Correlation Of EPCs With Repair And Regeneration Of Injuried Tissue After Traumatic Brain Injury In Rat

Background:In 1997, Asahara et al reported for the first time about endothelial progenitor cells (EPCs) that differentiated from participated in vasculogenesis in the animal hindlimb ischemic model. Circulating blood contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells in vitro and in vivo. These cells have been termed endothelial progenitor cells (EPCs). The isolation of EPCs by adherence culture or magnetic microbeads has been described. CD34+-enriched mononuclear cells in peripheral blood include a hematopoietic stem cell population, and were shown to be incorporated into neovascularization. This finding, that circulating EPCs may home to sites of neovascularization and differentiate into endothelial cells in situ, is consistent with "vasculogenesis," a critical paradigm for embryonic neovascularization, and suggests that vasculogenesis and angiogenesis may constitute complementary mechanisms for postnatal neovascularization. There has been extensive research on how CD34+ cells or specifically EPCs contribute to the process of tissue repair in ischemic injury such as myocardial infarction and stroke. Previous reports demonstrating therapeutic potential of EPC transplantation in animal models of hindlimb and myocardial ischemia opened the way to the clinical application of cell therapy.Objective1. To study on the isolation, culture and identification of EPCs from rat peripheral blood, and provided basic research for neurologic research.2. The study was designed to test the hypothesis that CD34+ cells correlate with posttraumatic angiogenesis and tissue repair.3. To test the hypothesis that angiogenesis correlate with posttraumatic neurogenesis and tissue repair.Methods1.The mononuclear cells were isolated from rat peripheral blood by Ficoll density gradient centrifugation and were cultured in vitro in a special medium(EGM-2). The expression of CD133,CD34,vWF were assessed by immunofluorescent staining and flow cytometer. The biological functions of endothelial cells were examined by the adsorption of ulex-agglutinin (UEA) labeled by fluorescein isothiacyanate (FITC) and Dil-Ac-LDL internalization on 7th day.2. The rats were randomly assigned to control and TBI group (49 rats in each group), each of which further divided into 7 subgroups (7 rats in each subgroup). Fluid percussion injury was performed over the right parietal lobe in TBI groups. Blood samples (1 ml) were collected from retro-orbital venous plexus at baseline,24,48,72, 120, and 168 hrs after TBI from only one subgroup from TBI groups and control group. CD34+ cells in peripheral blood were evaluated by flow cytometry. Rats in control and TBI groups were sacrificed before and 1st,3rd,7th,14th, and 21st days after TBI (7 rats in each time point). brains were collected and processed for 5 mm coronal paraffin sections through the TBI zone and used for immunohistochemistry staining to access changing of the CD34+ cells in brain tissue, further to test the hypothesis that CD34+ cells correlate with posttraumatic angiogenesis and tissue repair.3.42 Adult male Wistar rats were divided into two groups:(1) control group (n=21); (2) TBI group (n=21), Brdu, Brdu/Neun were measured by immunohistochemistry on frozen sections and paraffin sections 3rd,7th,14th days after TBI. CD34 and CD31 were measured by immunohistochemistry on paraffin sections 0,1st,3rd,7th,14th, and 21st days after TBI in the second section. Further to test the hypothesis that angiogenesis correlate with posttraumatic neurogenesis and tissue repair.Results1. The cells attached after 2 days, exhibited the clone-like morphology after 5d cultivation and proliferated faster then. Immunofluorescent staining showed that the adherent cells were positive for CD133,CD34 and vWF. The cells could take up DiI-acLDL and bind to FITC-UEA-1, and showed double-positive fluorescence. The CD34 and CD133 double-positive ratio of cultured cells were 36.5%, and the CD34 positive ratio were 85.4% by flow cytometer analyzing.2. The number of circulating CD34+ cells and CD34 and CD133 double-positive cells (per 1×104 mononuclear cells) increased 24 hrs in rats being exposed to TBI and those underwent only surgery. The number of CD34+ cells in the injured tissue and ipsilateral hippocampus was greater than that in control group. In the TBI group, CD34+ cells increased rapidly, reaching plateau at about 1 week after TBI and then declined, whereas MVD showed a steady increase during the same period.3. The number of CD34+ cells and MVD in the injured tissue and ipsilateral hippocampus was greater than that in control group. The number of BrdU+ cells and BrdU/NeuN+ cells was greater than that in control group.Conclusion1. Relatively purified EPCs can be obtained by certain procedure of isolation and culture from rat peripheral blood, which provides the basic for clinical application of EPCs transplantations.2. CD34+ cells correlate with posttraumatic angiogenesis and tissue repair. Our data strongly indicate that angiogenesis in the TBI zone originate as the result of homing, migration, proliferation and differentiation of CD34+ cells. Circulating CD34+ cells may play an important role in regeneration of brain injury. Mobilized CD34+ cells eventually contribute to the formation of new vessel in the injured tissue, a critical event for neural recovery after traumatic injury.3. Angiogenesis correlate with posttraumatic neurogenesis and tissue repair. Angiogenesis and neurogenesis take part in the regeneration of TBI brain together.