| Background:Ischemic stroke is the second leading cause of mortality worldwide.The systematic study of the treatment and mechanism of cerebral ischemia requires a stable and reproducible model of global cerebral ischemia.Although mice and gerbils are used in many models,but a reliable model of global cerebral ischemia in rats is also needed,and needs to be further improved.The four-vessel occlusion method is a widely used in the global cerebral ischemia model.Permanent occlusion of the bilateral vertebral arteries is the key to eventual success of this model.However,it is impossible to visualize the vertebral artery directly through the alar foramen during surgery in rats.Therefore,the selection of successful model relies on righting reflex and EEG results.This study needs to establish a stable model of global cerebral ischemia in rats that is the foundation for subsequent experiments.The blood-brain barrier is a diffusion barrier between plasma and cerebral cells,and is essential to maintain homeostasis and normal function of the central nervous system.The endothelial cells of blood-brain barrier differ functionally and morphologically from those of noncerebral capillaries.In many nervous system diseases,the integrity of the blood-brain barrier will be destroyed,such as ischemic stroke.The effects of cerebral ischemia on blood-brain barrier has been widely studied.A series of events,which leads to disruption of tight junctions and increased blood-brain barrier permeability is induced by cerebral ischemia and hypoxia.Many studies have shown that estrogen can protect brain tissue from ischemia to prevent the destruction of the blood-brain barrier.The protective effect of estrogen is mediated by estrogen receptors(ER?,ER?,GPER-1).Studies have shown that ER? and ER? were responsible for the protective effects of estrogen on tight junctions protein of vascular endothelial cells after cerebral ischemia.However,the roles of GPER-1 in the blood-brain barrier after cerebral ischemia is unclear.Purpose:1.To explore the location of the vertebral artery in rats,and to look for the best part for occluding the vertebral artery.2.To establish a stable model of the global cerebral ischemia,and to explore the best ischemic time and best ischemic brain region.3.To explore whether GPER-1 activation can preserve tight junctions integrity after global cerebral ischemia,and to evaluate proteins expression of GPER-1 and VEGF-A in the CA1 after global cerebral ischemia.4.To explore whether GPER-1-mediated blood-brain barrier protection is associated with the inhibition of VEGF-A expression.Methods:1.CTA,DSA,and latex perfusion method are used to show the location of the vertebral artery in rats.2.We established a new global brain ischemia model in rats.The survival rates of the global cerebral ischemic rats were observed with increased duration of ischemia(10min,20 min,30min).After perfusion-fixation,the rat brains were removed.The neuronal injury was observed in multiple brain regions of rats by nissl staining and TUNEL labeling staining.In the neurological deficit score and tape removal test,the rat behavior change after global cerebral ischemia is observed.3.We used western blot technique to observe Ig G extravasation and tight junction proteins levels in the ischemic CA1,and to evaluate proteins expression of GPER-1 and VEGF-A in the CA1 after 20 min global cerebral ischemia.4.We used western blot and immunofluorescence technique to observe whether GPER-1 activation can decrease blood-brain barrier breakdown and increased tight junction proteins levels in ischemic CA1.In addition,we observed whether the GPER-1 agonist can prevent the increase of VEGF-A protein in the ischemic CA1.Results1.The results of CTA,DSA,and latex perfusion showed that the vertebral artery is outside the atlantoaxial joint between the first and second transverse processes.We established a novel global cerebral ischemia model in rats with visualized occlusion of the vertebral artery and the common carotid arteries.2.Survival analyses indicated that 10 min ischemia already decreased the survival rate to 93% throughout 7 days after surgery.Rats subjected to 20 min ischemia showed a survival rate around 80%.Rats subjected to 30 min ischemia disclosed a survival rate around 50%.20 min is the best ischemia time in the new global cerebral ischemia model.3.The results of nissl staining showed that significant loss of CA1 neurons was observed at day 3 after 20 min global ischemia.No further decrease in neurons counts was observed between 3 and 7 days after 20 min ischemia.4.Tape test and TUNEL analysis confirmed neuronal damage to the cortex and striatum on days 3 and 7 after global ischemia.Therefore,this new global cerebral ischemia model may exhibit neuronal damage in areas relevant to sensorimotor integration.5.Western blot results showed that both occludin and claudin-5 proteins expression were reduced at 6 h in ischemic CA1,but their expression were significantly reduced at 24 h after global ischemia.The tight junction proteins levels were significantly reduced at 24 h after global ischemia.Therefore,the effect of G1 was examined at 24 h after global ischemia.6.Western blot and immunofluorescence results showed that reduced occludin and claudin-5 proteins levels were prevented by G-1 at 24 h after global ischemia.In addition,G-1 can significantly decrease VEGF-A protein levels in ischemic CA1.Conclusions1.New global cerebral ischemia model can induce reproducible delayed neuronal injury in CA1 region,and nerve functional disturbance in rats.2.New global cerebral ischemia model can induce blood-brain barrier breakdown in CA1.In addition,tight junction proteins levels were significantly reduced at 24 h after global ischemia.3.The GPER-1 agonist can significantly increase the levels of tight junction proteins in the ischemic CA1.The GPER-1 agonist can prevent the increase of VEGF-A protein in the ischemic CA1. |
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