Michael S. Gold, Ph.D. - US grants

DESCRIPTION: The pain and hyperalgesia, associated with tissue injury and inflammation is among the most common reasons people seek medical treatment. Hyperalgesia is associated with the sensitization of primary afferent nociceptors. Hyperalgesic inflammatory mediators (HIM's) released following injury act on nociceptor terminals to increase their excitability. HIM's such as Prostaglandin E2 sensitize nociceptors both in vivo and in vitro. Evidence suggests that HIM-induced sensitization may involve the activation of either protein kinase A- or protein kinase C-dependent second messenger pathways. Using a model for the nociceptor terminal in vitro, it has been demonstrated that HIM's modulate a tetrodotoxin resistant voltage-gated Na current (TTX-R Ina) in a manner consistent with nociceptor sensitization. Thus, changes in this current constitute a final step in the transduction pathway underlying the effects of inflammatory mediators in nociceptors. Using patch clamp electrophysiology and Ca imaging techniques in combination with a series of specific pharmacological agents to identify the cellular components, and therefore second messenger pathway utilized by HIM's, the following hypotheses will be tested: 1) HIM-induced modulation of TTX-R I involves the activation of a protein kinase A-dependent second messenger pathway: 2) HIM-induced modulation of TTX-R Ina involves the activation of a protein kinase C dependent second messenger pathway; 3) HIM-induced modulation of TTX-R Ina involves an interaction between PKA- and PKC-dependent second messenger pathways. These studies will provide the first detailed analysis of the second messenger pathway underlying inflammatory mediator-induced nociceptor sensitization. By increasing our understanding of the neurobiology of nociceptor sensitization, results from these studies have the potential to facilitate the development of novel therapeutic approaches to the treatment of pain. This is particularly true with respect to TTX-R Ina, because this current appears to be unique to primary afferent nociceptors. Thereby, manipulations targeting TTX-R Ina have the potential for providing the most effective pain relief known, with the smallest number of side effects.

DESCRIPTION: The pain and hyperalgesia, associated with tissue injury and inflammation is among the most common reasons people seek medical treatment. Hyperalgesia is associated with the sensitization of primary afferent nociceptors. Hyperalgesic inflammatory mediators (HIM's) released following injury act on nociceptor terminals to increase their excitability. HIM's such as Prostaglandin E2 sensitize nociceptors both in vivo and in vitro. Evidence suggests that HIM-induced sensitization may involve the activation of either protein kinase A- or protein kinase C-dependent second messenger pathways. Using a model for the nociceptor terminal in vitro, it has been demonstrated that HIM's modulate a tetrodotoxin resistant voltage-gated Na current (TTX-R Ina) in a manner consistent with nociceptor sensitization. Thus, changes in this current constitute a final step in the transduction pathway underlying the effects of inflammatory mediators in nociceptors. Using patch clamp electrophysiology and Ca imaging techniques in combination with a series of specific pharmacological agents to identify the cellular components, and therefore second messenger pathway utilized by HIM's, the following hypotheses will be tested: 1) HIM-induced modulation of TTX-R I involves the activation of a protein kinase A-dependent second messenger pathway: 2) HIM-induced modulation of TTX-R Ina involves the activation of a protein kinase C dependent second messenger pathway; 3) HIM-induced modulation of TTX-R Ina involves an interaction between PKA- and PKC-dependent second messenger pathways. These studies will provide the first detailed analysis of the second messenger pathway underlying inflammatory mediator-induced nociceptor sensitization. By increasing our understanding of the neurobiology of nociceptor sensitization, results from these studies have the potential to facilitate the development of novel therapeutic approaches to the treatment of pain. This is particularly true with respect to TTX-R Ina, because this current appears to be unique to primary afferent nociceptors. Thereby, manipulations targeting TTX-R Ina have the potential for providing the most effective pain relief known, with the smallest number of side effects.

nerve injury; craniofacial; oral facial pain; neural plasticity; neurons; afferent nerve; trigeminal nerve; inflammation; neurophysiology; innervation; chemical hypersensitivity; neurochemistry; electrophysiology;

