Spinal cord decompression reduces rat neural cell apoptosis secondary to spinal cord injury

Spinal cord decompression reduces rat neural cell apoptosis secondary to spinal cord injury 1 first-author †E-mail:xukan@medmail.com.cn †E-mail:xukan@medmail.com.cn xukan@medmail.com.cn 1 1 1 2 1 Department of Orthopaedic Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Orthopaedic Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Pathology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Pathology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Objective: 2 To determine whether spinal cord decompression plays a role in neural cell apoptosis after spinal cord injury. Study design: We used an animal model of compressive spinal cord injury with incomplete paraparesis to evaluate neural cell apoptosis after decompression. Apoptosis and cellular damage were assessed by staining with terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and immunostaining for caspase-3, Bcl-2 and Bax. Methods: 2 Experiments were conducted in male Sprague-Dawley rats ( n =78) weighing 300~400 g. The spinal cord was compressed posteriorly at T10 level using a custom-made screw for 6 h, 24 h or continuously, followed by decompression by removal of the screw. The rats were sacrificed on Day 1 or 3 or in Week 1 or 4 post-decompression. The spinal cord was removed en bloc and examined at lesion site, rostral site and caudal site (7.5 mm away from the lesion). Results: 2 The numbers of TUNEL-positive cells were significantly lower at the site of decompression on Day 1, and also at the rostral and caudal sites between Day 3 and Week 4 post-decompression, compared with the persistently compressed group. The numbers of cells between Day 1 and Week 4 were immunoreactive to caspase-3 and B-cell lymphoma-2 (Bcl-2)-associated X-protein (Bax), but not to Bcl-2, correlated with those of TUNEL-positive cells. Conclusion: 2 Our results suggest that decompression reduces neural cell apoptosis following spinal cord injury. Spinal cord injury Decompression Apoptosis Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) Caspase-3 B-cell lymphoma-2 (Bcl-2) Bcl-2-associated X-protein (Bax) INTRODUCTION 1 Recent reports have shown that apoptosis occurring secondary to spinal cord injury (SCI) contributes to further neural damage and functional loss following spinal cord trauma (Crowe et al., 1997 1997 ; Shuman et al., 1997 1997 ; Emery et al., 1998 1998 ). Apoptosis is determined by a genetic program, dependent on active protein synthesis, and characterized by nuclear fragmentation and appearance of apoptotic bodies seen as small basophilic material within the nucleus or as basophilic material extruded from the cell within the blebs of the cytoplasm (Liu et al., 1997 1997 ). The neurological status after spinal cord trauma primarily depends on the extent of neural element loss and function of the residual neural tissue. Hence, it is important to reduce any secondary neural damage, including apoptosis, which occurs after the initial traumatic SCI. Several experimental studies have suggested that decrement of neural cell apoptosis plays an important role in the neurological recovery process following incomplete SCI (Wada et al., 1999 1999 ; Springer et al., 1999 1999 ; Saito et al., 2000 2000 ). There is compelling evidence from laboratory studies in animal models that persistent compression of the spinal cord is a potentially reversible form of secondary injury. The severity of SCI in animal models is related to the force and duration of compression, displacement, impulse, and kinetic energy (Dolan et al., 1980 1980 ; Guha et al., 1987 1987 ; Panjabi, 1987 1987 ; Turker et al., 2005 2005 ; Kasahara et al., 2006 2006 ). Numerous experimental studies of decompression after SCI have been performed in various animal models (Aki and Toya, 1984 1984 ; Nyström and Berglund, 1988 1988 ; Delamarter et al., 1995 1995 ; Carlson et al., 1997 1997 ; Baydin et al., 2007 2007 ). These studies have consistently shown that neurologic recovery is enhanced by early decompression, while there are few reports about the role of the decompression concerning with the dynamic change of the apoptosis after SCI. Dimar et al.