Effects Of Myocardial And Subarachnoid CGRP Gene Transduction On Myocardial Ischemia/Reperfusion Injury In Diabetic Rats

Objective:1.To develop a rat model of diabetic sensory neuropathy by intraperitoneal injection of streptozotocin(STZ)to investigate the changes of cardiac function during acute myocardial ischemia/reperfusion(I/R)and the extent of myocardial I/R injury in diabetic sensory neuropathy rats,and the expression of calcitonin gene-related peptide(CGRP)in myocardium,spinal cord,and dorsal root ganglion in ischemic region.2.Lentivirus was used as a vector to transduct CGRP gene by myocardial injection and subarachnoid injection,to evaluate the feasibility of the effects on cardiac function and myocardial ischemia/reperfusion injury in diabetic neuropathy rats,the changes of CGRP transcription and expression in ischemic myocardium,spinal cord and dorsal root ganglion.Methods: The study was carried out in a series of experiments:1.The establishment of the model of diabetic rat with sensory neuropathy.Twenty-four healthy male Sprague-Dawley rats(200~220 g)were randomly divided into control group(Control group)and diabetic group(DM group),with regular feed for 9 weeks.At the end of the first week,fasting for 12 hours,STZ(50 mg/kg)was intraperitoneally injected to induce islet ?-cell damage.Fasting blood glucose was measured at regular intervals after 24 hours.Fasting blood glucose value was higher than 16.7mmol/L for 7 times that was successfully established as diabetic rats.Both groups were measured for body weight and tail flick reflex latency once a week and their changes over time were compared.At the 9th weekend,left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP),HR,LV+dp/dtmax and LV-dp/dtmax were continuously recorded to compare the cardiac function during acute myocardial I/R.Changes in cardiac function during acute myocardial ischemia/reperfusion were compared.Myocardial infarction area was examined using Evans Blue and TTC staining.The expression of CGRP protein in Ischemic myocardium,serum,spinal cord,and dorsal root ganglion were measured and analyzed by enzyme-linked immunosorbent assay(ELISA).2.Experimental study of myocardial CGRP gene transduction on myocardial ischemia/reperfusion injury in diabetic rats.The experiment was divided into 5 groups: control group,diabetic(DM)group,DM myocardial injection(Sham)group,DM myocardial injection control(MC)group,DM cardiac transduction(ME)group.The same feeding conditions lasted 10 weeks.At the end of the first week,CGRP gene was transfected into the heart muscle with mechanical ventilation by chloral hydrate abdominal anesthesia.At the end of the second week,a diabetic rat model was established by intraperitoneal injection of STZ(50 mg/kg).The latency period of rat tail flick reflex was measured every week,and the changes with the course of disease were compared.At the end of the tenth week,real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the transcript levels of CGRP in ischemic myocardium,spinal cord and dorsal root ganglia.EGFP expression was observed using a fluorescence microscope.The rest of the experiment is the same as the first part3.Experimental study of subarachnoid space CGRP gene transduction on myocardial ischemia/reperfusion injury in diabetic rats.The experiment was divided into 5 groups: Control group,Diabetes(DM)group,DM subarachnoid injection(Sham)group,DM subarachnoid injection control(SC)group,DM subarachnoid transduction(SE)group.At the end of the first week,CGRP gene was transfected into the subarachnoid space under chloral hydrate anesthesia.The feeding conditions,the experimental period,and follow-up experiments are the same as in the second part.Result:1.Diabetes duration lasted 8 weeks in DM group,body weight was significantly lower than that in the Control group(P<0.01).After 4 weeks of intraperitoneal injection of STZ,tail-flick latency which reflects the function of sensory nerve in model group was significantly