Alan Randich, PhD - US grants

Afferent neural activity arising from peripheral baroreceptors monitoring cardiovascular function not only produces CNS-mediated reflex adjustments of the circulation, but also activates CNS systems that inhibit pain. The primary goals of this proposal are to identify medullary and spinal cord structures of this cardiovascular pain regulatory network in the rat, establish the efficacy of putative transmitters of primary baroreceptor afferents in producing hypoalgesia or a diminished sensitivity to pain, and determine the range of influence of this network. The regions of the medulla and spinal cord under investigation include the nucleus tractus solitarius (NTS), nucleus raphe magnus (NRM), lateral reticular nucleus (LRN), dorsolateral funiculus (DLF), and ventrolateral funiculus (VLF) based upon their established roles in both cardiovascular and nociceptive function. The importance of these regions in the production of hypoalgesia will be determined through the use of electrolytic and mechanical lesions, and pressure microinjection of pharmacologic agonists and antagonists. Hypoalgesia will be induced by several peripheral cardiovascular stimulus operations including physiological (volume expansion), pharmacological (administration of veratrum alkaloids, enkephalinamide, phenylephrine, or norepinephrine), or electrical activation of cardiopulmonary or sinoaortic baroreceptor afferents. Pain sensitivity will be indexed through behavioral responses in tail-flick, hot-plate, and flinch-jump tests. These experiments will serve to delineate the nature of cardiovascular-pain regulatory interactions and thereby establish how physiological stimuli activate endogenous systems that inhibit pain. These studies will also clarify the mechanisms which mediate a diverse range of phenomena including stress-induced analgesia, the action of peripherally circulating enkephalins in producing analgesia, and the inhibition of cardiac pain.

Afferent neural activity arising from peripheral mechanoreceptors monitoring cardiopulmonary function not only produces well-characterized CNS-mediated reflex adjustments of the circulation, but also activates CNS systems that modulate noxious somatosensory input. The present proposal is based upon the hypothesis that peripheral and central substrates of baroreceptor reflex control of the circulation are physiologically linked to systems involved in the centrifugal modulation of pain. Thus, the primary objective of this proposal is to parametrically evaluate the modulation of spontaneous and noxious-evoked activity of spinal dorsal horn neurons by manipulations which increase afferent activity of the vagal nerve trunk in the rat. This will be accomplished by extracellular recording of antidromically- identified class 2 ("wide dynamic range") and class 3 ("nociceptive specific") neurons of the lumbar spinal dorsal horn activated by either tibial nerve stimulation (A-fiber and A- and C-fiber intensities) or noxious heat stimulation applied to the plantar surface of the hindlimb footpad. Activation of vagal afferents will be effected by two manipulations established as antinociceptive in the rat: electrical stimulation of the vagal trunk or intravenous administration of (D-ala2)- methionine enkephalinamide. The specific aims of the present proposal are: (1) to provide converging electrophysiological support for existing behavioral data on cardiopulmonary modulation of somatosensory input in the rat; (2) to evaluate the organization of medullary substrates mediating inhibition of spinal nociceptive transmission produced by activation of vagal afferents; and (3) assess potential interactions between known medullary substrates of descending spinal inhibition and those activated by vagal afferents.

This proposal is based upon the view that information derived from receptors located in the heart, lungs, and subdiaphragmatic regions, and transmitted via the vagal nerves to the CNS, interacts with excitatory and inhibitory CNS systems that modulate the processing of noxious input. The general objectives of the present proposal are to continue the analysis of this network by both extending behavioral analyses of vagal modulation of noxious somatosensory input and providing converging electrophysiological data supporting this view. This will be accomplished by (1) establishing the relative aversive-appetitive characteristics of cervical and subdiaphragmatic vagal stimulation in behavioral tests of escape and preference, (2) characterizing the effects of electrical stimulation in primary (nucleus tractus solitarius - NTS) and secondary (nucleus reticularis ventralis pars beta - NRV pars beta) sites of termination of vagal afferents in either facilitating or inhibiting the tail-flick reflex evoked by noxious heat, (3) establishing the spinal neurochemical basis of the nociceptive effects demonstrated in these escape, preference, and tail-flick experiments by intrathecal administration of receptor antagonists, (4) more fully characterizing midbrain and medullary substrates of the vagal network by assessing the effect of selective destruction of cell bodies with ibotenic acid on the capacity of electrical stimulation of the vagus to either facilitate or inhibit the tail-flick reflex, and (5) providing converging support for the role of the vagal network in the processing of noxious input by electrophysiological recordings of responses of class 2 and class 3 lumbar spinal dorsal horn neurons to either tibial nerve stimulation (A- and C-fiber intensities) or noxious heating of the foot during either electrical stimulation or microinjections of glutamate in the NTS and NRV regions.

