The Effect And Mechanism Of Delta-opioid Receptors Treating The Retinal Injury Caused By Ischemic With Acupuncture

Objective: Previous studies on cognitive dysfunction caused by ischemic brain injury often ignored the impact of visual impairment caused by modeling on cognitive dysfunction.In this study,the traditional craniotomy electrocoagulation model of focal cerebral ischemia and 4VO global cerebral ischemia were modified,and the modified craniotomy ischemia reperfusion model,the modified four vessel continuous occlusion model and the modified four vessel interrupted occlusion model were established.The modified craniotomy ischemia reperfusion model,modified four vessel continuous occlusion model and four vessel interrupted occlusion modified were established to improve the traditional craniotomy electrocoagulation model of focal cerebral ischemia and 4VO global cerebral ischemia.In addition,classical Longa modeling and the bilateral common carotid artery ligation model of SD and Wistar rats were selected to compare the differences between the main brain injury and retinal injury in the above ischemic model.On this basis,Longa suture method was selected as the animal model of cerebral ischemia with retinal injury in rats.DOR(delta opioid receptor)antagonist naltrindole was injected into the vitreous cavity.Through histopathological analysis and behavioral test,the protective effect of electroacupuncture by "Shui Gou" and "Jing Ming" acupoints in rats was observed,and the relationship between the protective effect of electroacupuncture and DOR was discussed.Methods: Part I: comparative study on retinal injury in rats with cerebral ischemia In the first part of the study,we firstly improved the model of classical craniotomy cerebral ischemia model to establish a new model of cerebral ischemia reperfusion in rats.In view of the high mortality rate of Pulsinelli four vessel occlusion model,incomplete electrocoagulation,vulnerability to brain stem and spinal cord injury and other defects,technical innovation was carried out to establish a new four vessel continuous occlusion model and a four vessel interrupted occlusion model for subsequent studies.On this basis,classic Longa suture model and two-vessel occlusion(2VO)model in SD and Wistar rats was chosen.The differences of major cerebral ischemia and retinal injury in the above models were compared.Each model respectively was divided into control group,model group D1,model group D3,model group D7,model group D14,model group D28.On day 1,3,7,14,28,brain and optic nerve were removed and stained with hematoxylin and eosin(HE)and luxol fast blue(LFB)for histopathological analysis.After HE staining,histopathological observation was performed on the isolated eyeball samples.The thickness of retinal cell layers and the cell density of retinal ganglion cells(RGCs)were analyzed.The light and dark box was used to detect the changes in the light recognition ability of the model rats 28 days after surgery,and the Y maze test was used to detect the changes in the learning and memory ability 28 days after surgery.The histopathological examination results of brain,optic nerve and retina were combined with the neurobehavioral examination results of rats,so as to evaluate the difference between brain injury and retinal injury of the six models.Part II: the role of delta-opioid receptors in electroacupuncture in the treatment of retinal injury associated with cerebral ischemia reperfusion in rats In the second part of the study,30 rats were randomly divided into Sham group(Sham),MCAO model group(MCAO),MCAO model electroacupuncture group(MCAO-E),MACO model pseudoelectroacupuncture group(MCAO-S),and MCAO model electroacupuncture + antagonist naltrindole group(MCAO-E-N).The sham group,MCAO group,MCAO-E group and MCAO-S group were injected with normal saline 10 ?l in vitreous cavity 30 min before the modeling,and MCAO-E-N group was injected with 100 n M antagonist naltrindole in the vitreous cavity.The acupoints of electroacupuncture were selected as "Shui Gou" and "Jing Ming".In MCAO-E and MCAO-E-N groups,electroacupuncture therapy was started at 30 min after ischemia and continued until 30 min after reperfusion.In the MCAO-S group,only the acupoints of rats were punctured without electricity.After 24 h,the tissue samples were removed.Frozen sections of the brain were stained with crystal violet(CV)staining to observe histopathological changes of cerebral infarction and calculate the volume ratio of cerebral infarction.HE staining was used for histopathological observation of isolated eyeball samples and thickness analysis of retinal cell layers and cell density analysis of retinal ganglion cells.The positive expressions of glial fibrillary acidic protein(GFAP)and glutamine synthetase(GS)were observed and mean optical density value was analyzed by retinal immunofluorescence staining.The improvement effect of electroacupuncture on retinal injury was evaluated and explored whether the improvement effect is related to DOR.Results:Part I: comparative study on retinal injury in rats with cerebral ischemia Improved method of model craniotomy postoperative brain tissue pathology inspection found that the model of cerebral infarction area is limited to the cortex.The histopathological changes of striatum,hippocampus,cerebral white matter were not observed.Neurological impairment of learning memory and lighting recognition ability was not observed by the Y-maze and light-dark box test 28 days after surgery.Using the classic Longa modeling method,the obvious ischemic changes of cerebral cortex and striatum were observed one day after the operation,which even damaging the hippocampus and white matter,and then the ischemic injury presented progressive aggravation.HE staining test of the retina,postoperative 1 day outer nuclear layer(ONL)appeared significant thickening(P ? 