The expression of caspase-1 after acute spinal cord injury in rats
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Objective To investigate the expression of cysteinyl aspartate specific protease-1(caspase-1) after acute spinal cord injury in rats and its significance.Methods Totally 36 healthy adult SD rats were randomly divided into the injury group and the control group.Each group was further divided into 3 time points: 8h,3d and 7d,6 rats were sacrificed at each time point.The control group only underwent T8 and T9 laminectomy without injury.In the injury group the model of acute spinal cord injury was induced with Nystrom's method.The spinal cord was dissected for morphological study by HE staining and the expression of caspase-1 at each time point was detected by immunohistochemical method.Results The low expression of caspase-1 was observed in the control group.The expression of caspase-1 was higher in the spinal cord injury group than in the control group at 8h,went on increasing and peaked at 3d,and slightly decreased at 7d.The level of caspase-1 in the spinal cord injury group was obviously higher than that in the control group at each time point(P0.01).Conclusion The expression of caspase-1 increases quickly after acute spinal cord injury,which may play an important role in the pathogenesis of acute spinal cord injury.Topics:
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[Objective]To study the protective mechanisms of neurotrophin-3 (NT-3)on spinal cord injury.[Method]using the method of Allen's (WD) to make the animal model of acute spinal cord injury of rats, 105 SD rats were randomly divided into three groups: control group, experimental group and sham group. A thin plastic tube was situated in subarachnoid space below the injury level for perfusion. Rats in experimental group received NT-3 20μl(including NT-3 200ng) from the tube at 0, 4, 8, 12, 24h and 3d, 7d after injury; the saline-treated animals received the equal volume of normal saline at the same time as control. The animals in sham group only received opening vertebral plate and putting tube in subarachnoid space. The rats were sacrificed at 4,8,12,24h and 3, 7, 14d postinjury (n=5). The expression levels of Fas protein in rats spinal cord were detected by immunohistochemistry assay. [Result]The levels of Fas protein in control group were significantly increased as compared with those in sham group, and the level reached peak at 24h after spinal cord injury. The levels of Fas protein in NT-3 group were significantly decreased as compared with those in control group.[Conclusion]NT-3 can protect spinal cord from injury in vivo. One of mechanisms is that inhibits abnormal expression of Fas protein,then inhibits apoptosis after spinal cord injury.
Subarachnoid space
Neurotrophin-3
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Objective:To observe relationship and significance of changes in the expression and the level of Caspase-8 in the tissue and blood plasma after rat spinal cord was acutely injured.Methods:Eighty-four Wistar rats were randomly divided into seven groups with 12 in each group,and one group was used as control group and the 6 others were prepared into acute spinal cord injury models,as 0h,6h,12h,24h,48h and 72h injury groups.The caspase-8 value in the tissue and blood plasma at different time points was detected after spinal cord injury by applying immunohistochemical method and ELISA.Results:Soon after acute spinal cord injury,the tissue and blood plasma Caspase-8 showed the positive expression,at 24h-48h,the Caspase-8 expression in the tissue and blood plasma reached a peak,at and 72h after injury began to decrease but still higher than that of the normal control group.Conclusion:At the early stage of the spinal cord injury,the Caspase-8 expression in the tissue and blood plasma is significantly increased,with a corresponding relation to time,which will help the selection of the opportunity of the clinic intervention in acute spinal cord injury .
Cord blood
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Objective To investigate the effects of exogenous transforming growth factor β1 on the apoptosis of nerve cells and its functions following spinal cord injury in rats.Methods Ninety-six male Wistar rats were randomly divided into 4 groups:group A (control group),group B (spinal cord injured group),group C (spinal cord injuried +TGF-β1 treated group),group D (spinal cord injuried +anti-TGF-β1 treated group).The rat model of spinal cord injury was found by the Allen's method.In group C and D,drugs were injected into subarachnoid cavity continuously by minipump.The changes of spinal cord were observed by HE staining.Nerve cells apoptosis was detected by transferase-medi-ated dUTP nick end labeling (TNUEL) method and the expression of cell apoptosis factor Fas by immunohistochemistry staining.Functional recovery of hind limbs was measured by Basso-Beattie-Bresnahan locomotor open field behavioral rating test.Results The expression of TGF-β1and Fas increased following spinal cord injury.The amount of TGF-β1and Fas reached the peak 7days and 1day after injury.The apoptosis of neuron had increased and peaked 8 hours after injury.The apoptosis of neuroglia cells had increased and peaked 7 dayss after injury.The slight changes was found in spinal cord in group C.Both of the number of apoptosis of nerve cells and expression of the apoptosis factor (Fas) decreased significantly; compare with group B and D.The BBB score was higher in group C than that in group B and D.Conclusion TGF-β1 can improve the functional recovery of spinal cord by inhibiting the nerve cell apoptosis after the spinal cord injury.
