Valproic acid protects neurons and promotes neuronal regeneration after brachial plexus avulsion.

Valproic acid has been shown to exert neuroprotective effects and promote neurite outgrowth in several peripheral nerve injury models. However, whether valproic acid can exert its beneficial effect on neurons after brachial plexus avulsion injury is currently unknown. In this study, brachial plexus root avulsion models, established in Wistar rats, were administered daily with valproic acid dissolved in drinking water (300 mg/kg) or normal water. On days 1, 2, 3, 7, 14 and 28 after avulsion injury, tissues of the C5-T1 spinal cord segments of the avulsion injured side were harvested to investigate the expression of Bcl-2, c-Jun and growth associated protein 43 by real-time PCR and western blot assay. Results showed that valproic acid significantly increased the expression of Bcl-2 and growth associated protein 43, and reduced the c-Jun expression after brachial plexus avulsion. Our findings indicate that valproic acid can protect neurons in the spinal cord and enhance neuronal regeneration following brachial plexus root avulsion.

INTRODUCTION

Brachial plexus root avulsion is the most serious type of peripheral nerve injury. It is encountered commonly in victims of traffic accidents and children after obstetric complications, leading to total paralysis of the corresponding muscle groups and severe sensory deficits in the arms on the lesioned side. There is currently no satisfactory treatment for brachial plexus root avulsion and its clinical prognosis is poor. Peripheral nerve injury is the damage to the axons of corresponding neuronal cell bodies in the spinal cord, which may cause neuronal impairment or death[1]. It has been confirmed that injury close to the neuronal cell bodies causes more neuronal death, so brachial plexus avulsion inevitably causes the greatest degree of neuronal death in the spinal cord, leading to poor regeneration of axons. The loss of neurons significantly restricts muscle reinnervation after a surgical repair of the ventral root connection. After peripheral nerve injury, the survival of motor neurons is an essential prerequisite of neural regeneration and functional recovery[2]. Therefore, an effective agent that reduces neuronal death and promotes the regeneration of surviving neurons is very important and valuable for brachial plexus root avulsion treatment. Neurotrophic factors should be a good choice for treatment, because they exert various beneficial neuronal roles in vitro and in vivo. However, the clinical effects of neurotrophic factors are unsatisfactory.

As an anticonvulsant and mood-stabilizing drug, valproic acid has been widely and safely used clinically for decades[3]. Because of its numerous advantages relative to neurotrophic factors, such as its rapid penetration of the blood-brain barrier and its safety for clinical use, a growing number of studies have investigated the neuro-protective effects of valproic acid in nerve injury[456]. For example, valproic acid can enhance the survival rate of damaged neurons after sciatic nerve transection in rats[7], and promote axonal regeneration and functional recovery following nerve repair[4]. However, it is unknown whether valproic acid can exert its neuro-protective effects in brachial plexus root avulsion, which induces much more neuronal injury and death. There are also few reports on the role of valproic acid in the promotion of neuronal regeneration. Thus, we designed this study to clarify these problems.

In this study, we examined Bcl-2 and c-Jun expression to reflect neuronal survival in the spinal cord after brachial plexus root avulsion and after treatment with valproic acid. As an anti-apoptotic gene, Bcl-2 exerts a protective role in the central nervous system and significantly protects motor neurons against cell death resulting from peripheral nerve injury[8910]. C-Jun is a component of the Ap-1 transcription factor, and is one of the earliest and most consistent markers for neurons that respond to axon interruption. The level of c-Jun expression can reflect the extent of the damage to neurons after axonal injury[111213]. So, we examined Bcl-2 and c-Jun expression to demonstrate the protective effect of valproic acid on spinal cord neurons after brachial plexus root avulsion. In addition, growth associated protein 43 (GAP-43) was also examined to verify the regeneration ability of spinal cord neurons after brachial plexus avulsion followed by administration of valproic acid. GAP-43 belongs to the calcium-activated protein family, and is the fast axonal transport cell membrane phospholipid acid protein in the nervous system. GAP-43 expression is highly involved in neuronal and axonal regeneration in adult animals[1415]. The content of GAP-43 in the spinal cord neurons usually increases many folds after axonal injury, and then gradually drops to a low level following the completion of neural regeneration[16]. Thus, GAP-43 is a reliable marker of neural regeneration.