0.05),postoperative 3 days appeared inner nuclear layer(INL)vacuolation in two rats,other phases were not appeared obvious histopathological changes of the retina.Neurological impairment of learning memory and lighting recognition ability was not observed by the Y-maze and light-dark box 28 days after surgery.In the two vessel occlusion model of SD rats,the areas of cerebral ischemic injury were mainly white matter,especially the optic tract.On the 28 th day after surgery,ischemic changes in the hippocampus CA1 region were found in two rats,while no significant ischemic changes were observed in the cortex and striatum.Ischemic injury of optic nerve fibers was observed one day after surgery,followed by progressive aggravation 28 days later.Retinal histopathological changes were observed 1 day after surgery,mainly in ganglion cell layer(GCL)and ONL,followed by progressive aggravation of ischemic changes.Retinal damage was obvious in the 28 days after surgery,mainly in GCL,inner plexiform layer(IPL)and outer plexiform layer(OPL).The number of RGCs decreased,and the cell density was significantly lower than that of the sham group(P < 0.001).The Y-maze test showed that the rats had learning and memory impairment on the 28 th day after surgery In the model of two vessel occlusion in Wistar rats,the areas of cerebral ischemic injury were mainly white matter,especially the optic tract,and the neuron damage could be observed 3 days after the surgery,while no significant ischemic changes were observed in the hippocampus,cortex and striatum at all stages.Ischemic injury of nerve fibers was observed in the optic nerve 1 day after surgery,followed by progressive aggravation of ischemic injury 28 days later.The retinal histopathology change can be seen one day after surgery,especially in GCL,IPL,INL,ONL.The ischemic changes were gradually aggravated,and the retinal damage was obvious 28 days after the surgery,mainly in GCL,IPL,INL,OPL,photoreceptor layer(PL)and retinal pigment epithelium(RPE).Total thickness and the retinal cells layer thickness have a significant difference compared with the sham group.RGCs number decreases,while cell density compared with the sham group had a significant difference(P < 0.001).The light-dark box test shows that rats have visual impairment 28 days after surgery.In the modified four vessel continuous occlusion model,the vertebral artery passing between the first cervical vertebra and the second cervical vertebra was separated and ligation.After 24 hours,the common carotid artery were occluded for 20 min by artery clamp.Postoperative cerebral ischemic injury mainly occurred in the hippocampal CA1 area.One day after surgery,pyramidal cell ischemic injury began to be observed in the hippocampal CA1 area,and then the injury continued to worsen.There was no ischemic change in the hippocampal CA3 and DG area,cortex,striatum and white matter area.Histopathological changes were not observed in the optic nerve or retina.The Y maze test 28 days after surgery showed that the rats had learning and memory impairment.In the modified four vessel interrupted occlusion model,the vertebral artery passing between the first cervical vertebra and the second cervical vertebra was separated.Artery clips were used to occlude bilateral vertebral artery and common carotid artery,causing ischemia for 5min,then reperfusion for 5min and circulation for 3 times.Post operative cerebral ischemic injury mainly occurred in the hippocampal CA1 area.One day after surgery,ischemic injury of pyramidal cells was observed in the hippocampal CA1 area,and then the injury continued to worsen.No ischemic changes were observed in neurons in the hippocampal CA3 and DG areas.There were no ischemic changes in cortex,striatum and white matter.Histopathological changes were not observed in the optic nerve or retina.The Y maze test 28 days after surgery showed that the rats had some learning and memory impairment.Part II: the role of delta-opioid receptors in electroacupuncture in the treatment of retinal injury associated with cerebral ischemia reperfusion in rats In the second part of the study,brain CV staining 24 h after Longa suture model showed no significant difference in cerebral infarction volume between MCAO-E,MCAO-S,MCAO-E-N group and MCAO group(P > 0.05).Retinal pathology analysis shows that ONL,INL cell layer of MCAO group,MCAO-S group,MCAO-E-N group thicken,compared with the sham group with a significant difference(P ? 0.05),while MCAO-E group having no change.GFAP immunofluorescence detection results showed that GFAP was only weakly expressed in the GCL/NFL layer of the MCAO-E group,and highly expressed in the GCL/NFL layer of the MCAO group,MCAO-S group,and MCAO-E-N group.GS immunofluorescence detection results showed that GS was highly expressed in GCL/NFL,IPL,INL,OPL,ONL layers of MCAO group,MCAO-S group and MCAOE-N group.MCAO-E group only found GS expression in GCL/NFL,INL,ONL layer of the retina.It is suggested that electroacupuncture can improve the retinal injury,which can be reversed by naltrindole.Conclusions: 1.The results of the ischemia model showed that cortical injury was observed in the craniotomy ischemia reperfusion model,gray matter(hippocampus)injury was observed in the modified four vessel continuous occlusion model and four vessel interrupted occlusion model,without white matter or retinal injury.In the 2VO of SD rat model,gray matter(hippocampus)and white matter injury were observed with retinal injury.In the 2VO of Wistar rat model,white matter injury was observed with retinal injury,while gray matter(hippocampus)was not.Cortical,striatum,and even gray matter(hippocampus)and white matter lesions were observed with retinal injury in the Longa suture occlusion model.2.Needling "Shui Gou","Jing Ming" to treat cerebral ischemia with retinal damage shows that electroacupuncture has obvious neural protection.However,DOR antagonist naltrindole injected into vitreous cavity can reverse the protective effect of electroacupuncture,showing that DOR maybe a key factor in reducing the retinal damage using electroacupuncture.