Transplantion Co-Cultivated BMSCs And Mononuclear In The Brain Stem To Promote The Repair Of Facial Nerve Axotomy And Intravital Imaging By MRI

Background and Objective:The functional disability or never recovery of facial expression muscle caused by facial nerve axotomy may influence the life quality of patient severely. Facial nerve in situ restoration is the main clinical treatment at present. Although postoperative facial expression muscle function of patients recovers satisfactorily, some people still feel obvious difference compared with pre-injury, and the restoration and reconstruction of facial nerve is a difficult problem in the fields of plastic surgery and neurology at present.Facial motoneuron (FMN)apoptosis may appear after the facial axonal injury, and keeping the injured neuron in living condition is the foundation of the fuctional recovery.40% of FMN in the nerve nuclei can live with the help of fuctional peripheral immune system and/or neurotrophic factor after the axonal injury, namely the FMN of environmental dependency. To explore the mechanism of neuron apoptosis after axonal injury, to change the neural-inflammatory microenvironment, and to prevent the quantity of the facial motoneuron (FMN)apoptosis are some of the research hotspots now. Research shows transplanted bone marrow mesenchymal stem cells (BMSCs)can turn into neuron-like cells or neuronal stem cells in the microenvironment of central nervous system, replace the apoptotic or necrotic neurons, and secret many kinds of neurotrophic factors to promote the neuron regeneration. Hence usage of the cells can be a treatment for the nervous system disease or injury. But many studies have shown the low survival rate of transplanted stem cells partly because of the local injured inflammatory microenvironment, hypoxia, and hypoperfusion. The damage signal of the neural broken ends enters FMN along the proximal axons after facial nerve axonal injury, which leads to disequilibrium and pro-inflammatory state of the immuno-inflammatory microenvironment in facial nerve nucleus, and the generated chemokines act on peripheral T cells to produce the autoimmune response. CD4+ and CD25+regulatory T cells are subsets of the class of T cells, they play an important role in maintaining the balance of immuno-inflammatory microenvironment of facial nerve nucleus, have protective effect on the apoptotic neurons, and act on endogenous neural stem cells to differentiate into nerve cells for the benefit of FMN. But the pro-inflammatory microenvironment of the facial nerve nucleus has an adverse impact on stem cells survival, and the stem cells can not make up the loss of FMN. Research shows BMSCs have immunosuppressive function, they can inhibit the proliferation of T cells, regulate the immune responses, reduce the degrees of responses, increase the proportion of CD4+ and CD25+regulatory T cells under mixed lymphocyte culture in vitro.Based on the above theory, our team proposed to find the best proportion of the mixed SD rat BMSCs and spleen mononuclear lymphocytes culture, used stereo-taxic technique to transplant the co-cultivated cells into the facial nerve nucleus of the brain stem. It could increase the proportion of CD4+ and CD25+regulatory T cells of facial nerve nucleus to improve the neuro immune-inflammatory microenvironment, alse provide a microenvironment for the benefit of BMSCs chemotaxis, living, and secretion of neurotrophic factors, save the FMN of environmental dependency. The research can provide the foundation for the clinical restoration and reconstruction of the facial nerve injury.To study the transplanted stem cells distribution, migration, differentiation and homing in body can help to understand the repair mechanism of stem cells. In this study, we use MR molecular imaging marker, including SPIO and GFP, to mark BMSCs, and use histologic tracing and vivo MR tracer method to demonstrate the BMSCs homing phenomenon. This can provide a theoretical basis for transplanted cells in vivo tracing.Methods:1. The BMSCs from SD rat tibiofibula bone marrow were isolated, cultured and identified in vitro. After marking the BMSCs and GFP transfected BMSCs by different concentrations of SPIO, we examined the proliferative activity and cell cycle of BMSC/GFP-BMSCs to make sure the influence of SPIO on cellular activity. MR imaging was used in vitro to know the marked cells MR signal intensity. The marked cells were induced to differentiated into osteoblast, fat, and nerve-like cells to know the influence of SPIO on cellular differentiation.2. After co-culturing BMSCs and mononuclear lymphocyte in different proportions, we examined the growth inhibitory effect of BMSC on CD8+T cells and the influence on the proportion of CD4+CD25+regulatory T cells. Protein chip was used to screen the lysate of the co-cultured and single-cultured cells and1 differential proteins of supernatant. We examined the influence of mononuclear lymphocytes on the differentiation of BMSC into facial neuron, and found the optimum proportion of two kinds of cells co-cultured in vitro, which provided the cells transplant co-cultured in vivo with objective indicator.3.4 days later after the establishment of the model of right facial nerve injury, we used the stereo-taxic technique to transplant the co-cultivated and single-cultivated cells according to different proportions into the facial nerve nucleus of the brain stem.4. In vivo MR tracing and histologic tracing of facial nerve nucleus at any time point after transplant showed BMSCs homing phenomenon, we examined the immune-inflammatory microenvironment related cytokines (the