Gerald F. Gebhart - US grants

The objectives of the proposed experiments are (1) to determine the selectivity for nociceptive input of the spinal inhibition descending from the brain stem, correlating the spinal inhibition with the analgesias produced by focal electrical stimulation and morphine given at selected sites in the brain stem, and (2) to clarify the organization in the brain stem of descending systems of analgesia/inhibition. Rats will be chronically implanted with 26 guage guide cannula(e), through which either a stimulating electrode or injection cannula can be passed. Both the electrodes and injection cannulae will be of identical length to permit stimulation and morphine administration at identical sites in the brain stem. The effect(s) of stimulation and morphine given in the periaqueductal gray, lateral mesencephalic reticular formation, nucleus raphe magnus, and lateral medullary reticular formation on both noxious (radiant heat, electrical C-fiber activation) and non-noxious (brush, electrical A Alpha, Beta-activation) evoked spinal neuronal activity will be quantitatively examined. At all four sites in the brain stem, morphine will be compared with stimulation. In other experiments, stimulation at one site will be compared with stimulation at another site. Significantly, prior to the electrophysiologic evaluation of spinal neurons, the analgetic efficacy of stimulation and morphine will be evaluated in several analgesiometric tests organized at different levels of the CNS to allow correlation of the analgesia produced with the descending inhibitory effects subsequently examined electrophysiologically in the same animals. The study will determine whether there exists a descending system(s) which is selectively analgetic and further our understanding of the brain stem pathways and mechanisms of spinal inhibition.

The experiments are aimed at a better understanding of: 1) Brain stem sites and transmitters involved in nociception and antinociception; 2) morphine's interaction with transmitters at extra hypothalamic sites for the secretion of luteinizing hormone; and 3) the role played by substance P in nociception and antinociception. The experiments employ: intracerebral drug microinjections; electrophysiologic/microiontophoretic drug evaluations, and assays for tissue contents of serotonin, norepinephrine, substance P and luteinizing hormone.

pain;

Our current understanding of pain mechanisms is based principally on experimental investigations of cutaneous pain. Less well understood are mechanisms of deep pain arising from joints, muscle and particularly the viscera. Visceral pain in general is more important clinically than is cutaneous pain, but we know considerably less about visceral pain mechanisms and its control than we do about cutaneous pain mechanisms. Over the past 10 years or so, there has been increased interest in deep pain mechanisms, and we and other laboratories have contributed to improved understanding of visceral pain mechanisms. Specifically, this project has in the past addressed cardiac, esophageal, gastric and colonic nociception. Colorectal distension (CRD) is the model with which we have worked most and which has been best characterized. Experiments proposed for the next project period will continue to examine mechanisms of visceral nociception and its modulation. Specific Aims include: (1) quantitative characterization of responses of myelinated and unmyelinated least splanchnic and pelvic nerve sensory afferent fibers to graded CRD; (2) modulation of sensory afferent fiber responses to CRD by opioid and somatostatin receptor agonists; (3) evaluation of the central (spinal) mediators of visceral nociceptive transmission; and (4) assessment by intracellular analysis of the cellular properties of spinal units that respond to CRD. These Specific Aims are a logical extension of the current project and will continue important investigations into the mechanisms of visceral pain and its modulation. The proposed experiments comprise a quantitative, parametric examination of visceral sensory afferent and spinal cord physiology and pharmacological modulation of visceral nociception that will lead to better understanding of the mechanisms and control of visceral pain.

DESCRIPTION (provided by applicant): Numerous studies have contributed significantly to our understanding of nociception-related circuitry in the rostroventral medulla (RVM). Work supported by this award has characterized facilitation and inhibition of spinal sensory transmission from the RVM that involves distinct, independent systems likely associated with neurons in RVM that are physiologically and functionally differentiated. Electrophysiololgical experiments have identified cells in RVM that differ based on response to noxious heating of the tail (in vivo) or effect of a mu opioid agonist (in vitro). Neither model of RVM circuitry arising from these studies is fully explanatory of the descending effects produced from RVM. This proposal describes experiments to more fully characterize responses of cells in RVM to non-opioid agonists. Specific Aim 1 will use whole cell patch clamp methods to record cells in vitro in an RVM slice preparation. Because cholecystokinin (CCK), neurotensin, glutamate and GABA play important roles in RMV-produced modulation of nociception, we will examine their effects on cell currents and synaptic transmission in RVM. The axonal projections of recorded cells in RVM will be known by content of retrograde tracer and RVM slices will be fixed and processed after recordings for determination of glutamic acid decarboxylase, tryptophan hydroxylase, enkephalin and/or CCK content. Despite evidence that the RVM plays a role in hyperalgesia, no information is available as to how the electrophysiologic and pharmacological character of RVM cells changes as a consequence of persistent peripheral tissue insult. Specific Aim 2 will reproduce experiments in specific aim 1, but RVM slices in these experiments will be taken from animals with persistent peripheral insult: complete Freund's adjuvant inflammation of a hindpaw, spinal nerve ligation (Chung model), or trinitobenzenesulfonic acid produced colon inflammation. We hypothesize that changes in the electrophysiologic properties and pharmacologic modulation of currents and/or synaptic transmission will be apparent in RVM cells after persistent peripheral insult consistent with alterations in excitability of RVM cells. The working hypothesis guiding these studies is that differences exist between cells in RVM that have yet to be determined. Better appreciation of these differences will enhance our understanding of the contributions these cells make to nociceptive processing, including their role in mechanisms of hyperalgesia.

