The Cerebral Vasospasm And Expression Of HIF-1α And VEGF In Experimental Subarachnoid Hemorrhage Rat

Subarachnoid hemorrhage (SAH) is, in most cases, caused by rupture of intracranial aneurysm. Cerebral vasospasm (CVS) is the most frequent and severe complication after SAH. CVS occurs in 70% of patients with SAH.SAH-induced cerebral vasospasm can lead to ischemic injury or death and is responsible for serious neurological disability and mortality.It is generally accepted that the release of blood in the subarachnoid space contributes to the development of vasospasm and neurological deficits. To date, however, the mechanism of CVS after SAH remains poorly understood. Efforts to elucidate these mechanisms have focused on vasospasm of the large conducting cerebral arteries, but it is clear that this type of vasospasm cannot fully account for the occurrence of delayed ischemic neurological deficits. Disturbances in cerebral arteriolar function, in addition to large vessel vasospasm, may be responsible for ischemia after SAH. However, data regarding change of microcirculation after SAH are controversial. Hypoxia-inducible factor 1α(HIF-1α) has been identified as a critical mediator of adaptive responses to reduced oxygen availability through its transcriptional regulation of genes that encode proteins required for tissue oxygen delivery, vascularization,and energy metabolism. A key angiogenic stimulator is vascular endothelial growth factor(VEGF), which promotes vessel permeability,cell proliferation, and migration of endothelial cells and inhibits apoptosis.VEGF can play a role in promoting angiogenesis, moderating cerebral ischemic damage, and reducing infarct size and edema formation, its direct neurotrophic effects may be independent of angiogenesis. However, at present study on the expression of both HIF-1αand VEGF in SAH is rarely reported.Objective: In this experimental study, an endovascular perforation model was used to induce subarachnoid hemorrhage in male Sprague-Dawley rat. The aim of this study was to observe morphologic and ultrastructural changes of cerebral arteriolar and neurons in different phases, offering the data of morphologic changes for studying mechanism of CVS. In this study, we examined the expression of HIF-1αand VEGF a rat SAH model by immunohistochemical staining. The aim is to find the regularity of expression and the correlations associated with the morphologic changes, which will provide theoretical principle for treating SAH-induced CVS in the clinical application.Methods:1 Three experimental groups of healthy adult male Sprague-Dawley rats weighing from 260 to 280 g were used: non-operated controls(normal,n=5), sham operated controls (n=5), and SAH groups (n=45). The SAH groups were divided into 9 categories:1h,3h,6h,9h,12h,24h,48h,5d,7d after operation. An endovascular perforation model was used to induce subarachnoid hemorrhage in SAH groups.Sham-operated rats underwent same procedures except that the suture was withdrawn after the resistance was felt.2 All animals were anesthetized , by means of transthoracic cannulation of the left ventricle, they were perfused with phosphate-buffered saline solution and reperfused with a mixture of 4% paraformaldehyde, The brains were collected to observe blood distribution after subarachnoid hemorrhage.To observe the morphologic changes and ultrastructural changes of cerebral arteriolar and neurons by light microscopic and transmission electron microscopy (TEM) .3 To examine the expression of HIF-1αand VEGF a rat SAH model by immunohistochemical staining and measure the means optical density(MOD)of the positive cell staining which accomplished with Image-Pro Plus 5.0 image analytical system.4 Statistical analysis was performed by The software package SPSS 15.0. All values are expressed as means±SE. Data were analyzed with one-way ANOVA . When multiple comparisons were indicated, the Student-Newman-Keuls test. We utilized Pearson Correlation to correlate results. Any probabilities less than 5% (P value<0.05) were considered to be significant.Results:1 Brain gross inspection:The widespread distribution of blood was seen in carotid cistern, chiasmatic cistern and less blood on the frontoorbital surface.In 3h to 24h SAH group, the blood was diffused from pavimentum cerebri to the frontoorbital surface and cerebral convexities. The blood in subarachnoid space of 48h SAH group was less than the prior group, but blood clots can be obviously seen in the fourth ventricle. A few dotty blood clots were observed on day 5 in rats of SAH group, a small quantity blood clots in the basal cisterns. The blood in subarachnoid space was disappear in SAH 7d group. There were no blood in subarachnoid space of both non-operated controls and sham operated controls group.2 Under the light microscope,the morphologic changes of cerebral neurons mostly included,the cerebral neurons swollen and degeneration,brain edema,the perivascular space of cerebral arteriolar became widen. Slight morphologic change was observed right after 3h of SAH group. The peak of brain edema can be found in 24h to 48h SAH group. No abnormal findings were seen in cerebral arteriolar and neurons of both non-operated controls and sham operated controls group.3 Under the electron microscope the ultrastructural changes of cerebral neurons mostly included the chromatin became concentrated , heterochromatin could be seen, and the mitochondria became swollen, vacuoles could be seen,rough endoplasmic reticulum(RER) degranulation. The close connection between the endothelial cells of cerebral arteriolar disappeared,the mitochondria became swollen, the vessel cavity became narrow or even vascular occlusion. Slight change was observed right after 1 h of SAH group,significant change was observed at 6h,and most obvious change was observed between 24h and 48h. No abnormal findings were seen in cerebral arteriolar and neurons of both non-operated controls and sham operated controls group.4 In accordance with the results obtained by immunohistochemistry, HIF-1αexpression was rarely detected in the cerebral cortex neurons at 1h or 3h,but the activities of HIF-1αin 1h to 3h SAH group were no statistically significant compared to the non-operated controls group(P>0.05). The expression of HIF-1αexpression in 6h SAH group was statistically significant compared to the non-operated controls group(P<0.05). HIF-1αexpression in the SAH group increased after 6h, reached its peak at 24h, lasted to 48h, and returned to normal level at 7d. The expression of HIF-1αexpression in the 7d SAH group was no statistically significant compared to the non-operated controls group(P>0.05). HIF-1αexpression was rarely in cerebral cortex neurons of both non-operated controls and sham operated controls group. There was no statistically significant in the expression of HIF-1αbetween normal control group and sham operated group(P>0.05).5 In accordance with the results obtained by immunohistochemistry, VEGF expression in the 1h SAH group transiently increased then decreased in the 3h,6hSAH group. The expression VEGF expression in the 9h SAH group increased again. The expression of VEGF in the 9h SAH group was statistically significant compared to the non-operated controls group(P<0.05),reached its peak at 24h, lasted to 48h, and returned to normal level at 7d. VEGF expression was rarely in cerebral cortex neurons of both non-operated controls and sham operated controls group. The expression of VEGF in the 7d SAH group was no statistically significant compared to the non-operated controls group(P>0.05). There was no statistically significant in the expression of VEGF between normal control group and sham operated group(P>0.05).6 The positive relation of HIF-1αand VEGF expression in cerebral cortex is significant(r=0.804, P<0.05).Conclusion:1 An endovascular perforation model is available by observing dynamically distribution characteristic of blood in subarachnoid space, observing the morphologic and ultrastructural changes of cerebral arteriolar and neurons. This model is considered to be the most suitable to study mechanisms of SAH.2 The injury of the cerebral microvascular endothelial could be the initiating factor of microcirculatory dysfunction,which is the direct reason that causes ischemic neurological deficits after SAH.3 The expressions of HIF-1αand VEGF were increased in cerebral cortex neurons after SAH. The correlation between HIF-1αand VEGF was significantly positive. They play an important role in the pathophysiological course of brain ischemic after SAH.