Cerebral Lymphatic Blockage The Cerebral Ischemic Injury And The Deriorates The Damage Of CSF On PC12 Cells And Hippocampal Neurons After Subarachnoid Hemorrhage

BACKGROUD:Acute cerebrovascular disease is one of the first three top causes of the death in the world. Among the total, the incidence of subarachnoid hemorrhage is second only to cerebral infarction and cerebral hemorrhage. Most SAH is cause by the cerebral rupture of aneurysm. The mortality of SAH is over 25%, and around 1/3 survivals's depend on others because of neurologic impairment ,which bring albatross to their family and the society. Rebleeding and secondary cerebral ischema are the main complications of SAH and contribute to the sever outcome. In spite of the fact that there is a drastic drop in rebleeding due to the progress of the surgical management of cerebral aneurism, the outcome of SAH dose not impove because of a concurrent increase in the occurrence of secondary cerebral ischemia. Thus, understanding of the pathogenesis, preventionand management of secondary cerebral ischemia are of great importance in improving the outcome of patients with SAH.Over the past several decades, the main cerebral arterial spasm, namely cerebral vasospasm(CVS), has become a focus. Research shows that the time course and severity of CVS are not completely parallel relation with secondary cerebral ischemia. In spite of CVS, the factor (such as cerebral micrangium vasospasm and abnormal regulate function of microcirculation etc.) are concerned with development of secondary cerebral ischemia. Neuronic death include apoptosis and necrosis. Mammalian cell apoptosis is regulated by Bcl-2 and caspase proteins, apoptosis protein activating factor (Apaf-1).It has been proved that macromolecular substance in brain and subarachnoid space could be drainage into extracranial lymphatic vessels and lymph nodes, by technique of macromolecular substance tracing in any kind of animals including of human. After occurrence of the secondary cerebral ischema following SAH, mass macromolecular substance (such as plasma protein etc.)can pass through the impaired blood-brain barrier into brain tissue. Because of damaged cell or ischemia metabolism fall, mass cell cleaved product and peptides are product and rapidly increased in brain tissue. Accumulation of above macromolecular substance can damage directly, or induce the brain edema by osmotic pressure of brain tissue rising, even cerebral hernia, which lead to neurologic impairment or die.OBJECTIVES:Firstly, to investigate the effects of the cerebral lymphatic drainage pathway on the drainage of macromolecular substance and secondary cerebral ischemia. Secondly, to investigate that the cerebral lymphatic blockage aggravate the injury of CSF to PC12 cells and hippocampal neurons after subarachnoid hemorrhage (SAH) and the mechanism concerned.METHODS:1. Forty New Zealand adult albino rabbits were randomized into blank control group, normal CSF group, SAH CSF group and SAH+CLB CSF group, (10 in each group, respectively). The CLB model was induced by blockage the cerebral lymphatic and remove lymphatic nodes, the SAH model by 2-injection of blood into cistem magna. In sham group, the equivalent volume of normal saline was injected into cistem magna. Withdraw CSF and determinate ET-1 and TP in the 5th day of the second blood-injection of cistem magna. Apoptosis and cell injury in the cerebral cortex and hippocampal neurons were detected by TUNEL method.2. CSF was withdrawed and then was added into PC12 cell and hippocampal neurons culture medium in the 5th day of the second blood-injection into cistem magna. Cells were randomized into blank control group (F-12 Ham's/DMEM), normal CSF group, SAH CSF group and SAH+CLB CSF group. MTT assay were used to record cell proliferation and cellular activity after 0.5h, 1h, 2h, 4h of intervention. LDH releasing rate in cells were recorded respectively to evaluate the injury lever at the same time. Immunocytochemistry was performed to detect the expression of apoptosis related proteins (Bax, Hsp70 etc.) in PC12 cells and hippocampal neurons. Flow cytometry method was used to evaluate the cell apoptosis. The changes of the concentration of intracellular free Ca2+ and H+ analyzed by confocal laser scanning microscope(CLSM). RESULTS:1. There is no obvious difference in TP and ET-1 of CSF between normal CSF group and SO group. The content of TP and ET-1 in SAH group and SAH+CLB group was increased compared with that in normal CSF group and SO group. Both were significantly more elevated in SAH+CLB group than that in SAH group.2. Scattered apoptotic cells were observed in SAH group, and a devil of apoptotic cells were observed in SAH+CLB group. Apoptotic cells were centralized in cerebral cortex, hippocampus, basal ganglia, choroids plexus and endyma etc. Of the total, hippocampus, cortex were more visible.3. (1) MTT and LDH releasing rate results showed that normal CSF did not damage the cells significantly contrasted to normal control group. Cell viability was reduced and LDH releasing rate increased in SAH group and SAH+CLB group. The changes in SAH+CLB group were the most obvious.(2) Immunohistochemical results showed that Bax, Hsp70, Caspase-3,Bcl-2 were positive in SAH group and SAH+CLB group, the expression of Bax and Caspase-3 were increased in the latter, which depended on the action time. Two hours after intervention the expression of Hsp70 and Bcl-2 reached the peak level in SAH group, while 1h in SAH+CLB group. It changes with time.(3) Immunofluorescence staining of cytoskeleton showed that cells morphology were completed with the cytoplasm green staining by fiber bunchiness and nuclear red staining by homogenizing. While the cytoplasm in SAH group and SAH+CLB group stains weak with the nuclear splitting.(4) The flow cytometry showed that there is no apoptosis cell in normal control group and normal CSF group, while the apoptosis rate was significantly higher in SAH+CLB group than in SAH group.(5) Confocal laser scanning microscope (CLSM) showed that the expression of Ca2+ and H+ was weak in normal control group and normal CSF group and intense in SAH group and SAH+CLB group. While the expression of Ca2+ and H+ was more intense in SAH+CLB group than SAH group.CONCLUSIONS:1. Cerebral lymphatic blockage increase the content of TP and ET-1 in CSF and aggravate the cerebral ischemia imjury following SAH.2. Cerebral lymphatic blockage aggravate the injury of CSF to PC12 cells and ippocampal neurons after SAH by up-regulating Bax and Caspase-3 protein expression, down-regulating Bcl-2 and Hsp70 expression.3. Cerebral lymphatic blockage aggravate the injury of CSF to PC12 cells and hippocampal neurons after SAH by intracellular acidification and intracellular calcium overload.