( 1999 1999 ) used the New York University (NYU) weight drop model to produce thoracic SCI in rats and an epidural spacer placed adjacent to the contusion to mimic the effect of persisting compression. Neurologic recovery was significantly dependent on time to decompression, with significant differences seen in all experimental groups. This study provides the strongest experimental evidence to date of a clear beneficial effect of spinal cord decompression after SCI. It is not so easy to control the spacer in the right position above the dura matter. So, another new mechanical compressive model should be established in order to observe the histopathology after the spinal cord decompression. To investigate the neural cell apoptosis after decompression, we extended these studies by examining the histopathological effect of spinal cord compression and decompression in a rat model. Thus, in the present experimental study, we used a rat SCI model with acute spinal cord compression/decompression to investigate the dynamic changes in the populations of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL)-, caspase-3-, B-cell lymphoma-2 (Bcl-2)- and Bcl-2-associated X-protein (Bax)-positive cells, representing apoptotic cells, within the spinal cord after compression/decompression. MATERIALS AND METHODS 1 Experimental animals and surgical preparation 2 The experimental study was conducted in 78 Sprague-Dawley rats weighing 300~400 g [(350±30) g]. Following anaesthesia by an intraperitoneal injection of sodium pentobarbiturate (0.05 mg/g body weight), the rats were fixed prone on a surgical table. A limited laminectomy was performed at T10 level to expose the dura mater and the spinal cord, using a micro-airtome under a surgical microscope without damaging the spinal cord. A custom-made mini-screw (2 mm in diameter, 2 mm long, with a flat and smooth screw tip) was screwed slowly and vertically on the midline spinal cord for 1-mm depth through the dura mater (Fig. 1a 1a ). The rats were then divided into three subgroups: Group A ( n =24), decompression at 6 h after compression; Group B ( n =24), decompression at 24 h after compression; Group C ( n =24), persistent compression. Following the compression, the surgical wound was closed. In Groups A and B, 6 and 24 h later, respectively, the rats were re-anaesthetised to remove the screw inserted into the spinal cord. The wound was sutured in layers, while in rats of Group C, the screw was kept on the dura mater for persistent compression. The remaining six rats that suffered from the laminectomy at the same level served as controls ( n =6). The animal was then allowed to recover from anaesthesia and housed under a 12 h-12 h light-dark cycle in a bio-clean room with free per os access to water and food. All surgeries and animal care strictly followed the Guidelines and Policies for Rodent Survival Surgery of the University (School of Medicine, Zhejiang University, China). Immunohistochemical studies 2 Rats that were subjected to decompression and persistent compression were anaesthetised and then sacrificed on Day 1 or 3, or in Week 1 or 4 ( n =6 at each time interval) using intracardiac perfusion with normal saline followed by 4% (w/v) paraformaldehyde in phosphate buffered saline (PBS) at pH 7.4. For histological evaluation, a 15-mm spinal cord segment with the site of injury at its centre was cut and placed overnight in the same fixative in a refrigerator at 4 °C, and further embedded in paraffin. Transverse sections (4 μm thick) were serially prepared from the lesion centre, 7.5 mm rostral and caudal from the centre (Fig. 1b 1b ). TUNEL staining 2 Paraffin-embedded cord sections were deparaffinized