Experimental Study The Neuroprotective Role Of Melatonin On Early Brain Injury In Male Rats After Subarachnoid Hemorrhage

Part I:The Neuroprotective effects of melatonin on early brain injury in male rats after subarachnoid hemorrhageObjective:To investigate neuroprotection of melatonin on early brain injury in male rats after subarachnoid hemorrahage, such as the clinical behavior scale, brain edema, and blood-brain barrier (BBB) impairment.Methods:Forty-eight health male Sprague-dawley(SD) rats were assigned randomly into following groups:Control group (n=12), SAH group (n=12), SAH+vehicle group (n=12), SAH+melatonin group (n=12). All SAH animals were subjected to injection of0.3ml fresh arterial, non-heparinized blood into prechiasmatic cistern in20seconds. Male rats were given150mg/kg injections of melatonin at post-SAH hours2h, and24h. Brain samples adjacent to the clotted blood were extracted at48h after SAH. Control animals underwent exactly the same procedure as described above with the exception that no blood was injected intracisternally. The clinical behaviors function after experimental SAH were observed; cerebral edema and blood-brain barrier permeability were detected at48h after experimental SAH.Results:Impact of Melatonin on Clinical Behavior Function after Experimental SAH. As compared with control group, clinical behavior function impairment caused by SAH was evident in SAH subjects (P<0.01). No significant difference was seen between the SAH group and SAH+vehicle group (P>0.05). Melatonin-treated rats showed better performance in this scale system than vehicle-treated rats at24h after SAH, and the difference was statistically significant (P<0.01),. Significant increase (P<0.05) in water content was detected in the brain samples at48hr after SAH when compared with rats in control group. The mean value of brain water content in the cortex was decreased by melatonin administration (P<0.05) as compared with SAH+vehicle group. The pattern of EB extravasation in SAH or SAH+vehicle group demonstrated a significant increase (P<0.01) in BBB permeability to EB relative to rats of control group. Administration of melatonin significantly inhibited EB extravasation(P<0.05) after SAH.Conclusions:1. As compared with control group, clinical behavior function impairment caused by SAH was evident in SAH subjects. SAH group significantly attenuate blood-brain barrier (BBB), increase brain edema compared to control group.2. Post-SAH melatonin treatment significantly ameliorated the EBI, such as the clinical behavior scale, brain edema, and blood-brain barrier (BBB) impairment. It suggests that melatonin could attenuate EBI in this rat SAH model.. Part Ⅱ:Melatonin administration modulates cortical Nrf2-AREsignaling pathway after subarachnoid hemorrhage in male ratsObjective:The aim of the current study was to investigate whether melatonin administration modulated Nrf2-ARE pathway signaling pathway in the brain at the early stage (EBI) of SAH.Methods:Forty-eight male Sprague Dawley rats weighing from300to350g were randomly divided into control group (n=12), SAH group (n=12), SAH+vehicle group (n=12) and SAH+melatonin group (n=12).All SAH animals were subjected to injection of0.3ml fresh arterial, non-heparinized blood into prechiasmatic cistern in20seconds. Male rats were given150mg/kg injections of melatonin at post-SAH hours2hr and24hr. Rats of SAH+vehicle group received equal volumes of vehicle (1%ethanol) at corresponding time points. Brain samples (adjacent to the clotted blood) were extracted at48h after SAH. Control animals underwent exactly the same procedure as described above with the exception that no blood was injected intracisternally.We measured the expressions of Nrf2and HO-1by western blot; and Nrf2and HO-1expressions by immunohistochemistry.;the mRNA levels of HO-1、NQO1and GST-α1by TR-PCR; NQO1and GST-a1enzyme activity was determined by emzyme activity assay; Some cortex was taken for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method.Results:Western Blot Analysis for Detecting Nrf2and HO-1Expressions after SAH. These proteins were expressed at a low level in the rat brains of control group. The levels of Nrf2and HO-1were significantly increased in the cortex in SAH+vehicle group as compared with that of control groups (P<0.05). The protein expressions had no significant difference between SAH group and SAH+vehicle group (P>0.05). After melatonin injections, the increased expression of Nrf2and HO-1was markedly further induced in animals of SAH+melatonin group (P<0.01). Immunohistochemical study showed that positive Nrf2and HO-1were mainly located at the neurons, with little expression at glial cells. The immunoreactivity of Nrf2andHO-1was weak in the cortex samples in control group with only a few Nrf2-or HO-1-positive cells in the brain. More Nrf2and HO-1positively immunostained neurons appeared in SAH group and SAH+vehicle group. In SAH+melatonin group, the number of Nrf2-or HO-1-positive cells was more than SAH+vehicle group. Both Nrf2and HO-1immunoreactivities of parenchymal cells in the cortex were progressively induced by melatonin therapy at48hr after blood injection(P<0.01). The mRNA of these proteins expressed at a low level in the rat brains of control group. The levels of HO-1, NQO1and GST-a1mRNA were significantly increased in the cortex in SAH and SAH+vehicle groups as compared with that of control group (P<0.01). The mRNA expressions had no significant difference between SAH group and SAH+vehicle group (P>0.05). The mRNA expressions of HO-1, NQO1, and GST-α1in the brains of SAH+melatonin group were significantly up-regulated than those of the SAH+vehicle group. In SAH+melatonin group, the cortical activity of NQO1and GST-a1was markedly up-regulately as compared with that of SAH+vehicle group(P<0.01). Few TUNEL-positive apoptotic cells were found in the control group rat brains. In SAH and SAH+vehicle groups, the apoptotic index in the cortex was found to be significantly increased compared with that in control animals (P<0.01). There was no statistically significant difference between SAH group and SAH+vehicle group (P>0.05). In SAH+melatonin group, when compared with that in the SAH+vehicle group, the apoptotic index in the studied cortex wassignificantly decreased (P<0.01).Conclusions:SAH could induce an activation of Nrf2-ARE signaling pathway that might play a central role in the antioxidant and detoxifying enzymes response that leads to secondary insults after SAH. The therapeutic benefit of post-SAH melatonin administration might be due to its salutary effect on modulating Nrf2-ARE signaling pathway.