Experimental Study On The Effects Of Transplanting NSCs Combined With The Chitosan Porous Scaffold Into Cerebral Cortex Lesion Of Rats With Traumatic Brain Injury

Part one Study of the Preparation and Biocompatibility of the Porous Chitosan Scaffold in vitroObjective: To study the preparation of the porous chitosan scaffold, evaluate the biocompatibility of the porous chitosan scaffold to NSCs, study how to influence the differentiation of NSCs by the ectogenic NGF in the microenvironment of the scaffold, explore the potential of chitosan to be applied to nerve tissue engineering. Methods: The porous chitosan scaffold was made by Freezing-drying technique, the porosity rate was calculated by alcohol displacement method and the average diameter of the scaffold was measured by image processing. The NSCs come from rat fetal cortex were cultured in scaffold and were divided into NSCs+scaffold group and NSCs+scaffold+NGF group. In the NSCs+ scaffold group, DMEM/F12 free-serum medium has been added. In the NSCs+scaffold+NGF group, DMEM/F12 free-serum medium and NGF have been employed. Two weeks later, Nissl staining and immunohistochemistries of MAP-2, GFAP and CNP were applied to detect the differentiation of the NSCs. Then image processing and statistical analysis about the cell numbers, the cell bodies'areas and the cells'perimeter were carried out. Results: The results showed that the porosity rate of the scaffold was 90% and the average diameter was 50-350μm; NSCs in the scaffold could differentiate into neurons, astrocytes and oligodendrocytes; The numbers of the MAP-2 positive neurons were more and perimeter were longer in NSCs+ scaffold +NGF group than those in NSCs+ scaffold group. Conclusion: The results suggest that the porous chitosan scaffold has good NSCs biocompatibility, the ectogenic NGF can promote NSCs to differentiate into neurons, and the chitosan has the potential to be applied to nerve tissue engineering.Part two Study of Transplanting NSCs Combined with the Chitosan Porous Scaffold into Cerebral Cortex Lesion of Rats with Traumatic Brain Injury in vivoObjective: To explore the therapeutic effects of transplanting NSCs combined with the chitosan porous scaffold into cerebral cortex lesion in rats with traumatic brain injury. Methods: The NSCs come from rat fetal cortex were labeled with BrdU. Sprague-Dawley(SD) rat models with TBI were made by Feeney's method. The TBI rats were randomly divided into injury-control group, NSCs+scaffold group and NSCs+scaffold+NGF group. The rats were immediately debrided after injury, then NSCs combined with the chitosan porous scaffold were transplanted into injured cerebral cortex in NSCs+ scaffold group, NSCs combined with the chitosan porous scaffold and NGF were transplanted into injured cerebral cortex in NSCs+ scaffold +NGF group. Rats of three groups were injected with BDA at 7 days before 1, 2, and 3 months postop, then tested for cognitive function using step-through passive avoidance test and active avoidance test, then the rats were perfused and fixed, and the cryostat sections of brain were prepared. Nissl staining, GFAP immunofluorescence and BDA histochemistry were used for brain sections. NF-200/BrdU and GFAP/BrdU double immunofluorescence were employed to detect the differentiating of the NSCs into neurons and astrocytes respectively in NSCs+scaffold group and NSCs+scaffold+NGF group. Results:①Significant improvement on cognitive function were observed at 1, 2, and 3 months after transplantation in two transplanting groups, and the cognitive function improvement of rats in NSCs+scaffold+NGF group were more obvious than that of rats in NSCs+scaffold group.②A cavity formed in the ipsilateral injured cortex and the ipsilateral hippocampus were depauperated in Nissl staining slices of injury-control group, but hippocampus were not depauperated evidently and there were grafts in the injured cortex sites in Nissl staining slices of two transplanting groups.③The number of GFAP positive cells near damaged area of the ipsilateral injured cortex in two transplanting groups were less than that of injury-control group at 1, 2, and 3 months postop.④Some NF-200 and BrdU double-labeled cells were detected in the transplantation zone of two transplanting groups at 1, 2, and 3 months postop. The number of NF-200 and BrdU double-labeled cells with more and longer processes in NSCs+scaffold+NGF group were more than that of NSCs+scaffold group.⑤Some GFAP and BrdU double-labeled cells were detected in the transplantation zone of two transplanting groups at 1, 2, and 3 months postop. The number and appearance of GFAP and BrdU double-labeled cells had no differences between two transplanting groups.⑥Results of BDA histochemistry showed that the processes of some cells in the transplantation zone of two transplanting groups had extended into the brain tissue of host at 3 months postop. The neurons in the transplantation zone of NSCs+scaffold+NGF group extended more processes into the host brain tissue. Conclusion: Cognitive function of rats with TBI can be improved by transplanting NSCs combined with the chitosan porous scaffold into injured cerebral cortex. The grafts can moderate depauperating of the ipsilateral injured hippocampus , and inhibit gliosis near damaged area of the ipsilateral injured cortex. Transplanted NSCs in the scaffold can survive and differentiate into neurons and astrocytes in vivo. Ectogenic NGF can promote NSCs to differentiate into neurons and speed up the differentiated neurons to connect with the host brain and accelerate the rats'functional recovery following TBI.