Spinal Cord Ischemia In Rabbits Research Articles

Cinnamaldehyde has ameliorative effects on rabbit spinal cord ischemia and reperfusion injury

Cinnamaldehyde has ameliorative effects on rabbit spinal cord ischemia and reperfusion injury

Necrostatin-1 Attenuates Delayed Paraplegia after Transient Spinal Cord Ischemia in Rabbits by Inhibiting the Upregulation of Receptor-Interacting Protein Kinase 1 and 3

Delayed-onset paraplegia is a disastrous complication after thoracoabdominal aortic open surgery and thoracic endovascular aortic repair. Studies have revealed that transient spinal cord ischemia caused by temporary occlusion of the aorta induces delayed motor neuron death owing to apoptosis and necroptosis. Recently, necrostatin-1 (Nec-1), a necroptosis inhibitor, has been reported to reduce cerebral and myocardial infarction in rats or pigs. In this study, we investigated the efficacy of Nec-1 in delayed paraplegia after transient spinal cord ischemia in rabbits and assessed the expression of necroptosis- and apoptosis-related proteins in motor neurons. This study used rabbit transient spinal cord ischemia models using a balloon catheter. They were divided into a vehicle-treated group (n=24), Nec-1-treated group (n=24), and sham-controls (n=6). In the Nec-1-treated group, 1mg/kg of Nec-1 was intravascularly administered immediately before ischemia induction. Neurological function was assessed using the modified Tarlov score, and the spinal cord was removed 8hr and 1, 2, and 7days after reperfusion. Morphological changes were examined using hematoxylin and eosin staining. The expression levels of necroptosis-related proteins (receptor-interacting protein kinase [RIP] 1 and 3) and apoptosis-related proteins (Bax and caspase-8) were assessed using western blotting and histochemical analysis. We also performed double-fluorescence immunohistochemical studies of RIP1, RIP3, Bax, and caspase-8. Neurological function significantly improved in the Nec-1-treated group compared with that in the vehicle-treated group 7days after reperfusion (median 3 and 0, P=0.025). Motor neurons observed 7days after reperfusion were significantly decreased in both groups compared with the sham group (vehicle-treated, P<0.001; Nec-1-treated, P<0.001). However, significantly more motor neurons survived in the Nec-1-treated group than in the vehicle-treated group (P<0.001). Western blot analysis revealed RIP1, RIP3, Bax, and caspase-8 upregulation 8hr after reperfusion in the vehicle-treated group (RIP1, P=0.001; RIP3, P=0.045; Bax, P=0.042; caspase-8, P=0.047). In the Nec-1-treated group, the upregulation of RIP1 and RIP3 was not observed at any time point, whereas that of Bax and caspase-8 was observed 8hr after reperfusion (Bax, P=0.029; caspase-8, P=0.021). Immunohistochemical study revealed the immunoreactivity of these proteins in motor neurons. Double-fluorescence immunohistochemistry revealed the induction of RIP1 and RIP3, and that of Bax and caspase-8, in the same motor neurons. These data suggest that Nec-1 reduces delayed motor neuron death and attenuates delayed paraplegia after transient spinal cord ischemia in rabbits by selectively inhibiting necroptosis of motor neurons with minimal effect on their apoptosis.

Cinnamaldehyde has ameliorative effects on rabbit spinal cord ischemia and reperfusion injury

Cinnamaldehyde has ameliorative effects on rabbit spinal cord ischemia and reperfusion injury

Therapeutic effects of syringaldehyde on spinal cord ischemia in rabbits.

