of Medicine, Chiba University, Chiba, Japan

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1 Inhibiting Nerve Growth Factor or Its Receptors Downregulates Calcitonin Gene-Related Peptide Expression in Rat Lumbar Dorsal Root Ganglia Innervating Injured Intervertebral Discs Sumihisa Orita, 1 Seiji Ohtori, 1 Maiko Nagata, 2 Manato Horii, 2 Masaomi Yamashita, 1 Kazuyo Yamauchi, 1 Gen Inoue, 1 Munetaka Suzuki, 1 Yawara Eguchi, 1 Hiroto Kamoda, 1 Gen Arai, 1 Tetsuhiro Ishikawa, 1 Masayuki Miyagi, 1 Nobuyasu Ochiai, 1 Shunji Kishida, 1 Masashi Takaso, 1 Yasuchika Aoki, 1 Kazuhisa Takahashi 1 1 Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, Inohana, Chuo-ku, Chiba , Japan, 2 School of Medicine, Chiba University, Chiba, Japan Received 19 February 2010; accepted 22 March 2010 Published online 24 May 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI /jor ABSTRACT: Nerve growth factor (NGF) and its dual structurally unrelated receptors, tropomyosin-related kinase A (TrkA) or p75 neurotrophin receptor (p75 NTR ), cause the pathogenesis of discogenic pain. To investigate the sensory innervation of injured rat lumbar intervertebral disc (IVD), we examined the expression of neuropeptides such as calcitonin gene-related peptide (CGRP) at dorsal root ganglia (DRG) by inhibiting NGF or its dual receptors. Sprague Dawley rats with multiply punctured L5 L6 IVD were used. Six experimental groups were prepared: naïve, sham control, and four agent-treated groups with punctured IVD (vehicle, anti-ngf antibody, anti-trka antibody, and anti-p75 NTR antibody). Retrograde neurotracer Fluoro-Gold (FG) was applied together except for the naïve group. Their lumbar DRG were harvested and immunolabeled for CGRP. FG-labeled DRG neurons were most prevalent at L1 and L2 DRG, and the proportion of FG-labeled CGRP-immunoreactive DRG neurons in the vehicle group was significantly elevated (p < 0.05) compared with the sham group, while those of antibody-treated groups, especially in the anti-p75 NTR group, significantly decreased compared with the vehicle group (p < 0.05). Direct intradiscal application of antibody to NGF or its receptors suppressed CGRP expression, and p75 NTR antagonism induced the most profound suppression Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J. Orthop. Res. 28: , 2010 Keywords: discogenic low back pain; calcitonin gene-related peptide (CGRP); nerve growth factor (NGF); tropomyosin-related kinase A (TrkA); p75 neurotrophin receptor (p75 NTR ) Lumbar intervertebral disc (IVD) is a source of low back pain. Previous studies demonstrated that degenerative IVD expresses proinflammatory cytokines such as tumor necrosis factor (TNF)- 1 and nerve growth factor (NGF) 2 and induces sensory nerve ingrowth into the injured sites to transmit nociceptive sensation. 3 NGF is one of the neurotrophins involved in certain chronic inflammatory or neuropathic pain states. 4 9 NGF produced at the inflamed or degenerative site of IVD is one of the causes for discogenic low back pain based on evidence that the significant dominant population of the sensory innervation derived from dorsal root ganglia (DRG) for IVD is sensitive to NGF. 10 We demonstrated that the innervation of L5 L6 IVD consists of multisegmental innervation through paravertebral sympathetic trunks and direct innervation through sinuvertebral nerves on the posterior longitudinal ligament. 11,12 Among these DRG neurons, All protocols for animal procedures were reviewed and approved by the ethics committees of our institution (Ethics Committees, Graduate School of Medicine, Chiba University), and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (1996 revision). None of the authors have any conflict of interest or disclosures to report in relation to this work. Correspondence to: Sumihisa Orita (T: ; F: ; sumihisa@silver. .ne.jp) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. small-sized neurons are mainly involved in pain perception. Some small neurons are NGF-dependent for activation and survival and contain inflammatory painrelated neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP). 