Role Of Endoplasmic Reticulum Stress In Brain Damage After Cardiopulmonary Resuscitation

[Objective] Recent studies have demonstrated that endoplasmic reticulum (ER) stress is an essential signaling event for neuronal injury resulting from ischemia/reperfusion, and inhibition of ER stress may be used as therapeutic target for neuroprotection. In this study, we investigated whether ER stress mediated apoptosis is activated in the brain after cardiac arrest (CA) and resuscitation. Furthermore, to determine whether inhibition of ER stress may attenuate the severity of brain damage after resuscitation, a specific ER stress inhibitor, salubrinal, was administered before CA.[Methods] In the first experiment, Male Sprague-Dawley rats were subjected to6min cardiac arrest (CA) and then resuscitated successfully. Animals were decapitated at1,3,6,12and24h after return of spontaneous circulation (ROSC). Brain tissues were analysis by RT-PCR and western blot. In the second experiment, Male Sprague-Dawley rats were randomly divided into three groups:(1) Sham group:animals received the same procedures except cardiac arrest;(2) Sal+CPR group:Salubrinal (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO) and was administered at the dose of1mg/kg (ip)30min before induction of CA (Sal+CPR group). Rats were subjected to6minutes cardiac arrest induced by transcutaneous electrical epicardium stimulation and then resuscitated successfully;(3) An equal does of DMSO was injected as control animals (DMSO+CPR). Rats were subjected to6minutes cardiac arrest induced by transcutaneous electrical epicardium stimulation and then resuscitated successfully. Neurological outcomes were assessed at24h after CA.①ER and mitochondrial damage in the three groups was studied by transmission electron microscopy.②TUNEL stain was employed to observe the neurocyte apoptosis in the hippocampal CA-1sector from three groups.③IHC stain was employed to observe the changes of glucose-regulated protein78(GRP78), X-box binding protein1(XBP-1), C/EBP homologous protein (CHOP) and caspase12in the hippocampal CA-1sector.④The expression of eukaryotic translation initiation factor2subunit a (eIF2a)、phosphorylate eukaryotic translation initiation factor2subunit a (p-eIF2a)、CHOP and caspase12were detected using western blot in the cortex.⑤After the isolation of cortex mitochondria, mitochondrial permeability transition pore (mPTP) opening was detected by monochromator microplate reader.⑥Cortex single cell suspension were prepared immediately at24h after CPR, Intracellular Ca2+concentration in the three groups were detected by monochromator microplate reader.⑦The MDA and SOD levels in the cortex of three groups were evaluated by a monochromator microplate reader.[Results] In the first experiment, we found that the mRNA and protein levels of GRP78, XBP-1, CHOP and caspase12were significantly elevated after resuscitation. In the first experiment,①In DMSO+CPR and Sal+CPR groups, the NDS were increased after CA(P<0.01), while animals treated with Sal revealed an improved neurological outcome(P<0.05).②Compared with sham group, the TUNEL positive cells were both increased in DMSO+CPR and Sal+CPRgroups(P<0.01). Compared with DMSO+CPR group, the TUNEL positive cells in Sal+CPR group was decreased (P<0.05).③Compared with Sham group, endoplasmic reticulum was damaged both in DMSO+CPR and Sal+CPR groups. However, Sal pretreatment reduced the damage.④Changes in the GRP78, XBP-1, CHOP and caspase12proteins were confirmed by IHC. Compared with those from the sham group, samples from the DMSO+CPR group exhibited notably higher immunoreactivity for XBP-1, CHOP and caspase12proteins. However, this increase was significantly ameliorated by Sal. Sal increased GRP78protein expression at24h after ROSC compared to the DMSO+CPR group.⑤Compared with Sham group, mitochondria was damaged both in DMSO+CPR and Sal+CPR groups. However, Sal pretreatment reduced the damage.⑥When compared with Sham group, the expression of p-eIF2α/eIF2α、CHOP and caspase12were both increased in DMSO+CPR and Sal+CPR groups (P<0.01, respectively). Compared with DMSO+CPR group, the expression of CHOP and caspase12(P<0.01, respectively) were decreased and p-eIF2a/eIF2a expression was enhanced in Sal+CPR group.⑦When compared with Sham group, the opening of brain mPTP was increased both in DMSO+CPR and Sal+CPR groups(P<0.01). Compared with DMSO+CPR group, Sal exhibited inhibition effect on mPTP opening(P<0.01).⑧When compared with Sham group, intracellular Ca2+concentration was increased in DMSO+CPR and Sal+CPR group (P<0.01). Compared with DMSO+CPR group, intracellular Ca2+concentration was decreased in Sal+CPR group (P<0.01).⑨Compared with Sham group, the expression of SOD in DMSO+CPR and Sal+CPR groups was decreased (P<0.01). Compared with DMSO+CPR group, the expression of SOD in Sal+CPR group was obviously increased (P<0.01).⑩Compared with Sham group, the expression of MDA in DMSO+CPR and Sal+CPR groups was increased (P<0.01). Compared with DMSO+CPR group, the expression of SOD in Sal+CPR group was obviously decreased (P<0.01).[Conclusions] Our study provides the evidence that ER stress-induced CHOP and caspase12apoptotic pathways are activated in hippocampus after ROSC. Sal pretreatment attenuated post-resuscitation brain injury in a rat model of CA. The protective effects may be though inhibiting ER stress response, apoptosis inhibition and enhancing GRP78expression. Thus, amelioration of ER stress-induced apoptosis could be a novel strategy for preventing and treating post-cardiac arrest brain injury.