Objectives:To investigate the effects of syringaldehyde (SA) on the antioxidant and oxidant system in spinal cord ischemia (SCI).Methods:These study and experiments were conducted at Medical Research Center, Çanakkale Onsekiz Mart University, Çanakkale, Turkey, between 2014-2018. Eighteen New Zealand White adult male rabbits were randomly divided into 3 groups (n=6). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), myeloperoxidase (MPO) activities, and malondialdehyde (MDA) levels were measured in the spinal cord tissues. Degenerated neurons, hemorrhage and inflammatory cell migration in the spinal cord were investigated histopathologically. Expressions of neuronal nitric oxide synthase (nNOS), caspase-3, and nuclear factor-κB (NF-κB) were evaluated immunohistochemically. Clinically, it was evaluated with Modified Tarlov score.Results:Biochemically, there was an expected decrease in SOD, CAT, and GPx enzyme activities in ischemia groups, there was also an increase in MPO activity at the same time. When the enzyme activities spinal cord ischemia/reperfusion (SCI/R)+SA, control and SCI/R groups were compared, the difference was found to be statistically significant (p<0.05). Glutathione peroxidase enzyme activity levels were very low in ischemia group compared to the significant increase in the SA group (p<0.05). Histopathologically, when SCI/R and SCI/R+SA groups were compared, there were statistically significant differences in the number of degenerative neurons and amount of hemorrhage; this comparison shows the significance of treatment in terms of inflammatory cell migration (p<0.05). The expressions of nNOS, caspase-3, and NF-κB were found significantly increased in SCI/R group compared to the control group (p<0.05). Syringaldehyde treatment decreased nNOS, caspase-3, and NF-κB expressions immunohistochemically. Clinical evaluation showed improvement in the SA-treated group.Conclusion:Syringaldehyde therapy administered for protective purposes may reduce oxidative stress, degenerative changes and inflammatory cell migration in the ischemic spinal cord.

The ameliorative effect of ozone therapy on spinal cord ischemia in rabbits

The ameliorative effect of ozone therapy on spinal cord ischemia in rabbits

Tat-HSP70 protects neurons from oxidative damage in the NSC34 cells and ischemic damage in the ventral horn of rabbit spinal cord

Heat shock protein 70 (HSP70) is an ATP-dependent molecular chaperone, and it has been shown that its levels increase after exposure to various types of stress, including ischemia. In the present study, we investigated the effects of HSP70 against H2O2-induced neuronal stress in NSC34 cells and against spinal cord ischemia in rabbits. Tat-HSP70 proteins facilitated the intracellular delivery of HSP70 into the NSC34 cells and enabled them to cross the blood-brain barrier in the rabbit spinal cord. Tat-HSP70 was effectively transduced into NSC34 cells in a concentration- and time-dependent manner, while control-HSP70 protein could not be delivered intracellularly at any concentration or time after treatment. Treatment with Tat-HSP70 reduced the generation of reactive oxygen species and cell death induced by H2O2, while the control-HSP70 did not show any significant effect on the NSC34 cells exposed to H2O2. In rabbit spinal cord, the administration of Tat-HSP70 showed significant amelioration of neurological defects and neuronal death in the ventral horn of spinal cord. In addition, Tat-HSP70 treatment significantly reduced lipid peroxidation and increased Cu, Zn-superoxide dismutase activities in the spinal cord, but glutathione peroxidase and Mn-superoxide dismutase activities remained unchanged. These results suggest that Tat-HSP70, not control-HSP70, decreases cell damage by reducing oxidative stress in NSC34 cells and rabbit spinal cord, and it can be employed for the reduction of neuronal damage caused after spinal cord ischemia.

Development of a modified model of spinal cord ischemia injury by selective ligation of lumbar arteries in rabbits.

Experimental study. The aim of this study is to develop a modified model of spinal cord ischemia in rabbits. Shenzhen Key Laboratory of Spine Surgery, Shenzhen, China. In total, 20 New Zealand rabbits were divided into the following four groups according to the level of ligation of bilateral lumbar arteries: (1) group A, sham group, no ligation, n=5; (2) group B, ligation of bilateral lumbar arteries at three levels (L2-L4, n=5); (3) group C, ligation of bilateral lumbar arteries at four levels (L2-L5, n=5); and (4) group D, ligation of bilateral lumbar arteries at five levels (L1-L5, n=5). The latency of motor-evoked potentials was measured intraoperatively and the modified Tarlov grades were scored, followed by a histological observation of spinal cord, on the seventh day after surgery. All 10 rabbits in Group A and Group B were electrophysiologically, neurologically and histologically normal. In Group C, moderate spinal cord ischemia injury was found in three of five rabbits: they had prolonged latency of motor-evoked potentials and neuronal karyopyknosis in the anterior horn of spinal cord, and the average Tarlov score was 4.2±0.8. In Group D, severe spinal cord ischemia injury was recorded in all the five rabbits: the latency of motor-evoked potential prolonged in one rabbit, whereas the waveform disappeared in four rabbits; loss of neurons and vacuolation of gray matter were seen in spinal cord sections, and the average Tarlov score was 0.6±0.9. Selective ligation of lumbar arteries was a modified method to induce feasible and reproducible model of spinal cord ischemia in rabbits.

