Effect of Methylcobalamin on Voltage-Gated Sodium Channels (VGSCS) Expression in Neuropathic Painanimal Model

1Department of Neurology, School of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia. 2Department of Neurology, Dr. Zainoel Abidin Hospital, Banda Aceh, Indonesia. 3Department of Neurology, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia. 4Department of Physiology, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia. 5Department of Pathology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. *Corresponding author E-mail: endangmutiawati@unsyiah.ac.id

Neuropathic pain is defined as a sensation arises from the results of a lesion or disease of the peripheral or central somatosensory nervous system; some of the examples are postherpetic neuralgia, painful polyneuropathy, trigeminal neuralgia, and post-stroke pain. 1 Burning pain, painful sensitivity to touch, and pain attacks are the common complains reported and these significantly reduce the quality of life of the patients and impose economic burdens on individuals and society. 2,3 The worldwide prevalence of chronic neuropathic pain ranging between 6.9% and 10%. 4 The pathophysiology neuropathic pain is complex and not completely understood. 5 One of pathophysiology mechanisms is alteration of ion channels within the affected neurons, leading to altered electrical excitability of sensory neurons. 5,6 Voltage-gated sodium channels (VGSC), one of the ion channels, allow rapid influx of sodium, causes depolarization of action potentials in excitable cells. 7 VGSCs are integral membrane glycoproteins on neurons and alteration of this ion channel such as overexpressed on neurons is critical for development of pain sensation of neuropathic pain. 6,8,9 Regardless of its origin (peripheral or central), the pharmacotherapy alternatives of neuropathic pain are similar. Tricyclic antidepressants, selective serotonin-norepinephrine reuptake inhibitors, and anticonvulsants are the first choice of drugs for neuropathic pain, while opioids can only be used when other drugs have not been effective or in need of a more rapid onset of pain relief. 10 Methylcobalamin, an activated form of vitamin B12, exerts neuronal protection by promoting regeneration of injured nerves and reduces glutamate-induced neurotoxicity. 11 Recent experimental and clinical studies suggested that methylcobalamin also has potential analgesic effects on neuropathic pain by improving nerve conduction, promoting regeneration of injured nerves, and preventing spontaneous discharges of injured sensory neurons. 11,12 Despite of rigorous studies on the efficacy of methylcobalamin as the treatment for pain, there is still no study investigating the effect of methylcobalamin on VGSC expression in neuropathic pain. This study sought to assess the effect of methylcobalamin on VGSC expression on nerves of neuropathic pain animal model.

Study setting
A study to assess the effects of methylcobalamin on expression of VGSCs on injured neurons was conducted in nerve ligation-induced neuropathic pain rats. The methylcobalamin was administrated in three different doses, intramuscularly, twice a week for 14 weeks. Apart from assessing the expression of VGSC by immunohistochemistry (IHC), the effect of methylcobalamin in reducing neuropathic pain was also evaluated by assessing the neuropathic pain behavior (mechanical allodynia) in animals.

Animals and neuropathic pain induction
Twenty male Sprague-Dawley rats, 2 months old, weighing 150-250 g, were used. The animals went through an adaptation process for a week prior the study under laboratory conditions (temperature 23±1 o C, 60% of humidity, and 12h light-dark cycle), and were fed ad libitum as explained previously. 13 To induce the neuropathic pain, segmental spinal nerve ligation (SNL) technique 14 was adopted. The ligation was conducted on lumbar 5 (L5) nerve. Briefly, the animals were anesthetized and the left hind paw was shaved and sterilized. A 5cm longitudinal incision midway of the left tight was performed. A 0-6 silk thread was inserted to the nerve distal and the nerve was ligated tightly as described previously. 15 The open wound was washed with penicillin-streptomycin solution and closed by thoroughly stitching the muscles using 4-0 chromic cat-gut thread and 0-3 cotton thread for the skin.