Pain arising from the viscera is associated with a variety of disorders. The major goal of this research proposal is to improve our understanding of the peripheral mechanisms of visceral pain under normal physiological and pathophysiological conditions. Single fiber studies will be undertaken to understand the role of primary visceral afferents in gastrointestinal (GI) pain. The aims involve characterization of responses of muscle and mucosal afferents in the vagus and splanchnic nerves of the rat to mechanical, thermal and chemical stimuli under normal physiological and inflammatory conditions. Experiments will address the following aims: 1) Quantitative characterization of vagal and splanchnic nerve afferents in these two pathways to graded distension will be compared to understand their relative roles in regulatory functions and nociception. The locations of mechanosensitive receptive fields in the stomach and small intestine will be determined. 2) Determine response-specificity of vagal and splanchnic nerve afferents and determine whether mechanically-insensitive afferents innervating the upper GI tract are truly silent or are chemo- and/or thermo-sensitive. 3) Quantitatively characterize the mechano, thermo- and chemo- sensitive properties of vagal and splanchnic nerve afferents innervating the chronically inflamed stomach and small intestine. 4) Quantitatively characterize the mechano, thermo- and chemo- sensitive properties of vagal and splanchnic nerve afferents innervating the chronically inflamed stomach and small intestine. This proposal represents the first systematic investigation of the possible roles of primary afferents in upper GI pain. The study will provide information about the adequate natural stimuli of the afferents ad about changes in response characteristics during inflammation.

pain; visceral afferent nerve; gastrointestinal disorder; inflammation; gastrointestinal system; temperature; splanchnic nerves; mucosa; gastrointestinal motility /pressure; chemical stimulation; intestine disorder; stomach disorder; intestinal mucosa; small intestines; muscle; vagus nerve; sensory mechanism; laboratory rat;

Many disorders of urinary bladder sensation, which is common in interstitial cystitis, bladder outlet obstruction, and bladder over activity, including idiopathic detrussor instability, are characterized by pain and discomfort and enhanced sensitivity to stimuli (i.e., bladder hypersensitivity). Inflammation is present in many, but not all major bladder disorders and interstitial cystitis, for example, often presents without demonstrable organic cause (i.e., there is no apparent mechanical, biochemical or inflammatory condition to explain the symptoms). Visceral hypersensitivity thus can differ from somatic hyperalgesia, which is typically associated with tissue injury and inflammation. Because the anatomical organization of innervation and adequate noxious stimuli for the viscera are unlike the innervation and adequate noxious stimuli in the somatic realm, peripheral mechanisms of visceral hypersensitivity differ from those of somatic hyperalgesia and are not well understood. The long term objective of this research program is to understand peripheral mechanisms of bladder hypersensitivity. The current application proposes to establish functional relevance of stimuli and treatments before subsequent examination of peripheral contributions to the development of bladder hypersensitivity. The peripheral receptors to be examined for contributions to bladder hypersensitivity include ASIC3, TRPV1, P2X2-3 and PAR2, all of which have been implicated in visceral hypersensitivity. For each of these receptors, in normal and hypersensitive conditions (e.g., cyclophosphamide-induced cystitis), we will study: 1) their contribution to bladder voiding and reflex micturition to functionally evaluate bladder hypersensitivity, 2) mechano- and chemo-sensitivity of pelvic nerve and lumbar splanchnic nerve fiber terminals in the bladder using an in vitro bladder-nerve preparation, and 3) whole cell currents and excitability of bladder sensory neurons, identified by content of retrograde tracer. The overall hypothesis is that these four ligand-gated ion channels contribute to mechano-transduction in the urinary bladder. We also hypothesize that protons and/or endogenous mediators (e.g., ATP,mast cell tryptase) contribute to bladder hypersensitivity and can do so even in the absence of frank tissue damage, which is relevant to interstitial cystitis and other so-called "functional" visceral disorders.