in two changes of xylene for 5 min each. The sections were rinsed sequentially in 100%, 95% and 75% ethanol before being incubated with 20 g/ml proteinase K (Roche Applied Science, Penzberg, Germany) for 5 min to strip off nuclear proteins. TUNEL was performed using in situ cell death detection kit (Lot No. 1-684-817, Roche Applied Science, USA). After rinsing in PBS twice for 10 min, the area around the sample was dried, and the sections were incubated with the TUNEL reaction mixture for 60 min at 37 °C in a humidified atmosphere in the dark. The slides were rinsed three times in PBS to stop the reaction. The sections were colorized with horseradish peroxidase after incubation with a secondary anti-fluorescein-peroxidase-conjugate (Converter-peroxidase) in a humidified chamber for 30 min at 37 °C, and then the sections were counterstained with haematoxylin. Caspase-3, Bcl-2, and Bax staining 2 The paraffin-embedded spinal cord sections were deparaffinized using a standard procedure described above before rinsing in PBS three times for 10 min. After aspirating the excess liquid from the slides, the sections were incubated for 10 min in 0.1% (w/v) hydrogen peroxidase in methanol to quench endogenous peroxidase activity. The slides were rinsed twice in PBS each for 5 min and then incubated in normal non-specific goat serum for 10 min at room temperature, followed by overnight incubation with primary antibodies (caspase-3, Bcl-2, and Bax; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C. The sections were then rinsed in PBS three times each for 5 min, incubated with biotin-conjugated secondary antibody for 30 min, again rinsed in PBS for three times each for 5 min, and incubated with avidin biotin enzyme reagent for 30 min. The sections were colorized with 3,3′-diaminobenzidine tetrahydrochloride in PBS at pH 7.4 containing 0.01% (v/v) H 2 O 2 , immersed in PBS to stop the reaction when the desired stain intensity appeared, counterstained with haematoxylin, and mounted according to the standard procedures. Neural cell count 2 Three individuals including a pathologist not participating in the study counted the number of neural cells labelled by TUNEL staining in the posterior column (pc), lateral column (lc), anterior column (ac), anterior horns (ah), and posterior horns (ph) at three axial levels: Site a, the level 7.5 mm rostral to the centre of the lesion site; Site b, the centre of the lesion site; and Site c, the level 7.5 mm caudal to the centre of the lesion site. All sections were examined under an Olympus BX51 microscope (Olympus, Tokyo, Japan) and photographed by a DP11 digital camera (Olympus). The number of positive cells in white matters (WM) was calculated as the sum of pc, lc and ac, and in grey matters (GM) as the sum of ah and ph, respectively. Statistical analysis 2 Data are expressed as mean± SD . The numbers of positive cells in the WM and GM were compared among the three experimental groups (decompression at 6 h, 24 h and persistent compression) using one-way analysis of variance (ANOVA) (Bonferroni’s method) with the SPSS 11.0 version software on an IBM Thinkpad 2672GCC computer (Armonk, NY, USA). Differences were considered statistically significant at the level of P < 0.05. RESULTS 1 Histopathology of the sham controls 2 There were no histopathological abnormalities in sham-operated rats. The TUNEL-, caspase-3-, Bcl-2- and Bax-positive cells were sparse in both the GM and the WM. The topographic distribution of these cells was similar at the rostral site (Site a), lesion center (Site b) and caudal site (Site c). Topographic distribution of TUNEL-positive cells 2 Fig. 2 2 shows that the changes in the distribution of TUNEL-positive cells at Sites a, b and c from Day 1 to Week 4 after decompression in Groups A, B and C. In general, there were some similar changes among these three groups. The number of TUNEL-positive cells increased on Day 1 after decompression at