Administration Of 17β-estradiol Or The Selective GPR30 Agonist G1 Provides Neuroprotection After Cardiac Arrest On Mice

Background:According to the 2013 update of Heart Disease and Stroke Statistics from the American Heart Association (AHA), out-of-hospital cardiac arrest (CA) has an overall incidence of 359,400 per year and only 9.5% of patients survivedt. However, about 70% of these patients remain moderate to severe cognitive deficits three months after CAt. Over decades, a host of putative neuroprotective strategies failed to demonstrate clinical benefit except for mild therapeutic hypothermia.Recently, the role of estrogen in central nervous system (CNS) has been intensively addressed. It has been shown that estrogen reduces the infarction volume in middle cerebral artery occlusion (MCAO) and mortality in global cerebral ischemia (GCI). Epidemiological studies also disclose that the incidence of stroke in pre-menopausal women is less compared to the same age-group men. However, there is no significant difference between men and post-menopause women. It is postulated that estrogen might exert neuroprotective effects by regulating estrogen synthesis and the expression of estrogen receptors (ER). Estrogen might provide neuroprotection through the following mechanisms:genome signaling pathways, non-genome signaling pathways, anti-oxidation, regulation of cell apoptosis, anti-excitotoxicity and regulation of mitochondrial function et al. G-protein coupled estrogen receptor 1 (GPER1/GPR30) is a newly identified G protein coupled receptor that binds to estrogen with high affinity. Activation of GPR30 results in both rapid nongenomic signaling events and transcriptional regulation. There is evidence showing that GPR30 agonist G1 reduces infarct volume in the cortex and striatum following experimental stroke. Our previous study indicates that chronic pretreatment with 17β-estrodiol (E2) or GPR30 selective agonist G1 is protective in cardiac arrest-induced global ischemia. A recent study shows that the neuroprotective effects of exogenously administered estrogen are mediated at least partly by GPR30 activation. However, it remains unclear whether activation of GPR30 is an alternative approach to neuroprotection following cerebral ischemia. Moreover, it remains unclear if the neuroprotection of E2 is mediated by GPR30 activation. The neuroprotective effects of intravenous treatment with E2 or Gl within a clinically relevant therapeutic window have not yet been studied in models of CA. It is important to explore the effect of E2 and GPR30 on global ischemia induced by CA which might provide alternative approach to neuroprotection. In this study, we investigated the effects of E2, G1 and the potential mechanism of E2’s neuroprotection against global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) in CA/CPR models. By using a selective antagonist of GPR30 (G15), we also clarified the possible mechanisms by which E2 exerts its neuroprotection.Objective:In a global cerebral ischemia mouse model induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR), we investigated the neuroprotective effect of 170-estradiol and GPR30’s selective agonist G1, and studied the underlying mechanisms of E2 using a GPR30’s selective antagonist G15.Methods:Adult (8-12 weeks old,20-25g) male mice were subjected to cardiac arrest induced by potassium chloride injection followed by resuscitation with mechanical ventilation, epinephrine and chest compressions. Thirty minutes after resuscitation, mice were treated with E2(100μg/kg), G1(200μg/kg), G15(300μg/kg), E2+G15(E2 100μg/kg, G15 300μg/kg) or vehicle. A neurological score was observed on each mouse 24,48 and 72h after CA/CPR. CA1 hippocampal injury was assessed 3 days after cardiac arrest by hematoxylin and eosin (H&E). Western blot was used to evaluate the effects of CA/CPR and G1 treatment on phosphorylation of ERK1/2.Results:Our data showed that body weight, mean ischemia time and epinephrine dose were not different among experimental groups (P>0.05). Neurofunctional consequence was no difference among each group in same time point (P>0.05). Body weight didn’t change significantly between treatment groups during the 3 day observation period (P>0.05). The mortality of neuronal cell in the hippocampal CA1 region in each treated group was 51.4± 5.0%(vehicle treated groupl, n=9),18.9±2.6%(E2 treated group, n=14),51.3+8.7% (vehicle treated group2, n=9),20.5±3.2%(G1 treated group, n=9),57.3±13.3%(G15 treated group, n=4),46.5±11.9%(E2 and G15 treated group, n=6). The mortality of neuronal cell in the hippocampal CA1 region in E2 treated group was reduced compared to the vehicle treated group 1 (P=0.000). The mortality of neuronal cell in the hippocampal CA1 region in E2 treated group was reduced compared to the vehicle treated group 2 (P=0.000). Compared to vehicle treated group 1, G1 significantly decreased the mortality of neuronal cell in the hippocampal CA1 region (P=0.001). Compared to vehicle treated group 2, G1 significantly decreased the mortality of neuronal cell in the hippocampal CA1 region (P<0.001). However, there was no significant difference in CA1 neuronal damage between the G15 treated group and the vehicle treated group (P>0.05). Similarly, there was no significant difference in CA1 neuronal damage between the E2+G15 treated group and the vehicle treated group (P>0.05). There was no significant difference in CA1 neuronal damage between the G1 treated group and the E2 treated group (P>0.05). The mortality of neuronal cell in the CA1 region in E2 treated group was reduced compared to the E2 combined G15 treated group (P=0.004).Compared to E2 combined G1 treated group, E2 significantly reduced the mortality of neuronal cell in the CA1 region (P=0.011).The mortality of neuronal cell in the CA1 region in E2 treated group was reduced compared to the G15 treated group (P=0.001). Compared to G15 treated group, E2 significantly reduced the mortality of neuronal cell in the CA1 region (P=0.002). The level of phosphorylation of ERK1/2 was reduced after GCI compared to sham group (P<0.05) and G1 maintained the level of phosphorylation of ERK1/2 following CA/CPR (P<0.05).Conclusion:Administration of E2 or the selective GPERl agonist G1 decreased neuronal damage and exerted neuroprotection following CA/CPR. However, G15, the selective GPR30 antagonist, significantly blocked the neuroprotection of E2 in neuronal cells of CA1 region, indicating that E2 exerted neuroprotection via GPR30 activation. The selective GPR30 agonist G1 protected neurons by maintaining the level of pERK1/2.