Effect Of Experimental Subarachnoid Hemorrhage On Behaviors And Hippocampal Structure Of Rats

Subarachnoid hemorrhage(SAH)is one of the clinical common critical diseases.SAH is a syndrome caused by the blood from the diseased blood vessels from the bottom or surface of brain rupture flows directly into the subarachnoid space.According to the bleeding site and volume,the treatment effect is quite different.The common complications of SAH are rebleeding,vasospasm and hydrocephalus,etc.Some of the survivors of SAH may experience progressive ventricular enlargement,cortical atrophy,cognitive dysfunction and even dementia.Alzheimer’s disease,named as AD,which is also called as presenile and senile dementia,is the most common clinical type of dementia.Successive studies have found that it is a neurodegenerative disease with the main clinical manifestations of progressive learning,memory and cognitive dysfunction.The pathogenesis of AD is still not very clear nowadays and there is no clear and effective method for preventing and treating it.We wonder whether the presence of these sequelae of SAH had similar pathological mechanism with the occurrence and development of AD?As we all know,the normal cerebrospinal fluid(CSF)circulation plays an irreplaceable role in the transportation of brain metabolites such as lipid,protein and other macromolecular substances and maintaining the internal environment of central nervous system relatively stable.Based on that,we speculate that whether the chronic CSF circulation disorder or hydrocephalus caused the abnormal transportation of brain metabolites and then induced the pathological changes in brain tissues and brain function impairment? This study simulated SAH,the experimental rat model of chronic dysfunction of CSF circulation was established,and the behavior changes of laboratory animals,mitochondrial transmembrane potential in the hippocampal cortex neurons,microscopic and ultrastructural changes of hippocampal cortex,lateral ventricular ependyma and choroid plexus,the changes of protein expression and were observed and measured.This research was performed in three parts as follows.Part Ⅰ The establishment of a rat model of experimental subarachnoid hemorrhage and the observation of the influence on behaviors of ratsObjective: This experiment aimed to study the brain damage following experimental subarachnoid hemorrhage(SAH)and noxious effect on cognitive function in rats,providing the behavioral evidence for the study on the mechanism of hippocampal cortex,lateral ventricular ependyma and choroid plexus damage caused by experimental SAH.Methods: Sixty adult SD rats of both sexes,with average weight of 300.40 ± 44.50 g,were selected.The 60 rats were randomly divided into test group and control group,with 30 rats in each group.In the test group,the rats were anesthetized with 10% chloral hydrate which was intraperitoneally injected.And then the autologous arterial blood 0.30 ml was injected into the pillow large pool(cerebellomedullary cistern)to establish the animal model of experimental SAH.In the control group,the equal volume of normal saline was injected into the pillow large pool instead,and the other procedures were similar to those previously described in the test group.Ten rats in each group were randomly selected at the same time and fed routinely after operation.The statistical analysis of the general behaviors,neurological function score and behavior study were carried out at 2,4 and 6 months after operation,and the related data were finally analyzed.Results: Compared with the control group,the rats in the test group appeared a series of neurological function impairment syndromes,such as spirit languish,somnolence,diet decreasing and physical activity decreasing and so on,P<0.05,the difference was statistically significant.With the prolongation of the feeding time,these phenomena were more obvious,P<0.05,and the difference was statistically significant.In the Morris water maze test,the spatial learning and memory abilities were significantly decreased in the test group as compared with control group,P<0.05,and the difference was statistically significant.With the prolongation of the feeding time,the learning,memory,activity ability and cognitive function were gradually degraded in the test group when compared with control group,P<0.05,and the difference was statistically significant.Part Ⅱ Morphological changes of hippocampal cortex and lateral ventri-cular ependyma and choroid plexus induced by experimental subarachnoid hemorrhage in ratsObjective: This experiment aimed to observe experimental subarachnoid hemorrhage(SAH)induced microscopic and ultrastructural changes of hippocampal cortex,lateral ventricular ependyma and choroid plexus,and the mitochondrial transmembrane potential(MTP)changes of hippocampal cortex neurons in the rats,providing morphological evidence for the mechanism of SAH-induced injury to hippocampal cortex,lateral ventricular ependyma and choroid plexus.Methods: Ninety adult SD rats of either sex,with average weight of 300.20 ±27.50 g,were selected.The rats were randomly divided into test group and control group,with 45 rats in each group.In the test group,after the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate,the autologous arterial blood 0.30 ml was injected into the pillow large pool to simulate the animal model of experimental SAH.In the control group,the equal volume of normal saline was injected into the pillow large pool instead,and the other procedures were similar to those previously described in the test group.Ten rats in each group were randomly selected and fed routinely after operation.At 2,4 and 6 months after operation,the aortic perfusion fixation was performed to prepare the specimens for light microscope(LM),scanning and transmission electron microscope(SEM,TEM)observation.Five rats in each group were randomly selected at the same time and sacrificed,brains were removed and bilateral hippocampi were isolated for detection of the MTP in hippocampal cortex neurons(by flow cytometry)and for observation of apoptosis in hippocampal cortex neurons(with fluorescence microscope).By comparing the difference between the two groups at different time points,the influence of experimental