This study aims to investigate the effects of valproic acid on Bcl-2, c-Jun and GAP-43 expression in spinal cord neurons after brachial plexus avulsion, in a broader attempt to explore whether valproic acid can protect neurons against death and promote neuronal regeneration.

RESULTS

Quantitative analysis of experimental animals

A total of 130 healthy adult male Wistar rats were used in this study. Five rats were randomly selected for the normal control group (without any treatment). The remaining 125 rats were subjected to models of brachial plexus root avulsion. Five rats were excluded because of accidental death after the surgery. After the surgery, the surviving 120 rats were randomly divided into two groups: injury group and valproic acid group, with 60 rats in each group. In the valproic acid group, rats were orally administered with valproic acid dissolved in drinking water[417] daily. Rats in the injury group were intragastrically administered with clean water. All five rats in the normal control group were sacrificed at the beginning of the study. In the other two groups, five rats were selected from each group on days 1, 2, 3, 7, 14 and 28 post-surgery to harvest spinal cord samples, for real-time PCR and western blot assay, respectively. All the involved 125 rats were included in the final analyses.

Valproic acid increased Bcl-2 mRNA and protein expression in the C5–T1 spinal cord segments of brachial plexus avulsion rats

Real-time fluorescent quantitative PCR was used to detect Bcl-2 mRNA expression in the C5–T1 spinal cord. The results of Bcl-2 mRNA levels in the injury group and valproic acid group were represented in relation to the normal group (normal group: 1.000 ± 0.242). Subsequent to brachial plexus root avulsion, Bcl-2 mRNA expression in the spinal cord increased immediately at day 1, reached a peak at day 7, and then decreased gradually, but remained high until day 28. Compared with the injury group, Bcl-2 mRNA levels in the valproic acid group were significantly increased at days 2, 3 and 7 (P < 0.05; Figure 1).

Figure 1

Real-time PCR for Bcl-2 mRNA expression in C5–T1 spinal cord after brachial plexus root avulsion followed by valproic acid treatment.

β-Actin was used as an internal reference. The Bcl-2 mRNA level was measured in comparison to the normal group and calculated after adjusting for β-actin using the 2–ΔΔ Ct method. Data are expressed as mean ± SD of five rats from each group at each time point. Statistical significance was determined using independent samples t-test. aP < 0.05, vs. injury group.

Western blot assay was used to detect Bcl-2 protein expression in the C5–T1 spinal cord. In the normal group, there was a weak band positive for Bcl-2 protein, with a expression (relative absorbance ratio) of 0.033 ± 0.010. After brachial plexus avulsion, the change in Bcl-2 protein expression was similar with that of Bcl-2 mRNA in the injury and valproic acid groups. In both the injury and valproic acid groups, the Bcl-2 protein bands became stronger and the expression began to increase from day 1 post-surgery, peaked at day 7, and then decreased until day 28. At days 2, 3 and 7, the Bcl-2 protein levels in the valproic acid group were significantly higher than that in the injury group (P < 0.05; Figure 2).

Figure 2

Western blot assay for Bcl-2 protein expression in C5–T1 spinal cord after brachial plexus root avulsion followed by valproic acid treatment.

(A) The Bcl-2 bands were amplified at different times after surgery. β-Actin immunoblots were used to confirm equal protein loading.

(B) Quantification of Bcl-2 protein expression. The relative Bcl-2 expression was the absorbance value ratio of Bcl-2 protein band to β-actin. Data are expressed as mean ± SD of five rats from each group at each time point. Statistical significances were determined using independent samples t-test. aP < 0.05, vs. injury group.

