Expression And Significance Of Growth-associated Protein 43 In A Rat Model Of Intervertebral Disc Inflammation

Discogenic low back pain is a common disease in clinic orthopaedics, which can develop into chronic low back pain that is very difficult to treat effectively. With the life and work quickened, it has become one of the major illness in modern life. However, the pathogenesis of the disease is unclear at present. In the early, we do not have effectively and specificly measure to treat. In the later, removal of intervertebral disc or interbody fusion is the major measure, but the effect of removal disc is bad, and it is easy to recur also. Interbody fusion is not only lose the function of motion segment, but also accelerate a degenerative change at unfused adjacent levels. Intervertebral disc prosthesis can not settle this problem because of the speciality of the materials and the diffculty to fix on the interbody. Therefore, it is important to explore the mechanism of discogenic low back pain and look for new therapeutic measure.Generally, nerve fibers are only presented in the outermost parts of the annulus fibrosus of the healthy human intervertebral disc. However, current studies have indicated that some nerve fibers are present in the inner parts of the annulus fibrosus, even in the nucleus pulposus of the specimens from patients with chronic low back pain. These findings suggest that nerve fibers are present in painful intervertebral discs, and this may be one of the causes of discogenic pain. It is also shown that the nerve fibers observed in the human symptomatic disc expresse Growth-associated protein 43(GAP-43). Since there are no nerve fibers in the inner layers of asymptomatic discs, the nerve fiber growth is likely to beassociated with the mechanism of discogenic pain.Recently, it has been demonstrated that the levels of pro-inflammatory mediators are higher in the degenerated discs from patients with chronic low back pain than in asymptomatic discs, suggesting that inflammatory changes occur in the painful discs. These findings indicate that persistent inflammation in the painful intervertebral disc may be one of the causes of chronic discogenic pain. At present, the research of discogenic low back pain mainly focus on inflammatory change after disc degeneration and it is lack in a systemic and in-deph research on the nerve ingrowth into degenerative disc.GAP-43 is a nerve-special protein, which is correlated with the course of nerve growth, nerve nutrition and regeneration and its expression is implicated in axonal growth and synaptic plasticity. Recently, many reserarch have demonstrated that GAP-43 may be associated with mechanisms of discogenic low back pain. However, at present, the pathophysiological role of expression of GAP-43 in disc inflammation has not been examined. So in this study, the expression of GAP-43 was investigated in DRGs and discs in a rat lumbar discinflammation model.In the paper, the study was separated into three parts. Part one: The construction and observation of the model of intervertebraldisc inflammation in ratObjection: To observe the change of the inflammation in intervertebral disc at different time points after the L5,6 disc of rat was injected with complete Freund's adjuvant.Methods: A total of 17 Male Sprague-Dawley rats weighting 200-250g were used and random grouped into experiment (n=12), control (n=4) and normal control group(n=1). A total of 50μl complete Freund's adjuvant were used to the L5, 6 intervertebral disc of rat in experimental group, and the same volume of saline were also applied to the control. After the days of 1, 3, 7, 14, the disc of L5, 6 were harvested respectively, then HE-staining was adopted to detect the change ofinflammation.Result: At 1 day after the surgery, a lot of inflammatory cells infiltrated in the disc and tissue edema was detected. Inflammatory cells Infiltration decreased while fibrous tissue hyperplasia was found at 3 day. Inflammatory cells Infiltration evidently decreased and part fibrous tissurre collagenation was found at 7 day. At 14 day, fibrous tissue collagenation was seen evidently while inflammatory tissue evidently decreased. In the control, only some inflammatory cells were found at punctured point at 1 day. There were cartilage and fibrous tissue in the normal intervertebral disc.Conclusion: Injecting CFA into intervertebral disc could mimic the inflammatory model in rat.Part two: Fluorescent immunohistochemistry detection of GAP-43 in a rat model of intervertebral disc inflammationObjective: To detect the expressive change of GAP-43 in the DRGs and discs of rats in disc inflammation at different time points using Fluorescent immunohistochemisty.Methods: A total of 61 Male Sprague-Dawley rats weighting 200-250g were used and random grouped into experiment(n=24), control(n=24), normal control (n=4) and injecting NGF group(n=9).After 7 days of application of F-G as tracer, a total of 50μl complete Freund's adjuvant were used to the experimental L5, 6 intervertebral disc, and the same volume of