13,14 Among them the neuropeptide CGRP produces hyperalgesia via both protein kinase A and C second-messenger pathways, which suggests that elevated CGRP expression produces pain. 15 NGF acts by binding with two structurally unrelated receptors: tropomyosin-related kinase A (TrkA), which is specific for NGF, and p75 neurotrophin receptor (p75 NTR ), which has low-affinity for NGF and common to other neurotrophins TrkA at the peripheral nerve terminal is involved in the production of an NGF TrkA complex that is retrogradely transported to affect and modulate changes in gene expression in the DRG soma producing SP and CGRP. 20 Thus, degenerative IVD with increased production of NGF and nerve fibers expressing TrkA leads to activation of primary afferent neurons to cause discogenic pain We reported that p75 NTR as well as TrkA are expressed on DRG neurons innervating IVD, 24 suggesting that p75 NTR may be involved in discogenic pain pathogenesis. We now aimed to investigate the involvement of NGF and its receptors in discogenic pain through inhibiting each molecule in rodent models. Furthermore NGF antagonism produces adverse events such as upper respiratory infection, paresthesia, and headache. 23 We 1614

2 NGF INHIBITION ATTENUATES CGRP PRODUCTION 1615 therefore also examined pathological upper respiratory tract infection. METHODS All protocols were approved by our ethics committees (Graduate School of Medicine, Chiba University), and followed NIH Guidelines for Care and Use of Laboratory Animals (1996 revision). Experimental Groups Sixty female Sprague Dawley rats weighing g (CLEA, Japan, Tokyo) were equally divided into six groups: naïve, sham control (sham), and four agent-treated groups (vehicle, anti-ngf, anti-trka, and anti-p75 NTR groups). In groups treated with the agents, L5 L6 IVDs were multiply punctured to cause degeneration, 25,26 and crystals of the neurotracer Fluoro-Gold (FG; Fluorochrome, Denver, CO) were applied intradiscally with the following agents as appropriate: 15 l sterilized saline in the vehicle group, 3 g anti-ngf antibody (Santa Cruz, Delaware, CA), anti-trka antibody (Chemicon, Temecula, CA), and anti-p75 NTR antibody (Santa Cruz). The appropriate volume of anti-ngf antibody was determined from a previous report, 27 and the volumes of antibodies against the two receptors were suggested by the anti-ngf antibody volume. For the sham groups, the L5 L6 IVD was exposed and FG alone applied to the surface. Retrograde FG Labeling and Induction of L5 L6 Intervertebral Disc Degeneration All rats except the naïve group were anesthetized with an intraperitoneal injection (i.p.) of sodium pentobarbital (40 mg/kg) and treated aseptically throughout the experiments. A midline abdominal incision was made, and the retroperitoneum was exposed. Then the ventral portion of the L5 L6 IVD was exposed. For the treated groups requiring a punctured IVD (vehicle, anti-ngf, anti-trka, or anti-p75 NTR ), the exposed portion of the IVD was punctured 10 times with a 23-gauge needle to a depth of about 1.5 mm. Then crystals of FG were applied into the L5 L6 IVD by filling a needle tip with FG and the agent appropriate to each group. The application site was immediately sealed with cyanoacrylate-based glue to prevent leakage of the FG. After confirmation that none had leaked into peripheral tissues, the surface of the abdomen was closed with suture thread. In the sham group, FG was applied on the surface of the L5 L6 IVD. These procedures were performed according to our previously reported method. 