Site b, but there were only a few such cells at Sites a and c at that time. From Day 3 to Week 4 after decompression, the number of TUNEL-positive cells decreased gradually at Site b, while they tended to increase at Sites a and c in the WM of all three groups. The peak numbers of TUNEL-positive cells in GM area at Site b were noted on Day 1 after decompression, while the peak numbers of TUNEL-positive cells in WM at Sites a and c were in Week 1 after decompression. In Week 4 after decompression, the TUNEL-positive cells were still detected at all the three sites in all the three groups but their numbers decreased. We also found that the number of the TUNEL-positive cells in the sham controls was so small, so we did not put them into the statistical analysis. On Day 1 after decompression, a significant decrease of TUNEL-positive cells was observed at WM and GM areas of Site b ( P =0), and a significant reduction of these cells was still noted at this site on Day 3 post-decompression, and especially by 6 h after decompression ( P =0.03). On the other hand, a significant decrease of TUNEL-positive cells was observed at WM and GM areas at Site c ( P =0.01). In Week 1 after decompression, a significant reduction of TUNEL-positive cells was still noted at Site b ( P =0.02), while a significant decrease of these cells was observed in the WM of the peripheral sites, especially by 6 h after decompression ( P =0 at Site a; P =0.01 at Site c). In Week 4 after decompression, a significant reduction of TUNEL-positive cells was observed at Site b ( P =0.02), as well as at Sites a ( P =0.01) and c ( P =0.03) (Fig. 3 3 ). Caspase-3, Bcl-2 and Bax immunohistochemistry 2 We also stained the spinal cord tissues for caspase-3, Bcl-2 and Bax to confirm the TUNEL findings. Changes in immunoreactivity were examined in the GM between Day 1 and Week 4 after injury. In general, the numbers of caspase-3- and Bax-positive cells were similar to those of TUNEL-positive cells. In contrast, a reverse pattern was found with regard to the number of Bcl-2-positive cells. Fig. 4 4 shows a typical example of staining for TUNEL, caspase-3, Bcl-2, and Bax in Week 4 after the injury in the anterior horn of Site c of Groups A, B, and C. At that stage, the numbers of TUNEL-, caspase-3-, and Bax-positive cells were significantly lower in Groups A and B than in Group C. However, the numbers of Bcl-2-positive cells were significantly higher in Groups A and B. DISCUSSION 1 Apoptosis in traumatic spinal cord injury 2 Recent studies have shown that apoptosis plays an important role in determining neurological outcome following SCI (Crowe et al., 1997 1997 ; Emery et al., 1998 1998 ). It is well known that neural cell loss in SCI occurs both at the time of injury and secondarily over a period of days to weeks after the traumatic event. Neuronal cell death occurs mainly by necrosis; however, it continues throughout a much more extensive axial section in the spinal cord WM for a period of up to several weeks in a process referred as secondary injury (Katoh et al., 1996 1996 ; Crowe et al., 1997 1997 ; Liu et al., 1997 1997 ; Springer et al., 1999 1999 ; Saito et al., 2000 2000 ). In these studies, visible signs of oligodendrocyte apoptosis appear within 24 h and continue for at least 3 weeks after injury. A histological study of human SCI reported the existence of apoptotic oligodendrocytes from 3 h to at least 8 weeks after the injury (Emery et al., 1998 1998 ). In the present study, we investigated apoptosis with special references to cellular morphology, nuclear chromatin staining with the TUNEL method, and immunoreactivity of three apoptosis-related proteins (caspase-3, Bcl-2, and Bax). We found that on Days 1 and 3 after spinal cord injury, the number of TUNEL-positive cells was high at the lesion site, whereas only a few of these cells were found at the sites rostral and caudal to the lesion. However, in Week 1 after injury, the number of