study, leukocytes (in particular polymorphonuclear cells) were detected in the microvessels (capillaries and venules) of the ischemic hemisphere as early as 30 minutes after the arterial occlusion. Numbers of intravascular neutrophils peaked at 12 hours, whereas intraparenchymal granulocytes were most numerous at 24 hours; a few granulocytes were visible in the brain infarct as late as day 7. Circulating monocytes were first detected within the capillaries/venules of the ischemic area after 4 to 6 hours. Platelet aggregates were more abundant in the arterial than the venous side of the circulation, and luminal obstruction of arteries by platelet aggregates became noticeable only 48 hours after the arterial occlusion. Fibrin thrombi were conspicuous for their absence. These observations provide the background for studies that will attempt to unravel the relationship between the biological responses of leukocytes and neuronal necrosis secondary to focal ischemia.
39652740
Marked Induction of Calcium-Independent Nitric Oxide Synthase Activity after Focal Cerebral Ischemia
We studied the effect of focal cerebral ischemia on inducible (iNOS) and constitutive (cNOS) nitric oxide synthase enzymatic activities in the affected brain. The middle cerebral artery (MCA) was occluded in spontaneously hypertensive rats. Animals were killed 1, 2, 4, and 7 days later. cNOS and iNOS enzymatic activities were determined in the infarcted cortex using the assay of Bredt and Snyder. cNOS was assayed in the presence of calcium, whereas iNOS was assayed in the absence of calcium and in the presence of tetrahydrobiopterin. The validity of the iNOS assay was verified in rats treated with bacterial lipopolysaccharide. In these animals, the magnitude of the induction of iNOS enzymatic activity in lung, spleen, and brain paralleled the expression of iNOS mRNA, assessed by reverse-transcription polymerase chain reaction. After MCA occlusion, calcium-dependent (cNOS) activity was markedly reduced only in lesioned cerebral cortex at days 1–7 (p < 0.001; analysis of variance and Tukey's test). In contrast to cNOS, calcium-independent (iNOS) activity was induced substantially in the infarct (p < 0.005) but not in the contralateral intact cortex (p > 0.05). iNOS activity peaked at day 2 and was not different from baseline at day 7 (p > 0.05). No NADPH diaphorase-positive neurons were observed in the area of the lesion at days 1–7. Macrophages appeared at day 2 and invaded the infarcted tissue by day 7. At this time, numerous glial fibrillary acidic protein-positive astrocytes were observed within the lesion. The results suggest that the decline in calcium-dependent (cNOS) activity reflects loss of NOS neurons within the lesion. The induction of calcium-independent activity is likely to reflect induction of iNOS in nonneuronal cells. Sustained nitric oxide production by iNOS may contribute to the late phase of tissue damage in focal cerebral ischemia.
14759429
Inflammatory Mediators and Stroke: New Opportunities for Novel Therapeutics
Contrary to previous dogmas, it is now well established that brain cells can produce cytokines and chemokines, and can express adhesion molecules that enable an in situ inflammatory reaction. The accumulation of neutrophils early after brain injury is believed to contribute to the degree of brain tissue loss. Support for this hypothesis has been drawn from many studies where neutrophil-depletion blockade of endothelial-leukocyte interactions has been achieved by various techniques. The inflammation reaction is an attractive pharmacologic opportunity, considering its rapid initiation and progression over many hours after stroke and its contribution to evolution of tissue injury. While the expression of inflammatory cytokines that may contribute to ischemic injury has been repeatedly demonstrated, cytokines may also provide “neuroprotection” in certain conditions by promoting growth, repair, and ultimately, enhanced functional recovery. Significant additional basic work is required to understand the dynamic, complex, and time-dependent destructive and protective processes associated with inflammation mediators produced after brain injury. The realization that brain ischemia and trauma elicit robust inflammation in the brain provides fertile ground for discovery of novel therapeutic agents for stroke and neurotrauma. Inhibition of the mitogen-activated protein kinase (MAPK) cascade via cytokine suppressive anti-inflammatory drugs, which block p38 MAPK and hence the production of interleukin-1 and tumor necrosis factor-α, are most promising new opportunities. However, spatial and temporal considerations need to be exercised to elucidate the best opportunities for selective inhibitors for specific inflammatory mediators.
19764521
The Two Patterns of Reactive Astrocytosis in Postischemic Rat Brain
The distribution and time course of postischemic astrocyte hypertrophy and hyperplasia and the relationship to neuronal viability or necrosis was studied in rats subjected to 30 min of carotid and vertebral artery occlusion followed by reperfusion from 3 h to 5 weeks. Intermediate filaments (IFs) were evaluated by electron microscopy, IF proteins by immunohistochemistry, and astrocyte division by [3H]thymidine uptake. Glial fibrillary acidic protein (GFAP) increased in damaged and nondamaged brain regions by 2 days and was associated with cell enlargement, increases in IF, and transformation of GFAP-negative into GFAP-positive glia. Cell hypertrophy and increased GFAP persisted only in regions of neuronal necrosis whereas the number and size of GFAP-positive astrocytes returned to control levels in nondamaged regions by 2 weeks. Astrocyte hyperplasia was not seen until 3 days and was confined to damaged brain regions. Vimentin-positive astrocytes were numerous by 2 days in damaged brain and remained only in those regions at 5 weeks. The data demonstrate that reactive astrocytosis develops in undamaged brain, but is reversible with prolonged survival, whereas reactive astrocytosis that accompanies structural brain damage persists for prolonged periods and is associated with hyperplasia, as well as hypertrophy. In addition, the results show that astrocyte expression of vimentin is more specific than GFAP in identifying regions of permanent ischemic injury during the early postischemic period.
32713676
Ischemia-Induced Neuronal Cell Death, Calcium Accumulation, and Glial Response in the Hippocampus of the Mongolian Gerbil and Protection by Propentofylline (HWA 285)
The localization and timing of cellular calcium loading and glial cell reaction in relation to selective death of hippocampal neurons was studied in Mongolian gerbils following transient forebrain ichemia. Two days after a 5-min period of ischemia, heavy calcium staining was histochemically demonstrated in circumscribed groups of nerve cells, located in the transition zone between the CA1 and CA3 areas. This preceded complete neuronal cell death that was quantitatively assessed by measuring the intensity of Nissl staining. After a 12-min period of ischemia, extensive calcium loading was observed in conjunction with severe neuronal damage throughout the CA1 region as well in the dorsal nuclei of the thalamus. The extent of calcium staining decreased with time and was not seen at stages later than 7 days. Already at 2 days after a 5-min period of ischemia, a strong increase of glial fibrillary acidic protein immunoreactivity was seen. This indicates a marked and early hypertrophy of astrocytes that was not accompanied by an obvious proliferation. Neither the astrocytic response nor the neuronal calcium accumulation were observed in gerbils pretreated with propentofylline, HWA 285 (10 mg/kg, i.p.) 15 min before bilateral carotid artery occlusion. Also, the decrease of Nissl staining in the CA1 area after 5 and 12 min of ischemia was considerably less pronounced and did not significantly differ from sham-operated controls.
