Experimental Study Of O2A Cell Transplantation In Acute Experimental Allergic Encephalomyelitis C57BL/6J Mice

Multiple sclerosis (MS), which is an acquired, immune-induced, chronic demyelination disease of the central nervous system, is often found in young adults, and can often lead to the disability of neurological function. Its most frustrating aspect is the inadequacy of the healing response of remyelination^1-2. But as the disease progresses, the number of lesions in which demyelination persists increases, significantly contributing to clinical deterioration. And clinically, it is difficult to diagnose, manage and treat.Objective: To investigate the effect of the myelin-forming cells transplantation on the animal model of MS - experimental allergic encephalomyelitis (EAE), and the effect of 3% hypoxia, several cytokines on the proliferation of the transplantation cells- O2A cells (Oligodendrocyte-and-type 2-Astrocyte cells), which may be a necessary explore for clinical therapy for MS.Methods: 1. The effects of 3% hypoxia and several cytokines on the proliferation of O2A cells were studied: ( i) O2A cells from neonatal SD rat cortex were cultured, purified and identified by cell surface markers in vitro.( ii) In the continuous hypoxia group, purified O2A cells which having been cultured for 2 days were put into 3% oxygen condition for 11k 21k 31k 41k 8h> 121k 241k 48h and 72h, respectively, and were then put into 20% oxygen condition until the 9th day. In the hypoxia-reoxygenation group, purified O2A cells which having been cultured for 2 days were put into 3% oxygen condition for 11k 21k 3h and 4h .respectively ,for l-4d, and were then incubated in the 20% condition till the 9th day. In the cytokine group, there were 16 subgroups as follows: blank control(no cell), control(with cells, no cytokines), PDGF, bFGF, IL-6, IFN- β1, GDNF, PDGF+bFGF, PDGF+IL-6, PDGF+GDNF, bFGF+IL-6, bFGF+GDNF, IL-6+GDNF, PDGF+bFGF+IL-6, PDGF+bFGF+GDNF, PDGF +bFGF+IL-6+GDNF, the concentration of cytokines was lOng/ml. (iii) The effect of hypoxia and/or cytokines on O2A cells proliferation were testified by MTT assay and cell number calculating. (iv)The HIF-1α mRNA expression of O2A cells in different oxygen conditions were testified by semi-quantitative RT-PCR.2. 30 four to 6-week-old female C57BL/6J mice were randomly divided into 4 groups: the control group, the EAE group, the EAE+sham group and the EAE+cell transplantation group, with 6 mice in each group. The effects of cell transplantation were evaluated by the neurological function scoring of the mice and the histopathology of the mice cryostat brain sections (35-40 μm thick). The cryostat brain sections were stained with hematoxylin-eosin, BrdU, FITC, and Gal-C, O4, A2B5 to assess inflammation, cell proliferation, transplantation cells and the differentiation of oligodendrocyte lineage, respectively.Results: 1. In the hypoxia-reoxygenation group, the proliferation of O2A cells was enhanced, especially in the 3h for 3d subgroup. And continuous hypoxic conditions over 24h inhibited the proliferation of O2A cells. 2. These cytokines and several their combinations promoted the proliferation of O2A cells, especially in the subgroups with the combinations of PDGF, bFGF and IL-6. 3. O2A cells transplantation therapy improved the neurological function scoring of the EAE mice. 4. The histopathology results showed that O2A cells transplantation therapy significantly promoted the repair of the neurological deficit of the EAE mice, and the BrdU+and Gal-C+cells increased significantly in the cells transplantation group, whereas there was no statistical difference for 04+and A2B5+cells among the EAE group, the EAE+sham group and the EAE+cell transplantation group. 5. No tumor tissue or tumor cells were found in mice brains after 8 weeks of cells transplantation.Conclusions: 1. For the first time we find that short hours of hypoxia-...