Key words:
Spinal cord injuries; Transforming growth factor-batal; Apoptosis
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Effect of hyperbaric oxygen on MMP9/2 expression and motor function in rats with spinal cord injury.
To study the effect of hyperbaric oxygen intervention on the microenvironment of nerve regeneration after spinal cord injury modeling and to explore the possible mechanism of nerve regeneration and functional recovery in rats with spinal cord injury. In 98 adult female SD rats, 90 successful models were obtained, which were divided into sham group, spinal cord injury group and hyperbaric oxygen group using randomized block method, 30/group. Spinal cord injury rat model was established in accordance with the modified Allen method. Motor function was assessed at the time points of before modeling, one day, three days, one week, two weeks, three weeks and four weeks after modeling respectively by BBB rating, inclined plane test and improved Tarlov score. At 3 days after modeling, apoptosis of neuronal cells in spinal cord injury region in experimental group was detected by TUNEL method; gene and protein expression of MMP9/2 in spinal cord injury and surrounding tissues was detected by RT-PCR and Western blot assay. At 4 weeks after modeling, histopathological morphological changes in spinal cord injury were observed by HE staining; fluorogold retrograde tracing was used to observe the regeneration and distribution of spinal cord nerve fibers and axon regeneration was observed by TEM. The three motor function scores in hyperbaric oxygen group at each time point after two weeks of treatment were significantly increased compared with spinal cord injury group (P < 0.05). At 3 d after modeling, apoptosis index in hyperbaric oxygen group were significantly lower than those in spinal cord injury group (P < 0.05). At 72 h after modeling, compared with spinal cord injury group, MMP9/2 gene and protein expression in hyperbaric oxygen group was significantly lower (P < 0.05). At four weeks after modeling, fluorogold positive nerve fibers were the most sham group, followed by hyperbaric oxygen group and spinal cord injury group in order; the differences among the groups were statistically significant (P < 0.05). Under TEM, newborn unmyelinated and myelinated nerve fibers could be observed in the middle cross-section in the sham group and hyperbaric oxygen group; unmyelinated and myelinated nerve fibers in hyperbaric oxygen group were more than those in spinal cord injury group. Hyperbaric oxygen therapy played a protective effect on spinal cord injury through reducing apoptosis of neuronal cells and expression of MMP9/2 gene and protein in rats with spinal cord injury.
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Objective:To explore the Parecoxib on apoptosisfollowingspinal cord injury in different time in rats model of spinal cord injury.Methods:72 healthyratswerechosen andtheirbody weight varied from 250 gram to 310 gram.The rats were divided into two groups randomly:controlled group(36)and cox-2 group(36).All of rats spinal cord injury model were established with ALLEN bump,equipment(25g cm).The model of COX-2 group injected parecoxib(5mg/Kg) every day until they were sacrificed.The model of controlled group injected NS(0.5ml/d)every day until they were sacrificed.Fabrication of tissue section:after spinal cord injury 1d,3d,5d,8d,14d,21d,we sacrificed rats inbatch.4% formaldehydum polymerisatum were pured into the cardiac muscle and fixed for 24h.Then obtain the specimen of spinal cord from T5 to T13.Establishing slices,HE dyeing,bcl-2 dectation,terminal doxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL).Results:Bcl-2 dectation:Duringthefirst day after spinal cord injury,we found many bcl-2 protein in the spinal cord tissue.In control group,they reached the peak on the third day after spinal cord injury.In COX-2group,they reached the peak on the eight hday after spinal cord injury,Bcl-2protein were moreinglial cells than those innervecells.They begn to decrease after the for teenthday of spinalcordinjury(P0.05).Tunel dectation:In control group,after 24 hours,there many positive ceils,most of them are gliao cells,it reached the peak after 3-8 day,and then,they were decreased gradually,in cox-2 group,positivecells obviouslylessthanthoseOfcontrolgroup(P0.05).Conclusion:Fromthe lnvestigate,we proved that parecoxib could improve the microcirculation,get rid of oxygen free radieal,resist oxygenation,so as to restrainthe apoptosis of nerve cell and glial cell,lesson spinal cordinJury,improveTOTecoverspinalnerve.Therefore,duringearly time of spnalcord injury,we made use of parecoxib with other medicine,it should lesson spinal cord injury and provide a new methodfortreatment of spinal cord injury.
