ALK5 Signaling Pathway Mediates Brain Plasticity And Functional Recovery After Cerebral Ischemia/Reperfusion Injury In Rats

Stroke has become a global disease with an increasing incidence.Every year,about 44 million people worldwide suffer from disability due to stroke,and more than 80% of them are ischemic stroke.At present,the most effective treatment for ischemic stroke is to quickly restore blood supply.Therefore,intravenous thrombolysis and endovascular intervention have been widely used and developed.However,with the development of thrombolytic technology,the incidence of cerebral ischemia/reperfusion injury is increasing,and the neurological dysfunction after injury is becoming more and more prominent.Improving the functional recovery after cerebral ischemia/reperfusion injury has become a key problem to be solved urgently.However,the mechanism of cerebral ischemia/reperfusion injury is complicated,the investigation of its mechanism is very limited,and there is no satisfactory treatment strategy for it.Therefore,this study will focus on the recovery of neurological function after cerebral ischemia/reperfusion injury,and strive to find a possible effective treatment.Part one: Expression characteristics of ALK5 in a rat model of cerebral ischemia/reperfusion injuryObjective: To investigate the expression and localization of activin receptor-like kinase 5(ALK5)in brain tissues of rats with cerebral ischemia/reperfusion(I/R)injury.Methods:1.Sprague-Dawley(SD)rats were subjected to right middle cerebral occlusion/reperfusion(MCAO/R)by using suture-occluded method.2.Western blotting and immunohistochemistry were used to detect the expression changes of ALK5 at 24 h and 14 d after the establishment of the model.3.Double immunofluorescence labeling was used to detect the cellular localization of ALK5 in I/R injured cortex.Results:1.After reperfusion and resuscitation,SD rats were included in the experiment according to Longa-Z score(the scores ? 1 and ?3).TTC staining showed that there was a definite infarct,and the model was successfully established.2.At 24 h and 14 d after cerebral I/R injury in SD rats,the expression of ALK5 in the ipsilateral cortex was significantly higher than that in the sham group(p<0.05).3.In the ipsilateral cortex,ALK5 co-localized with beta-III tubulin(TUBB3)positive cells,but not with glial fibrillary acidic protein(GFAP).Conclusion:In the ipsilateral cortex of SD rats with cerebral I/R injury,the expression of ALK5 increased significantly,mainly in the cell membrane and cytoplasm of neurons.The significant differences of ALK5 expression between I/R group and Sham group suggest that ALK5 may play an important role in cerebral I/R injury.Part two: Effects of ALK5 on brain plasticity and motor function recovery after cerebral I/R injuryObjective: To clarify the role of ALK5 in cerebral I/R injury,we will evaluate the effects of ALK5 on motor neurological dysfunction caused by cerebral I/R injury.Based on the results,we speculate that the effects of ALK5 on motor function may be related to its regulation of brain plasticity.Therefore,in this part of the study,we will focus on the regulation of ALK5 on brain plasticity after cerebral I/R injury.Methods:1.Lentiviruses(LV)carrying ALK5-RNAi or ALK5-CDS were constructed.Lentiviruses were then stereotactically injected into the ischemic ipsilateral sensorimotor cortex of SD rats to intervene in the expression of ALK5.The intervention efficiency of LV was detected by immunofluorescence and WB.2.The modified neurological severity score(mNSS)and the adhesive-removal somatosensory test(ARST)were used to assess the changes in neurological function.3.The co-localization of ALK5 with Nestin(neural stem cell marker)or DCX(neuroblasts and immature neuron markers)was detected by double immunofluorescence labeling to reflect the effects of ALK5 on neural regeneration after cerebral I/R injury.4.To detect the expression changes of growth associated protein-43(GAP43)and neurofilament-200(NF200)after LV intervention in ALK5,so as to reflect the regulation of ALK5 on axonal regeneration after cerebral I/R injury.5.Bioin-ylated dextran amine(BDA)was stereotactically injected into the ischemic contralateral sensorimotor cortex.The neonatal nerve fibers acrossed the midline of corpus callosum and the new axonal sprouting of CRT acrossed the midline were traced to reflect the regulation of ALK5 on axonal reorganization after cerebral I/R injury.6.Golgi–Cox staining and Sholl analysis were performed to evaluate dendritic plasticity.Results:1.LV-ALK5-RNAi or LV-ALK5 were successfully transduced into neurons and efficiently reduced or improved