2397030
Fas/FasL-mediated apoptosis and inflammation are key features of acute human spinal cord injury: implications for translational, clinical application
The Fas/FasL system plays an important role in apoptosis, the inflammatory response and gliosis in a variety of neurologic disorders. A better understanding of these mechanisms could lead to effective therapeutic strategies following spinal cord injury (SCI). We explored these mechanisms by examining molecular changes in postmortem human spinal cord tissue from cases with acute and chronic SCI. Complementary studies were conducted using the in vivo Fejota™ clip compression model of SCI in Fas-deficient B6.MRL-Fas-lpr (lpr) and wild-type (Wt) mice to test Fas-mediated apoptosis, inflammation, gliosis and axonal degeneration by immunohistochemistry, Western blotting, gelatin zymography and ELISA with Mouse 32-plex cytokine/chemokine panel bead immunoassay. We report novel evidence that shows that Fas-mediated apoptosis of neurons and oligodendrocytes occurred in the injury epicenter in all cases of acute and subacute SCI and not in chronic SCI or in control cases. We also found significantly reduced apoptosis, expression of GFAP, NF-κB, p-IKappaB and iba1, increased number of CD4 positive T cells and MMP2 expression and reduced neurological dysfunction in lpr mice when compared with Wt mice after SCI. We found dramatically reduced inflammation and cytokines and chemokine expression in B6.MRL-Fas-lpr mice compared to Wt mice after SCI. In conclusion, we report multiple lines of evidence that Fas/FasL activation plays a pivotal role in mediating apoptosis, the inflammatory response and neurodegeneration after SCI, providing a compelling rationale for therapeutically targeting Fas in human SCI.
13007112
The bright side of the glial scar in CNS repair
Following CNS injury, in an apparently counterintuitive response, scar tissue formation inhibits axonal growth, imposing a major barrier to regeneration. Accordingly, scar-modulating treatments have become a leading therapeutic goal in the field of spinal cord injury. However, increasing evidence suggests a beneficial role for this scar tissue as part of the endogenous local immune regulation and repair process. How can these opposing effects be reconciled? Perhaps it is all a matter of timing.
11872811
Cell death in models of spinal cord injury.
Current treatments for acute spinal cord injury are based on animal models of human spinal cord injury (SCI). These models have shown that the initial traumatic injury to cord tissue is followed by a long period of secondary injury that includes a number of cellular and biochemical cascades. These secondary injury processes are potential targets for therapies. Continued refinement of rat and mouse models of SCI, along with more detailed analyses of the biology of the lesion in these models, points to both necrotic and apoptotic mechanisms of cell death after SCI. In this chapter, we review recent evidence for long-term apoptotic death of oligodendrocytes in long tracts undergoing Wallerian degeneration following SCI. This process appears to be related closely to activation of microglial cells. It is has been thought that microglial cells might be the source of cytotoxic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), that kill oligodendrocytes. However, more recent evidence in vivo suggests that TNF-alpha by itself may not induce necrosis or apoptosis in oligodendrocytes. We review data that suggests other possible pathways for apoptosis, such as the neurotrophin receptor p75 which is expressed in both neurons and oligodendrocytes after SCI in rats and mice. In addition, it appears that microglial activation and TNF-alpha may be important in acute SCI. Ninety minutes after a moderate contusion lesion, microglia are activated and surround dying neurons. In an 'atraumatic' model of SCI, we have now shown that TNF-alpha appears to greatly potentiate cell death mediated by glutamate receptors. These studies emphasize that multiple mechanisms and interactions contribute to secondary injury after SCI. Continued study of both contusion models and other new approaches to studying these mechanisms will be needed to maximize strategies for acute and chronic therapies, and for neural repair.
15571749
Apoptotic and Anti‐Apoptotic Mechanisms Following Spinal Cord Injury
A number of studies have provided evidence that cell death from moderate traumatic spinal cord injury (SCI) is regulated, in part, by apoptosis that involves the caspase family of cysteine proteases. However, little or no information is available about anti-apoptotic mechanisms mediated by the inhibitors of apoptosis (IAP) family of proteins that inhibit cell death pathways. In the present study, we examined caspase and IAP expression in spinal cords of rats subjected to moderate traumatic injury. Within 6 h after injury, caspase-8 and−9 (2 initiators of apoptosis) were predominantly present in gray matter neurons within the lesion epicenter. By 3 days following spinal cord injury (SCI), caspase-8 and−9 immunoreactivity was localized to gray and white matter cells, and by 7 days following SCI, both upstream caspases were expressed in cells within white matter or within foamy macrophages in gray matter. Caspase-3, an effector caspase, was evident in a few fragmented cells in gray matter at 24 h following injury and then localized to white matter in later stages. Thus, distinct patterns of caspase expression can be found in the spinal cord following injury. XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the cord. Immunoblots of spinal cord extracts revealed that the processed forms of caspases-8 and−9 and cleavage of PARP are present as early as 6 h following trauma. The expression of caspases corresponded with the detection of cleavage of XIAP into 2 fragments following injury. cIAP-1 and cIAP-2 expression remained constant during early periods following SCI but demonstrated alterations by 7 days following SCI. Our data are consistent with the idea that XIAP may have a protective role within the spinal cord, and that alteration in cleavage of XIAP may regulate cell death following SCI.
42316563
Degeneration and regeneration of axons in the lesioned spinal cord.
For many decades, the inability of lesioned central neurons to regrow was accepted almost as a "law of nature", and on the clinical level, spinal cord and brain lesions were seen as being irreversible. Today we are starting to understand the mechanisms of neuronal regeneration in the central nervous system and its presence in the periphery. There is now a rapid expansion in this field of neuroscience. Developmental neurobiology has produced tools and concepts that start to show their impact on regeneration research. This is particularly true for the availability of antibodies and factors and for the rapidly growing cellular and molecular understanding of crucial aspects of neurite growth, guidance, target finding, and synapse stabilization. New cell biological concepts on the mechanisms of neuron survival and death and on the interaction of inflammatory cells with the central nervous system also find their way into the field of spinal cord and brain lesions and have, indeed, led already to new therapeutic approaches. This review briefly summarizes the current knowledge on the mechanisms involved in degeneration and tissue loss and in axonal regeneration subsequent to spinal cord lesions, particularly in mammals and humans.
