Effects of ginsenoside Rb1 on spinal cord ischemia-reperfusion injury in rats.

BACKGROUND: The aim of this study was to evaluate the effects of different doses of ginsenoside Rb1 (GRb1) pretreatment on spinal cord ischemia-reperfusion (SCII) in rats and explore the potential mechanisms about the expression of survivin protein after the intervention. METHODS: A total of 90 healthy adult Sprague-Dawley (SD) rats were randomly divided into six groups: sham-operated (n = 15), SCII model (n = 15), and GRb1-treated groups (n = 60). The GRb1-treated group was divided into four subgroups: 10 mg/kg, 20 mg/kg, 40 mg/kg, and 80 mg/kg (n = 15). The corresponding dose of GRb1 was injected intraperitoneally 30 min before operation and every day after operation. Forty-eight hours after model establishment, the neurological function of hind limbs was measured with Basso, Beattie, and Bresnahan (BBB) scale. The superoxide dismutase (SOD) and malondialdehyde (MDA) levels in serum and spinal cord tissue were detected respectively. The expression of survivin protein was observed by immunofluorescence staining. HE and TUNEL staining were used to observe neural cell injury and apoptosis, respectively, in the spinal cord of rats with SCII. RESULTS: The intervention of different doses of GRb1 could increase SOD activity and decrease MDA content in serum and spinal cord tissue, increase survivin protein expression, and decrease neuronal apoptosis. It was dose-dependent, but there was no significant change between 40 mg/kg and 80 mg/kg. CONCLUSIONS: GRb1 could reduce the cell apoptosis induced by SCII through inhibiting oxidative stress. It can also inhibit apoptosis by promoting the expression of Survivin protein. Ginsenoside Rb1 had a dose-dependent protective effect on SCII in the dose range of 10 mg/kg-40 mg/kg.

Electroacupuncture preconditioning and postconditioning inhibit apoptosis and neuroinflammation induced by spinal cord ischemia reperfusion injury through enhancing autophagy in rats.

Electroacupuncture (EA) has beneficial effects on spinal cord ischemia reperfusion (I/R) injury, but the underlying mechanisms are not fully understood. This study aimed to investigate the role of autophagy in the protection of EA preconditioning and postconditioning against spinal cord I/R injury. For this, spinal cord I/R injury was induced by 14min occlusion of the aortic arch, and rats were treated with EA for 20min before or after the surgery. The expression of autophagy components, light chain 3 and Beclin 1, was assessed by Western blot. The hind-limb motor function was assessed using the Basso-Beattie-Bresnahan (BBB) criteria, and motor neurons in the ventral gray matter were counted by histological examination. The apoptosis of neurocyte was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The expression of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and matrix metalloproteinase-9 (MMP-9) was also measured using Western blot or enzyme-linked immunosorbent assay (ELISA). Either EA preconditioning or postconditioning enhanced autophagy, and minimized the neuromotor dysfunction and histopathological deficits after spinal cord I/R injury. In addition, EA suppressed I/R-induced apoptosis and increased in the expression of TNF-α, IL-1ß, and MMP-9. In contrast, the autophagic inhibitor (3-methyladenine, 3-MA) inhibited the neuroprotective effects of EA. Moreover, 3-MA increased the apoptosis and the expression of TNF-α, IL-1ß, and MMP-9. In summary, these findings suggested that EA preconditioning and postconditioning could alleviate spinal cord I/R injury, which was partly mediated by autophagy upregulation-induced inhibition of apoptosis and neuroinflammation.

Electroacupuncture pretreatment attenuates spinal cord ischemia-reperfusion injury via inhibition of high-mobility group box 1 production in a LXA4 receptor-dependent manner.

Paraplegia caused by spinal cord ischemia is a severe complication following surgeries in the thoracic aneurysm. HMGB1 has been recognized as a key mediator in spinal inflammatory response after spinal cord injury. Electroacupuncture (EA) pretreatment could provide neuroprotection against cerebral ischemic injury through inhibition of HMGB1 release. Therefore, the present study aims to test the hypothesis that EA pretreatment protects against spinal cord ischemia-reperfusion (I/R) injury via inhibition of HMGB1 release. Animals were pre-treated with EA stimulations 30min daily for 4 successive days, followed by 20-min spinal cord ischemia induced by using a balloon catheter placed into the aorta. We found that spinal I/R significantly increased mRNA and cytosolic protein levels of HMGB1 after reperfusion in the spinal cord. The EA-pretreated animals displayed better motor performance after reperfusion along with the decrease of apoptosis, HMGB1, TNF-α and IL-1ß expressions in the spinal cord, whereas these effects by EA pretreatment was reversed by rHMGB1 administration. Furthermore, EA pretreatment attenuated the down-regulation of LXA4 receptor (ALX) expression induced by I/R injury, while the decrease of HMGB1 release in EA-pretreated rats was reversed by the combined BOC-2 (an inhibitor of LXA4 receptor) treatment. In conclusion, EA pretreatment may promote spinal I/R injury through the inhibition of HMGB1 release in a LXA4 receptor-dependent manner. Our data may represent a new therapeutic technique for treating spinal cord ischemia-reperfusion injury.

