The role of excitotoxicity in secondary mechanisms of spinal cord injury: a review with an emphasis on the implications for white matter degeneration

Following an initial impact after spinal cord injury (SCI), there is a cascade of downstream events termed 'secondary injury', which culminate in progressive degenerative events in the spinal cord. These secondary injury mechanisms include, but are not limited to, ischemia, inflammation, free radical-induced cell death, glutamate excitotoxicity, cytoskeletal degradation and induction of extrinsic and intrinsic apoptotic pathways. There is emerging evidence that glutamate excitotoxicity plays a key role not only in neuronal cell death but also in delayed posttraumatic spinal cord white matter degeneration. Importantly however, the differences in cellular composition and expression of specific types of glutamate receptors in grey versus white matter require a compartmentalized approach to understand the mechanisms of secondary injury after SCI. This review examines mechanisms of secondary white matter injury with particular emphasis on glutamate excitotoxicity and the potential link of this mechanism to apoptosis. Recent studies have provided new insights into the mechanisms of glutamate release and its potential targets, as well as the downstream pathways associated with glutamate receptor activation in specific types of cells. Evidence from molecular and functional expression of glutamatergic AMPA receptors in white matter glia (and possibly axons), the protective effects of AMPA/kainate antagonists in posttraumatic white matter axonal function, and the vulnerability of oligodendrocytes to excitotoxic cell death suggest that glutamate excitotoxicity is associated with oligodendrocyte apoptosis. The latter mechanism appears key to glutamatergic white matter degeneration after SCI and may represent an attractive therapeutic target.     

Molecular mechanisms of Fas-mediated cell death in oligodendrocytes

Oligodendrocyte cell death is a significant component of the secondary damage following spinal cord injury (SCI) and other neurodegenerative disorders. However, the mechanisms underlying oligodendroglial apoptotic cell death and the potential relationship to Fas receptor (FasR) activation require further clarification. Here, using MO3.13, a human oligodendroglial cell line, we show clear evidence of apoptosis upon exposure to soluble Fas ligand (sFasL). Apoptosis was linked to caspase-8, -9, and -3 activity and resulted in DNA fragmentation detected by deoxynucleotide transferase dUTP nick end-labeling (TUNEL). Dissipation of mitochondrial membrane potential (DeltaPsim) was an early event and temporally coincided with mitochondrial outer membrane permeability (MOMP), demonstrated by the presence of cytochrome c and apoptosis inducing factor (AIF) in cytosolic fractions. Pretreatment with 100 microM of the caspase inhibitor zVAD-fmk prior to sFasL exposure reduced caspase activation, the dissipation of DeltaPsim, MOMP, and apoptotic cell death. These data provide clear evidence that Fas activation induces apoptosis in oligodendrocytes signaling through intrinsic and extrinsic events. Moreover, we provide evidence for the first time that AIF may play a role in caspase-independent apoptotic execution following Fas activation of oligodendrocytes. These data also add to an emerging body of evidence, which strongly implicates Fas-mediated apoptosis of oligodendrocytes as a potential mediator in the pathobiology of a variety of neurological disorders, including SCI.     

Cellular localization of tumor necrosis factor-alpha following acute spinal cord injury in adult rats

Posttraumatic inflammatory reaction may contribute to secondary injury after traumatic spinal cord injury (SCI). Expression of tumor necrosis factor-alpha (TNF-alpha), a key inflammatory mediator, has been demonstrated in the injured cord. However, the specific cell types that are responsible for TNF-alpha expression after SCI remain to be identified. In the present study, cellular sources of TNF-alpha were examined in rats that received a spinal cord impact injury at the 9th thoracic (T9) level. Here we demonstrate that, within hours after SCI, increased TNF-alpha immunoreactivity was localized in neurons, glial cells (including astrocytes, oligodendrocytes, and microglia), and endothelial cells in areas of the spinal cord adjacent to the lesion site. Myelin breakdown was noted in oligodendrocytes that are immunopositive for TNF-alpha. In sham-operated controls, a low level of TNF-alpha immunoreactivity was detected. In antigen-absorption experiments, no TNF-alpha immunoreactivity was detected, indicating the specificity of TNF-alpha immunocytochemistry in the present study. Results suggest that various cell types, including neurons, glial cells, and vascular endothelial cells, contribute to TNF-alpha production in the injured cord.     