2232621
Contribution of Fas ligand to T cell-mediated hepatic injury in mice.
BACKGROUND & AIMS
Fas has been implicated in liver damage. The aim of this study was to investigate the role of its ligand to induce hepatocyte death and liver damage in T cell-dependent hepatitis.


METHODS
Fas ligand-mediated lysis of primary hepatocytes from C57BL/6 wild-type, Fas ligand-deficient gld, and Fas-deficient lpr mice and concanavalin A-induced hepatitis in these mice were assessed.


RESULTS
Freshly isolated hepatocytes from wild-type or gld mice, but not those from lpr mice, were susceptible to Fas ligand-mediated lysis. When concanavalin A was intravenously administered into wild-type mice, they developed acute hepatic injury with massive degenerative changes in hepatocytes. In contrast, both gld and lpr mice had lower aminotransferase levels with milder histological changes. Reverse-transcription polymerase chain reaction and flow cytometric analysis showed that Fas ligand was induced in the liver shortly after the concanavalin A injection and was predominantly expressed on intrahepatic T cells. Administration of monoclonal antibody neutralizing mouse Fas ligand could reduce the aminotransferase increase.


CONCLUSIONS
The results indicate that Fas ligand plays a role in the T cell-dependent hepatitis induced by concanavalin A administration.
9196594
Fas engagement induces neurite growth through ERK activation and p35 upregulation
Fas (also known as CD95), a member of the tumour-necrosis receptor factor family of 'death receptors', can induce apoptosis or, conversely, can deliver growth stimulatory signals. Here we report that crosslinking Fas on primary sensory neurons induces neurite growth through sustained activation of the extracellular-signal regulated kinase (ERK) pathway and the consequent upregulation of p35, a mediator of neurite outgrowth. In addition, functional recovery after sciatic nerve injury is delayed in Fas-deficient lpr mice and accelerated by local administration of antibodies against Fas, which indicates that Fas engagement may contribute to nerve regeneration in vivo. Our findings define a role for Fas as an inducer of both neurite growth in vitro and accelerated recovery after nerve injury in vivo.
34295099
CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site.
Injury to the central nervous system initiates an uncontrolled inflammatory response that results in both tissue repair and destruction. Here, we showed that, in rodents and humans, injury to the spinal cord triggered surface expression of CD95 ligand (CD95L, FasL) on peripheral blood myeloid cells. CD95L stimulation of CD95 on these cells activated phosphoinositide 3-kinase (PI3K) and metalloproteinase-9 (MMP-9) via recruitment and activation of Syk kinase, ultimately leading to increased migration. Exclusive CD95L deletion in myeloid cells greatly decreased the number of neutrophils and macrophages infiltrating the injured spinal cord or the inflamed peritoneum after thioglycollate injection. Importantly, deletion of myeloid CD95L, but not of CD95 on neural cells, led to functional recovery of spinal injured animals. Our results indicate that CD95L acts on peripheral myeloid cells to induce tissue damage. Thus, neutralization of CD95L should be considered as a means to create a controlled beneficial inflammatory response.
2454543
Caspase Activation Is Required for T Cell Proliferation
Triggering of Fas (CD95) by its ligand (FasL) rapidly induces cell death via recruitment of the adaptor protein Fas-associated death domain (FADD), resulting in activation of a caspase cascade. It was thus surprising that T lymphocytes deficient in FADD were reported recently to be not only resistant to FasL-mediated apoptosis, but also defective in their proliferative capacity. This finding suggested potentially dual roles of cell growth and death for Fas and possibly other death receptors. We report here that CD3-induced proliferation and interleukin 2 production by human T cells are blocked by inhibitors of caspase activity. This is paralleled by rapid cleavage of caspase-8 after CD3 stimulation, but no detectable processing of caspase-3 during the same interval. The caspase contribution to T cell activation may occur via TCR-mediated upregulation of FasL, as Fas-Fc blocked T cell proliferation, whereas soluble FasL augmented CD3-induced proliferation. These findings extend the role of death receptors to the promotion of T cell growth in a caspase-dependent manner.
25681066
FAS deficiency reduces apoptosis, spares axons and improves function after spinal cord injury
After spinal cord injury (SCI), apoptosis of neurons and oligodendrocytes is associated with axonal degeneration and loss of neurological function. Recent data have suggested a potential role for FAS death receptor-mediated apoptosis in the pathophysiology of SCI. In this study, we examined the effect of FAS deficiency on SCI in vitro and in vivo. FAS(Lpr/lpr) mutant mice and wildtype background-matched mice were subjected to a T5-6 clip compression SCI, and complementary studies were done in an organotypic slice culture model of SCI. Post-traumatic apoptosis in the spinal cord, which was seen in neurons and oligodendrocytes, was decreased in the FAS-deficient mice both in vivo and in vitro particularly in oligodendrocytes. FAS deficiency was also associated with improved locomotor recovery, axonal sparing and preservation of oligodendrocytes and myelin. However, FAS deficiency did not result in a significant increase in surviving neurons in the spinal cord at 6 weeks after injury, likely reflecting the importance of other cell death mechanisms for neurons. We conclude that inhibition of the FAS pathway may be a clinically attractive neuroprotective strategy directed towards oligodendroglial and axonal preservation in the treatment of SCI and neurotrauma.
