Protective effects of Batroxobin on spinal cord injury in rats

Expansion of the secondary injury following primary spinal cord injury is a major pathological event that increases destruction in the spinal cord, so measures to reduce secondary injury are needed. Our previous study demonstrated that, at the front of the expanding secondary injury in the spinal cord, there is an ischemic area in which many neurons can still be rescued. Therefore, enhancement of blood circulation in the cord may be helpful, and indeed, we found that a traditional Chinese medicine, shu-xue-tong, efficiently reduces the secondary injury. The aim of the present study was to investigate the effect of reducing fibrinogen with Batroxobin, a drug widely used clinically for ischemia, in rats with spinal cord contusion. We found that both 2 and 4 Batroxobin units (BU)/kg efficiently decreased the plasma fibrinogen, and 2 BU/kg significantly increased spinal blood flow, enhanced neuronal survival, mitigated astrocyte and microglia activation, and improved locomotor recovery. However, 4 BU/kg had no effect on the secondary spinal cord injury. These data suggest that Batroxobin has multiple beneficial effects on spinal cord injury, indicating a potential clinical application.     

Neuroprotective effects of tamoxifen on experimental spinal cord injury in rats

The aim of this study was to evaluate the effects of tamoxifen on tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) levels and ultrastructural changes in rats with spinal cord injury. Rats were divided into four groups: control group (laminectomy only), trauma group (laminectomy + spinal trauma), tamoxifen group (laminectomy + spinal trauma + tamoxifen), and vehicle group (laminectomy + spinal trauma + vehicle). Spinal cords were extirpated at the T₇–T₁₂ level and tissue samples from the spinal cords were gathered for TNF-α and IL-1β measurements at 1 and 6 hours. Spinal cords harvested at 6 hours were evaluated for ultrastructural changes. TNF-α and IL-1β levels at 6 hours were significantly lower in the tamoxifen group than in the trauma group. Electron microscopic examination of tissue from the trauma group revealed gross cell deformities with widespread edema of all structures as well as severe edema in the neuropil. At 6 hours after trauma, these ultrastructural changes were less marked in the tamoxifen group. Our findings support a neuroprotective and restorative role for tamoxifen in the context of secondary pathological biochemical events after SCI.     

肾源性细胞因子促红细胞生成素对急性脊髓损伤大鼠细胞凋亡的影响**★

目的：促红细胞生成素为肾源性细胞因子，在大鼠急性脊髓损伤后对脊髓神经功能具有保护作用。实验拟证明不同时间硬膜外腔注射后其对脊髓神经细胞凋亡的影响。 方法：实验于2007-01/04在苏州大学附属第二医院动物实验室完成。①实验材料：清洁级成年雄性SD大鼠48只，体质量(270 ±10) g；实验用人重组促红细胞生成素为日本麒麟啤酒株式会社制造，规格3 000 IU /支。②实验分组及处理：将大鼠随机分为正常组6只，假手术组6只（仅切除椎板），生理盐水组18只，实验组18只。按改良Allen打击法建立大鼠脊髓不完全损伤模型。实验组分别于大鼠脊髓损伤后1，6，24 h（每个时间点6只），于硬膜外腔注射重组人促红细胞生成素5 000 u/（kg?bw）；生理盐水组于相同时间予以相同体积生理盐水。③实验评估：通过动物神经运动功能BBB评分及斜板试验评价神经损伤程度；苏木精－伊红染色法观察组织学改变，并采用原位末端标记法标记法检测脊髓神经细胞凋亡数。 结果：①神经行为学评分：正常组、假手术组大鼠双下肢运动功能正常；与生理盐水组相比,脊髓损伤后实验组1,6及24 h神经运动功能有改善；斜板角度及BBB评分均明显提高，差别有显著性意义(P < 0.05)。②组织学苏木精－伊红染色结果：实验组组织学破坏改变明显较生理盐水组轻。③凋亡神经细胞及计数结果：实验组脊髓神经细胞凋亡指数均明显下降，差异有显著性(P < 0.05)。 结论：早期应用外源性促红细胞生成素对脊髓不完全损伤后引起的神经运动功能损害具有保护作用,能明显改善脊髓损伤后的临床功能表现。此种保护作用与促红细胞生成素能够抑制神经细胞凋亡有关。     

Protective effects of gonadal hormones on spinal motoneurons following spinal cord injury