23660609
Molecular profile of reactive astrocytes—Implications for their role in neurologic disease

 Abstract
 
 The central nervous system responds to diverse neurologic injuries with a vigorous activation of astrocytes. While this phenomenon is found in many different species, its function is obscure. Understanding the molecular profile characteristic of reactive astrocytes should help define their function. The purpose of this review is to provide a summary of molecules whose levels of expression differentiate activated from resting astrocytes and to use the molecular profile of reactive astrocytes as the basis for speculations on the functions of these cells. At present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial fibrillary acidic protein. In vivo, this increase in glial fibrillary acidic protein-positive cells reflects predominantly phenotypic changes of resident astroglia rather than migration or proliferation of such cells. Upon activation, astrocytes upmodulate the expression of a large number of molecules. From this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system by participating in diverse biological processes. For example, upregulation of proteases and protease inhibitors could help remodel the extracellular matrix, regulate the concentration of different proteins in the neuropil and clear up debris from degenerating cells. Cytokines are key mediators of immunity and inflammation and could play a critical role in the regulation of the blood-central nervous system interface. Neurotrophic factors, transporter molecules and enzymes involved in the metabolism of excitotoxic amino acids or in the antioxidant pathway may help protect neurons and other brain cells by controlling neurotoxin levels and contributing to homeostasis within the central nervous system. Therefore, an impairment of astroglial performance has the potential to exacerbate neuronal dysfunction. Based on the synopsis of studies presented, a number of issues become apparent that deserve a more extensive analysis. Among them are the relative contribution of microglia and astrocytes to early wound repair, the characterization of astroglial subpopulations, the specificity of the astroglial response in different diseases as well as the analysis of reactive astrocytes with techniques that can resolve fast physiologic processes. Differences between reactive astrocytes in vivo and primary astrocytes in culture are discussed and underline the need for the development and exploitation of models that will allow the analysis of reactive astrocytes in the intact organism.
 

22500418
The Progression and Topographic Distribution of Interleukin-1β Expression after Permanent Middle Cerebral Artery Occlusion in the Rat
The cytokine interleukin-1 (IL-1) has been implicated in the exacerbation of ischemic damage in the brains of rodents. This study has ascertained the cellular localization and chronologic and topographic distribution of pro/mature interleukin-1β (IL-1β) protein 0.5, 1, 2, 6, 24, and 48 hours after ischemia by subjecting rats to permanent unilateral occlusion of the middle cerebral artery. Interleukin-1β was localized immunocytochemically in vibratome sections of perfusion-fixed brains. The cells that expressed IL-1β had the morphologic features of microglia and macrophages. Interleukin-1β was first detected 1 hour after occlusion in ipsilateral meningeal macrophage-like cells. By 6 hours, pro/mature IL-1β-immunoreactive (IL-1βir) putative microglia were present in the ischemic cerebral cortex, corpus callosum, caudoputamen, and surrounding tissue. By 24 and 48 hours after ischemia, the number and spread of IL-1βir cells increased greatly, including those resembling activated microglia and macrophages, as the core of the infarct became infiltrated. Interleukin-1βir cells also were present in apparently undamaged tissue, adjacent to the lesion ipsilaterally, and contralaterally in the cerebral cortex, dorsal corpus callosum, dorsal caudoputamen, and hippocampus. These results support the functional role of IL-1 in ischemic brain damage and reveal a distinct temporal and spatial expression of IL-1β protein in cells believed to be microglia and macrophages.
54352955
Human brain S100β and S100β mRNA expression increases with age: Pathogenic implications for Alzheimer's disease
S100 beta is a neurite extension factor that has been implicated in the development of neuritic plaques in Alzheimer's disease. We analyzed the expression of S100 beta and its encoding mRNA, using immunohistochemistry, enzyme-linked immunosorbent assay, and Northern blot analysis, in postmortem brain tissue from 26 neurologically normal patients, aged 1-80 years. Tissue levels of S100 beta and S100 beta mRNA, as well as the number of S100 beta-immunoreactive (S100 beta +) astrocytes, increased with advancing age (r = 0.60, p = 0.008; r = 0.65, p = 0.007: and r = 0.73, p = 0.001, respectively). In patients more than 60 years old, the number of S100 beta + astrocytes and the tissue levels of S100 beta and S100 beta mRNA were significantly higher than those in patients less than 60 years of age (p = 0.001, p = 0.035, and p = 0.047, respectively). All of these values, however, were significantly less than those found in Alzheimer patients (p < 0.05 or better). Our findings, together with the known functions of S100 beta, suggest that age-related increases in S100 beta expression are important in the pathogenesis of Alzheimer's disease and may explain in part the increased incidence of this disease with advancing age.
35342347
Cloning and expression of the human S100 beta gene.
S100 protein is a low molecular weight, EF-hand, Ca2(+)-binding protein widely distributed and conserved in the central nervous system of vertebrates. The gene coding for the beta subunit of human S100 protein (S100 beta) has been recently mapped to chromosome 21. In order to study the expression of this gene in normal and abnormal brain development, we have isolated and characterized overlapping genomic clones spanning the region coding for human S100 beta and its flanking sequences. The intron-exon organization of the human S100 beta gene is similar to that of the genes coding for several other members of the S100 protein subfamily of EF-hand proteins. The human S100 beta gene is composed of 3 exons, the first of which specifies the 5'-untranslated region, while the second and third each encode a single EF-hand, Ca2(+)-binding domain. The promoter region contains several potential regulatory transcription elements including the cAMP-responsive elements CRE and AP-2. A novel sequence motif, the S100 protein element, situated in close proximity to the TATA box of the genes of several members of the S100 protein subfamily, has been identified. In addition, multiple repeats with similar nucleotide sequence and location to the recently reported beta globin direct repeat elements have been also found in the human S100 beta promoter. A full length (17.3 kilobases) copy of the human S100 beta gene was constructed and transfected into rat glioma C6 cells. Stable transfectants were shown to express correctly initiated transcripts of the human S100 beta gene, indicating that the cloned sequences contain functional regulatory transcription elements.
40211639
Cytokine‐stimulated astrocytes damage human neurons via a nitric oxide mechanism
Astrocytes have been reported to play a neuropathogenic role within the brain, although little is known about the mechanism underlying astrocyte‐mediated neuronal injury. We investigated the hypothesis that cytokine‐stimulated astrocytes adversely affect neuronal cell survival via generation of the free radical nitric oxide (NO). Primary human astrocytes produced substantial amounts of NO in response to interleukin (IL)‐1α or IL‐1β, which was blocked by the NO synthase inhibitor NG‐monomethyl‐L‐arginine (NMMA). IL‐1β‐induced NO production was markedly potentiated by interferon (IFN)‐γ. IL‐1 receptor agonist protein (IRAP) totally blocked NO generation by cytokine‐stimulated astrocytes. Using reverse transcription‐polymerase chain reaction and sequencing analyses of the astrocyte NO synthase gene, we found a single band encoding for a 615 bp product that was identical to the corresponding sequence reported for human hepatocytes. Treatment of human fetal brain cell cultures with IL‐1β plus IFN‐γ resulted in marked neuronal loss, as assessed by microscopic analysis and measurement of lactate dehydrogenase release. This cytokine‐induced neuronal damage was blocked by simultaneous treatment of the brain cell cultures with NMMA or IRAP, suggesting a critical role of IL‐1. These findings indicate that cytokine‐stimulated astrocytes are neurotoxic via a NO‐mediated mechanism and point to potential new therapies for neurodegenerative disorders that involve cytokines and reactive astrocytes. © 1996 Wiley‐Liss, Inc.
