| Part Ⅰ Experiment in vitroObjective:Confirmed that the cells of rat spinal cord neurons containing the calcium sensing receptor(CaSR)and calpain and investigate the role of CaSR and calpain in apoptosis induced by anoxia/reoxygenation(A/R)in rat spinal cord neurons by using of CaSR agonist(Gd Cl3)and CaSR inhibitor(NPS2390)on the anoxia/reoxygenation(A/R)model of rat spinal cord neurons in vitro.Methods:1.Culture of spinal cord neurons through the acquisition of newborn SD rat spinal cord tissue and identified rat spinal cord neurons(Neurofilament 200).Immunofluorescence confirmed the existence of CaSR and calpain in rat spinal cord neurons.2.Establishment of a rat model of anoxia/reoxygenation(A/R)in rat spinal cord neurons using cultured SD rat spinal cord neurons.That is,the use of 95% nitrogen into the cell culture plate with a closed air conditioning box,culture incubator for 1 hours at 37 degrees Celsius.Then the cells were cultured in Neurobasol-A solution containing 2%B27 and 10% fetal bovine serum for 24 hours.3.Effects of CaSR agonist(Gd Cl3)and CaSR inhibitor(NPS2390)on anoxia/reoxygenation(A/R)model of rat spinal cord neurons: randomly divided into four groups: normal control group(control);anoxia/reoxygenation group(A/R);anoxia/reoxygenation+CaSR activator group(A/R+Gd Cl3)and anoxia/ reoxygenation +CaSR inhibitor group(A/R+NPS2390).The changes of intracellular free calcium in spinal cord neurons were detected by laser scanning confocal microscopy(LCSM);the expression levels of CaSR,calpain and apoptosis related proteins Bax and Caspase-3 were detected by Western blotting;the apoptosis of spinal cord neurons in each group was observed by situ end labeling technique(TUNEL)of fragmental DNA.Result:1.CaSR and calpain were expressed in cytoplasm of rat spinal cord neurons.2.The model of anoxia/reoxygenation(A/R)in neonatal rat spinal cord neurons(SD)was successfully established.The expression of CaSR and calpain in anoxia/reoxygenation group was significantly higher than that in normal control group.3.Intracellular free calcium concentration in spinal cord neurons detected by confocal laser scanning microscopy(LCSM).The concentration of free calcium in anoxia/reoxygenation group(A/R)was significantly higher than that in normal control group(control),which was lower than that of anoxia/reoxygenation+activator group(A/R+ Gd Cl3)and higher than anoxia/reoxygenation+inhibitor group(A/R+NPS2390)group.The results of Western blotting and Tunel staining showed that the expression level of apoptosis related protein Bax and Caspase-3 in anoxia/reoxygenation group(A/R)was higher than that in normal control group(control),and the apoptosis was increased,which was lower than that of anoxia/reoxygenation+activator group(A/R+Gd Cl3)and higher than anoxia/reoxygenation+inhibitor group(A/R+NPS2390)group.Conclusion:CaSR and calpain are widely expressed in the neurons of spinal cord of rats,and they are expressed in the cytoplasm of neurons.The expression of CaSR and calpain was increased in rat spinal cord neurons during anoxia/reoxygenation(A/R)injury.The use of CaSR agonist(Gd Cl3)can increase the overload of calcium,thereby increasing the apoptosis of spinal cord neurons during anoxia/reoxygenation injury.On the contrary,the use of CaSR inhibitor(NPS2390)can reduce the intracellular calcium concentration,so that the expression of calpain decreased,thus reducing the apoptosis of spinal cord neurons during anoxia/reoxygenation injury.Part Ⅱ Experiment in vivoObjective:Confirm that the rat spinal cord tissue containing calcium sensing receptor(CaSR)and calpain and study the role of CaSR and Calpain in inducing apoptosis in rat spinal cord ischemia reperfusion injury(SCIRI).Methods:1.Extract of spinal cord tissue from healthy adult SD rats.Immunohistochemical staining were used to observe the expression of CaSR and calpain in spinal cord tissues.2.Adult SD rats were treated with modified Zivin method to establish the model of spinal cord ischemia-reperfusion injury(SCIRI)in rats.The effectiveness of the SCIRI model in vitro is evaluated by TUNEL staining and BBB scale.3.The SD rats were randomly divided into normal control group(sham operation group),model group of spinal cord ischemia reperfusion injury(SCIRI),SCIRI model+CaSR agonist group(SCIRI+ Gd Cl3)and SCIRI model +CaSR inhibitor group(SCIRI+NPS2390).The expression of CaSR/calpain in spinal cord of each group was compared by immunohistochemistry staining and Western blotting.TUNEL staining was used to observe the apoptosis of spinal cord and BBB scale was used to compare each group of SD rats after the operation.Result:1.CaSR and calpain were expressed in the front horn,posterior area of gray matter and the boundary areas between gray and white matter of the spinal cord in rats,and they were expressed in cytoplasm of rat spinal cord neurons.2.The model of spinal cord ischemia-reperfusion injury(SCIRI)in adult SD rats was established successfully with modified Zivin method,and the TUNEL staining and the BBB scale showed that the apoptosis of SCIRI model group was higher than that of sham operation group.3.Compared with spinal cord ischemia-reperfusion injury(SCIRI)model group,TUNEL staining in the SCIRI model+CaSR agonist group(SCIRI+ Gd Cl3)showed that the apoptosis of spinal cord tissue was further increased.The expression of CaSR and calpain in the SCIRI model+CaSR inhibitor group(SCIRI+NPS2390)significantly decreased by immunohistochemistry staining and Western blotting,TUNEL staining showed that the apoptosis in spinal cord of rats can be reduced,while the BBB scale can improve in rats.Conclusion:CaSR and calpain were expressed in the front horn,posterior area of gray matter and the boundary areas between gray and white matter of the spinal cord in rats,and they were expressed in cytoplasm of rat spinal cord neurons.The expression of CaSR and calpain in spinal cord ischemia-reperfusion injury(SCIRI)was higher than that of sham operation group.CaSR agonist(Gd Cl3)can increase the apoptosis of spinal cord ischemia-reperfusion injury.On the contrary,the use of CaSR inhibitor(NPS2390)can decrease the expression of calpain,reduce the apoptosis of spinal cord ischemia-reperfusion injury,and alleviate the spinal cord injury in rats. |
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