| Objective:To explore the effect of optogenetic regulation of the albumin neurons(CC-Parv)GABA loop connecting the sensory motor cortex of the two hemispheres via the corpus callosum on the motor function of rats with ischemic stroke based on the theory of mutual inhibition of the two cerebral hemispheres(IHI).Use magnetic resonance imaging and molecular biology methods to explore the relationship between the excitation/inhibition(E/I)balance of the both sides of cerebral hemispheres and the recovery of motor function after stroke,in order to provide important scientific basis for the application of IHI theory in the clinical rehabilitation of stroke.Methods:(1)The model group were injected optogenetic virus 3 weeks before ischemic stroke model building.And the ischemic stroke model were prepared according to the improved thread plug method.The animal magnetic resonance T2WI scan and Zea-Loga score were used for model testing.(2)On the fouth day after modeling,the optogenetic group used continuous light stimulation with a wavelength of 550 nm,intensity of 5m W,and a stimulation interval of 10 ms,and the intervention was performed for 2 weeks,once a day,with continuous stimulation for15 minutes each time.The sham intervention group were stimulated with 473 nm wavelength light,and other parameters were the same as the optogenetic group.The sham operation group and the model group were kept in common cages,only grabbing under the same conditions,and no treatment was given.(3)Confocal laser scanning was used to determine the CC-Parv GABA loop to test the optogenetic animal model;the modified neurological deficit score(mNSS)was used to evaluate the neurological deficit of rats;the open field test and the Catwalk gait analyzer were used to test the change of motor function of rats;animal magnetic resonance spectroscopy was used to observe the changes of excitatory/inhibitory nerve substance Glu/GABA metabolism in the sensory motor cortex;Western Blot analysed the expression of key enzymes and receptor of glutamate/γ-aminobutyric acid;magnetic resonance T2WI detected cerebral infarction volume after ischemic stroke;Tunel staining observed neuronal apoptosis after ischemic stroke.Results:(1)Optogenetic virus CC-Parv GABA loop detection.The green fluorescent retro-tracing transmonosynaptic virus fPV-CRE injected in iM1 projected to the cM1 region,and the co-targeted with the red fluorescent light-sensitive protein in the cM1 region was CC-Parv GABA loop that optogenetics regulated.This indicated that the animal model of optogenetics was successfully constructed.(2)The effect of optogenetic regulation of the CC-Parv GABA loop on the neurological function score in rats with ischemic stroke.Before intervention,the mNSS scores of rats in each group except the sham operation group increased,and there was no statistical difference between the groups(P>0.05).After the intervention,the score of the optogenetic group was lower than that of the model group and the sham intervention group(P>0.05)(3)The effect of optogenetic regulation of CC-Parv GABA loop on motor function in rats with ischemic stroke.Cat Walk gait analysis results showed that there were statistically significant differences in the indicators such as,standing time,stride length,swing speed,swing time of the right forelimb,total passing time and average speed(P<0.05)of the model group,optogenetic group and sham intervention group compared with that of sham operation group before intervention,while the maximum contact area was not significantly different from that of the sham operation group(P<0.05);after intervention compared with the model group and the sham intervention group,the optogenetic group had shorter right forelimb standing time(P<0.01),longer stride length(P<0.01),faster swing speed(P<0.01),and reduced swing time(P<0.01),the overall average speed increased(P<0.01),and the total passage time decreased(P<0.05 compared with the model group,P<0.01 compared with the sham intervention group),but there was no significant difference in the maximum contact area(P<0.05).The open field experiment showed that the total exercise distance of the model group,the optogenetic group and the sham intervention group was significantly lower than that of the sham operation group(P<0.01)before the intervention;2 weeks after the intervention,the total exercise distance of the optogenetic group increased compared with the model group and the sham intervention group(P<0.01).(4)The effect of optogenetic regulation of CC-Parv GABA loop on nerve damage in rats with ischemic stroke.T2WI analysis showed that there was no significant difference in cerebral infarction volume between the model group,the optogenetic group and the sham intervention group before intervention(P>0.05).After intervention,the infarct volume of the optogenetic group reduced significantly compared with the model group and the sham intervention group(P<0.01).Tunel staining results showed that the IOD of the optogenetic group was lower than that of the model group and the sham intervention group(P<0.01)after 2 weeks of optogenetic intervention,and the optogenetic group,model group and sham intervention group were all higher than that of the sham operation group(P<0.01).(5)The effect of optogenetic regulation of CC-Parv GABA loop on the neurochemical metabolism of two hemispheres of ischemic stroke model rats.MRS results showed that in iM1 area,the concentration of Glu in the model group,optogenetic group and sham intervention group was lower than that in the sham operation group before intervention(P<0.05),the concentration of GABA increased(P<0.01/0.05).Compared with the sham operation group,the concentration of NAA in the three groups with successful models was also significantly reduced(P<0.01),and there was no significant difference in the concentration of Cho in each group(P>0.05);after the intervention,the concentration of Glu increased(P<0.01/0.05)and the concentration of GABA decreased(P<0.01)in the optogenetic group compared with the model group and the sham intervention group,the concentration of NAA increased in the optogenetic group compared with the model group(P<0.01),and there was an upward trend compared with the sham intervention group,but there was no statistics significance.There was no significant difference in the concentration of Cho among the four groups(P>0.05).In cM1 area,the concentration of Glu in the model group,optogenetic group and sham intervention group was higher than that in sham operation group before intervention(P<0.05),and the concentration of GABA of optogenetic group was lower than model group and sham intervention group after intervention(P<0.01).Before intervention,the ratio of Glu between iM1 and cM1 in the model group,optogenetic group and sham intervention group was lower than that in the sham operation group(P<0.05),and the ratio of GABA between iM1 and cM1 increased(P<0.05).After the intervention,the ratio of Glu in iM1 and cM1 in the optogenetic group was higher than that in the model group(P<0.05);the ratio of GABA in iM1 and cM1 in the model group and sham intervention group was higher than that in the sham operation group(P <0.01),but the difference between the three groups was not statistically significant.(6)The effect of optogenetic regulation of CC-Parv GABA loop on the expression of excitatory/inhibitory related proteins in ischemic stroke model rats.Western blot results showed that the expression of Kv1.1 channel protein in the optogenetic group was up-regulated compared with the model group and the sham intervention group,and the difference was statistically significant(P<0.05).There was no statistically significant difference between the sham operation group and the optogenetic group(P>0.05).And the content of NMDAR and AMPAR in the optogenetic group was higher than that in the model group and the sham intervention group(P<0.05),and the excitatory transmitter receptor content in the model group was lower than that in the sham operation group(P<0.05 or P<0.01).The content of AMPAR in the sham intervention group was lower than that in the sham operation group(P<0.01),and the NMDAR showed a downward trend,but there was no statistically significant difference from the sham operation group.The content of GAD67 in the optogenetic group was significantly lower than that in the model group and the sham intervention group(P<0.05),and the content of GAD67 in the model group and the sham intervention group was higher than that in the sham operation group(P<0.05).Conclusions:Optogenetic regulation of CC-Parv GABA loop enhanced the excitability of the affected hemisphere and improved the nerve damage and motor function after ischemic stroke thereby providing important experimental basis for the application of IHI theory in the rehabilitation of motor function after stroke. |
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