CGRP Expression in Joint Afferents Compared to All Other

In the C7 DRG, 41.5±5.4% of all of the neurons were CGRP-positive. However, 54.4±15.3% of CTb-positive neurons at that level expressed CGRP (Table 4.1), and this difference in the ratios of CGRP-positive neurons between these two populations of neurons was significant (p=0.0084). This trend was also observed in each of the groups but was not significant for any of the groups. Interestingly, the average cross-sectional area of neurons positive for both CTb and CGRP at the C7 level (724±133µm2) was significantly smaller (p=0.0005) than the average area of all the CGRP-positive neurons in the C7 DRG (892±116µm2) (Table 4.2). Although this relationship was consistent for all of the experimental groups, the differences within each group were not significant.

Data summarizing the ratio of CGRP-positive neurons among all afferents as well as specifically among joint afferents are included in Appendix H.

4.5. Discussion

These data characterize a multi-segmental innervation of the C6/C7 facet joint in the rat and demonstrate that the joint innervation is unchanged at day 7 after painful mechanical joint loading (Tables 4.1 & 4.2). The applied distraction of 0.47±0.05mm in the current study is in close agreement with a previously identified distraction magnitude (0.49±0.09mm) that was found to be sufficient to induce sustained behavioral sensitivity, while a lower magnitude of distraction (0.19±0.03mm) does not induce even transient mechanical sensitivity (Dong et al., 2011). In that context, it is not likely that the joint distractions used in this study (~ 0.5mm) are induced by the normal head movements in the rat, though the physiological range of C6/C7 facet joint distraction during normal movement has not been defined explicitly. Of the spinal levels analyzed, the greatest number of neurons with projections to the C6/C7 joint had cell bodies in the C7 DRG, followed by the C8, C6, and C5 DRGs (Table 4.1). This trend in the segmental joint innervation is maintained despite an injury-induced increase in sensitivity to mechanical stimulation of the forepaw (Figure 4.1; Table 4.1). Although painful injury does not alter the percentage of joint afferents expressing CGRP in the C7 DRG, greater than one-half of the joint afferents are peptidergic (Table 4.1), but only slightly more than 40% of all neurons in the C7 DRG are peptidergic. Further, the average cell body is smaller for the peptidergic joint afferents (724±133µm2) than for all of the peptidergic neurons (892±116µm2) in the C7 DRG (Table 4.2). Although a previous study defined the C5/C6

facet innervation with or without a complete disruption of its capsule (Ohtori et al., 2003), that study did not quantify pain. This study is the first to characterize the innervation of the C6/C7 facet joint in the context of injury-induced pain, and by doing so suggests that future studies to identify the cellular responses to painful injury to this joint should be directed at assessments of the C7 spinal level.

The distribution pattern of neurons innervating the C6/C7 facet joint identified here is consistent with studies characterizing innervation of other cervical facet joints in that joint afferents originate from multiple spinal levels with one level (C7 in this case) having a dominant number of neurons (Ohtori et al., 2001; Ohtori et al., 2003). Indeed, multi-segmental innervation of facet joints is also evident in humans in which the lower cervical facets receive fibers from the medial branches of the dorsal rami above and below the joint (Barnsley and Bogduk, 1993). The finding that the most C6/C7 joint afferents originate in the C7 and C8 DRGs (Table 4.1) supports the observation of forepaw hypersensitivity (Figure 4.1) since the C7 and C8 dermatomes in the rat extend from the neck to the forepaw (Takahashi and Nakajima, 1996). Further, neurons innervating lumbar facet joints have been identified with dichotomizing axons projecting to peripheral targets (Sameda et al., 2001; Umimura et al., 2012), suggesting that some neurons innervating the C6/C7 facet joint may also possess dichotomizing axons extending into the forelimb and contributing to referred pain. Studies using multiple retrograde tracers are necessary to determine the incidence of dichotomizing axons projecting to the C6/C7 facet joint and forepaw. Nevertheless, these data indicate that the C7 spinal level is likely a major contributor to C6/C7 facet joint-mediated pain.

