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

Objective: To investigate neuroprotection of Tamoxifen on early brain injury in malerats after subarachnoid hemorrahage, such as brain edema, and blood-brain barrier (BBB)impairment.Methods: forty-eight health male Sprague-dawley(SD) rats were assigned randomlyinto following groups: Control group（n=12）, SAH group（n=12）, SAH+vehicle group（n=12）, SAH+Tamoxifen group（n=12）.All SAH animals were subjected to injectionof0.3ml fresh arterial, non-heparinized blood into suprachiasmatic cistern in20seconds.Male rats were given5mg/kg injections of tamoxifen at post-SAH hours2h,24h and36h.Brain samples adjacent to the clotted blood were extracted at48h after SAH. Controlanimals underwent exactly the same procedure as described above with the exception thatno blood was injected intracisternally. Cerebral edema and blood-brain barrier permeabilitywere detected at48h after experimental SAH.Results: Significant increase (P <0.01) in water content was detected in the brainsamples at48h after SAH when compared with rats in control group. The mean value ofbrain water content in the cortex was decreased by tamoxifen administration (P <0.01) ascompared 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 of EBrelative to rats of control group. Administration of tamoxifen significantly inhibited EBextravasation(P <0.01) after SAH. Conclusions:1. As compared with control group, clinical behavior functionimpairment caused by SAH was evident in SAH subjects. SAH group significantlyattenuate blood-brain barrier (BBB), increase brain edema compared to control group.2.Post-SAH tamoxifen treatment significantly ameliorated the EBI, such as the clinicalbehavior scale, brain edema, and blood-brain barrier (BBB) impairment. It suggests thatTamoxifen could attenuate EBI in this rat SAH model. Objective: The aim of the current study was to investigate whether tamoxifenadministration modulate TLR4/NF-κB signaling pathway in the brain at the early braininjury (EBI) of SAH.Methods: Sixty male Sprague Dawley rats weighing from300to350g wererandomly divided into control group（n=15）, SAH group（n=15）, SAH+vehicle group（n=15） and SAH+tamoxifen group（n=15）.All SAH animals were subjected toinjection of0.3ml fresh arterial, non-heparinized blood into suprachiasmatic cistern in20seconds. Male rats were given5mg/kg injections of tamoxifen at post-SAH hours2h24hand36h. Rats of SAH+vehicle group received equal volumes of vehicle (1%ethanol) atcorresponding time points. Brain samples（adjacent to the clotted blood） were extracted at48h after SAH. Control animals underwent exactly the same procedure as described abovewith the exception that no blood was injected intracisternally.We measured the expressionsof TLR4, NF-κB, intercellular adhesion molecule-1(ICAM-1) by Western Blot；andTLR4、NF-κB and ICAM-1expressions by Immunohistochemistry.NF-κB DNA bindingactivity were quantified by EMSA.Results: Western Blot Analysis for detecting TLR4, NF-κB and ICAM-1expressionsafter SAH. These proteins were expressed at a low level in the rat brains of control group. The levels of TLR4, NF-κB and ICAM-1were significantly increased in the cortex inSAH group and SAH+vehicle group as compared with that of control group (P <0.01). Theprotein expressions had no significant difference between SAH group and SAH+vehiclegroup (P>0.05). After tamoxifen injections, the decreased expression of TLR4, NF-κBand ICAM-1was markedly further induced in animals of SAH+tamoxifen group (P<0.01).Immunohistochemical study showed that positive TLR4, NF-κB and ICAM-1were mainlylocated at the neurons, with little expression at glial cells. The immunoreactivity of TLR4,NF-κB and ICAM-1was weak in the cortex samples in control group with only a fewTLR4, NF-κB and ICAM-1positive cells in the brain. More TLR4, NF-κB and ICAM-1positively immunostained neurons appeared in SAH group and SAH+vehicle group. InSAH+tamoxifen group, the number of TLR4, NF-κB and ICAM-1positive cells was lessthan SAH group and SAH+vehicle group. TLR4,NF-κB and ICAM-1immunoreactivities of parenchymal cells in the cortex were progressively induced bytamoxifen therapy at48h after blood injection(P <0.01). EMSA autoradiography of NF-κBDNA binding activity in the brain was shown that Low NF-κB binding activity (weakEMSA autoradiography) was found in the control group. Compared with the control group,NF-κB binding activity significantly increased (P <0.01) in SAH group and SAH+vehiclegroup. There was no statistically significant difference between SAH group and SAH+vehicle group (P>0.05). In SAH+tamoxifen group, NF-κB binding activity wassignificantly down-regulated (P <0.01)after tamoxifen injections.Conclusions: SAH could induce an activation of TLR4/NF-κB signaling pathway thatmight play a central role in the inflammatory response that leads to secondary insults afterSAH. The therapeutic benefit of post-SAH tamoxifen administration might be due to itssalutary effect on modulating TLR4/NF-κB signaling pathway.