| Objectives:Brachial plexus root avulsion is a severe peripheral nerve injury that causes physical andpsychological disability. At present, with advanced microsurgery techniques, nerveanastomosis, nerve grafting and so on are available surgical options for treating this devastatinginjury. However, death of a major neuronal pool and complex interventions are usuallyassociated with poor results. Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug,has been found exerting neuroprotection. It has been reported to enhance neurite outgrowth andto protect neurons from death in vitro. But the potential neuroprotective actions of VPA,especially its neuroprotection of the spinal cord following brachial plexus root avulsion, havenot yet well studied. After brachial plexus root avulsion, we harvest spinal cord (C5-T1) forWestern-blot, immunohistochemistry, real-time quantitative PCR, Nissl staining and TUNELanalysis to study the neuroprotection role of valproic against the brachial plexus root avulsioninduced motoneurons death. We hope the results can serve as a theoretical underpinning for thetreatment of brachial plexus root avulsion with VPA.Methods:Two hundred and eighty eight Wistar rats were randomly divided into three groups: shamoperation, control group (rats have brachial plexus root avulsion), VPA treated group (rats havebrachial plexus root avulsion and were orally administrated daily with VPA dissolved indrinking water at300mg/kg). The rats were sacrificed at day1,2,3,7,14and28after surgeryfor harvesting C5-T1tissue samples. We used Nissl staining and TUNEL to analyze thepercentage of surviving motoneurons and apoptotic cells, and transmission electron microscopewas used to observe the ultrastructure in motoneurons and gliacytes. We usedimmunohistochemistry to observe distribution rule of c-Jun and Bcl-2in neuron, real-timequantitative PCR and Western blot to measure the mRNA and protein quantity of c-Jun andBcl-2. Results:1. Dramatic decrease of motoneurons in the spinal cord was resulted by brachial plexus rootavulsion, the percentage of surviving motoneurons was significantly increased at day14(p<0.05) and28(p<0.01).2. At day1after avulsion, no TUNEL stained motoneurons were observed. TUNEL positive(apoptotic) motoneurons appeared at day2, followed by a gradual change until day28. Thepercentage of apoptotic motoneurons in VPA group was significantly decreased at day3(p<0.01) and7(p<0.01) as compared to control group.3. Ultrathin sections were cut and collected on copper grids for counterstaining with uranylacetate and lead citrate. The cavitates were formed and organelles were disappeared in thecytoplasm or nuclear matrix of motoneurons on the injured side of control group after avulsion.In VPA group, however, a number of mitochondria, rough endoplasmic reticulums, andribosomes, as well as a regular contour of the nuclear membrane were observable. Afteravulsion, the cytoplasm and nuclei in abnormal gliacytes on the injured side of control groupwas electron dense with a hypertrophied and sub-marginalized nucleolus. In contrast, thecytoplasm and nuclei of gliacytes on the injured side of VPA group had a normal appearancewith more rough endoplasmic reticulum and ribosome.4. c-Jun expressing in motoneurons:(1) c-Jun expressed in the nucleus.(2) Compared withsham operation, the percentage of c-Jun positive motoneurons was increased. At day1(p<0.05), day2(p<0.01), day3(p<0.05), and day7(p<0.05) after avulsion, the percentage ofc-Jun positive motoneurons in VPA group was relatively less than that in control group. thequantity of c-Jun in control group was increased from day1, with a peak at day3, and thendescended sharply at day14after which the values had a plateau. The quantity of c-Jun proteinwas significantly decreased in VPA group than in control group at day1(p<0.05), day2(p<0.05), day3(p<0.01) and day7(p<0.05).(3) Compated with sham operation, the quantityof c-Jun mRNA in control group was increased from day1, with peak at day3, and thendescended sharply at day14, after which the values had a plateau. the quantity of c-JunmRNA was significantly decreased in VPA group than in control group at day1(p<0.05), day2(p<0.05), day3(p<0.01) and day7(p<0.05).5. Bcl-2expressing in motoneurons:(1) Bcl-2expressed in the cytoplasm of motoneurons. (2) Compared with sham operation, the percentage of c-Jun and Bcl-2positive motoneuronswas increased. The percentage of Bcl-2positive motoneurons was significantly larger in VPAgroup than in control group at each time point except at day28(day1p<0.05, day2p<0.01,day3p<0.01, day7p<0.01, day14p<0.05). The quantity of Bcl-2in VPA group was increasedfrom day1, with a peak at day7, and then descended sharply at day14, after which the valuesalso had a plateau. The quantity of Bcl-2protein was significantly increased in VPA group thanin control group at day2(p<0.01), day3(p<0.05) and day7(p<0.05).(3) Compated withsham operation, the quantity of Bcl-2mRNA in VPA group was increased from day1to day3with peak at day7, and then descended sharply at day14, after which the values also had aplateau. The quantity of Bcl-2mRNA was significantly increased in VPA group than in controlgroup at day2(p<0.05), day3(p<0.05) and day7(p<0.05).Conclusions:1. The improved animal model of brachial plexus root avulsion has fewer side effects, andhas the same mechanism of avulsion in practice.2. The results showed that brachial plexus root avulsion induced dramatic death ofmotoneuronts, and with VPA administration, the survival of motoneurons was promoted andthe cell apoptosis was inhibited.3. The present study reported that brachial plexus root avulsion decreasing c-Jun expressionand increasing Bcl-2expression. Motoneurons were protected by VPA against brachial plexusroot avulsion induced death through both decreasing c-Jun expression and increasing Bcl-2expression directly or indirectly. |
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