TUNEL-positive cells decreased at the lesion site, but increased at the peripheral sites. In Week 4 post-SCI, the number of TUNEL-positive cells diminished at the rostral and caudal sites especially in the WM. These findings are in agreement with those of previous studies (Li et al., 1996 1996 ; Liu et al., 1997 1997 ), reflecting the natural history of the pathophysiology of SCI, and suggest that changes in the number of TUNEL-positive cells during SCI are related to apoptosis. The process of apoptosis is complex and involves the upregulation of numerous key molecules (Springer et al., 1999 1999 ). Therapeutic benefits may be achieved by inhibiting such upregulation to prevent apoptosis. Apoptosis secondary to spinal cord injury and effect of decompression 2 It is still not clear whether decompression surgery reduces neural cell apoptosis secondary to SCI. In the present study, we found that the number of TUNEL-positive cells in the lesion site was significantly reduced by decompression on Day 1 and Day 3, and in Week 1 and Week 4 after injury, compared with that of persistently-compressive group. Moreover, on Day 3 and in Week 1 and Week 4 after injury, the numbers of TUNEL-positive cells at the rostral and caudal sites were significantly lower in the spinal cord decompressed rats compared with that of the persistently-compressed rats. These findings suggest that decompression surgery suppresses the onset of neural cell apoptosis secondary to SCI. However, a serious drawback of TUNEL staining is that not all TUNEL-positive cells are apoptotic cells; DNA fragmentation also occurs in accidental cell death process. Thus, we studied caspase-3, Bcl-2 and Bax immunoreactivities in the GM. Our results confirm that the immunoreactivities of caspase-3 and Bax correlated with the changes noted in TUNEL-positive cells, indicating that the observed cellular changes reflect apoptosis of neural cells. The genetic and environmental factors that trigger neuronal apoptosis may be different in various physiological and pathological settings. The biochemical alterations that occur during the early stages of apoptosis may induce mitochondrial dysfunction either directly or indirectly. The Bcl-2 family of proteins includes both pro- and anti-apoptotic members (Pellegrini and Strasser, 1999 1999 ). The best-studied anti-apoptotic member in neurons is Bcl-2, and pro-apoptotic members include Bax- and Bcl-associated death promoter. Previous studies showed that overexpression of Bcl-2 in cell cultures and in transgenic mice increases resistance of neurons to death induced by excitotoxic, metabolic and oxidative insults relevant to stroke and other disorders (Martinou et al., 1994 1994 ). Conversely, neurons lacking Bax are protected against apoptosis (White et al., 1998 1998 ). Other mechanisms that regulate the early stages of apoptosis include various caspases, the prostate apoptosis response-4 protein and telomerase. Some caspases are activated during the early phase of apoptosis, such as caspase-3. Activation of caspase-3 enhances the cleavage of DNA, nuclear lamins, cytoskeletal components and proteins that inhibit apoptosis (Hengartner, 2000 2000 ). While several mechanisms could explain the effect of decompression on the recovery of SCI, we demonstrated in the present study that decompression results in reduction of TUNEL-positive cells following SCI, especially in the early period following decompression (6 h). This decompression-related mechanism might positively improve the clinical outcome after SCI. CONCLUSION 1 The beneficial effects of decompression of the mechanically-injured spinal cord may reflect, at least in part, changes in the apoptotic cell population at the site of the lesion. Surgical decompression may have favourable effects on the outcome of SCI by reducing the number of apoptotic cells at the lesion site as well as the surrounding areas. References T Aki , S Toya . Experimental study on changes of the spinal-evoked potential and circulatory dynamics following spinal cord compression and decompression . Spine , 1984 . 