11,12 Immunohistochemistry of the DRGs One week after the surgery, rats were anesthetized and perfused transcardially with 0.9% saline, followed by 500 ml of 4% paraformaldehyde in phosphate buffer (0.1 M, ph 7.4). Bilateral DRGs from L1 to L6 levels were harvested and immersed in buffered paraformaldehyde for 12 h at 4 C, and then in phosphate-buffered saline (PBS) containing 20% sucrose for 20hat4 C. Subsequently, they were embedded in OCT Tissue Tec (Sakura Finetek Japan, Tokyo) and frozen in liquid nitrogen. Each sample was sectioned at 10 m thickness on a cryostat (Leica Microsystems, CM3050S, Wetzlar, Germany), and then endogenous tissue peroxidase activity was quenched by soaking the sections for 30 min in 0.3% hydrogen peroxide solution in PBS. The sections were then treated for 90 min at room temperature with a blocking solution consisting of PBS containing 0.3% Triton X-100 and 3% skim milk. They were processed using a rabbit antibody to CGRP (1:1,000; Immunostar, Hudson, WI) diluted with blocking solution for 20 h at 4 C. After incubation with the primary antibody, sections were incubated with Alexa 488-conjugated goat anti-rabbit IgG (1:1,000; Molecular Probes, Eugene, OR). After each step, the sections were rinsed three times in PBS and observed with a fluorescence microscope (Olympus, Tokyo, Japan) in a treatment-blinded manner to count the number of FG-labeled DRG neurons and FG-labeled and CGRP-immunoreactive (- ir) DRG neurons. The proportion of FG-labeled CGRP-ir DRG neurons to the number of FG-labeled DRG neurons was calculated. Possible Respiratory Tract Infection Caused by Inhibiting the NGF System The trachea was harvested from rats in the agent-treated groups, and embedded in paraffin for hematoxylin and eosin (HE) staining to look for histological evidence of upper respiratory infection. Statistical Analysis The proportions of FG-labeled CGRP-ir DRG neurons in each DRG were compared using Welch s unpaired t-test. The distribution of the FG-labeled DRG neurons and CGRP-ir DRG neurons was analyzed with a non-repeated measures ANOVA with Bonferroni post hoc correction. p < 0.05 was considered statistically significant. RESULTS FG-Labeled DRG Neurons FG-labeled DRG neurons were present in bilateral DRG from L1 to L6 in the FG-labeled groups (Fig. 1A). No lateral bias in the number of FG-labeled DRG neurons existed in any DRG from the same level. Small-, intermediate-, and large-sized DRG neurons were included in each specimen, and small-sized DRG neurons were counted. Figure 2 shows the segmental distribution of FG-labeled DRG neurons, indicating that the numbers of FG-labeled neurons at L1, L2, and L3 were significantly higher than in the other levels, and predominant at levels L1 and L2. CGRP Expression in Lumbar DRG Neurons CGRP-ir DRG neurons were observed in bilateral DRGs from L1 to L6 in all experimental groups (Fig. 1B). No lateral bias in the number of CGRP-ir DRG neurons existed in any DRG at the same level. Table 1 shows the segmental distribution of CGRPir DRG neurons. Figure 3 shows the average CGRP expression through the lumbar DRGs in each group determined from Table 1. The decreasing incidence of CGRP expression in each group was not significantly different among all of the DRGs compared with the control group, which allowed us to average the proportion of CGRP expression. The distribution in each DRG did not show a significant difference in any group. The proportions of CGRP expression showed no significant difference between the sham (26.3 ± 4.3%) and naïve (25.0 ± 2.8%) groups. CGRP expression was significantly elevated in the vehicle group (54.0 ± 5.9%, p < 0.05). CGRP expression was decreased in the antibody-treated groups, and significant differences existed among groups (anti-ngf