2337572
Determination of S‐100 and Glial Fibrillary Acidic Protein Concentrations in Cerebrospinal Fluid After Brain Infarction
Background and Purpose We initiated the present study to evaluate the clinical value of consecutive concentration determinations of S-100 and glial fibrillary acidic proteins in cerebrospinal fluid from patients with brain infarction. Methods We took sequential samples of cerebrospinal fluid from 28 patients within 48 hours, at 7 days, and at 18-21 days after the ictus. We measured astroglial protein concentrations using an enzyme-linked immunosorbent assay and also determined size of the infarction (computed tomography), clinical state of the patient (simplified activities of daily living test), blood-brain barrier dysfunction (cerebrospinal fluid/serum albumin ratio), and a myelin marker (myelin basic protein). Results We found a transient increase of both proteins in the cerebrospinal fluid during the first week after the ischemic stroke (p<0.05). This increment was significantly correlated with the size of the infarction and the clinical state of the patients. Conclusions Transient release of astroglial proteins into the cerebrospinal fluid possibly reflects initial focal ischemic damage and, in the later phase, ongoing destruction of astroglial cells in the penumbra zone. We suggest that determinations of cerebrospinal fluid astroglial protein concentrations can be used to estimate ischemic brain damage, which should be of particular value in clinical trials of pharmacological agents, such as calcium antagonists, on stroke patients.
25470356
Serial measurement of interleukin-6, transforming growth factor-beta, and S-100 protein in patients with acute stroke.
BACKGROUND AND PURPOSE
Cytokine changes in patients with acute stroke have been insufficiently studied. The purpose of this study was to delineate the characteristics of serial changes of serum interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) in patients with cerebral infarction and intracerebral hemorrhage.


METHODS
We serially (within 24 hours, at day 3, and at day 7) measured the serum levels of IL-6 and TGF-beta in 29 patients with acute stroke (10 with large cortical cerebral infarction, 9 with subcortical small infarction, and 10 with intracerebral hemorrhage). As an index of brain damage. S-100 protein was also measured. Twelve age-matched healthy subjects were tested as a control.


RESULTS
S-100 protein was detected in only 11 patients with large infarction or intracerebral hemorrhage. Its level peaked at day 3 in patients with infarction, whereas it peaked within 24 hours in those with intracerebral hemorrhage. The level of IL-6 was most markedly elevated at day 1, which tended to decrease thereafter. However, its level remained significantly elevated compared with that of the control group even at day 7. The level of TGF-beta was significantly decreased at day 1 and day 3 and tended to return toward the control value thereafter. The levels of both cytokines were not significantly different among the three different stroke subtypes and were not correlated with the number of blood leukocytes and platelets.


CONCLUSIONS
The alteration of IL-6 and TGF-beta levels, which occurs rapidly after acute stroke regardless of the subtype, may reflect the changing immunological-inflammatory status of these patients and does not appear to reflect merely the consequence of the brain damage.
38359577
Regional Heterogeneity in the Response of Astrocytes following Traumatic Brain Injury in the Adult Rat
The regional distribution and temporal appearance of astrocytes expressing glial fibrillary acidic protein (GPAP), S100 protein, and vimentin were determined in a nonpenetrating lateral fluid percussion (LFP) brain injury model. Following injury, reactive astrocytes were observed in the subcortical white matter tracts as early as 1 day, in the hippocampus and injured cortex by 3 days, and in the thalamus by 1 week. Reactive astrocytes in the injured cortex, subcortical white matter tracts, and CA3 region of the hippocampus were all vimentin positive at 1 month post-injury. These astrocytes had a distinct morphology characterized by an enlarged cell body and long intertwined processes. In contrast, reactive astrocytes in the thalamic nuclei never expressed vimentin, and displayed an enlarged cell body with thick shortened processes. An increase in S100 protein was detected in all reactive astrocytes following LFP brain injury. Quantitative assessment of GFAP, S100, and vimentin polypeptides confirmed the immunohistochemical evaluation. Our data indicate that although astrogliosis mirrors the spatial pattern of post-traumatic neuronal cell loss, the expression of vimentin and the cellular morphology of the cells were regionally distinct, suggesting that astrogliosis may be modulated by present in the post-traumatic brain.
14980523
Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease.
Interleukin 1, an immune response-generated cytokine that stimulates astrocyte proliferation and reactivity (astrogliosis), was present in up to 30 times as many glial cells in tissue sections of brain from patients with Down syndrome and Alzheimer disease compared with age-matched control subjects. Most interleukin 1-immunoreactive glia in Down syndrome and Alzheimer disease were classified as microglia. The number of interleukin 1 immunoreactive neurons did not appear to differ in Down syndrome and Alzheimer disease compared with control brain. Numerous temporal lobe astrocytes in Alzheimer disease and postnatal Down syndrome were intensely interleukin 1-, S-100-, and glial fibrillary acidic protein-immunoreactive and had reactive structure. Interleukin 1 levels in Alzheimer disease temporal lobe homogenates were elevated, as were the levels of S-100 and glial fibrillary acidic protein, two proteins reportedly elevated in reactive astrocytes. These data suggest that increased expression of S-100 in Down syndrome, resulting from duplication of the gene on chromosome 21 that encodes the beta subunit of S-100, may be augmented by elevation of interleukin 1. As a corollary, the astrogliosis in Alzheimer disease may be promoted by elevation of interleukin 1.
29689983
S-100 protein: serum marker of focal brain damage after ischemic territorial MCA infarction.
BACKGROUND AND PURPOSE
Elevations of protein S-100 (S-100) in cerebrospinal fluid and serum have been reported after cerebral infarctions. The aim of our study was to evaluate the time course of serum S-100 concentrations after territorial middle cerebral artery (MCA) infarctions in correlation with clinical data and prognosis.


METHODS
S-100 serum levels were serially determined in 26 patients with an acute infarction in the territory of the MCA at day 0 (within 12 hours after onset of symptoms), day 1 (24 hours after stroke onset), and days 2, 3, 4, 5, 7 or 8, and 10 after stroke and in 26 age- and sex-matched control subjects. S-100 assays were performed using a two-site radioimmunoassay technique. The clinical status was documented using the Scandinavian Stroke Scale. The functional deficit 4 weeks after stroke onset was scored by use of the modified Rankin scale. A cranial computed tomography (CCT) was performed initially and at day 4 or 5.


RESULTS
Elevated concentrations of S-100 (> 0.2 microgram/L) were observed in 21 of 26 patients with MCA infarction but in none of the control subjects. S-100 levels peaked at days 2 and 3 after stroke. The S-100 concentrations in serum were significantly higher in patients with severe neurological deficits at admission, with extensive infarctions and a space-occupying effect of ischemic edema as compared with the rest of the population. S-100 values were not significantly correlated with the functional prognosis.


CONCLUSIONS
Presence of S-100 in serum after ischemic stroke may be due to combined leakage out of necrotic glial cells and passage through an impaired brain-blood barrier, indicating severe ischemic cell injury. Therefore, S-100 in serum can be used as a peripheral marker of ischemic focal brain damage and may be helpful for therapeutic decisions in acute ischemic stroke.