9 ( 8 ): 800 - 809 . 6528294 DOI: 10.1097/00007632-198411000-00006 http://doi.org/10.1097/00007632-198411000-00006 . Experimental study on changes of the spinal-evoked potential and circulatory dynamics following spinal cord compression and decompression A Baydin , C Cokluk , K Aydin . A new minimally invasive experimental spinal cord injury model in rabbits . Minim Invasive Neurosurg , 2007 . 50 ( 3 ): 170 - 172 . 17882754 DOI: 10.1055/s-2007-985375 http://doi.org/10.1055/s-2007-985375 . A new minimally invasive experimental spinal cord injury model in rabbits GD Carlson , Y Minato , A Okada , CD Gorden , KE Warden , JM Barbeau , CL Biro , E Bahnuik , HH Bohlman , JC Lamanna . Early time-dependent decompression for spinal cord injury: vascular mechanisms of recovery . J Neurotrauma , 1997 . 14 ( 12 ): 951 - 962 . 9475376 DOI: 10.1089/neu.1997.14.951 http://doi.org/10.1089/neu.1997.14.951 . Early time-dependent decompression for spinal cord injury: vascular mechanisms of recovery MJ Crowe , JC Bresnahan , SL Shuman , JN Masters , MS Beattie . Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys . Nat Med , 1997 . 3 ( 1 ): 73 - 76 . 8986744 DOI: 10.1038/nm0197-73 http://doi.org/10.1038/nm0197-73 . Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys RB Delamarter , J Sherman , JB Carr . Pathophysiology of spinal cord injury: recovery after immediate and delayed decompression . J Bone Joint Surg Am , 1995 . 77 ( 7 ): 1042 - 1049 . 7608226 . Pathophysiology of spinal cord injury: recovery after immediate and delayed decompression JR Dimar , SD Glassman , GH Raque , YP Zhang , CB Shields . The influence of spinal canal narrowing and timing of decompression on neurologic recovery after spinal cord contusion in a rat model . Spine , 1999 . 24 ( 16 ): 1623 - 1633 . 10472095 DOI: 10.1097/00007632-199908150-00002 http://doi.org/10.1097/00007632-199908150-00002 . The influence of spinal canal narrowing and timing of decompression on neurologic recovery after spinal cord contusion in a rat model EJ Dolan , CH Tator , L Endrenyi . The value of decompression for acute experimental spinal cord compression injury . J Neurosurg , 1980 . 53 ( 6 ): 749 - 755 . 7441334 . The value of decompression for acute experimental spinal cord compression injury E Emery , P Aldana , MB Bunge , W Puckett , A Srinivasan , RW Keane , J Bethea , AD Levi . Apoptosis after traumatic human spinal cord injury . J Neurosurg , 1998 . 89 ( 6 ): 911 - 920 . 9833815 . Apoptosis after traumatic human spinal cord injury A Guha , CH Tator , L Endrenyi , I Piper . Decompression of the spinal cord improves recovery after acute experimental spinal cord compression injury . Paraplegia , 1987 . 25 ( 4 ): 324 - 339 . 3627821 . Decompression of the spinal cord improves recovery after acute experimental spinal cord compression injury MO Hengartner . The biochemistry of apoptosis . Nature , 2000 . 407 ( 6805 ): 770 - 776 . 11048727 DOI: 10.1038/35037710 http://doi.org/10.1038/35037710 . The biochemistry of apoptosis K Kasahara , T Nakagawa , T Kubota . Neuronal loss and expression of neurotrophic factors in a model of rat chronic compressive spinal cord injury . Spine , 2006 . 31 ( 18 ): 2059 - 2066 . 16915089 DOI: 10.1097/01.brs.0000231893.21964.f2 http://doi.org/10.1097/01.brs.0000231893.21964.f2 . Neuronal loss and expression of neurotrophic factors in a model of rat chronic compressive spinal cord injury K Katoh , T Ikata , S Katoh , Y Hamada , K Nakauchi , T Sano , M Niwa . Induction and its spread of apoptosis in rat spinal cord after mechanical trauma . Neurosci Lett , 1996 . 216 ( 1 ): 9 - 12 . 8892379 DOI: 10.1016/0304-3940(96)12999-2 http://doi.org/10.1016/0304-3940(96)12999-2 . Induction and its spread of apoptosis in rat spinal cord after mechanical trauma GL Li , G Brodin , M Farooque , K Funa , A Holtz , WL Wang , Y Olsson . Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord . J Neuropathol Exp Neurol , 1996 . 55 ( 4 ): 280 - 289 . 8786386 DOI: 10.1097/00005072-199603000-00003 http://doi.org/10.1097/00005072-199603000-00003 . Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord XZ Liu , XM Xu , R Hu , C Du , SX Zhang , JW McDonald , HX Dong , YJ Wu , GS Fan , MF Jacquin , 等 . Neuronal and glial apoptosis after traumatic spinal cord injury . J Neurosci , 1997 . 17 ( 14 ): 5395 - 5406 . 9204923 . Neuronal and glial apoptosis after traumatic spinal cord injury JC Martinou , M Dubois-Dauphin , JK Staple , I Rodriguez , H Frankowski , M Missotten , P Albertini , D Talabot , S Catsicas , C Pietra . Overexpression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia . Neuron , 1994 . 13 ( 4 ): 1017 - 1030 . 7946326 DOI: 10.1016/0896-6273(94)90266-6 http://doi.org/10.1016/0896-6273(94)90266-6 . Overexpression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia B Nyström , JE Berglund . Spinal cord restitution following compression injuries in rats . Acta Neurol Scand , 1988 . 78 ( 6 ): 467 - 472 . 3223233 DOI: 10.1111/j.1600-0404.1988.tb03689.x http://doi.org/10.1111/j.1600-0404.1988.tb03689.x . Spinal cord restitution following compression injuries in rats MM Panjabi . Experimental spinal cord trauma: a biomechanical viewpoint . Paraplegia , 1987 . 25 ( 3 ): 217 - 220 . 3601430 . Experimental spinal cord trauma: a biomechanical viewpoint M Pellegrini , A Strasser . A portrait of the Bcl-2 protein family: life, death, and the whole picture . J Clin Immunol , 1999 . 19 ( 6 ): 365 - 377 . 10634210 DOI: 10.1023/A:1020598632068 http://doi.org/10.1023/A:1020598632068 . A portrait of the Bcl-2 protein family: life, death, and the whole picture N Saito , T Yamamoto , T Watanabe , Y Abe , T Kumagai . Implications of p53 protein expression in experimental spinal cord injury . J Neurotrauma , 2000 . 17 ( 2 ): 173 - 182 . 10709875 DOI: 10.1089/neu.2000.17.173 http://doi.org/10.1089/neu.2000.17.173 . Implications of p53 protein expression in experimental spinal cord injury SL Shuman , JC Bresnahan , MS Beattie . Apoptosis of microglia and oligodendrocytes after spinal cord contussion in rats . J Neurosci Res , 1997 . 50 ( 5 ): 798 - 808 . 9418967 DOI: 10.1002/(SICI)1097-4547(19971201)50:5<798::AID-JNR16>3.0.CO;2-Y http://doi.org/10.1002/(SICI)1097-4547(19971201)50:5<798::AID-JNR16>3.0.CO;2-Y . Apoptosis of microglia and oligodendrocytes after spinal cord contussion in rats JE Springer , RD Azbill , PE Knapp . Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury . Nat Med , 1999 . 5 ( 8 ): 943 - 946 . 10426320 DOI: 10.1038/11387 http://doi.org/10.1038/11387 . Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury M Turker , G Tezeren , M Tukenmez , S Percin . Indirect spinal canal decompression of vertebral burst fracture in calf model . Arch Orthop Trauma Surg , 2005 . 125 ( 5 ): 336 - 341 . 15843943 DOI: 10.1007/s00402-005-0816-4 http://doi.org/10.1007/s00402-005-0816-4 . Indirect spinal canal decompression of vertebral burst fracture in calf model S Wada , K Yone , Y Ishidou , T Nagamine , S Nakahara , T Niiyama , T Sakou . Apoptosis following spinal cord injury in rats and preventative effect of N -methyl-D-aspartate receptor antagonist . J Neurosurg Spine , 1999 . 91 ( 1 Suppl. ): 98 - 104 . . Apoptosis following spinal cord injury in rats and preventative effect of N-methyl-D-aspartate receptor antagonist FA White , CR Keller-Peck , CM Knudson , SJ Korsmeyer , WD Snider . Widespread elimination of naturally occurring neuronal death in Bax-deficient mice . J Neurosci , 1998 . 18 ( 4 ): 1428 - 1439 . 9454852 . Widespread elimination of naturally occurring neuronal death in Bax-deficient mice 10.1631/jzus.B0820161 Project (No. Y207216) supported by the Natural Science Foundation of Zhejiang Province, China 2008-05-13 2009-01-20 2008-09-23 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 10