longer than that of the Control group(P<0.05).Compared with the control group,HR,LVSP,+dP/dtmax,-dP/dtmax significantly declined(P<0.05),and LVEDP significantly rised in the DM group after ischemia/reperfusion(P<0.05).The percentage of myocardial infarction area(IS/AAR%)in DM group was significantly greater than that in Control group(P<0.01).CGRP transcription level in myocardial tissue(P<0.01),serum(P< 0.01),1-5 thoracic spinal cord(P<0.05)and 1-5 thoracic dorsal root ganglia(DRG)(P<0.05)in the ischemic region were significantly lower than those in the Control group.2.Transduction of CGRP Gene by Myocardial Point Injection.Compared with Sham group,HR,LVSP,+dP/dtmax,-dP/dtmax in ME group rised significantly(P<0.05),and LVEDP declined significantly(P<0.05).In IS/AAR%,ME group was greater than Control group but smaller than DM,Sham,and MC groups(P<0.05).Compared with the Control group,CGRP transcription levels in ischemic myocardium,1-5 thoracic spinal cord,1-5 thoracic DRG,and 1-5 lumbar DRG in DM,Sham and MC groups were significantly decreased(P<0.05),and the CGRP transcription level in the ischemic myocardium and 1-5 thoracic DRG increased significantly in the ME group(P<0.05).Compared with DM,Sham and MC groups,CGRP transcription level in thoracic spinal cord of ME group 1-5 increased significantly(P<0.05).Compared with the DM,Sham,and MC groups,CGRP in ischemic myocardium(P<0.01),serum(P<0.01),1-5 thoracic spinal(P<0.05)and 1-5 thoracic DRG(P<0.05)of the Control group and the ME group were significantly higher,and the expression of CGRP in ischemic myocardium was significantly higher and in 1-5 thoracic spinal was significantly lower than that in the Control group(P<0.05).3.Transduction of CGRP gene by subarachnoid injection.Compared with the Control group,the latency of the tail-flick reflex in the other 4 groups of diabetic rats was significantly prolonged(P<0.01),among them compared with the Sham group,the tail-flick latency was significantly shorter in the SE group in 4-6 weeks(P<0.05).Compared with Sham group,there was no significant difference in HR,LVSP,+dP/dtmax,-dP/dtmax,and LVEDP in SE group(P>0.05).Compared IS/AAR%,the difference between SE and Sham groups was not statistically significant(P>0.05).The transcript levels of CGRP in ischemic myocardium in the SE group was lower than those in the Control group(P<0.05)but higher than that in Sham group(P>0.05).The transcript levels of CGRP in thoracic DRG and lumbar DRG from 1 to 5 were significantly higher than those in the Sham group(P<0.05).Compared with DM,Sham and SC groups,serum CGRP levels were significantly higher in the the Control group(P<0.01)and the SE group(P<0.05).The content of CGRP in DRG of lumbar segments from 1 to 5 in SE group was higher than that in other 4 groups(P<0.05).Conclusion:1.Intraperitoneal injection of STZ can establish a rat model of diabetes effectively.With the prolongation of the disease process,diabetic rats showed obvious sensory neuropathy,and the thermal pain threshold increased.2.Compared with normal rats,cardiac dysfunction during myocardial ischemia/reperfusion in rats with diabetic sensory neuropathy was more severe,more sensitive to ischemia/reperfusion injury,and significantly less endogenous CGRP.3.Transduction of CGRP gene by myocardial infusion can effectively reduce cardiac function injury in diabetic heart during ischemia/reperfusion and cardiac ischemia/reperfusion injury in diabetic rats.It may be related to the up-regulation of exogenous CGRP expression in ischemic myocardium and dorsal root ganglion.4.Subarachnoid injection of CGRP gene may delay sensory neuropathy to a certain extent and slowing down the thermal pain threshold,which may be associated with upregulation of exogenous CGRP expression in dorsal root ganglia.But it can not change the course of diabetic sensory neuropathy and reduce cardiac dysfunction and ischemia/reperfusion injury during cardiac ischemia/reperfusion in diabetic rats.