大鼠脊髓损伤中的细胞凋亡及甲基强的松龙的干预作用

目的：探讨脊髓损伤（SCI）继发损伤机制,研究损伤脊髓细胞的凋亡及其意义,观察甲基强的松龙（MP）对细胞凋亡的影响。方法：使用改良Allen法制作大鼠急性SCI模型,实验分3组,假损伤（脊髓未受打击）,损伤组及MP治疗组,采用HE,荧光Hoechst 33342,TUNEL(末端脱氧核苷转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记技术）等技术观察SCI后4h,8h,3d,7d,14d,21d及28d时损伤中心及邻近节段脊髓细胞的凋亡,治疗组损伤后30min给予大剂量MP,比较MP治疗组与损伤组脊髓细胞凋亡的变化,同时平行观察大鼠神经学和组织学恢复情况及两组神经丝蛋白（NF）含量的变化。结果：假损伤组各检测方法未见脊髓细胞凋亡,损伤组大鼠急性SCI后1d开始出现脊髓细胞凋亡,3d达高峰,自损伤中心向头尾端递减分布,持续21d,MP治疗组在伤后3d及7d凋亡脊髓细胞较损伤组显著减少,神经学恢复及组织学评分较损伤组有显著性提高,结论：凋亡是SCI后脊髓神经元死亡的一种重要方式,在继发性损伤中起极为重要的作用。MP的治疗作用可能与其干预SCI后细胞凋亡有关。     

Spinal cord injury induces lesional expression of the proinflammatory and antiangiogenic cytokine EMAP II

Inflammatory cellular responses to spinal cord injury are promoted by proinflammatory messengers. We have analyzed expression of endothelial monocyte activating polypeptide II (EMAP II), a proinflammatory, antiangiogenic cytokine in rats after spinal cord injury (SCI) in comparison to normal rat spinal cords. Immunohistochemical analysis demonstrated a highly significant (p < 0.0001) accumulation of EMAP II(+) microglia/macrophages at the lesion site compared to remote areas and uninjured controls. After peaking at day 3, EMAP II(+) microglia/macrophage cell numbers declined gradually until day 28 after SCI-but still remained elevated. Further, EMAP II(+) cells formed clusters in perivascular Virchow-Robin spaces reaching a maximum at day 3. Prolonged accumulation of EMAP II(+), ED1(+) microglia/macrophages suggest a role of EMAP II in the pathophysiology of secondary injury following SCI.     

Brain-derived neurotrophic factor suppresses delayed apoptosis of oligodendrocytes after spinal cord injury in rats

We evaluated the effect of brain-derived neurotrophic factor (BDNF) on cell death after spinal cord injury. A rat spinal cord injury model was produced by static load, and continuous intrathecal BDNF or vehicle infusion was carried out either immediately or 3 days after the injury. Cell death was examined by nuclear staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After injury, typical apoptotic cells were observed. Double staining with TUNEL and specific cell markers revealed that, soon after the injury, the apoptotic or necrotic cells at the injury site were neurons and microglia. One week after the injury, apoptotic oligodendrocytes, but not apoptotic astrocytes, were observed in the white matter rostral and caudal to the injury site, whereas few apoptotic cells were found in the gray matter. The immediate BDNF treatment significantly reduced the number of TUNEL-positive cells in the adjacent rostral site 1 and 2 weeks after the injury, and in the adjacent caudal site 3 days and 1 week after the injury, even though there was no significant difference between BDNF-treated and control rats at the injury site itself. In addition, similar antiapoptotic effects were observed in these regions 1 week after injury in rats that received BDNF treatment from the third day after injury. These findings suggest that BDNF suppresses delayed apoptosis of oligodendrocytes after spinal cord injury, for which even delayed injections are effective. BDNF administration may therefore be useful for the clinical treatment of spinal cord injury through the suppression of secondary events.     

