Effects Of Fastigial Nucleus Electric Stimulation On Synaptic Ultrastructure And Synaptophysin Expression In Hippocampus Of Neonatal Rat

Background and ObjectiveNeonatal hypoxic-ischemic brain damage(HIBD) is an important neonatal disease.It is a major cause of disability in children.So far there is no ideal treatment. The pathogenesis and treatment of HIBD is focus all the time.Synapses is the most important part for communicating information between neurons.Synaptophysin(p38) is a molecular of 38KD of the calcium-binding glycoprotein.It involves in the neurotransmitter acetylcholine,glutamate release and regulation of synaptic plasticity,and synaptophysin(p38) is closely related to learning and memory activites.Its product positioning can quantitatively reflect the distribution and density of synapses.One of the important synaptic plasticity and nerve reconstruction symbols is p38.The early intervention research of neonatal hypoxic-ischemic brain injury is hotspot in recent years.Electrical stimulation as a physical therapy has been used in clinical to promote nerve rehabilitation.But the ultrastructure of nerves reaserch after ischemic brain injury is less.By observing neurons,synaptic ultrastructure and the expression of synaptophysin changes,we explore the mechanisms of nerve injury and repair function reconstruction after FNS. Materials and method1.180 healthy seven-day-old newborn SD rats,female or male,twelve to sixteen gram,were randomly divided into three groups:control group(n=60),model group(n=60) and electric stimulation group(=60).Every group was randomly divided into 3days group(n=15),7days group(n=15),14days group(n=15) and 21days group(n=15)again.2.The preparation of HIBD model:seven-day-old newborn SD rats,inhaled ether to anesthesia,were incisioned from the left neck after disinfection,abrnptioed left common carotid artery,then,ligationed with thread and sutured the incision,put into hypoxic box,inhalted 92%N₂ and 8%O₂ for two hours.Control group only separated from the left common carotid artery but did not ligation and hypoxia.3.The method of electric stimulation:electric stimulation group was used electric stimulation for half an hour,one time everyday after surgery.The electrical stimulation intensity was 3V,and frequency was 70HZ,so that the lightest tremor in rats physically is appropriate.The sham operated group and model group were not used electric stimulation but catched to fix in the corresponding period.4.Sample collection and preparation:Fastigial nucleus electric stimulated for a long time 3d,7d,14d,21d.Electrical stimulation to stimulate the end of the period with a corresponding take group 5 rats were killed cut the chest,exposing the heart,by the left ventricular perfusion 1g / L glutaraldehyde,until the right atrial appendage outflow of clarified liquid,with 10g / L of glutaraldehyde fixation quickly check the minds of Mace into 1 mm×1 mm×2 mm slice into 40g / L of glutaraldehyde fixed preservation,after the application of electron microscope to observe the brain cortex neurons and synaptic ultrastrncture changes.The remaining rats would be killed and the brain tissue would be got after cardiac perfusion in corresponding period.The changes of synaptophysin expression in neonatal rat brain was observed by immunohistochemistry staining assay.5.Index measurement1)Synaptophysin(p38) immunohistochemistry staining:positive expression of synaptophysin were brown-yellow color.Pathological image analysis system measured p38 slices of hippocampal CA1 area absorbance value(OD).Each slice was measured absorbance value from five horizons and chosen the mean.Simultaneously the same slice of the corpus callosum OD was measured as the background OD values, with correction of absorbance values of COD(measured value-background value) for comparison and analysis,in order to avoid staining of non-specific staining caused by error.2) Hippocampal neurons and synaptic ultrastructure observation:the glutaral dehyde-fixed samples by PBS after washing liquid,added 1%osmic acid in the post-fixed by dehydration,embedded,polymerization,50nm ultra-thin sections, acetate uranium and lead citrate double staining Electronics.H-7500 type transmission electron mic- roscope observe the fiber neurons and synaptic ultrastructure of change.6.All of the datas would be described by(?)±s and analyzed by SPSS 15.0.More than a few means would be compared by one-way analysis of variance and the least significant difference between two groups would be used.The mean difference was significant at the 0.05 level.ResultsHippocampal neurons and synaptic ultrastructure1) HIBD group the neuronal shrinkage,the amount of organelles decreased, cytoplasm obviously edema,swellen mitochondria obviously.2) HIBD group the synapse quantity decreased,synaptic space fusion,synaptic vesicle obviously decreased.3) Interference group different times mitochondria hydrops gradually lighten, synaptic space gradually cleared,synaptic vesicle increased,pathological changes obviously lessen compared to hypoxic-ischemic group at the same time,and no apparent abnormality compared to control group at the 21d.Synaptophysin(p38) immunohistochemistry1) There were no marked difference of immunohistochemically reactived COD of p38 among the three groups at 3d(P＞0.05).2) The corrected opticaldensities(COD)of immunoreactive products of the hippocampal p38 were significantly decreased in model group as compared with electric stinulation group at 14d,21d(P＜0.05).Conclusion1.Electric stimulation can promote the ultramicrostructures recovery of HIBD rats.2.Electric stinulation could improve the synaptic reconstruction of HIBD rats.