8886467
The Role of Fas-Mediated Apoptosis After Traumatic Spinal Cord Injury
Study Design. Functional recovery and histopathological change after spinal cord injury in the Fas-deficient mice and the wild-type mice were investigated. Objectives. To investigate the role of the Fas/Fas ligand (FasL) system as a signal transduction pathway leading to apoptosis after spinal cord injury. Summary of Background Date. Apoptosis observed after spinal cord injury has recently gained widespread interest as a cause of collateral damage after the initial injury. Apoptosis mediated by the Fas antigen in the postischemic brain or spinal cord was reported. Recently, the upregulation of Fas after spinal cord injury was also reported. However, the influence of Fas-mediated apoptosis on the extent of the secondary spinal cord injury has not yet been clarified. Methods. We investigated Fas-mediated apoptosis after spinal cord injury and examined the behavioral changes and the histopathological changes after spinal cord injury using MRL/Mp-lpr/lpr (MRL/lpr) mice, which were Fas-deficient mutant mice, and MRL/Mp-+/+ (MRL/+) mice, which were Fas-positive wild-type mice. Results. Locomotor recovery after spinal cord injury in MRL/lpr mice was superior to that observed in MRL/+ mice. In addition, the damaged area in MRL/lpr mice was significantly smaller than that seen in MRL/+ mice. Further, the frequency of apoptotic cells in the injured spinal cord of MRL/lpr mice was significantly less than that in MRL/+ mice. Conclusion. We demonstrated the appearance of Fas-mediated apoptosis in the spinal cord after spinal cord injury. In addition, we elucidated for the first time that Fas-mediated apoptosis following spinal cord injury played an important role in the spinal cord damage and the ultimate neurologic injury.
43581299
Involvement of mitochondrial signaling pathways in the mechanism of Fas‐mediated apoptosis after spinal cord injury
Activation of the Fas receptor has been recently linked to apoptotic cell death after spinal cord injury (SCI). Although it is generally considered that Fas activation mediates apoptosis predominantly through the extrinsic pathway, we hypothesized that intrinsic mitochondrial signaling could be involved in the underlying mechanism of Fas‐induced apoptosis after SCI. In the present study, we utilized the FejotaTM clip compression model of SCI at T5–6 in C57BL/6 Fas‐deficient (lpr) and wild‐type mice. Complementary studies were conducted using an in vitro model of trauma or a Fas‐activating antibody to induce apoptosis in primary neuronal–glial mixed spinal cord cultures. After in vivo SCI, lpr mice, in comparison with wild‐type mice, exhibited reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)‐positive cells at the lesion, reduced expression of truncation of Bid (tBid), apoptosis‐inducing factor, activated caspase‐9 and activated caspase‐3, and increased expression of the antiapoptotic proteins Bcl‐2 and Bcl‐xL. After in vitro neurotrauma or the induction of Fas signaling by the Jo2 activating antibody, lpr spinal cord cultures showed an increased proportion of cells retaining mitochondrial membrane integrity and a reduction of tBid expression, caspase‐9 and caspase‐3 activation, and TUNEL‐positive cells as compared to wild‐type spinal cord cultures. The neutralization of Fas ligand (FasL) protected against traumatically induced or Fas‐mediated caspase‐3 activation and the loss of mitochondrial membrane potential and tBid expression in wild‐type spinal cord cultures. However, in lpr spinal cord cultures, FasL neutralization had no protective effects. In summary, these data provide direct evidence for the induction of intrinsic mitochondrial signaling pathways following Fas activation after SCI.
11607372
Inhibition of Fas-mediated apoptosis through administration of soluble Fas receptor improves functional outcome and reduces posttraumatic axonal degeneration after acute spinal cord injury.
Fas receptor activation has been implicated in inflammatory responses, programmed cell death, Wallerian degeneration in neural injury and the axotomy induced death of motoneurons. Recent work using transection models of spinal cord injury (SCI) demonstrated that neutralization of Fas ligand with antibodies may promote axonal regeneration and functional recovery. Moreover, recent studies from our laboratory in mutant mice with deficient expression of Fas, show reduced cell death and enhanced behavioural recovery after SCI. The present paper examines the effects of soluble Fas receptor (sFasR) administration on inhibition of Fas receptor-Fas ligand interaction in the setting of acute SCI in vitro and in vivo. An in vitro model of SCI demonstrated that sFasR administration decreases cell death as assessed by propidium iodide fluorescence. Furthermore, in a moderately severe in vivo clip compression model of SCI at C7-T1, we demonstrate that subarachnoid infusion of sFasR results in increased neuron and oligodendrocyte survival, improved tissue and long tract axonal preservation, reduced apoptotic cell death and enhanced functional neurological outcome after acute SCI. These results strongly suggest that inhibiting Fas receptor activation is neuroprotective after acute SCI and that this strategy may have important translational significance.
31681786
Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury
The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.
3332405
Human neuropathological and animal model evidence supporting a role for Fas-mediated apoptosis and inflammation in cervical spondylotic myelopathy.
Although cervical spondylotic myelopathy is a common cause of chronic spinal cord dysfunction in humans, little is known about the molecular mechanisms underlying the progressive neural degeneration characterized by this condition. Based on animal models of cervical spondylotic myelopathy and traumatic spinal cord injury, we hypothesized that Fas-mediated apoptosis and inflammation may play an important role in the pathobiology of human cervical spondylotic myelopathy. We further hypothesized that neutralization of the Fas ligand using a function-blocking antibody would reduce cell death, attenuate inflammation, promote axonal repair and enhance functional neurological outcomes in animal models of cervical spondylotic myelopathy. We examined molecular changes in post-mortem human spinal cord tissue from eight patients with cervical spondylotic myelopathy and four control cases. Complementary studies were conducted using a mouse model of cervical spondylotic myelopathy (twy/twy mice that develop spontaneous cord compression at C2-C3). We observed Fas-mediated apoptosis of neurons and oligodendrocytes and an increase in inflammatory cells in the compressed spinal cords of patients with cervical spondylotic myelopathy. Furthermore, neutralization of Fas ligand with a function-blocking antibody in twy/twy mice reduced neural inflammation at the lesion mediated by macrophages and activated microglia, glial scar formation and caspase-9 activation. It was also associated with increased expression of Bcl-2 and promoted dramatic functional neurological recovery. Our data demonstrate, for the first time in humans, the potential contribution of Fas-mediated cell death and inflammation to the pathobiology of cervical spondylotic myelopathy. Complementary data in a murine model of cervical spondylotic myelopathy further suggest that targeting the Fas death receptor pathway is a viable neuroprotective strategy to attenuate neural degeneration and optimize neurological recovery in cervical spondylotic myelopathy. Our findings highlight the possibility of medical treatments for cervical spondylotic myelopathy that are complementary to surgical decompression.
