The Therapy Of Intraventricular Erythropoietin-Infusion On Traumatic Brain Injury In Rats

Objective Traumatic brain injury (TBI) has become the main form of acquired brain injury in youth.There is still lack of effective intervention means for post-traumatic neurological and cognitive dysfunction. The neuroprotective effects of exogenous EPO in various diseases of central nervous system were confirmed in experimental animal models. However, EPO’s mechanism of action is not fully clarified. Moreover as a hematopoietic stimulating hormone, EPO hematological side effects often limit the clinical application of EPO. Recent studies have shown that erythropoietin (EPO) and erythropoietin receptor (EPOR) is widespread in central nervous tissues. Exogenous EPO in the central nervous system play a neuroprotective role. However, the molecular mechanism of EPO effect is still not clear. CD133positive cells as a surface glycoprotein has an important role for stem and progenitor cell chemotactic migration and may plays an important role in the process of posttraumatic nerve repair. This experiment was designed to explore the drug’s effectiveness of EPO intracranial injection, the effect of EPO intraventricular infusion on CD133positive cells in wound tissue following TBI, and the mechanism of action of EPO in the treatment of TBI.Study1:Intraventricular erythropoietin-infusion improved sensorimotor functional outcome and spatial learning.Method For the detection of effects of EPO intraventricular injection on post-traumatic angiogenesis and neurological,40adult male Wistar rats was used, the rat fluid percussion brain injury model was established, and the lateral ventricle micro osmotic pump was implanted24hours after trauma. The animals were randomised into one of the following treatment groups:Sham group, TBI group, EPO intraventricular infusion, saline intraventricular infusion, EPO intraperitoneal injection, saline intraperitoneal injection. The vascular density in rat injured area was detected using immunohistochemical method35days after trauma. Brdu positive cells were detected using immunofluorescent technique in rat brain tissue. The hemoglobin concentration and hematocrit of intraventricular administration group and intraperitoneal group were detected in rat. At35days after trauma motor and cognitive function in rats after brain trauma were detected using the Morris water maze and improved neurological score.Results1The score values of mNSS in EPO intraventricular perfusion group were significantly reduced compared with TBI group and TBI+saline intraventricular perfusion group4to35days after trauma (P<0.05). The error probability by foot fault EPO in intraventricular perfusion group was decreased compared with TBI group and TBI+saline intraventricular perfusion group7to35days after trauma (P <0.05).2Morris water maze test showed that escape latencies in EPO intraventricular perfusion group were significantly lowered compared with TBI group32to35days after trauma (P<0.05).3The cell count of rat hippocampal dentate gyrus and CA3region in TBI+EPO intraventricular infusion group was higher than TBI group35days after brain trauma (P<0.05). The lesioned ipsilateral hippocampal and cortical Brdu positive cells in TBI+EPO intraventricular perfusion rats was significantly higher than that of group TBI35days after brain trauma (P<0.05).4The lesioned ipsilateral hippocampal and cortical vWF positive cells in TBI+EPO intraventricular perfusion rats was significantly higher than that of group TBI35days after brain trauma (P<0.05).5Compared with TBI group and TBI+saline intraventricular perfusion group, hemoglobin concentration in EPO intraventricular perfusion group increased slightly but not statistically significant4to7days after trauma (P>0.05), while in EPO intraperitoneal injection group, the hemoglobin concentration and hematocrit increased significantly4,7,14and21days after trauma (P<0.05).Conclusion EPO intraventricular perfusion can increase cell proliferation in rats after brain trauma, increased cortical and hippocampal microvessel density in wound area, promoted tissue repair, improved the recovery of movement, sensory and cognitive dysfunction.Study2:Intraventricular erythropoietin-infusion increased CD133positive cells in injured brain tissues.Method In order to explore the influence of EPO intraventricular injection on CD133antigen, VEGF and SDF-1expression in traumatic zone,55adult male Wistar rats were used in the experimental study, the rat fluid percussion brain injury model was established, and the lateral ventricle micro osmotic pump was implanted24hours after trauma. The randomized grouping design is the same as study1. CD133positive cells in injured area in rat were observed using immunohistochemical detection35days after trauma. The immunofluorescence double staining technique was used for detecting the colocalizations of CD133with EPOR, CD133with VEGF, and CD133SDF-1expression. The enzyme-linked immunosorbent assay (ELISA) was used to detect VEGF and SDF-1protein concentration in rat brain post trauma. The RT-PCR analysis was used to detect VEGF and SDF-1gene transcription level in traumatic brain tissue.Results1CD133+cell counts in hippocampal dentate gyrus, CA3and cerebral cortex area in TBI+EPO intraventricular perfusion group were higher than group TBI35days after trauma (P<0.05).2Immunofluorescence double staining experiments show that CD133+cells in the wound area can express EPO receptors, VEGF and SDF-1.3At35days after brain trauma, hippocampal and cortical VEGF and SDF-1concentration in lesioned side in TBI+EPO intraventricular perfusion rats was significantly higher than that in TBI group (P<0.05); VEGF and SDF-1concentration in lesioned ipsilateral hippocampus and cortex TBI+EPO in intraventricular perfusion rats was significantly higher than that of TBI+EPO intraperitoneal injection group (P<0.05). RT-PCR results showed that35days after trauma lesioned ipsilateral hippocampal and cortical VEGF and SDF transcription level in TBI+EPO intraventricular perfusion rats was significantly higher than that of group TBI (P<0.05); TBI+EPO intraventricular perfusion rats lesioned ipsilateral hippocampal and cortical VEGF and SDF transcription level was significantly higher in TBI+EPO intraperitoneal injection group (P<0.05). Conclusion EPO intraventricular perfusion can increase CD133+cells infiltration in the wound area of cerebral cortex and increased VEGF and SDF-1concentration in brain tissue following TBI.