Rheumatoid arthritis (RA) is caused by aberrant attack of the joints by native inflammatory system. This can lead to joint destruction and pain that can be debilitating. Increased angiogenesis and innervation by nociceptive afferent fibers are characteristic features of RA joints, which in addition to the elevated levels of a wide variety of inflammatory mediators, are thought to play an important role in the pathogenesis of chronic inflammatory pain associated with RA. Interestingly, a recent report indicates that C–type natriuretic peptide (CNP) is increased in the blood serum of RA patients. Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Many biological effects of NPs are mediated by guanylate cyclase (GC)–coupled NP receptors, NPR–A and NPR–B, whereas the third NP receptor, NPR–C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR–C can couple to specific Gái–βã–mediated intracellular signaling cascades in numerous cell types. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain largely unknown. In Aim 1, I show that CNP acutely sensitized the excitation of mouse dorsal root ganglia (DRG) sensory neurons that is dependent on the transient receptor potential vanilloid–1 (TRPV1). CNP potentiated capsaicin– and proton–activated TRPV1 currents in cultured mouse DRG neurons and increased neuronal firing frequency, an effect that was absent in DRG neurons from TRPV1−/−mice. Further, CNP injection into mouse hind paw led to the development of thermal hyperalgesia, which was absent in TRPV1−/−mice. In Aim 2, I dissected the signaling mechanism underlying TRPV1 sensitization by CNP. My results show that all 3 functional NPRs are expressed in mouse DRG neurons; however NPR–A/B–cGMP signaling is not involved in CNP–mediated sensitization of TRPV1. Interestingly, I observed that sensitization of TRPV1 by CNP is dependent on protein kinase C (PKC) activity. Furthermore, I found that NPR–C is co–expressed in TRPV1–expressing mouse DRG neurons and can be co–immunoprecipitated with Gαi, but not with Gαq/11 or Gαs subunits. CNP treatment induced translocation of PKCå to the plasma membrane of these neurons, which was attenuated by pertussis toxin pre–treatment. Accordingly, CNP–induced sensitization of TRPV1 was attenuated by pre–treatment of DRG neurons with the specific inhibitors of Gβã, phospholipase–Cβ (PLCβ) or PKC, but not of protein kinase A (PKA), and by mutations at two PKC phosphorylation sites, S502 and S800, in the TRPV1 protein. Furthermore, the development of thermal hyperalgesia in CNP–injected hindpaw was attenuated by administration of specific inhibitors of Gβã or PKC. Thus, my work identifies the Gβã–PLCâ–PKC–dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity. Such signaling cascade could presumably constitute one of the mechanisms underlying chronic inflammatory joint pain associated with RA.

Functional magnetic resonance imaging (fMRI) studies performed to date have focused on brain structures rostral to the thalamus, although the first level of sensory information and pain transmission occurs at the spinal cord (SC). The primary goal of this project is to map activity using fMRI, from the entire cervical SC and brainstem following innocuous and noxious stimuli before and after peripheral sensitization in normal human volunteers. This study is unique in that it determines functional activity throughout the lower neural axis in response to mechanical stimuli that are perceived as painful only after sensitization. Functional MRI studies of the SC were carried out in 18 healthy individuals in a 3T Siemens Magnetom Trio. Innocuous touch and brush (n=8), and noxious touch (n=10) stimuli were applied before and after peripheral sensitization. Peripheral sensitization was induced by topical application of capsaicin. Functional image data spanned from the C7/T1 disc to the superior edge of the thalamus and analyzed using a general linear model to discriminate signal intensity changes from physiological motion. Normalized results were combined to demonstrate the number of volunteers showing activity at each location on a voxel-by-voxel basis. Areas of activity were superimposed onto anatomical transverse drawings and identified visually with comparison to several stereotaxic atlases. The results from this study confirm previous reports that a non-noxious stimulus translates into a pain response after peripheral sensitization. The brush stimulus, before sensitization activated areas in the ipsilateral dorsal horn (DH), gracile and cuneate nuclei in the medulla and areas surrounding the dorsal column medial lemniscal pathway. Peripheral sensitization produced activity in the contralateral ventral horn (VH), typical of a pain response. The innocuous von Frey stimulus produced activity in typical sensory centres in the DH and brainstem before sensitization, and areas more consistent with a noxious response after sensitization. When examining equi-nociceptive stimuli in a control versus sensitized state, the noxious touch stimuli showed similar activation patterns even though the force of the filaments were different. In all experiments there was indication of descending modulation as activity was observed in the periaqueductal gray, midbrain red nuclei and pontine reticular formation. This study demonstrates how non-painful and pain information is transmitted from the dorsal spinal horn to the brain in healthy individuals and how peripheral sensitization induces changes in non-noxious stimuli that correlate with pain sensory transmission. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-24 20:13:08.655