Spinal cord injury(SCI) results in lesions that destroy tissue and disrupt spinal tracts, producing deficits in locomotor and autonomic function. The majority of treatment strategies after SCI have concentrated on the damaged spinal cord, for example working to reduce lesion size or spread, or encouraging regrowth of severed descending axonal projections through the lesion, hoping to re-establish synaptic connectivity with caudal targets. In our work, we have focused on a novel target for treatment after SCI, surviving spinal motoneurons and their target musculature, with the hope of developing effective treatments to preserve or restore lost function following SCI. We previously demonstrated that motoneurons, and the muscles they innervate, show pronounced atrophy after SCI. Importantly, SCI-induced atrophy of motoneuron dendrites can be attenuated by treatment with gonadal hormones, testosterone and its active metabolites, estradiol and dihydrotestosterone. Similarly, SCI-induced reductions in muscle fiber cross-sectional areas can be prevented by treatment with androgens. Together, these findings suggest that regressive changes in motoneuron and muscle morphology seen after SCI can be ameliorated by treatment with gonadal hormones, further supporting a role for steroid hormones as neurotherapeutic agents in the injured nervous system.     

腺苷预处理对大鼠脊髓缺血再灌注损伤保护作用的实验研究

目的探讨腺苷预处理对脊髓缺血再灌注损伤的保护作用。方法制作大鼠脊髓缺血再灌注损伤模型。72只SD大鼠随机分为3组:假手术组(A组)、缺血再灌注组(B组)、腺苷预处理组(C组),再按缺血再灌注后不同时间把各组随机分成3个亚组。通过BBB神经评分观察3组大鼠的神经症状;应用HE染色评价脊髓内的病理组织学变化;并测定3组大鼠不同时间点的SOD、MDA含量。结果 A组、C组在各个时间点的BBB评分明显高于B组(P<0.05);与B组相比,A组、C组细胞形态学变化轻,脊髓组织的SOD含量明显增高(P<0.05),MDA明显降低(P<0.05)。结论腺苷预处理通过提高SOD水平,降低氧自由基和脂质过氧化对大鼠脊髓缺血再灌注损伤进行保护,使神经损伤症状得到一定程度的恢复。     

半乳糖凝集素-1对急性脊髓损伤模型大鼠的保护作用及其机制

目的研究半乳糖凝集素-1(Galectin-1)对大鼠急性脊髓损伤的保护作用及其机制。方法 45只SPF级SD大鼠随机平均分为Galectin-1组、甲泼尼龙(MP)组和生理盐水对照组(生理盐水组)。以Allen's法打击脊髓造模,分别在术后1 d、7 d、14 d每组各取5只大鼠,先做BBB评分及斜板试验,然后处死大鼠取损伤处脊髓组织进行苏木精-伊红(HE)染色及胶质纤维酸性蛋白(GFAP)免疫组化染色检测,同时用酶联免疫吸附测定(ELISA)法检测血清白细胞介素-10(IL-10)的含量。结果术后1 d,3组大鼠BBB评分、斜板试验结果及血清IL-10含量的差异无统计学意义(均P0.05);术后7 d、14 d,MP组和Galectin-1组的BBB评分、斜板试验结果及血清IL-10含量均较生理盐水组高(均P0.05),而MP组与Galectin-1组之间的差异均无统计学意义(均P0.05)。HE染色显示术后各时间点,Galectin-1组和MP组脊髓损伤肿胀及出现核固缩或解离神经元的数量少于生理盐水组。术后1 d,3组大鼠脊髓星形胶质细胞(GFAP表达阳性)数量无明显区别;术后7 d、14 d,Galectin-1组及MP组明显多于生理盐水组,而Galectin-1组与MP组则无明显差别。结论Galectin-1对大鼠急性脊髓损伤具有一定的保护作用,其机制可能是通过增强机体IL-10的表达,从而抑制损伤后的炎症反应而发挥脊髓保护作用的。     

