The Research Of Human Umbilical Cord Blood Mononuclear Cells,Umbilical Cord Mesenchymal Stem Cells Transplantation For Hypoxic-ischemic Brain Injury In Neonatal Rat Model

ObjectiveThe aim of this study was to assess whether hUCB,hUCMSCs or the combination of hUCB and hUCMSCs could effectively migrate to the neonatal rat HI brain and differentiate into neural cells intraperitoneally or intravenously, and the effect of the grafts on motor performance and brain morphologic changes. And we determined which is the better route of administration to cure hypoxic-ischemic brain injury in neonatal rat model, intraperitoneally or intravenously. What's more, we determined which is the best cell type to cure hypoxic-ischemic brain injury in neonatal rat model, the hUCB, the hUCMSCs or the combination of hUCB and hUCMSCs.Methods1. The Levine model, modified by Rice et al. was used to achieve reproducible hypoxic-ischemic injury in neonatal rats. Seven-day-old Sprague-Dawley rat pups were deeply anesthetized and the right common carotid artery double-ligated and severed. To introduce systemic hypoxia, the pups were subsequently placed in a hypoxia chamber with a constant flow of 02 (8%) and N2 (92%) partially immersed in a water bath at 37℃for 3 h..3 weeks later, we do the body weight analysis, behavioral tests and morphologic study to judge the model.2. At day 7 after HI injury,8 randomly selected HI-injured animals received 100uL suspension of 1×106 hUCB via an intraperitoneal injection. Another group of 8 randomly selected HI-injured animals received intravenous infusion (via the jugular vein) of 1x106 viable hUCB suspended in 10μL PBS using a micropipette with ultrafine tip.2 weeks after cell transplantation, body weight analysis, behavioral tests including the footprint analysis and the elevated body swing test (EBST), morphologic study and Immunofluorescent staining were performed in a strictly blinded fashion.3. At day 7 after HI injury, in order to determined which is the best cell type, the hUCB cells, the hUCMSCs or the combination of hUCB and hUCMSCs, we assigned another two study groups according to the initial results:HI- hUCMSCs- intravenous group and HI-Combine-intravenous group. In the HI- hUCMSCs- intravenous group, randomly selected 8 HI-injured animals received intravenous infusion of 1x106 viable hUCMSCs suspended in 10μL PBS. And in the HI-Combine-intravenous group, randomly selected 8 HI-injured animals received 10uL suspension of 1x106 the mix of hUCB and hUCMSCs via an intravenous injection.2 weeks after cell transplantation, body weight analysis, behavioral tests including the footprint analysis and the elevated body swing test (EBST), morphologic study and Immunofluorescent staining were performed in a strictly blinded fashion.Results1. The mean body weight of that HI-vehicle group was significantly lower than that of the sham group; the digit distance 1 to 4 and the step length of of the left forepaw (contralateral to the lesion) revealed a highly significant reduction compared with the sham group; Hyposic-ischemic brain damage resulted in significantly motor asymmetry; the lesioned hemisphere became atrophic after HI, and its area was decreased2. Intraperitoneal (IP) or Intravenous (IV) injection of hUCB significant improved the body weight at 2 weeks after injection. The mean digit distance and the mean step length of the left forepaw was longer in the transplanted HI-injured groups than in the HI+Vehicle group. And transplanted HI-injured animals exhibited significantly reduced motor asymmetry. The area ratio of the right hemispheric was restored significantly. What's more, there was difference between intraperitoneally (IP) and intravenously (IV) hUCB-transplanted animals in the area ratio of the right hemispheric analysis. Intravenously hUCB-transplanted animals restored better than those intraperitoneally. 2 weeks after injection, we were able to detect a few grafts in the brains of HI-injured animals, delivered via either the intraperitoneal or the intravenous route. Of note, trasplanted cells colabeled with the astrocyte marker GFAP and the nuclei marker DAPI in rats receiving cells.3. Intravenous (Ⅳ) injecton of hUCB or hUCMSCs or the combination of hUCB and hUCMSC significantly improved the body weight at 2 weeks after injection. The mean digit distance and the mean step length of the left forepaw was more in the transplanted HI-injured groups. Transplanted HI-injured animals exhibited significantly reduced motor asymmetry, and the area ratio of the right hemispheric was restored significantly too. What's more, there was significant difference between hUCMSCs-transplanted animals and the combinition-transplanted animals in motor asymmetry analysis and the area ratio of the right hemispheric analysis. The combinition-transplanted animals restored better than those hUCMSCs-transplanted animals or hUCB-transplanted animals.2 weeks after injection, we were also able to detect a few grafts in the brains of HI-injured animals. Of note, trasplanted cells colabeled with the astrocyte marker GFAP and the nuclei marker DAPI in rats receiving cells.ConclusionIt was a stable and reliable method to construct cerebral palsy models in neonatal rats using unilateral common carotid artery ligation and hypoxia. hUCB and hUCMSCs could be candidates for tne treatment of cerebral palsy models in neonatal rats. Intraperitoneal (IP) injection was better than Intravenous (Ⅳ) injection to cure hypoxic-ischemic brain injury in neonatal rat model.Injecton of the combination of hUCB and hUCMSC was better than injecton of hUCB or hUCMSC alone to cure hypoxic-ischemic brain injury in neonatal rat model.