56635318
S-100 Protein and Neuron-Specific Enolase Concentrations in Blood as Indicators of Infarction Volume and Prognosis in Acute Ischemic Stroke
Background and Purpose Better techniques are needed to monitor infarction volume and predict neurological outcome after ischemic brain infarction. We evaluated the usefulness of serial measurements of S-100 protein versus neuron-specific enolase (NSE) in blood samples from patients with acute stroke. Methods Using nonisotopic sandwich immunoassays, we measured plasma concentrations of S-100 protein and NSE on admission and on days 3, 4, 7, and 14 after infarction in 44 patients (age range, 22 to 86 years; mean age, 65.1 years; 12 female, 32 male). Infarct volume was measured by volumetric CT on day 4 after ictus, and clinical outcome was assessed at discharge from hospital with the Activities of Daily Living Scale and 6 months after infarction with the Glasgow Outcome Scale. Results Peak blood levels of S-100 protein were found on day 2.5±1.3, and peak levels of NSE were found on day 1.9±0.8 after infarction. Peak plasma levels of S-100 protein correlated well with infarct volume (r=.75, P<.001) and with ...
54405925
S100β protects hippocampal neurons from damage induced by glucose deprivation
Abstract S100β is a calcium-binding protein elevated in Down's syndrome and Alzheimer's disease. Previous studies have demonstrated that S100β is trophic for several neuronal populations. We tested the influence of S100β on hippocampal neurons. The initial response included a rapid increase in [Ca2+]i similar to that elicited by S100β in other populations. S100β also substantially decreased cell death and loss of mitochondrial function resulting from glucose deprivation. Therefore, S100β exerts a neuroprotective influence on CNS neurons, suggesting that its elevation in neurological disorders may be a compensatory response.
25247930
Reactive glia as rivals in regulating neuronal survival
Reactive gliosis is a response noted after nearly every type of CNS injury and involves both activated microglia and astroglia. Although many investigators believe that reactive glia in some way regulate the survival of injured neurons, the influence of glial elements upon damaged neural tissues remains uncertain. To examine relationships between reactive glia and neurons, secretion products from both microglia and astroglia are tested for their effects upon the survival of cultured neurons. Microglia are found to secrete neurotoxic agents, while astroglia are a source of neuronotrophic factors. Similar patterns of soluble factor production are noted for astroglia‐rich or microglia‐rich regions of rat neocortex damaged by ischemia. These observations suggest that microglia and astroglia compete for control of neuronal survival. Importantly, microglial neurotoxins might hinder the recovery of neurologic function at sites of inflammation.
39930913
S100β Induces Neuronal Cell Death Through Nitric Oxide Release from Astrocytes
Abstract: The glial‐derived neurotrophic protein S100β has been implicated in the development and maintenance of the nervous system. S100β has also been postulated to play a role in mechanisms of neuropathology because of its specific localization and selective overexpression in Alzheimer's disease. However, the exact relationship between S100β overexpression and neurodegeneration is unclear. Recent data have demonstrated that treatment of cultured rat astrocytes with high concentrations of S100β results in a potent activation of inducible nitric oxide synthase (iNOS) and a subsequent generation of nitric oxide (NO), which can lead to astrocytic cell death. To investigate whether S100β‐induced NO release from astrocytes might influence neurons, we studied S100β effects on neuroblastoma B104 cells or primary hippocampal neurons co‐cultured with astrocytes. We found that S100β treatment of astrocyte‐neuron co‐cultures resulted in neuronal cell death by both necrosis and apoptosis. Neuronal cell death induced by S100β required the presence of astrocytes and depended on activation of iNOS. Cell death correlated with the levels of NO and was blocked by a specific NOS inhibitor. Our data support the idea that overexpression of S100β may be an exacerbating factor in the neurodegeneration of Alzheimer's disease.
30577671
S-100β protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT1A-receptor agonist, Bay x 3702
The serotonin (5-HT)(1A) receptor agonists have already been shown to protect cultured neurons from excitotoxic as well as from apoptotic damage [B. Ahlemeyer, J. Krieglstein, Stimulation of 5-HT(1A) receptors inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo, Brain Res. 777 (1997) 179-186. ; B. Ahlemeyer, A. Glaser, C. Schaper, I. Semkova, J. Krieglstein, The 5-HT(1A) receptor agonist, Bay x 3702, inhibited apoptosis induced by serum deprivation in cultured neurons, Eur. J. Pharmacol. 370 (1999) 211-216.; J.H.M. Prehn, M. Welsch, C. Backhauss, J. Nuglisch, F. Ausmeier, C. Karkoutly, J. Krieglstein, Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia, Brain Res. 630 (1993) 110-120.; I. Semkova, P. Wolz, J. Krieglstein, Neuroprotective effect of 5-HT(1A) receptor agonist, Bay x 3702, demonstrated in vitro and in vivo, Eur. J. Pharmacol. 359 (1998) 251-260.; B. Suchanek, H. Struppeck, T. Fahrig, The 5-HT(1A) receptor agonist, Bay x 3702, prevents staurosporine-induced apoptosis, Eur. J. Pharmacol. 355 (1998) 95-101.] and to increase the release of the neurotrophic protein, S-100beta [P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, Stimulation of astroglial 5-HT(1A) receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology, Brain Res. 497 (1989) 80-86. ; P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, S-100 protein is released from astroglial cells by stimulation of 5-HT(1A) receptors, Brain Res. 528 (1990) 155-158.]. In this study, we tried to find out whether S-100beta can protect cultured neurons from glutamate- and staurosporine-induced damage and whether the neuroprotective activity of the highly selective 5-HT(1A) receptor agonist, Bay x 3702, is mediated by an induction of S-100beta. Extracellularly added S-100beta (1-10 ng/ml) reduced staurosporine-induced damage in pure neuronal cultures from chick embryo telencephalon as well as in mixed neuronal/glial cultures from neonatal rat hippocampus. In addition, S-100beta (1 ng/ml) reduced neuronal death induced by exposure to glutamate (0.25 mM, 30 min) in mixed neuronal/glial cultures from neonatal rat hippocampus. In cultured rat cortical astrocytes, a 24 h-treatment with Bay x 3702 (1 nM) increased the S-100beta content in the culture medium from 2.2+/-0.3 (controls) to 6.2+/-0.7 ng/ml. In the adult rat, a 4 h-infusion of 4 microg/kg Bay x 3702 (i.v.) was found to increase the S-100beta content in the striatum 6 h after the beginning of the infusion to 153+/-37 microg/g compared with 60+/-20 microg/g in vehicle-treated rats. Bay x 3702 had no effect on the S-100beta content in the rat hippocampus. Finally, we tried to block the protective effect of Bay x 3702 against staurosporine-induced damage in mixed neuronal/glial cultures from rat neonatal hippocampus by anti-S-100beta antibodies. We found only a partial blockade, although the antibodies fully blocked the antiapoptotic effect of S-100beta itself demonstrating that the antibody was effective in blocking neuroprotection by S-100beta. Thus, we conclude that S-100beta was able to protect cultured neurons against glutamate- and staurosporine-induced damage. Furthermore, S-100beta mediated partially the protective effect of the 5-HT(1A) receptor agonist, Bay x 3702, against staurosporine-induced apoptosis in mixed neuronal/glial cultures from neonatal rat hippocampus.
