The Change Of Cerebral Cortex Ultramicrostructure And Brain Tissue Energy Metabolism Expression Of COX-4 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 brain tissue severe IDEM effect or tardive brain injured or cerebral infarction and is responsible for causing death or disabled. 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 nerve cell energy metabolism and 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 the nerve cell energy metabolism and ischemia after SAH. However, data regarding change of microcirculation after SAH are controversial. Cytochrome c oxidase 4, (COX-4)is believed to one of key point component in mitochondrial respiratory chain which is encoded by nDNA. the molecule of COX-4 is biggest in 10 COX subunit which is encoded by nDNA.( MW or so17kD). COX4 is one of significant subunit which is encoded by nucleus. They are synthesize in kytoplasm and input chondriosome, which to combine mtDNA encoded subunit to shape heloenzyme. COX-4 have two isomer: COX4-1 and COX4-2, which to combine ATP,to restrain and to adjust COX activity when ATP/ADP ratio step up and to adjust Significant effect in cellula nervosa energy metabolism. However, at present study on the expression of COX-4 in nerve cell energy metabolism 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 nerve cell energy metabolism and mechanism of CVS after SAH. In this study, we examined the expression of COX-4 in nerve cell energy metabolism after 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=6), sham operated controls (n=6), and SAH groups (n=54). 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 chondriosome,morphologic changes and ultrastructural changes of cerebral arteriolar and neurons by transmission electron microscopy (TEM) .3 We use immunohistochemistry technique to detect the expresseion of protein level of COX-4 in nerve cell after SAH. We execute the procedure according to illustration of kit. we observe positive cell in the field of vision of 10 high power lens (10×40) by optical microscope.Results:1 Brain gross inspection:In 1h to 3h SAH group 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 24h-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 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.3 COX-4 protein in rats after SAH nerve cells expression of energy metabolism in surgical group: COX-4 expression was increased detected in the cerebral cortex neurons and reached its peak at 1h, the activities of COX-4 in 1h group were statistically significant compared to the non-operated controls group. After this the expression of COX-4 begin to cut down. The expression of COX-4 decreased in 12h after SAH group than 1h, compared with the control group but there are still significant. COX-4 expression in the SAH group in 24h appear first nadir, then COX-4 expression was increased .In 48h appear one small peak, then COX-4 expression was breakdown. 7d was no statistically significant compared to the controls group. There was no statistically significant in the expression of COX-4 between normal control group and sham operated group.Conclusion:1 The dysfunction of the nerve cell energy metabolism and cerebral microvascular,which is the direct reason that causes ischemic neurological deficits after SAH.2 The expressions COX-4 were increased in cerebral cortex neurons after SAH. The correlation of COX-4 was significantly positive. They play an important role in the pathophysiological course of brain ischemic after SAH.