Effects of Ginkgo biloba extract on spinal cord ischemia-reperfusion injury in rats.

BACKGROUND: We aimed to assess the biochemical and histopathologic effects of Ginkgo biloba extract (EGb) in an ischemia-reperfusion (IR) model of spinal cord ischemia induced by cross-clamping of the infrarenal abdominal aorta. METHODS: A total of 24 Sprague-Dawley rats were divided into 3 groups as group 1: control (sham laparotomy), group 2: IR, and group 3: IR+EGb treatment (IR+T) group. All subjects were euthanized 2 days postsurgery and their spinal cords were removed. Tissue malondialdehyde, superoxide dismutase, glutathione (GSH), and glutathione peroxidase levels were measured, and the spinal cord tissue samples were examined histopathologically. RESULTS: No significant difference was detected in ischemia markers between control, IR, and IR+T groups, with the exception of GSH, which was significantly lower in the IR group. GSH levels in group 1 and group 3 were similar. The group 2 displayed significant ischemic damage to the medulla spinalis. This damage was less pronounced in group 3 compared with group 2 only, but in extent and intensity comparable with the controls. CONCLUSIONS: Although we were not able to demonstrate a uniform effect of EGb on biochemical markers of IR injury, the histopathologic data appear to show a protective effect conferred on the spinal cord tissue by EGb.

Effects of curcumin on acute spinal cord ischemia-reperfusion injury in rabbits. Laboratory investigation.

OBJECT: The object of this study was to conduct a prospective, randomized, laboratory investigation of the neuroprotective effects of curcumin functionally, biochemically, and histologically in an experimental acute spinal cord ischemia-reperfusion injury on rabbits. METHODS: Eighteen rabbits were randomly assigned to 1 of 3 groups: the sham group, the ischemia-reperfusion group, or the curcumin group. Spinal cord ischemia was induced by applying an infrarenal aortic cross-clamp for 30 minutes. At 48 hours after ischemia, neurological function was evaluated with modified Tarlov criteria. Biochemical changes in the spinal cord and plasma were observed by measuring levels of malondialdehyde (MDA), advanced oxidation protein products (AOPP), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), nitrite/nitrate, and tumor necrosis factor-α (TNF-α). Histological changes were examined with H & E staining. Immunohistochemical staining with antibodies against caspase-3 was performed to evaluate cell apoptosis after ischemia. RESULTS: In the curcumin group, neurological outcome scores were statistically significantly better compared with the ischemia-reperfusion group. In the ischemia-reperfusion group, MDA, AOPP, and nitrite/nitrate levels were significantly elevated in the spinal cord tissue and the plasma by the induction of ischemia-reperfusion. The curcumin treatment significantly prevented the ischemia-reperfusion-induced elevation of nitrite/nitrate and TNF-α. In addition, the spinal cord tissue and the plasma SOD, GSH, and CAT levels were found to be preserved in the curcumin group and not statistically different from those of the sham group. Histological evaluation of the tissues also demonstrated a decrease in axonal damage, neuronal degeneration, and glial cell infiltration after curcumin administration. CONCLUSIONS: Although further studies including different dose regimens and time intervals are required, curcumin could attenuate a spinal cord ischemia-reperfusion injury in rabbits via reducing oxidative products and proinflammatory cytokines, as well as increasing activities of antioxidant enzymes and preventing apoptotic cell death.

Supplement zinc as an effective treatment for spinal cord ischemia/reperfusion injury in rats.

