Overview: Our research program addresses basic molecular and physiological processes of nociceptive transmission in the central nervous system, and new ways to effectively treat intractable pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that innervate the skin and deep tissues and their connections in the dorsal spinal cord, which is the first site of synaptic information processing for pain. Our research has identified the DRG and spinal cord as loci of neuronal plasticity and altered gene expression in persistent pain states. The mechanisms of transduction of physical pain stimuli are also under investigation by examining events in damaged or inflamed peripheral tissue and using reductionistic approaches such as cloned thermal and chemo-responsive ion channels expressed in heterologous cell systems or naturally expressed in primary cultures of dorsal root ganglion. Our goals are (1) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (2) mechanisms underlying human chronic pain disorders, and (3) to use this knowledge to devise new treatments for pain. ? New Treatments for Pain: We address the new treatment goal by a translational research and human clinical trials program aimed at developing new analgesic treatments for severe pain. The current approach is based on our studies of pain transduction through the vanilloid receptor 1 (TRPV1). This molecule is a heat-sensitive calcium/sodium ion channel and converts painful heat into nerve action potentials by opening the pore of the ion channel which then depolarizes pain-sensing nerve endings and triggers an action potential that is conducted to the spinal cord. Channel opening is also stimulated by capsaicin, a vanilloid chemical and the active ingredient in hot pepper. We use a very potent vanilloid analog called resiniferatoxin (RTX) to """"""""prop open"""""""" the ion channel thereby causing calcium cytotoxicity and death of a specific class of pain-sensing neurons. This proved to be a very effective means of pain control in several pre-clinical models. To effect the transition to a human clinical trial we have established an inter-Institute working group with NIDA's Division of Pharmaco-Therapeutics and Medical Consequences of Drug Abuse to bring this novel treatment to human clinical trial. The working group consists of experts on chemistry and manufacture, toxicological, neurobiological, medical, and regulatory affairs as well as anesthesiologists, pharmacologists and pathologists from our group. We have also established mechanisms for obtaining the natural product from which the active drug is extracted and procedures for isolation, purification and formulation of the drug product that are compliant with Food and Drug Administration (FDA) regulations. We are presently finalizing the toxicology study and preparing the Investigational New Drug Application for submission to the FDA. We are also finalizing the human clinical protocol with the NCI's IRB. The RTX cell deletion treatment will first be tested for its ability to control cancer pain. If it is safe and effective, we shall conduct a second protocol for treatment of head and neck cancer and then explore control of of other chronic pain conditions such as arthritis, trigeminal neuralgia and chronic neuropathic pain problems. ? Clinical Pain Mechanisms: Chronic neuropathic pain conditions, either in the body or the oro-facial region, are difficult to treat effectively and to perform pre-clinical research on since informative animal models are not available. One of these neuropathic pain problems is Complex Regional Pain Syndrome (CRPS, formerly called reflex sympathetic dystrophy). This syndrome arises subsequent to a nerve injury or trauma. We are testing the hypothesis that the injury provokes an autoimmune response to one or more proteins from small diameter pain-sensing axons (C-fiber neurons). to the immune system that becomes an autoantigen. We have established a very sensitive assay to test for the presence of autoantibodies to C-fiber components in the serum of patients with CRPS. We shall compare the results to patients with other autoimmune diseases that exhibit neuropathic pain to determine if there is an overlap in antigen profiles indcative of a general mechanism or common denominator, and to patients that have experienced a nerve injury but do not develop CRPS to establish clinical specificity. One autoimmune disorder is Sjogren's Syndrome. Approximately 30 % of patients develop a small fiber neuropathy which progresses in another 30% to a mixed fiber neuropathy. Thus, C-fibers appear to be an autoimmune target in this syndrome and we are collaborating with the Gene Therapy and Therapeutics Branch, NIDCR to examine sera from Sjogren's patients to test against our antigen panel. If there is some success, we shall expand the analyses other cohorts of chronic pain patients and patients with nerve damage to establish whether the underlying mechanisms are generalized or specific to these types of nerve injury-induced neuropathies.? Basic Pain Mechanisms: Underlying the translational studies are our investigations of molecular regulation of gene expression, neuronal function, and mechanisms of pain transduction. We are systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal spinal cord. This approach provides a comprehensive and informative view of nociceptive process. The dorsal ganglion is quit small and its inclusion is, in part, is made possible by the extensive use of reverse-transcription-PCR, which allows us to make multiple measurements on small tissue samples like the dorsal root ganglion. Our studies reveal the dynamic modulation of gene expression at all three steps in a more complex fashion than previously hypothesized. We have examined novel molecules as well as neuropeptide, cytokine and chemokine expression and identified prominent roles for new, key molecules with distinct combinatorial patterns. In addition to pain, these studies fundamentally explore the molecular basis of synaptic plasticity. We hypothesize modularity in neuronal responses to new levels of synaptic or pharmacological input (e.g. learning, neurological disorders, drug abuse). A set of """"""""generic"""""""" alterations is combined with modulation of circuit-specific genes to meet the demands generated by the new level of stimulation. Understanding the molecular repertoire will lead to a deeper understanding of molecular mechanisms that trigger and sustain chronic pain and possibly other chronic disorders of the nervous system. The functional implications of our studies on cytokines are being explored using behavior and a new technique of in vivo microscopy. We are trying to evaluate the effects of C-fiber activation on immune cell dynamics and endothelial responses. We are examining neurogenic inflammation and plasma extravasation following C-fiber activation. These studies provide information about basic parameters of stimulation and timing and demonstrate in real time the interactions between the C-fiber afferents and the vascular elements. We shall also explore the actions of C-fiber activation on responses of circulating neutrophils and monocytes. Here we hope to obtain a fundamental understanding of the relationships between tissue damage, inflammation and pain sensation.
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