18487487
Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death
Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.
27980405
Development and characterization of a novel, graded model of clip compressive spinal cord injury in the mouse: Part 1. Clip design, behavioral outcomes, and histopathology.
In order to take advantage of various genetically manipulated mice available to study the pathophysiology of spinal cord injury (SCI), we adapted an extradural clip compression injury model to the mouse (FEJOTA mouse clip). The dimensions of the modified aneurysm clip blades were customized for application to the mouse spinal cord. Three clips with different springs were made to produce differing magnitudes of closing force (3, 8, and 24 g). The clips were calibrated regularly to ensure that the closing force remained constant. The surgical procedure involved a laminectomy at T3 and T4, followed by extradural application of the clip at this level for 1 min to produce SCI. Three injury severities (3, 8, and 24 g), sham (passage of dissector extradurally at T3-4), and transection control groups were examined (n = 12/group). Quantitative behavioural assessments using the Basso, Beattie, and Bresnahan (BBB; H > 46; df = 4; p < 0.001; Kruskal-Wallis one-way ANOVA) and inclined plane (IP; F = 123; df = 4; p < 0.0001; two-way repeated measures ANOVA) tests showed a significant graded increase in neurological deficits with increasing severity of injury. By day 14, the motor recovery of the mice plateaued. Qualitative examination of the injury site morphology indicated that microcystic cavitation, degenerating axons, and robust astrogliosis were characteristic of the murine response to clip compressive SCI. Morphometric analyses of H&E/Luxol Fast Blue stained sections at every 50 microm from the injury epicenter indicated that with greater injury severity there was a progressive decrease in residual tissue (F = 220, df = 3; p < 0.0001; two-way ANOVA). In addition, statistically significant differences were found in the amount of residual tissue at the injury epicenter between all of the injury severities (p < 0.05, SNK test). This novel, graded compressive model of SCI will facilitate future studies of the pathological mechanisms of SCI using transgenic and knockout murine systems.
44491225
Development and characterization of a novel, graded model of clip compressive spinal cord injury in the mouse: Part 2. Quantitative neuroanatomical assessment and analysis of the relationships between axonal tracts, residual tissue, and locomotor recovery.
A detailed examination of the histopathological features of the clip compression injury in mice was performed to understand the relationships between neurological function and existing pathology of the spinal cord. Adult, female CD1 mice underwent three grades of extradural clip compression injury (3-g, 8-g, and 24-g FEJOTA mouse clips), transection, and sham injury at T3-4. Quantitative behavioural assessments were performed for 4 weeks following SCI. After 4 weeks, Fluoro-Gold was introduced caudal to the SCI site, at T9, and was retrogradely transported for 5 days to the origin of spared axons through the injury site. Counts of retrogradely labeled neurons in the brain-stem, midbrain, and sensory-motor cortex indicated that the number of intact descending axons that traversed the lesion decreased with increasing injury severity (F > 28; df = 4; p < 0.0001; one-way ANOVA). Independent linear correlation analyses were performed between indices of neurological recovery (BBB and IP test), counts of retrogradely labeled neurons and morphometric assessments of normal residual tissue at the injury epicenter. The BBB test correlated strongly with the amount of residual tissue at the injury epicenter (R = 0.945, df = 28, p < 0.0001). Counts of neurons retrogradely labeled with Fluoro-Gold were also strongly correlated with the BBB scores. The extrapyramidal (raphespinal, reticulospinal, vestibulospinal, and rubrospinal) tracts had Pearson correlation coefficients (R) of 0.814, 0.812, 0.813, and 0.747, respectively (df = 28, p < 0.0001). The pyramidal (corticospinal) tract had a correlation of R = 0.747, df = 28, p < 0.0001 with the BBB scores. The IP scores also correlated strongly with the persistence of extrapyramidal (raphespinal, reticulospinal, vestibulospinal and rubrospinal) tracts with correlation coefficients of 0.801, 0.782, 0.790, and 0.836, respectively (df = 28, p < 0.0001). These data indicate that the counts of retrogradely labeled neurons at the origin of distinct descending motor pathways are predictors of the variance of the functional recovery measured by the BBB and IP tests following spinal cord injury. In addition, we provide a detailed neuroanatomical study of clip compression injury in mice that can be used to study the molecular mechanisms of SCI in knockout and transgenic mice.
19730250
Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.
Genetically engineered mice are used extensively to examine molecular responses to spinal cord injury (SCI). Inherent strain differences may confound behavioral outcomes; therefore, behavioral characterization of several strains after SCI is warranted. The Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) for rats has been widely used for SCI mice, but may not accurately reflect their unique recovery pattern. This study's purpose was to develop a valid locomotor rating scale for mice and to identify strain differences in locomotor recovery after SCI. We examined C57BL/6, C57BL/10, B10.PL, BALB/c, and C57BL/6x129S6 F1 strains for 42 days after mild, moderate, and severe contusive SCI or transection of the mid thoracic spinal cord. Contusions were created using the Ohio State University electromagnetic SCI device which is a displacement-driven model, and the Infinite Horizon device, which is a force-driven model. Attributes and rankings for the Basso Mouse Scale for Locomotion (BMS) were determined from frequency analyses of seven locomotor categories. Mouse recovery differed from rats for coordination, paw position and trunk instability. Disagreement occurred across six expert raters using BBB (p < 0.05) but not BMS to assess the same mice. BMS detected significant differences in locomotor outcomes between severe contusion and transection (p < 0.05) and SCI severity gradations resulting from displacement variations of only 0.1 mm (p < 0.05). BMS demonstrated significant face, predictive and concurrent validity. Novice BMS raters with training scored within 0.5 points of experts and demonstrated high reliability (0.92-0.99). The BMS is a sensitive, valid and reliable locomotor measure in SCI mice. BMS revealed significantly higher recovery in C57BL/10, B10.PL and F1 than the C57BL/6 and BALB/c strains after moderate SCI (p < 0.05). The differing behavioral response to SCI suggests inherent genetic factors significantly impact locomotor recovery and must be considered in studies with inbred or genetically engineered mouse strains.
