The Mechanism Of Anti-apoptosis And Chronic Stress In The Treatment Of Rat Spinal Cord Injury With Allogenic Rat Bone Mesenchymal Stem Cells Transplantation

Part I : Co-culture and multilineage differentiation potentialcharacterization of allogenic rat bone mesenchymal stem cellsObjective To investigate the Co-culture of allogenic SD rat bone mesenchymal stem cells (BMSCs) in vitro, and to perform multilineage differentiation potential characterization.Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were obtained and Wright-Gemsa staining were performed for morphological observation; and to identify the surface marker of CD29, CD45, CD90 by flow cytometry; moreover, the passage cells were induced to differentiate into osteoblast and adipocyte respectively, then performed alkaline phosphatase, Von Cossa and oil red staining characterization.Results The primary cells of complete marrow direct culture method are uneven in shape, but with excellent reproductive activity; the cells obtained by density gradient method are fairly uniform in shape, but with lower reproductive activity; after 3 passages, the cells obtained by these two methods both got relative purification. The co-cultured cells were good in reproductive activity and relatively uniform in shape, without cell death because of immunologic rejection. After Wright-Gemsa staining, the cells displayed typical spindle-shaped cells; and the co-cultured cells were positive for CD29 and CD90, but negative for CD45 by flow cytometry. After induction, the co-cultured cells appeared morphological changes of osteoblast and adipocyte, and were confirmed be osteoblast by alkaline phosphatase staining and Von Cossa staining; be adipocyte by oil red staining.Conclusion By use complete marrow direct culture method and density gradient method, after 3～5 passages, can obtain relative purified BMSCs. The co-cultured allogenic BMSCs were good in growth, without immunologic rejection, imply the weak immunogenicity of BMSCs. In vitro, the co-cultured rat BMSCs can be induced osteoblast and adipocyte, indicate the multilineage differentiation potential of BMSCs.Part II: Neural differentiation potential characterization of allogenicrat bone mesenchymal stem cellsObjective To investigate the Co-culture of allogenic SD rat bone mesenchemal stem cells (BMSCs) in vitro, and to perform neural differentiation potential characterization.Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were obtained and induced to differentiate into neurocyte, then performed immunocytochemistry of Nestin, NSE, GFAP.Results After induction, the co-cultured cells appeared morphological changes of neuron and glial cell, and were confirmed by Nestin, NSE, GFAP immunostaining.Conclusion The co-cultured allogenic BMSCs can be induced into neuron and glial cell in vitro, indicate that in appropriate circumstance, BMSCs can be induced into neurocyte in vivo.Part III: Migration of allogenic rat bone mesenchymal stem cells toinjured sites through cerebrospinal fluid in rat with spinal cord injuryObjective To investigate the migration of allogenic rat bone mesenchymal stem cells to injured sites through cerebrospinal fluid in rat with spinal cord injury. Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were labeled by transfection of recombinant adenovirus containing enhanced green fluorescence protein (EGFP) gene (Ad5F35-EGFP). A total of 20 adult male Sprague-Dawley (SD) rats weight 222-289 g were divided into 3 groups: control group, n=4; model group, n=8; treatment group, n=8. The rats in model and treatment group were performed a partial low thoracic spinal cord injury (SCI) by modified Allen's method (weight drop method) at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs (EGFP transfected) or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by Basso-Beatie-Bresnahan (BBB) scale. Half of the rats in each group had spinal cord tissue harvested at day 7 after cell transplantation. The other were harvested at day 14 after cell transplantation. The spinal cord segments containing the injured sites was removed from the spinal column after perfusion. Spinal cords were postfixed in 4% paraformaldehyde overnight and half-cut longitudinally in the sagittal direction. One half of the spinal cord was transferred to 30% sucrose solution, frozen, and cut in a cryostat at 10μm thickness for the examination of migration of transplanted cells to injured sites under fluorescent microscopy. The other half of the spinal cord was paraffin-embedded and sectioned in the same size as above for hematoxylin and eosin staining for the examination of cell aggregation in injury sites under optical microscope.Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. 24h after transfection through Ad5F35-EGFP adenovirus, some BMSCs expressed GFP under fluorescent microscope, 48h after transfection, a great many BMSCs expressed GFP. After transplantation of EGFP-BMSCs following a subarachnoid injection, BBB scale score of the animals in treatment group increased progressively. 