细胞自噬在脊髓损伤中作用的研究进展

近年来,细胞自噬在脊髓损伤中的研究逐渐成为热点,但脊髓损伤后早期自噬激活所起的作用尚有争议。其原因在于细胞自噬在脊髓损伤中的作用具有两面性:一方面自噬能诱导自噬性细胞死亡的发生并参与细胞凋亡的发生,另一方面自噬能促进受损变性蛋白的代谢以及抑制细胞凋亡。然而究其对脊髓损伤后修复的利弊作用,早期自噬激活的程度具有决定作用,脊髓损伤后适当地上调自噬水平可促进受损变性蛋白的代谢并抑制细胞凋亡,而过度激活自噬可能引发自噬性细胞死亡。     

Role of Rho-associated coiled-coil containing protein kinase in the spinal cord injury induced neuropathic pain

BACKGROUND CONTEXTSpinal cord injury (SCI) can lead to increased phosphorylation of p38 in spinal cord microglia. This is one of the main causes for the development of persistent pain. Recently, we reported our study on the activation of p38 mitogen-activated protein kinases (MAPK) in spinal microglia, which has been considered the key molecule for the onset and maintenance of neuropathic pain after peripheral nerve injury, using a rat model. We also reported that the RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) pathway mediates p38 activation in spinal microglia in peripheral nerve injury. But the precise mechanisms of neuropathic pain induced by SCI are still unclear.PURPOSEThis study aimed to examine the activation of microglia and the p38 MAPK expression in the lumbar spinal cord after thoracic SCI in rats, and the correlation to the therapeutic effect of ROCK inhibitor ripasudil in rats with SCI.STUDY DESIGNMale Sprague–Dawley rats underwent thoracic (T10) spinal cord contusion injury using an Infinite Horizon impactor device. SCI rats received ROCK inhibitor ripasudil (24 nmol/day or 240 nmol/day) from just before SCI to 3 days after SCI.METHODSThe mechanical threshold in the rat's hind paws was measured over four weeks. Morphology of microglia and phosphorylation of p38 (p-p38) in the lumbar spinal cord and were analyzed using immunohistochemistry.RESULTSThe p-p38 positive cell and Iba1 (a maker of microglia) positive area were significantly increased at the lumbar spinal dorsal horn (L4–5) 3 days and 7 days after SCI compared with the sham-control (p<.05), whereas phosphorylated p38 was co-localized with microglia. Three days after SCI, the intensity of phosphorylated p38 and Iba1 immunoreactive cells in the dorsal horn was significantly lower in the ripasudil treated groups than in the saline group. However, administration of ROCK inhibitor did not affect the numbers of microglia. Moreover, the withdrawal threshold of the ripasudil-treated rats was significantly higher than that of the saline-injected rats on 14 days and 28 days after SCI.CONCLUSIONSOur results suggest that activation of ROCK in spinal cord microglia is likely to have an important role in the activation of p38 MAPK, which has been considered as a key molecule that switches on neuropathic pain after SCI. Inhibition of ROCK signaling may offer a means in developing a novel neuropathic pain treatment after SCI. It may help patients with neuropathic pain after SCI.CLINICAL SIGNIFICANCEThe findings in the present study regarding intracellular mechanisms suggest that modulation of ROCK signaling may be a focus for novel treatment for neuropathic pain after SCI.     

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.     

Neuroprotective effects of recombinant thrombomodulin in controlled contusion spinal cord injury implicates thrombin signaling