Gastric dysreflexia after acute experimental spinal cord injury in rats

Abstract  Gastric reflexes are mediated mainly by vago-vagal reflex circuits in the caudal medulla. Despite the fact that brainstem vago-vagal circuitry remains intact after spinal cord injury (SCI), patients with SCI at the cervical level most often present gastric stasis with an increased risk of reflux and aspiration of gastric contents. Using a miniature strain gauge sutured to the gastric surface; we tested gastric motility and reflexive gastric relaxation following oesophageal distension (oesophageal-gastric relaxation reflex) in animals 3 days after a severe spinal contusion at either the third or ninth thoracic spinal segment (acute T3- or T9 SCI, respectively). Both basal gastric motility and the oesophageal-gastric relaxation reflex were significantly diminished in animals with T3 SCI. Conversely, both basal gastric motility and the oesophageal-gastric relaxation reflex were not significantly reduced in T9 SCI animals compared to controls. The reduced gastric motility and oesophageal-gastric reflex in T3 SCI rats was not ameliorated by celiac sympathectomy. Our results show that gastric stasis following acute SCI is independent of altered spinal sympathetic input to the stomach caudal to the lesion. Our data suggest that SCI may alter the sensitivity of vagal reflex function, perhaps by interrupting ascending spinosolitary input to brainstem vagal nuclei.     

Neuroprotective effects of infliximab in experimental spinal cord ischemic injury

Reactive oxygen species (ROS) have been implicated in the pathogenesis of spinal cord injury after both ischemia–reperfusion (I/R) and trauma. This experimental study was designed to investigate the potential effects of infliximab, an anti-tumor necrosis factor-α agent, on I/R injury of the rabbit spinal cord. Eighteen New Zealand white rabbits were divided into three groups, each consisting of six rabbits: sham (no I/R), I/R, and infliximab (I/R + infliximab). Spinal cord ischemia was induced by applying an infrarenal aortic cross clamp for 30 minutes. At 48 hours after ischemia, animals were functionally evaluated using the Tarlov score. Changes in the spinal cord were observed by measuring tissue levels of malondialdehyde (MDA), glutathione (GSH), advanced oxidation protein products (AOPP), and superoxide dismutase (SOD) and by evaluating hematoxylin–eosin-stained sections. At 48 hours after ischemia, the Tarlov scores in the infliximab group were higher than those of the I/R group, MDA and AOPP levels in the I/R group were significantly higher than those in the sham and infliximab groups (p < 0.05), and SOD levels in the infliximab group were significantly higher than those in the I/R and sham groups (p < 0.05). The sham group had higher GSH levels than the infliximab group; however, the difference was not statistically significant (p > 0.05). Histological examination revealed that the infliximab group had significantly less vascular proliferation, edema, and neuron loss than the I/R group. These results indicate that infliximab may protect the spinal cord against injury in a rabbit I/R model.     

Protective role of astragalus injection in spinal cord ischemia-reperfusion injury in rats

Objectives:To investigate the neuroprotective effect of Astragalus injection in a spinal cord ischemia-reperfusion (I/R) injury model.Methods:A total of 27 Sprague Dawley rats were randomly divided into 3 groups: control group (n=3), I/R group (n=12), and Astragalus injection group (Ast group, n=12). Spinal cord ischemia was induced by occlusion of the abdominal aorta above the right renal artery for 32 min. Animals in the Ast group were administered Astragalus injection (6.42 mL/kg) at 30 min before the induction of ischemia. After reperfusion for 8, 12, 24, or 48 hours, the serum neuron-specific enolase (NSE) concentration was measured by enzyme-linked immunosorbent assay (ELISA) and the aquaporin-4 (AQP4) protein level was detected by western blotting.Results:The pathological changes, as assessed by hematoxylin and eosin (HE) staining, were milder in the spinal cords of the Ast group compared to the I/R group. Enzyme-linked immunosorbent assay demonstrated that the NSE concentration of the Ast group was significantly lower than that of the I/R group (p<0.05). However, the NSE concentrations of the I/R and Ast groups were significantly higher than that of the control group (p=0.05). Additionally, the expression of AQP4 in the Ast group was lower than that of the I/R group at each time point.Conclusion:These findings indicate that Astragalus injection has a neuroprotective effect in spinal cord I/R injury by decreasing the AQP4 expression.