40255082
Production of nitric oxide by glial cells: Regulation and potential roles in the CNS
Roles proposed for nitric oxide (NO) in CNS pathophysiology are increasingly diverse and range from intercellular signaling, through necrotic killing of cells and invading pathogens, to the involvement of NO in apoptosis and tissue remodeling. In vitro evidence and observations from experimental animal models of a variety of human neuropathologies, including stroke, indicate that glial cells can produce NO. Regulation of at least one of the NO synthase genes (NOS‐2) in glia has been well described; however, apart from hints emerging out of co‐culture studies and extrapolation based upon the reactivity of NO, we are a long way from identifying functions for glial‐derived NO in the CNS. Although the assumption is that NO is very often cytotoxic, it is evident that NO production does not always equate with tissue damage, and that both the cellular source of NO and the timing of NO production are important factors in terms of its effects. With the development of strategies to transfer or manipulate expression of the NOS genes in specific cells in situ, the ability to deliver NO into the CNS via long‐lived chemical donors, and the emergence of more selective NOS inhibitors, an appreciation of the significance of glial‐derived NO will change. GLIA 29:1–13, 2000. © 2000 Wiley‐Liss, Inc.
23179052
Expression of nitric oxide synthase (NOS)-2 following permanent focal ischemia and the role of nitric oxide in infarct generation in male, female and NOS-2 gene-deficient mice
Considerable evidence implicates nitric oxide (NO) in the pathological events following cerebral ischemia and, depending on the enzyme/cell source, NO is considered to be either damaging or protective. As a role for the enzyme nitric oxide synthase (NOS)-2 in permanent focal ischemia is not clear, we examined its expression following permanent middle cerebral artery occlusion in mice. At 24 h after occlusion, NOS-2 was expressed in cells infiltrating the infarct, while at later times, there was also expression in astrocytes around the infarct. To reveal a role for NO derived from this source, we compared infarct size in male and female mice with littermates in which the NOS-2 gene was disrupted. No differences were found between gender and genotype at 24 h. At 72 h, the infarct was increased in male mice, but not in females or in either gender with the gene disruption. These results suggest that NOS-2 plays a role in the later development of the infarct in male mice. Female mice are protected either against the damaging effects of NO, or because NOS-2 expression/activity is modulated by steroids.
32527134
N omega-nitro-L-arginine attenuates early ischemic neuronal damage of prolonged focal cerebral ischemia and recirculation in rats.
The present study aimed to examine the effects of N omega-nitro-L-arginine (LNA) on the early ischemic neuronal damage (EIND). All the experiments were carried out under general anesthesia, maintaining the blood gases and the body temperature within the physiological ranges. The local CBF, the topographically corresponding cortical specific gravity, and the volume of EIND were determined in each rat, which was subjected to prolonged or temporary occlusion of middle cerebral artery (MCA) using our original miniclip. Significant cortical edema developed only in the brain area where the local CBF value was below 200 ml 100 g-1 min-1. The prolonged MCA occlusion for 1, 2, and 4 h induced a time-dependent increase in the severity of cortical edema and the volume of EIND. Removal of the clip invariably induced recirculation. Compared to that induced by 4 h prolonged ischemia, the brain damage was improved by 1 h MCA occlusion followed by 3 h recirculation, whereas it was significantly worsened by 2 h ischemia followed by 2 h recirculation. While LNA [1 mg, i.p., given two times during the experiment] only partially inhibited the activity of brain nitric oxide synthase, it remarkably ameliorated EIND of both prolonged ischemia and recirculation in this model. The above findings indicate the pathogenic role of nitric oxide in prolonged ischemia as well as recirculation.
6961974
Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.
We have examined the incidence and size of infarction after occlusion of different portions of the rat middle cerebral artery (MCA) in order to define the reliability and predictability of this model of brain ischemia. We developed a neurologic examination and have correlated changes in neurologic status with the size and location of areas of infarction. The MCA was surgically occluded at different sites in six groups of normal rats. After 24 hr, rats were evaluated for the extent of neurologic deficits and graded as having severe, moderate, or no deficit using a new examination developed for this model. After rats were sacrificed the incidence of infarction was determined at histologic examination. In a subset of rats, the size of the area of infarction was measured as a percent of the area of a standard coronal section. Focal (1-2 mm) occlusion of the MCA at its origin, at the olfactory tract, or lateral to the inferior cerebral vein produced infarction in 13%, 67%, and 0% of rats, respectively (N = 38) and produced variable neurologic deficits. However, more extensive (3 or 6 mm) occlusion of the MCA beginning proximal to the olfactory tract--thus isolating lenticulostriate end-arteries from the proximal and distal supply--produced infarctions of uniform size, location, and with severe neurologic deficit (Grade 2) in 100% of rats (N = 17). Neurologic deficit correlated significantly with the size of the infarcted area (Grade 2, N = 17, 28 +/- 5% infarction; Grade 1, N = 5, 19 +/- 5%; Grade 0, N = 3, 10 +/- 2%; p less than 0.05). We have characterized precise anatomical sites of the MCA that when surgically occluded reliably produce uniform cerebral infarction in rats, and have developed a neurologic grading system that can be used to evaluate the effects of cerebral ischemia rapidly and accurately. The model will be useful for experimental assessment of new therapies for irreversible cerebral ischemia.
45664097
Identification of dying cells--in situ staining.
Publisher Summary The basic requirements for any methodology of programmed cell death (PCD) detection include (1) resolution at the individual cell level, and (2) in situ applicability while preserving the tissue architecture. To investigate PCD in its physiological context, the chapter develops a method that satisfies both criteria that is referred to as “terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL).” This technique is based on the observation that PCD is associated with DNA degradation. Researchers have come to consider the appearance of the ladder of nucleosomal DNA on agarose gels as the hallmark of PCD. The TUNEL method relies on the in situ labeling of DNA breaks in individual nuclei in tissue sections processed through the routine procedures of histopathology. TUNEL relies on the specific binding of TdT to exposed 3′-OH ends of DNA followed by the synthesis of a labeled polydeoxynucleotide molecule. Nuclear DNA on histological sections is first exposed by proteolytic treatment; then TdT is used to incorporate biotinylated deoxyuridine into the sites of DNA breaks. The signal is amplified by avidin-peroxidase, enabling conventional histochemical identification of PCD by light microscopy.
14670460
Measurement of S-100 Protein in Human Blood and Cerebrospinal Fluid: Analytical Method and Preliminary Clinical Results
An immunofluorometric sandwich assay for determination of S-100 protein in cerebrospinal fluid (CSF) and blood is described. The lower detection limit was 0.015 micrograms/l of S-100 protein. Intra-assay and inter-assay imprecision (coefficients of variation, CVs) were 2.1 to 3.2% and 7.8 to 11.6%, respectively. S-100 protein recovery in cerebrospinal fluid was 94 to 103%. In blood the recovery varied from 67 to 96%, depending on blood samples used and the concentration of S-100 protein. The best recovery in blood was found using heparinized plasma. In healthy subjects 0.098 +/- 0.11 micrograms/l (mean +/- SD) of S-100 protein was detected (n = 30). In the CSF of otherwise healthy patients undergoing a myelography for lumbar pain 1.43 +/- 0.49 micrograms/l (mean +/- SD) of S-100 protein was found. Preliminary results from longitudinal studies on S-100 protein in neurosurgical patients indicate a positive correlation between S-100 protein blood levels and clinical course. Thus, the determination of S-100 protein in blood appears to be helpful in the monitoring of patients with neuronal damage.