Although the central nervous system (CNS) of mammals has had poor prospects for regeneration, recent studies suggest this might improve from blocking "secondary cell loss" or apoptosis. In this regard, intravenous activated protein C (aPC) improved neurologic outcomes in a rat compression spinal cord injury (SCI) model. Protein C activation occurs when the serine protease thrombin binds to the cell surface proteoglycan thrombomodulin (TM) forming a complex that halts coagulation. In culture, rTM blocks thrombin's activation of protease-activated receptors (PARs), that mediate thrombin killing of neurons and glial reactivity. Both PAR1 and prothrombin are rapidly upregulated after contusion SCI in rats, prior to peak apoptosis. We now report neuroprotective effects of intraperitoneal soluble recombinant human rTM on open-field locomotor rating scale (BBB) and spinal cord lesion volume when given 1 h after SCI. BBB scores from four separate experiments showed a 7.6 +/- 1.4 absolute score increase (p < 0.05) at 3 days, that lasted throughout the time course. Histological sections at 14 days were even more dramatic where a twofold reduction in lesion volume was quantified in rTM-treated rats. Thionin staining revealed significant preservation of motor neuronal profiles both at, and two segments below, the lesion epicenter. Activated caspase-3 immunocytochemistry indicated apoptosis was quite prominent in motor neurons in vehicle (saline) controls, but was dramatically reduced by rTM. Microglia, increased and activated after injury, were reduced with rTM treatment. Taken together, these and previous results support a prominent role for coagulation-inflammation signaling cascades in the subacute changes following SCI. They identify a neuroprotective role for rTM by its inhibition of thrombin generation and blockade of PAR activation.     

Activation of microglial cells and complement following traumatic injury in rat entorhinal-hippocampal slice cultures

The complement cascade has been suggested to be involved in development of secondary brain damage following traumatic brain injury (TBI). Previous studies have shown that reactive microglia are involved in activation of the complement cascade following various injuries to the nervous system. Macrophages seem to have a significant role in this process, but it is still unclear whether these cells, as well as the complement components, are derived from reactive microglia or if these biological events only can occur as a result from the influx of plasma and monocytes via a disrupted blood-brain barrier (BBB). The aim of this study was to investigate the response of microglial cells and the complement system in the absence of plasma/blood components following a standardized crush injury in an entorhinal-hippocampal slice culture. There was a clear increase in complement component C1q and C5b-9-IR (Membrane Attack Complex, MAC) in the area near the crush injury. MAC-IR appeared as numerous dots in clusters which co-localized with anti-NeuN labelled neurons in the injury border zone. Complement C1q-IR co-localized with reactive microglia, co-labelled with OX42 antisera. These findings show activation of the complement cascade near the injury zone and in particular, formation of MAC at the surface of neurons in this area. There was a distinct activation of microglial cells (OX42-IR) near the site of injury, as well as an increase in ED-1 expressing macrophages. In the absence of blood and plasma components it is likely that ED-1-labelled cells represent reactive microglia transformed into macrophages. In addition, Neurons (Neun-IR) near the injury were found to co-localize with clusterin-IR indicating upregulation of a defense system to the endogenous complement attack. The present study provides evidence that microglia and complement is activated in the injury border zone of the tissue slice in a similar fashion as in vivo following TBI, despite the absence of plasma/blood products and cells. These findings support the hypothesis that reactive microglia have a key role in complement activation following TBI by local synthesis of complement with a potential impact on development of secondary neuronal insults.     

Caspase activation in neuronal and glial apoptosis following spinal cord injury in mice

The involvement of caspases in apoptosis after spinal cord injury (SCI) was investigated in adult mouse spinal cord after contusion. Sections of spinal cord were processed for staining 7 days after SCI with the fluorescent dye Hoechst 33342, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL), and immunostaining with an antibody (CM1) recognizing activated caspase-3. Caspase-3- and caspase-8-like enzyme activities were measured colorimetrically at 8 hours to 7 days after SCI using the specific substrates Asp-Glu-Val-Asp-p-nitroanilide and Ile-Glu-Thr-Asp-p-nitroanilide, respectively. Hoechst 33342 staining showed small, bright areas in fragmented nuclei. Double labeling with TUNEL plus immunostaining with cell type-specific markers identified TUNEL-positive neurons stained by anti-neuronal nuclear protein/neurons antibody, and TUNEL-positive oligodendrocytes stained by anti-cyclic nucleotide 3'-phosphohydrolase antibody. Double labeling with CM1 and cell-type specific markers similarly identified CM1-positive neurons and oligodendrocytes. Caspase-8-like enzyme activity was increased significantly on days 3 and 7 (p < 0.01), whereas caspase-3-like activity increased on day 7 (p < 0.01). Intraventricular injection of a nonspecific tetrapeptide caspase inhibitor or a specific tetrapeptide inhibitor of caspase-3 just after SCI reduced enzyme activity at 7 days. Apoptotic cells were identified with TUNEL staining in both neurons and oligodendrocytes in mice after SCI, which also showed activated caspase-3. Increased caspase-3- and caspase-8-like activity was detected in the injured spinal cord on days 3 and 7. Caspase protease activities may be involved in delayed neuronal and glial apoptosis after SCI.     