24264666
Increased activity of caspase 3 and caspase 8 in anti‐Fas‐induced apoptosis in lymphocytes from ageing humans
We have previously shown an increased susceptibility of T cell subsets to anti‐Fas‐induced apoptosis in human ageing [ 1]. In this study, we have examined the role of downstream mediators, including caspases, in Fas‐mediated apoptosis in lymphocytes from ageing humans. The cleavage activity of caspase‐8 and caspase‐3 was compared between ageing and young subjects at different times following anti‐Fas treatment, using colorimetric detection analysis. The expression of Fas‐associated death domain (FADD), caspase‐8, and caspase‐3 in lymphocytes was compared at the protein level using Western blotting, and at the mRNA level by Northern blot analysis. In lymphocytes from ageing subjects, there was an early increase in the cleavage activity of caspase‐8 and caspase‐3 compared with young controls. Furthermore, increased protein expression of FADD, caspase‐8 and caspase‐3 at the basal level was observed in lymphocytes from ageing humans. Our results suggest that the altered expression and activity of molecules in the Fas/FasL signalling pathway may play a role in increased Fas‐induced apoptosis and T cell deficiency in ageing humans.
8818645
Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat
Apoptosis or programmed cell death has been reported after CNS trauma. However, the significance of this mechanism in the pathophysiology of spinal cord injury, in particular at the cervical level, requires further investigation. In the present study, we used the extradural clip compression model in the rat to examine the cellular distribution of apoptosis following cervical spinal cord injury, the relationship between glial apoptosis and post-traumatic axonal degeneration and the possible role of apo[apoptosis]-1, CD95 (FAS) and p75 in initiating post-traumatic glial apoptosis. In situ terminal-deoxy-transferase mediated dUTP nick end labeling revealed apoptotic cells, largely oligodendrocytes as identified by cell specific markers, in grey and white matter following spinal cord injury. Apoptotic cell death was confirmed using electron microscopy and by the demonstration of DNA laddering on agarose gel electrophoresis. Beta-amyloid precursor protein was used as a molecular marker of axonal degeneration on western blots and immunohistochemistry. Degeneration of axons was temporally and spatially co-localized with glial apoptosis. FAS and p75 protein expression was seen in astrocytes, oligodendrocytes and microglia, and was also seen in some apoptotic glia after cord injury. Both FAS and p75 increased in expression in a temporal course, which mirrored the development of cellular apoptosis. The downstream caspases 3 and 8, which are linked to FAS and p75, demonstrated activation at times of maximal apoptosis, while FLIP-L an inhibitor of caspase 8, decreased at times of maximal apoptosis. We conclude that axonal degeneration after traumatic spinal cord injury is associated with glial, in particular oligodendroglial, apoptosis. Activation of the FAS and p75 death receptor pathways may be involved in initiating this process.
12729259
Apoptosis after traumatic human spinal cord injury.
OBJECT
Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated.


METHODS
The authors examined the spinal cords of 15 patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1beta-converting enzyme/Caenorhabditis elegans D 3 (ICE/CED-3) family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the 15 spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody.


CONCLUSIONS
These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of caspase-3 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.
43027489
FasL, Fas, and death-inducing signaling complex (DISC) proteins are recruited to membrane rafts after spinal cord injury.
The Fas/CD95 receptor-ligand system plays an essential role in apoptosis that contributes to secondary damage after spinal cord injury (SCI), but the mechanism regulating the efficiency of FasL/Fas signaling in the central nervous system (CNS) is unknown. Here, FasL/Fas signaling complexes in membrane rafts were investigated in the spinal cord of adult female Fischer rats subjected to moderate cervical SCI and sham operation controls. In sham-operated animals, a portion of FasL, but not Fas was present in membrane rafts. SCI resulted in FasL and Fas translocation into membrane raft microdomains where Fas associates with the adaptor proteins Fas-associated death domain (FADD), caspase-8, cellular FLIP long form (cFLIPL ), and caspase-3, forming a death-inducing signaling complex (DISC). Moreover, SCI induced expression of Fas in clusters around the nucleus in both neurons and astrocytes. The formation of the DISC signaling platform leads to rapid activation of initiator caspase-8 and effector caspase-3, and the modification of signaling intermediates such as FADD and cFLIP(L) . Thus, FasL/Fas-mediated signaling after SCI is similar to Fas expressing Type I cell apoptosis.
37985094
Mechanism of Destructive Pathologic Changes in the Spinal Cord Under Chronic Mechanical Compression
Study Design. A histologic and histochemical study was performed both in the autopsy of a human patient with cervical spinal cord compression caused by ossification of the posterior longitudinal ligament and in a tiptoe-walking Yoshimura mouse model of progressive cervical cord compression. Objectives. To clarify the mechanism of destructive pathologic changes in the spinal cord under chronic mechanical compression. Summary of Background Data. Under chronic compression, the spinal cord exhibits destructive changes considered to be causes of profound and irreversible motor paresis. Recently, some investigators have found that apoptosis in acute spinal cord injury induces both secondary degeneration at the site of injury and chronic demyelination of tracts away from the site of injury. However, the mechanism responsible for these destructive spinal cord changes under chronic compression remains unclear. Methods. The spinal cord was examined histologically, and an attempt was made to detect apoptotic cells using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling in both the autopsy of a human patient and tiptoe-walking mice exhibiting spinal cord compression. Results. Apoptotic cells were observed in the chronically compressed spinal cord in both the autopsy of a human patient and model mice. In tiptoe-walking mice exhibiting spinal cord compression, descending degeneration in the anterior and lateral columns and ascending degeneration in the posterior column were observed. The distribution of oligodendrocytes with positive results from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling was similar to that for degeneration of the long tracts. Conclusions. Spinal cord cell apoptosis may produce destructive changes in the spinal cord under chronic compression, with a resulting irreversible neurologic deficit.