82800085
Early and Late Neuronal Damage Following Cerebral Ischemia
The hippocampus is one of the most vulnerable structures in the brain to ischemia. It is been widely believed that the hippocampus is most easily subject to neuronal death and “sclerosis” following only a brief period of ischemia of merely a few minutes. However, neurophysiologists have long been using the hippocampal slice method for their experiments. They know that their in vitro slice preparation of the hippocampus obtained mainly from rodent species is surprisingly normal and remains normal for many hours (Lynch, 1980). In this procedure, hippocampal tissue is unavoidably exposed to brief ischemia during preparation until it is cut into slices. The experience of neurophysiologists may, therefore, confirm that hippocampal neurons, if any, are only on rare occasions destroyed rapidly following brief ischemia. These seemingly contradictory observations may be resolved by the recognition of delayed neuronal death in the hippocampus. Most of the hippocampal CA1 neurons die following brief ischemia, but cell disintegration takes place very slowly (Kirino, 1982; Pulsinelli et al., 1982). It takes almost days until overt morphological signs of neuronal death appear. As the brain is subjected to longer ischemia, this neuronal cell alteration develops faster following recirculation (Kirino and Sano, 1984a). This general tendency of neuronal pathology is describled by Ito et al. and called “maturation phenomenon” (Ito et al., 1975). In the rodent hippocampus, delayed neuronal death is most commonly encountered in animals which survive longer than a few days following ischemia. On the other hand, ischemia which is long enough to cause widespread acute cell change in the hippocampus is usually incompatible with animal survival.
14183072
Delayed Reduction of Ischemic Brain Injury and Neurological Deficits in Mice Lacking the Inducible Nitric Oxide Synthase Gene
Inducible nitric oxide synthase (iNOS), an enzyme that produces toxic amounts of nitric oxide, is expressed in a number of brain pathologies, including cerebral ischemia. We used mice with a null mutation of the iNOS gene to study the role of iNOS in ischemic brain damage. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCA). In wild-type mice, iNOS mRNA expression in the post-ischemic brain begun between 24 and 48 hr peaked at 96 hr and subsided 7 d after MCA occlusion. iNOS mRNA induction was associated with expression of iNOS protein and enzymatic activity. In contrast, mice lacking the iNOS gene did not express iNOS message or protein after MCA occlusion. The infarct and the motor deficits produced by MCA occlusion were smaller in iNOS knockouts than in wild-type mice (p < 0.05). Such reduction in ischemic damage and neurological deficits was observed 96 hr after ischemia but not at 24 hr, when iNOS is not yet expressed in wild-type mice. The decreased susceptibility to cerebral ischemia in iNOS knockouts could not be attributed to differences in the degree of ischemia or vascular reactivity between wild-type and knockout mice. These findings indicate that iNOS expression is one of the factors contributing to the expansion of the brain damage that occurs in the post-ischemic period. iNOS inhibition may provide a novel therapeutic strategy targeted specifically at the secondary progression of ischemic brain injury.
5614553
Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament
The present experiments were designed to study under what circumstances middle cerebral artery (MCA) occlusion by an intraluminal filament technique leads to hyperthermia and what the mechanisms are. We found that permanent MCA occlusion by this technique lead to a rise in body (core) temperature to 39.0-39.5 degrees C during the first 2-4 h, and to sustained hyperthermia thereafter (38.5-39.0 degrees C). After 2 h of transient MCA occlusion hyperthermia could only be avoided if anesthesia (with control of temperature) was maintained for 2 h of ischemia and 1 h of recirculation or, in unanesthetized animals, if external cooling was maintained for 2 h of ischemia and 2 h of recirculation. Control of temperature only during ischemia did not prevent a postischemic rise in temperature. One hour of MCA occlusion had less effect on body temperature. Results are presented which suggest that the hyperthermia observed is due to an interference, by the intraluminal filament, of circulation to hypothalamic centers regulating body temperature. It is speculated that the hyperthermia induced may blunt or obliterate the effect of drugs, normally considered to ameliorate brain damage due to focal ischemia.
43825171
Gene Expression Induced by Cerebral Ischemia: An Apoptotic Perspective
The flow of new information on gene expression related to apoptosis has been relentless in the last several years. This has also been the case with respect to gene expression after cerebral ischemia. Many of genes associated with an apoptotic mode of cell death have now been studied in the context of experimental cerebral ischemia from the immediate early genes through modulating genes such as bcl-2 to genes in the final execution phase such as interleukin-1β converting enzyme (ICE)-related proteases. It was impossible to adequately cite all primary reports on these subjects. However, many excellent reviews have appeared in the last year, which together, cover all these areas of interest. In this review, we have elected to cite only reports published since January 1996 and use an extensive collection of reviews (indicated in italics) to guide the reader to the earlier literature. Our intent is to provide the reader with a timely and useful analysis of the current state of the art. It is hoped that this approach does not cause offense with our colleagues whose contributions before 1996 laid the foundation for much of this work.
9378297
Multiple Molecular Penumbras after Focal Cerebral Ischemia
Though the ischemic penumbra has been classically described on the basis of blood flow and physiologic parameters, a variety of ischemic penumbras can be described in molecular terms. Apoptosis-related genes induced after focal ischemia may contribute to cell death in the core and the selective cell death adjacent to an infarct. The HSP70 heat shock protein is induced in glia at the edges of an infarct and in neurons often at some distance from the infarct. HSP70 proteins are induced in cells in response to denatured proteins that occur as a result of temporary energy failure. Hypoxia-inducible factor (HIF) is also induced after focal ischemia in regions that can extend beyond the HSP70 induction. The region of HIF induction is proposed to represent the areas of decreased cerebral blood flow and decreased oxygen delivery. Immediate early genes are induced in cortex, hippocampus, thalamus, and other brain regions. These distant changes in gene expression occur because of ischemia-induced spreading depression or depolarization and could contribute to plastic changes in brain after stroke.
10724097
Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models
Huntington disease (HD) is an inherited neurodegenerative disorder characterized by selective death of striatal medium spiny neurons. Intrastriatal injections of glutamate receptor agonists (excitotoxins) recapitulate some neuropathological features of this disorder. Although this model suggests that excitotoxic injury may be involved in HD, the exact mechanisms of cell death in HD and its models are unknown. The present study was designed to test the hypothesis that HD can develop via the activation of an apoptotic mechanism of cell death and to examine whether excitotoxic striatal lesions with quinolinic acid in rats represent accurate models of HD. To characterize cell death, we employed DNA electrophoresis, electron microscopy (EM), and the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)- biotin nick end labeling (TUNEL) method for the in situ detection of DNA strand breaks. In the neostriatum of individuals with HD, patterns of distribution of TUNEL-positive neurons and glia were reminiscent of those seen in apoptotic cell death during normal development of the nervous system; in the same areas, nonrandom DNA fragmentation was detected occasionally. Following excitotoxic injury of the rat striatum, internucleosomal DNA fragmentation (evidence of apoptosis) was seen at early time intervals and random DNA fragmentation (evidence of necrosis) at later time points. In addition, EM detected necrotic profiles of medium spiny neurons in the lesioned rats. In concert, these results suggest that apoptosis occurs in both HD and excitotoxic animal models and that apoptotic and necrotic mechanisms of neuronal death may occur simultaneously within individual dying cells in the excitotoxically injured brain. However, the distribution of dying neurons in the neostriatum, the degree of glial degeneration, and the involvement of striatofugal pathways are very different between HD and excitotoxically damaged striatum. The present study suggests that multiple methods should be employed for a proper characterization of neuronal cell death in vivo.