Spinal cord ischemia and the resulting paraplegia are devastating complications that can occur following thoracic and thoracoabdominal aortic interventions as well as decompression spinal canal stenosis.1,2 In addition to the physical impact, paraplegia is usually associated with psychological and economic burdens that affect patients as well as their family members. The past few decades have witnessed considerable medical advances that have decreased the rate of spinal cord ischemia and paraplegia.3,4,5 Various methods have been proposed to reduce the risk of paraplegia, including ischemic preconditioning, moderate hypothermia, antioxidants, steroids, and calcium channel blockers.6 Nevertheless, none of these methods can completely prevent the development of paraplegia, and the current incidence rate ranges from 5% to 10%. Astragalus injection is an aqueous solution extracted from the Chinese herb Astragalus membranaceus.7 The main components of Astragalus injection include Astragalus saponins (astragaloside I, astragaloside II, and astragaloside IV) and flavonoids (formononetin, ononin, calycosin, and isoflavane).8 Astragalus injection is a stable pharmaceutical product; the concentration of Astragaloside IV (C41H68O14) in Astragalus injection should be no less than 0.08 mg/mL, according to the quality control standard of the Ministry of Health of the People’s Republic of China.9 Astragalus has been widely used in China for a wide array of conditions, including the treatment of diabetes and stroke.10,11 Additionally, recent reports have suggested a possible neuroprotective effect for Astragalus injection.12,13 Therefore, in this study, we aimed to investigate the neuroprotective function of Astragalus injection in spinal cord ischemia-reperfusion (I/R) injury in rats and to explore the underlying mechanism.     

Protective effects of adenoviral cardiotrophin-1 gene transfer on rubrospinal neurons after spinal cord injury in adult rats

Cardiotrophin-1 (CT-1), a muscle-derived cytokine, supports the survival of motoneurons in vivo and in vitro. The present study investigated whether adenoviral huCT-1 gene transfer protected injured neurons from cell death or atrophy and promoted regeneration of rubrospinal tract (RST) after spinal cord injury in adult rats. Administration of the adenoviral CT-1 vector (Adv-CT1) to C3-4 lateral funiculus hemisection cavity, that completely interrupted RST, led to sustained CT-1 expression. Providing Adv-CT1, which rescued 20% of neurons, could prevent the loss of injured rubrospinal neurons 8 weeks post-injury. Retrograde tracing with FluoroGold showed that 1.2% of RST neurons regenerated at least two segments caudal to the injury site. Anterograde tracing with biotinylated dextran amine revealed that the RST axons terminated in white matter and gray matter. Behavioral testing revealed a significant functional recovery in limb usage. This observation indicated that adenoviral CT-1 gene transfer into the injured cord promoted survival and regeneration of rubrospinal neurons in adult rats.     

Early protective effects of Iloprost after experimental spinal cord injury

Abstract

Serial magnetic resonance (MR) imaging has not yet been validated in the therapy of experimental intracerebral hematomas in a rat model. It is possible to test the effect of local fibrinolysis and aspiration on the clot volume using serial magnetic resonance imaging and different MR-sequences. Experiments were carried out in 22 male Sprague-Dawley rats. Intracerepra I hematoma was produced by injection of fresh autologous blood into the caudate nucleus using a double injection technique. Thirty minutes later 70 rats were treated by injecting 12 µl of recombinant tissue plasminogen activator. MR-imaging was performed immediately after generation of the hematoma and after clot lysis. The clot volume measured in the magnetic resonance images was compared with that obtained in stained histological serial sections at the end of the experiment. Serial MR scanning demonstrated a significant reduction (p<0.07) of hematoma volume after fibrinolysis followed by aspiration of the blood clot. The best correlation between MR- and histological volumetry was found on RF-spoiled FLASH 2D-images. This study documents the efficacy of MRI in detecting and delineating the size of acute intracerebral hematomas and its time course. Local fibrinolysis and aspiration can be simulated in an experimental rat model. [Neural Res 1998; 20: 349-352]     

Protective effects of human umbilical cord mesenchymal stem cell vein transplantation against spinal cord ischemia/reperfusion injury in rats