7 days after grafting procedure, the animals of model group and treatment group reached a mean value of 6.00±1.60 and 8.13±1.96 in the BBB scale respectively, still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group, with significant difference (P<0.05). Under fluorescent microscope, both of control group and treatment group animals' sections expressed GFP 7 and 14 days after grafting procedure, but the GFP expression was negative in model group. HE staining showed that 7 and 14 days after grafting procedure, sections of control group exhibited normal neural morphological change, while huge cell aggregations were observed on the surface of the spinal cord facing the subarachnoid space in treatment group, cell aggregations could not be seen in the model group.Conclusion EGFP-BMSCs following a subarachnoid injection could migrate from a lumbar site into the injured low thoracic spinal cord through the cerebrospinal fluid, and could improve the hind limb motor function of SCI rats. This would potentially establish a simpler, less invasive procedure for BMSCs transplantation to spinal cord injury by lumbar puncture in clinical application.Part IV: The influence of allogenic bone mesenchymal stem cellstransplantation to the neurocyte apoptosis of injured rat spinal cord.Objective To investigate a potential anti-apoptotic mechanism of transplanted BMSCs for rat SCI.Methods A total of & male SD rats weight 90-102 g were used for BMSCs harvest, and a total of 84 adult male SD rats weight 235-319 g were divided into 6 groups depending on the timing of the lumbar injection after thoracic SCI, n=14 in every group: Group A1, control group, day 14 post; Group A2, control group, day 28 post; Group B1, model group, day 14 post; Group B2, model group, day 28 post; Group C1, treatment group, day 14 post; Group C2, treatment group, day 28 post. The rats in model and treatment group were performed a partial low thoracic spinal cord injury (SCI) by modified Allen's method at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by BBB scale. Rats were anesthetized at day 14, 28 postoperatively according to the timing point of group. Every 8 rat spinal cord segments containing the injured sites or counterparts in each group were harvested after perfused with normal saline followed 4% paraformaldehyde intracardially. Spinal cords were postfixed in neutral formalin for 24-48h and were paraffin-embedded and sectioned at 4 urn thickness. Hematoxylin and eosin staining were performed for histological observation. Neurocyte apoptosis was examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling ( TUNEL) staining, the expression of Bax and Bc1-2 protein were tested with immunohistochemistry. 5 rat spinal cord segments containing the injured sites or counterparts in each group were harvested directly, to investigate the mRNA expression of Bax and Bcl-2 by reverse transcriptase -polymerase chain reaction (RT-PCR). 1 rat spinal cord segments containing the injured sites or counterparts in each group were harvested directly, a 4 1mm×1mm×1mm spinal tissue were cut from the center of injured sites, for ultramicrostructural observation under transmission electron microscope (TEM).Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. After transplantation of BMSCs, BBB scale score of the animals in treatment group increased progressively, but still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group from 7 day after grafting procedure, with significant difference (P<0.01). HE staining showed that 14 and 28 days after grafting procedure, sections of control group exhibited normal neural morphological change, while cavities of SCI were observed in those of the model and treatment group, the model group was more significant than treatment group. Under TEM, 14 and 28 days after grafting procedure, sections of control group exhibited few apoptotic neurocyte, while in the sections of model and treatment group, a lot of apoptotic and necrotic neurocytes were observed, the model group was more significant than treatment group. The TUNEL staining showed that the TUNEL positive cells in model group were higher than those in control group and treatment group at 14 day after injection, with significant difference (P < 0.01), and positive cells in treatment group were higher than those in control group as well, with significant difference (P<0.01), the TUNEL positive cells mainly distributed in white matter. The positive cells declined at 28 day after injection, model group were still higher than those in the other two groups, but without significant difference. The result of immunostaining against Bax and Bcl-2 demonstrated that model group showed higher Bax positive expression than those in the other two groups, with significant difference (P<0.01) at 14 day after injection, and Bax positive cells in treatment group were higher than those in control group as well, with significant difference (P<0.01), which on the contrary in Bcl-2 positive expression, but without significant difference. The Bax positive cells declined at 28 day after injection, model group were still higher than those in the other two groups, but without significant difference (P>0.05), the change of Bcl-2 positive expression was insignificant. The RT-PCR of Bax and Bcl-2 demonstrated that model group showed higher Bax gene positive expression than those