Administration of methylcobalamin
The animals were randomly divided into 4 groups of 5; one control group and three methylcobalamin groups (M 50 , M 100 , M 150 ). Control group was given 0.9% sodium chloride intramuscularly, while M 50 , M 100 and M 150 groups were received methylcobalamin 50µg, 100µg, and 150µg, respectively, intramuscularly, twice a week for 14 weeks.

Mechanical allodynia assessment
To assess the effect of methylcobalamin on neuropathic pain, mechanical allodynia was evaluated in animals. Mechanical allodynia was assessed using the von Frey filaments (BiosebLab, France), by pressing an actuator filament slowly against the hind paw until it buckles, with a frequency of 1/s and each intensity was repeated for 10 times. Mechanical allodynia was defined when 5 out of 10 of specific size von Frey stimuli caused the rats to withdraw the paw. Mechanical allodynia was assessed on the 1 st , 3 rd , 5 th , 7 th day, and every weekend afterwards as suggested previously. 16 Three indicators were used: (a) onset time, the length of the time from L5 ligation to time of the first onset of mechanical allodynia appeared; (b) von Frey filament size, the size of von Frey filament which induced mechanical allodynia in the first onset; and (c) duration of mechanical allodynia, the length of the time between the first time when neuropathic pain appeared and when it resolved.

Evaluation of VGSC expression
After 14 weeks of treatment, all animals were deeply anesthetized and then sacrificed via cervical dislocation per protocol. 17 The surgical wound was re-opened and the ligated nerve was extracted together with nearby tissues, and kept in preservative solution (saturated picric acid, formalin (37-40%), and glacial acetate acid with proportion of 15:5:1). The nerve tissues were then dehydrated, processed and cut at the required thickness followed standard operation procedure for histological preparation technique 18 . These nerve tissues where then stained with IHC to assess VGSC expression. IHC staining was conducted using primary anti-pan Na V antibody (Alomone Labs, Jerusalem, Israel). The staining was visualized by using horseradish peroxidase (HRP) -chromogen 3,3'-diaminobenzidine (DAB) detection IHC kit (ab64259, Abcam). All procedures were performed based on the manufacturer's instructions.
The expression of VGSC was divided into two: active and inactive VGSC. Active VGSC indicating that the VGSC was open and therefore Na V antibody could enter to neuron cells and bind with epitope, which located intracellular loop between domains III and IV domain of VGSC (i.e. cytoplasm colored as brownish after secondary training). Inactive VGSC indicating that the GSC was closed and therefore the Na V antibody could not enter the neurons (i.e. cytoplasm does not turn brownish secondary training). Interpretation of VGSC expression was conducted by two pathologists.

Statistical analysis
To compare the effect of methylcobalamin on mechanical allodynia between groups ANOVA and independent t-test were employed. The expression of VGSC from IHC was analyzed descriptively. Significance was assessed at á=0.05 and analyses were conducted using SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA).

Mechanical allodynia Onset of mechanical allodynia
The first observed mechanical allodynia was in day 3 for control and M 50 , and day 5 for M 100 and M 150 . On day 14, mechanical allodynia was observed in all animals within all groups. The mean onset time of mechanical allodynia was 5.4±1.6 days for control group, 4.6±2.1 days for M 50 , 9.0±4.6 days for M 100 , and 8.6±4.9 days for M 150 . Statistical analysis indicated the mean onset time had no different among groups.

von Frey filament size
Our study showed that the size of von Frey filament which induced the pain on controlled group was smaller compared to the ones used for methylcobalamin groups, with mean 14.6±1.9, 18.0±1.4, 17.4±0.8 and 15.8±1.3 for control, M 50 , M 100 , M 150 , respectively (Table 1). Statistical analysis indicated that there was significant different among the four groups (p=0.007). Significant different only observed between control and M 50 group (p=0.013), and between control and M 100 group (p=0.019).