2330826
Thromboembolic Events Lead to Cortical Spreading Depression and Expression of c-fos, Brain-Derived Neurotrophic Factor, Glial Fibrillary Acidic Protein, and Heat Shock Protein 70 mRNA in Rats
The hypotheses that cerebral embolic events lead to repetitive episodes of cortical spreading depression (CSD) and that these propagating waves trigger the expression of c-fos, brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and heat shock protein 70 (HSP70) mRNA were tested. Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) (n = 18) or sham (n = 8) procedures. In a subgroup of rats (n = 5), laser-Doppler flowmetry probes were placed overlying the right parietal cortex to record CSD-like changes in cortical blood flow during the initial 2-hour postinjury period. Rats were killed by decapitation at 2 or 24 hours after CCAT, and brains were processed for in situ localization of the gene expression. Two to five intermittent transient hyperemic episodes lasting 1 to 2 minutes were recorded ipsilaterally after CCAT. At 2 hours after CCAT, the widespread expression of c-fos and BDNF mRNAs was observed throughout the ipsilateral cerebral cortex. Pretreatment with the N-methyl-d-aspartate receptor blocker MK-801 (2 mg/kg) 1 hour before CCAT reduced the expression of BDNF mRNA expression at 2 hours. At 24 hours after CCAT, increased expression of GFAP mRNA was present in cortical and subcortical regions. In contrast, multifocal regions of HSP70 expression scattered throughout the thrombosed hemisphere were apparent at both 2 and 24 hours after injury. These data indicate that thromboembolic events lead to episodes of CSD and time-dependent alterations in gene expression. The ability of embolic processes to induce widespread molecular responses in neurons and glia may be important in the pathogenesis of transient ischemic attacks and may influence the susceptibility of the postembolic brain to subsequent insults including stroke.
22426215
Cortical Spreading Depression Increases Protein Synthesis and Upregulates Basic Fibroblast Growth Factor
Protective effects of cortical spreading depression (CSD) against ischemic damage have been demonstrated in cortex when elicited at either 24 h or 3 days prior to ischemia. The present study was carried out to investigate possible mechanisms of neuroprotection following CSD. In Sprague-Dawley rats, 5 M KCl, 5 M NaCl, or physiological saline was applied to the cortex for 1 h. Repetitive CSD waves were elicited only in the KCl group. Measurements of cerebral glucose utilization demonstrated a marked reduction in affected cortex and subcortical regions in both the NaCl and the KCl groups, whereas cortical and hippocampal protein synthesis was discretely increased only in the KCl group. Immunohistochemistry of GFAP demonstrated a rapid activation in reactive astrocytes at 3 days in the KCl group whereas only a discrete activation was observed in the NaCl group. Similar changes were observed for basic fibroblast growth factor. Our results suggest that CSD-induced ischemic tolerance is not due to a reduction in energy metabolism but rather is associated with an upregulation of trophic factors and glial cell activation which might provide a mechanism for a long-lasting neuroprotection.
22305900
Reactive Astrocytosis from Excitotoxic Injury in Hippocampal Organ Culture Parallels that Seen in Vivo
Reactive astrocytes influence not only the severity of brain injury, but also the capacity of brain to reshape itself with learning. Mechanisms responsible for astrogliosis remain unknown but might be best studied in vitro, where improved access and visualization permit application of modern molecular and cellular techniques. We have begun to explore whether gliosis might be studied in hippocampal organotypic cultures (HOTCs), where potential cell-to-cell interactions are preserved and the advantages of an in vitro preparation are still realized. Following HOTC exposure to N-methyl-D-aspartate (NMDA), dose-dependent changes occurred in the optical density (OD) values for the astrocytic immunohistochemical [immunostaining (IS)] markers glial fibrillary acidic protein (GFAP) and vimentin. Exposure of HOTCs to NMDA (10 μM) caused selective death in the CA1 hippocampal region and the dentate gyrus. It also significantly increased GFAP IS and vimentin IS OD values in these regions. Increased GFAP IS and vimentin IS OD values were also seen in CA3, a hippocampal region that displayed no cell death. Light microscopic examination revealed hypertrophied GFAP and vimentin IS cells, characteristic of reactive astrocytes. Cellular proliferation, as assessed by proliferating cell nuclear antigen IS, was also significantly increased in all three of these hippocampal regions. In contrast, exposure of HOTCs to a noninjurious level of NMDA (1 μM) caused only minor changes in GFAP IS and vimentin IS OD values but a significantly reduced cellular proliferation in all HOTC regions. These results show that reactive astrocytosis from excitotoxic injury of HOTC parallels changes seen in vivo after global ischemia. Furthermore, since resting astroglia within HOTCs are also similar to their counterparts in vivo, HOTCs may be used to examine mechanisms by which these cells are transformed into reactive species within tissue that resembles intact brain.
83579076
Glial fibrillary acidic protein is necessary for mature astrocytes to react to β‐amyloid
Upregulation of the glial fibrillary acidic protein (GFAP) in astrocytes is a hallmark of the phenomenon known as reactive gliosis and, yet, the function of GFAP in this process is largely unknown. Our previous studies have shown that mature astrocytes react vigorously to substrate bound β‐amyloid protein (BAP) in a variety of ways (i.e., increased GFAP, enhanced motility, unusual aggregation patterns, inhibitory ECM production). In order to uncover which, if any, of these phenomena are causally related to the function of GFAP, primary cortical astrocytes from transgenic mice lacking GFAP were cultured on BAP substrates at low or high density and at various lengths of time following in vitro maturation. Differences between mutant and control cells became progressively more obvious when cells were matured in vitro for two weeks or longer and especially in cultures that were at high density. Mature control astrocytes show a dramatic response to BAP by aggregating into a meshwork of rope‐like structures that completely bridge over the peptide surface. In marked contrast, mature GFAP‐null astrocytes initiate the response much more slowly and had a much reduced ability to aggregate tightly. Furthermore, we prepared hippocampal slice cultures from GFAP−/− and GFAP+/+ mice and compared their astrocytic responses to injected BAP. GFAP−/− astrocytes of hippocampal slice cultures failed to form a barrier‐like structure around the edge of the BAP deposit as did GFAP+/+ astrocytes. Our data suggest that GFAP may be essential for mature astrocytes to constrain certain types of highly inflammatory lesions in the brain. GLIA 25:390–403, 1999. © 1999 Wiley‐Liss, Inc.