3 1616 ORITA ET AL. Figure 1. Representative fluorescence photomicrographs of L2 DRGs in the anti-ngf group. (A) FG-labeled DRG neurons, (B) CGRP-ir DRG neurons. (A) and (B) are from the same section. The scale bars are 200 m. Arrows in (A) and (B) show FG-labeled CGRP-ir DRG neurons ± 3.7%, anti-trka 28.6 ± 2.1%, and anti-p75 NTR 27.8 ± 2.4%, respectively) compared with the vehicle group. CGRP expression in DRG from the anti-ngf group was significantly higher than in the other two antibodytreated groups (p < 0.05). There were no significant differences in CGRP expression in DRG between the anti-trka group and the anti-p75 NTR group. CGRP-ir FG-Labeled DRG Neurons FG-labeled CGRP-ir DRG neurons innervating the L5 L6 IVD were present in all the DRG (Fig. 4). DRG neurons from L1 to L3 DRG were significantly higher in all experimental groups. Table 2 shows the segmental distribution of CGRP-ir FG-labeled DRG neurons. Figure 4 shows the average CGRP expression through the FG-labeled DRGs in each group calculated from Table 2. The proportion of FG-labeled CGRP-ir DRG neurons was significantly higher (34.1 ± 3.5%; average of all DRG) in the vehicle group than in the other groups (p < 0.05). Among the antibody-treated groups, no significant difference existed between the proportion in the anti-ngf group (21.6 ± 2.5%) and the anti-trka group (22.6 ± 3.1%). The proportion of the FG-labeled CGRP-ir DRG neurons in the anti-p75 NTR group was Figure 2. The segmental distribution of FG-labeled DRG neurons in the groups with FG-labeled IVD. No significant difference existed among the groups at each DRG level. The proportions of FG-labeled DRG neurons from L1 to L3 were significantly higher than at other levels, especially in L1 and L2 (p < 0.05). significantly decreased compared with the other two antibody-treated groups (13.6 ± 3.0%, p < 0.05). Confirmation of Pathological Upper Respiratory Tract Inflammation No evidence of infection such as infiltration of inflammatory cells or hyperplasia of mucous cells was observed in any of the treated groups (i.e., vehicle, anti-ngf, anti- TrkA, or p75 NTR group, Fig. 5). DISCUSSION The present study demonstrated that inhibiting NGF or its receptors suppresses CGRP expression in lumbar DRG innervating injured IVD in rats. Of the two NGF receptors, TrkA and p75 NTR, inhibiting p75 NTR produced the most profound suppression. FG-Labeled DRG Neurons Innervating L5 L6 Intervertebral Discs L1 L3 DRG were found to predominantly innervate the L5 L6 IVD. Previous reports indicate that the sensory fibers from Th13 to L2 DRG innervate the dorsal portion of the L5 L6 IVD through the paravertebral sympathetic trunks, while those from the L3 to L6 DRG innervate through the sinuvertebral nerves. 11 Here we showed few FG-labeled DRG neurons in the L4 L6 DRG, suggesting that sinuvertebral nerves affected few elements. Suppressed CGRP Expression in Lumbar DRG Neurons The elevated CGRP expression in the vehicle group may suggest its contribution to the pathogenesis of discogenic pain. CGRP expression in the antibody-treated groups decreased both in the total DRG neurons and in the FG-labeled DRG neurons. The decreased CGRP expression at all DRG levels may be due to systemic spread of agents in addition to direct transmission via DRG nerve fibers. In FG-labeled CGRP-ir DRG neurons, the expression was significantly lower in the anti-p75 NTR group than in the anti-ngf and anti-trka groups, which should be important in discussing their role. The NGF trka signaling complex plays an important role along pathways in peripheral nerves to the

4 NGF INHIBITION ATTENUATES CGRP PRODUCTION 1617 Table 1. Proportion of CGRP-ir DRG Neurons to Total DRG Neurons (Mean ± SE (%)) Group L1 L2 L3 L4 L5 L6 Average Non-punctured Naïve 25.4 ± ± ± ± ± ± ± 2.8 Sham 27.2 ± ± ± ± ± ± ± 4.3 Punctured Vehicle 63.7 ± ± ± ± ± ± ± 3.9 Anti-NGF 41.3 ± ± ± 5.0, 36.6 ± 3.8, 34.4 ± 4.4, 31.1 ± ± 3.7 Anti-TrkA 29.9 ± 2.3, 29.5 ± 1.9 **, 27.0 ± 1.7, 28.5 ± 2.5, 28.8 ± 2.5, 27.8 ± ± 2.1 Anti-p75 NTR 33.9 ± 2.1, 29.7 ± 1.2, 30.2 ± 4.2, 27.2 ± 2.5, 22.8 ± ± ± 2.4 There was no significant difference among the L1 L6 DRGs within each group. The proportions in the agent-treated groups were significantly higher than those in the control group at each DRG level (p < 0.05, text in bold). The proportions were significantly higher than those of the other groups at all DRG