RhEPO Enhances The Mobilization Of EPCs And Improves The Neurofunctional Recovery In TBI Rats

Objective:To observe the effect of different doses of recombinant human erythropoietin (rhEPO) on the biological characters of the in vitro cultured endothelial progenitor cells (EPCs) from the rats bone marrow, the effect of rhEPO on the mobilization of their EPCs from bone marrow and the repairing of the neuro-angiogenesis in the injured brain tissue in traumatic brain injury (TBI) rats and investigate the effect of rhEPO on the neurol functional and spatial memeory recovery in TBI rats.Methods:Bone marrow was obtained from both femurs and tibials of Wistar rats, and then the mononuclear cells were isolated by density gradient centrifugation, which were cultured in vitro and treated with a serial of doses of rhEPO (0,4,8 U/mL).7 days after culture, the adherent cells were identified as the EPCs by double staining with FITC-UEA-1 and Dil-Ac-LDL, and then doublely stained with CD133,VEGFR-2 and CD34. Additionally, the functions of EPCs were evaluated using adhesion test, migration assay and proliferation experiment. In vivo studies,30 male Wistar rats were randomly devided into sham-operated group, traumatic brain injury with saline-treated group (TBI+Saline Group) and TBI with recombinant human erythropoietin group (TBI+rhEPO Group). Each rat in the TBI+rhEPO group received rhEPO intraperitonieally injection just after TBI at a dose of 5000U/kg/day for 7 consecutive days. While the rat in the RBI+saline group only receive the saline injection with the eaual volumne. Then lml of blood was collected in EDTA tubes from retro-orbital venous plexus by glass capillary before and 1,4,7,14,25 day after TBI, then EPCs levels were measured by flow cytometry labeled with CD34 and CD133. At the same time, the mNSS was used to evaluate the functional recovery induced by rhEPO at 1,4,7,14,21,25 day after TBI, and morris water maze was used for investigating the memory recovery at 21-25 days post-TBI. The neo-vascular endothelial marker CD34 in brain was measured with immunohistochemistcal staining at 1,4,7,14,25 days after TBI in another three groups of 51 wistar rats. A correlationship analysis were performed to investigate the relationship between the circulating EPCs level and the CD34+ staining level in the injured brain tissue and between the CD34+ staining level of the injured brain tissue and the spatial memory improvement degree of the TBI rats.Results:The attached cells became spindled like and clonal morphology after 3 days culture in vitro, forming a tube-like structure at 7th day and presenting as a typical cobblestone appearance with cell conjugation at 14th day. The immuno-fluorescence staining confirmed that the EPCs were double-staining positive with both FITC-UEA-1 and Dil-Ac-LDL and also double-positive either with both of CD133 and CD34 or with both of CD34 and VEGFR-2. The rhEPO was demonstrated to improve capacities of adhesion, migration and proliferative in a dose-dependent manner. In vivo studies showed that the circulating EPCs level decreased at 3 hours at first and peaked at 6 hours(P<0.05), which then decreased gradually to the normal levle 24 hours later after TBI. While the circulating EPCs from the rhEPO-treated rats increased gradually and reaching a plateau around 7th day after rhEPO treatment, which was significantly higher than the one from saline-treated rats (P<0.05). At the same time, the CD34+ cells in the injury brain tissues and DG areas were observed to increase steadily in both saline-treated and rhEPO-treated TBI rats when compared with the one from the sham-injured rats(p<0.05), with the most significant CD34+ cells detected in brain tissues of the rhEPO-treated TBI rats (P<0.05), and there were strong positive correlation between the changes of circulating EPCs and the changes of CD34+ cells of boundary zone and dentate gyrus in the rhEPO-treated rats. The spatial learning ability was significantly improved in rhEPO-treated rats at the 24 and 25 day after TBI (P<0.05), compared with the saline-treated rats, and there were strong positive correlation between the changes of time spent in the target zone and the changes of CD34+ cells of boundary zone and dentate gyrus in the rhEPO-treated rats The mNSS was performed to assay the neuro-function status of the TBI rats to reveal that the rhEPO-treated rats scored higher than the saline-treated ones at 14,21 and 25 days after TBI(P<0.05).Conclusion:rhEPO significantly enhance the adhesion, migration and proliferation capacities of EPCs in vitro in dose-dependent manner and mobilze EPCs from bone marrow to circulation in TBI rats. The administration of rhEPO induces significant angiogenesis, which is enhanced after TBI in the injured brain,resulting in neuro-function improving and spatial memory ability recovery in TBI rats. The degree of the neurofunction improvement is positive related to the angiogenesis power. Thus a new strategy to treat TBI patients is rationally indicated.