Experimental Study Of The Expression And Effects Of Erythropoietin And Its Receptor In Spinal Cord And Peripheral Nerve System In Rat

Part one: Expression of EPO and its receptors in the spinal cord,dorsal root ganglia and sciatic nerve and their changes after injury of rat sciatic nervesObjective To investigate the expression and the pattern of EPO and its receptors (EPOR) in the spinal cord, the dorsal root ganglia and the sciatic nerve after sciatic nerves crushed injury in rats.Methods Thirty-six male adult Sprague-Dawley rats were divided randomly into normal group,control group and experimental group. The rats of experimental group were received crushed to bilateral sciatic nerves and L4～L6 spinal cords , dorsal root ganglias and sciatic nerves were harvested respectively at 1d,7d,14d and 28d after operation. Immunohistochemical method and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) for erythropoietin and erythropoietin receptor and image analysis were used to observe the expression of EPO and EPOR.Results EPO and EPOR were present in uninjured and injured sciatic nerves. They were also expressed in normal dorsal root ganglias. After crush injury, EPO expression is increased and EPOR is decreased.EPO immunostaining and RT-PCR were unoberserved in motor neurons of spinal cord in normal group and experimental group,however, EPOR were noted in motor neurons of spinal cord in normal group and after sciatic nerves crush injury.Conclusion The results reveal the favorable effects of EPO on nerve regeneration from the angles of central neurons, which establishes the experimental and theoretical foundation in the further treating peripheral nerve injuries with EPO.Part two: Effect of target muscle injection of recombinant human erythropoietin on peripheral nerve regeneration in ratsObjective To investigate the effect of target muscle injection of recombinant human erythropoietin(rh-EPO)on the nerve regeneration of adult rats sciatic nerves. Methods 12 healthy male Sprague-Dawley rats were involved and right sciatic nerve crushed model was used.The experimental rats were divided randomly into two groups: the EPO group and the control group, 6 rats in each group. Rh-EPO 2500U/kg was injected daily into the gastrocnemius in EPO group , and normal saline was injected into the gastrocnemius every day after operation in control group. At days 7,14 and 21 after operation, the sciatic function index (SFI) was determined by walking tract analysis. At 21 days after operation,the muscle wet weights were measured,and the spinal cord segments (L4～L6 ),the distal site of injured sciatic nerve and the injured gastrocnemius muscle were observed by light microscope.The count of motor neurons of anterior horn , the number of myelinated fibers , the cross section areas of gastrocnemius muscle cells and other parameters were determined.Results At day 7 after operation , SFI scores had no significant difference between two groups;at day 14 and 21 after operation ,SFI score of EPO group was significantly higher than that of the control group(P<0.01). At day 21 after operation,the gastrocnemius muscle wet weights in the EPO group and the control group were 554.27±51.96(mg) and 451.67±66.79(mg) respectively, with significant difference between the EPO group and the control group(P<0.05). The numbers of motor neurons of anterior horn, myelinated nerve fibers,diameter of axon,thick of myelin sheath and the cross section areas of gastrocnemius muscle cells in EPO group were significantly superior to those in control group(P< 0.05 ,P<0.01).Conclusion Target muscle injected rh-EPO can promote the injured nerve regeneration and improve the recovery of their function.Part three: The effect of exogenous erythropoietin on the denervated muscle atrophy Objective To study the effect of exogenous recombinant human EPO (rh-EPO)on the denervated muscular atrophy.Methods 24 male Sprague-Dawley rats were involved. Model of denervated gastrocnemius muscle was set up after sciatic nerves were transected at the right lower limb. The rats were randomly divided into two groups: the EPO group and the control group. Rh-EPO 2500U/kg was injected daily into the denervated gastrocnemius in EPO group , and normal saline was injected into the denervated gastrocnemius in control group.At 2 and 4 weeks after operation, the muscle wet weight, the muscle cell diameter and cross section areas, the protein amount, the percentage of the apoptotic muscle cells and Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities were observed.Results At 2 and 4 weeks after operation, the muscle wet weight, Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities,the muscle cell diameter and cross section areas and the protein amount were significantly greater in EPO group than those in control group, however, the percentage of the apoptotic muscle cells was significantly smaller in EPO group than that in control group .Conclusions Exogenous erythropoietin can delay the denervated muscle atrophy.