Effect And Mechanism Of Necrostatin-1 On Synaptic Protection In Hippocampus After SAH In Rats

BackgroundsSubarachnoid hemorrhage(SAH)is a devastating form of stroke that leads to a high mortality and disability rate.It was traditionally believed that vasospasm is the key factor which leads to its unfavorable prognosis.Increasing evidence has proved that early brain injury(EBI)contributes mostly to unfavorable outcomes after SAH,including the direct injury and the secondary pathophysiological changes of the brain within 72 hours after the occurrence of SAH.Recently,A previously unknown mechanism of regulated cell death known as necroptosis has recently been reported known as necroptosis,which was also involved in EBI.Necroptosis was a kind of caspase-independent programed cell death involved in many diseases including SAH.Necrostatin-1(nec-1),a specific and potent inhibitor of necroptosis,can attenuate brain impairments after SAH.However,the effect of nec-1 on the hippocampus and its neuroprotective impact on synapses after SAH is not well understood.ObjectivesOur present study was designed to investigate the potential effects of nec-1 administration cn synapses and its relevant signal pathway in early brain injury after SAH.Methods1.A total of 159 rats were randomly divided into three groups:necroptosis after SAH,the protective effect of nec-1 for hippocampus,and the potential relationship between nec-1 and CREB-BDNF pathway.An experimental SAH model was made using a double blood injection model.Nec-1 was administrated via intraperitoneal injection after SAH.2.Western blotting and immunofluorescence was used to detect the necroptosis via RIP1 and RIP3 at 24 hours after SAH.3.Neurobehavior scores and brain edema were detected at 24 hours after SAH occurred.4.Hematoxylin and eosin staining,Nissl staining,silver staining and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)was used to observe the morphological changes in hippocampus.5.The protective effect of nec-1 on synapses was evaluated using western blotting and electron microscopy.6.Western blotting was used to detect the cAMP responsive element binding(CREB)protein and brain-derived neurotrophic factor(BDNF),and we used transmission electron microscopy and TUNEL to detect the protective effects of nec-1 when a specific inhibitor of CREB,known as 666-15,was used.Results1.In the SAH group,RIP1 and RIP3 significantly increased in the hippocampuswhile the treatment of nec-1 could reduce the expression of RIP 1 and RIP3.2.Injection of nec-1 alleviated brain edema and improved neurobehavior scores,compared with those in the SAH group.3.Compared with Sham group,in SAH group,the amount of neuron decreased,and the apoptotic cells increased.The structure of hippocampus is destroyed.In the Sham+nec-1 group,the damage to neurons was attenuated,and hippocampus structure also improved.4.According to TEM images,the synapses in hippocampus were normal,presynaptic membrane,postsynaptic membrane and synaptic cleft were observed clearly.The structure of synapse was damaged after SAH.Compared to the SAH group,the damage to synapses was alleviated after nec-1 injection according to TEM images.5.Synaptic proteins were decreased after SAH occurred.However,the nec-1 treatment could increase the synaptic protein expression.6.Phospho-CREB and BDNF expression decreased after SAH.Nec-1 treatment significantly enhanced the levels of phospho-CREB and BDNF compared with those in the SAH group.The protective effect of nec-1 could hindered by 666-15.ConclusionsIn this study,we report that SAH induced necroptosis because of a subsequent increase in the levels of RIP 1 and RIP3;nec-1 treatment could effectively reduce brain edema and increase neurobehavioral score.However,inhibition of necroptosis by nec-1 ameliorated the morphological damage to the hippocampus.In addition,the number of synaptic-associated proteins were reduced after SAH but obviously increased again after an injection of nec-1.Furthermore,p-CREB and BDNF levels were enhanced,and a specific CREB inhibitor could counteract the effect of nec-1,indicating that CREB-BDNF could be one potential pathway influenced by nec-1 treatment.