Effect of early evaluation of CSEP and DNEP on spinal cord ischemic injury: an experimental study

Objective To investigate the effect of early evaluation of cortical somatosensory evoked potentials(CSEP)and desending neurogenic motor evoked potentials(DNEP)in spinal cord ischemic injury by observing the early change characteristics of CSEP and DNEP of spinal cord ischemia in rabbits. Methods To establish a rabbit model with different degree of permanent spinal cord ischemic injury through the ligation of different levels of lumbar arteries. The rabbits were divided into 5 groups(ligation of the left renal artery and the different levels of lumbar arteries between arterial bifurcations respectively, including 1 to 5-artery group). After modeling, CSEP and DNEP were continuously recorded for 60 min. After 2 days, the samples of ischemic central area were selected for HE staining. The motor function score Tarlov criteria were used to assess the motor function. The relationship between the amplitude variation of CESP and DNEP and the motor function after ligation were analyzed. Results There were statistical significance in the percentages of the CSEP amplitude and preoperative baseline amplitude between the 4- and 5-artery groups compared with before ligation(P 0.05). There were statistical differences in the percentages of the DNEP amplitude and preoperative baseline amplitude among the 3-, 4- and 5 artery groups compared with before ligation(all P 0.05). Compare with before ligation, there were statistical significances in the motor function 2 days after ligation between the 4- and 5-artery group(all P 0.05). The amplitude variation of CSEP and DNEP was positively correlated with the motor function 2 days after ligation of blood vessels(r=0.97, P<0.001; r=0.95, P<0.001). Conclusions Both CSEP and DNEP can predict the motor function after spinal cord injury. DNEP is more sensitive to spinal cord ischemic injury and is more specific to motor function. It can find ischemic injury in the early spinal cord ischemia. Key words: Spinal cord ischemia; Evoked potentials, somatosensory; Evoked potentials, motor; Rabbits

The major cholesterol metabolite cholestane-3β,5α,6β-triol functions as an endogenous neuroprotectant.

Overstimulation of NMDA-type glutamate receptors is believed to be responsible for neuronal death of the CNS in various disorders, including cerebral and spinal cord ischemia. However, the intrinsic and physiological mechanisms of modulation of these receptors are essentially unknown. Here we report that cholestane-3β,5α,6β-triol (triol), a major metabolite of cholesterol, is an endogenous neuroprotectant and protects against neuronal injury both in vitro and in vivo via negative modulation of NMDA receptors. Treatment of cultured neurons with triol protects against glutamate-induced neurotoxicity, and administration of triol significantly decreases neuronal injury after spinal cord ischemia in rabbits and transient focal cerebral ischemia in rats. An inducible elevation of triol is associated with ischemic preconditioning and subsequent neuroprotection in the spinal cord of rabbits. This neuroprotection is effectively abolished by preadministration of a specific inhibitor of triol synthesis. Physiological concentrations of triol attenuate [Ca(2+)]i induced by glutamate and decrease inward NMDA-mediated currents in cultured cortical neurons and HEK-293 cells transiently transfected with NR1/NR2B NMDA receptors. Saturable binding of [(3)H]triol to cerebellar granule neurons and displacement of [(3)H]MK-801 binding to NMDA receptors by triol suggest that direct blockade of NMDA receptors may underlie the neuroprotective properties. Our findings suggest that the naturally occurring oxysterol, the major cholesterol metabolite triol, functions as an endogenous neuroprotectant in vivo, which may provide novel insights into understanding and developing potential therapeutics for disorders in the CNS.

