Study On The Relativity Between Astrocyte And Synaptic Reconstruction After Ischemia As Well As The Influence Of Acupuncture In Rats

Searching for effective treatment and explain its mechanism are the investigating center in the field of brain research.Brain plasticity especially synaptic plasticity has becoming focus of research which provides much proof to the study of ischemia mechanisms.The researching to astrocyte has gotten good results in recent years.Astrocyte not only have plasticity itself it can also adapt to the activity of synapse by changing its function so as to regulate synaptic plasticity in some extend.We try to clarify the specific path and mechanism of ischemia treated with Acupuncture by the investigation to the synaptic plasticity and the influence of astrocytes.Some aspects are related in following text.1.Literature researchThese parts discuss the nominating of stroke in ancient Chinese medicine.The etiology and pathology of stroke and the ancient records relating to Acupuncture treatment were also discussed which can guide us to deep recognizing to ancient background of stroke.In recent years the researching to cerebral vascular disease had found that brain function and organization had ability to self-recovery and reconstruction at different times. In text the brain plasticity interfering by Acupuncture as well as the astrocyte and its reaction after ischemia were also summarized which can put theory foundation to farther research about the ischemic mechanism treated with Acupuncture.2.Experiments researchPurposeIn this study the changes of synaptic ultrastructure at different times,the dynamic changes of glial gap junction and glial glutamate transporter as well as the influence of Acupuncture on above by using Baihui(GV20)and Dazhui(GV14)acupoints were observed. Methods(1)Ninety Wistar rats were randomized into sham-operated group,model group and eletroacupuncture(EA)group.Each group was observed at 1hour,1day,3days,1week and 3weeks after ischemia and EA,respectively.Heat-coagulation-induced occlusion of the middle cerebral artery was performed to establish the model of focal cerebral ischemia.The middle cerebral arteries were exposed without operation in sham-operated group.EA was delivered to Baihui(GV20)and Dazhui(GV14)in EA group.(2)Neurofunctional scores and pathomorphology observation:observations of behavior performance refer to the methods used by Berderson and Belyayev at 1day,3days, 1week and 3weeks.The pathomorphology of ischemic area dyed by the way of HE were observed by at 1hour,1day,3days,1week and 3weeks.(3)Synaptic ultrastructure:3 rats of each group in 15 groups were chosen at random 1hour,1day,3days,1week and 3weeks.The rats were fixed by intracardial perfusing.The brains were exposed and the samples were taken from ischemic area of cerebral cortex. Electronmicrographs of synapses were observed at magnification of 1500 by electronmicroscopy JEM-1200EX.Synaptic Number Density(Nv),Surface Density(Sv), the thickness of postsynaptic density(PSD),the width of synaptic cleft,the length and curvature of synaptic interface were measured to observe the changes and the effects of EA.(4)The expression of glial gap junction CX43 and CX43mRNA:90 rats were intracardially perfused at 1hour,1day,3days,1week and 3weeks.CX43 and CX43mRNA were detected with immunohistochemical method and in situ hybridization(ISH).(5)The expression of GLT-1(glial glutamate transporter-1)and GLT-1mRNA:90 rats were intracardially perfused at 1hour,1day,3days,1week and 3weeks.GLT-1 and GLT-1mRNA were detected with immunohistochemical method and in situ hybridization(ISH).(6)The correlating analysis between synaptic ultrastructure and CX43,GLT-1:taking canonical correlation analysis in same time and same group using Stata10.0 to analyze between Synaptic Number Density(Nv),the thickness ofpostsynaptic density(PSD)and CX43,GLT—1.Results(1)Neurofunctional scores:there represent neurological deficits in different extent of model group and EA group.For lday,most have 2 marks of neurofunctional scores.The scores of model group increased from 1day to 3days.With strengthen of dysfunction and less of rat's activities,the scores of model group decrease gradually at 1week and 3weeks. There have significant differences of model group compared with sham-operated group from lday to 3weeks(P＜0.05,P＜0.01).With the ongoing of brain injury,the neurofunctional scores of EA group decreased,and neurofunction improved.The scores had significant differences between EA and model group from 3days to 3weeks.