[unreadable] DESCRIPTION (provided by applicant): Pain resulting from inflammation of is one of the most common reasons people seek medical attention. Inflammatory pain is associated with the sensitization of primary afferent neurons innervating injured tissue; i.e., those arising from trigeminal ganglia (TG) or dorsal root ganglia (DRG). Pain arising from specific structures such as the colon or TMJ is often the most difficult pain to treat possibly reflecting the unique properties of these afferents and/or the unique structures these afferents innervate. Nevertheless, our present understanding of the neurobiology of sensory neurons and their response to injury is derived largely from studies on somatic afferents. [unreadable] [unreadable] In normal tissue, voltage- and Ca2+-activatedchannels present in the plasma membrane of the afferent terminal control afferent excitability. Primary afferent neurons are hyper-excitable in the presence of persistent inflammation. However little is known about the mechanisms underlying this increase in excitability? This is particularly true for visceral and joint afferents given the dearth of data on the basic membrane properties of these afferents innervating normal tissue. One class of ion channels that may be particularly important for the expression of inflammation-induced hyperexcitability is Ca2+activated K+(CaK) channels. Inhibition of CaK channels appears to underlie sensitization of vagal afferents following airway inflammation and our preliminary data suggest that these channels are likely to contribute to inflammation-induced changes in the excitability DRG and TG neurons innervating several structures. Importantly, there have been only two studies on the function of CaK channels in sensory neurons from naive animals and none on the function of CaK channels in sensory neurons from inflamed animals. Therefore, we propose to test the following hypotheses: 1) that the distribution and functional role of CaK channels varies with respect to target of innervation (i.e., colon, TMJ, muscle and skin); and 2) that inflammation results in changes in the pattern of expression of CaK channels, which underlies changes in excitability that are unique to specific targets of innervation. We will test these hypotheses in experiments described under 2 Specific Aims employing a combination of retrograde tracing, in vitro patch-clamp electrophysiology, Ca2+imaging and RT-PCR analysis on adult rats either in the presence of absence of inflammation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): GABA-A receptor (GAR) mediated presynaptic inhibition of afferent input to the spinal cord is critical for the establishment of nociceptive threshold. In the absence of injury, spinal administration of GAR antagonists produce hyperalgesia and allodynia whereas GAR agonists are analgesic. We recently demonstrated, however, that in the presence of persistent inflammation, there is a shift in GAR signaling such that activation of spinal GARs actually contributes to inflammatory hyperalgesia: spinal administration GAR antagonists reverse inflammatory hyperalgesia while GAR agonists exacerbate it. Our preliminary data suggest this shift reflects a presynaptic change in GAR signaling which includes the emergence of distinct GAR receptor subunits and a depolarizing shift in the anion equilibrium potential (Eanion). A depolarizing shift in Eanion may enable GAR activation to become excitatory. However, our recent data also indicate that midazolam, a benzodiazepine receptor agonist, retains analgesic efficacy in the presence of inflammation, indicating that a depolarizing shift in Eanion alone is insufficient to account for the inflammation-induced shift in GAR signaling. The apparent shift in spinal GAR signaling has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, we have proposed a series of experiments described under 4 specific aims, designed to identify mechanisms underlying spinal GAR signaling in the presence and absence of inflammation. In Specific Aim 1, we will characterize inflammation-induced changes in the biophysical properties and pharmacology of GAR mediated currents and the regulation of anion homeostasis in cutaneous sensory neurons. In Specific Aim 2, we will characterize inflammation-induced changes in the expression and distribution of GAR subunits in cutaneous sensory neurons. In Specific Aim 3, we will assess the functional consequences of inflammation-induced changes in GAR subunit expression and intracellular anion homeostasis. And in Specific Aim 4, we will determine the extent to which specific GAR subunits in cutaneous sensory neurons mediate the emergence of pronociceptive actions of GAR signaling in the presence of inflammation. The experiments described under these 4 aims, involving an array of approaches ranging from behavioral pharmacology to viral vector mediated manipulation of gene expression, are designed to test the central hypothesis that persistent inflammation results in changes in GAR signaling that reflect a combination of changes in Eanion and GAR subunit expression in primary afferent neurons. PUBLIC HEALTH RELEVANCE: 3-aminobutyric acid-A (GABA-A) receptors on the central terminals of primary afferent neurons appear to play critical role both in the inhibition of acute pain and the maintenance of persistent pain in the presence of injury. The mechanisms underlying this shift in GABA-A receptor signaling remain to be identified, but it has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, with the ultimate goals of both minimizing deleterious consequences of general anesthetics and identifying novel targets for therapeutic interventions, we have proposed a series of experiments designed to identify mechanisms underlying the shift in spinal GABA-A receptor signaling in the presence of inflammation.