Valproic acid decreased c-Jun mRNA and protein expression in the C5–T1 spinal cord of brachial plexus avulsion rats

C-Jun mRNA expression in the C5–T1 spinal cord was detected by real-time PCR. The results in the normal group were tested to determine the basic level of c-Jun mRNA expression, and were used as a reference without statistical analysis. The c-Jun mRNA expression levels in the injury and valproic acid groups were measured in relation to the normal group (normal group: 1.000 ± 0.213). After brachial plexus avulsion, c-Jun mRNA expression in the injury and valproic acid groups increased from day 1 and was higher at each time point than the normal levels. The results in the injury and valproic acid groups both showed an up-down trend that peaked at day 3, and then decreased until day 28. The c-Jun mRNA levels in the valproic acid group was lower than that in the injury group at days 1, 2, 3 and 7 (P < 0.05; Figure 3).

Figure 3

Real-time PCR for c-Jun mRNA expression in C5–T1 spinal cord after brachial plexus root avulsion followed by valproic acid treatment.

β-actin was used as an intrinsic reference. The c-Jun mRNA level was measured compared to the normal group and calculated after adjusting for β-actin using the 2–ΔΔCt method. Data are expressed as mean ± SD of five rats from each group at each time point. Statistical significances were determined using independent samples t-test. aP< 0.05, vs. injury group.

The c-Jun protein expression in the C5–T1 spinal cord was detected by western blot assay. Results showed that there was a very low level of c-Jun protein expression in the normal group (0.289 ± 0.030). Subsequent to brachial plexus avulsion, c-Jun protein levels in the spinal cord increased immediately in the injury and valproic acid groups, reached peaks at day 3, and then decreased until the end of the experiment. Furthermore, the levels of c-Jun protein expression in the valproic acid group were significantly lower than that in the injury group at days 1, 2, 3 and 7 (P < 0.05; Figure 4).

Figure 4

Western blot assay for c-Jun protein expression in C5–T1 spinal cord after brachial plexus root avulsion followed by valproic acid treatment.

(A) The c-Jun bands were amplified at different times after surgery. β-actin immunoblots were used as the internal references.

(B) Quantification of c-Jun protein expression. The relative c-Jun expression was the absorbance value ratio of c-Jun protein to β-actin. Data are expressed as mean ± SD of five rats from each group at each time point. Statistical significances were determined using independent samples t-test. aP < 0.05, vs. injury group.

Valproic acid increased GAP-43 mRNA and protein expression in the C5–T1 spinal cord of brachial plexus avulsion rats

Real-time PCR was used to detect GAP-43 mRNA expression in the C5–T1 spinal cord. Results showed that there was a low level of GAP-43 mRNA expression in the normal rats (1.00 ± 0.232). GAP-43 mRNA expression in the injury group gradually increased from day 1 post-surgery, peaked at day 14, and then decreased. GAP-43 mRNA expression in the valproic acid group had a similar curve with the injury group. However, the GAP-43 mRNA expression levels in injury and valproic acid groups were higher than the normal group, even at day 28 post-surgery, but the increases in the valproic acid group were much more evident than the injury group. There were significant differences between the injury group and valproic acid group from day 2 to day 28 (P < 0.05; Figure 5).

Figure 5

Real-time PCR for growth associated protein 43 (GAP-43) mRNA expression in C5–T1 spinal cord after brachial plexus root avulsion followed by valproic acid treatment.

β-actin was used as an intrinsic reference. The GAP-43 mRNA level was measured in comparison to the normal group and calculated after adjusting for β-actin using the 2–ΔΔ Ct method. Data are expressed as mean ± SD of five rats from each group at each time point. Statistical significances were determined using independent samples t-test. aP < 0.05, vs. injury group.

