| Objective: Make a model of rat cerebral ischemia for 2 hours and reperfusion for 4 hours,namely the middle cerebral artery occlusion(MCAO)model,from N-methyl-D-aspartate receptor 3(N-methyl-D-aspartate Receptor 3,NR3)to investigate the role of NR3 subunit in neuroprotective effect of clonidine on acute focal cerebral ischemia in rats.Methods: 10-week-old male SD rats were selected and divided into sham operation group,ischemia reperfusion(I/R)group,clonidine 20 μg/kg group,clonidine 40 μg/kg group,yohimbine 5 mg/kg + clonidine 40 μg/kg group,and MK-801 positive control group;one week of pre-administration,the sham operation group and the ischemic group were given normal saline,the other groups were given corresponding drugs.(1)Making rat MCAO model,then neurobehavioral scores and TTC staining were performed after 2 hours of ischemia and 4 hours of reperfusion.(2)HE staining was used to observe the changes of neuron morphology in cortex and hippocampus of each group of rats after acute focal cerebral ischemia.(3)Real-time fluorescence quantitative PCR was used to detect the expression levels of NR3 A and NR3 B m RNA in cortex and hippocampus of each group of rats after acute focal cerebral ischemia.(4)Immunohistochemical technique was used to detect the expression levels of NR3 A and NR3 B molecules in the cortex and hippocampus of each group after acute focal cerebral ischemia.(5)Immunoblotting technique was used to detect the expression levels of NR3 A and NR3 B total protein in cortex and hippocampus of rats after acute focal cerebral ischemia.(6)Immunoblotting technique was used to detect the expression levels of NR3 A and NR3 B cell membrane proteins in the cortex and hippocampus of rats after acute focal cerebral ischemia.RESULTS:(1)TTC staining results showed that the infarct volume of the ischemic group increased significantly compared with the sham operation group(P<0.01),the infarct volume of the clonidine 20 μg/kg group,the clonidine 40 μg/kg group and the MK-801 group significantly decreased compared with the ischemic group(P<0.05,P<0.01),compared with the clonidine 20 μg/kg group,the infarct volume of the clonidine 40 μg/kg group was further reduced(P<0.05),and the infarct volume significantly increased with the addition of yohimbine(P<0.05).Neurological function scores results showed: the neurological function score of the ischemic group significantly increased(P<0.01),compared with the ischemic group,the neurological function score of the clonidine 40μg/kg group was significantly reduced(P<0.05).(2)HE staining results showed that the ischemic group in cortex and hippocampus had ischemic changes,increased the numbes of damaged neurons(P<0.01),disordered cell arrangement and irregular shape.In the clonidine 20 μg/kg group and clonidine 40 μg/kg group,neuronal damage in the cortex and hippocampus was alleviated,cells were arranged neatly,cell vacuoles were reduced,and the numbers of damaged neurons were reduced(P<0.01).Compared with the clonidine 20μg/kg group,the neuronal damage in the cortex and hippocampus of the clonidine 40 μg/kg group was further reduced(P<0.01).Compared with the clonidine 40 μg/kg group,the yohimbine + clonidine 40 μg/kg group had more severe neuronal damage(P<0.01),disordered cell arrangement and irregular shape in the cortex and hippocampus.(3)Real-time fluorescence quantitative PCR results showed: the expressions of NR3 A and NR3 B m RNA in cortical tissue increased after ischemia(P<0.01);The expressions decreased after using clonidine(P<0.01,P<0.05),while the hippocampus showed opposite trend with the cortex;after adding yohimbine,the expressions of NR3 A,NR3B m RNA in cortex and hippocampus decreased(P<0.01).(4)Immunohistochemical results showed:NR3A and NR3 B were mainly expressed in the cell membrane and cytoplasm.In cortex and hippocampus,the expressions of NR3 A and NR3 B decreased after ischemia(P<0.01),the expressions increased after using clonidine(P<0.01,P<0.05),but decreased after adding yohimbine(P<0.01,P<0.05).(5)Immunoblotting results of NR3 A,NR3B total proteins showed: in cortex and hippocampus,the expressions of total protein of NR3 A and NR3 B decreased after ischemia(P<0.01),and the expressions of NR3 A,NR3B total proteins increased in cortex after using clonidine(P<0.01),While in hippocampus the expressions of NR3 A and NR3 B total proteins increased in the clonidine 20 μg/kg group(P<0.01),but decreased after the addition of yohimbine in cortex and hippocampus(P<0.01).(6)Immunoblotting results of NR3 A,NR3B cell membrane proteins showed: the expressions of cortical NR3 A,NR3B and hippocampal NR3 B cell membrane protein and the ratio of membrane protein to total protein decreased after ischemia(P<0.01,P<0.05),but increased after clonidine(P<0.01),while the expressions decreased after adding yohimbine(P<0.01);the expressions of in hippocampus NR3 A membrane protein and the ratio of membrane protein to total protein increased after ischemia(P<0.01),the expression of in hippocampus NR3 A ratio of membrane protein to total protein increased in clonidine40 μg/kg group;after adding yohimbine,the expressions of hippocampal NR3 A cell membrane protein and the ratio of membrane protein to total protein decreased(P<0.01). |
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