(2)Synaptic ultrastructure:At early stage,sypnapses of cerebral cortex were damaged and synaptic structure broke.The Nv and Sv of model group decreased significantly at 1hour,1day and 3days(P＜0.01,compared with sham-operated group of same stage), increased at 1week and which still had significant differences compared with sham-operated group at 3weeks(P＜0.01).The Nv and Sv of EA group increased significantly compared with model group at lday(P＜0.01)and which still higher than model group significantly at 3 days and 1week (P＜0.001).The thickness of PSD in model group decreased at all stages(P＜0.05,compared with sham-operated group of same stage).There had no difference between EA and sham-operated group at all stages,but the differences between EA and model group were significant(P＜0.05,P＜0.01).The width of synaptic cleft decreased significantly at 1hour,1day and 3days in the model group.There had significant differences of model group compared with sham-operated group from lday to 3weeks(P＜0.05,P＜0.01).The width of synaptic cleft decreased at 1hour in the EA group and which had significant differences compared with sham-operated group(P＜0.05).There had no difference between EA and sham-operated group at 1day and the width of synaptic cleft of EA group were higher than sham-operated group at 1week and 3weeks.EA group had significant differences compared with model group from 3day to 3weeks(P＜0.05,P＜0.01).The curvature of synaptic interface decreased obviously at,3days,1week and 3weeks in the model group and there had significant differences compared with sham-operated group(P＜0.05,P＜0.01).It decreased at 1day in the EA group,but there had less decrease compared with model group at all stages and there had significant differences between these two groups(P＜0.05,P＜0.01).(3)The expression of glial gap junction CX43 and CX43mRNA:The CX43 expressions of model group with immunohistochemical method had no change at 1hour, decreased at 1day,drop down to most degree at 3days and didn't recover at 1week.There had significant differences compared with sham-operated group from 1day to 1week (P＜0.05,P＜0.01).The CX43 expressions of EA group decreased markedly at 1day,3days and 1week,but which higher than model group.There had significant differences compared with model group at these stages(P＜0.05,P＜0.01). The CX43mRNA expressions of model group and EA group with ISH method had no differences compared with sham-operated group.The CX43mRNA expressions of model group decreased at 3days and 1week(P＜0.001,compared with sham-operated group of same stage).The expressions of EA group were higher than model group(P＜0.01, compared with model group at 3days and lweek). (4)The expression of GLT-1 and GLT-1mRNA:The GLT-1 expressions of model group with immunohistochemical method decreased at 1hour,reduced mostly and 3days and recovered at 1week,which There had significant differences compared with sham-operated group(P＜0.001).The GLT-1 expressions of EA group decreased at 1day,but were higher than model group.At 3days the GLT-1 expressions of EA group increased obviously than model group(P＜0.001,compared with sham-operated group,P＜0.01, compared with model group at 1day and 3days).The expression GLT-1mRNA of model group with ISH method decreased at 1day, reduced mostly at 3days.Both groups increased at 1week and there had significant differences compared with sham-operated group from 1day to 3days.The expressions of EA group were higher than model group from 1day to 1week.(5)The correlating analysis between synaptic ultrastructure and CX43,GLT-1:There have correlation between Synaptic Number Density(Nv)and GLT-1 at 1hour.There have correlation between the thickness of postsynaptic density(PSD)and GLT-1 at 1day. Synaptic Number Density(Nv)and CX43 have correlation at 3days.At 3weeks They all have correlation of Synaptic Number Density(Nv),the thickness of postsynaptic density (PSD)and CX43,GLT—1.After electroacupuncture they all have correlation of,at 1hour as well as 1week.Synaptic Number Density(Nv),the thickness of postsynaptic density(PSD) and GLT—1 at 3days and Synaptic Number Density(Nv),CX43 have correlation at 3weeks.Conclusion(1)The models were made successfully according to the results of neurofunctional scores,which indicate that EA could ameliorate and reduce neurological injury.(2)From the early stage and with the prolongation of ischemia lesion,synaptic structures were damaged.The amount and area of synapses,the thickness of PSD,the width of synaptic cleft and the curvature of synaptic interface had dynamic changes.EA could promote the reconstruction of synapses from early stage by influence the synaptic ultrastructure.(3)The decreasing of CX43 expression was one mechanism of ischemic aggravation. EA could improve the expression of CX43 which provide environmental and material basis to the recovery of.(4)The decreasing of GLT-1 expression could reduce the reabsorbing of glutamate by astrocytes which could cause the accumulation of glutamate.EA could improve the expression of GLT-1.These were the key effects to maintain the morphology and function of synapses.(5)At different stages after ischemia,the changes of CX43,GLT-1 have the correlating with synaptic ultrastructure.After electroacupuncture they still have correlation. The recovery of ischemia after acupuncture was the results of correlating between astrocytes and synapses.Astrocytes could promote Synaptic Reconstruction.From above conclusion we considered initially acupuncture could start the adjustment of neuron-glial network which could promote the synaptic reconstruction.These were the key mechanisms of treating ischemia by acupuncture.