GAP-43 protein expression in the C5–T1 spinal cord was detected by western blot assay. In the normal group, there was a small GAP-43-positive band with a low absorbance value ratio (0.039 ± 0.020). After brachial plexus avulsion, GAP-43 protein expression in the spinal cord increased in the injury and valproic acid groups, reached peaks at day 14, and then decreased until day 28. The absorbance value ratio of GAP-43 protein bands in the valproic acid group was significantly higher than that in the injury group from days 2 to day 28 (P < 0.05; Figure 6).

Figure 6

Western blot assay for growth associated protein 43 (GAP-43) protein expression in C5–T1 spinal cord after brachial plexus root avulsion followed by valproic acid treatment.

(A) After brachial plexus root avulsion, the GAP-43 protein bands were amplified at different time points. GAPDH immunoblots were used to as an internal reference.

(B) Quantification of GAP-43 protein expression. The relative GAP-43 expression was the absorbance value ratio band of GAP-43 protein to GAPDH. Data are expressed as mean ± SD of five rats from each group at each time point. Statistical significances were determined using independent samples t-test. aP < 0.05, vs. injury group.

DISCUSSION

In this study, we established a brachial plexus root avulsion model in rats, and detected the gene transcription and protein expression of Bcl-2, c-Jun and GAP-43 in the C5–T1 spinal cord segments. Our findings indicate that oral administration of valproic acid has neuro-protective effects that cause a significant increase in Bcl-2 expression while decreasing c-Jun expression, and enhances neuronal regeneration by up-regulating GAP-43 expression after brachial plexus root avulsion.

Avulsion of the brachial plexus causes serious disability in the arm and the current treatments are still far from satisfactory. Compared with other types of nerve damage (such as clamping and cutting), root avulsion causes a larger amount of neuronal death as the damage site is close to neuronal cell bodies in the spinal cord[1819]. In adult animals, over 80% of injured motor neurons exhibit retrograde cell death in the model of ventral root avulsion[20]. The loss of neurons leads to very poor neural regeneration and functional recovery[1920]. An increasing number of neurotrophic factors have been found to support motor neuron survival and enhance axonal regrowth in a variety of experimental paradigms[212223]. However, because neurotrophins are peptides, their benefits are largely limited by their poor ability to cross blood-nerve and blood-brain barriers and by enzymatic degradation. Besides, long-term and frequent administration with high-dose neurotrophic factors may have unexpected side effects.

The discovery of the neuroprotective effects of valproic acid brings hope to this problem because valproic acid is an inorganic small molecule compound that can easily penetrate the blood-brain barrier and that is not easily degraded by enzymes, thereby giving it a great advantage over neurotrophic factors[3424]. The safety of valproic acid has been widely validated because it has been used clinically for antiepileptic therapy for more than 30 years. Clinical studies have found that valproic acid can relieve symptoms of neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and spinal muscular atrophy[25]. In vitro experiments have shown that valproic acid can prevent neuronal apoptosis and exert neurotrophic effects through the activation of multiple signal transduction pathways[2627282930]. However, there has been no report about the effect of valproic acid in brachial plexus root avulsion, which causes much more neuronal death than other types of peripheral nerve damage[4].

Neuronal death from brachial plexus avulsion in adult animals is caused by apoptosis and necrosis of neurons[2031]. Bcl-2, an anti-apoptotic gene, strongly inhibits cell apoptosis, promotes signal transduction and inhibits free radical damage[3233]. It is highly expressed in the developing nervous system and significantly decreases in the adult central nervous system, but retains a certain degree of expression in the peripheral nervous system[34]. In many different models, Blc-2 over-expression has been demonstrated to protect neurons from death after peripheral nerve injury[353637].