Description (provided by applicant): Current treatment of severe dental pain includes root canal therapy or tooth extraction. Obtaining adequate anesthesia for these invasive procedures is often difficult. Failure to obtain complete or profound anesthesia is observed in as many as 18% of patients undergoing root canal treatment. The result is that a significant number of patients must withstand intense pain for the duration of the procedure. In addition to several long-term deleterious consequences of such painful experiences, the fact that the inflamed pulp is more difficult to anesthetize suggests that more local anesthetic (LA) will be employed in an effort to obtain a sufficient level of anesthesia, thereby increasing the likelihood of systemic and/or local toxicity. Whereas a number of explanations for LA failure have been proposed, none are able to account for the nature and extent of LA failure observed in the presence of pulpitis. In the present application, we propose to test a novel hypothesis concerning the basis of LA failure in the presence of pulpal inflammation: that LA failure is due to a change in voltage-gated Na+ channel (VGSC) subunits along the axons of pulpal afferents. Importantly, these changes may also play a significant role in the pain associated with inflammation within the oral cavity. We proposed to test this hypothesis in experiments described under 3 Specific Aims in which we will: determine whether the pulpal inflammation-induced decrease in LA sensitivity in the rat reflects a change in the biophysical properties, relative density and/or pattern of expression of VGSC subunits in pulpal afferents (Specific Aim 1);determine whether there is an association between pulpal inflammation, a decrease in LA sensitivity and changes in the relative density of VGSC subunits in human teeth (Specific Aim 2);and evaluate the basis for a causal link between pulpal inflammation, changes in VGSC subunit expression and changes in LA sensitivity in the rat (Specific Aim 3). Importantly, results from these experiments will not only enable us to determine whether changes observed in the rat may contribute to LA failure in humans, but will enable us to establish a causal link between changes in subunit expression and LA failure. Results from these experiments may not only enable identification of ways to make root canal treatment a more tolerable procedure, but may yield novel therapeutic interventions for the treatment of pain associated with both chronic inflammation and nerve injury. PUBLIC HEALTH RELEVANCE: As many as 18% of the 16 million Americans who undergo root canal therapy every year are subjected to intense pain both during and after the procedure because it is difficult, and in some cases impossible to obtain an adequate level of anesthesia with local anesthetics in the presence of acute inflammation of the tooth pulp. We propose to test a novel hypothesis that the decrease in local anesthetic sensitivity observed in the presence of an inflamed tooth reflects a change(s) in the biophysical properties, density and/or relative distribution of voltage-gated Na+ channels in the sensory neurons that innervate the inflamed tooth. Because the changes in VGSCs observed in the presence of inflammation may also contribute to pain associated with persistent inflammation, identification of mechanisms underlying the change in local anesthetic sensitivity may not only enable more tolerable dental procedures, but may yield novel therapeutic interventions for the treatment of pain associated with both chronic inflammation and nerve injury.