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Objective:To observe the expression of heat shock protein 70(HSP70) in rats with acute spinal cord injury.Methods:Fifty-six Wistar rats were randomly divided into 7 groups.Six groups were prepared into acute spinal cord injury models and the other group was used as control.The animal models of spinal cord injury were established at 2h,6h,12h,24h,48h and 72h respectively after injury.The HSP 70 expression in the spinal cord from each group at different time points was studied by method of west-blot.Results:The expression of HSP70 was already found at 2h after the spinal cord injury,reached the peak at 24h and lasted to the time point of 72h after injury.Conclusion:The HSP70 expression is the response of the body to stress.HSP70 is obviously increased in the gray nucleus of the spinal cord from 2h to 72 h after acute injury.HSP 70 may play a role in the prevention of the spinal cord from secondary lesion.
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Objective:To investigate the effect of the cyclosporine A(CsA) on the expression of cycloxygenase-2(Cox-2) and tumor necrosis factor-α(TNF-α) in acute spinal cord injury,and the mechanism of CsA on spinal cord injury in rat.Method:180 rats were randomly divided into control group,injury group and the CsA-intervention group with 60 rats in each group.An impact of 2.5g·cm was used to induce spinal cord injury in rat both in injury group and in intervention group.In CsA-intervention group,CsA(2.5mg/kg) was administrated in rats through caudal vein injection 1h after injury,then repeated per 12h after injury,while the control group and injury group received the same dose of normal saline at the same time point.The injured spinal cord segment was collected and processed pathologically at 2h,6h,12h,24h,4h8 and 72h after operation.HE staining and immunohistochemistry staining(TNF-α by the EnVision detection system and Cox-2 by the SP detection system) were performed for every specimen.The expressions of Cox-2 and TNF-α were analyzed quantitatively by HPIAS-2000 high-resolution color graphic pathology report management system.Result:HE staining showed normal of rat spinal cord in control group without bleeding and necrosis.Edema and hemorrhage instead of necrosis in spinal cord gray matter cauld be seen in 2h,6h after injury both in injury group and in intervention group.A wide range of focal hemorrhage and formation of cysts after bleeding occurred in 12h or 24h after injury in spinal cord gray matter.Neurons appeared as swelling,part of nucleus concentrated,HE staining increased,numerous red blood cells and myelin slight swelling were seen in the white matter as well.Neurons appeared pyknosis,necrosis,dissolution in 48h or 72h after injury in the spinal cord gray matter.Microglial cell proliferation and neutrophil infiltration were evidenced in the injured spinal cord.Numerous red blood cells and myelin swelling were seen in the white matter.A great quantity of emptiness,inflammation cell infiltration and microglial cell proliferation were evidenced.Pathological severity at any time point in intervention group was milder than injury group.The expression of Cox-2 remained uncertainty in control group and reached peak at 6h after injury,then decreased.Cox-2 expression in injury group in 72h was higher than control group(P0.05),while was equal to intervention group in 48h after injury(P 0.05),Cox-2 expression in intervention group was lower than injury group at each time point(P0.05).The expression of TNF-α remained uncertainty in control group.The expression of TNF-α was detected 2h after injury in injury group and the intervention group,which reached peak at 12h after injury,then decreased.The expression of TNF-α in injury group was higher than control group(P0.05) in 72h after injury.The expression of TNF-α in intervention group resumed to the baseline level of the control group(P0.05) 72h after injury.TNF-α expression in intervention group was lower than injury group at each time point(P0.05).Conclusion:CsA down-regulate the expression of Cox-2 and TNF-α significantly in SCI rats,which can alleviate the secondary spinal cord injury.
