| Objectives Adipose-derived mesenchymal stem cells(ADSCs)from SD rats were isolated and cultured,after identifying the morphology and phenotype of cells,they were transplanted to treat intracerebral hemorrhage(ICH)rats,to explore whether ADSCs transplantation plays a neuroprotective role and its possible mechanism in ICH rats based on its effects on autophagy.Methods 1 Isolation and culture of rat ADSCs:Ten healthy SD rats aged about 6-8weeks were randomly selected to separate periinguinal fat in a sterile environment,and ADSCs were cultured in vitro and cultured to the fourth generation.The surface markers of ADSCs were measured by flow cytometry.2 Grouping and model preparation:190adult male healthy SD rats were randomly divided into three groups:sham operation group(sham group),intracerebral hemorrhage model group(ICH group):Autologous caudal artery blood was injected into the basal ganglia(blood injection volume 55μL)ICH model was prepared by the method,and the group of intracerebral hemorrhage model+adipose-derived mesenchymal stem cells(ADSCs group):Two hours after ICH model was successfully established,and the right striatum of rats was injected with 5μL contains 1×10~5 ADSCs.Each group was divided into five subgroups at different time points,namely 12h,1d,3d,5d and 7d.3 Test indicators are as follows:(1)The neurological function of rats was scored by Zea-longa scoring method;(2)HE staining was used to observe the pathological changes and cell survival of basal ganglia after operation;(3)The expression of ADSCs surface marker CD29 in damaged brain tissue was detected by immunofluorescence;(4)Nissl staining was used to observe the changes of Nissl bodies in neurons in basal ganglia of the rat brain under optical microscope,and the undamaged neurons were counted;(5)The water content of brain tissue was measured by the dry wet weight method;(6)Immunohistochemical staining was used to detect the expression of autophagy-related proteins Beclin1 and LC3,AKT and m TOR phosphorylated positive cells;(7)Western blot was used to detect the expression of 1d and 3d autophagy-related proteins Beclin1 and LC3,AKT/m TOR signal pathway-related proteins in the brain tissue.Results 1 ADSCs detection results:The flow cytometry detection found positive expression of adhesion molecules CD29 and CD90 and negative expression of CD45 in cultured rat inguinal adipocytes.2 The survival of ADSCs in the rat brain was observed under HE staining microscope.Immunofluorescence detected the positive expression of CD29,the surface marker of ADSCs in the brain tissue.3 Compared with sham group,the neurological score of ICH group was significantly higher(P<0.05),and behavioral changes occurred in the rats in ICH group at each observation time point,such as turning left,dumping and even paralysis.Compared with ICH group,the Zea-longa score of rats in ADSCs group was significantly lower(P<0.05).4 After dissecting the brain tissue around the needle eye of the rats,it can be seen with the naked eye that sham group presented normal brain tissue.In ICH group and ADSCs group:there was brain tissue injury around the basal ganglia,and there were points and flake hemorrhage in the ventricle.Compared with ICH group,the condition of ADSCs group was significantly improved.Under the HE staining microscope,the brain tissue structure of sham group was complete and no pathological changes were found.In ICH group,there was obvious space occupying effect around the basal ganglia,a large number of red blood cells,enlarged cell space,edema,inflammatory cell infiltration and degeneration and necrosis of a large number of nerve cells.The pathological changes in ADSCs group became significantly less than those in ICH group.5 The Nissl staining microscope was used to count the undamaged neurons.Compared with sham group,the neuronal cell structure in ICH group was seriously damaged,the cells shrank,the nuclei disintegrated,the volume decreased,and the Nissl bodies were obviously lost(P<0.05).Compared with ICH group,Nissl bodies of nerve cells in the brain tissue of ADSCs group were significantly increased(P<0.05).6 Compared with sham group,determination of brain water content found the content of ICH group began to increase at 12h,reached peak at 3d and then decreased.The difference at each time point was statistically significant(P<0.05).Compared with ICH group,the brain water content of ICH rats at each time point after ADSCs transplantation decreased significantly(P<0.05).7 The results of immunohistochemistry showed that there was no significant change in the expression of LC3II/I,Beclin1,p-AKT and p-m TOR at each time point in sham group;compared with sham group,the expression of LC3,Beclin1,p-AKT and p-m TOR positive cells in ICH group increased significantly from 12h,reached peaked at 3d and then decreased gradually;compared with ICH group,the expression of LC3 and Beclin1 positive cells in ADSCs group decreased significantly,while the expression of p-AKT and p-m TOR positive cells increased significantly.8 The proteins in the brain tissue at 1d and 3d with the immunoprotein imprinting showed that the results of LC3 demonstrated two double bands LC3I and LC3II with different depths,and the ratio of p-AKT/AKT and p-m TOR/m TOR represented the phosphorylation level of AKT and m TOR.Compared with sham group,the contents of LC3II/I,Beclin1,p-AKT/AKT and p-m TOR/m TOR protein in ICH group increased,and the difference was statistically significant(P<0.05).Compared with ICH group,the protein expression of LC3II/I and Beclin1 in ADSCs group decreased significantly(P<0.05),and the protein expression levels of p-AKT/AKT and p-m TOR/m TOR increased significantly(P<0.05).Conclusions 1 Autophagy is activated in neurons in basal ganglia of ICH rats,and the secondary injury of the brain tissue is aggravated with the enhancement of autophagy activity.2 After ADSCs transplantation,it can improve the neurological symptoms of ICH rats and reduce the secondary brain tissue injury around hematoma by inhibiting autophagy activities.3 ADSCs transplantation may inhibit autophagy activities through the AKT/m TOR pathway,so as to reduce the degree of secondary brain injury and promote the recovery of neurological functions in ICH rats.Figure 19;Table 3;Reference 106. |
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