| Intracerebral hematoma(ICH)is one of the most common diseases of neurosurgery,and it is a killer which seriously threatens human health in modern society.Although neurosurgical hematoma removal surgery can be performed as a major treatment to save the lives of patients with ICH,patients will still have serious neurological dysfunctions,which make it difficult for them to return to society.Repairing and remodeling the corticospinal tract in the damaged brain tissue after ICH is the anatomical pathology basis for the recovery of limb movement disorders in patients,and the rupture and injury of axon directly affects the function of the corticospinal tract.Although it is almost impossible for the damaged neurons to regenerate,the neuronal axons can show some regenerative capacity.Bone marrow-derived mesenchymal stem cells(BMSCs)are one type of mesoderm-derived pluripotent stem cells which are isolated from the bone marrow.They have the ability to expand in vitro,and they can develop into not only the mesoderm-derived fat,bone,cartilage and other tissues,but also internal and external embryonic layers-derived vascular endothelium,nerves and other tissues.BMSCs have good ability to proliferate and are easy to be isolated and cultured.Autologous transplantation of BMSCs has no immunogenicity and is free from ethical restriction.BMSCs are more and more often applied in the experiment on the treatment of acute brain injury related diseases.Currently,the ability of BMSCs transplantation has been reported promoted axonal regeneration in vitro and in animal experiments.Autophagy is a cell metabolism process which maintains the stability of the internal environment through degradation of protein aggregates and damaged organelles.It has been confirmed that ICH can lead to the activation of autophagy.Recent studies have found the enhancement of cortical neurons autophagy level promotes the regeneration of axon and reduces the formation of axonal retraction ball.On the basis of comprehensive retrieval of literature and preliminary work,we speculate that BMSCs transplantation can effectively improve neurological dysfunction and promote axonal regeneration.The mechanism may be related to the activation of neurons autophagy mediated by PI3K-Akt-m TOR pathway.To prove this hypothesis,this study elaborates in the following three parts.Part one Neuroprotective effect of BMSCs transplantation in rats after intracerebral hemorrhageObjective: To confirm BMSCs transplantation can provide neuroprotective effect and improve neurological dysfunction in rats after ICH.Methods: Ninty-eight adult female SD rats were randomly divided into three groups: sham operation group(Sham group);intracerebral hemorrhage group(ICH group);intracerebral hemorrhage + BMSCs group(BMSCs group).The BMSCs of 3-week-old male SD rats were isolated and cultured to the third generation.The surface markers of BMSCs were identified by flow cytometry.The ICH model of female SD rats was reproduced by injecting blood.We injected 2?L suspension of BMSCs of which the concentration was 1×108/m L into the intracerebral ventricle 1 hour after reproducing the model.The following items were detected: 1.Use HE staining to observe the pathological change and cells survival state of the damaged brain tissue in different groups 3 days after the operation.2.Detect the distribution of Sry positive cells in the brain tissue around the hematoma before modeling operation and 12 hours to 7 days after the operation by using immunofluorescence.3.Use TUNEL staining to show the brain tissue apoptosis in different groups from one to seventh day after the operation.4.Measure the water content in brain tissue to show the conditions of intracerebral edema from one to seventh day.5.Before modeling operation and 12 hours to 7 days after the operation,perform the placement test,corner test and NSS score to detect the neurological dysfunction changes in rats.The experimental results were analyzed using Image Lab5.1,Image J and other analysis systems.The experimental results were presented in the form of mean ± standard deviation,and analyzed using statistical software SPSS 17.0.P < 0.05 was considered statistically significant.Results: 1 BMSCs morphological observation: 1 day after inoculation,primary BMSCs slowly adhered to the wall.The nucleuses of BMSCs placed in the middle.The cell body is small and the shape is round or fusiform.The refractive attribute is relatively strong and the proliferation method is scattered clonal proliferation.After the growth inhibition period,the cells arranged in parallel and presented rapid swirling aggregation growth.The shapes of cells became more symmetry afterwards,which is mostly fusiform.2 The results of BMSCs surface markers identified by flow cytometry: We identified the surface markers of the third-generation BMSCs,the results of flow cytometry showed that the positive expression rate of CD106 on BMSCs was 74.56%.The positive expression rate of CD29 was 76.11%.The expressions of CD106 and CD29 were positive.The positive expression rate of CD54 was 5.05%,and for CD45 it was 3.88%.The expressions of CD54 and CD45 were negative.3 Preparation of rats ICH model: By general observation of the brain tissue of ICH rats,we could see the injection site of the