OBJECTIVE: Brain-derived neurotrophic factor (BDNF) plays a key role in the pathophysiology process and therapy of spinal cord injury (SCI). Accordingly, zinc regulates the expression of BDNF and its receptor in the central nervous system, the mechanism of which is still unknown. The present study investigates whether supplement zinc could reduce neurological damage in a rat model, with spinal cord ischemia-reperfusion (I/R) injury and how the effect of zinc transporter 1(ZnT-1) was involved. METHODS: 100 Sprague-Dawley male rats were randomly and evenly divided into four groups. They were subjected to spinal cord ischemia by clamping the abdominal aorta for 45 min. Rats in the zinc-deficient dietary model group (ZD), zinc-adequate dietary model group (ZA), and zinc-high dietary model group (ZH) were given free access to purified diet, containing 5, 30, or 180 mg Zn/kg. Sham operation rats were subjected to laparotomy without clamping of the aorta and were fed by ZA diet (30 mg Zn/kg). Neurological function was scored by Tarlov's score. The spinal cord segments (L5) were harvested for histological examination, auto-metallographic (AMG) analysis, myeloperoxidase (MPO) activity analysis, expression of ZnT-1 and BDNF. RESULTS: The rats in the ZH group have shown the higher neurological scores, slighter histological changes and the attenuated MPO activity, compared with those in the ZD and ZA groups at the four observation time points (p<0.05). The AMG staining density in the ZH group was significantly higher than that of ZD group in 14 days later after the operation. Compared with other groups, ZH group's expression of Zn-T1 and BDNF were significantly increased, and was positively correlated with the same time points after surgery (Spearman rho=0.403, p=0.0152.) CONCLUSION: These findings suggest that zinc supplement can significantly reduce the spinal cord I/R injury in rats. The mechanism may be related with restraining the MPO activity and increasing of ZnT-1, which promoted the synthesis and release of BDNF.

Evaluation of the neuroprotective effect of minocycline in a rabbit spinal cord ischemia model.

OBJECTIVE: To investigate whether postischemic administration of minocycline attenuates hind-limb motor dysfunction and gray and white matter injuries after spinal cord ischemia. DESIGN: A prospective, randomized, laboratory investigation. SETTING: Laboratory in university, single institution. PARTICIPANTS: Male New Zealand White rabbits. INTERVENTION: 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. MEASUREMENT AND MAIN RESULTS: 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. CONCLUSIONS: 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.

The change of the spinal cord ischemia-reperfusion injury in mitochondrial passway and the effect of the Ginkgo biloba extract's preconditioning intervention.

In order to explore whether the apoptosis in ischemia-reperfusion injury could be affected by Ginkgo biloba extract (GBE) and the free radical scavenger GBE could suppress this affection. Rabbits were randomly divided into sham group, ischemia group, ischemia-reperfusion group (1, 6, 24, 48 h), the drug group (1, 6, 24, 48 h). Measure the rate of apoptosis by flow cytometry, the caspase 9 and apoptosis-inducing factor (AIF) in the cytoplasm and serum by ELISA. Compared with the sham group and ischemia group, the reperfusion group increased the rate of apoptosis, the caspase 9 and AIF in serum have a peak at 24 h after reperfusion, in the cytoplasm the peak at 6 h.GBE inhibit performance has the systemic and local aspects. The apoptosis of nerve cells after spinal cord ischemia-reperfusion has the relationship with the mitochondrial caspase-dependent and caspase-independent pathways and both the local and systemic role. GBE inhibits nerve cell apoptosis by these ways.

Morphological changes of the neural cells after blast injury of spinal cord and neuroprotective effects of sodium beta-aescinate in rabbits.

OBJECTIVE: Explosive blast neurotrauma is becoming more and more common not only in the military population but also in civilian life due to the ever-present threat of terrorism and accidents. However, little attention has been offered to the studies associated with blast wave-induced spinal cord injury in the literatures. The purpose of this study is to report a rabbit model of explosive blast injury to the spinal cord, to investigate the histological changes, focusing especially on apoptosis, and to reveal whether beta-aescinate (SA) has the neuroprotective effects against the blast injury. METHODS: Adult male New Zealand white rabbits were randomly divided into sham group, experimental group and SA group. All rabbits except the sham group were exposed to the detonation, produced by the blast tube containing 0.7 g cyclotrimethylene trinitramine, with the mean peak overpressure of 50.4 MP focused on the dorsal surface of T9-T10 level. After evaluation of the neurologic function, spinal cord of the rabbits was removed at 8 h, 1, 3, 7, 14 or 30 days and the H&E staining, EM examination, DNA gel electrophoresis and TUNEL were progressively performed. RESULTS: The study demonstrated the occurrence of both necrosis and apoptosis at the lesion site. Moreover, the SA therapy could not only improve the neurologic outcomes (P<0.05) but also reduce the loss of motoneuron and TUNEL-positive rate (P<0.05). CONCLUSIONS: In the rabbit model of explosive blast injury to the spinal cord, the coexistent apoptotic and necrotic changes in cells was confirmed and the SA had neuroprotective effects to the blast injury of the spinal cord in rabbits. This is the first report in which the histological characteristics and drug treatment of the blast injury to the spinal cord is demonstrated.