levels (p < 0.01). The proportions were significantly lower than those of the sham group and the vehicle group (p < 0.05). No significance among the marked groups in the same DRG level. Figure 3. Average CGRP expression in the experimental groups. CGRP expression was significantly elevated in both the sham and the vehicle groups (p < 0.05). Significant decreases were observed in the groups treated with antibodies compared with the sham and vehicle groups (p < 0.05), while no significant difference existed between the anti-trka and the anti-p75 NTR group. cell bodies of nociceptive neurons, leading to alterations in gene expression of CGRP. 28 p75 NTR is essential for formation of this signaling complex, while it does not serve as a functional receptor for signal transduction itself. 29,30 Furthermore several reports have described the interactions between TrkA and p75 NTR ; p75 NTR interacts with Trks to enhance neurotrophin affinity and specificity of binding to Trks Neither TrkA nor p75 NTR form high-affinity NGF binding sites when expressed alone, but coexpression of the two receptors results in formation of high-affinity NGF binding sites. 34 These receptors are coimmunoprecipitated and closely located, 35 and this coexistence of p75 NTR accelerates the on-rate of binding of NGF to TrkA compared with TrkA alone. 36 The result of the present study demonstrates that p75 NTR may regulate the sensitivity to NGF. Theories exist regarding the interaction between p75 NTR and TrkA. The ligand-passing model is a model in which NGF rapidly associates with the extracellular Figure 4. Average CGRP expression in the FG-labeled DRG neurons in the experimental groups. CGRP expression of FG-labeled neurons was elevated the most in the vehicle group, and decreased in the antibody-treated groups. The expression in the anti-p75 NTR group was more suppressed than in the other antibody groups (p < 0.05), between which there was no significant difference.

5 1618 ORITA ET AL. Table 2. Proportion of FG-Labeled CGRP-Immunoreactive DRG Neurons Per Total FG-Labeled DRG Neurons from L1 to L6 (Mean ± SE (%); N.D., No Data) Group L1 L2 L3 L4 L5 L6 Average Non-punctured Naïve N.D. N.D. N.D. N.D. N.D. N.D. N.D. Sham 18.7 ± ± ± ± ± ± ± 3.5 Punctured Vehicle 52.4 ± ± ± ± ± ± ± 3.5 Anti-NGF 33.7 ± 2.8, 33.1 ± 2.6, 27.7 ± 2.6, 12.0 ± 2.1, 10.0 ± 2.5, 13.3 ± ± 2.5 Anti-TrkA 37.3 ± 3.4, 35.5 ± 3.0, 31.0 ± 4.2, 14.3 ± 2.8, 6.7 ± 2.5, 10.8 ± ± 3.1 Anti-p75 NTR 22.2 ± 1.9, 21.2 ± 2.4, 15.9 ± 2.9, 6.7 ± 4.4, 7.5 ± 4.0, 8.3 ± ± 3.0 The proportions in the vehicle or antibody-treated groups were significantly higher than those of the sham group at each DRG level (p < 0.05, text in bold). The proportions in the antibody groups were significantly lower than those of the vehicle group (p < 0.01). No significance among the marked groups in the same DRG level. The proportions were significantly lower than those of the other antibody groups (p < 0.05). Figure 5. No evidence of active infection, such as infiltration of inflammatory cells or hyperplasia of mucous cells was observed in any of the treated groups (i.e., vehicle, anti-ngf, anti-trka, or p75 NTR groups). HE staining of tracheal sections. The scale bars are 100 m. domain (ECD) of p75 NTR for its fast association rate and is then presented to the ECD of TrkA with a much slower association rate. 37,38 Such theories reflect the complex interaction of TrkA and p75 NTR, which remains to be investigated. However, p75 NTR can play an essential role in affecting NGF-triggered TrkA signaling pathways that mediate neuropathic pain. Another possible reason for the significant decrease of CGRP expression after inhibiting p75 NTR is the low affinity that enables it to bind other neurotrophins such as brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NGF. BDNF modulates and mediates nociceptive sensory inputs and pain hypersensitivity by being released in the dorsal horn to modulate or even mediate nociceptive sensory inputs and pain hypersensitivity. 