Autophagy-mediated stress response in motor neurons after hypothermic spinal cord ischemia in rabbits

The development of spinal cord injury is believed to be related to the vulnerability of spinal motor neurons to ischemia. However, the mechanisms underlying this vulnerability have not been fully investigated. Previously, we reported that spinal motor neurons are lost likely due to autophagy and that local hypothermia prevents such spinal motor neuron death. Therefore, we investigated the role of autophagy in normothermic and hypothermic spinal cord ischemia using an immunohistochemical analysis of Beclin 1 (BCLN1; B-cell leukemia 2 protein [Bcl-2] interacting protein), Bcl-2, and γ-aminobutyric acid type-A receptor-associated protein (GABARAP), which are considered autophagy-related proteins. We used rabbit normothermic and hypothermic transient spinal cord ischemia models using a balloon catheter. Neurologic function was assessed according to the Johnson score, and the spinal cord was removed at 8hours and 1, 2, and 7days after reperfusion, and morphologic changes were examined using hematoxylin and eosin staining. A Western blot analysis and histochemical study of BCLN1, Bcl-2, and GABARAP, and double-labeled fluorescent immunocytochemical studies were performed. There were significant differences in the physiologic function between the normothermic model and hypothermic model after the procedure (P< .05). In the normothermic model, most of the motor neurons were selectively lost at7days of reperfusion (P< .001 compared with the sham group), and they were preserved in the hypothermic model (P= .574 compared with the sham group). The Western blot analysis revealed that the sustained expression of the autophagy markers, BCLN1 and GABARAP, was observed (P< .001 compared with the sham group) and was associated with neuronal cell death in normothermic ischemic conditions. In hypothermic ischemic conditions, the autophagy inhibitory protein Bcl-2 was powerfully induced (P< .001 compared with the sham group) and was associated with blunted expression of BCLN1 and GABARAP and neuronal cell survival. The double-label fluorescent immunocytochemical studyrevealed that immunoreactivitiy for BCLN1, Bcl-2, and GABARAP was induced in the same motor neurons. These data suggest that the prolonged induction of autophagy might be a potential factor responsible for delayed motor neuron death, and the induction of the autophagy inhibitory protein Bcl-2 using hypothermia might limit autophagy and protect against delayed motor neuron death.

Neuroprotective effects of progesterone in spinal cord ischemia in rabbits

BackgroundThis experimental study was performed to investigate the neuroprotective effects of progesterone on spinal cord ischemia in rabbits. MethodsEighteen female New Zealand white rabbits were used in this study. Rabbits were randomized into 3 groups. Spinal cord ischemia was induced by clamping the abdominal aorta from a point just inferior to the left renal artery to the aortic bifurcation for a period of 30 minutes. Group 1 served as the control group, and groups 2 and 3 received intraperitoneal progesterone immediately after the onset of reperfusion, at a dose of 8 mg/kg. Two hours after reperfusion, the animals in group 1 were killed. Four hours after reperfusion, the animals in group 2 were killed, and 6 hours after reperfusion, the group 3 rabbits were killed. Spinal cords were removed and fixed in 10% formalin in a phosphate buffer. Neuronal injury was evaluated by a pathologist who was blinded to the treatment groups, and 5 sections per animal were evaluated. The number of intact large motor neuron cells in the ventral grey matter region was counted. ResultsThe analysis revealed that the average mean arterial pressure for group 1 was significantly higher than that for group 2, and the mean sacrificed pressure value for group 1 was significantly higher than that for group 3 (P < .05). The number of intact neurons in group 1 was significantly lower than the number of intact neurons found in both groups 2 and 3 (P < .05). No other statistically significant differences were found between the groups. ConclusionThe findings from the present study indicate that progesterone effectively protects the spinal cord tissues against ischemic damage in the setting of decreased perfusion.

Evaluation of the Neuroprotective Effect of Minocycline in a Rabbit Spinal Cord Ischemia Model

To investigate whether postischemic administration of minocycline attenuates hind-limb motor dysfunction and gray and white matter injuries after spinal cord ischemia. A prospective, randomized, laboratory investigation. Laboratory in university, single institution. Male New Zealand White rabbits. Spinal cord ischemia was induced by an occlusion of the infrarenal aorta for 15 minutes. The groups were administered minocycline 1 hour after reperfusion (M-1; n = 8), minocycline 3 hours after reperfusion (M-3; n = 8), saline 1 hour after reperfusion (control [C]; n = 8), or saline and no occlusion (sham; n = 4). Minocycline was administered intravenously at 10 mg/kg 6 times at 12-hour intervals until 60 hours after the initial administration. Hind-limb motor function was assessed using the Tarlov score. For histologic assessments, gray and white matter injuries were evaluated 72 hours after reperfusion using the number of normal neurons and the percentage of areas of vacuolation, respectively. Motor function 72 hours after reperfusion was significantly better in group M-1 than in group C. The number of neurons in the anterior horn was significantly larger in group M-1 than in groups M-3 or C but did not differ significantly between groups M-3 and C. No significant difference was noted in the percentage of areas of vacuolation among the ischemia groups. Minocycline administration beginning at 1 hour after reperfusion improved hind-limb motor dysfunction and attenuated gray matter injury in a rabbit spinal cord ischemia model.