10341470
Presence and significance of CD-95 (Fas/APO1) expression after spinal cord injury.
OBJECT
A glycoprotein, CD95 (Fas/APO1) is widely considered to be implicated in the development of apoptosis in a number of tissues. Based on the hypothesis that apoptosis is related to cell death after spinal cord injury (SCI), the authors studied the presence and distribution of CD95 (Fas/APO1)-positive cells in injured spinal cord tissue for the purpose of determining the significance of this protein during the early phases of SCI.


METHODS
The presence and distribution of cells showing positive immunostaining for CD95 (Fas/APO1) were studied 1, 4, 8, 24, 48, and 72 hours and 1, 2, and 4 weeks after induction of experimental SCI in rats. Studies were conducted using a monoclonal antibody to the CD95 (Fas/APO1) protein. Positivity for CD95 (Fas/APO1) was observed in apoptotic cells, mainly in the gray matter, 1 hour after trauma, and the number of immunostained cells increased for the first 8 hours, at which time the protein was expressed in both gray and white matter. From 24 to 72 hours postinjury, the number of immunostained cells decreased in the gray matter, but increased in the white matter. From then on, there were fewer CD95 (Fas/APO1)-positive cells, but some cells in the white matter still exhibited positive immunostaining 1 and 2 weeks after injury. At 4 weeks, there remained no CD95 (Fas/APO1)-positive cells in injured spinal cord.


CONCLUSIONS
These findings indicate that CD95 (Fas/APO1) is expressed after SCI, suggesting a role for this protein in the development of apoptosis after trauma and the possibility of a new therapeutic approach to SCI based on blocking the CD95 (Fas/APO1) system.
5730467
Inflammation and apoptosis: linked therapeutic targets in spinal cord injury.
The secondary cascade of cell death that follows central nervous system (CNS) injury or ischemia has long been considered a target for neuroprotective agents aimed at sparing tissue and function. Recently, several laboratories have shown remarkable protection and recovery of function in rodent models of spinal cord injury using treatments that target components of the CNS inflammatory response. The use of minocycline, an antibiotic that reduces microglial activation, antibody blockade of the CD95 (FAS) ligand and the blockade of glycosphingolipid-induced iNOS (inducible nitric oxide synthase) have recently been shown to reduce neuronal and glial apoptosis with concomitant improvement in neurological function, and appear to enhance the efficacy of cell transplantation strategies.
24108787
Post-traumatic inflammation following spinal cord injury
Inflammatory reaction following a spinal cord injury (SCI) contributes substantially to secondary effects, with both beneficial and devastating effects. This review summarizes the current knowledge concerning the structural features (vascular, cellular, and biochemical events) of SCI and gives an overview of the regulation of post-traumatic inflammation.
500587
Cellular inflammatory response after spinal cord injury in sprague‐dawley and lewis rats
The distribution of microglia, macrophages, T‐lymphocytes, and astrocytes was characterized throughout a spinal contusion lesion in Sprague‐Dawley and Lewis rats by using immunohistochemistry. The morphology, spatial localization, and activation state of these inflammatory cells were described both qualitatively and quantitatively at 12 hours, 3, 7, 14, and 28 days after injury. By use of OX42 and ED1 antibodies, peak microglial activation was observed within the lesion epicenter of both rat strains between three and seven days post‐injury preceding the bulk of monocyte influx and macrophage activation (seven days). Rostral and caudal to the injury site, microglial activation plateaued between two and four weeks post‐injury in the dorsal and lateral funiculi as indicated by morphological transformation and the de‐novo expression of major histocompatibility class II (MHC II) molecules. Similar to the timing of microglial reactions, T‐lymphocytes maximally infiltrated the lesion epicenter between three and seven days post‐injury. Reactive astrocytes, while present in the acute lesion, were more prominent at later survival times (7–28 days). These cells were interspersed with activated microglia but appeared to surround and enclose tissue sites occupied by reactive microglia and phagocytic macrophages. Thus, trauma‐induced central nervous system (CNS) inflammation, regardless of strain, occurs rapidly at the site of injury and involves the activation of resident and recruited immune cells. In regions rostral or caudal to the epicenter, prolonged activation of inflammatory cells occurs preferentially in white matter and primarily consists of activated microglia and astrocytes.
2340208
Matrix metalloproteinases and their inhibitors in human traumatic spinal cord injury
BackgroundMatrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that degrade the extracellular matrix and other extracellular proteins. Studies in experimental animals demonstrate that MMPs play a number of roles in the detrimental as well as in the beneficial events after spinal cord injury (SCI). In the present correlative investigation, the expression pattern of several MMPs and their inhibitors has been investigated in the human spinal cord.MethodsAn immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type.ResultsIn the unlesioned human spinal cord, MMP and TIMP immunoreactivity was scarce. After traumatic SCI, a lesion-induced bi-phasic pattern of raised MMP-1 levels could be found with an early up-regulation in macrophages within the lesion epicentre and a later induction in peri-lesional activated astrocytes. There was an early and brief induction of MMP-2 at the lesion core in macrophages. MMP-9 and -12 expression peaked at 24 days after injury and both molecules were mostly expressed in macrophages at the lesion epicentre. Whereas MMP-9 levels rose progressively from 1 week to 3 weeks, there was an isolated peak of MMP-12 expression at 24 days. The post-traumatic distribution of the MMP inhibitors TIMP-1, -2 and -3 was limited. Only occasional TIMP immuno-positive macrophages could be detected at short survival times. The only clear induction was detected for TIMP-3 at survival times of 8 months and 1 year in peri-lesional activated astrocytes.ConclusionThe involvement of MMP-1, -2, -9 and -12 has been demonstrated in the post-traumatic events after human SCI. With an expression pattern corresponding largely to prior experimental studies, they were mainly expressed during the first weeks after injury and were most likely involved in the destructive inflammatory events of protein breakdown and phagocytosis carried out by infiltrating neutrophils and macrophages, as well as being involved in enhanced permeability of the blood spinal cord barrier. Similar to animal investigations, the strong induction of MMPs was not accompanied by an expression of their inhibitors, allowing these proteins to exert their effects in the lesioned spinal cord.