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Objective To study the effect of LY294002 on the expression of Caspase-3 after acute spinal cord injury. Methods 104 SD healthy adult rats were chose,and randomly divided into the sham group(8),the spinal cord injury group(48),LY-294002 group(48) on average.The spinal cord injury(SCI) was induced with Nystrom's way by the moderate compression at the level of T8 and T9 spinal cord.The expression of Caspase-3 was observed by immunochemistry and western blot methods. Results There were few expression of Caspase-3 positive cells in sham group by immunohistochemical study.The number of positive cells of Caspase-3 was increased obviously at 8 hours,got to a peak at 3 days,and was lowest at 7 days.Compared with SCI group,The number of positive cells of Caspase-3 at the same time point was significantly lower in injury sites of rats in LY-294002 group.There were few expression of Caspase-3 in sham group by western blot.The expression of Caspase-3 was increased obviously at 8 hours,got to a peak at 3 days,and was lowest at 7 days.Compared with SCI group,The expression of Caspase-3 at the same time point was significantly lower in injury sites of rats in LY-294002 group,and agree to the result of immunochemistry.Conclusion LY-294002 could downregulate the expression of Caspase-3 of rat after acute spinal cord injury
Immunochemistry
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Objective To study the effect of Ginsenosides on protecting the neuron after spinal cord injury. Methods Modified Allen impaction methods were used to establish spinal cord injury model in rats. Rats were randomly divided into three groups:control group,injury group and Ginsenosides group,36 rats per each group. The rats were sacrificed at 6h,12h,1d,3d,7d and 14d,and T9 spinal cord specimen was obtained. Parallel tilt plate test and BBB evaluation were performed to observe the outcome of neural functional recovery; TUNEL methods was used to detect the apoptosis cells in spinal cord; Western blot was used to detect the expression level of Bcl-2 and Bax protein; RT-PCR was performed to detect the mRNA expression of Caspase-3 in spinal cord. Results Some degree of neural functional recovery was found in injury group and Gs group during the 14 days' observation,scores of Gs group were significantly higher than that of injury group (P0.05 or P0.01); The TUNEL-positive cells showed a maximum presence at 3d after surgery,and decreased significantly at every time point in Gs group compared with injury group (P0.01); At every time point after surgery,the expression of Bcl protein in Gs group was higher than that in injury group (P0.01),12h、1d、3d、7d、14d after surgery,the expression of Bax protein in Gs group was lower than that in injury group (P0.05 or P0.01); The mRNA expression of Caspase-3 in Gs group were lower than that in injury group at every time point after surgery (P0.01). Conclusion Gs has the neuroprotective effect after spinal cord injury. Inhibiting apoptosis of neuron after spinal cord injury via enhancing the rate of Bcl-2/Bax and impressing the mRNA expression of Caspase-3 may be one of possible mechanisms.
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Objective To investigate the expression and significance of Slit2 in different time point after the spinal cord injury of rats.MethodSeventy adult wistar rats were randomly divided into three groups: spinal cord injury by fully transection on T10 level spinal cord (Group A); laminectomy and the spinal cord uninjuried (Group B); natural without operation (Group C).Then the rats were sacrificed and the spinal cord was taken out fresh quickly on different time-point(12h, 1, 3, 5, 7d after operation). The tissues perfusion by formaldehyde were taken out on 3, 5, 7, 14d after operation. The expressions of Slit2 were tested by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical measurement. By means of above,the author investigated the expressions and location of Slit2 after the spinal cord injury.All data were statisticsed by SPSS 11.5 software.ResultThe expression of Slit2 mRNA of appeared in the spinal cord tissue 12 hours after injury, reached peak on the 3rd day, declining gradually later. The positive expression of Slit2 located in the cytoplast on oligodendrocyte and astrocyte.The positive cells were found at 3d after spinal cord injury, reached peak on 7d after injury, declining after 14d. The change of Slit2 was correlative with the rehabilitation and regeneration of the axon on the forepart period.ConclusionAs an important factor in axonal growth-guidance,the author crewed that Slit2 may be participated in the regeneration and rehabilitation of axons after the spinal cord injury.
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