saline were also applied to the control. After the days of 1, 3, 7, 14, T13-L5 DRGs and discs were harvested respectively (normal control group were harvested at 3 day), then Fluorescent immunohistochemistry were adopted to detect the expression of GAP-43 with either CGRP or IB4, NGF with CGRP.After 7 days of application of F-G as tracer, the injections of rat NGF (1μg or 10μg reconstituted NGF in 30 301μl of saline) were applied to L5, 6 intervertebraldisc (n=3, resecptively), the same volume of saline were also injected to the control (n=3). After the 3 days, T13-L5 DRGs were harvested respectively, then Fluorescent immunohistochemistry were adopted to detect the expression of GAP-43. Result:1. The number of GAP-43 immunoreactive (GAP-43-IR) cells in the DRGs increase was seen as early as 1 day after surgery, reached to the peak at 3 day, subsequent decreased. There was a significant difference between inflammatory group and the control at 3 day (p<0.01).2. The number of NGF immunoreactive (NGF-IR) cells in the DRGs increase was seen as early as 1 day after surgery, subsequent decreased. There was a significant difference between inflammatory group and the control at 1 day3. The number of GAP-43 immunoreactive (GAP-43-IR) cells in the DRGs increase was seen in 1μg and 10μg group at 3 days afer rat NGF (1μg or 10μg reconstituted in 30 301μl of saline) were applied to L5,6 intervertebral disc. There was a significant difference between 10μg group and 1μg group or the control4. With the double-staining of GAP-43 with CGRP or IB4, 85.8%(28 of 33) of the GAP-43-ir neurons expressed CGRP while none shown binding IB4 in the control group. In the inflammatory group, 89.5% (43 of 48) of the GAP-43-ir neurons labeled by F-G application were positive for CGRP while none bound IB4.5. With the cross-section area of the GAP-43-ir DRG neurons innervating the lumbar intervertebral disc, in the control group, GAP-43-ir was observed only in the small F-G-labled neurons while in inflammatory group some GAP-43-ir were larged. However, there was no significant difference in the mean cross-section area of the F-G-labled neurons and GAP-43-ir neurons between inflammatory and control groups.6. Nerve fibers were observed only in the outer layer of the annulus fibrosusin both groups. GAP-43-ir nerve fibers were occasionally found (8 of the 24 discs) in the control group but were more frequently observed in the inflammatory group (17 of the 24 discs). CGRP-ir nerve fibers were evident in all samples in both groups. In contrast, no IB4-positive nerve fibers were detected in the disc in either group.Conclusion: Inflammation of intervertebral disc induced an increased expression of NGF at discs and DRGs; Inflammation of intervertebral disc induced an increased expression of GAP-43 in discs and DRGs. There was a time lag between the expression of GAP-43 and NGF; The possible correlation between them was demonstrated by the expression of GAP-43 up-regulated differently after disc was injected with different dose of NGF; Most GAP-43-ir neurons were small nociceptive ones and small nociceptive DRG neurons were more likely to grow into the inflammatory disc than large proprioceptive DRG neurons.Part three: Expression of GAP-43 in mRNA levels in DRG neurons of a rat model of intervertebral disc inflammationObjective: To detect the expressive change of GAP-43 in mRNA levels in the DRG of rats in disc inflammation at different time points using in situ hybridization histochemisty(ISHH) and RT-PCR..Methods: A total of 51 Male Sprague-Dawley rats weighting 200-250g were used and random grouped into experimental group (n=24, half for ISHH and RT-PCR), control group (n=24, half for ISHH and RT-PCR) and normal control (n=3, for RT-PCR). A total of 50μl complete Freund's adjuvant were used to the experimental L5, 6 intervertebral disc, and the same volume of saline were also applied to the control. After the days of 1, 3, 7, 14, T13-L5 DRGs were harvested respectively, then in situ hybridization histochemisty (ISHH) and RT-PCR were adopted to detect the expression of GAP-43 in mRNA levels.Result:1. In situ hybridization histochemisty revealed that the positive particle of GAP-43 was located in cytokine. Most GAP-43-ir neurons were middle and small neuron cells, and their staining was deeper and immunoreaction was stronger than the large ones.2. The expression of GAP-43mRNA in DRGs increase was seen as early as 1 day and the difference was significant compared to the control (p<0.01), subsequent decreased. There was not significant difference between inflammatory group and the control at other time points.Conclusion: Inflammation of intervertebral disc induced an increased expression of GAP-43 in mRNA levels in DRGs. There was a time lag between the increased mRNA for GAP-43 and increased expression of GAP-43 protein, which is consistent with the expressed sequence between them. Most GAP-43-ir neurons were small nociceptive ones and small nociceptive DRG neurons were more likely to grow into the inflammatory disc than large proprioceptive DRG neurons.