SAH on the structure of brain tissues was revealed.Results: In the test group,the LM observation showed that the lateral ventricular cavity was gradually enlarged,the ependymal cells turned to pleormorphic even squamous shape from cuboidal shape with the prolongation of feeding time.In the hippocampal cortex,the paravascular space was widened around small blood vessels,the hippocampal pyramidal layer was thinned,the number of nerve cells was decreased,and the arrangement was disordered.This condition was more obvious over the feeding time.The SEM observation showed crater sag appeared on the surface of choroid plexus epithelial cells,the plush atrophied,distorted or even fell off.The cilia and microvilli of ependymal cells surface in lateral ventricle decreased markedly.The TEM observation showed that a lot of pinocytosis bubbles emerged in choroid plexus epithelial cells,the shape of cell bodies and nucleus was irregular,nuclear heterochromation in the pyramidal cells increased obviously,swelling mitochondria and neurofibrillary tangle were observed in the neurons,and lipoprotein deposited in the neuropil and around the blood vessel.When the rats were fed to postoperative month 6,this phenomenon was more obvious.The decrease in microvilli on ependymal cells surface was more marked,and a lot of lipoprotein deposited around the blood vessel.The detection of JC-1 flow cytometry showed that the MTP depolarization phenomenon in hippocampal cortex neurons was serious,compared with the control group,P<0.05,the difference was statistically significant,suggesting that the different degrees of pathological changes of mitochondria were found in the neurons,and cell apoptosis phenomenon was obvious.This phenomenon was gradually aggravated over the feeding time,P<0.05,and the difference was statistically significant.However,in the control group,no obvious pathological changes of hippocampal cortex,lateral ventricular ependyma and choroid plexus and MTP in hippocampal cortex neurons were observed at 2,4 or even 6 months after operation.Part Ⅲ The changes in the expression of inflammatory and autophagy associated proteins in the hippocampal cortex in a rat model of experimental subarachnoid hemorrhageObjective: This experiment aimed to further investigate the effect of experimental subarachnoid hemorrhage(SAH)on the hippocampal cortex the important brain region for learning and memory through the immunohistochemistry,Western blot and TUNEL assays based on the two experiments mentioned above,revealing the relationship between experimental SAH and Alzheimer’s disease(AD).Methods: Ninety adult SD rats of either sex,with average weight of 289.40 ± 45.50 g,were selected.The rats were randomly divided into test group and control group,with 45 rats in each group.In the test group,after the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate,the autologous arterial blood 0.30 ml was injected into the pillow large pool to simulate the animal model of experimental SAH.In the control group,the equal volume of normal saline was injected into the pillow large pool instead,and the other procedures were similar to those previously described in the test group.Ten rats in each group were randomly selected and fed routinely after operation.At 2,4 and 6 months after operation,10 rats in each group were randomly selected,and the aortic perfusion fixation was performed to prepare the specimens.The expression of Aβ,IL-1β and IBA-1 in the hippocampal cortex was determined by immunohistochemistry,and the apoptosis in pyramidal cells in the hippocampal cortex was detected by TUNEL.Five rats in each group were randomly selected at the same time and sacrificed,brains were removed and bilateral hippocampi were isolated for detection of the expression of IL-1β and autophagy-related proteins(Atg-5,Beclin-l and LC-3)in the hippocampal cortex.The related data were recorded and analyzed.Results:1 The results of Western blot showed that compared with the control group,the expression of IL-1β,Atg-5,Beclin-l and LC3-II/LC3-I in the hippocampal cortex was significantly increased,P<0.05,and the difference was statistically significant;the expression presented a rising tendency over the feeding time,P<0.05,and the difference was statistically significant in the test group.2 The results of immunohistochemistry showed that compared with the control group,the expression of amyloid-β(Aβ)protein,IL-1β and IBA-1 in the hippocampal cortex was markedly increased,and the expression tended to increase over the feeding time after experimental SAH in the test group.3 The results of TUNEL detection showed that compared with the control group,the apoptosis in pyramidal cells in the hippocampal cortex was increased,P<0.05,the difference was statistically significant;this phenomenon was gradually aggravated over the feeding time and peaked when fed to 6 months after experimental SAH in the test group,P<0.05,and the difference was statistically significant.Conclusions:1 The method of single injection of the autologous arterial blood into the pillow large pool can successfully simulate the rat model of experimental SAH,the pathological changes are consistent with behaviors of rats,and it has reliable basis and high significance for clinical research.2 Experimental SAH could lead to decrease in learning,memory and activity abilities of rats,and the condition was gradually getting worse with the prolongation of the feeding time,indicating that experimental SAH may induce pathological damage to the structure such as the lateral ventricular ependyma,choroid plexus and hippocampal cortex of rats in the test group.3 Experimental SAH could induce the microscopic and ultrastructural changes of hippocampal cortex,lateral ventricular ependyma and choroid plexus,could lead to depolarization of MTP and increased apoptosis or necrosis in hippocampal cortex neurons.4 Experimental SAH could lead to increased expression of inflammatory factors in hippocampal cortex,enhanced cell autophagy,Aβ protein deposition and apoptosis in pyramidal cells in the hippocampal cortex,and this condition were aggravated over the feeding time after experimental SAH.Experimental SAH had a certain connection with AD,but the mechanism still needs further study to explain.