DESCRIPTION (provided by applicant): Persons with chronic abdominal and pelvic pain disorders comprise a large proportion of patients seeking relief from gastroenterologists, urologists and gynecologists. Two common pelvic disorders, painful bladder syndrome (PBS) and irritable bowel syndrome (IBS), are characterized by altered bladder/bowel habits (e.g., urge, frequency and, in IBS, stool consistency), pain and hypersensitivity. These patients typically exhibit significantly lower response thresholds to provocative stimuli (e.g., cystometry, rectal distension), complain of increased sensitivity during normal organ function, and exhibit increased tenderness in areas of somatic referral which, in addition, are expanded in size. Interestingly, PBS and IBS exist in the absence of an apparent pathobiological cause and are thus characterized as 'functional.' It is widely assumed that functional disorders reflect altered CNS processing, but considerable evidence suggests that persistent afferent drive contributes significantly to the recurrent, unexplained pain and hypersensitivity that characterize them. The objective of this proposal is to determine the mechanisms by which pain and hypersensitivity arise and is maintained as a necessary step in developing better informed and more successful management strategies. The Aims include: 7 Aim 1: characterize the proportions of mechano-sensitive and insensitive afferents in electrophysiological experiments in the both the lumbar splanchnic and pelvic nerve innervations of the bladder and colorectum. 7 Aim 2: evaluate organ hypersensitivity in behavioral experiments, after which the proportions of bladder and colorectal mechanically insensitive afferents (MIAs) will be determined in bladder and colorectum removed from hypersensitive mice. We hypothesize the proportions of MIAs will be significantly reduced in organs from hypersensitive mice relative to normal controls. 7 Aim 3: identify the 'chemotypes' of mechano-sensitive and insensitive bladder and colorectal dorsal root ganglion neurons in both the lumbar splanchnic and pelvic nerve pathways. We hypothesize that there are significant differences in chemotype between MIAs and mechanosensitive afferents as well as between different mechanosensitive afferents and, further, between organs.

Mechanisms of Migraine Migraine is a debilitating episodic pain disorder for which there are no consistently effective therapeutic interventions. It is also one of the most prevalent pain disorders afflicting as many as 10% of the general adult population and 18% of women. Identification of novel approaches for the treatment of migraine is therefore highly significant. The prevailing weight of evidence indicates that the primary afferent neurons innervating the dura and dural vasculature are the source of the pain of a migraine attack. The present proposal is therefore focused on components of the dura that can influence afferent activity. These components include resident and recruited immune cells in the dura and the dural vasculature. We will also study the afferents themselves. We have proposed to exploit two unique features of migraine as a means to identify mechanisms that enable the initiation of a migraine attack. One is that stress is the most common trigger of a migraine attack. A second is that migraine attacks occur during relaxation phase after stress has ended. We propose that sympathetic post-ganglionic neurons (SPGN) in the dura serve as a link between stress and migraine because they are a critical component of the stress response system, the dura is heavily innervated by SPGN terminals, and all three dural components to be studied are regulated by mediators released from SPGN terminals. Finally, we also propose that sex is a critical factor that influences the link between stress and migraine because of the higher prevalence of migraine in women and the fact that gonadal hormones, in particular estrogens co- regulate each of the dural components to be studied. Thus, the central hypothesis of this proposal is that that stress drives sex- and SPGN-dependent changes in the regulation of dural immune cells, vasculature and primary afferents, that set the stage for the initiation of a migraine attack. This hypothesis will be tested in experiments described under three specific aims. In the first, we will determine the impact of sex, SPGN innervation, and persistent stress on resident and recruited immune cells in the dura. In the second, we will determine the impact of sex and persistent stress on SPGN-dependent regulation of the dural vasculature. In the third, we will determine the impact of sex and persistent stress on SPGN-dependent changes in voltage-gated Ca2+ currents in dural afferents and dural afferent excitability. The proposed experiments will not only provide valuable insight into the neurobiology of the dura, a structure critical for the health of the brain, but suggest novel approaches for the treatment of migraine enabling the prevention an attack altogether.