modeling surgery was right above the caudate nucleus.Transverse incision of rat intracerebral tissue was made at the level of basal ganglia.We could see that the hematoma was in the basal ganglia area,with local brain injury,intraventricular hemorrhage and subarachnoid hemorrhage.The brain tissue of rats in Sham group had normal histological morphology and intercellular space.Neurons were arranged in an orderly manner and the nucleuses were symmetry.The intercellular space in ICH group increased.Obvious edema could be seen in the damaged brain tissue around the hematoma.The body of neurons shrinked and cytoplasmic acidophilia increased,together with pyknosis.The morphological changes of brain tissue in BMSCs group were similar to those in ICH group,but the peripheral tissue edema was mild,and there were obviously less degenerated and necrotic neurons.4 The distribution of BMSCs in the damaged brain tissue around the hematoma: Before BMSCs transplantation,immunofluorescence results showed no Sry positive cells from male mice.12 hours later,the Sry protein presented granulated expression in the nucleus of BMSCs.After that,the number of positive cells increased gradually,and peaked at 3 days after transplantation.Sry positive cells were still visible in the damaged brain tissue around hematoma after 14 days.5 TUNEL staining detecting results: The expression of TUNEL positive cells were visible in Sham group,which is scattered and lightly stained.In ICH group,TUNEL positive cells presented in a clustered appearance from the 1 to 7 days,of which the integral optical density was higher than that in Sham group,and the difference was significant(P < 0.05).The expression of TUNEL positive cells in BMSCs group was similar to that in ICH group,but the integral optical density was significantly lower compared to ICH group(P < 0.05).6 Brain tissue water content detecting: The brain tissue water content of Sham group had no significant change.Compared with Sham group,the brain tissue water content of ICH group was significantly higher at 1,3 and 7 day after modeling surgery(P < 0.05).The water content of brain tissue in BMSCs group was lower than that in ICH group at 1,3 and 7 day,and the difference was significant(P < 0.05).7 Evaluation of neurological dysfunction after ICH: The forelimb placement rate and corner rate of BMSCs group were higher than those of ICH group on the 3,7 and 14 day after ICH surgery of rats(P < 0.05),and the NSS score was significantly lower than that of ICH Group(P < 0.05).Summary: The methods of primary culture,passage and identification of BMSCs were established.The ICH model of rat was successfully established by injecting blood.The brain tissue around the hematoma is damaged after ICH,leading to neurological dysfunction in ICH rats.After BMSCs transplantation therapy,BMSCs play a certain role in neuroprotection,reducing the damage extent of brain tissue around hematoma,improving brain edema and neurological dysfunction of ICH rats.Part two Effects of BMSCs transplantation on autophagy and axon regeneration in rats with intracerebral hemorrhage through PI3K-Akt-m TOR pathwayObjective: To explore whether BMSCs transplantation after ICH in rats can affect autophagy and axon regeneration in the damaged brain tissue by intervention of LY294002,the inhibitor of PI3K-Akt-m TOR pathway.Methods: One hundred and two female SD rats were randomly divided into four groups: sham operation group(Sham group);intracerebral hemorrhage group(ICH group);intracerebral hemorrhage + BMSCs transplantation group(BMSCs group);intracerebral hemorrhage + BMSCs transplantation + LY294002 intervention group(LY294002 group).Methods of animal model preparation and BMSCs transplantation were the same as those in the first part.The following items were detected at 12 hours,1,3,7 and 14 days after the surgery: 1.Use transmission electron microscopy to observe ultrastructure of the damaged brain tissue in rats after ICH and detect autophagosome.2.Use western-blot to detect the expression of the axon regeneration related proteins GAP-43 and SCG10,autophagy related protein LC3 and p62,pathway protein p-Akt in the damaged brain tissue after ICH.3.Use immunohistochemisty method to detect the expression and localization of GAP-43 in the damaged brain tissue.4.Use immunofluorescence to observe the confocal results of p-Akt and m TOR in the damaged brain tissue.The methods of measurement recording and statistical analysis of results were the same as that in the first part.Results: 1 Transmission electron microscopy results: 3 days after ICH modeling surgery,many swelling and deformed mitochondria could be observed around the nucleus in the shrinked neurons.At this time there were also damaged mitochondria and other cell structures surrounded by double membrane-like structure,which entered the lysosome and fused with it,resulting in the final formation of autophagic lysosome.2 Results of p-Akt western-blotting analysis: p-Akt protein was expressed at a low level in Sham group.In the damaged brain tissue of rats in the ICH group,p-Akt rose at 12 hours after the modeling surgery and peaked at 3 days.After that,it gradually decreased.At each time point,the ratios of p-Akt to Akt in ICH group were higher than those in Sham group(P < 