The Repair Effect Of Valproic Acid On Spinal Cord Injury In Rats And Related Mechanism

Spinal cord injury (SCI) is a catastrophic event that imposes an enormous medical, psychological, social, and economic impact on individuals, families and society.Multiple studies have demonstrated that there is only peripheral nerve regeneration but no regeneration of central nervous system(CNS). Damaged axons in the adult CNS are unable to sponta- neously regenerate upon injury, in contrast to neurons In the peripheral or embryonic nervous system. Such a failure in regeneration is believed to arise from both a decline in the intrinsic growth state of mature neurons,as well as the presence of a nonpermissive environment in the injured adult CNS preventing axon growth.In addition to the physical barrier formed by the astrocytic scar tissue that develops at the lesion site,Recent researches indicate that CNS regeneration occurs at appropriate condition, which depends on the intrinsic properties of central neurons and the surrounding environment. Although many ways to promote the sprouting of axons, most of the regenerated axons grow surrounding the cell or tissue transplantation are and few nerve fibers will enter the distal end through the injured region. therefore, it become more difficult to establish the connections between functional axons. Drug intervention may be an easy method if it is useful. At present, drugs including therapeutic vaccination, mythylprednisolone (MP) are used to improve the intrinsic regeneration ability of neurons, increase the cAMP level and deactivate Rho. Data have shown that there is not enough evidence indicating that MP is obviously effective for acute SCI. Application of neurotrophic factors (NTF) in local CNS of adult rats promote the survival of injured neurons and the growth of injured axons.However, as a result of the great molecular weight of NTF families, it is hard for them to go through the blood brain barrier(BBB).And its protection for neurons and growth promotion for axons are greatly limited because of the enzyme degradation. Therefore, it is very important to find a chemical drug which is capable of going through BBB and stable, nontoxic, as well as with a similar function as NTF. Valproic acid (VPA), a short-chain fatty acid, is capable of going through BBB very fast. It has been used for about 40 years as a first line medicine for epilepsy and anti-convulsive treatment clinically with an approved security and reliability. Although there are profound and extensive researches on VPA, the corresponding mechanism is still not fully illuminated. Recent studies showed that VPA had an ability of protection and nutrition as well as an anti-apoptosis effect on neurons, it could antagonize the nervous toxicity caused by lipopolysaccharide (LPS); intracellular oxidization was down-regulated by VPA; inflammatory factor level was down-regulated as well by VPA to protect the dopamine neurons and reduce the proliferation of astrocyte and gitter cells; VPA could promote the growth of nerve including growth cone to spread and preventing its collapse; it could obviously promote the growth of injured peripheral nerve, and accelerate its myelinization. What's more, VPA could promote the differentiation of neurons from cultured neural stem cells (NSCs).After their differentiation, VPA was more effective in promoting the growth of nerve than BDNF and NT3.There are few researches on the function of VPA on SCI, such as whether it can protect the neural neurons, promote the growth of axons in the remaining neurons and inhibit the proliferation of glial cells. This study aims to investigate the repair function of VPA in the rats after SCI and related mechanism, which will provide treatment strategy for SCI, as well as the preliminary exploration of the cause for respiratory system complications after SCI.Main Methods and TechniquesThe experiment was divided into four parts1. After myeloid tissue were taken from fetus rats with 13-16 days of pregnancy, NSCs were cultured in Neural basal medium (NB) in vitro and identified by immunofluorescence with Nestin staining. Culture media containing 1.0mM VPA was applied for NSCs culture when they were differentiating.After their differentiation, double-label immunofluorescence with anti-GFAP and anti-NF-200 was performed to observe the effect of VPA on the differentiation of NSCs.2. Allen's injury model of T10 spinal cord was established in Wistar rats with strike force of 25 g·cm. The experimental animals were divided into control group (group A), simple operation group (group B), group with MP treatment after injury (group C), and group with VPA treatment after injury (group D). For group C, first administration of MP (30mg/kg) was taken within half hour after injury, followed by 23 successive administration of MP (5.4mg/kg/h) for every each hour. The rats were treated with VPA(300mg/kg/d) by twice daily intraperitoneal injections for 8 weeks after injury in group C. Equal amount of normal saline was provided for the rats in B group every day as that for group D, observation of the rats lasted for 8 weeks. BBB score , electrophysiology and BDA nerve tracer technique were used to evaluate the repair function of VPA on nervous system after SCI.3. Allen's injury model was established by strike in rats, the animals were divided into control group (group A),only SCI group (group B), and VPA treatment group (group C). For every,1d,3d,1w,2w,4w, the protection of VPA on nerve cells after SCI was shown by pathological observation and TUNEL technology eletronic microscope; immunohistochemistry with Nestin staining, double-label immunofluorescence with anti-Brdu and anti-NF-200 were taken to observe the effect of VPA on endogenous NSCs; Brdu immunochemistry and double-label immunofluorescence with anti-GFAP and anti-NF-200 were taken to observe the protection of VPA on the neurons and its