The Restoration Effect Of The Exosomes Derived From High-expression BDNF MSCs In MCAO Rats

Objective: Based on constructing the rat model of Middle cerebral artery occlusion(MCAO)and simulating human ischemic stroke disease,the therapeutic effect of two treatment: high-expression-BDNF MSCs and their exosomes,were compared in MCAO rats.And the effect of brain restoration and possible mechanism from the exosomes derived highexpression-BDNF MSCs is also explored in MCAO rats.Method: Male SD rats weighting vary 250g-300 g were selected and employed the Longe method for MCAO model of SD rat.The modeled MCAO rats were randomly divided to groups as follow(n=8): Group MCAO,Group high-expression-BDNF MSCs transplantation,Group high-expression-BDNF MSCs-Exo transplantation,and a blank control group was also set up.After successful modeling for 2 hours and reperfusion for 24 h,each group was transplanted.The Longa scores of each group were observed on the 1st,3rd,7th and 14 th day after successful modeling.After 14 days,the rats in each group were breaken its neck to death.The infarc volume and microscopic structure of brain tissue in each treatment group was calculated by HE staining,so as to evaluate the improvement of neurological deficits among groups.The expression of Ki-67 protein positive cells in each group of brain tissue was counted by immunofluorescence staining,in order to observe the proliferation of brain cells in brain tissue and evaluate the improvement level of cerebral repair in each group.The expression of BDNF,TrkB,PI3 K and pAkt was detected by WB.The data and map were analyzed by Graphpad prism8.0 software.Result:1.Isolation and extraction of exosomes:(1)The classical exosomes membrane structure of exosomes were shown in the transmission electron microscopy;(2)Identification of exosomes-specific Markers by WB: CD63 and TSG101;(3)The results of BDNF-Elisa derived from exosomes show that the content of BDNF in the exosomes from high-expression-BDNF MSCs is significantly higher than that from natural-expression-BDNF MSCs(P<0.01).Moreover,the content of BDNF in the exosomes from low-expression-BDNF MSCs is significantly lower than that from naturalexpression-BDNF MSCs(P<0.05).2.The neurobehavioral function recovery of rats is evaluated by longa test:(1)On the 1d,there is no apparent statistics significance for the scores in each group(P>0.05);(2)Compared with the scores Longa of Group MCAO,the scores of each transplant treatment group on the 3d,7d and 14 d show a significant decreasing trend(P<0.05);(3)There is no statistics significance for the trend difference between the Group high-expressionBDNF MSCs and Group high-expression-BDNF MSCs-Exo transplantation(P>0.05).3.The infarct degree of the rats in each group after treatment: Compared with the Group MCAO(P<0.01),the infarct area decrease significantly in both treatment group.There is no apparent statistics significance for the area difference between Group high-expression-BDNF MSCs and Group high-expression-BDNF MSCs-Exo transplantation(P>0.05).4.Morphological manifestations of brain tissue by microscope(4×10):(1)In Group MCAO,a large number of disordered nerve cells are observed.In addition,the quality edema,deep staining of cytoplasm of neurons,and the nuclear fragmentation or dissolution are also discovered;necrotic neurons are reduced and interstitial sparse hydrophobic swelling is improved to some extent each transplantation group.(2)The improvement in high-expression-BDNF MSCs and high-expression-BDNF MSCs-Exo transplantation group demonstrates the most obvious change,the structure of neurons is relatively intact,and the degree of interstitial edema is lighter.5.The expression of Ki-67 protein positive cells in immunofluorescence staining of each group:(1)The expression rate of Ki-67 protein positive cells in MCAO group is significantly increased(P<0.05),while the high expression of BDNF-MSCs group and their exosomes transplantation group is further significantly increased(P<0.01).There is no significance for the difference in the expression rate of Ki-67 protein positive cells between the high expression BDNF-MSCs group and exosomes transplantation group(P>0.05).6.Results of BDNF,pTrkB,PI3 K and pAkt protein detected b y WB:(1)The expression of BDNF in the MCAO group is signifi cantly down-regulated(P<0.01),while the expression of BDNF in ea ch transplantation group is up-regulated(P<0.01).There is no signifi cance for the up-regulation trend difference between high-expression-BDNF MSCs transplantation group and high-expression-BDNF MS Cs-Exo transplantation group(P>0.05).(2)The expression of pTrkB in the MCAO group is significantly down-regulated(P<0.01),while t he expression of BDNF in each transplantation group is up-regulate d(P<0.05).There is no significance for the up-regulation trend diffe rence between high-expression-BDNF MSCs transplantation group an d high-expression-BDNF MSCs-Exo transplantation group(P>0.05).(3)The expression of PI3 K in the MCAO group is significantly d own-regulated(P<0.01),while the expression of PI3 K in each transpl antation group is significantly up-regulated(P<0.05).There is no si gnificance for the up-regulation trend difference between high-expres sion-BDNF MSCs transplantation group and high-expression-BDNF MSCs-Exo transplantation group(P>0.05).(4)The expression of pAk t in the MCAO group is significantly down-regulated(P<0.01),while the expression of pAkt in each transplantation group is significantly up-regulated(P<0.05).There is no significance for the up-regulatio n trend difference between high-expression-BDNF MSCs transplantat ion group high-expression-BDNF MSCs-Exo transplantation group(P>0.05).Conclusion: 1.With the level of BDNF in MSCs increasing,the BDNF contained in the produced exosomes increased consistently.2.Transplantation of exosomes from high-expression-BDNF MSCs have an effect on ameliorating neurological deficits in MCAO rats,which may protect brain via activation of the PI3K/Akt signaling pathway.