Both the ratio of CGRP-positive joint afferents and their phenotype are unchanged by injury (Figure 4.2; Tables 4.1 & 4.2). This finding is surprising since several studies have identified a shift in the phenotypic expression of pain-associated proteins like CGRP and brain-derived neurotrophic factor towards larger-diameter afferents in response to facet inflammation or traumatic injury (Ohtori et al., 2002; Ohtori et al., 2003). Despite the lack of change in the phenotype of joint afferents, injury- induced behavioral sensitivity may still result from afferent sensitization. Joint inflammation sensitizes afferents innervating the inflamed joint such that the threshold to activation is decreased and normally innocuous stimuli can be perceived as painful (Guilbaud et al., 1985; Schaible and Grubb, 1993; Schaible et al., 2009). Moreover, neuropeptides such as CGRP contribute to joint inflammation-induced pain and spinal neuronal sensitization (Schaible et al., 2009), demonstrating a role for neuropeptides in inflammatory joint pain. While it is unlikely that the discs and other spinal ligaments contribute to pain in this model, previous work with this same injury model demonstrated that intra-articular injection of an NSAID abolishes facet joint injury-induced pain (Dong et al., 2011). Joint inflammation is associated with hyperexcitability of the afferents innervating the joint and pain (Guilbaud et al., 1985; Tachihara et al., 2007), and inflammation contributes to facet joint loading-induced pain (Dong et al., 2011). Combining those observations with the findings of the current study that greater than 50% of joint afferents express CGRP supports a contribution of joint afferents to pain after facet joint distraction. Yet, the subpopulations of joint afferents contributing to injury-induced pain still remain unknown. CGRP- and substance P-containing fibers have been identified in human cervical facet capsular ligaments (Kallakuri et al., 2004;

Kallakuri et al., 2012), supporting the assertion that peptidergic afferents likely mediate pain in this joint. In the C7 DRG in the rat, CGRP-positive neurons account for a greater percentage of neurons innervating the C6/C7 joint than they do among all neurons in the C7 DRG (Table 4.1). Taken together, these data indicate that peptidergic joint afferents may make a greater contribution to facet joint pain than other neuronal subpopulations. Studies specifically investigating the roles of these and other populations of joint afferents in joint injury would determine their relative contributions to facet-mediated pain, helping to identify specific mechanisms contributing to joint pain as potential targets for its treatment.

Although these data provide insight into the innervation of the C6/C7 facet joint in the rat from C5 to C8, additional spinal levels also may contain joint afferents. In fact, Ohtori found that the C5/C6 facet joint in the rat contains fibers originating in the DRGs from C3-T3, although the vast majority originates in the cervical DRGs (Ohtori et al., 2003). Nevertheless, the C6/C7 facet joint is likely innervated by additional neurons with cell bodies in the upper thoracic DRGs. Only the right DRGs were analyzed in this study, despite the application of a bilateral joint distraction; there is not expected to be differences based on sides since this injury is symmetric (Lee et al., 2004). Of note, CTb may not label all joint afferents because not all sensory neurons express the ganglioside GM1, to which CTb binds. Since the majority of sensory neurons (85% of small, 45% of medium, and 60% of large diameter neurons) do express GM1 (Gong et al., 2002), the joint afferent count data (Table 4.1) likely represent the majority of neurons innervating the C6/C7 facet joint. However, it is possible that some neurons, especially among the larger myelinated neurons, may not be labeled by CTb because GM1 is not universally

expressed. The use of additional and distinct retrograde neuronal tracing agents would provide a more robust characterization of the full extent of the facet joint’s innervation. However, because nociceptors have primarily small or medium diameter cell bodies (Merighi et al., 2008) and GM1 is expressed on 85% and 45% of these afferents, respectively (Gong et al., 2002), the majority of the nociceptive afferents are likely captured using the current technique. Further, although no visible leakage of the CTb solution from the facet joint was observed immediately after injection, a small amount may have leaked from the joint into the surrounding soft tissues. Nonetheless, any such leakage likely had only a minimal impact on the neuronal counts since the number of labeled neurons innervating the facet joint in our study is consistent with those reported in a study without joint injury in which cyanoacrylate was applied as a joint sealant (Ohtori et al., 2003).

This study identified no differences in the ratio or cross-sectional area of CGRP- positive joint afferents after injury; however, other peptides such as substance P may be differentially upregulated in these neurons. Previous work using this model identified increased substance P and the prostaglandin E2 receptor, EP2, in the DRG after painful joint injury (Lee and Winkelstein, 2009; Kras et al., 2013a), supporting that additional targets may be upregulated by afferents after injury. The lack of change in the ratio and cross-sectional area of CGRP-positive joint afferents observed in this study after injury (Tables 4.1 & 4.2) may be due to the small sample sizes. Indeed, a previous study by Ohtori required nearly twice as many rats in each group to identify changes in the ratio and size of joint afferents expressing CGRP after a joint capsule transection compared to controls (Ohtori et al., 2003). Additional studies including larger group sizes are

necessary to verify our pilot studies finding that the ratio and cross-sectional area of the peptidergic joint afferents are unchanged by painful facet joint distraction. Despite these known injury-induced changes in the DRG, the specific roles of joint afferents in the generation and maintenance of facet-mediated pain are unknown.