2094605
The cellular inflammatory response in human spinal cords after injury.
Spinal cord injury (SCI) provokes an inflammatory response that generates substantial secondary damage within the cord but also may contribute to its repair. Anti-inflammatory treatment of human SCI and its timing must be based on knowledge of the types of cells participating in the inflammatory response, the time after injury when they appear and then decrease in number, and the nature of their actions. Using post-mortem spinal cords, we evaluated the time course and distribution of pathological change, infiltrating neutrophils, monocytes/macrophages and lymphocytes, and microglial activation in injured spinal cords from patients who were 'dead at the scene' or who survived for intervals up to 1 year after SCI. SCI caused zones of pathological change, including areas of inflammation and necrosis in the acute cases, and cystic cavities with longer survival (Zone 1), mantles of less severe change, including axonal swellings, inflammation and Wallerian degeneration (Zone 2) and histologically intact areas (Zone 3). Zone 1 areas increased in size with time after injury whereas the overall injury (size of the Zones 1 and 2 combined) remained relatively constant from the time (1-3 days) when damage was first visible. The distribution of inflammatory cells correlated well with the location of Zone 1, and sometimes of Zone 2. Neutrophils, visualized by their expression of human neutrophil alpha-defensins (defensin), entered the spinal cord by haemorrhage or extravasation, were most numerous 1-3 days after SCI, and were detectable for up to 10 days after SCI. Significant numbers of activated CD68-immunoreactive ramified microglia and a few monocytes/macrophages were in injured tissue within 1-3 days of SCI. Activated microglia, a few monocytes/macrophages and numerous phagocytic macrophages were present for weeks to months after SCI. A few CD8(+) lymphocytes were in the injured cords throughout the sampling intervals. Expression by the inflammatory cells of the oxidative enzymes myeloperoxidase (MPO) and nicotinamide adenine dinucleotide phosphate oxidase (gp91(phox)), and of the pro-inflammatory matrix metalloproteinase (MMP)-9, was analysed to determine their potential to cause oxidative and proteolytic damage. Oxidative activity, inferred from MPO and gp91(phox) immunoreactivity, was primarily associated with neutrophils and activated microglia. Phagocytic macrophages had weak or no expression of MPO or gp91(phox). Only neutrophils expressed MMP-9. These data indicate that potentially destructive neutrophils and activated microglia, replete with oxidative and proteolytic enzymes, appear within the first few days of SCI, suggesting that anti-inflammatory 'neuroprotective' strategies should be directed at preventing early neutrophil influx and modifying microglial activation.
1829691
Matrix Metalloproteinase-2 Facilitates Wound Healing Events That Promote Functional Recovery after Spinal Cord Injury
Matrix metalloproteinases (MMPs) are proteolytic enzymes that are involved in both injury and repair mechanisms in the CNS. Pharmacological blockade of MMPs, limited to the first several days after spinal cord injury, improves locomotor recovery. This beneficial response is, however, lost when treatment is extended beyond the acutely injured cord to include wound healing and tissue remodeling. This suggests that some MMPs play a beneficial role in wound healing. To test this hypothesis, we investigated the role of MMP-2, which is actively expressed during wound healing, in white matter sparing and axonal plasticity, the formation of a glial scar, and locomotor recovery after spinal cord injury. MMP-2 increased between 7 and 14 d after injury, where it was immunolocalized in reactive astrocytes bordering the lesion epicenter. There was reduced white matter sparing and fewer serotonergic fibers, caudal to the lesion in injured MMP-2 null animals. MMP-2 deficiency also resulted in increased immunoreactivity to chondroitin sulfate proteoglycans and a more extensive astrocytic scar. Most importantly, locomotion in an open field, performance on a rotarod, and grid walking were significantly impaired in injured MMP-2 null mice. Our findings suggest that MMP-2 promotes functional recovery after injury by regulating the formation of a glial scar and white matter sparing and/or axonal plasticity. Thus, strategies exploiting MMPs as therapeutic targets must balance these beneficial effects during wound healing with their adverse interactions in the acutely injured spinal cord.
29186328
Loci predisposing to autoimmunity in MRL-Fas lpr and C57BL/6-Faslpr mice.
Background genes determine the incidence and severity of lymphoaccumulation and histopathologic manifestations of systemic autoimmunity in mice homozygous for the apoptosis-defective Faslpr mutation. By interval mapping of 274 F2 mice intercrossed between MRL-Faslpr (severe disease) and C57BL/6-Faslpr (minimal disease), four loci were identified with significant linkage to lymphadenopathy and/ or splenomegaly on chromosomes 4, 5, 7, and 10, which were named lupus in (MRL-Faslpr x B6-Faslpr)F2 cross1-4 (Lmb1-4), respectively. Lmb1, -2, and -3 were also linked to the production of anti-dsDNA antibodies, but not glomerulonephritis, whereas Lmb4 was associated with glomerulonephritis. Lmb2, -3, and -4 were inherited from the MRL background, but interestingly, Lmb1 was derived from the C57BL16-Faslpr. Nevertheless, each locus, regardless of the strain of origin, appeared to act in an additive manner, although certain combinations were more effective. Only a single suggestive locus on chromosome 1 could be correlated with arthritis. The identification of loci with highly significant linkage to disease manifestations in Faslpr strains will make it possible to map and clone new genetic defects contributing to autoimmunity.
41822527
Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury
Trauma to the central nervous system (CNS) triggers intraparenchymal inflammation and activation of systemic immunity with the capacity to exacerbate neuropathology and stimulate mechanisms of tissue repair. Despite our incomplete understanding of the mechanisms that control these divergent functions, immune-based therapies are becoming a therapeutic focus. This review will address the complexities and controversies of post-traumatic neuroinflammation, particularly in spinal cord. In addition, current therapies designed to target neuroinflammatory cascades will be discussed.