0.05).The trend of p-Akt in BMSCs group was similar to that in ICH group,but the ratio of p-Akt to Akt further increased at each time point(P < 0.05).However,compared with BMSCs group,the ratio of p-Akt to Akt decreased in LY294002 group and the difference was significant.3 The immunofluorescence confocal results of p-Akt(red fluorescence)and m TOR(green fluorescence): In Sham group the expressed red fluorescence and green fluorescence were relatively less,and they overlapped,showing a small amount of yellow.After 3 days,slightly stronger red and green fluorescence were visible in the damaged brain tissue around hematoma in ICH modeling group than those in Sham group,and they overlapped,showing orange.After the intervention of LY294002,the inhibitor of PI3K-Akt-m TOR pathway,the red and green fluorescence were significantly lower than that in BMSCs group,and the overlapping fluorescence of orange was also dimmer.4 The analysis results of LC3 western-blotting: The LC3 II protein in Sham group expressed at a low level.The ratio of LC3 II to LC3 I in the damaged brain tissue in ICH group increased at 12 hours after operation and reached the peak at 3 days.It decreased afterwards but was still higher than that in Sham group(P < 0.05).The changing tendency of the ratio of LC3 II to LC3 I in BMSCs group was similar to that in ICH group,but the expression amount was higher than that in ICH group,and the difference was significant(P < 0.05).Compared with BMSCs group,the intervention of LY294002 could significantly reduce the ratio of LC3 II to LC3I(P < 0.05).5 The analysis results of p62 western-blotting: There was no significant difference in the expression of p62 protein in Sham group.1 day after the operation,the expression of p62 in the damaged brain tissue in ICH group was lower than that in Sham group.At 3 days,it reached minimum and slightly increased after that,but still lower than that in the Sham group(P < 0.05).The tendency of p62 expression in BMSCs group was similar to that in ICH group,but it was significantly lower than that in ICH group at each time point(P < 0.05).After the intervention of LY294002,the expression of p62 was significantly higher than that in BMSCs group(P < 0.05).6 The analysis results of GAP-43 immunohistochemisty: The GAP-43 positive cells could be seen in Sham group.1 to 7 days after the operation,GAP-43 positive cells significantly increased in the ICH group,and the immunoreactivity was significantly higher compared with the Sham group(P < 0.05).The immunoreactivity of GAP-43 positive cells in BMSCs group became higher than that in ICH group from 3 and 7 days(P < 0.05).The immunoreactivity of GAP-43 positive cells in the LY294002 intervention group was significantly lower than that in BMSCs group(P < 0.05).7 The analysis results of GAP-43 western-blotting: There was no obvious difference in the expression of GAP-43 in Sham group.The expression of GAP-43 in the damaged brain tissue increased at 3 days after the modeling surgery.Although it slightly decreased on the 7 and 14 days,it was still higher than that in the Sham group,and the difference was significant(P < 0.05).The expression tendency of GAP-43 in BMSCs group was similar to that in ICH group,but the amount of expression increased at each time point(P < 0.05).After LY294002 intervention,the expression of GAP-43 was significantly lower than that in BMSCs group(P < 0.05).8 The analysis results of SCG10 western-blotting: The expression of SCG10 protein in Sham group was not significantly different.The amount of SCG10 in the damage brain tissue in ICH group increased at 12 hours after modeling surgery,and reached the peak at 3 days,then gradually decreased but was still higher than that in Sham group(P < 0.05).The expression tendency of SCG10 in BMSCs group was similar to that in ICH group,but the expression amount was lower at each time point,and the difference was significant(P < 0.05).Compared with BMSCs group,LY294002 intervention could significantly increase the expression intensity of SCG10(P < 0.05).Summary: The activation of autophagy and PI3K-Akt-m TOR pathway exists in the damaged brain tissue around hematoma after ICH.The PI3K-Akt-m TOR pathway is further activated after BMSCs transplantation.Autophagy flux increases in the damaged brain tissue around hematoma,while axonal regeneration enhance.PI3K-Akt-m TOR signal pathway regulates BMSCs transplantation on autophagy flux and on the expression of axon regeneration related proteins.Part three Study on the mechanism of bone marrow mesenchymal stem cells transplantation mediate axon regeneration by autophagy flux after intracerebral hemorrhage in ratsObjective: To make sure whether BMSCs transplantation mediate axon regeneration by enhancing neuronal autophagy flux in the damaged brain tissue around hematoma after ICH in rats,by the intervention of autophagy inhibitor 3MA and the autophagy activator Rapamycin(RAPA).Methods: Forty-five female SD rats were randomly divided into five groups: sham operation group(Sham group);intracerebral hemorrhage group(ICH group);intracerebral hemorrhage + BMSCs transplantation group(BMSCs group);intracerebral hemorrhage + BMSCs transplantation + 3-MA intervention group(3MA group);intracerebral hemorrhage + BMSCs transplantation + RAPA