A Pain Related Behavior and Intervertebral Disc Degeneration After Lumbar Facetectomy in the Rat - A New Animal Model for Degenerative Disc Disease
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Introduction Intervertebral disc degeneration is thought to contribute to low back pain, however, the pathophysiological mechanisms are still controversial. Despite the reports of many experimental animal models with regard to intervertebral disc degeneration, it is still unknown if these models show evidence of pain in the animals. We developed and published a rat model of intervertebral disc degeneration induced by chronic mechanical compression to tail, and demonstrated that application of the degenerated nucleus pulposus tissues to the lumbar nerve roots enhanced hyperalgesia [1]. Pain in this model is radicular pain but not low back pain in degenerative disc disease seen in human. In addition, animal models utilizing researches for intervertebral disc degeneration have been achieved by injury or invasion to the intervertebral discs such as punctures and direct compression [1 4]. Considering clinical relevancy and pathophysiology of discogenic pain, these experimental models are thought to be quite different from patients with degenerative disc disease. To elucidate mechanisms of low back pain due to degenerative disc disease, it is necessary to develop an animal model, in which the intervertebral disc does not receive direct invasion, and which shows evidence of pain. The purpose of the present study was to introduce an experimental animal model of intervertebral disc degeneration with possible pain behavior induced by lumbar facetectomy. Materials and Methods The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee in our institute. Twenty male Sprague-Dawley rats, each weighing about 250 g were used. All surgical procedures were performed with the rats anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Rats were divided into three experimental groups. In the sham group (n = 6), only exposure of bilateral facet joints at the L4-5 level was performed. Ten rats underwent complete resection of bilateral L4-5 facet joints without neural tissue injuries (the experimental group). The operative fields were closed in layers with 4-0 nylon sutures. Naïve rats were used for the control (n = 4). After the surgery, behavioral analysis under a spontaneous walking was performed in all rats of each group utilizing the Cat-Walk method [5] over time. As a radiological analysis, dynamic lumbar lateral X-rays of flexion and extension were taken at the time when behavioral changes were observed. We compared the disc height and intervertebral disc mobility using a remote computer workstation (Virtual Place Lexus 64, Aze Co Ltd, Tokyo, Japan). The anterior, middle and posterior disc heights at the L4-5 level were measured and the intervertebral disc mobility was calculated as a ratio of the anterior intervertebral height to the posterior intervertebral height at the flexion and extension positions. The intervertebral disc mobility was evaluated as the flexion and extension ratios, respectively. After taking X-rays, the lumbar spine was harvested. After fixation in 10% neutral buffered formalin, the specimens were decalcified in 10% ethylene-diaminotetraacetic acid (EDTA) solution and then embedded in paraffin wax. The specimens were sectioned at the L4-5 level in the sagittal plane. At least 10-15 histological slides were prepared for each sample with 2-3 paraffin sections of 6m thickness per slide. Each stain was repeated twice to confirm the reproducibility of results. Hematoxylin-Eosin and safranin O fast green stainings were used to evaluate histological changes of the intervertebral disc and matrix synthesis among the groups. For immunohistochemistry, the following antibodies were used: monoclonal rat anti—aggrecanase-1 (ADAMTS4), anti-matrix metallopeptidase (MMP) 13, anti-Interleukin (IL) -1β and antitumor necrosis factor (TNF)-α. Each of the sections used for immunohistochemistry was graded blinded and in agreement by two investigators. Relationships between behavioral changes including a pain-related behavior, radiological and histological changes were examined. Data obtained from behavioral measurements were analyzed by ANOVA and Student's t-test. P values < 0.05 were considered significant. Results All rats in each group did not show any motor paresis and stress reaction during the experimental period. In the walking analysis, the reduction of the rate of step sequence regularity index was only seen in the experimental group three weeks after the surgery, compared with the control and sham groups. After that, the rate of step sequence regularity index in the experimental group returned to normal and there were no difference in this rate among three groups. At seven weeks postoperatively, however, a reduction of the swing speed of legs, the average speed, the stride length and the rate of step sequence regularity index, an increase in the number of steps, and a decrease in optical intensity of posterior feet were observed in the experimental group, but not in the control and sham groups. There were statistically significant differences in these behavioral observations among three groups (P<0.05). In the experimental group only, walking with legs apart was also seen 7 weeks after the surgery (P<0.05). Radiographic analysis revealed that the disc height ratio and extension ratio of the experimental group was larger than the control and sham groups (P<0.05), and that anterior osteophyte formation at the L4-5 level was seen in 80% of the experimental group. There was no translational instability such as slippage of the vertebral body at the L4-5 level. In the histological and immunohistochemical analysis, the existence of diffuse nucleus pulposus cells in the matrix was found in these groups. The cellularity of the nucleus pulposus was decreased in the experimental group. Decrease in extracellular matrix content and increase in ADAMTS4, MMP13, IL1β and TNF-α immunoreactivities were seen in the experimental group, compared with the control and sham groups. Discussion In the present study, we found that lumbar facetectomy resulted in abnormal walking patterns 7 weeks after the surgery and degeneration and expression of inflammatory cytokines in the intervertebral disc at the facetectomy level. This is a first report of the animal model in which the intervertebral disc has no surgical invasion directly at the operation, and which shows evidence of abnormal walking pattern. In addition, we considered the possibility that there were relationships between the gait abnormalities observed by the walking analysis system and radiological changes of intervertebral disc in the experimental group. Changes of the disc height and an increase in the extension ratio seen in radiological examination suggest segmental instability of the lumbar spine and might result in biomechanical and biological abnormalities of the intervertebral disc. Among abnormal walking patterns, a decrease in optical intensity of posterior feet observed in the experimental group is thought to be a pain related behavior observed in neuropathic pain models [6]. A clinical study demonstrated that an etiology of chronic low back pain might be neuropathic pain [7]. Collectively, the intervertebral disc abnormality after lumbar facetectomy might induce neuropathic pain. This finding suggests that we consider pain secondary to intervertebral disc degeneration in no relation to nerve root irritation. In future, it is necessary to perform quantitative molecular biological analysis of degenerative intervertebral disc and to make clear pathophysiological mechanisms of abnormal walking pattern in this model. This animal model may be useful to elucidate mechanisms of degenerative disc disease with chronic low back pain seen in human. Conclusions We developed an animal model, in which lumbar facetectomy resulted in delayed intervertebral disc degeneration and abnormal walking pattern in the rat. This model has no direct invasion to the intervertebral disc and might showed evidence of neurogenic pain without nerve root irritation. References [1] Kawakami M, et al. JOR. 2003;21: 535. [2] Masuda K, et al. Spine. 2006;31:742. [3] Sobajima S, et al. Spine. 2005;30:15. [4] Lotz JC. Spine.2004;29:2742. [5] Vogelaar CF, et al. Brain Res. 2004;1027:67. [6] Vrinten DH, et al. Pain. 2003;102:203. [7] Freynhagen R, et al. Curr Med Res Opin. 2006;22:1911. Significance: We developed a rat model, in which the intervertebral disc did not receive direct invasion, and which showed evidence of pain, to elucidate mechanisms of low back pain due to degenerative disc disease. In this animal model, we suggest that delayed intervertebral disc degeneration results in neurogenic pain without nerve root irritation.
[1] R. Baron,et al. painDETECT: a new screening questionnaire to identify neuropathic components in patients with back pain , 2006, Current medical research and opinion.