BACKGROUND: The majority of studies addressing spinal cord ischemia/reperfusion injury (SCIRI) have focused on drugs, proteins, cytokines, and various surgical techniques. A recent study reports that human umbilical cord mesenchymal stem cell (hUCMSC) transplantation achieves good therapeutic effects, but the mechanisms underlying nerve protection remain poorly understood.OBJECTIVE: To observe survival of transplanted hUCMSCs in SCIRI rat models and the influence on motor function in the hind limbs, to determine interleukin-8 expression and cellular apoptosis in spinal cord tissues, and to verify the hypothesis that hUCMSC transplantation exhibits protective effects on SCIRI.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Laboratory of the Department of Orthopedics in the First Affiliated Hospital of Soochow University,China between January 2007 and December 2008.MATERIALS: hUCMSCs were harvested from umbilical cord blood of healthy pregnant women after parturition in the Obstetrical Department of the First Affiliated Hospital of Soochow University, China. Rabbit anti-human BrdU monoclonal antibody was provided by DAKO, USA. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kit and enzyme-linked immunosorbent assay (ELISA) Kit were purchased by Wuhan Boster, China. METHODS: A total of 72 healthy, Wistar, adult rats were randomly assigned to three groups: sham-surgery, model, and transplantation, with 24 rats in each group. SCIRI was induced in the model and transplantation groups via the abdominal aorta block method. The inf rarenal abdominal aorta was not blocked in the sham-surgery group. Prior to abdominal aorta occlusion, 0.2-0.3 mL bromodeoxyuridine (BrdU)-labeled hUCMSCs suspension (cell concentration 5 × 10~3/μL) was injected through the great saphenous vein of the hind limb, and an equal volume of physiological saline was administered to the model and sham-surgery groups.MAIN OUTCOME MEASURES: Pathological observation of rat spinal cord tissues was performed by hematoxylin-eosin staining at 6, 24, and 48 hours post-surgery. Immunohistochemistry was applied to determine hUCMSCs survival in the spinal cord. The amount of cellular apoptosis and interleukin-8 expression in spinal cord tissues was assayed utilizing the TUNEL and ELISA methods, respectively. Motor function in the hind limbs was evaluated according to Jacob's score. RESULTS: Numerous BrdU-positive cells were observed in spinal cord tissues from the transplantation group. The number of apoptotic cells and interleukin-8 levels significantly decreased in the transplantation group (P < 0.05), pathological injury was significantly ameliorated, and motor function scores significantly increased (P < 0.05) compared with the model group. CONCLUSION: Via vein transplantation, hUCMSCs were shown to reach and survive in the injury area. Results suggested that the transplanted hUCMSCs contributed to significantly improved pathological changes in the injured spinal cord, as well as motor function, following SCIRI. The protective mechanism correlated with inhibition of cellular apoptosis and reduced production of inflammatory mediators.     

Comparison of the effects of octreotide and melatonin in preventing nerve injury in rats with experimental spinal cord injury.

In this study, we aimed to investigate the biochemical and histopathological protective effects of octreotide and melatonin in an experimental model of spinal cord injury. Fifty- six male albino Wistar rats were divided into four groups. Rats in the G1 group (n=7; control group) did not undergo any treatment except for anesthesia prior to being killed. Rats in the G2 group (n=7) underwent laminectomy and aneurysmal clip application at the T4-5 level. G3 group rats (n=14) were either treated with a 7.5 mg/kg intraperitoneal dose of melatonin (Sigma, St. Louis, MO, USA) immediately after laminectomy, then the same dose again on the day following injury (G3a), or given three equal doses over 10 days to achieve a total dose of 7.5 mg/kg/day (G3b). G4 group rats (n=14) were either treated with a 30microg/kg intraperitoneal dose of octreotide (Sandostatin; Novartis, Istanbul, Turkey) immediately after laminectomy, then the same dose again on the day following injury (G4a), or given three equal doses over 10 days to achieve a total dose of 30miocrog/kg/day (G4b). Rats in the G3 and G4 groups were sacrificed on days 1 and 10 after spinal cord injury (n=7 at each time point) and spinal cord samples were obtained. Tissue malonyldialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels were assayed. G3a, G3b and G4b had significantly lower levels of MDA than G2 (p<0.01). G3b had significantly higher SOD and GSH-Px levels than G2 (p<0.01). Histopathologically, melatonin significantly reduced necrosis and degeneration in both the initial and late stages (p<0.01). Octreotide had significant effects on necrosis and degeneration during the late stages, and on edema and congestion in both the initial and the late stages of injury (p<0.01). Melatonin was found to be superior to octreotide with respect to the prevention of congestion, edema, axonal degeneration and necrosis.     

Effects of progesterone on experimental spinal cord injury

Progesterone has been proposed to be protective to the central nervous system following injury. This study assessed progesterone supplementation in the setting of contusional spinal cord injury in male and female rats. Short-term (5 days of either 4 or 8 mg/kg progesterone) and long-term (14 days of either 8 or 16 mg/kg progesterone) therapy failed to show any significant alteration in locomotor functioning and injury morphometrics after 21 days. This study does not support progesterone as a potential therapeutic agent in spinal cord injury.     

Vascular permeability in experimental spinal cord injury

Following spinal cord injury in rats there was a time-dependent change of vascular permeability as reflected by extravasation of ¹²⁵I-labelled serum albumin. The change of vascular permeability correlated with tissue calcium and water accumulation suggesting that cord exposure to plasma calcium as a consequence of vascular injury may contribute to the progressive post-traumatic cord necrosis.     