In this study, low Bcl-2 expression was found in C5–T1 spinal cord segments of the normal group. In the injury and valproic acid groups, after the brachial plexus were avulsed, Bcl-2 expression was significantly elevated compared with the normal group. The increasing Bcl-2 expression should be contributed to the surviving neurons as there was a large number of neuronal death caused by avulsion injury. The induction of Bcl-2 expression after peripheral nerve injury has been shown in many studies[3438]. For example, Natsume et al[39] administered exogenous Bcl-2 to rats with L4–6 root avulsion injury and the number of surviving motor neurons in the anterior horn of the spinal cord were significantly increased. Valproic acid has also been shown to play a neuro-protective role by increasing GAP-43 and Bcl-2 expression[40]. The results in the present study are in agreement with previous reports. The increase in Bcl-2 expression in the spinal cord induced by valproic acid has two possible explanations. Firstly, valproic acid may have reduced neuronal death, thereby increasing the number of surviving neurons. Secondly, valproic acid may also promote the survival of neurons that highly express Bcl-2. Whatever mechanism of neuro-protective activity of Bcl-2, valproic acid protects spinal cord neurons after brachial plexus root avulsion by upregulating Bcl-2 expression at both the mRNA and protein levels.

The c-Jun expression in neurons was elevated in several nerve injury paradigms, but the role of it remains unclear. Some studies have shown that the induction of c-Jun is related with neuronal death, whereas others have demonstrated its role in nerve regeneration[414243]. Thus, its effect on neuronal apoptosis is currently unknown and could be bipotential[11134243]. The c-Jun and JNK/c-Jun pathway is an attractive candidate as a sensor and trigger for axonal response[44]. Axonal injuries near the neurons can strongly stimulate c-Jun expression in neurons. Furthermore, there is some correlative evidence between the high expression of c-Jun and a variety of neurodegenerative insults, including ischemia, ionizing radiation and the formation of plaques in Alzheimer's disease[4546]. There is also a rapid increase of phosphorylated c-Jun in injured motor neurons in all lesions, which can induce death of spinal neurons, such as root avulsion. Taken together, neuronal death caused by nerve injury has close relations with the induction of c-Jun phosphorylation[47]. Although the mechanism and effect of high c-Jun expression in neurons after nerve injury is unclear, we tentatively suggest that c-Jun is a marker of neuronal injury or death, where higher c-Jun expression represents increased neuronal injury.

In the normal group, c-Jun expression is very low. High expression of c-Jun after brachial plexus root avulsion indicates that root avulsion caused massive death of neurons in the spinal cord, with the most serious neural death appearing at day 3 after injury. Zhou et al[48] showed that brachial plexus root avulsion leads to c-Jun expression at 4 hours after injury. Wu demonstrated that c-Jun phosphorylation correlates with motor neuron death, because root avulsion in adult rats induced phosphorylation of c-Jun in motor neurons and almost all of these injured neurons died thereafter[47]. These reports support the results of the present study. Valproic acid down-regulated c-Jun expression at each time point compared with the injury group. According to the above-mentioned reports, the lower expression of c-Jun in the spinal cord caused less neuronal death. The results of this study clearly show that valproic acid can protect neuronal death and support increased neuronal survival. Furthermore, the down-regulation of c-Jun expression at day 1 post-surgery suggests that valproic acid exerted its role on c-Jun rapidly and directly. This result also supports the effectiveness of oral administration of valproic acid dissolved in conventional drinking water for rats.

GAP-43 was found by Skene et al in the early 1980s and is now considered to be an intrinsic determinant of neuronal development and regeneration[144950]. Once the nerve is damaged, GAP-43 is synthesized by the neuronal cell body, then transported to the tip of the axonal regeneration site by fast axoplasmic transport and fixed on the cytoplasmic side of the growth cone. Through this the axonal growth cone can extend easily and the nerve can regenerate. Thus, GAP-43 has been shown to promote neuronal regeneration[5152].