39,40 It is also expressed in annulus fibrosus and nucleus pulposus cells of human IVD, and its levels increase significantly according to the severity of disc degeneration. 41,42 NT-3 correlates with the peripheral neuropathic pain by suppression of transient receptor vanilloid-1 and sodium channel expression. 43 Thus p75 NTR can induce more profound suppression of CGRP expression in the FG-labeled neurons by inhibiting neurotrophins besides NGF. Clinical Relevance of Anti-NGF Therapy Possible NGF antagonism has been postulated to be a highly effective and safe therapeutic approach for many pain states. For example, anti-ngf treatment reduces pain in both non-malignant pathological states such as fracture-induced skeletal pain 44 and bone cancer pain 45 in mice. Furthermore, NGF antagonism reduces pain from knee osteoarthritis. 46 The present study demonstrates that inhibiting the NGF system has a potential attenuation of pathological pain in degenerative IVD. Inhibiting NGF or its receptors can bring about adverse

6 NGF INHIBITION ATTENUATES CGRP PRODUCTION 1619 events, likely due to its important role in homeostasis. The present study showed no evidence of upper respiratory infection described in another report, possibly due to the difference in administration: intravenous injection versus direct single intradiscal application. The exact onset period of respiratory infection as an adverse event of NGF antagonism remains unknown, while it occurs at the rate of 7.3%. 46 However, at least at 1 week after administration, histology of the respiratory tracts showed no evidence of inflammation or infection, including no findings of possible infection. A more definitive study would require more animals and closer observation for a longer time-course after administration of the inhibitory antibodies. Previous studies indicated the involvement of NGF in the prevention of respiratory tract infection. Othumpangat et al. 47 reported that overexpression of NGF and TrkA may play a crucial role in protection against virusrelated infection in the respiratory tract using human tracheal and bronchial epithelial cell lines, and implied that its inhibition amplifies programmed cell death in infected bronchial epithelium. They also concluded that pharmacologic modulation of NGF expression may offer a promising new approach for the management of common respiratory infections. We should, therefore, consider these adverse events when discussing anti- NGF therapy. Nevertheless, NGF antagonism has great potential for pain treatment. The present study has some limitations. First, the punctured IVD model itself remains controversial. We have to consider the effect of injury in discussing the CGRP expression. The injury induced by puncture may bring about neovascularization during its repairing process that may affect the innervation of the injured site. These changes in vascularization and innervation may bring about more CGRP expression than might be found in naturally occurring degenerative states. Second, we were uncertain of the amount of NGF or its receptors in the injured IVD. Third, we only investigated CGRP expression in the DRG neurons. We did not investigate the sensory nerve endings at the IVD or at the dorsal horn of spinal cord. Fourth, we did not investigate a dose-dependent effect of the antibodies. If anti-ngf therapy were to advance to human trials, it will be important to define the lowest dose efficacious in reducing discogenic pain without significant unwanted side effects. A further investigation of the changes of NGF and its receptors in a future study would be desirable. Finally, we did not conduct any behavioral studies of pain reduction. Further study is needed to clarify these points. In conclusion, the increased production of CGRP was observed in DRG neurons innervating injured IVD in rats. 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