Study Of The Relationship Between Delta Opioid Receptor In Ischemic Injury And Electroacupuncture Anti-ischemic Efficacy In MCAO Rats

Ischemic stroke is a high-incidence disease in clinic.Studying the mechanism of ischemic brain injury and neuronal protection in stroke has been a major focus on clinical research,however,clinically effective and safe methods for reducing ischemic brain injury have not yet been well established.Evidence has accumulated to indicate that opioids in the body are involved in the pathogenesis of stroke.Opioids have a wide range of physiologic and behavioral effects on pain perception,mood,motor control and autonomic function.Their effects are mediated by opioid receptors.In addition,previous studies have suggested that delta opioid receptors protect neurons against ischemia/hypoxic stress.For example:1.The high density and high affinity of delta-opioid receptors may be important in the tolerance to hypoxia in the turtle brain.2.Systemic administration of opioid receptor agonists prolonged the survival period during hypoxia in mice in whole animal experiments and this protection is selectively blocked by delta- but not by mu- or kappa-opioid receptor antagonists.3.Delta-,but not mu- and kappa- opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury.These findings support the concept that the d-opioid receptor plays an important role in neuronal protection.Brain-derived neurotrophic factor(BDNF) is a member of the neurotrophin family.BDNF is recognized to play an important role in the neuroprotection. Activation of cAMP-response-element-binding protein(CREB) after focal cerebral ischemia increase in CREB phosphorylation can increases BDNF expression and stimulates neuroprotection in the adult dentate gyrus.It is reported that delta-opioid receptor agonist[D-Pen(2,5)]enkephalin(DPDPE) produced a dose-dependent increase in CREB phosphorylation and the effect was reversed by Naltrindole,which is a highly selective deltaopioid receptor antagonist.Intracerebroventricular administration of a selective non-peptidic DOR agonist(+)BW373U86,increased BDNF mRNA expression in the frontal cortex through a DOR-mediated mechanism because these effects were blocked by the DOR antagonist Naltrindole,but not by the micro-opioid receptor(MOR) antagonist naltrexone(NTX) or the kappa-opioid receptor antagonist nor-binaltorphimine.Therefore,to ensure the role of DOR in MCAO/reperfusion injury and to investigate its mechanism from p-CREB/CREB and BDNF in the pathogenesis of stroke is the main target of the present study.In traditional Chinese medicine,acupuncture for ischemic stroke is much better than other treatments with convenience availability,cheapness and convinced by both experts and patients of ischemic stroke all over the world.Compared with the increasingly application of acupuncture in the treatment of stroke,the basic studies of these protective mechanisms are still limited so far.Numerous reports have indicated that Electroacupuncture(EA) can activate the endogenous opioid system during the treatments.It is reported that the EA preconditioning reduced brain infarct volume,and administration of Naltrindole attenuated the brain infarct volume reduction induced by EA preconditioning.It is reported that EA may increase expression of BDNF in the peripheral cortex of the infarction,and it would be protective effect against cerebral ischemia.In the previous work of our laboratory,we detected that EA could enhance extracellular BDNF level during ischemia and reperfusion.EA can reverse the decreased levels of p-CREB in rat model of depression and increase the BDNF expression.Then,another part of our studies was to explore the relationship between DOR and EA in MCAO/reperfusion injury,and to investigate its mechanism involving p-CREB/CREB and BDNF in the pathogenesis of stroke.It is well documented that neurogenesis exists in the adult mammalian brain throughout the life,and it is strengthened by cerebral ischemia.Adult neurogenesis as a treatment to replace dying neurons is therefore a potentially significant therapeutic strategy for ischemic injury.It is reported recently that DOR is related with neurogenesis,the subpopulations of neurons,astrocytes,and oligodendrocytes in the SVZ and striatum differentially express mu-,delta-,and/or kappa-receptor immunoreactivity in a cell type-specific and developmentally regulated manner.The selective delta-opioid receptor antagonist Naltrindole,increase astrogliosis in the cingulate cortex,but SNC-80 decrease