The low expression of GAP-43 in the normal group was consistent with the conclusion of other studies[1553]. Brachial plexus avulsion may stimulate long-term regeneration of neurons in the spinal cord. As noted above, c-Jun expression in the spinal cord increased immediately after brachial plexus avulsion and peaked at day 3, which indirectly reflects that the neuronal death began to increase. Despite the large amount of neuronal death, GAP-43 expression in the spinal cord still gradually ascended and peaked at day 14. The seemingly paradoxical results indicate that the surviving neurons with mild retrograde injury entered an active regenerative state. Studies have shown that GAP-43 expression increased immediately, and soon disappeared when the nerve injury site is far away from the neurons, while damage close to neurons produce a more sustained expression of GAP-43[5455]. This partly explains the long period of GAP-43 expression found in this study after brachial plexus avulsion.

Results of the valproic acid group showed that oral administration of valproic acid after brachial plexus avulsion can significantly improve GAP-43 expression in neurons. Previous studies also support these results, where an in vitro study showed that after cells were cultured with medium containing valproic acid for 5 days, GAP-43 expression in SH-SY5Y cells increased approxmately 4.5-fold as compared with the injury group[4056]. As noted above, valproic acid can reduce neuronal death by promoting Bcl-2 expression and lowering c-Jun expression. The up-regulation of GAP-43 expression in the valproic acid group may be attributed to increased neuronal survival compared with the injury group. However, as the total number of dead neurons increased from days 1 to 28 after brachial plexus injury, and the GAP-43 expression gradually ascended over time, this does not seem possible. Thus, the increase in GAP-43 expression must be ascribed to the activation of injured neurons. However, there were some limitations with this study. Firstly, we did not directly detect the number of apoptotic or surviving neurons in the spinal cord. We did not use histological methods, such as immunohistochemical staining, to determine whether the changes caused by valproic acid on Bcl-2, c-Jun and GAP-43 expressions occurred in neurons or other cells of the spinal cord. In addition, the mechanism through which valproic acid exerts its role on Bcl-2, c-Jun and GAP-43 expression should be further explored.

In summary, valproic acid increased Bcl-2 expression and decreased c-Jun expression in the spinal cord after brachial plexus root avulsion. Valproic acid has a more significant effect on neurons within 7 days post-injury, indicating that it may play an early protection role. Another finding is that valproic acid up-regulated GAP-43 expression in the spinal cord after brachial plexus root avulsion, and this improvement lasted to day 28 post-injury. We conclude that valproic acid protects neurons from cell death and enhances their regeneration ability after brachial plexus root avulsion in rats. Although the mechanism of this process is unclear and needs further study to clarify, the present study gives strong support for the clinical use of valproic acid in patients with brachial plexus root avulsion.

MATERIALS AND METHODS

Design

A randomized controlled animal study.

Time and setting

Experiments were performed from May to December in 2012. Animal operations were performed at the Experimental Animal Centre, Jilin University, China. Bcl-2, c-Jun and GAP-43 detection were performed at the Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Cytology and Genetics, Northeast Normal University, China.

Materials

Animals

Adult male Wistar rats were provided by the Experimental Animal Centre of Jilin University, China (clean grade, license No. SCXK (Ji) 2012-0003). All rats were healthy, weighing 250–300 g and aged 8–10 weeks. All animals were housed under 12-hour light/dark cycle and standard housing conditions (relative humidity 60%, temperature 22°C) and allowed free access to food and water with one rat per cage. All procedures conducted were in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[57].

Drugs

Valproic acid was purchased from Sigma (St. Louis, MO, USA; lot numbers: 47H3481), stored at –4°C and used within 6 weeks.

Methods

Procedure of brachial plexus avulsion

The surgical procedures were undertaken in batches by the same surgeon within 1 week. Rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (2.5 mL/kg) and then stabilized on the operating table in a supine position. The neck area was sterilized with 0.5 iodophor, followed by 75% alcohol. An approximately 2 cm oblique supraclavian incision was made, and the right brachial plexus was exposed by separating the superficial and deep pectoral muscles with fine forceps. Then, the procedures were taken under the surgical microscope (Zhenjiang Zhongtian Company, Zhenjiang, Jiangsu Province, China) with 10 × magnification. The trunks of brachial plexus were identified firstly, and then the C5–T1 nerve roots were dissociated to where they exit the vertebral foramen. With a micro-hemostat forceps, the C5–T1 nerve roots were avulsed one by one from the intervertebral foramen by a steady moderate traction (Figure 7A). The avulsed ventral and dorsal roots, and dorsal root ganglia can be identified easily under a microscope (Figure 7B). All avulsed roots, as well as a 1 cm segment of distal nerve, were cut away from the brachial plexus to confirm success of the avulsion. Finally, the skin was sutured by 4/0 MERSIKL.