12011315
Exacerbation of Ischemic Brain Damage by Localized Striatal Injection of Interleukin-1β in the Rat
Interleukin-1β (IL-1β) has been implicated in ischemic brain damage. The site of action of IL-1β in such damage is not known, but we have demonstrated previously that injection of the interleukin-1 receptor antagonist (IL-1ra) in the striatum but not the cortex of rats inhibits damage caused by permanent middle cerebral artery occlusion. The present study investigated the site of action of IL-1β on ischemic damage by examining the effects of intracerebroventricular, striatal, or cortical injection of recombinant IL-1β at the onset of permanent middle cerebral artery occlusion in the rat. Intracerebroventricular injection of IL-1β (2.5 ng) significantly increased infarct volume in the striatum (35%, P < 0.0001) and in the cortex (44%, P < 0.0001) compared with vehicle treatment. Direct injection of IL-1β into the striatum also increased infarct volume in both the striatum (36%, P < 0.0001) and the cortex (38%, P < 0.0001), whereas injection of IL-1β into the cortex failed to affect infarct volume in either the striatum or the cortex. Cortical injection of a higher dose of IL-1β (20 ng) also failed to affect ischemic damage in either the striatum or the cortex. Injection of IL-1β into the striatum contralateral to the infarction had no effect on striatal damage in the ischemic hemisphere, but did increase cortical damage by 18% (P < 0.0001). In separate groups of animals, IL-1β (2.5 ng) was injected into either the striatum or the cortex, and body temperature was recorded continuously in conscious free-moving animals by remote telemetry. Injection of IL-1β at either site failed to influence body temperature, suggesting that exacerbation of brain damage by striatal injection of IL-1β is not caused by effects on body temperature. These results imply that IL-1β exacerbates ischemic damage by specific actions in the striatum where it can influence damage at distant sites in the cortex.
21584791
Intracerebral administration of interleukin-1beta and induction of inflammation, apoptosis, and vasogenic edema.
OBJECT
The proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) are produced intracerebrally in brain disorders such as trauma, ischemia, meningitis, and multiple sclerosis. This investigation was undertaken to analyze the effect of intracerebral administration of IL-1beta and TNFalpha on inflammatory response, cell death, and edema development.


METHODS
Intracerebral microinjections of these cytokines were administered to rats. The animals were killed 24 or 72 hours after the injections, and their brains were analyzed by using deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) with digoxigenin-labeled deoxyuridine triphosphate, immunohistochemical studies, and brain-specific gravity measurement. The IL-1beta induced a transient inflammatory response (p < 0.001) and TUNEL staining (p < 0.001), indicating cell death, in intrinsic central nervous system (CNS) cells and infiltrating inflammatory cells. In 73.8+/-6.77% of the TUNEL-positive cells, small, fragmented nuclei were found. All TUNEL-positive cells expressed the proapoptotic gene Bax, and 69.6+/-4.6% of the TUNEL-positive cells expressed the antiapoptotic gene Bcl-2; the Bax expression was stronger than the Bcl-2 expression. Taken together, the data indicate that cell death occurred via the apoptotic pathway. The TNFalpha did not induce inflammation or DNA fragmentation within the analyzed time period. Both IL-1beta (p < 0.001) and TNFalpha (p < 0.01) caused vasogenic edema, as measured by specific gravity and albumin staining. The edematous effect of TNFalpha persisted 72 hours after injection (p < 0.01), whereas the IL-1beta-treated animals had normalized by that time.


CONCLUSIONS
Intracerebral inflammation, death of intrinsic CNS cells, and vasogenic edema can be mediated by IL-1beta, and TNFalpha can cause vasogenic edema. Suppression of these cytokines in the clinical setting may improve outcome.
54276207
Basic fibroblast growth factor-2 and interleukin-1β regulate S100β expression in cultured astrocytes
Abstract Basic fibroblast growth factor and interleukin-1 β are known to regulate the expression of other trophic factors and to stimulate reactive gliosis in vivo . S100 β is a glial-specific putative neurotrophic factor and has been considered a marker of the reactive status of astrocytes. Therefore, we tested the hypothesis that basic fibroblast growth factor-2 and interleukin-1 β achieve their effects by altering S100 β gene expression in cultured rat astrocytes using an RNase protection assay. Short-term treatment with basic fibroblast growth factor-2 produced a transient decrease in S100 β messenger RNA which was followed by an increase after longer term treatment. In contrast, both short- and long-term treatment with interleukin-1 β suppressed S100 β messenger RNA. We measured levels of S100 β nuclear primary transcript to assess whether alterations in transcriptional rate explain the changes in messenger RNA. Our results indicate that changes in transcription account for changes in steady state levels of messenger RNA since basic fibroblast growth factor-2-induced changes in S100 β primary transcript temporally preceded changes in messenger RNA. We further measured intracellular S100 β protein levels by enzyme-linked immunosorbent assay to determine whether changes in gene expression were translated into parallel changes in protein. Our results clearly demonstrate that basic fibroblast growth factor-2 and interleukin-1 β influence the expression of the S100 β gene, that this regulation appears to occur at the level of transcription, and that alterations in messenger RNA are sometimes, but not always, reflected in changes at the level of protein. These observations suggest that basic fibroblast growth factor-2 may amplify its trophic effects, in part, by influencing the expression of another trophic factor.
19170468
Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia.
Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 micrometer) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2 (PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.
37264907
Spreading depression is not associated with neuronal injury in the normal brain
This study was performed in order to evaluate whether waves of spreading depression (SD) induces irreversible neuronal injury. SD was elicited by topical application of 3 M KCl to the exposed cortex for 4-5 h and the resulting change of the cortical electrical potential showing the occurrence of SD, was recorded by glass microelectrodes. Histological examination of cerebral cortex revealed no signs of neuronal injury outside the area of KCl application as examine after 4 days recovery. The results indicate that recurrent waves of SD do not induce irreversible neuronal injury in the otherwise normal rat brain.
24261909
Glutamate Does Not Mediate Acute Neuronal Damage after Spreading Depression Induced by O2/Glucose Deprivation in the Hippocampal Slice
This study argues that, in contrast to accepted excitotoxicity theory, O2/glucose deprivation damages neurons acutely by eliciting ischemic spreading depression (SD), a process not blocked by glutamate antagonists. In live rat hippocampal slices, the initiation, propagation, and resolution of SD can be imaged by monitoring wide-band changes in light transmittance (i.e., intrinsic optical signals). Oxygen/glucose deprivation for 10 minutes at 37.5°C evokes a propagating wave of elevated light transmittance across the slice, representing the SD front. Within minutes, CA1 neurons in regions undergoing SD display irreversible damage in the form of field potential inactivation, swollen cell bodies, and extensively beaded dendrites, the latter revealed by single-cell injection of lucifer yellow. Importantly, glutamate receptor antagonists do not block SD induced by O2/glucose deprivation, nor do they prevent the resultant dendritic beading of CA1 neurons. However, CA1 neurons are spared if SD is suppressed by reducing the temperature to 35°C during O2/glucose deprivation. This supports previous electrophysiologic evidence in vivo that SD during ischemia promotes acute neuronal damage and that glutamate antagonists are not protective of the metabolically stressed tissue. The authors propose that the inhibition of ischemic SD should be targeted as an important therapeutic strategy against stroke damage.