The neurobiological mechanisms underlying chronic pain in bone-metastasized breast and prostate cancer are not well understood although it is hypothesized that factors released in the tumor microenvironment may modulate sensory nociceptive sensory nerve fibers innervating the bone increasing pain sensation. Advanced metastatic breast and prostate cancer cells secrete high levels of parathyroid hormone-related peptide (PTHrP), which plays a critical role in metastasis to bones and subsequent tumor growth. PTHrP can activate parathyroid hormone receptor 1 (PTH1R), which signaling can activate either protein kinase C (PKC) and/or protein kinase A (PKA) depending on the tissue type. Both of these kinases are well known to modulate the nociceptive ion channel transient receptor potential vanilloid member 1 (TRPV1) due to which PTHrP constitutes an intriguing candidate that could modulate nociceptors, for pain sensitization related to cancer. TRPV1 can be activated by temperatures greater than 43°C and moderately acidic pH, less than pH 6. However, PKC and PKA phosphorylation of TRPV1 can potentiation channel activity by reducing the temperature of activation to 37°C and proton activation to pH 6.8 resulting in a channel that is constitutively active at body temperature or in the mildly acidic tumor microenvironment. Additionally, Src kinase, which under certain circumstances can be activated by PKC, can increase trafficking of TRPV1 to the plasma membrane, and enhance TRPV1-mediated signaling. Therefore, I hypothesize that PTHrP can sensitize TRPV1 and lead to an increase in nociceptive signaling. First I show that intraplantar PTHrP injection causes a TRPV1-dependent increase in thermal and mechanical hypersensitivity in mice. PTHrP treatment of cultured mouse dorsal root ganglion (DRG) neurons enhances TRPV1 activation and increases action potential firing, which was dependent on PKC activation. Furthermore, co-injection of PKC inhibitor attenuated PTHrP-induced thermal hypersensitivity. I also observed that PTHrP activated Src which led to an increase in the number of TRPV1-responsive neurons and an increase in TRPV1 protein level in the plasma membrane. While investigating the role of PTHrP-induced Src phosphorylation of TRPV1 I made a startling observation. Inhibition of Src phosphorylation of TRPV1 completely abolished PKC-induced potentiation of TRPV1. I found that Src phosphorylation of TRPV1 regulated PKC-induced potentiation of channel activity elicited by bradykinin, nerve growth factor and PMA. However, it did not regulate PKA induced potentiation of TRPV1 channel activity. In summary, my results suggest that PTHrP in the tumor microenvironment could induce constitutive pathological sensitization of adjacent nociceptive sensory fibers via upregulation of TRPV1 function, trafficking and expression. These actions are dependent on Src and PKC phosphorylation of TRPV1. Additionally, I found that Src regulates PKC-induced phosphorylation of TRPV1 by PTHrP as well as other inflammatory mediators, suggesting a crucial role for Src in PKC-induced sensitization of TRPV1.

No / Chronic pain is currently under-diagnosed and under-treated, partly because doctors' training in pain management is often inadequate. This situation looks certain to become worse with the rapidly increasing elderly population unless there is a wider adoption of best pain management practice. This paper reviews current knowledge of the development of chronic pain and the multidisciplinary team approach to pain therapy. The individual topics covered include nociceptive and neuropathic pain, peripheral sensitization, central sensitization, the definition and diagnosis of chronic pain, the biopsychosocial model of pain and the multidisciplinary approach to pain management. This last section includes an example of the implementation of a multidisciplinary approach in Belgium and describes the various benefits it offers; for example, the early multidimensional diagnosis of chronic pain and rapid initiation of evidence-based therapy based on an individual treatment plan. The patient also receives continuity of care, while pain relief is accompanied by improvements in physical functioning, quality of life and emotional stress. Other benefits include decreases in catastrophizing, self-reported patient disability, and depression. Improved training in pain management is clearly needed, starting with the undergraduate medical curriculum, and this review is intended to encourage further study by those who manage patients with chronic pain.