Study On The Expression Level Of Asymmetric Dimethylarginine (ADMA) After Subarachnoid Hemorrhage And The Correlation Between ADMA And Cerebral Vasospasm In Rabbit

BACKGROUND AND PURPOSES:There is high incidence of cerebral vasospasm (CVS) occurred after subarachnoid hemorrhage (SAH), it is one of the leading causes of mobility and mortality which seriously influences the long term prognosis and life quality after SAH. How to prevent and reverse CVS has already turned into the important tasks that all medical researchers must confront, it will be of great significance in life quality improvement of patients with SAH.There has been over a hundred years of research on CVS, in which some chemokines and proinflammatory mediators had been proved to play the important role in CVS mechanism, such as hemoglobin, endothelin(ET), nitric oxide(NO), protein kinase C(PKC) and kinaseρ. More and more researchers gradually have reached the consensus that CVS was induced by a synergistic action of multiple factors, although these multiple factors which induced and sustained CVS have not been fully recognizedRecent research has showed that the degree of cerebral vasospasm after SAH was negatively related to the concentration of NO, a kind of vasodilator substance which fluctuated regularly. NO induces the concentration fluctuation of the vasoactive substances such as ET-1 and prostacyclin by activating cGMP-dependent or cGMP-independent pathway, it hyperpolarizes the arteriolar smooth muscle cell membrane and dilates the smooth muscle cell. As an important transmitter of intracellular signals, NO plays a pivotal role in regulating the vasodilation of cerebral vessels.As for the reasons which induce NO decrease, there are two main aspects: one is the chemical precipitation induced by oxyhemoglobin (OxyHb) which released from the erythrocytes lysis, the other is the decrease of synthesis ability of endothelial nitric oxide systhase (eNOS), and moreover the latter became the hot research topic.As a specific inhibitor of eNOS, ADMA can effectively inhibit intracellular NOS activity of endothelial cells and macrophages. In recent years, after more attentions about ADMA were taken on the pathogensis researchs of cardiovascular system and renal disease, ADMA has been widerly considered to be a new predictor of vascular endothelial disfunction, but so far little research advances about ADMA have been achieved in cerebral vascular disease. In our research, we dectected the dynamic changes of ADMA, NO and eNOS in SAH animal model, and aimed to further explore the regulatory mechanism of ADMA in cerebral vasospasm.METHODS:1. The experimental cerebral vasospasm rabbit model was achieved by twice injections of fresh autologous arterial blood into the cisterna magna of each animal.2. The experimental animals were randomly divided into 3 groups: control group, sham-operated group and SAH group.3. The behaviors of experimental model after SAH were observed.4. The velocity of basilar artery (BA) was measured by transcranial Doppler (TCD) at the 1st, 4th, 7th after twice injections.5. The experimental animals were selected randomly for heart perfusion at each observation time, and then the samples of BA were harvested for HE staining and electron microscopic observation.6. The concentrations of ADMA in serum and cerebral spinal fluid (CSF) were measured by ELISA method at each observation time.7. At the same time, the concentrations of NO in CSF were measured by nitrate reductase method.8. The expression of eNOS in intima of BA was detected by immunohistochemistry method.RESULTS:1. The two-hemorrhage model established by injecting autologous arterial blood into cisterna magna 48 hours apart was a successful symptomatic SAH rabbit model, and its rate of success was 71.9%.2. There was some ethological abnormal in SAH group compared with control group and sham-operated group, such as shaggy hair, anorexia, huddling, activity decrease.3. There was no significant difference between the velocities of BA in control group and in sham-operated group, while the velocities in SAH group were higher than that in control group, especially at the 4th day the velocity reached the peak(P<0.05).4. Morphological structural changes of the basilar arterial wall in SAH group were detected by light microscope and transmission electron microscope, including vessel wall incrassation, vessel stenosis, and endothelial cell degeneration, swelling, vacuolation, and mitochondria condensation, artery corrugation, disorder of vascular smooth muscle cell (VSMC) structure.5. There was no significant difference among the serum concentrations of ADMA at each observation time in SAH group, so did in the other two group(P>0.05). The change of the ADMA in the CSF was synchronal with the velocity of basilar artery. At the 4th day after twice injections, the concentration reached the peak and was correlated positively with the velocity of basilar artery. There was significant difference between the concentrations in SAH group and in control, sham-operated group(P<0.05).6. The change of the NO in CSF was regular. At the 4th day after twice injections, the concentration reached the bottom and was correlated negatively with ADMA level(P<0.05).7. There was no significant difference of eNOS expression detected by IHC within 7 days after SAH(P>0.05)。CONCLUSIONS:1. The two-hemorrhage model established by twice injecting antilogous arterial blood into cistern magna was a successful symptomatic SAH model with notable behavioral changes.2. The velocities of BA in SAH group were increased regularly after the injection than that before injection, and reached the highest level at the 4th day.3. There were some morphological structural changes on the basilar arterial wall in SAH group, such as vessel wall incrassation, vessel stenosis, endothelial cell lesions and disorder of VSMC structure.4. The ADMA concentrations of CSF in SAH group were increased significantly after the injection which reached the highest level at the 4th day, while there was no change happened in serum.5. The expression of eNOS in basilar artery was decreased after subarachnoid hemorrhage, while within 7 days after operation no significant change of eNOS expression was detected;6. The concentration of NO in CSF after SAH was closely related to CVS, it was decreased significantly after the injection than that before injection, and increased gradually after reaching the lowest level at the 4th day. The concentration of ADMA in CSF after SAH was negatively related to the concentration of NO, so the expression of ADMA in CSF might play a pivotal role in the regulation mechanism of NO synthesis.