astrogliosis.MCAO mediates the phosphorylation of CREB which drives a prominent synthesis of BDNF and this BDNF synthesis induces neurogenesis.Electroacupunctrue enhances striatal neurogenesis in adult rat brains after a transient cerebral middle artery occlusion and up-regulates BDNF.Then,the third part of our studies was to explore the relationship berween DOR and EA in neurogenesis after MCAO/reperfusion injury,and to investigate its mechanism from p-CREB/CREB and BDNF in the neurogenesis after stroke.By using the molecular biological techniques,including immunohistochemistry and Western-Blot,the present study was designed to explore:(1) the role of DOR and the mechanism in MCAO/reperfusion injury with p-CREB/CREB and BDNF;(2) the role of DOR in EA anti-ischemic efficacy and the mechanism with p-CREB/CREB and BDNF;(3) the role of DOR and EA in neurogenesis,and the relationship between p-CREB/CREB and BDNF.The results are following:1.Role of DOR and its mechanism in MCAO/reperfusion injury with p-CREB/CREB and BDNF1.1 Effects of DOR antagonist(Naltrindole) and agonist(TAN-67) on MCAO/reperfusion ratsRats were divided into eight groups:sham group,middle cerebral occlusion group(MCAO),MCAO+TAN67 3 mM group,MCAO+TAN67 6mM group, MCAO+TAN67 10 mM group,MCAO+Naltrindole 2mM group,MCAO+Naltrindole 5mM group group,MCAO+Naltrindole 10mM group.Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.1.1.1 Effects of Naltrindole and TAN-67 on rCBFRegional cortical focal cerebral blood flow(rCBF) of ischemic hemisphere was monitored by laser Doppler Flowmetry.Steady state baseline values were recorded before MCAO so that all regional CBF(rCBF) data were expressed as percentages of the respective basal values.The rCBF was monitored continuously during focal ischemia till the first 30 min of reperfusion in all experimental groups.When the middle cerebral artery is obstructed successfully,local blood flow decreased immediately to lower than 30%of pre-ischemia(baseline) and persisted lower level during the whole ischemia stage and even to stage of reperfusion.The results indicated that no significant differences were observed between the MCAO (aCSF+MCAO),[MCAO+Naltrindole(2 mM,5mM,10 Mm)]and[MCAO+TAN-67 (3mM,6 mM,20 mM)]groups.1.1.2 Effects of Naltrindole and TAN-67 on Infarct VolumeAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Rats were received intracerebroventricular injection with DOR antagonist(Naltrindole,2 mM,5mM,10 mM) and agonist(TAN-67,3mM,6 mM,20 mM) or vehicle,starting at 30 min before MCAO and continuing until the time of death.ICV injection was confirmed by the use of fluorescent dye DiI.Infarct volume was quantified by cresyl violet-stained brain sections at 24 h of reperfusion with Leica Q500 IW Image Processing System. The results showed TAN-67 6 mM treatment decreased infarct volume,whereas NAI 10 mM treatment increased infarct volume,compared with the MCAO groups.1.1.3 Effects of Naltrindole and TAN-67 on Neurological scoresAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Neurological deficits were evaluated at 24 h of reperfusion with 0～5 grade method.MCAO+TAN-67 6 mM group's neurological scores were the highest,and MCAO+Naltrindole 10 mM group's neurological scores were the lowest.1.1.4 Effects of Naltrindole and TAN-67 on DOR protein expressionBased on the effects of Naltrindole and TAN-67 on infarct volume,the experiment animals were divided into six groups:sham group,middle cerebral occlusion group(MCAO),MCAO+TAN67 6 mM group,TAN67 6mM group (without MCAO),MCAO+TAN67 10 mM group,Naltrindole 10 mM group(without MCAO).Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.DOR protein levels at 24 h of reperfusion were measured by laser scanning confocal microscope(LSCM). DOR-positive cells,exhibiting neuronal-like morphology,were abundant in the frontoparietal cortex and lateral caudate putamen in the sham operated groups,but relatively rare in the MCAO groups.Western blot analysis indicated that DOR protein level(36KD and 60KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum. MCAO+NAI 10 mM groups resulted in a significant decrease in the ipsilateral striatum compared with the sham groups;MCAO+TAN-67 6 mM partly inhibited the MCAO-induced decrement experession of DOR protein(36KD and 60KD) in the ipsilateral striatum.DOR protein level(48KD) was not changed in the ipsilateral striatum.Naltrindle 10 mM and TAN-67 6 mM(i.c.v) without MCAO have no effects to DOR protein level(36KD,48KD and 60KD) compared with the sham groups. 