influence on astrocyte.4. Allen's injury model of T10 spinal cord was established in Wistar rats with strike force of 25 g·cm.The experimental animals was divided into the control group (group A) and the experimental group (group B), ( group A only removed the vertebral plate, while SCI in group.) According to different time point, every group was divided into 1d, 3d, 1w, 2w, 4w. After the pulmonary tissue was taken followed by SCI, the pulmonary morphological changes were shown in rats by general samples and pathological observation, edema extent of pulmonary tissue was decided by testing the wet/dry weight ratio.Main Results and Conclusion1. In the culture of NSCs, the number of differentiated neurons was obviously increased in the groups cultured with serum medium containing 1.0mM VPA compared with the control group (p<0.01), while the ratio of astrocyte was less than the control group (p<0.01), which suggested that VPA could promote the differentiation of the NSCs into neurons and inhibit its differentiation into astrocyte.2. At 8w After SCI, BBB score was 21 in group A; 17.46±1.98 in group B: 17.53±1.76 in group C and 19.87±1.89 in group D (p<0.05). The delitescence of MEP was 11.06±1.18 in group B, while it was 10.88±1.42 in group C and 8.90±0.80 in group D(p<0.05). The delitescence of SEP was 15.75±1.04, and 15.09±1.32, 13.25±0.89 in group B, C, D respectively (p<0.05). BDA nerve tracing demonstrated that the number of corticospinal tract labeled by DBA was 32.23±3.21 at 8 weeks in group A while it was 17.52±3.98 in group B,18.09±4.21 in group C, and 25.26±4.84 in group D (p<0.05),which demonstrated VPA has certain repair function on the nerve repair of rats after SCI. However, MP had no obvious effect on the functional restoration of nervous system in rats after SCI(p>0.05).3. Three days after SCI, the apoptotic index in rats reached the peak, the apoptotic index in group C was 25.24±4.05, while it was 38.21±4.55 in group B which was obviously higher than the group C (p<0.05), followed by the subsequent decrease. The scanning record of electron microscope showed neurons characteristized as chromatin margination in the nucleus, as well as enlarged nucleus, more apoptotic bodies in rats from group B than those from group C. The study indicated that VPA could inhibit the apoptosis of neurons, which might be the mechanism for VPA to promote the restoration of the nerve function after SCI.4. 24h after SCI, there was no obvious difference of Nestin expression between the rats from group B and the ones from group C; there were more Nestin positive cells surrounding the central canal in the rats from C group than the ones from B group 1w after SCI(p<0.05)and there was consecutive high expression of Nestin until 4 w after SCI, the expression still existed at 8w after SCI. The expression of Nestin obviously decreased, with seldom expression or nearly no expression at all at 8 w after SCI in the rats from B group (p<0.05). There were many cells with positive Nestin expression surrounding the central canal proved that initiation of Nestin expression in cells occurred as result of the injury after SCI. The double-label immunofluorescence with anti-Brdu and anti-NF-200 showed that there were cells with co-expression of Brdu and NF-200 surrounding the central canal,which indicating that SCI could induce the development and regeneration of spinal cord. The study demonstrated that VPA could excite the NSCs and promote their differentiation into neurons, which may be another mechanism for its promotion of the function restoration of nervous system. Besides, results also showed that glial cells including oligodendrocyte, gitter cells and oligodendrocyte not only provided nutrition support for neurons, but also transmit the signals among neurons.5. Three days after SCI, both the number of Brdu positive cells in group B and group C increased. But the increase in group B was more obvious than that in the group C(p<0.05). However, the two groups had few cells with positive expression of Brdu 4w after SCI, with no significant difference between the results of the two groups(p>0.05).The immunofluorescence with anti-GFAP and anti-NF-200 double label showed the following results: the number of neurons in rats from group B decreased significantly, as well as many astrocyte in a disorder arrangement. There were more neurons and relatively fewer astrocyte in rats from group C than the ones from group B (p<0.05).The result of this study demonstrated that VPA could inhibit the proliferation of astrocyte and relieve the hyperplasy of glial scar after SCI in rats, which laid foundation for the regenerated axons to enter the distal end through the injured region.6. Results from general sample observation in rats after SCI: the pulmonary hemorrhage and edema occurred 12h after SCI, which became most severe 3d after SCI,which began to relieve 1w later and recovered to be normal after 4w. Microscope observation: capillary vessels engorgement in pulmonary alveoli occurred 6h after SCI in the experimental group, there was little hemorrhage in the capillary vessels of pulmonary alveoli, the severity of hemorrhage and edema reached the peak after 3d , and began to relieve 1w later. 24h after injury, the wet/dry weight ratio in injury group was significant higher than the control group(p<0.05), which turned out to be most obvious after 3 d (p<0.01). Results demonstrated that: there were different levels of pulmonary hemorrhage and edema in rats after SCI; pulmonary hemorrhage and edema might be the pathological basis for the early respiratory failure; and might be the main reason for the early death of rats. The five steps treatment method pays no attention to the prevention and treatment for complications. The occurrence of changes in lung indicates that the clinicians should pay attention to the pulmonary changes when they are rescuing the emergent SCI patients.