Duration of mechanical allodynia
Our data suggested that the duration of mechanical allodynia was different among groups. In control group, mechanical allodynia was observed in all animals till day 42 while mechanical allodynia already resolved in all animals of M 150 group in day 42 (Fig.1). On the last day of the examination, day 52, one animal from control and M 50 group still exhibited mechanical allodynia.
In average, the longest and the shortest duration of mechanical allodynia was observed    (Table 1). However, the significant different found between control and M 150 group only (p=0.027).

VGSC expression
Using IHC staining, the expression VGSC protein on the effected nerves was classified as positive (cytoplasm colored into brownish indicates VGSCs were open) and negative (cytoplasm unstained indicating VGSC were closed or inactive). Figure 2 presented ICH of VGSC expression from nerve tissue from control group (positive VGSC expression) and M 100 group (negative VGSC expression). Our data showed that all nerve tissues from all animals within control group were expressed VGSC and two animals (40%) of M 50 group also had positive VGSC expression. All nerve tissues from all animals of both M 100 and M 150 group, had negative expression of VGCS.

DISCUSSION
SNL procedure is standard technique to induce neuropathic pain, 14,15 adopting this technique ensured the production of neuropathic pain in animal model. Accumulation of VGSC will induce ectopic pacemaker, whose together with sensitization of other receptors (mechanical, thermal, or chemical) will induce neuropathic pain. 19 This process requires some time and neuropathic pain does not directly arise after neural injury. 20 When we measure the onset time, our data found there was no significant different of onset time of mechanical allodynia between control and treatment groups. However, a previous study found administration of methylcobalamin on dorsal root ganglion significantly delay the early onset of pain. 11 One of the possible reasons is the onset time of mechanical allodynia (representing neuropathic pain) was not measured every day in the current study. Therefore, although the assessment time was followed the previous study, 16 we might failed to measure the exact onset time of mechanical allodynia among groups.
Unlike the onset time of mechanical allodynia, our study found the duration of mechanical allodynia reduced as methylcobalamin dose increased suggesting that methylcobalamin enabled to reduce neuropathic pain. In the same study it was demonstrated also that methylcobalamin significantly shorten the duration of mechanical allodynia by lowering spike amplitude of ectopic discharge. 11 Furthermore, the present study also found that the bigger size of von Frey filament was required to induce pain in methylcobalamin groups compare to control group, suggesting that methylcobalamin could increase the pain threshold. Altogether, our data suggest that methylcobalamin could improve the outcome of neuropathic pain.
To the best of our knowledge, this was the first study assessing the effect of methylcobalamin on VGSC expression in neuropathic pain animal model. Our study indicated that methylcobalamin reduced the expression of VGSC on injured nerves highlighting a potential mechanism of methylcobalamin in reducing neuropathic pain. VGSC is a critical development of pain sensation in neuropathic pain 6,8,9 making VGSC is one of the main therapeutic target of chronic neuropathic pain. 21 Our study highlights that methylcobalamin improves the neuropathic pain through downregulation and/or inactivation of VGSC on the neurons.
This study had some limitations that need to be discussed. The number of animals was relatively small for each group; therefore, further study with bigger number of samples is warranted. In our study, the expression of VGSC was measured without specify the VGSC family. Further analysis to determine whether methylcobalamin effects specific VGSC family only maybe required. In this study the expression of VGSC was evaluated by IHC staining only making a further study using multiple approaches to measure VGSC expression is crucial to elucidate the finding of this study.

CONCLUSION
This present study suggests that methylcobalamin improve the symptoms of SNL-induced neuropathic pain animal model by shorten the duration of pain and increasing pain threshold. This improvement might be associated with reduction of VGSC expression on affected nerves.

ACKNOWLEDGMENTS
We would like to thank to the patients and HT Editorial Service for the assistance during manuscript preparation.

Funding Source
This study received no external funding.

Ethical Approval
This study received no funding. The protocol of this study was approved by Institutional Review Board of the School of Medicine, Universitas Syiah Kuala, Banda Aceh, in accordance to Indonesian national legislation.

Conflict of Interest
Authors do not have any conflict of interests.

Authors' Contributions
Conceptualization