Intrathecal Injection of Hepatocyte Growth Factor Gene-modified Marrow Stromal Cells Attenuates Neurologic Injury Induced by Transient Spinal Cord Ischemia in Rabbits

Our previous studies showed that transfer of hepatocyte growth factor (HGF) gene or transplantation of marrow stromal cells (MSCs) remarkably attenuated neurologic injuries after spinal cord ischemia. We sought to investigate a novel neuroprotective strategy of transplantation of human HGF gene-modified MSCs on ischemic spinal cords. Human HGF gene was transferred into MSCs in vitro. The HGF gene-modified MSCs were transplanted by means of intrathecal injection. Two days later, spinal cord ischemia was induced by occlusion of the infrarenal aorta with a balloon catheter for 40 or 50 min. Hind-limb motor function was assessed during a 14-day recovery period with Tarlov criteria, and then histologic examination was performed. Human HGF was detected in the cerebrospinal fluid from 2 to 16 days after transplantation of HGF gene-modified MSCs. Compared with the controls, transplantation of HGF gene-modified MSCs or MSCs alone significantly improved the Tarlov scores 1, 2, 7, and 14 days after spinal cord ischemia of 40 or 50 min (P < 0.01, respectively) and increased the number of intact motor neurons in the lumbar spinal cord (P < 0.01, respectively). When the ischemic period was extended to 50 min, the Tarlov scores and the number of intact motor neurons of rabbits transplanted with HGF gene-modified MSCs were markedly higher than those of the rabbits transplanted with MSCs only (P < 0.05, respectively). Transplantation of HGF gene-modified MSCs induces powerful neuroprotection on spinal cords against ischemia-reperfusion injury and is more therapeutically efficient than transplantation of MSCs only.

Effects of xenon on ischemic spinal cord injury in rabbits: a comparison with propofol

Xenon has been shown to reduce cellular injury after cerebral ischemia. However, the neuroprotective effects of xenon on ischemic spinal cord are unknown. The authors compared the effects of xenon and propofol on spinal cord injury following spinal cord ischemia in rabbits. Thirty-two male New Zealand white rabbits were randomly assigned to one of three groups. In the xenon and propofol group, 70% of xenon and 0.8 mg/kg/min of propofol were administered 30 min before an aortic occlusion and maintained until the end of the procedure. The aortic occlusion was performed for 15 min. In the sham group, the aorta was not occluded. After an assessment of the hind limb motor function using the Tarlov score (0=paraplegia, 4=normal) at 48 h after reperfusion, gray and white matter injuries were evaluated based on the number of normal neurons in the anterior spinal cord and the percentage areas of vacuolation in the white matter, respectively. In the xenon and propofol groups, the Tarlov score and the number of normal neurons were significantly lower than those in the sham group, whereas the percentage areas of vacuolation were similar among the three groups. There were no significant differences in Tarlov scores and the number of normal neurons between the xenon and the propofol groups. The results indicated that 70% of xenon has no additional neuroprotective effects on ischemic spinal cord injury in rabbits compared with propofol.

Autophagy-mediated stress response in motor neuron after transient ischemia in rabbits

Spinal cord injury is considered to be related to a vulnerability of spinal motor neurons to ischemia. However, the mechanisms underlying this vulnerability are not fully understood. We investigated the role of autophagy, which is an intracellular bulk degradation process, at motor neuron as a potential mechanism of neuronal death by immunohistochemical analysis for microtubule-associated protein light chain3 (LC3) and gamma-aminobutyric-acid type-A-receptor-associated protein (GABARAP) which are considered as markers of autophagy. We used a rabbit spinal cord ischemia model with the use of a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 minutes of transient ischemia, and histologic changes were examined with hematoxylin-eosin staining. Western blot analysis for LC3 and GABARAP, temporal profiles of LC3 and GABARAP immunoreactivity, and double-label fluorescence immunocytochemical studies were performed. In the ischemia group, about 85% of motor neurons were preserved until 2 days after reperfusion, but were selectively lost at 7 days (P < .001 compared with sham group). Western blot analysis demonstrated slight immunoreactivity for LC3 and GABARAP in the sham-operated spinal cords. In contrast, the ischemia group LC3 and GABARAP immunoreactivity became apparent at 8 hours after reperfusion. With quantitative analysis we found that ischemia affected expression profiles of LC3-II and GABARAP. At 8 hours after reperfusion, co-labeling of LC3 and GABARAP were observed in the same motor neurons that eventually died. These data suggest that autophagy was induced in motor neurons by transient spinal cord ischemia in rabbits.