Low-dose edaravone injection into the clamped aorta prevents ischemic spinal cord injury.

Previous studies have indicated that high-dose intravenous edaravone (3-10mg/kg) protects against ischemic spinal cord injury. This study examined whether direct injection of low-dose edaravone into the clamped segment of the aorta prevents ischemic spinal cord injury. Spinal cord ischemia was induced in rabbits by aortic clamping below the renal artery and above the aortic bifurcation for 15min at normothermia. In groups A and B, 3 and 1mg/kg of edaravone, respectively, was injected into the clamped segment of the aorta immediately following aortic clamping. In group C, saline was injected. Neurological function was assessed at 8, 24, and 48hr and 7 days after reperfusion with Tarlov criteria. The spinal cord was histologically examined at 7 days with hematoxylin-eosin staining and in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The Tarlov score remained grade 4 throughout the period in groups A and B, whereas it dropped to grade 0 or 1 at 7 days in group C, significantly higher in the former two groups. The number of intact motor neurons was significantly greater in groups A and B with less necrotic motor neurons than in group C. There was no significant difference in terms of spinal cord protection between groups A and B. There was no TUNEL-positive neuron in any group, indicating the absence of apoptosis. Low-dose intra-aortic edaravone injection prevents immediate neuronal injury by reducing neuronal cell damage in the early stage as well as delayed neuronal injury at 7 days.

Effects of clotrimazole on experimental spinal cord ischemia/reperfusion injury in rats.

OBJECTIVE: The effect of clotrimazole was examined using a spinal cord ischemia/reperfusion model. METHODS: Twenty albino Wistar rats weighing 234 +/- 12.3 g were used in this study. Rats were anesthetized intraperitoneally with 50 mg/kg ketamine HCl. All animals underwent laparotomy under aseptic conditions. Abdominal aortas of the animals in all but the sham group were exposed. After opening the retroperitoneum, the infrarenal abdominal aorta was clipped for 45 minutes to produce ischemia/reperfusion injury. Polyethylene glycol (PEG, 1 mL) was administrated to the vehicle group. PEG (1 mL) and clotrimazole (30 mg/kg) were administered intraperitoneally in the clotrimazole group. Total laminectomy of T8-T12 was performed on all rats under a microscope. Spinal cords were excised for a length of 2-cm rostrally and 1-cm caudally to the injury site and deep frozen at -76 degrees C for biochemical studies. The levels of malondialdehyde, glutathione-peroxidase, superoxide dismutase, and catalase were measured as an indicator of ischemia level. The most cranial part of the specimens was evaluated morphologically. RESULTS: Treatment with clotrimazole significantly decreased malondialdehyde, glutathione-peroxidase, superoxide dismutase, and catalase levels in comparison with other groups (P = 0.008). Morphologic evaluation revealed that clotrimazole protected the axons and their myelin sheaths from ischemic damage. CONCLUSION: This study showed the neuroprotective effects of clotrimazole on spinal cord ischemia/reperfusion injury.

Extract EGb 761 pretreatment limits ubiquitin positive aggregates in rabbit spinal cord neurons after ischemia-reperfusion.

1. Ubiquitin immunohistochemistry was used for investigation of time dependent changes of ubiquitin in the nerve cells reacting to ischemic/reperfusion damage. In the rabbit spinal cord ischemia model a period of 30 min ischemia followed by 24 and 72 h of reperfusion caused neuronal degeneration selectively in the ventral horn motor neurons as well as interneurons of the intermediate zone. 2. Ubiquitin aggregates were accumulated in the neurons of lamina IX and the neurons of intermediate zone destined to die 72 h after 30 min of the spinal cord ischemia. 3. The activation of ubiquitin hydrolytic system is related to a defective homeostasis and could trigger different degenerative processes. Having in mind this, we used EGb 761 to rescue the motor neurons and interneurons against ischemia/reperfusion damage. Our results show that after 30 min of ischemia and 24 or 72 h of reperfusion with EGb 761 pre-treatment for 7 days the vulnerable neurons in the intermediate zone and lamina IX exhibit marked elevation of ubiquitin-positive granules in the cytoplasm, dendrites and nuclei. Abnormal protein aggregates have not been observed in these cells. 4. The rabbits were completely paraplegic after 30 min of ischemia and 24 or 72 h of reperfusion. However, after 7 days EGb 761 pre-treatment, 30 min of ischemia and 24 or 72 h of reperfusion the animals did not show paraplegia. 5. Evaluated ubiquitin-positive neurons of the L(5)-L(6) segments showed significant decrease in number and significant increase of density after 30 min of ischemia followed by 24 h and mainly 72 h of reperfusion. Ubiquitin immunohistochemistry confirmed the protective effect of EGb 761 against ischemia/reperfusion damage in the rabbit spinal cord.