Traumatic brain injury induces prolonged accumulation of cyclooxygenase-1 expressing microglia/brain macrophages in rats

Inflammatory cellular responses to brain injury are promoted by proinflammatory messengers. Cyclooxygenases (prostaglandin endoperoxide H synthases [PGH]) are key enzymes in the conversion of arachidonic acid into prostanoids, which mediate immunomodulation, mitogenesis, apoptosis, blood flow, secondary injury (lipid peroxygenation), and inflammation. Here, we report COX-1 expression following brain injury. In control brains, COX-1 expression was localized rarely to brain microglia/macrophages. One to 5 days after injury, we observed a highly significant (p < 0.0001) increase in COX-1+ microglia/macrophages at perilesional areas and in the developing core with a delayed culmination of cell accumulation at day 7, correlating with phagocytic activity. There, cell numbers remained persistently elevated up to 21 days following injury. Further, COX-1+ cells were located in perivascular Virchow-Robin spaces also reaching maximal numbers at day 7. Lesion-confined COX-1+ vessels increased in numbers from day 1, reaching the maximum at days 5-7. Double-labeling experiments confirmed coexpression of COX-1 by ED-1+ and OX-42+ microglia/ macrophages. Transiently after injury, most COX-1+ microglia/macrophages coexpress the activation antigen OX-6 (MHC class II). However, the prolonged accumulation of COX-1+, ED-1+ microglia/macrophages in lesional areas enduring the acute postinjury inflammatory response points to a role of COX-1 in the pathophysiology of secondary injury. We have identified localized, accumulated COX-1 expression as a potential pharmacological target in the treatment of brain injury. Our results suggest that therapeutic approaches based on long-term blocking including COX-1, might be superior to selective COX-2 blocking to suppress the local synthesis of prostanoids.     

Activation of complement pathways after contusion-induced spinal cord injury

Previous studies have shown that a cellular inflammatory response is initiated, and inflammatory cytokines are synthesized, following experimental spinal cord injury (SCI). In the present study, we tested the hypothesis that the complement cascade, a major component of both the innate and adaptive immune response, is also activated following experimental SCI. We investigated the pathways, cellular localization, timecourse, and degree of complement activation in rat spinal cord following acute contusion-induced SCI using the New York University (NYU) weight drop impactor. Mild and severe injuries (12.5 and 50 mm drop heights) at 1, 7, and 42 days post injury time points were evaluated. Classical (C1q and C4), alternative (Factor B) and terminal (C5b-9) complement pathways were strongly activated within 1 day of SCI. Complement protein immunoreactivity was predominantly found in cell types vulnerable to degeneration, neurons and oligodendrocytes, and was not generally observed in inflammatory or astroglial cells. Surprisingly, immunoreactivity for complement proteins was also evident 6 weeks after injury, and complement activation was observed as far as 20 mm rostral to the site of injury. Axonal staining by C1q and Factor B was also observed, suggesting a potential role for the complement cascade in demyelination or axonal degeneration. These data support the hypothesis that complement activation plays a role in SCI.     