the expression of ALK5(p<0.05),respectively.2.ALK5 knockdown was not conducive to the recovery of neurological function,while overexpression of ALK5 can promote the recovery of neurological function.And we observed a significant continuous improvement trend in functional recovery from 7 d to 14 d(p<0.05).3.After cerebral I/R,ALK5 colocalized with Nestin,and also colocalized with DCX.4.WB analysis showed that knockdown of ALK5 inhibited Gap43 expression,while overexpression of ALK5 increased Gap43 expression.Immunohistochemical staining of NF-200 showed that compared with the MCAO/R groups,the LV-ALK5-RNAi group displayed worsened NF-200 positive fibers,while the fibers in the LV-ALK5 group were better(p < 0.05).5.LV-ALK5-RNAi-treated rats displayed a lower density of crossing midline BDA-positive fibers;the LV-ALK5 group displayed a higher density of BDA-positive fibers crossing the midline.6.Golgi–Cox staining showed that the length and distribution of dendrites were significantly decreased after MCAO/R.Compared with the MCAO/R groups,the LV-ALK5-RNAi group was worse and the LV-ALK5 group was better(p<0.05).Fourteen days after MCAO/R,the recovery was poor in the LV-ALK5-RNAi group and obvious in the LV-ALK5 group.Conclusion:ALK5 may be closely related to neural regeneration after cerebral I/R injury,which promotes axonal plasticity and dendritic plasticity.ALK5 may play a critical role in improving long-term recovery of neurological function by promoting brain plasticity after cerebral I/R injury.Part three: ALK5 promotes brain plasticity after cerebral I/R injury by activating Smad2/3 and Gadd45bObjective: Previous studies have suggested that ALK5 can promote the expression of growth arrest and DNA-damage inducible gene b(Gadd45b)by activating Smad2/3.Our previous studies have shown that Gadd45 b can promote axonal plasticity and functional recovery after cerebral ischemia.Based on this,we speculate that ALK5 may promote the expression of Gadd45 b by activating Smad2/3 to regulate brain plasticity after cerebral I/R injury,thereby improving neurological functional recovery.In addition,our previous studies have found that vagus nerve stimulation(VNS)can promote functional recovery after cerebral ischemia.Therefore,we speculate that VNS may play a key role in clinical improvement of neurological functional recovery by regulating ALK5/Smad2/Smad3/Gadd45 b signaling pathway.Methods:1.LV-ALK5-RNAi was stereotaxically injected into the ischemic ipsilateral sensorimotor cortex to inhibit the expression of ALK5.The phosphorylation level of Smad2/3 and the expression level of Gadd45 b in the brain tissues of the virus injection areas were detected by WB at 24 h and 14 d after models established.2.LV-ALK5 was stereotaxically injected into the ischemic ipsilateral sensorimotor cortex to inhibit the expression of ALK5.The phosphorylation level of Smad2/3 and the expression level of Gadd45 b in the brain tissues of the virus injection areas were detected by WB at 24 h and 14 d after models established.3.Co-Immunoprecipitation(Co-IP)was used to detect the interaction between ALK5 and Gadd45 b after cerebral I/R injury.4.After models successfully established,the rats were given VNS intervention for 30 min/d for 14 days.The physiological parameters of SD rats should be monitored during the operation,and attention should be paid to the prevention of infection.Results:1.WB analysis showed that LV-ALK5-RNAi significantly inhibited the phosphorylation level of Smad2/3 and the expression of Gadd45b(p< 0.05)while effectively knocking down the expression of ALK5.2.WB analysis suggested that LV-ALK5 significantly promoted the phosphorylation level of Smad2/3 and the expression of Gadd45b(p< 0.05)while significantly promoting ALK5 expression.3.Co-IP results showed that there may be an interaction between ALK5 and Gadd45 b at 24 h and 14 d after cerebral I/R injury.4.WB results suggested that VNS intervention could promote the expression of ALK5 and further promote the phosphorylation of Smad2/3 and the expression of Gadd45b(p<0.05).The effect of VNS intervention was comparable to that of LV-ALK5(p>0.05).Conclusion:ALK5 expression level was positively correlated with Smad2/3 phosphorylation level and Gadd45 b expression level.After cerebral I/R injury,there might be interaction between ALK5 and Gadd45 b.Therefore,we suggest that ALK5 may promote the expression of Gadd45 b by regulating the phosphorylation level of Smad2/3,thereby regulating the brain plasticity after cerebral I/R injury.After cerebral ischemia,VNS treatment may improve neurologic function by regulating the ALK5/Smad2/Smad3/Gadd45 b signaling pathway.