Antioxidant therapy in experimental spinal cord injury

We have tested the capacity of several compounds with in vitro and/or in vivo antioxidant or antilipolytic activity to ameliorate locomotor function in cats subjected to static loading (i.e. compression) injury of the spinal cord. These include the synthetic glucocorticoid, methylprednisolone sodium succinate (MP), and the new 21-aminosteroid antioxidant, U74006F. Treatment of spinal cord-injured cats with high doses of MP promoted or spared locomotor function and preserved spinal cord tissue. Extending these findings in cats to humans, it was recently demonstrated that high doses of MP administered within 8 h of injury significantly improved neurologic recovery in human spinal cord-injured patients. The compound U74006F is one of a series of 21-aminosteroids that, unlike MP, lack glucocorticoid, mineralocorticoid or other hormonal activity yet are potent inhibitors of lipid perioxidation. Over a 100-fold range of doses, U74006F promoted recovery of locomotor function in spinal cord-injured cats. The lowest effective dose for U74006F was 100 times lower than the maximally effective dose for MP. The efficacy of U74006F is unchanged if treatment is initiated within 4 h of injury. However, if treatment is delayed for 8 h, the therapeutic potency of U74006F is substantially reduced. These findings suggest that antioxidant therapy can successfully limit the effects of both experimental and clinical spinal cord injury especially if the treatment is initiated shortly after injury.     

甲基强的松龙对急性脊髓损伤神经元保护作用的实验研究

目的探讨甲基强的松龙对急性脊髓损伤(ASCI)后神经元是否具有保护作用。方法大鼠随机分为2组，即模型组和正常对照组(N组)，模型组建立脊髓半横断损伤模型后又分两组，即激素治疗组(M组)，腹腔注射甲基强的松龙(MP)；模型对照组(B组)术后不做处理。每组大鼠观察损伤后3d、7d、15d和30d。取脊髓损伤部位标本做光镜病理组织学检查，观察正常神经元和变性神经元的数量变化。结果(1)B组在ASCI早期脊髓损伤区的灰白质被明显的破坏，有出血及坏死，可见多量空泡状细胞和溃变的神经纤维，部分组织液化。在损伤灶内见正常神经元及神经纤维的数量稀少，多数神经元呈现不同程度的变性乃至坏死。随着时间的延长，神经元数量进一步减少，胶质细胞增生形成疤痕，或者液化形成囊腔。病变常常累及对侧组织，而M组的脊髓损伤区组织结构的变化与B组基本相同。(2)在ASCI后3d，M组的正常神经元数量少于B组，变性神经元数量与B组相近；在损伤7d以后，随着时间的延长，B组的变性神经元数量减少的同时，正常神经元的数量稍有减少，而M组的变性神经元数量减少的同时，正常神经元数量有所增加。结论(1)MP对ASCI后的继发性组织结构破坏无明显的改善作用；(2)MP在ASCI早期能促使神经元的死亡，但在后期能增加正常神经元的数量，其机理有待于进一步研究。     

Proteolytic enzymes in experimental spinal cord injury

Experimental spinal cord injury was produced in rats by dropping a 10 g weight from 30 cm upon dura-invested exposed spinal cord. Proteolytic activities at neutral (pH 7.6) and acid (pH 5.5 and 3.6) pH were determined in whole homogenate and the cytosolic fraction of the lesion (lumbar) and cervical control segments. The enzyme activity was monitored by SDS-PAGE analysis of the extent of substrate myelin basic protein (MBP) degradation. Activities (neutral and cathepsin B-like) in the sham-operated spinal cord were lower than those of cervical autologous control at 24 h after injury. The increase in neutral proteinase activity was progressive and greater in the lesion than the autologous control. A 61.5% +/- 3.5 loss of MBP was observed at 2 h following injury and increased at 24 h (78.2% +/- 3.4). The loss of MBP coincided with the appearance of several low molecular weight peptides. The cathepsin B-like and cathepsin D activities were also increased in the lesion but to a lesser extent than the neutral proteinase. The neutral proteinase and cathepsin B-like activity were inhibited by leupeptin and not by pepstatin while the converse obtained for cathepsin D activity. The release of neutral proteolytic activity which is nonlysosomal in origin suggests a novel hypothesis for the mechanism of traumatic axon-myelin injury.