Figure 7

Brachial plexus root avulsion model in rats (× 10).

Surgical procedures were performed under a surgical microscope. C5–T1 roots of the right brachial plexus were avulsed from the intervertebral foramen. Ventral roots (black arrows) and dorsal roots (white arrows) can be easily observed.

Animals were allowed to recover for 1–2 hours and then returned to the house with one rat in each cage.

After the brachial plexus root avulsion was made, the right front legs of the rats were immediately paralyzed. Several days later, there would be varying degrees of ulcers and autophagy on the right arms.

Drug administration

Rats in the injury group were allowed free access to a standard diet of pellets with clean water. In the valproic acid group, rats also had free access to food and water, but were orally administrated daily with valproic acid dissolved in drinking water at 300 mg/kg per day, a dose equivalent to that for the antimanic action at plasma level about 50 mg/mL without clinical toxicity[417].

Preparation of C5–T1 spinal cord

After anesthetization with intraperitoneal injection of 10% chloral hydrate (2.5 mL/kg), the rats were stabilized in a prone position on the table and sacrificed by cutting off the femoral artery. Then the back neck area of rats was disinfected and a longitudinal incision was made. After separation and removal of the muscle, a laminectomy was performed, and the C5–T1 spinal cord segments on the avulsed side were harvested, marked and placed in liquid nitrogen for real-time PCR and western blot assay.

Real-time PCR for Bcl-2, c-Jun and GAP-43 mRNA expression

Primers were designed and obtained by Huada Zhongtian Biotechnologies Company (Changchun, Jilin Province, China). β-actin was used as an internal control. Primers were designed as follows: Bcl-2: forward primer 5′-TCT GTG GAT GAC TGA GTA CCT GAA C-3′, length 25 bp; reverse primer 5′-AGA GAC AGC CAG GAG AAA TCA AAC-3′, length 24 bp. c-jun: forward primer 5′-CTG ATC ATC CAG TCC AGC AA-3′, length 20 bp; reverse primer 5′-TGG CTA TGC AGT TCA GCT AGG-3′, length 21 bp. GAP-43: forward primer 5′-GCC ATA TGC TGT GCT GTA TGA GA-3′, length 23 bp; reverse primer 5′-TCA GGC ATG TTC TTG GTC A-3′, length 19 bp. β-actin: forward primer 5′-AGG GAA ATC GTG CGT GAC-3′, length 18 bp; reverse primer 5′-CGC TCA TTG CCG ATA GTG-3′, length 18 bp.

