| Mesenchymal stem cells (MSCs), also called bone marrow stromal cells (BMSCs), are a kind of multipotential adult stem cell, apart from hematopoietic stem cells in bone marrow, and are the hot spot and leading edge in stem cell investigation. MSCs are convenient to be harvested, easy to be isolated and cultured, effective to be expanded and purified, and keep multipotential after multigeneration in vitro. Their genetic background is stable. There will be weak immunologic rejection after transplanted in vivo. After gene transfection in vitro, they could express exogenous gene lastingly. During the last few years, however, it has been reported that MSCs have the ability to differentiate into ectodermal and endodermal cells except mesodermal cells. Recently, neural transdifferentiation of MSCs especially provoke extensive interest. It has been reported frequently that autologous or allogeneic MSCs transplanted by local, intraperitoneal or intravenous injection into various neuronal degeneration animal models (such as focal cerebral ischemia, cerebral hemorrhage, cerebral trauma, Parkinson's disease and so on) could home to injury area, survive for a long time, transdifferentiate into neurons or neurogliocytes and even significantly improve neural function. However, report on the application of MSCs inhypoxic-ischemic induced diffuse neuronal degeneration disease is rare. Furthermore, the neural protection effect of rodents derived MSCs have been extensively conformed, but there is few research about the biological behavior of human derived MSCs (hMSCs) in vivo. Because of the unique immunological characteristics of MSCs, heterogeneic MSCs were transplanted intracerebrally into the rats with perfect immunological function, in a few of researches. Consequently, grafted heterogeneic MSCs could also survive for long-term, without significant immunological rejection. Thus, in this study, we decided to investigate without immunodepressant, whether hMSCs after being intracerebrally xenotransplanted into hypoxic-ischemic brain damage (HIBD) rats with perfect immunological function could survive, migrate, transdifferentiate and have therapentic benefit.Experimental animals were randomly divided into four groups: hMSCs-treated group (n=18), control group (n=18), HIBD only group (n=6) and sham-operated group (n=6). In the first three groups, HIBD animal models were built in one month old Wistar rats according to the method described by Rice. hMSCs were isolated and purified by density gradient centrifugation and their adherence to culture flask. hMSCs were expanded passage by passage, and passage 3-5 cells were prelabeled with pyrimidine analogue: 5-bromodeoxyuridine (BrdU) for 72h, then they were digested and collected. The concentration was adjusted to 5><104 cells/ul, and about 5><105 hMSCs inlOul were injected slowly into the left dorsal hippocampus of HIBD rats in hMSCs-treated group. In control group, HIBD rats received PBS of the same volume, hi HIBD only group and sham-operated group, rats did not receive any transplantation.Rats in hMSCs-treated group and control group were allowed to survive for 0 day, 3 days, 1 weeks, 2 weeks and 4 weeks. Rats in sham-operated group and HIBD only group were allowed to survive for 4 weeks. Rats of the four groups received behavior test (alternative electro-stimulus Y-maze) 4 weeks after transplantation before sacrificed, which show that the space memory capacity of rats in hMSCs-treated group is significantly better than that of rats in control group, however, between hMSCs-treated group and sham-operated group or between control groupand HIBD only group, there is no significant difference. After rats were sacrificed, the FFPE brain tissue sections were prepared for HE staining. Pathological changes in control group and HIBD only group are similar: bulks of neural cells necrosis and neuropil cavitations formation in cortex and hippocampus. The normal architecture of the CA1 area of hippocampus even the whole hippocampus disappear, and lots of gliocytes proliferate. In hMSCs-treated group and sham-operated group rat brains, there are also some necrosis neural cells, but the architecture keeps normal, and there is no cavitation. FFPE brain tissues from all rats were prepared for IHC staining of BrdU, Nestin, neurofilament (NF) and glial fibrillary acidic protein (GFAP). Results show that: one week after transplantation hMSCs migrate mainly along the ventricular system, a few of hMSCs migrate along the corpus callosum to the opposite side. Four weeks later, hMSCs migrate to the parenchyma and distribute throughout the cerebra. Three days after transplantation, we could detect that some hMSCs express GFAP in the local area of transplantation and a few of cells express NF near blood vessel. We do not detected Nestin positive cells at every time point. Four weeks after transplantation, BrdU and GFAP co-express cells increased, which mainly distribute in the local area of transplantation, the cortex, hippocampus and ependymal layer. In summary, several conclusions are reached:1. We succeeded in building HIBD model of childhood Wistar rats with diffuse lesion, especially in the cortex and hippocampus of the ligature side, according to the method described by Rice.2. hMSCs could survive at least for 4 weeks in xenogenic (rat) brains with pefect immunological function, without immunodepressant , and do not result in graft related death in rats.3. After transplanted intracerebrally, hMSCs could migrate to the lesion area.4. Under the microenvironment of hypoxic-ischemic induced brain damage, hMSCs could differentiate into mature neurons and neurogliocytes.5. Four weeks after transplantation, hMSCs could significantly reduce HIBD induced neurological defects. |
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