1.2 Effects of DOR antagonist(Naltrindole) and agonist(TAN-67) on p-CREB/CREB protein expression in MCAO ratsAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion,p-CREB/CREB protein levels at 24 h of reperfusion were measured by laser scanning confocal microscope(LSCM).p-CREB (green)/CREB(red)-positive cells were abundant in the frontoparietal cortex and lateral caudate putamen in the sham operated groups,but relatively rare in the MCAO groups.Western blot analysis indicated that p-CREB/CREB protein level(43KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum. MCAO+NAI 10 mM groups resulted in a significant decrease in the ipsilateral striatum compared with the sham groups;MCAO+TAN-67 6 mM partly inhibited MCAO-induced decrement of p-CREB/CREB protein expression in the ipsilateral striatum.Naltrindle 10 mM and TAN-67 6 mM(i.c.v) without MCAO have no effects on CREB protein level,compared with the sham(C) groups,p-CREB was low in MCAO+TAN67 6 mM,MCAO+NAI 10 mM groups.1.3 Effects of DOR antagonist(Naltrindole) and agonist(TAN-67) on BDNF protein expression in MCAO ratsThe animals were divided as the 1.2 groups.Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.BDNF protein levels at 24h of reperfusion were measured by laser scanning confocal microscope(LSCM). BDNF-positive cells were abundant in the frontoparietal cortex and lateral caudate putamen in the sham operated groups,but relatively rare in the MCAO groups.Triple fluorescent signals also showed DOR,CREB,and BDNF co-localized in neurons.Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that BDNF protein level(21KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.MCAO+NAI 10 mM groups resulted in a significant decrease in the ipsilateral striatum compared with the sham groups; MCAO+TAN-67 6 mM partly inhibited MCAO-induced BDNF protein expression decrease in the ipsilateral striatum.Naltrindle 10 mM and TAN-67 6 mM(i.c.v) without MCAO have no effects to BDNF protein level. Summary:1.Activation of DOR with DOR agonist TAN-67(6 mM i.c.v.) reduced MCAO-induced brain ischemia injury.DOR antagonist Naltrindole(10 mM i.c.v.),aggravated MCAO-induced brain ischemia injury.This study demonstrates that activation of DOR in the brain protect neurons from MCAO-induced brain ischemia injury.DOR might be one of the endogenous protective factors in the anti-ischemic efficacy.2.In the MCAO rats,DOR expression in cytoplasm and membrance were all decreased,the Naltrindole did not change the protein in 48KD, but inhibited the 60KD protein in the membrance.It shows that the DOR expression in different area of cell may have different function and the neuroprotective function of DOR may be related with its distribution.3.The content of p-CREB/CREB and BDNF in the striatum decreased after MCAO/reperfusion injury,DOR antagonist can downregulation the degree of CREB and BDNF further,DOR agonist can increase DOR expression.It shows that DOR might be one of the key protective factors in the anti-ischemic efficacy2.Role of DOR in EA anti-ischemic efficacy and the mechanism with p-CREB/CREB and BDNFBased on the 1.1.2 exprements,2 mM Naltrindole could be used in EA experiment.Rats were divided into five groups:sham group,MCAO group,EA group, MCAO+2 mM Naltrindole +EA group.EA was delivered to "ShuiGou(GV 26) and NeiGuan(PC 6)".2.1 Effects of EA,MCAO,MCAO+2 mM Naltrindole +EA on Infarct Volume and neurological scoresAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Rats(J) were received intracerebroventricular injection with Naltrindole(2mM),starting at 30 min before MCAO.At the beginning of reperfusion EA treatment was given.Infarct volume was quantified by cresyl violet-stained brain sections at 24 h of reperfusion with Leica Q500 IW Image Processing System.The results showed EA treatment decreased infarct volume,and NAI 2 mM trEAtmeaent reversed EA anti-ischemic effects and increased infarct volume compared with the MCAO groups.Sham EA had no anti-ischemic effects.Furthermore,the neurological deficits analyzed at 24-hour reperfusion were medianed in the MCAO groups,with a mild neurological score of (2.76±0.28).The EA group's neurological scores were the highest and MCAO+2mM Naltrindole group's neurological scores were the lowest.2.2 Effects of EA,MCAO,MCAO+ Naltrindole 2 mM+EA on DOR protein expressionAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that DOR protein level(36KD and 60KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.MCAO groups resulted in a decrease in the ipsilateral striatum compared with the sham groups;EA increased