Intra-aortic injection of propofol prevents spinal cord injury during aortic surgery.

OBJECTIVE: We investigated whether propofol, a widely used anesthetic, injected into clamped aortic segments quickly attenuated transcranial spinal motor-evoked potential (MEP) amplitudes and protected against spinal cord injury during thoracoabdominal aortic surgery. METHODS: Eighteen beagle dogs were divided into three groups (n=6, each group): group 1 (20 ml of saline, intra-aortic injection), group 2 (1.5 mg/kg of propofol, intravenous injection), and group 3 (1.5 mg/kg of propofol, intra-aortic injection). Aortic cross-clamping was performed for 30 min. In each group, MEP amplitudes were recorded before, during, and after aortic cross-clamping. Tarlov score and histopathological examination were used to evaluate the protective effects of intra-aortic propofol injections. RESULTS: MEP amplitudes in group 3 attenuated to a value that was 60% of the control in just a minute after aortic cross-clamping, but maintained 40% of the control value during aortic cross-clamping. However, MEP amplitudes in groups 1 and 2 gradually attenuated and almost disappeared. Groups 1 and 2 amplitudes were lower than those in group 3, 30 min after aortic cross-clamping (p<0.001). Twenty-four hours after ischemia, the Tarlov score in group 3 was 3.5+/-0.5 and was higher than scores from groups 1 and 2, which were 0.5+/-0.5 and 1.3+/-1.2 (mean+/-SD, p<0.001, and p<0.001), respectively. Histopathologically, normal spinal cord motor neurons in group 3 were preserved to a significantly greater extent than in groups 1 and 2 (p=0.0031, and p=0.0282, respectively). There was a strong correlation between Tarlov scores at 24h and the number of normal motor neurons in the anterior horns of spinal cords (r=0.897; p<0.001). CONCLUSIONS: Intra-aortic propofol injections produce the quick suppression of MEP amplitudes and protect spinal cords from ischemia during aortic cross-clamping.

Effects of Ginkgo biloba extract on lipid peroxidation and apoptosis after spinal cord ischemia/reperfusion in rabbits.

OBJECTIVE: To study the effects of Ginkgo biloba extract (GBE) on lipid peroxidation and apoptosis after spinal cord ischemia/reperfusion (I/R) in rabbits. METHODS: Spinal cord I/R injury model was established according to the description of Erten et al. A total of 27 New Zealand white rabbits were divided into three groups randomly: a sham group (9 rabbits treated with sham operation but without aortic occlusion), a model group (9 rabbits treated with aortic occlusion and volume-matched saline), and a GBE group (9 rabbits treated with aortic occlusion and Ginaton (100 mg/kg) injected 30 minutes before aortic clamping and at the onset of reperfusion). The neurological outcomes were evaluated at 24 and 48 hours after reperfusion, respectively. The spinal cord malondialdehyde (MDA) level, superoxide dismutase (SOD) were then detected. Neural cell apoptosis was determined by terminal deoxynucleotidyl t-ransferase (TdT)-mediated dUTP-fluorescence nick end labeling (TUNEL) method and the expression of bcl-2 and bax were examined histologically in the spinal cord with immunohistochemistry. RESULTS: I/R produced a significant decrease in neurological scoring. The motor scores of the GBE group were significantly higher than those of the model group at 24 and 48 hours after reperfusion (P<0.05). Compared with the model group, GBE ameliorated the down-regulation of SOD and produced a significant reduction of the MDA level (P<0.01). The positive cells for TUNEL in the model group were much more than those of the GBE group (P<0.01). The bcl-2 was up-regulated after I/R, especially in the GBE group (P<0.01). The up-regulation of bax was greatly diminished by GBE (P<0.01). CONCLUSIONS: GBE has protective effects against spinal cord I/R injury, and the mechanism may be that it can scavenge oxygen free radicals and inhibit the apoptosis of neural cells.