Study On The Neuroprotection And Its Molecular Mechanisms Of Erythropoietin Against Brachial Plexus Root Avulsion-Induced Neurons Death In Rats

Objectives:Brachial plexus root avulsion is a severe peripheral nerve injury that causesphysical and psychological disability. However, death of a major neuronal pool andcomplex interventions are usually associated with poor results. Eythropoietin (EPO), acommon drug to increase the quantity of red blood cell, has been found exertingneuroprotection to central and peripheral nerve injury or disease. The neuroprotectiveand adverse effects of low dosage EPO for the spinal cord neuron following avulsionwill be researched. Furthermore, we hope to explore the mechanism associated withJNK/c-Jun apoptosis pathway.Methods:180Wistar rats were randomly divided into two groups: control group (avulsion+1ml normal saline s.c. on alternate days), EPO treated group (avulsion+1000U/kgEPO s.c. on alternate days). The rats were sacrificed at day0（before avulsion）,1,2,4,7and14after avulsion for harvesting C5-T1spinal samples. We used H-E, Nissl,TUNEL, and Cleaved-PARP stainings to analyze the survival and apoptotic neurons.Besides these indicators, we used immunohistochemistry, Western blot, real-timequantitative PCR to observe the p-JNK and c-Jun expression in neurons.Results:1. Dramatic decrease of neuron amount in the spinal cord was caused by avulsion;the percentage of survivial neurons was significantly decreased at7d and14d. EPOgroup has more survival neurons compared with C group.2. TUNEL stained neurons were observed. TUNEL positive (apoptotic) neuronsappeared at2d, followed by a gradual change until14d. The IOD of TUNEL in EPOgroup was significantly decreased at4d and7d as compared to control group.3. As a critical apoptotic factor, the IOD of c-PARP positive neurons in controlrats was increased from1d to3d, peaked at7d and then descended sharply at14d. The IOD of c-PARP was showed a pronounced decrease in EPO group than control ones at2d and4d.4. There are no significant differences on blood routine examination in twogroups at all time points.5. The expression of c-Jun in control group was increased from1d to7d, with apeak at2d. At day1,2,4,7after avulsion, the IOD of c-Jun in EPO group wasrelatively less than C group.6. The IOD of p-JNK in control group was increased from1d to7d, with a peakat2d, and then descended sharply at14d. At day1,2,4after avulsion, the IOD ofJNK in EPO group was relatively less than C group.7. Gradient centrifuge of spinal will enhance the quality and specificity of neuron,which will be the basis for the further research.Conclusions:1. The improved animal model of brachial plexus root avulsion is feasible andreliable, identified with the actural trauma mechanism.2. Brachial plexus root avulsion arroused serious death of neurons, and with EPOadministration, the survival rate of neurons was enhanced and the apoptosis wasalleviated.3. Brachial plexus root avulsion increase p-JNK and c-Jun expression.Down-regulation of the two proteins is the associated anti-apoptosis mechanism ofEPO.4.1000U/kg EPO sc on alternate days did not have significant erythropothesiseffect.