Valproic Acid Protects Neuron Against Death And Promotes The Neuronal Regeneration After Brachial Plexus Avulsion In Rats

Brachial plexus root avulsion is the most serious type of peripheral nerve injury. It isencountered commonly in victims of traffic accident and children after obstetriccomplications, leads to totally paralysis of the corresponding muscle groups and severesensory deficits in the arm on the lesion side. Compared with other types of nerve damage（such as clamping, cutting, etc.）, root avulsion cause a large number of neuronal death as thedamage site is closer to the neuronal cell bodies in the spinal cord. After peripheral nerveinjury, the survival of motoneurons is an essential prerequisite of nerve regeneration andfunction recovery. The loss of neurons may significantly restrict the achievement of themuscle reinnervation after a surgical repair of the ventral root connection. Therefore, aneffective agent to reduce the neuronal death and promote the survival neuron to regeneratewill be very important and valuable for brachial plexus root avulsion. An increasing numberof neurotrophic factors have been found to support the motoneuron survival and enhanceaxonal regrowth in a variety of experimental paradigms.However, as the neurotrophinsbelong to peptide, their benefits are largely limited by the poor ability to cross theblood-nerve and blood-brain barrier and by enzymatic degradation. Besides, a long time andfrequent administration with high-dose neurotrophic factor may have unexpected side effects.There is still a long way to go before the neurotrophin can be effectively used in clinicalpatients.The discovery of the neuroprotective effects of valproic acid brings hope to this problem.Since VPA is an inorganic compound with small molecules allowing easily penetrating theblood-brain barrier, and is not easily degraded by enzymes, it has a great advantage relative toneurotrophic factors. The security of VPA has been widely validated because it has been usedfor antiepileptic therapy clinically for more than30years, and approved by the FDA in1995for antidepressant. Clinical studies have found that VPA can protect neurons against death inthe sciatic nerve transection model in rats. However, as to the brachial plexus root avulsion, which induces much more neuronal injury and death, whether the VPAcan exert its protectiveeffect on the neuron? In order to clarify this problem, we designed this study.In the present study, after the brachial plexus root avulsions of rats were made, the ratswere orally administrated with water containing VPA. First, we detected the neuronalapoptosis in the spinal cord to verify the neuronal protection role of VPA. Then we detectedthe expression of nNOS and Ca²⁺concentration in the spinal cord neurons to explore theprotective mechanism of VPA. Besides, we want to know whether VPA can enhance theneural regeneration by detecting the GAP-43expression in spinal cord.Rats were randomly divided to sham group （exposure the brachial plexus root withoutinjury）, control group （brachial plexus root avulsion） and VPAgroup （administered with VPAafter avulsion）. At different time points, the apoptosis of neurons, expression of nNOS,intracellular Ca²⁺concentration and GAP-43expression were investigated. Apoptosis ofneurons was inspected by TUNEL method. Detection of nNOS used Real-time PCR, Westernblot method and immunohistochemical staining and GAP-43was by the Real-time PCR andWestern blot method. The intracellular Ca²⁺concentration were tested by Fura-2/AM method.Results were showed as follows. In the sham group, there was no neuron apoptosis,expression of nNOS and GAP-43in spinal cord are in low level, and intracellular Ca²⁺concentration has no obvious change at all time points. In the control group, within28daysafter injury, the proportion of apoptosis neurons, the expression of nNOS in the spinal cordand intracellular Ca²⁺concentration, as well as the expression of GAP43-all appear to dropafter rising first. The process of apoptosis of neuron spikes at D7; nNOS expression waspeaked at D2; intracellular Ca²⁺concentration peak appeared at D2, and GAP-43peakappeared at D14. The four indicators in the VPA group had similar changes and peak time asin the sham group. But relative to the control group, the VPA significantly reduced thenumber of apoptotic neurons at D3, significantly lowered the expression of nNOS at D1, D2,D3, and D7, significantly reduced intracellular Ca²⁺concentration at D2, D3, and D7, andobviously up-regulated the expression of GAP-43between D2and D28.The results indicated four conclusions as follows. First, VPA is able to protect neuronsagainst cell death induced by brachial plexus root avulsion in rats. Second, VPA may exertneuronal protection through decreasing the nNOS expression. Third, VPA may down regulate the intracellular Ca²⁺concentration to protect neurons. The down regulation of nNOS may beattributed to the decrease of intracellular Ca²⁺concentration induced by VPA. Four, VPA canpromote neural regeneration by up regulate the expression of GAP-43. The conclusionsprovide theory supports for the clinical application of VPAto treat nerve injury.