Autologous Transplantation Of Bone Marrow Stromal Cells For Treatment Of Traumatic Brain Injury In Rabbits

Traumatic brain injury (TBI) is the brain tissue damage caused by trauma. Among the death caused by vehicle accidents, the mortality ratio of TBI is second to none. So that TBI is a vital threat to the human beings. At the same time, the mutilation ratio of TBI is also very high. After TBI, the neurological dysfunction like hemiplegia, even persistent vegetable state (PVS) is often encountered. Thus, heavy burden is put on the family and the society. Therefore, TBI treatment is a serious topic of our world.But up to now, the traditional measures have brought little light to treatment of TBI. In the recent years, the method is enheartening the researchers that the transplanted adult stem cells can serve to substitute the injured neural cells or the damaged neural functions.Bone marrow stromal cells (BMSCs), a member of the family of adult stem cells, are the incompletely differentiated primitive cells in the bone marrow other than haemopoietic stem cells (HSCs). They can differentiate into many kinds of mesenchymal stem cells. And under certain condition, they can transdifferentiate into neural cells. It has been demonstrated that BMSCSs transplantation can improve the rehabilitation from neurological dysfunction in TBI models in rodents. And moreover, BMSCs can be easily and sufficiently harvested. Furthermore, autologous transplantation can be accompanied when the choice of seed-cells is BMSCs. By the way, ethical debate and immunological rejection caused by embryonic stem cells (ESCs) transplantation can be avoided. Therefore, BMSCs have developed into the optimal seed-cells for cell based therapy of TBI.At present, the ways that cells are delivered in the treatment of experimental TBI by adult stem cells transplantation are as following: injected into parenchyma of the cerebrum, injected into the lateral ventricle, injected into the remote vein, and relatively minor used methods like injected into artery, injected into abdominal cavity and injected into cisterna magna. Cells transplantation by these ways might have some shortcomings, such as aggravating brain injury, sending fewer cells to the injured site, and providing unsatisfied therapeutics. Thus, it's essential for us to create a new way for cells delivery that has many advantages compared to the traditional methods, such as bringing minimal invasion, sending more cells to the injured sits, and providing satisfied therapeutics.In the recent years, some researchers have detected that stem cells implanted through lumbar puncture could migrate to the injured cervical cord, and even more a few implanted cells could be identified in the brain tissues. And the neurological function was improved after cells based therapy in these studies. Thus, the idea is illuminated that lumbar puncture delivery might be a relatively optimal pathway for TBI therapy with BMSCs transplantation.In this study, BMSCs cultured in vitro, were the seed cells. And they were marked by the adeno-virus transfected green fluorescent protein (Ad-GFP). The characteristics of the cultured BMSCs were identified. Then TBI models of New Zealand rabbits were developed. The marked BMSCs were injected into the parenchyma of the cerebrum, the lateral ventricle, the vein and the subarachnoid cavity by lumbar puncture, respectively. The motor dysfunction was evaluated at several time points after cells transplantation. And the injured tissues were harvested for pathological examination. Finally, the results were compared within different groups.Part one: Investigation of BMSCs cultured in vitro and the configuration of goat anti-mouse IgG gold conjugate with atomic force microscopyObjective: To detect the adhesion device and its structure of bone marrow stromal cell, and to warrant the isolation and differentiation of the cell. To identify the configuration of goat anti-mouse IgG gold conjugate (IgG-gold).Methods: Bone marrows were harvested from the femurs of rabbits. And BMSCs were obtained with density gradient centrifugation. After cultured in primary for 1day, 7 days and 14 days, BMSCs were fixed with 2.5% glutaraldehyde for 5 minutes. Then the cells were cleansed thrice with double evaporated water. After the cells were open-air dried, they were scanned with atomic force microscope under contact mode respectively. The molecules of IgG-gold affiliated