? DESCRIPTION (provided by applicant) There are few if any consistently effective treatments for the visceral pain associated with inflammatory bowel diseases such as ulcerative colitis, or functional bowel disorders such as Irritable Bowel Syndrome. Given the prevalence of these disorders and the fact that pain is a primary complaint of those who suffer, identification of nove approaches for the treatment of visceral pain is highly significant. The focus central nervous system has been the focus of altered GABA signaling in the context of pain. Even for the primary afferents essential for transmitting the pain signal from the periphery, the focus has been on GABA regulation of central terminals. Indeed, we and others have described a number of changes in the central terminals of nociceptive afferents resulting in a shift in GABAA-receptor (GAR) signaling from inhibition to excitation, enabling GABA to contribute to the pain and hypersensitivity of inflammation. But what if GAR signaling at the peripheral terminals of nociceptive afferents also contributes to the regulation of nociceptive signaling? After all, GARs are also transported to the peripheral terminals of nociceptive afferents. In the colon, GABAergic enteric neurons, epithelial cells and endocrine cells are potential sources of GABA. Preliminary data indicate that in the absence of tissue injury, endogenous GABA released within the colon serves to attenuate the excitability of colonic afferents. Strikingly, there is a loss of periphera GAR inhibition of colonic afferents in a model of inflammatory colonic hypersensitivity (i.e. IBD-like) and the emergence of GAR-dependent excitation of colonic afferents non-inflammatory (i.e. IBS-like) persistent colonic hypersensitivity. These changes in GAR signaling are associated with a decrease in GABA synthetic enzymes in the IBD-like model and increases in GABA regulatory machinery in the IBS-like model. Thus, we hypothesize that a distinct pattern of changes in peripheral GAR signaling contributes to the colonic hypersensitivity observed in inflammatory and non- inflammatory visceral pain disorders. Experiments designed to test this hypothesis as well as identify mechanisms underlying the changes in GAR signaling are described under the following three Specific Aims. First, we propose to determine mechanisms underlying GAR-mediated suppression of afferent activity in the absence of tissue injury in the mouse colon. Second, we will determine the mechanisms underlying the changes GAR-mediated inhibition of colonic afferents in models of inflammatory and non-inflammatory colonic hypersensitivity. Third, we will establish a causal link between changes in GAR signaling mechanisms and colonic hypersensitivity. The proposed studies have the potential to transform the current view of GAR signaling to include the peripheral control of afferent excitability. More importantly, they may suggest novel approaches for the management of visceral pain.

Project Summary/Abstract The overarching goal of this training program is to train the next generation of pain researchers. We will continue to train both pre- (two per year) and post-doctoral (two per year) fellows in the fundamental principles of pain, as nociceptive signals arise and are modulated throughout the body and ultimately integrated in the brain to produce the sensory and emotional experience. We will continue to build on the training program developed through the first funding period of this grant around a combination of more formal coursework and less formal training experiences designed to provide not only a solid background in pain mechanisms and management, and a greater appreciation of the burden of pain, but in a variety of skills critical for career development including the use of cutting edge methodology, networking, presentations to both lay and expert audiences, and writing. We are committed to an integrated approach to the study of pain which is based on our belief that major breakthroughs in this field can only be achieved through multidisciplinary approaches. This is most clearly manifest in practice through interactions between [1] laboratories (horizontal integration) and [2] researchers and clinicians (vertical integration). Accordingly, horizontal and vertical integration are essential components of this training program, which consists of three core elements: 1) Research - Multidisciplinary research projects are not only encouraged, but expected, as is exposure to clinical management of pain/pain-related problems. Horizontal and vertical integration will be achieve both through the choice of project, shaped by a primary mentor with input from the executive committee, and through the formation of multidisciplinary mentoring committees which will include at least one clinical faculty member among a three to four-member committee. 2) Theory - Trainees participate in four required for-credit courses: Mechanisms and Clinical Presentation of Pain, Pain Journal Club, bi-weekly Current Research on Pain presentations, and Pain Models ? Rationale, Testing and Interpretation, as well as the monthly Pain Seminar Series, where trainees interact with prominent pain researchers. These courses serve as a primary venue to address issues of scientific rigor and responsibility as well as reinforce issues associated with the responsible conduct of research. Post-doctoral trainees will obtain additional training through their participation in the patient/family education program developed by the chronic pain clinic and offered every six weeks as an educational resource for pain patients and families. 3) Practice - Trainees will be exposed to the assessment, diagnosis and treatment of chronic pain patients through two (and for post-docs three) primary venues: 1) The last third of the course Mechanisms of Clinical Presentation of Pain is directed at assessment diagnosis and treatment of specific subpopulations/aspects of pain patients (visceral, headache, geriatric, etc); 2) in the course Pain Perspectives trainees shadow pain physicians as they interview, diagnose and manage pain patients; and 3) Post-docs participate in the presentation of didactic material to Regional and Chronic Pain Fellows, as well as participate in the semi-annual pain management forum for all fellows (clinical and basic) in pain related training programs.