in other two groups, but without significant difference (P>0.05) in relative optical density at 14 and 28 days after injection, which on the contrary in Bcl-2 positive expression, without significant difference (P>0.05) either.Conclusion Transplantation of BMSCs by a subarachnoid injection could improve the hind limb motor function of low thoracic SCI rats, and could reduce spinal neurocyte apoptosis, decrease the genic and proteinic expression of Bax, increase the genic and proteinic expression of Bcl-2, which implied a potential anti-apoptotic mechanism of transplanted BMSCs for rat SCI, the anti-apoptotic effect maybe achieved by the effect to apoptotic controlling gene Bax and Bcl-2.Part V: The influence of allogenic bone mesenchymal stem cells transplantation to the stress state and AMFA receptor protein expressionof brain in rat with spinal cord injury.Objective To investigate the effect of allogenic bone mesenchymal stem cells transplantation to the stress state and AMPA receptors protein expression of hippocampus and basolateral amygdale (BLA) in rat with SCI.Methods A total of 48 adult male SD rats weight 250-319 g were divided into 6 groups depending on the timing of the lumbar injection after thoracic SCI, n=8 in every group: Group A1, control group, day 14 post; Group A2, control group, day 28 post; Group B1, model group, day 14 post; Group B2, model group, day 28 post; Group C1, treatment group, day 14 post; Group C2, treatment group, day 28 post. The rats in model and treatment group were performed a partial low thoracic SCI by modified Allen's method at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by BBB scale. Rats were anesthetized at day 14, 28 postoperatively according to the timing point of group, to obtain blood for detection of plasmic ACTH and serumal corticosterone concentration by enzyme linked immunosorbent assay (ELISA), and to harvest brains after perfused with normal saline followed 4% paraformaldehyde intracardially, brains were postfixed in 4% paraformaldehyde overnight and were transferred to 30% sucrose solution, frozen, and cut in a cryostat at 30μm thickness for the immunohistochemistry staining of GluR1 and GluR2.Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. After transplantation of BMSCs, BBB scale score of the animals in treatment group increased progressively, but still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group from 7 day after grafting procedure, with significant difference (P<0.01). After low thoracic SCI and sham operation, all animals' body weight decreased because of operation interference, but increased gradually from then on. At timing point of day 7 (pre-grafting procedure), animals' body weight in the control group were higher than those in the model and treatment group, but without significant difference (P>0.05). After grafting procedure, all animals' body weight decreased again due to operation interference as before. Then all animals' body weight increased gradually. Animals' body weight in the control group were higher than those in the model and treatment group, and with significant difference (P<0.05或P<0.01) from 3 days after grafting procedure. From 7 days after grafting procedure, animals' body weight in the treatment group were higher than those in the model group, but without significant difference (P>0.05). At the timing point of 14 days after grafting procedure (21 days after SCI), the plasmic ACTH concentrations in the model group were higher than those in the other groups, which were on the contrary in the concentrations of serumal corticosterone. At the timing point of 28 days after grafting procedure (35 days after SCI), the plasmic ACTH concentrations in the model group decreased and near to normal level, but at the same time, the concentrations of serumal corticosterone increased. At the timing point of 14 days after grafting procedure, GluR1 positive cells of the model group increased in CA1, CA3, DG regions of hippocampus and BLA, with significant difference in CA1 (P<0.01); GluR2 positive cells had similar tendency, but without significant difference. At the timing point of 28 days after grafting procedure, GluR1 positive cells of the model group were higher than those of the control group in CA1, CA3, DG regions and of the treatment group in CA1, CA3 regions of hippocampus, with significant difference (P<0.05 or P<0.01, respectively); GluR1 positive cells of the model and treatment group were higher than their counterpart at the timing point of 14 days after grafting procedure, with significant difference (P<0.05 or P<0.01, respectively); GluR2 positive cells of the treatment group were higher than those of the control group in BLA, with significant difference (P<0.05); GluR2 positive cells had similar tendency in the other regions, but without significant difference.Conclusions Transplantation of BMSCs could improve the hind limb motor function of low thoracic SCI rats, and could relieve the stress state of SCI rats, which implied a potential anti-chronic stress mechanism of transplanted BMSCs for rat SCI, the anti-chronic stress effect maybe achieved by influence to the expression of AMPA receptor protein of GluR1 and GluR2.