4.6. Conclusions & Integration

By characterizing the segmental innervation of the C6/C7 facet joint and identifying a greater prevalence of neuropeptide expression among joint afferents compared to all other neurons in the DRG, this study has identified the C7 spinal level as most likely contributing to facet joint pain and provides direction for future studies investigating the cellular mechanisms underlying joint injury-induced pain. Because this study identified that more C6/C7 facet joint afferents originate from the C7 spinal level than any other level, studies in later chapters investigate cellular responses to injury at that level. In addition to CGRP and/or substance P, peptidergic afferents express the trkA receptor and are thus responsive to nerve growth factor (NGF) in the innervated tissue (Merighi et al., 2004; Pezet and McMahon, 2006). The abundance of peptidergic fibers innervating the C6/C7 facet joint (Figure 4.2; Table 4.1) suggests that the peptidergic afferents, and consequently NGF signaling, may make an important contribution to the development of joint pain. In fact, elevated levels of NGF are a common component of tissue inflammation (Amaya et al., 2004; Ma and Woolf, 1997; McMahon, 1996; Merighi et al., 2004), and Dong has recently shown joint inflammation is necessary for the maintenance of loading-induced facet joint pain (Dong et al., 2011), further supporting a potential role for NGF in facet pain.

Exposure to NGF has wide-ranging effects including: upregulation and increased release of neuropeptides and brain-derived neurotrophic factor (BDNF) (Merighi et al., 2004; Pezet and McMahon, 2006), hyperexcitability of spinal neurons (Hoheisel et al., 2007), and both mechanical and thermal behavioral sensitivity (Amaya et al., 2004; Malik-Hall et al., 2005). Nearly all of the aforementioned effects of NGF exposure have been documented in the rat following painful facet joint distraction (Dong et al., 2010; Kras et al., 2013c; Lee and Winkelstein, 2009; Quinn et al., 2010), including both mechanical (Figure 4.1) and thermal hypersensitivity (Figure 3.6). The previous findings of behavioral hypersensitivity in association with joint inflammation (Dong et al., 2013a) and spinal neuronal hyperexcitability (Crosby et al., 2013; Quinn et al., 2010) strongly implicate NGF as a key contributor to facet-mediated pain. In fact, elevated levels of NGF have been reported in painful arthritic joints in humans (Aloe et al., 1992; Barthel et al., 2009; Raychaudhuri et al., 2011; Saito et al., 2000), and recent studies found that intravenous anti-NGF treatment alleviates pain from osteoarthritis and low back pain (Brown et al., 2012; Katz et al., 2011; Lane et al., 2010), suggesting NGF has a role in joint pain. Therefore, in order to begin to define the mechanisms by which initial joint injury leads to persistent pain, studies in Chapter 5 will identify the role of intra-articular NGF in facet joint loading-induced pain, as well as the role of the peptidergic and non- peptidergic afferents in mediating the onset of mechanical and thermal hyperalgesia due to NGF injection in the facet. Further, increased expression of BDNF in primary afferents and second order sensory neurons occurs downstream of NGF signaling (Merighi et al., 2004; Pezet and McMahon, 2006). Because BDNF has been linked to inflammatory and neuropathic pain as well as hyperexcitability of spinal neurons (Grimsholm et al., 2008;

Lu et al., 2009; Mannion et al., 1999; Ohtori et al., 2002), Chapter 6 will characterize the expression of BDNF after painful facet joint injury and define its role in the maintenance of facet-mediated pain.

CHAPTER 5

Development of Injury-Induced Facet Joint Pain

and Central Sensitization: Contributions of Intra-

Articular Nerve Growth Factor

Kras JV, Kartha S, Winkelstein BA (2014). Intra-Articular Nerve Growth Factor Regulates Development, But Not Maintenance, of Injury-Induced Facet Joint Pain & Spinal Neuronal Hypersensitivity. Osteoarthritis and Cartilage, in revision.

5.1. Overview

Studies in Chapters 3 and 4 demonstrate facet joint distraction as inducing both mechanical and thermal hyperalgesia (Figures 3.5 & 3.6) and identify greater than 50% of facet joint afferents as peptidergic (Table 4.1). Despite evidence from those animal studies (Chapters 3 & 4), and from clinical studies (Lord et al., 1996; Manchikanti et al., 2002; van Eerd et al., 2010) that identifies the facet joint as a source of pain after mechanical neck injury, the local initiators of pain in the C6/C7 facet joint after its injury remain poorly defined. Inflammatory cascades, such as prostaglandin E2 (PGE2) signaling, are evident in the joint and spinal cord following painful facet joint injury at the time when the corresponding spinal neurons at the level of the injured joint are also hyperexcitable (Crosby et al., 2013, Dong et al., 2013a, Kras et al., 2014a). Nerve growth factor (NGF) is increased in inflamed tissues and is sufficient to induce both mechanical and thermal hypersensitivity and spinal neuronal hyperexcitability (Hoheisel et al., 2007;

