Experimental Study On Subarachnoid Hemorrhage-induced Delayed Cerebral Vasospasm

Objective: To establish the animal model of subarachnoid hemorrhage(SAH) -induced delayed cerebral vasospasm(DCVS) by modifying the two injections of nonheparinized autologous arterial blood into the cisterna magna. Using the animal model, we dynamically monitor the changes of cerebral blood fluid(CBF) and chose basilar artefies(BA) for morphometric Analysis and histopathological examination; To investigate the changes of the cerebral energy metabolism and the neuronal apoptosis in hippocampal CA1 region following SAil-induced DCVS, and explore the correlation with CBF changes; To further explore the pathogenesis of DCVS.Methods: (1) Establishment of animal model: Male Sprague-Dawley rats(n=50) were divided into five groups: control group and 1d,3d,5d,7d group following SAH. The animal model was induced by two injections of nonheparinized autologous arterial blood into the cistema magna. Five rats(n=5) were used for the general observation: Four rats were euthanized at 30 min after dobble-hemorrhage for blood distribution; One rat was injected twice into the cisterna magna with artificial cerebrospinal fluid for control.; The cortical CBF was measured by Laser Doppler Flowmetry(LDF); (2) Each group was sacrificed respectively, We chose basilar artedes(BA) for morphometdc Analysis with light microscope and for observation the changes of ultrastructure with transmission electron microscopy; (3) Microdialysis(MD) was applied to monitor the concentration of Glu,Lac,Pyr,Glut,Gly in the cerebral extracellular fluid(ECF)in each group, and calculated the I/P; (4) we chose hippocamp for the study on the neuronal apoptosis with TUNEL and observe the time course of apoptosiso Results:(1) The distribution of blood clot was seen in the basal cisterns, interpeduncular cisterns, on the base of the frontal lobe. Methylthioninium Chloride with blood was mainly observed in the subarachnoid space on the base of the brain, blood was seen in the ventricle and diastematia cistern as well. However, in control group none of the rats developed blood and no blood distribution was seen. the LDF measurements show severe vasospasm and there was an obviously decrease in CBF compared with baseline in each group following SAH(P＜0.05), especially in 5d group the CBF decreased significantly(P＜0.05); (2) Morphometric Analysis and histopathological examination: The basal artery revealed luminal narrowing, reduction in diameter, increased wall thickness, corrugation of the internal elastic lamina(IEL), and cellular proliferation were observed in the spastic BA as well, especially severe in 5d group(P＜0.05). In 1d,3d,5d,7d group, the mean diameter of BA was reduced from control levels by46.34%, 33.95%, 50.59%, 17.21%; the increases in wall thickness of BA were 98.55%, 75.58%, 159.27%, 19.21%; the mean luminal perimeter reduced by 56.30%, 45.97%, 62.50%, 25.77%. Apoptotic-like changes were noted in endothelial cells in each group following SAH and consisted of blebbing of the cell membrane, condensation of the cytoplasm, condensation of peripheral nuclear chromatin, and cytoplasmic vacuolization. The morphological changes in endothelial were most severe and wide spread in 5d group with some endothelial cells and smooth muscle cells showed evidence of necrotic .changes. (3) MDanalysis: [Glu],[Pyr] was lower ineach group following SAH, whereas L/P,[Glut] and [Gly] were significantly higher than that in control group(P＜0.05), at same time, L/P in 5d,7d group was higher than that in other groups(P＜0.05), [Lac],[Glut] and [Gly] were significantly elevated in 5d group(P＜0.05); Changes in CBF has significantly positive correlation with L/P,Lac,Glut and Gly (r=0.721, 0.477, 0.814, 0.718 P＜0.05), whereas, it has strongly negative correlation with Glu and Pyr (r=-0.447, -0.579; P＜0.05); (4) Neuron apoptosis in the hippocampal CA1 region was no found in control . the number of TUNEL—positive cells showed a significant increase in each SAIl group, especially in the 5d,7d group (P＜0.05).Conclusion: (1) The model of SAH was induced successfully and it can accurately simulate the time course of vasospasm in humans following SAH; (2) The course of CVS following SAH coincided with the progress of apoptosis changes in ehdothelial cells and proliferation of cells in the vascular wall. Endothelial apoptosis and cellular proliferation might play an important role in the pathogenesis of vasospasm; (3) MD markers are in good accordance with changes in CBF, MD is an ideal tool for dynamically monitoring neurochemical substances in the ECF following SAH and can indicate early the onset of delayed cerebral ischemia; (4) Neuron apoptosis in the hippocamp may play an important role in the pathogenesis of DIND.