Transplantation Of Olfactory Ensheathing Cells For Experimental Spinal Cord Injury

ObjectiveThe purpose of this experiment is 1. To evaluate the purification effect of the modified Nash' s method for primary culture of Olfactory ensheathing cells;2. To observe the therapeutic effect of primary cultured OECs on the left hemitransect-ed spinal cord of Wistar rats.Methods1. OECs were primary cultured with modified Nash method ,8-10 days later, the conformation of the cells were observed under phase contrast microscope, the cells were identified and the purity was counted with P75 immunos-taining.2. The left hemisection of T10 spinal cord of adult Wistar rats was made, whom were separated into the therapeutic and control groups. The therpeutic group: OECs were stereotoxically transplanted with microinjection method into the injuried area; the control group: the DF - 10S culture media were injected with the same method mentioned above. Eight weeks after transplantation, (1) the recovery of the motor function of the rats were evaluated with the modified Tarlov method and the result data were analyzed with Wilcoxon Mann - Whitney test; as well as (2) the regenerated axons with HRP anterograde tracing technique ; (3 ) the number of axons, the integrity of axonal sheathes and the differentiation of the OECs with transmission electron microscope (TME) ; (4) the survival and distribution of OECs in the spinal cord with P75 immunostaining and (5 ) the severity of spinal cord with HE staining.Results1. Eight to ten days later, there were three types of cells in the culture flasks; 1) long bipolar fusiform cells; 2) spherical cells; and 3) unipolar/mul-tipolar cells. The unipolar/multipolar cells were the main cell type in the purified cells, which had a long process and there was little branches or vesicals at the end of the process, the bodies of the cells were round or elliptical and had higher deopter with aureola surround, the cleveage phase and the other two cell types could been oberserved occasionally. The cells were identified with P75 antibody on the cell membrane and the purity is 92%.2. Eight weeks after transplantation, ( 1) the motor functions of the therapeutic group scored higher than those of the control group ( u = 2. 07, P < 0. 05) ; (2) the regenerated axons of therapeutic group penetrated the glial scar while those of control group did not; ( 3 ) the observation of ultrastructure under TME demonstrated that there were much more axons and better integrity of the axonal sheathes in the therapeutic group than those of control group and OECs exhibited Schwann cell - like and astrocyte - like, ensheathing the regenerated axons, while the number of axons in the control group was small and there were glial cells ensheathing the axons; (4) the immunostaining demonstrated that the OECs transplanted survived 8 weeks, evenly dispersed in the injuried area and ( 5 ) HE staining showed there were glial scar or cavity formation in the injuried area of spinal cord.Conclusions1. Based on the Nash' s method, OECs are separated and purified according to different adhesion rate, the culture time is shortened and no other biochemical reagents are used . The biological characters of the cells are well kept.2. The transplanted OECs can improve the motor function of rats, and enhance the regeneration and re - ensheathing of severed axons.