differential proteins screened by protein chip) of facial nerve nucleus, SDF-1, BDNF and the change of signaling pathway at any time point, then compared correlation between the change of immune-inflammatory microenvironment related cytokines and the order of magnitude of the neuron apoptosis in the facial nerve nucleus.Results:1. BMSCs were successfully isolated from SD rat tibiofibula bone marrow and cultured to the third generation.4 days later, marker CD29 and CD90 were examined positive, CD34 was negative. BMSC/GFP-BMSCswere successfully marked by different concentrations of SPIO (25μg/ml,50μg/ml,75μg/ml,100μg/ml, 0μg/ml, group A to E).Proliferative activity of BMSC/GFP-BMSCs in group D was examined the lowest (P<0.05).Cell cycle of group A to E showed no significant difference (P >0.05). There was no significant difference in signal intensity of in vitro MR imaging between group C and D, but they were lower than those in the other groups (P<0.05). Decrease in signal intensity of SWI was more obvious than that of T2*WI (P<0.05). The marked BMSC/GFP-BMSC were successfully induced to differentiated into osteoblast, fat, and nerve-like cells. The result showed SPIO of 75μg/ml had no influence on differentiation of BMSCs.2. Mononuclear lymphocytes and BMSCs were co-cultured in different proportions(single-cultured mononuclear lymphocytes, single-cultured mononuclear lymphocytes plus PHA,1:1,1:10,1:30,1:50, single-cultured BMSCs,1:1,10:1, 30:1,50:1, group A to K). CD4+T/CD8+T cells showed the highest proportion (P <0.05) when the ratio of monocytes to BMSCs was 1:30(co-cultured group 1). BMSCs were induced to differentiate into nerve-like cells when the ratio of monocytes to BMSCs was 30:1 (co-cultured group 1). The protein chip result showed the proteins up-regulated from the lysate of co-cultured and single-cultured cells were VEGF, BDNF, CXCR4, and CD34, proteins up-regulated from the supernatant were VEGF, TGF-β, IL-4, IL-10, and proteins down-regulated were IL-2, INF-γ. The expression quantity of Tub 3, Nestin, NSE, Syt lmRNA before induction by PCR increased significantly than those after induction.3. The models of unilateral facial high level injury of 150 SD rats were successfully established, and stereo-taxic technique was successfully used to transplant the cells into the facial nerve nucleus of the brain stem 4 days later.4. All the models were divided into 6 groups according to the different transplanted cells(negative control group, positive control group, single-cultured mononuclear lymphocytes, co-cultured group 1, single-cultured BMSCs, co-cultured group 2). The operation was proved successfully on the day of transplant by MR scans. The migratory paths of cells were marked by MR tracing, and GFP positive cells homing were shown by means of pathological section on day 3, day 7, day 14, day 21, and day 28 separately after transplant. The expression quantity of Caspase-3 and Bcl-2 of each group at any time point examined by Tunel staining and West-blot test showed the increase of apoptosis quantity from day 3 to day 21, the decrease of them after day 21, and the quantity of co-cultured group 2 were the least of all the groups (P<0.05). The expression quantity of the cytokines from the facial nerve nucleus of all the groups examined by West-blot and Q-PCR test increased from day 3 to day 14, reached the peak level from day 14 to day 21, and declined after day 21. The highest expression quantity of anti-inflammatory cytokines IL-4, IL-10, TGF-β1, and JAK/STAT6 signal pathway, chemokines SDF-1/CXCR 4 axis, BDNF, trkB/ERK signal pathway were observed in co-cultured group 2 (P<0.05), closely followed by co-cultured group 1 (P<0.05), and single-cultured BMSCs (P<0.05), the lowest was observed in positive control group (P<0.05). The lowest expression quantity of proinflammatory cytokines INF-γ, IL-2, IL-6 were observed in co-cultured group 2 (P <0.05), a little higher one was the co-cultured group 1 (P<0.05), and the highest one was the positive control group (P<0.05)Conclusions:1. The proliferation and differentiation of BMSCs are not affected by the mark of GFP and SPIO of 75μg/mL, and SWI sequence can show the signal intensity of BMSCs marked with GFP and SPIO of 75μg/mL more sensitively than T2WI.2. BMSCs can inhibit the proliferation of lymphocytes caused by PHA, increase the proportion of CD4+CD25+T cells and the ratio of CD4+T/CD8+T cells, promote the secretion of TGF-β, IL-4, IL-10, inhibit the secretion of IL-2、INF-γ, which changes the living microenvironment of BMSCs.(the best ratio of mononuclear lymphocytes to BMSCs is 1:30)3. Co-culture of BMSCs and lymphocyte promotes the BMSCs to differentiate into neuron-like cells, increases the secretion of BDNF、SDF-1、VEGF (the best ratio of monocyte to BMSC is 30:1)4. Transection of facial nerve at a high level of stylomastoid foramen can be used to establish a stable and repeatable model of unilateral facial nerve injury. The brain stem stereo-taxic transplant is feasible and reliable.5. The change of neuro-immune inflammatory microenvironment in facial nerve nucleus caused by facial nerve axonal injury can increase FMN apoptosis (the "drifting" of anti-inflammatory/pro-inflammatory microenvironment)6. In combined BMSCs and mononulear lymphocytes transplantation, IL-4 inhibits inflammatory response by the way of JAK/STST6 signal pathway, improves the neural-immune inflammatory microenvironment, promotes chemotaxis, differentiation, and secretion of BDNF of the transplanted BMSCs, and BDNF can provide a living environment for anti-neuron apoptosis by the way of trkB/erk signal pathway, decrease the neuron apoptosis.