Does high-dose opioid anesthesia exacerbate ischemic spinal cord injury in rabbits?

Intrathecal morphine given during a post-ischemic period has been reported to have the potential to exacerbate ischemic spinal cord injury. However, it remains unknown whether synthetic opioids administered systemically exacerbate ischemic injury. We sought to compare the damage of the spinal cord after transient spinal cord ischemia in rabbits anesthetized with three different regimens; isoflurane, fentanyl with isoflurane, and remifentanil with isoflurane. We assigned rabbits to three groups (n = 9 in each); an isoflurane group (isoflurane 1 minimum alveolar concentration [MAC]), a fentanyl group (isoflurane 0.5 MAC + 100 microg x kg(-1) i.v. fentanyl given over 30 min before aortic occlusion), and a remifentanil group (isoflurane 0.5 MAC + 1 microg x kg(-1) x min(-1) i.v. remifentanil started 30 min before aortic occlusion and maintained until 1 h after reperfusion). Spinal cord ischemia was produced by occluding the abdominal aorta for 13 min. Hindlimb motor function (score range: 4, normal to 0, paraplegia) was assessed daily for 7 days, and then the number of normal neurons in the anterior spinal cord was counted. Severe motor dysfunction (score < or = 1) was observed in seven, four, and five animals in the isoflurane, fentanyl, and remifentanil groups, respectively. There were no significant intergroup differences in neurological scores. There were no differences in the numbers of normal neurons among the three groups (22 +/- 22, 42 +/- 30, 33 +/- 28, respectively). Our results suggest that neither i.v. fentanyl nor i.v. remifentanil added to 0.5 MAC isoflurane exacerbated ischemic spinal cord injury in rabbits when compared to 1 MAC isoflurane.

Induction of Parkinson Disease-Related Proteins in Motor Neurons after Transient Spinal Cord Ischemia in Rabbits

The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We investigated a possible mechanism of neuronal death by immunohistochemical analysis for DJ-1, PINK1, and alpha-Synuclein. We used a 15-min rabbit spinal cord ischemia model, with use of a balloon catheter. Western blot analysis for DJ-1, PINK1, and alpha-Synuclein; temporal profiles of DJ-1, PINK1, and alpha-Synuclein immunoreactivity; and double-label fluorescence immunocytochemical studies were performed. Western blot analysis revealed scarce immunoreactivity for DJ-1, PINK1, and alpha-Synuclein in the sham-operated spinal cords. However, they became apparent at 8 h after transient ischemia, which returned to the baseline level at 1 day. Double-label fluorescence immunocytochemical study revealed that both DJ-1 and PINK1, and DJ-1 and alpha-Synuclein were positive at 8 h of reperfusion in the same motor neurons, which eventually die. The induction of DJ-1 and PINK1 proteins in the motor neurons at the early stage of reperfusion may indicate oxidative stress, and the induction of alpha-Synuclein may be implicated in the programmed cell death change after transient spinal cord ischemia.