DOR protein expression.NAI 2 mM reversed EA induced DOR protein(36KD and 60KD) expression increase in the ipsilateral striatum.DOR protein level(48KD) was not changed in the ipsilateral striatum.2.3 Effects of EA,MCAO,MCAO+Naltrindole 2 mM+EA on p-CREB/CREB protein expressionAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that p-CREB/CREB protein level(43KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.MCAO groups resulted in a decrease in the ipsilateral striatum compared with the sham groups;EA increased CREB protein expression.NAI 2 mM reversed EA induced CREB protein expression increase in the ipsilateral striatum,compared with the sham,EA,MCAO,MCAO+Naltrindole 2 mM+EA groups p-CREB was low.2.4 Effects of EA,MCAO,MCAO+Naltrindole 2 mM+EA on BDNF protein expressionAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 24-hour reperfusion.Western blot analysis indicated that BDNF protein level(21KD) was decreased at 24 hours of reperfusion after 1hr MCAO in the ipsilateral striatum.EA increased BDNF protein expression.NAI 2 mM reversed EA induced BDNF protein expression increase in the ipsilateral striatum. compared with the sham,EA,MCAO,MCAO+Naltrindole 2 mM+EA groups BDNF was low. Summary:1.EA on "ShuiGou(GV 26)-NeiGuan(PC 6)" acupoints had anti-ischmic efficacy in the early stage of stroke,that shows EA had neuroprotective efficacy.MCAO+2mM NAI reversed EA anti-ischmic efficacy.These results suggested that DOR might be one of the key factors in the anti-ischemic efficacy of EA in the early stage of stroke.2.In the MCAO rats,DOR expression in cytoplasm and membrance were all decreased,EA can increase DOR protein expression and MCAO+2mM NAI reversed EA induced DOR increase.The neuroprotective function of EA may be related with the changes of the DOR.3.The content of p-CREB/CREB and BDNF in the striatum decreased after MCAO/reperfusion injury,DOR antagonist can downregulation the degree of CREB and BDNF further,DOR agonist can increase DOR expression and the effects could be reversed by 2mM NAI.It shows that DOR might be communicated with CREB and BDNF in EA neuroprotectiv efficency.3.Effect of DOR and EA in neurogenesisRats were divided into seven groups:sham group,MCAO group,EA group, sham-EA group,MCAO+ 2 mM Naltrindole +EA group,MCAO+ 10 mM Naltrindole group,MCAO+ 6 mM TAN-67 group.EA was applied to"ShuiGou(GV 26)-NeiGuan (PC 6)" acupoints.Adult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 7 days reperfusion.3.1 Effects of EA,sham-EA,MCAO,MCAO+ Naltrindole 2 mM+EA,MCAO+ Naltrindole 10 mM,MCAO+ TAN-67 6 mM on Infarct VolumeAdult male Sprague-Dawley rats underwent 1-hour middle cerebral artery occlusion(MCAO) and 7 days reperfusion.Infarct volume was quantified by cresyl violet-stained brain sections at 7 days of reperfusion with Leica Q500 IW Image Processing System.The results showed EA and 6 mM TAN-67 treatment decreased infarct volume.NAI 10 mM treatment increased infarct volume compared with the MCAO groups.3.2 Effects of EA,sham-EA,MCAO,MCAO+Naltrindole 2 mM+EA,MCAO+ Naltrindole 10 mM,MCAO+ TAN-67 6 mM on BrdU immunostaining10 mM MCAO+Naltrindole promoted BrdU+ cells in the stratum EA and MCAO+TAN-67 6 mM decreased BrdU+ cells.Sham EA and MCAO+aCSF had no effect on BrdU immunostaining. Summary:1.Cumulative EA on "ShuiGou(GV 26)-NeiGuan(PC 6)" acupoints and TAN-67 had anti-ischmic efficacy in the late stage of stroke.NAI 2 mM reversed EA anti-ischmic efficacy,these results suggested that DOR might be one of the key factors in the anti-ischemic efficacy of EA in the late stage of stroke.2.BrdU positive cells were increased after MCAO/reperfusion injury,EA and TAN-67 can reduced the BrdU positive cells,Naltrindole increased BrdU positive cells.It shows that the proliferation after ischemia will be related with the degree of injury.Conclusion:1.Activation of DOR protected neurons against MCAO induced brain ischemia injury,whereas antagonist of DOR further enhanced ischemic injury.DOR might be one of the key factors in the anti-ischemic efficacy.2.Naltrindole reversed EA anti-ischmic efficacy.These results suggested that DOR might be one of the key factors in the anti-ischemic efficacy of EA.The change of CREB and BDNF might be one of the key factors in the anti-ischemic efficacy of DOR and EA in the stroke.3.BrdU positive cells were increased after MCAO/reperfusion injury,EA and TAN-67 can reduced the BrdU positive cells.That shows EA and DOR in neurogenesis after MCAO/reperfusion may be related with the degree of MCAO/reperfusion injury.