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

Part Ⅰ: The Neuroprotective effects of Dimethylfumarate on early braininjury in male rats after subarachnoid hemorrhageObjective: To investigate the Dimethylfumarate(DMF) treatment whethersignificantly ameliorated the early brain injury, as the clinical brain edema, blood-brainbarrier (BBB) and behavior scale impairment.Methods: Ninety-six health male Sprague-dawley(SD) rats were assigned randomlyinto two parts(48rats/part). One part we observe the behavior scaleand brain edema, theother one part we observe the blood-brain barrier (BBB) impairment. The following groups:Control group（n=12）, SAH group（n=12）, Vehicle+SAH group, DMF+SAH group（n=12）. The SAH animals were subjected to injection of0.3ml fresh arterial andnon-heparinized blood into prechiasmatic cistern in20seconds. Male rats were given15mg/kg via oral gavage of DMF at post-SAH hours1,12,24and36. At48h aftersubarachnoid hemorrhage, brain samples adjacent to the clotted blood were extracted. Noblood was injected intracisternall, control animals underwent exactly the same procedureas described above with the exception. The clinical behaviors function after experimentalSAH were observed; blood-brain barrier and cerebral edema permeability were detected at48h after experimental subarachnoid hemorrhage.Results: After Experimental SAH, impact of DMF on Clinical Behavior Function.Compared with control group, the clinical behavior function impairment caused by SAHwas evident in SAH subjects (P <0.01). No significant difference was seen between the Vehicle group and SAH group (P>0.05). The15mg/kg DMF via oral gavage treated ratsshowed better performance in this scale system than SAH group rats at48h after SAH andthe difference was statistically significant (P <0.01). In the brain samples at48h after SAHwhen compared with rats in control group, there have significant increase (P<0.01) inwater content was detected. The mean value of brain water content in the cortex wasdecreased by15mg/kg DMF administration (P<0.01) as compared with Vehicle group andSAH group. The results suggested that moderae DMF treatment could attenuate brainedema in this rat SAH model. The Blood-Brain Barrier influence. Rats in SAH groupdemonstrated a significant increase (P<0.01) in blood-brain barrier permeability to Evansblue relative to rats of control group. The DMF significantly inhibited Evans blueextravasation (P<0.05), indicating a reduced BBB opening in response to via oral gavageof DMF.Conclusions:1. The Vehicle group and SAH group increase brain edema,significantly attenuate blood-brain barrier compared to control group.2. Post-SAH via oralgavage of DMF treatment significantly ameliorated the early brain injury, such as theblood-brain barrier, clinical behavior scale and brain edema impairment. Via oral gavageof DMF could attenuate the early brain injury in this rat SAH model. Part II: Dimethylfumarate administration modulates corticalKeap1-Nrf2-ARE signaling pathway after subarachnoid hemorrhage inmale ratsObjective: We investigate whether Dimethylfumarate administration modulatedKeap1-Nrf2-ARE pathway signaling pathway in the brain at the early stage ofsubarachnoid hemorrhage.Methods:48male Sprague-Dawley (SD) rats weighing from280to320g wererandomly divided into4group. control group（n=12）, SAH group（n=12）, vehicle+SAHgroup（n=12） and DMF+SAH group（n=12）. Rats were injected of300μl autologous blood into the chiasmatic cistern. Rats in the DMF treated group were gavaged (15mg/kg)twice daily for2days after onset of the SAH. Cortical apoptosis, brain edema, neuralnecrosis, learning deficits, blood-brain barrier impairment, and changes in theKeap1-Nrf2-ARE pathway were assessed. The expressions of the mRNA levels ofGST-a1、NQO1and HO-1by RT-PCR; By emzyme activity assay the GST-a1and NQOlenzyme activity; Keap1、Nrf2and HO-1by immunohistochemistry and western blot; Somecortex was taken for terminal deoxynucleotidyl transferase-nediated dUTP nick endlabeling (TUNEL) and Fluoro-jade B method; Nuclear protein was determined by Nuclearprotein extract and electrophoretic mobility shift assay (EMSA); The levels ofinflammatory mediators were quantified using specific ELISA。Results: The levels of GST-a1, NQOl and HO-1mRNA were significantly increasedin the cortex in SAH and vehicle+SAH groups as compared with that of control group (P<0.01). The mRNA expressions had nosignificant difference between vehicle+SAH groupand SAH group (P>0.05). The mRNA expressions of GST-a1, NQOl and HO-1in thebrains of DMF+SAH group were significantly up-regulated than those of the vehicle+SAH group. In DMF+SAH group, the cortical activity of GST-a1and NQOl was markedlyup-regulately as compared with that of vehicle group (P<0.01). Immunohistochemistry andwestern blot analysis for detecting Keap1, Nrf2and HO-1expressions after subarachnoidhemorrhage. In the rat brains of control group, the proteins were expressed at a low level.The levels of Keap1, Nrf2and HO-1were significantly increased in the cortex in Vehiclegroup and SAH, After via oral gavage of DMF, the increased expression of Nrf2and HO-1was markedly further induced in animals of DMF group (P<0.01). In the control group, thefew TUNEL and Fluoro-jade B-positive apoptotic cells were found. In Vehicle and SAHgroups, In control animals, the apoptotic index in the cortex was found to be significantlyincreased (P<0.01). No statistically significant difference between SAH+vehicle groupand SAH group (P>0.05), The DMF group the apoptotic index in the studied cortexwassignificantly decreased (P<0.01). In the control group, low Nrf2binding activity (weakEMSA autoradiography) was found. Compared with the control group, Nrf2binding activity in the injured brain was significantly increased (P<0.01) in Vehicle and SAHgroups. In the DMF group, the Nrf2binding activity was significantly up-regulated(P<0.01) in the brain tissue surrounding the blood clot site after SAH. The cortical levelsof the three inflammatory cytokines were greatly increased after SAH (P<0.01), DMFadministration after SAH could lead to significantly decreased IL-1β, IL-6and TNF-αconcentrations (P<0.01).Conclusions:1. Up-regulate the protein expressions of Keap1、Nrf2and downstream factors(HO-1、NQO1、GST-α1), which could be markedly up-regulated by DMF therapy.2. The dimethylfumarate could induce an activation of Keap1-Nrf2-ARE signalingpathway in the rat brain. The DMF might play a central role in the anti-oxidative andanti-inflammatory effect that leads to better outcome after subarachnoid hemorrhage.