The goal of this project is to develop a rat model of temporomandibular joint disorder (TMJD) to address two questions that stand out among the many vexing problems facing the pain field. 1) Why do some patients not follow a ?normal? recovery trajectory after tissue injury, ultimately becoming what are now generally referred to as chronic pain patients? 2) Why do many chronic pain patients report such high levels of pain and pain related disability in the apparent absence of injury or disease in the painful tissue (a condition generally referred to as a functional pain disorder)? Both clinical and pre-clinical data indicate nociceptive input from a single peripheral site may be both necessary and sufficient to maintain widespread pain and/or hypersensitivity. Furthermore, there is a growing list of mechanisms underlying long term changes in cellular processes that may contribute to persistent activity in nociceptive afferents sufficient to maintain changes in the central nervous system (CNS) that may not be associated with conventional signs of injury or disease. Thus, we hypothesize that at least a fraction of functional pain disorders are due to changes in the periphery responsible for ongoing nociceptive input into the CNS, and that the magnitude of these changes accounts for differences in the time course of pain resolution, accounting for the subpopulation in which pain persists. We have chosen to focus on TMJD to test this hypothesis because: a) the prevalence and severity of the problem, b) that like many pain syndromes, the majority of patients with TMJ pain get better with time, and c) that TMJD is not only clearly associated with altered CNS processing, but is often present with a variety of other pain syndromes, consistent with the trajectory of a centralized pain syndrome. In addition, evidence from our rabbit TMJD model suggests that it may be the ideal model to address the two questions posed. With variability between animals in onset and recovery as well as magnitude of pain behavior, we would be able to identify mechanisms that contribute to the variability in pain behavior as well as the emergence of chronic pain. We therefore propose to adapt the model we originally developed in the rabbit to the rat, a species in which a far wider variety of nociceptive assays have been developed and for which there is a far greater number of research tools available, by assessing pain behavior, joint damage, TMJ afferent excitability, and inflammation. To establish the model and lay the groundwork for the causal links to be pursued in a larger application, we propose to determine the duration for the emergence and maintenance of hypersensitivity, condylar cartilage damage, afferent excitability, and inflammation, in both female and male rats.

SUMMARY Visceral pain is notoriously difficult to treat, often persisting long after the precipitating injury/disease is no longer evident. In this application we will explore a novel, multicellular peripheral circuit that we hypothesize explains many of the intractable features of chronic, visceral pain. We now know that epithelial-neuronal communication is widespread, with numerous epithelial cell types releasing neuroactive substances (e.g., ATP, ACh, 5HT, glutamate). This is particularly apparent in the colon where we have found that channelrhodopsin (ChR2) -induced activation of colon epithelial cells produces high frequency bursting of colon extrinsic primary afferent neurons (ExPAN?s), phenocopying physiologic stimuli and inducing robust behavioral responses (visceromotor responses (VMR), a validated assay of hypersensitivity). Building on these findings, new surprising data indicate colon epithelium also receives functional input from sympathetic neurons; activation of sympathetic projections to the colon induces large, phase-locked calcium signals in the epithelium. Closing the loop, we found that activation of ExPAN?s via colorectal distension (CRD) induces calcium signals in the post-ganglionic sympathetic neurons projecting to the colon, and that ChR2- induced activation of ExPAN?s induces cFos expression in these same neurons. That this multicellular circuit plays a role in visceral pain is supported further by preliminary data that shows that inflammation (acute and/or chronic) is correlated with increased signaling in all portions of this circuit. Thus, the goal of the proposed experiments is to test the hypothesis that persistent visceral hypersensitivity is due, at least in part, to amplification in an epithelial-ExPAN-sympathetic circuit such that it is possible to treat pain by breaking any limb of this feed-forward circuit (Fig.1). This hypothesis will be tested in 3 aims: Aim 1: Determine if persistent hypersensitivity induced in a model of IBD (DSS (dextran sulfate sodium)) is due to increased epithelial signaling and/or ExPAN excitability, Aim 2: Determine if DSS-induced inflammation increases the ability of ExPANs to activate sympathetic neurons in prevertebral sympathetic ganglion (PrSG) directly (via synapses in PrSG) or indirectly (via a spinal cord circuit) and, Aim 3 Determine the ability of sympathetic neurons to drive activity in epithelial cells in naïve mice and in the DSS model of IBD.