Lewin et al., 1994; McMahon, 1996). During development, most small diameter primary afferent neurons require NGF for survival (Merighi et al., 2008; Pezet and McMahon, 2006); however, mainly peptidergic afferents that are capable of transmitting nociceptive information continue to express the NGF receptor, trkA, and remain sensitive to NGF in the adult (Merighi et al., 2004). Because a high proportion of joint afferents are peptidergic (Table 4.1; Kras et al., 2013b), NGF may induce joint pain by sensitizing the facet joint afferents. Animal models of pain resulting from inflammation demonstrate increased NGF as contributing to the development and maintenance of pain (Amaya et al., 2004; Woolf et al., 1994). Both clinically and in pain models of inflamed or arthritic joints, elevated levels of NGF are identified in the painful joint, suggesting a possible role for intra-articular NGF in joint pain (Barthel et al., 2009; Orita et al., 2011; Surace et al., 2009). Although increases in NGF are associated with inflammation and inflammatory responses contribute to facet joint loading-induced pain (Dong et al., 2013a; Kras et al., 2014a; McMahon, 1996; Woolf et al., 1994), it is not known if intra-articular NGF induces and/or maintains pain. NGF is a target for ongoing development of clinical treatments in several other musculoskeletal pain modalities, such as low back and knee pain (Brown et al., 2012; Katz et al., 2011). As such, defining whether or not NGF has a similar role in injury-induced facet pain will help identify if NGF signaling is a common mechanism across a range of musculoskeletal pain modalities. In addition, similarities in the mechanisms of arthritis- and injury-induced joint pain could indicate that mechanical joint injury initiates a degenerative process similar to osteoarthritis. A role for NGF in facet pain would support expanding current anti-NGF pain therapies being developed to include facet-mediated pain and local anti-NGF delivery in the joint as a new treatment.

Studies presented in this chapter summarize experiments under Aim 2, and test the hypothesis that NGF increases in the facet joint after a painful joint distraction and is both necessary and sufficient for the initiation of both behavioral and spinal neuronal hypersensitivity after injury. As such, NGF expression is quantified within the facet joint as well as in the dorsal root ganglion (DRG) to determine if joint injury modulates local NGF levels. Further, behavioral responses are evaluated after intra-articular application of NGF (Aim 2a) to assess whether or not intra-articular NGF is sufficient to induce pain. In addition to behavioral outcomes, neuronal excitability is quantified in the spinal cord after application of NGF to the facet joint to determine if intra-articular NGF is sufficient to induce central sensitization, which is a state of increased neuronal excitability and synaptic efficacy that contributes to chronic pain (Latremoliere and Woolf, 2009). Based on those findings, targeted ablation of joint afferents involved in either peptidergic or non-peptidergic signaling is performed in separate groups of rats prior to NGF application to identify those neurons that contribute to NGF-mediated joint pain (Aim 2b). The role of intra-articular NGF in the development of facet joint distraction-induced pain and spinal hyperexcitability is evaluated using an anti-NGF antibody to locally block NGF signaling in the joint (Aim 2c). Additional studies in Aim 2c also apply the anti- NGF antibody to the facet joint after the initiation of loading-induced pain to determine if intra-articular NGF also maintains pain after joint injury.

5.2. Relevant Background

Musculoskeletal pain, especially joint and neck/back pain, is the most common type of chronic pain (Johannes et al., 2010; Pizzo et al., 2011). Among the fibers that

innervate articular joints, the Aδ- and C-fibers exhibit increased mechanosensitivity during joint inflammation and can be activated by normal joint motions (Guilbaud et al., 1985; Schaible et al., 2009). Joint inflammation has been shown to sensitize neurons in the spinal cord and to expand receptive fields to include adjacent non-inflamed tissues in rat models (Martindale et al., 2007; Schaible et al., 2009; Woolf and Wall, 1986), supporting a role for central sensitization in joint pain.

The facet joint is the most common source of pain in chronic neck pain patients (Manchikanti et al., 2002). Non-physiological loading of the facet joint activates nociceptors in its capsule (Lu et al., 2005) and induces hyperexcitability of spinal neurons and pain (Crosby et al., 2013; Dong et al., 2013b; Lee et al., 2008; Quinn et al., 2010). Moreover, intra-articular non-steroidal anti-inflammatory drug treatment alleviates pain after mechanical facet injury (Dong et al., 2013a), suggesting inflammatory contributions to loading-induced facet pain. Because arthritis-induced joint pain and injury-induced facet joint pain exhibit similar inflammatory and neuronal responses (Boettger et al., 2008; Dong et al., 2013a; Martindale et al., 2007; Quinn et al., 2010), common mechanisms may contribute to both types of joint pain. Yet, the local molecular mechanisms that lead to the onset of facet pain are not defined.

Nerve growth factor sensitizes adult sensory neurons, and is increased in inflamed tissues (McMahon, 1996). Injection of NGF into peripheral tissues in animals (Lewin et