intervention group(RAPA group).The following items were tested at 3 days after operation: 1.Use immunofluorescence to observe the confocal conditions of LC3 and cell markers in damaged brain tissue.2.Use western-blotting to detect the expression of GAP-43 and SCG10,autophagy-related proteins LC3 and p62,the expression of pathway protein p-Akt.3.Use immunohistochemistry to detect the expression and localization of GAP-43 in the damaged brain tissue.The methods of measurement recording and statistical analysis of results were same as that in the first part.1 The immunofluorescence confocal results of LC3 and cell markers: Red fluorescence labeled LC3,green fluorescence labeled neurons,magenta fluorescence labeled astroglia cells,and blue fluorescence labeled nucleus.In Sham group the expression of red fluorescence was relatively less,it overlapped with green fluorescence,showing a small amount of orange.More red fluorescence could be seen in the cytoplasm of damaged brain tissue in ICH group than that in the Sham group,and green fluorescence significantly reduced.They overlapped and showed orange,indicating neuron autophagy after ICH.Meanwhile,magenta fluorescence was more than that in Sham group,indicating astroglia cells proliferation after ICH.The red and green fluorescence in BMSCs group were more than those in ICH group,and they overlapped and enhanced orange,indicating BMSCs transplantation therapy could further activate neuronal autophagy and reduce death of neurons.At the same time,magenta fluorescence was less than that in ICH group,indicating that BMSCs could inhibit the proliferation of astroglia cells after ICH.After the intervention of autophagy inhibitor 3MA,the red fluorescence decreased compared with that in BMSCs group,while the green fluorescence slightly decreased.After overlapping,the orange color was also decreased,and the change of magenta fluorescence was not significantly.After the intervention of autophagy activator RAPA,the red fluorescence was significantly enhanced,and the green fluorescence was not significantly,after overlapping the orange color significantly increased and the magenta fluorescence has no obvious change.2 Results of autophagy-related protein LC3 and p62 western-blotting analysis: The ratio of LC3 II to LC3 I was significantly higher in the damaged brain tissue of ICH rats than that in the Sham group(P < 0.05),while the expression of p62 was significantly lower than that in sham(P < 0.05),indicating the activation of autophagy flux.BMSCs transplantation therapy could further increase the ratio of LC3 II to LC3I(P < 0.05),while the expression of p62 was further decreased(P < 0.05),indicating that BMSCs could enhance autophagy flux.After the intervention of autophagy inhibitor Results:3MA,autophagy was inhibited.The ratio of LC3 II to LC3 I decreased(P < 0.05),but the expression of p62 increased(P < 0.05).The autophagy activator RAPA had the opposite effects,leading to further increase of LC3 II to LC3 I ratio,and the expression of p62 further decreased(P < 0.05).3 Results of axon regeneration-related protein GAP-43 and SCG10 western-blotting analysis: The expression of GAP-43 and SCG10 in the damaged brain tissue in ICH rats was higher than those in the Sham group(P < 0.05).BMSCs transplantation therapy could further increase the expression of GAP-43(P < 0.05),while the expression of SCG10 decreased(P < 0.05).After intervention of autophagy inhibitor 3MA,the expression of GAP-43 decreased(P < 0.05),while the expression of SCG10 increased(P < 0.05).Autophagy activator had the opposite effect,leading to further increase of the expression of GAP-43,while the expression of SCG10 further decreased(P < 0.05).4 Results of GAP-43 immunohistochemistry detecting analysis: Sham group showed sporadic distribution of positive cells,and the color was very light.GAP-43 positive cells in the damaged brain tissue in ICH rats significantly increased and the coloration also enhanced,and the immunoreactivity significantly increased compared with Sham group(P < 0.05).GAP-43 positive cells further increased after BMSCs transplantation therapy,and the immunoreactivity further enhanced.The difference was significant(P < 0.05).After the intervention of autophagy inhibitor 3MA,the immunoreactivity of GAP-43 positive cells decreased compared to BMSCs group.The autophagy activator RAPA had no significant effect on GAP-43 positive cells.5 Results of p-Akt western-blotting analysis: 3 days after modeling surgery,the ratio of p-Akt to Akt in ICH group was significantly higher than that in Sham group(P < 0.05).BMSCs transplantation therapy could further increase the ratio of p-Akt to Akt(P < 0.05).However,autophagy inhibitor 3MA and autophagy activator RAPA had no significant effect on the ratio of p-Akt to Akt.Summary: BMSCs transplantation after ICH in rats could promote axon regeneration by enhancing the autophagy flux of neurons in the damaged brain tissue.Conclusion: BMSCs transplantation after ICH plays a role in neuroprotection,improving neurological dysfunction.The molecular mechanism may be associated with the activation of PI3K-Akt-m TOR pathway to promote the autophagy flux-mediated axon regeneration in the damaged brain tissue. |
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