25983844
Delivery of hyper‐interleukin‐6 to the injured spinal cord increases neutrophil and macrophage infiltration and inhibits axonal growth
Cytokine growth factors of the interleukin (IL)–6 family have recently been shown to play an important role in central nervous system (CNS) development, repair, and inflammation. These cytokines, which interact via specific membrane receptors, share a signal‐transducing receptor subunit, glycoprotein 130 (gp130). Gp130 is expressed by motoneurons in the gray matter of the rat spinal cord and by several brainstem nuclei that project to the spinal cord including the red, reticular, and vestibular nuclei. In this study, we examined whether stimulation of gp130 signaling, with the use of grafts of fibroblasts genetically modified to deliver the fusion protein, hyper‐IL‐6 (H‐IL‐6), which consists of the cytokine growth factor, IL‐6, and its α receptor, would elicit growth of injured spinal cord axons. Particular emphasis was placed on examining the potentially competing effects of growth factor versus proinflammatory influences of H‐IL‐6 in the context of spinal cord injury. Our results demonstrated that grafts delivering H‐IL‐6 induce a sixfold increase in the number of neutrophils (P < 0.05) and a twofold increase in the areas of spinal tissue occupied by macrophages and activated microglia (P < 0.01) at the site of the spinal cord injury when compared with control grafts. Of note, this augmentation in inflammatory cell infiltration correlated with a significant twofold increase in lesion size (P < 0.05) and a fourfold reduction in axonal growth (P < 0.01) at the lesion site. Thus, potential neurotrophic properties of this cytokine family of growth factors must be balanced against their inflammatory properties when considering therapeutic application to CNS injury. J. Comp. Neurol. 454:213–228, 2002. © 2002 Wiley‐Liss, Inc.
21387421
Effects of pro-inflammatory cytokines in experimental spinal cord injury
Following injury to the spinal cord, secondary tissue damage leading to massive additional tissue loss and inflammatory reactions as well as scar formation takes place. The precise functions and effects of the inflammatory cells and their secreted factors are largely unclear. The present study investigates whether the exogenous local administration of pro-inflammatory cytokines to mice after spinal cord injury can influence these intrinsic processes. A mixture of murine recombinant interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha) was administered to the lesioned spinal cord of adult mice. These cytokines provoked an increased recruitment and activation of macrophages and microglial cells in the lesion area when administered 1 day post lesion. In contrast, when administered 4 days after the lesion, recruitment of macrophages was slightly increased while activation of microglia was decreased as compared to controls. The amount of tissue loss 7 days after trauma was smaller in the animals receiving the cytokine mixture than in the mice receiving Ringer control solution on day 4 after lesion. Thus the role of the inflammatory response in spinal cord injury seems to be complex and well regulated. Anti-inflammatory cytokines and factors probably also contribute to the outcome of the damage following injury to the spinal cord.
38408471
Cytokine mRNA Profiles in Contused Spinal Cord and Axotomized Facial Nucleus Suggest a Beneficial Role for Inflammation and Gliosis
We have studied temporal mRNA expression patterns for interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and transforming growth factor-beta1 (TGF-beta1) in two rat injury paradigms with very different cellular inflammatory reactions: contussion of the spinal cord and axotomy of the facial nerve. Our comparative analyses using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) show an early and robust upregulation of IL-1beta, TNF-alpha, IL-6, and M-CSF mRNAs in spinal cord after contusion injury. Peak expression of these mRNAs was transient and returned to control levels by 24 h postinjury. In contrast, expression of IL-1beta and TNF-alpha mRNAs in the axotomized facial nucleus was minimal and delayed, and levels of M-CSF mRNA remained unaltered. Similar to injured spinal cord, the axotomized nucleus showed a dramatic and early upregulation of IL-6 mRNA, but unlike spinal cord, IL-6 mRNA levels subsided only gradually. Both injury paradigms showed gradually increasing levels of TGF-beta1 mRNA which were maximal at 7 days postinjury. RT-PCR analyses were also performed on isolated blood-borne mononuclear cells and neutrophils. The results showed that these cells contain high levels of IL-1beta and M-CSF mRNAs, moderate levels of TGF-beta and TNF-alpha mRNAs, and minimal levels of IL-6 mRNA. The RT-PCR analyses together with histological observations indicate that expression of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 is short-lived and self-limited after contusion injury, and that it occurs primarily within endogenous glial cells. Transient expression of these molecules likely triggers secondary events which may be beneficial to wound repair and regeneration.
25002762
Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats.
In these studies, we examined the neuroprotective effects of the potent antiinflammatory cytokine interleukin-10 (IL-10) following spinal cord injury (SCI). Neuroprotection was assessed by using behavioral and morphological end points. We hypothesized that injury-induced inflammation contributes to the resulting neuropathology and subsequent loss of function. Therefore, by attenuating injury-induced inflammation, we should promote functional recovery. The New York University device was used to induce moderate SCI and study the resulting inflammatory response and functional consequences of inhibiting this response in rats. We determined that SCI induces the expression of tumor necrosis factor-alpha (TNF-alpha) in the spinal cord and by SCI-activated monocytes isolated from the peripheral circulation. IL-10 (5.0 microg) administered 30 minutes after-injury significantly reduced the expression of TNF-alpha protein in the spinal cord and in vitro by SCI-activated monocytes. Next, we investigated whether IL-10 would improve functional recovery after SCI. Randomized, double-blinded studies demonstrated that a single injection of IL-10 significantly improves hind limb motor function 2 months after injury, as determined by the Basso, Beattie and Bresnahan (BBB) open-field behavioral test. IL-10-treated animals had a mean BBB score of 18.0+/-0.5 (SEM, n = 9) compared with a score of 12.9+/-0.6 (SEM, n = 9) for the saline-treated controls. Morphological analysis demonstrated that IL-10 reduces lesion volume by approximately 49% 2 months after injury. These data suggest that acute administration of IL-10 reduces TNF-alpha synthesis in the spinal cord and by activated macrophages, is neuroprotective, and promotes functional recovery following SCI.