A semiquantitative analysis of reactive astrogliosis demonstrates its correlation with the number of intact motor neurons after transient spinal cord ischemia

We evaluated the relationship between reactive astrogliosis and delayed motor neuron death after transient spinal cord ischemia in rabbits using a semiquantitative analysis of glial fibrillary acidic protein expression. Spinal cord ischemia was induced by means of balloon occlusion of the infrarenal aorta for 15 minutes at 39 degrees C in 18 New Zealand white rabbits. At 1, 3, and 7 days after reperfusion, 6 animals at each time point were killed, and the spinal cord was removed for histologic and immunohistochemical study. The variables analyzed were (1) neurologic function (Johnson score) at every 24 hours after reperfusion, (2) the number of intact motor neurons and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling-positive positive neurons, and (3) expression of glial fibrillary acidic protein in the gray and white matter, which was expressed as the percentage of stained area. All animals presented delayed motor neuron death. The number of intact neurons decreased correlatively with neurologic function. No obvious terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling-positive cells were observed. Glial fibrillary acidic protein expression increased with time in both the gray and white matter, representing the development of reactive astrogliosis. Significant correlation was found between glial fibrillary acidic protein expression and the number of intact motor neurons on the third day in both the gray (r(2) = 0.726, P = .031) and white (r(2) = 0.927, P = .002) matter. Reactive astrogliosis 3 days after transient spinal cord ischemia correlates with the number of intact motor neurons. Our method for semiquantitative analysis of reactive astrogliosis is simple and reproducible and seems useful for such experimental studies.

Ubiquitin-Mediated Stress Response in the Spinal Cord After Transient Ischemia

Vulnerability of motor neurons in the spinal cord against ischemia is considered to play an important role in the development of delayed paraplegia after surgery of the thoracic aorta. However, the reasons for such vulnerability are not fully understood. Recently, the ubiquitin system has been reported to participate in neuronal cell death. In the present study, we investigated the expression of ubiquitin system molecules and discussed the relationship between the vulnerability and the ubiquitin system after transient ischemia in the spinal cord. Fifteen minutes of spinal cord ischemia in rabbits was applied with the use of a balloon catheter. In this model, the spinal motor neuron shows selectively delayed neuronal death, whereas other spinal neurons such as interneurons survive. Immunohistochemical analysis and Western blotting for ubiquitin system molecules, ubiquitin, deubiquitylating enzyme (ubiquitin carboxy-terminal hydrolase 1), and ubiquitin-ligase parkin were examined. In cytoplasm, ubiquitin and ubiquitin carboxy-terminal hydrolase 1 were strongly induced both in interneuron and motor neuron at the early stage of reperfusion, but the sustained expression was observed only in motor neuron. Parkin was induced strongly at 3 hours after the reperfusion, but the immunoreactivity returned to the sham control level at 6 hours in both neurons. In the nuclei, ubiquitin, ubiquitin carboxy-terminal hydrolase 1, and parkin were strongly induced in interneuron, whereas no upregulation of these proteins was observed in motor neuron. These results indicate that the vulnerability of motor neuron of the spinal cord might be partially attributed to the different response in ubiquitin-mediated stress response after transient ischemia.

Co-application of ischemic preconditioning and postconditioning provides additive neuroprotection against spinal cord ischemia in rabbits

Postconditioning can induce cardioprotection against ischemia. However, the data on postconditioning of the spinal cord is very limited. We investigated here whether co-application of ischemic preconditioning (IPC) and postconditioning can provide additive neuroprotection against prolonged spinal cord ischemia. Spinal cord ischemia was produced in rabbits by infrarenal aortic occlusion with a balloon catheter for 30 min. The four treatment groups were control ( n = 10): no intervention; IPC ( n = 10): a 5-minute aortic occlusion was performed 20 min before the prolonged ischemia; Postcon ( n = 10): postconditioning comprised of four cycles of 1-minute occlusion/1-minute reperfusion was applied one minute after the start of reperfusion. IPC + postcon ( n = 11): both IPC and postconditioning were applied. Functional evaluation with Tarlov score was performed during a 14-day observation period. Neurologic impairment was noticeably attenuated in the IPC + postcon group (compared with the control group, P < 0.01, at day 1, day 2, day 7 and day 14, respectively), but not in either the IPC or Postcon group. Plasma malondialdehyde levels after reperfusion were significantly decreased to a similar extent in the IPC, Postcon and IPC + Postcon groups (compared with the control group ( P < 0.01). In the IPC + Postcon group, many more large motor neurons were preserved than in the control group ( P < 0.05) and white matter injury was also markedly attenuated as evidenced by reduction of the vacuolation area of the white matter ( P < 0.01) and decreased amyloid precursor protein immunoreactivity ( P < 0.01). From this, we conclude that the combination of IPC and postconditioning induces additive neuroprotective effects for spinal cord against ischemia and reperfusion injuries.