Role of vitamin D3 in Treatment of Lumbar Disc Herniation—Pain and Sensory Aspects: Study Protocol for a Randomized Controlled Trial

Background Vitamin D receptors have been identified in the spinal cord, nerve roots, dorsal root ganglia and glial cells, and its genetic polymorphism association with the development of lumbar disc degeneration and herniation has been documented. Metabolic effects of active vitamin D metabolites in the nucleus pulposus and annulus fibrosus cells have been studied. Lumbar disc herniation is a process that involves immune and inflammatory cells and processes that are targets for immune regulatory actions of vitamin D as a neurosteroid hormone. In addition to vitamin D’s immune modulatory properties, its receptors have been identified in skeletal muscles. It also affects sensory neurons to modulate pain. In this study, we aim to study the role of vitamin D3 in discogenic pain and related sensory deficits. Additionally, we will address how post-treatment 25-hydroxy vitamin D3 level influences pain and sensory deficits severity. The cut-off value for serum 25-hydroxy vitamin D3 that would be efficacious in improving pain and sensory deficits in lumbar disc herniation will also be studied. Methods/Design We will conduct a randomized, placebo-controlled, double-blind clinical trial. Our study population will include 380 cases with one-level and unilateral lumbar disc herniation with duration of discogenic pain less than 8 weeks. Individuals who do not have any contraindications, will be divided into three groups based on serum 25-hydroxy vitamin D3 level, and each group will be randomized to receive either a single-dose 300,000-IU intramuscular injection of vitamin D3 or placebo. All patients will be under conservative treatment. Pre-treatment and post-treatment assessments will be performed with the McGill Pain Questionnaire and a visual analogue scale. For the 15-day duration of this study, questionnaires will be filled out during telephone interviews every 3 days (a total of five times). The initial and final interviews will be scheduled at our clinic. After 15 days, serum 25-hydroxy vitamin D3 levels will be measured for those who have received vitamin D3 (190 individuals). Trial registration Iranian Registry for Clinical Trials ID: IRCT2014050317534N1 (trial registration: 5 June 2014)

In addition to the information described above, many studies about changes that occur in LDH have been done, as outlined below.
1. The contribution of inflammatory cytokines in the pathogenesis of LDH has been widely addressed in the literature. The herniated nucleus pulposus, either with immunogenic properties itself or by inducing an immunologic response in the nerve roots, dorsal root ganglia and surrounding muscles, is the starting point for the cascade of inflammation initiated through immune cell activation and infiltration and cytokine release . 2. Neuropathic pain involves the activation of neurons, glial cells and the immune system [185,186]. Dorsal root ganglia and dorsal roots play important roles in LDH, not only by the effect of released inflammatory cytokines but also by actively amplifying inflammation by producing proinflammatory cytokines and pain mediators that affect pain perception and nociception. Among these substances is brain-derived neurotrophic factor. Its receptor has been identified in intervertebral discs, with its expression being increased during inflammatory conditions such as LDH and its neuroimmunomodulatory role in the dorsal root of the spinal cord [185,[187][188][189][190][191][192][193][194][195][196][197][198][199][200][201][202][203][204]. The other factor is glial cell-derived neurotrophic factor (GDNF). It has been shown that GDNF reduces neuropathic pain states [188,190,[205][206][207][208]. Interestingly, vitamin D affects neuropathic pain by directly suppressing inducible nitric oxide that is expressed in glial cells [96,136] or by affecting other substances, such as reactive oxygen species or glutamate. Given the immunomodulatory action of vitamin D, it is possible that it could downregulate inflammatory chemokines released by glial cells [96,[185][186][187][188][189][209][210][211][212][213][214][215]. It has been suggested that vitamin D attenuates ischemia-induced brain injury that is thought to be mediated through upregulation of GDNF, in addition to its role in nitric oxide (NO) suppression [216]. The results of other studies support the hypothesis that GDNF is upregulated by vitamin D [90,94,96,190,217]. Interleukin 6 (IL-6) and tumor necrosis factor α produced by glial cells were shown to be downregulated by vitamin D [94,96,136], as were glial cell release of NO [188,218,219], prostaglandin [188], IL-1 and IL-6 [218], which, as described below, could be suppressed by vitamin D administration. Glial cells have glutamate receptors that are important in the process of nociception [220][221][222][223][224]. Therefore, vitamin D, through its immunoregulatory properties, affects another important cell population that is inflamed in disc herniation, either through suppressing neurotoxic agents or by its action on neurotrophins.

Detailed study of inflammatory cytokines and
Another interesting aspect of vitamin D deficiency is how it promotes skeletal muscle hypersensitivity and sensory hyperinnervation [275]. Vitamin D supplementation was shown to increase the diameter of type II muscle fibers [181,276]. It also influences transdifferentiation of muscle cells to adipose cells [277]. With regard to the presence of vitamin D receptor in skeletal muscles [151][152][153][154][155], its effect on muscle growth and proliferation [278][279][280][281][282] and the changes seen in muscles after LDH, we propose that vitamin D supplementation also influences muscle changes in this condition.

Methods/Design
Design of the study We will conduct a randomized, placebo-controlled, doubleblind clinical trial.

Statement of ethical approval
This study was approved by the local research ethics committee of Shiraz University of Medical Sciences, Shiraz, Iran (CT-P-92-6632).

