| Objective: Neonatal hypoxic-ischemic encephalopathy (HIE) do harm to the lives and health of newborn infants severely. It is the important etiology that leads to disability of children in our country. Mesenchymal stem cells (MSCs) could secrete a series of growth factors and neurotrophic factors. As well MSCs have the potential ability to differentiate to the neural cells in vitro and in vivo. Therefore we expect to transplant and directly induce MSCs to differentiate into neurons in order to cure HIE. We could take advantage of these traits to promote recovery of brain function and reduce the sequelae. Studies have shown that MSCs could enter the cerebral parenchyma and differentiate to neural cells through systemic infusion, but most of the researches aim at the adult stroke animal models. This study would use neonatal HIE models to test the hypothesis that MSCs could enter the brain of newborn Wistar rats through their blood--brain barrier (BBB) by intraperitoneal infusion, and observe the characteristics of the distribution and differentiation of MSCs in brain tissues, and then explore the effects of hypoxic-ischemic brain damage to the penetration and differentiation of MSCs.Methods: Isolation and purification of MSCs were established from the whole bone marrow of juvenile Wistar rats by removing the nonadher-ent cells in primary and passage cultures. For cellular identification, MSCs of three to five passages were continuously prelabeled with 5-bromo-2--deoxyuridine (BrdU) for 72 hours before transplantation. Animal models of HIE were built in 7-day-postnatal Wistar rats according to Rice method. Two hours after hypoxia-ischemia, rats with HIE group (n=8) were intrape-ritoneally infused MSCs (4 X 106, 0.5 ml). In control group (n=8), 7-day--postnatal normal Wistar rats were intraperitoneally infused with the same amount of MSCs. In 14 days after transplantation all rats were sacrificed and their cerebra were sectioned by cryomicrotome. Immunohistochemical staining with chromogen diaminobenzidine (DAB) was used to detect and measure the cells derived from MSCs, and study the characters of distribution. To determine the differentiation of the BrdU positive cells entering the brains, Immunofluorescence double labeling for BrdU and neural cells specific antigens was performed.Results: MSCs were distributed throughout the cerebra in both groupsat the 14th day after transplantation. The number of MSCs detected was 2415+226 in the control group, and 3626+461 in HIE group. There was a significant difference (t=6.68, P<0.05) . More BrdU reactive cells were observed in the right ischemic hemisphere (1904 + 267) than in the contralateral hemisphere (1723 + 204), (t=4.47, P<0.05). No significant difference was proved while comparing both cerebral hemispheres of the control group (t=0.31, P>0.05) . In the HIE group, MSCs distributed more extensively, and some focus aggregations of MSCs were found. A few MSCs expressed Nestin-protein marker of neural progenitor cells, and almost none of the MSCs which expressed proteins characteristic of neuron (e.g. NSE) and astrocyte (e.g. GFAP) was detected at the 14th day after transplantation.Conclusions: 1. MSCs can enter the cerebral parenchyma through BBB and migrate throughout the brain by intraperitoneal infusion. 2. Hypoxic-ischemic brain damage could improve MSCs entrance and migration. 3. Transplanted MSCs could not differentiate to adult neural cells without other interventions during 14 days after transplantation.
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