The Neuroprotective Role And Mechanism Of Aggf1 In Brain Injury After Subarachnoid Hemorrhage In Rats

Part One The expression of endogenous Aggf1 after SAHObjectives: To explore the expression of endogenous Aggf1 in protein level and its cellular localization after subarachnoid hemorrhage(SAH).Methods: The time course of endogenous Aggf1 in ipsilateral/left cerebral cortex was measured by Western blot analysis.Double immunofluorescence staining was performed to characterize the cellular localization of Aggf1 at 24 h after SAH.Results: There was a significant increase of Aggf1 level at 24 h,which peaked at 72 h after SAH when compared to sham group.Double immunofluorescence staining revealed that Aggf1 was mainly expressed in endothelial cells and astrocytes,as well as microglia in the ipsilateral basal cortex at 24 h after SAH.Conclusions: Expression of endogenous Aggf1 was markedly increased after SAH.Aggf1 was primarily expressed in endothelial cells and astrocytes,as well as microglia after SAH.Part Two The effects of exogenous rh-Aggf1 on brain injury and neuroinflammation after SAHObjectives: To explore the effects of exogenous rh-Aggf1 on neurological functions,brain water content,blood-brain barrier(BBB)permeability and neuroinflammation after SAH.Methods: Three different dosages of rh-Aggf1 dissolved in normal saline(NS)were administered via tail vein at 1 h after SAH.Aggf1 small interfering RNA(Aggf1 si RNA)was administered intracerebroventricularly(i.c.v)at 48 h before SAH induction.The foot-fault test was measured to assess the Motor sensory deficits at days 7,14,21 and the Morris water maze was performed to assess the spatial learning memory at days 21-25 after SAH.The neurological status was assessed using modified Garcia and beam balance,and SAH grade and brain water content were measured at 24 h after SAH.Evans Blue(EB)extravasation was performed to assess the BBB permeability.Immunofluorescence staining was conducted to assess the neutrophil infiltration.Furthermore,Western blot was used to measure the levels of TNF-α and IL-1β.Results: The neurological scores of modified Garcia and beam balance were significantly reduced,and Evans blue leakage and brain edema of both hemispheres was significantly increased at 24 h after SAH,but considerably reversed by administration of rh-Aggf1.Meanwhile,exogenous rh-Aggf1 reduced the number of infiltrating neutrophils and inhibit the expression of TNF-α and IL-1β.Conversely,these effects were abolished by Aggf1 si RNA.Aadministration of rh-Aggf1 significantly improved the neurological deficits in foot fault both in left forelimb and right forelimb.In the water maze test,the travel distance and escape latency for rats to find the platform were significantly increased after SAH.However,a significantly shorter distance on Block 2,3,4,5 and a decrease in time to find the platform at day 22,23,24,25 were observed after rh-Aggf1 treatment.Conclusions: Exogenous rh-Aggf1 treatment improved short-and long neurological functions,reduced brain edema and BBB permeability,and inhibited neutrophil infiltration and the release of inflammatory mediators.However,inhibition of endogenous Aggf1 aggravated brain injury and neuroinflammation.Part Three Aggf1 exerted neuroprotective roles via PI3K/Akt/NF-κB signaling pathway after SAHObjectives: To explore whether PI3K/Akt/NF-κB signaling pathway is involved in the Aggf1 induced neuroprotection after SAH.Methods: As described in part 2.PI3 K specific inhibitor LY294002 was administered i.c.v at 30 min before SAH induction.The exogenous rh-Aggf1 was administered via tail vein at 1 h after SAH.Neurological scores,brain water content were assessed.The expression of PI3 K,p-Akt,VE-cadherin,Occludin,Claudin-5,p-NF-κB p65,NF-κB p65,TNF-α,and IL-1β were measured by Western blot.Results: LY294002 significantly abolished the effects of rh-Aggf1 on neurological functions,brain water content,Albumin and MPO expression.Exogenous rh-Aggf1 increased PI3 K,p-Akt,VE-cadherin,Occludin,and Claudin-5 with a decrease in p-NF-κB p65,TNF-α,and IL-1β expression.However,LY294002 reversed rh-Aggf1 induced changes of downstream proteins.Conclusions: Exogenous Aggf1 treatment attenuated neuroinflammaiton and BBB disruption,improved neurological deficits after SAH in rats,at least in part through the PI3K/Akt/ NF-κB pathway.