Functional Recovery Of Rats With Spinal Cord Injury Treated By Neural Stem Cell Transplantation Combined With Low Intensity Ultrasound

PART ONE ISOLATION AND CULTIVATION OF NEONATAL RAT NEURAL STEM CELLSBackground and Objective Neural stem cell, the self-renewing multipotential cell, could generate neural tissue, including neurons, astrocytes and oligodendrocytes. It existed not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including human. Isolation and culture of NSC was the basement of the fundamental and applied research. Objective of this part was to obtain neonatal rat NSCs for further study on its proliferation and differentiation.Methods Isolation the primary NSCs from endbrain and midbrain of neonatal rats(in 24h after birth), the cells were suspended and cultured in serum free medium (containing bFGF and EGF ) for long-term survival in vitro. Technique of single-cell clone culture was used to confirm the character of self-renewing. Ability of multi-directional differentiation and specific antigen, nestin, were detected by immunocytochemistry. Cytometry was used to evaluate the proliferation capability of NSCs and the growth curve is plotted.Results NSCs could survive in vitro for long time. They showed multipotential of differentiate into neurons and glial cells and the ability of self-renewing and proliferating. The NSCs were positive for nestin expression.Conclusions The NSCs were isolated from endbrain and midbrain of neonatal rats. These cells could be cultured for long time in vitro. They expressed nestin and had the ability of self-renewing and multi-directional differentiation. Cells could expand in serum free medium plus mitogen factors.PART TWO FUNCTIONAL RECOVERY OF RATS WITH SPINAL CORD INJURY TREATED BY NEURAL STEM CELL TRANSPLANTATION COMBINED WITH LOW INTENSITY ULTRASOUNDBackground and Objective Recovery of spinal cord injury(SCI) was a great puzzle in the neuroscience research domain for a long time. At present neural stem cells(NSCs) were utilized to treat SCI and low intensity ultrasound(LIU) could improve the experimental growth of peripheral nerves. Objective of this part was to observe the function recovery of rats with spinal cord injury(SCI) transplanted by neural stem cells(NSCs) and combined with low intensity ultrasound(LIU) application .Methods Forty SD rats were randomly divided into four groups. Group A: control group,Group B: spinal cord injury group (SCI), Group C:spinal cord injury with NSCs transplantation(NSCs), Group D:NSCs transplantation combinded with low intensity ultrasound(NSCs+LIU) application, each group had 10 rats. The rats in the Group B,C and D were made into the models of T8 plane with full transection of spinal cord ;the gelatin sponge with the cultured NSCs suspension of rats was grafted into the injured spinal cord in the group C and D while the gelatin sponge with the equal volume cell culture solution was transplanted into the rats in the group B. The rats in the Group D were utilized ultrasonic therapy meanwhile. Two months after operation, the motor and conduction function of spinal cord was determined by using BBB scores, cortical somatosensory evoked potential (CSEP), electron microscope and immunohistochemistry.Results As assessed by BBB scores, the rats in the Group B,C and D recovered but not recovered to the normal leve1, the Group D was the best after operation, CSEP of the Group B was still disappeared. Meanwhile in group C and D, the CSEP wave appeared again,but the waveform of the Group C and D was prolonged and the amplitude of wave was degraded slightly.Axons and myelinsheath regrown in group C and D, and nerve cells were discovered in group D cheked by electronic microscope. The quantities of neurons of NSCs differentiation were more and their appearances were more regular by immunohistochemistry in the Group D.Conclusions NSCs transplantation combined with low intensity ultrasound can improve the partial recovery of motor and conduction function of injured spinal cord in rats.