Informed consent
Informed consent will be obtained from all participants.

Setting
We will recruit patients who have appointments at the neurosurgery outpatient departments of the universityaffiliated hospitals of Shiraz, Iran.

Participants
We will recruit 380 patients with LDH proven by physical examination and confirmed by magnetic resonance imaging.

Intervention
Patients in the intervention arm will receive single-dose intramuscular injections of 300,000 IU of vitamin D 3 (1 ml). Individuals will be informed about the nature of this study.

Inclusion criteria
The following are the inclusion criteria: 1. Single-level LDH 2. No coexistent or preexisting spine pathology (for example, spondylolysis, spondylolisthesis, infection, tumors, fracture) 3. Discogenic pain duration less than 8 weeks from onset to physician's evaluation

Exclusion criteria
The following are the exclusion criteria: 1. Daily supplementation of more than 800 IU of vitamin D 3 2. Serum calcium level above 10.5 mg/dl 3. Hypercalciuria (spot urine calcium/creatinine ratio above 0.4) 4. Lymphoma, sarcoidosis, tuberculosis (TB), hyperparathyroidism, celiac disease or malabsorption syndromes 5. History of kidney stones 6. History of inflammatory back pain 7. Impaired renal function tests (glomerular filtration rate less than 30 ml/min/1.73 m 2 ) 8. Impaired hepatic function tests 9. Abnormal serum phosphorus, alkaline phosphatase and parathyroid hormone values 10. Fasting blood sugar above 126 mg/dl 11. Previous spine surgery 12. History of trauma 13. Taking anticonvulsant, anti-TB medications or vitamin D 3 analogues 14. Cauda equine syndrome that requires emergency surgical decompression

Laboratory Assessments
The following laboratory workups will be performed for all included participants: serum 25-hydroxy vitamin D 3 level, serum calcium, serum phosphorus, alkaline phosphatase, parathyroid hormone, liver function tests (bilirubin (direct and total), alanine transaminase, aspartate transaminase, total protein, total albumin), blood urea nitrogen, creatinine, spot urine for calcium and fasting blood sugar. Clinic-based pre-intervention interviews and physical examinations will include the following:

Randomization
Patients will be categorized on the basis of their serum 25-hydroxy vitamin D 3 levels into three groups: Group 1: Optimum 25-hydroxy vitamin D 3 level (32 to 50 ng/ml) Group 2: Deficient 25-hydroxy vitamin D 3 level (less than 10 ng/ml) Group 3: Insufficient 25-hydroxy vitamin D 3 level (less than 32 ng/ml) Each of the groups will be randomized, based on randomly computer-generated numbers, into two groups to receive intramuscular injection of either 300,000 IU of vitamin D 3 (1 ml) or distilled water (1 ml). All patients will be prescribed daily 15 mg Meloxicam capsules. Our study population will be warned verbally and in writing about the potential for severe adverse side effects of vitamin D 3 (nausea, vomiting, abdominal pain, metallic taste, breathing difficulties). They will have access to emergency department care should side effects occur.
The study will last 15 days. After vitamin D 3 injection, patients will be contacted by telephone every 3 days to assess the sensory and pain effects of vitamin D 3 with the McGill Pain Questionnaire and the VAS (a total of five times). Participants will be provided with the VAS so that they can look at the scale and report their pain severity during the telephone interviews.
The following are the final post-treatment evaluations that will be carried out at the clinic:

McGill Pain Questionnaire 2. VAS (for low back pain and radicular pain) 3. Physical examination to detect any sensory deficits
Post-treatment 25-hydroxy vitamin D 3 levels (after 15 days) will be measured for those participants who have received vitamin D 3 (N = 190).

Statistical analysis
Data will be assessed by analysis of variance and paired tests.

Discussion
On the basis of the inflammatory nature of disc herniation and the immunomodulatory effects of vitamin D, as well as the existence of vitamin D receptors in various parts of areas that are affected in the process of disc herniation, we propose a novel role for vitamin D in the treatment of discogenic pain and sensory deficits related to this pathology. We hypothesized that vitamin D 3 plays a role in reducing the severity of discogenic pain and that vitamin D 3 can improve discogenic-related sensory deficits.
The following are our general objectives in this trial: 1. Effect of vitamin D 3 on discogenic pain 2. Effect of vitamin D 3 on discogenic sensory deficits 3. Effect of posttreatment 25-hydroxy vitamin D 3 level on pain and sensory deficit severity 4. Determining a cut-off level of 25-hydroxy vitamin D 3 that is efficient in improving pain and sensory deficits The following are our applicative objectives: 1. Proposing vitamin D 3 as part of medical treatment for LDH 2. Improving LDH patients' quality of life 3. Decreasing the economic and health burden of LDH Our ultimate goal in this study is to introduce a new treatment strategy for the treatment of discogenic pain.

Trial status
The study protocol has been approved by the Vice-Chancellor for Research of Shiraz University for Medical Sciences. Recruitment has not been initiated.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions MS contributed to the acquisition and study of background data, proposed the novel role for vitamin D in the treatment of lumbar disc herniation, suggested the design of the study and how it will be carried out, and helped develop the inclusion and exclusion criteria and laboratory studies. AH participated in the design of the study and how it will be carried out and developing the inclusion and exclusion criteria. Both authors read and approved the final manuscript.