DESCRIPTION (provided by applicant): Pain is the principal reason for seeking medical attention. Aim is not restricted to a single disease or medical discipline. Pain is, instead, distributed across the span of life, is experienced by all persons irrespective of status (economic, educational, etc.) or gender and arises from all tissues of the body. While acute episodes of pain typically resolve, many tissue insults lead to chronic pain states, and some pain syndromes are not associated with demonstrable pathology. The mechanisms that lead to tissue hypersensitivity and chronic pain, which negatively impacts quality of life, workplace productivity, interpersonal relationships, etc. at enormous cost, are poorly understood. Study of these mechanisms is the principal focus of research in the Pittsburgh Center for Pain Research (Center). Core faculty in the Center include those with a focus on transduction and signaling in nociceptors innervating superficial as well as deep tissues (e.g., muscle and viscera), circuitry and modulation of nociceptive input in the medullary and spinal dorsal horn, genetic mechanisms of individual variability in pain traits, and mechanisms that underlie sensory neuron development, differentiation and function in the adult nervous system. The Aim of the PROGRAM PLAN that is the basis of this application is to expand the breadth and depth of research expertise and research programs in the Center by recruitment through this RFA of a new junior faculty member into the Center. Core faculty has identified two broad areas of research important for growth of the Center: Translational Research, specifically epidemiology, clinical psychology, brain imaging or microneurography. Immunology or Molecular Biology We have identified areas for recruitment that will make the Center a better research and training environment and, because of our commitment to research excellence and collegiality;will enhance the research program and career development of the recruit as well. PUBLIC HEALTH RELEVANCE: Given the magnitude of personal and societal costs associated with ersistent pain states, its indiscriminate expression and the inability of the medical community to effectively manage many pain states, investing in the growth and breadth of a group of interactive, collaborative investigators in the Center - as requested in this application - addresses a fundamentally important public health issue.

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. PUBLIC HEALTH RELEVANCE: Painful bladder syndrome and irritable bowel syndrome are chronic, debilitating visceral disorders, more common in women, in which pain and hypersensitivity are the most troubling symptoms. These disorders are notoriously difficult to manage. The objective of this research is to determine the mechanisms by which organ pain and hypersensitivity arises and is maintained as a necessary step in developing better informed and more successful management strategies.

DESCRIPTION (provided by applicant): The overarching goal of this proposed training program is to integrate interdisciplinary and translational aspects of the current, rich training environment in pain research at the University of Pittsburgh. In addition to exposure of trainees to fundamental principles of pain mechanisms and pain presentation, we will establish new program elements (clinical experiences, multi-laboratory projects, courses) that are typically very difficult for individual investigators to support. We are committed to an integrated approach to the study of pain because major breakthroughs in this field can only be achieved through multidisciplinary approaches, and thus will incorporate and facilitate interactions between [1] laboratories (horizontal integration) and [2] researchers and clinicians (vertical integration). Accordingly, horizontal and vertical integration will be essential components of this training program, which will consist of three core elements: 1) Research - Multidisciplinary research projects will not only be encouraged, but expected, as will exposure to clinical management of pain/pain-related problems. These integrations will be achieved through formation of multidisciplinary mentoring committees which will include at least one clinical faculty member among a four-member committee. 2) Theory - Trainees will participate in four required for-credit courses: Mechanisms and Clinical Presentation of Pain, Pain Journal Club, bi-weekly Current Research on Pain presentations, a new course Pain Models - Rationale, Testing and Interpretation as well as the monthly Pain Seminar Series, where trainees interact with prominent pain researchers. 3) Practice - Trainees will be exposed to the assessment, diagnosis and treatment of chronic pain patients through a 2nd new course - Pain Perspectives - that will provide trainees the opportunity to shadow pain physicians as they interview, diagnose and manage chronic pain patients within the Pain Medicine Program at the University of Pittsburgh Medical Center. PUBLIC HEALTH RELEVANCE: Pain is the principal reason for seeking medical attention. It respects no boundaries, is not restricted to any one disease or medical discipline, is distributed across the life span and experienced by all irrespective of status (economic, educational, etc.) or gender. Many tissue insults lead to chronic pain states which are poorly managed because the mechanisms underlying pain are incompletely understood. Thus, there is a compelling need to enhance and broaden the training of the next generation of pain researchers.