Combined VEGF and PDGF treatment reduces secondary degeneration after spinal cord injury

Trauma to the spinal cord creates an initial physical injury damaging neurons, glia, and blood vessels, which then induces a prolonged inflammatory response, leading to secondary degeneration of spinal cord tissue, and further loss of neurons and glia surrounding the initial site of injury. Angiogenesis is a critical step in tissue repair, but in the injured spinal cord angiogenesis fails; blood vessels formed initially later regress. Stabilizing the angiogenic response is therefore a potential target to improve recovery after spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) can initiate angiogenesis, but cannot sustain blood vessel maturation. Platelet-derived growth factor (PDGF) can promote blood vessel stability and maturation. We therefore investigated a combined application of VEGF and PDGF as treatment for traumatic spinal cord injury, with the aim to reduce secondary degeneration by promotion of angiogenesis. Immediately after hemisection of the spinal cord in the rat we delivered VEGF and PDGF and to the injury site. One and 3 months later the size of the lesion was significantly smaller in the treated group compared to controls, and there was significantly reduced gliosis surrounding the lesion. There was no significant effect of the treatment on blood vessel density, although there was a significant reduction in the numbers of macrophages/microglia surrounding the lesion, and a shift in the distribution of morphological and immunological phenotypes of these inflammatory cells. VEGF and PDGF delivered singly exacerbated secondary degeneration, increasing the size of the lesion cavity. These results demonstrate a novel therapeutic intervention for SCI, and reveal an unanticipated synergy for these growth factors whereby they modulated inflammatory processes and created a microenvironment conducive to axon preservation/sprouting.     

Phenotypic changes in NG2+ cells after spinal cord injury

Following contusive spinal cord injury (SCI), 50% of oligodendrocytes in the residual white matter are lost within 24 h. NG2-expressing cell proliferation is maximal 3 days after SCI, and may be the source of mature oligodendrocytes and astrocytes that chronically replace those that were lost. We studied NG2(+) cells dissociated from the 3-day injured spinal cord for comparison with those from uninjured adult and early postnatal cords. After 24 h in serum-containing medium, we performed patch clamp analysis and immunocytochemistry for NG2 in combination with nestin (progenitors), and A2B5, O4, and O1 (oligodendrocyte lineage markers). We observed an NG2(+)/A2B5-/O4-/O1- population in both adult preparations. More than double the normal number of NG2(+) cells was isolated from the injured cord, but OX42(+) microglia/macrophages were the predominant cell type after injury. Most cells isolated at P7 were NG2-/A2B5(+), whereas those from the normal adult were NG2(+)/A2B5-. NG2(+) cells after SCI displayed altered voltage-gated potassium current profiles compared to normal adult and P7 animals. Additionally, less than 25% of adult cells (normal and injured) responded to GABA and glutamate, compared to 100% of P7 cells. Our results indicate that the adult NG2(+) cell pool is antigenically and physiologically different than the early postnatal pool, and that contusive injury induces changes in adult NG2(+) cells.     

Overexpression of SOD1 in transgenic rats attenuates nuclear translocation of endonuclease G and apoptosis after spinal cord injury

Spinal motor neurons are selectively vulnerable after spinal cord injury (SCI). Recent studies suggest they undergo apoptosis after caspase activation through a mitochondria-dependent apoptosis pathway, and that oxidative stress after SCI is likely to play a role. However, other signaling pathways of apoptosis that involve mitochondria have not been thoroughly studied after SCI. Apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are mitochondrial apoptogenic proteins that are capable of inducing neuronal apoptosis when translocated from mitochondria to nuclei through a caspase-independent pathway. In this study, we examined translocation of these proteins and apoptotic cell death of motor neurons. The role of oxidative stress was also studied using transgenic (Tg) rats that overexpress the intrinsic antioxidant copper/zinc-superoxide dismutase (SOD1). Western blots and an activity assay demonstrated that a greater amount of SOD1 and higher activity of SOD presented in mitochondria of Tg rats compared with wild-type (Wt) rats. Immunohistochemistry and Western blots showed translocation of EndoG and AIF from mitochondria to nuclei in motor neurons 1 day after SCI in both groups of rats. However, there was significantly less translocation of EndoG in the Tg rats compared with the Wt rats. Less apoptotic cell death was detected in the Tg rats than in the Wt rats 3 days after SCI. These results suggest that translocation of EndoG and AIF from mitochondria to nuclei may initiate a caspase-independent pathway of apoptosis. An increased level of SOD1 in mitochondria conceivably reduces oxidative stress, thereby attenuating EndoG translocation, and resulting in reduction of caspase-independent apoptosis.