on the new cleaved mica were scanned with atomic force microscope (AFM) under the physiological environment. The data was analyzed with the statistic software in the AFM.Results: Adhesion was the characteristic of bone marrow stromal cells in the given stage of their lives. And correspondingly every cell had the adhesion device. The configuration of IgG-gold molecule is an "ellipsoidal sphere" sized as 22.0nm×9.0nm×5.7nm.Conclusions: Atomic force microscope had special predominance in observation of the ultrastructure of cell surface. Bone marrow stromal cells had adhesion device with corresponding structure. IgG-gold has a steady uniform characteristic conformation. AFM can play an important role in detecting biomacromolecules at a nanometer scale resolution. Part two: study on green fluorescent protein, 5-bromodeoxyuridine and 5,6-carboxyfluorescein diacetate succinimidyl ester marking BMSCs in rabbitsObjective: To detect the applicability of BrdU, CFSE and GFP serving as labels of MSCs of rabbits.Methods: BMSCs of rabbits were labeled with BrdU, CFSE and GFP respectively. The efficiency of the three markers was quantified. Cell cycle and apoptosis ratio of labeled BMSCs were revealed, the growth curves were delineated, so as to identify whether the markers cast an influence on the cell growth characteristics in vitro. Furthermore, the traumatic brain injury model was introduced. And the labeled BMSCs were transplanted by means of lumbal puncture. 4 weeks later, the animals were sacrificed and the pathological sections underwent fluoroscopy.Results: The efficiency of BrdU labeling was 73.3%, that of GFP was 81.6%, and that of CFSE was 99.1%. The three markers had no effects on the growth characteristics of BMSCs of rabbits cultured in vitro. The transplanted labeled cells were detected at the site of the injured area. And CFSE leakage was encountered.Conclusions: BrdU, CFSE and GFP can serve as a label of BMSCs of rabbits. Each of BrdU and CFSE has its limitations.Part three: Autologous transplantation of bone marrow stromal cells for treatment of traumatic brain injury in rabbitsObjective: To establish a stable TBI model in rabbits. To identify the therapeutics of treatment of TBI with autologous BMSCs transplantation through different pathways in rabbits. To explore a minimally invasive method for stem cell transplantation. And to clarify whether the optimizing pathway of stem cell transplantation does exist or not.Methods: 45 New Zealand rabbits were randomly divided into five groups equally. BMSCs were injected into right ear-edge vein, subarachnoid cavity through lumbar puncture in one of the two treatment groups after TBI respectively. PBS was injected into subarachnoid cavity through lumbar puncture in another group. And BMSCs were injected into subarachnoid cavity through lumbar puncture in the last group without TBI. The last two groups together with the group receiving no engraftment after TBI served as control group. 72h after marked with Ad-GFP, 2×106BMSCs cultured for 10d in vitro, were harvested and suspended in 60μl sterile PBS. Moderate TBI models of rabbits were established. The cells were autologously implanted 24h after TBI. The motor dysfunction was assessed at 24h, 1w, 2w and 4w after cells transplantation respectively. And 2 models in each group were sacrificed at 1w, 2w and 4w after cells transplantation through cardio-perfusion with 4% paraformaldehyde (PFA). Then the brain specimen underwent frozen section. And the labeled cells were counted under fluorescence microscope.Results: The recover of motor dysfunction in treated groups were better than that in the control groups. And the rehabilitation of motor function in the group of BMSCs transplantation by lumbar puncture was more outstanding. The transplanted cells found at the injured sites were not the same. And from many to few, they are by turns: the group of transplantation by mtraparenchymal injection, by lumbar puncture, by intraventricle injection, by intravenous injection.Conclusions: A stable moderate TBI model of rabbits was established. BMSCs transplantation through various pathways can improve the motor dysfunction rehabilitation after TBI in rabbits. Among the several commonly used methods, the therapeutics of BMSCs injection into subarachnoid cavity by lumbar puncture is optimized. An effective and minimally invasive pathway for BMSCs transplantation to treat TBI has been explored.