Total RNA of C5–T1 spinal cord was extracted using Trizol reagent (Shanghai Sangon Company, Shanghai, China), and mRNA was isolated with a PolyA Ttract® mRNA Isolation System III (Promega, Madison, MA, USA). After the concentration and purity of mRNA were confirmed, cDNA synthesis was performed using SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Carlsbad, CA, USA) in a two-step reaction. The reverse transcription reactions contained 10 μL 2 × reverse transcription reaction mix, 2 μL reverse transcription enzyme mix, 1 μg RNA, and DEPC-treated water to a 20 μL final volume. Tube contents were gently mixed and incubated at 25°C for 10 minutes, at 50°C for 30 minutes. The reaction was terminated at 85°C at 5 minutes, and then chilled on ice. Then, 1 μL (2 U) of E. coli RNase H was added to the mixture and incubated at 37°C for 20 minutes. The cDNA copy number for each gene was detected using standard curves of the corresponding PCR product. Setup for the cDNA PCR reaction was standard for all samples. Each 50 μL reaction system contained 25 μL of FastStart Universal SYBR Green (ROX) (Roche, Basel, Switzerland), 0.5 μL (30 μmol/L) forward primers, 0.5 μL (30 μmol/L) reverse primers and 19 μL water (PCR-grade) and 5 μL cDNA. Real-time PCR was conducted with ABI PRISM 7900 HT Fast Real-Time PCR System (ABI, Carlsbad, CA, USA). Reaction conditions were as follows: 1 cycle of 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 59°C for 30 seconds, and 72°C for 20 seconds. Data were analyzed by software supplied by the PCR system that uses a modified 2-ΔΔCt method (ΔΔCtcontrol = (Ctcontrol – Ctβ-actin) – (Ctnormal – Ctβ-actin), ΔΔCtvalproic acid = (Ctvalproic acid – Ctβ-actin) – (Ctnormal – Ctβ-actin))[58].

Western blot assay for Bcl-2, c-Jun and GAP-43 protein expression

The spinal segments were ground in a mortar, then lysed with RIPA Lysis buffer on ice, and centrifuged at 16 099.2 × g for 3 minutes at 4°C. Supernatant protein was harvested, and protein concentration was determined using the Coomassie brilliant blue method. Proteins (50 μg) were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, MA, USA), and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), followed by separate incubation with primary antibodies, rabbit anti-rat monoclonal antibody anti-Bcl-2, anti-c-Jun, and anti-GAP-43 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, followed by incubation with the secondary antibody goat anti-rabbit IgG (1:1 000; Santa Cruz Biotechnology) for 1 hour at room temperature. Coloration was performed in accordance with the 3,3’-diaminobenzidine reagent kit instructions (Santa Cruz Biotechnology). The absorbance of the scanned bands was determined using ImageJ software (National Institutes of Health, Rockville, Maryland, USA). In the detection of Bcl-2 and c-Jun, β-actin was used as an internal reference. The ratio of the absorbance value between Bcl-2 or c-Jun and β-actin was used as the relative expression data. However, for the detection of GAP-43 protein, GAPDH was used as the internal reference, as the molecular weight of β-actin (42 kDa) is close to that of GAP-43 (43 kDa), which may cause confusion of the electrophoretic bands.

Statistical analysis

All data was statistically analyzed using SPSS 17.0 software (SPSS, Chicago, IL, USA). The data of real-time PCR and western blot assay were expressed as mean ± SD. Statistical significance between injury group and valproic acid group were determined using independent samples t-test. P < 0.05 was considered statistically significant.

Research background: There is currently no satisfactory treatment for brachial plexus root avulsion, and its clinical prognosis is poor. However, there has been no report about the effect of valproic acid in the treatment of brachial plexus root avulsion.

Research frontiers: The present study is the first to use brachial plexus avulsion model in rats to investigate the role of valproic acid on spinal cord neuronal injury. Furthermore, it is the first demonstration on the effect of valproic acid on Bcl-2 and c-Jun expressions in the spinal cord.

Clinical significance: Valproic acid can protect neurons in the spinal cord against cell death induced by brachial plexus root avulsion and can promote neural regeneration. The present study gave strong support for the clinical use of valproic acid in patients with brachial plexus root avulsion.

Academic terminology: The brachial plexus nerves, a group of important peripheral nerves that innervates the skeletal muscle of the upper limbs, shoulder, upper and lateral thoracic parts, is also responsible for the sensory functions in these areas, with complex components and branches.

Peer review: This study aims to observe the effect of valproic acid on Bcl-2 and c-Jun expression in the spinal cord after brachial plexus avulsion in animals. Results showed that valproic acid upregulated Bcl-2 expression and reduced c-Jun in spinal cord neurons, thus protecting neurons against brachial plexus avulsion. This is an interesting study and the methods are reliable, and the results may provide guidance for clinics.