DESCRIPTION (Adapted from the Investigator's Abstract): Tissue damage and nerve injury are often associated with inflammation. The common signs of inflammation include hyperalgesia, redness, swelling, and increased temperature. The overall goal of our research program is to determine the mechanisms responsible for the development and maintenance of primary hyperalgesia. The specific goals of this proposal are to (1) identify which peripheral afferents contribute to zymosan-induced mechanical and thermal hyperalgesia using single fiber recordings of low threshold mechanoreceptors (LTMs), high threshold mechanoreceptors (HTMs), A-mechanoheat nociceptors (AMHs), C-polymodal nociceptors (CPMs), and C-mechanonociceptors (CMNs), (2) determine how mechanical and thermal stimulus-response functions (SRFs) of nociceptive specific (NS) and wide dynamic range (WDR) spinal dorsal horn neurons are affected under conditions of zymosan-induced inflammation, (3) determine whether spinal administration of selective glutamate receptor antagonists differentially affect the altered stimulus-response characteristics of NS and WDR neurons to thermal and mechanical stimuli under conditions of zymosan-induced inflammation, and (4) determine whether zymosan-induced inflammation results in hyperexcitability of NS and WDR spinal dorsal horn neurons and if so, whether it can be sustained in the presence of glutamate receptor antagonists that block primary hyperalgesia to thermal and mechanical stimuli. These goals will be accomplished in electrophysiological and pharmacological studies of primary hyperalgesia in the rat. Intraplantar administration of the inflammatory agent zymosan will be used to produce primary hyperalgesia to thermal and mechanical stimuli. In each aim, complete SRFs to thermal (36 C - 50 C in 2 C increments) and mechanical stimuli (0.02 - 190 g) will be generated before and at hourly intervals following administration of zymosan. All units will be studied using a repeated measures design. These studies will advance our understanding of the mechanisms of thermal and mechanical primary hyperalgesia resulting from inflammation. Data obtained from the pharmacological studies will help resolve some of the current controversy over whether similar or different excitatory amino acids (EAAs) mediate the production and/or maintenance of mechanical and thermal hyperalgesia.

DESCRIPTION (provided by applicant): Pain originating from the urinary bladder is a common clinical entity affecting more than 50% of females at some time in their lives. Some conditions are easy to treat, but others, such as interstitial cystitis (IC) have proven resistant to diagnosis and treatment. There are multiple proposed etiologies for IC with the common theme of an eventual sensitization/activation of sensory elements: abnormalities in the periphery lead to central neurophysiological changes that become expressed as enhanced sensory (pain-urgency) and reflex responses (i.e. reduced bladder capacity) which may outlast "triggering" events within the bladder. In somatic systems, it has been demonstrated that exposure to painful stimuli during early life can produce permanent changes in the neuroanatomical and neurophysiological substrates that process nociceptive information. Since 10-28% of adult patients with IC report urinary bladder symptoms as children a logical avenue for exploration is an examination of the effects of early-in-life painful bladder experiences on bladder responses in adults since individuals who are "primed" for hypersensitivity of the bladder could have pain that is both easily triggered and manifested as a sustained response to normally self-limited events. The hypothesis central to these studies is: A peripheral and spinal neuronal sensitization process initiated by early-in-life, high-intensity primary afferent activation, can enhance susceptibility to pathological urinary bladder pain as an adult. This early-in-life process leads to a hypersensitive state that is manifested by lowered intravesical stimulus thresholds needed for pain evocation and augmented responses to supra-threshold stimuli. To test this hypothesis reflex, primary afferent neuronal and spinal neuronal responses to urinary bladder distension (UBD) will be characterized in rats, which are given high-intensity UBD and/or inflammatory stimuli (intravesical zymosan) in the neonatal, pre-pubescent or post-pubescent periods. These exploratory studies will lay the groundwork for potential, novel therapeutic modalities for the treatment of urinary bladder pain by identifying substrates of hypersensitivity development and thereby treatment of urinary bladder pain by identifying substrates of hypersensitivity development and thereby presenting targets for intervention. Translation to the treatment of IC would be highly probable.

DESCRIPTION (provided by applicant): Early-in-life exposure to painful and/or inflammatory stimuli can produce permanent changes in the neuroanatomical and neurophysiological substrates that process nociceptive stimuli in both somatic and gastrointestinal systems. We propose that early-in-life bladder inflammation also may predispose an individual to experience increased bladder sensitivity as an adult. As part of an NIDDK-funded R21 grant, preliminary studies showed that neonatal, but not adolescent, exposure to bladder inflammation led to a hypersensitive nociceptive and reflex state in female rats raised to adulthood, as manifested by increased visceromotor reflexes (VMRs) and arterial blood pressure (ABP) responses to phasic urinary bladder distention (UBD), decreased threshold for micturition reflexes in cystometrographic (CMC) analyses, and increased baseline frequency of micturition. These outcomes also are consistent with two primary symptoms of the painful bladder syndrome interstitial cystitis (IC): enhanced sensory (pain-urgency) and reflex responses (i.e., reduced bladder capacity) to bladder distention. These data served to generate the overall hypothesis of the present proposal. Specifically, we propose the following: An alteration in neuronal sensory substrates is initiated by early-in-life bladder inflammation. This process enhances susceptibility to the development of a hypersensitive state as an adult, especially when a second bladder insult occurs, and is manifested by lowered intravesical pressure thresholds for micturition reflexes and enhanced bladder-related nociceptive responses. This results in urinary dysfunction and pathological urinary bladder pain as an adult. The general hypothesis is tested in three specific aims that determine in a quantitative fashion the effect of neonatal, and in some cases adolescent, bladder inflammation on: (1) VMRs and c-fos expression to phasic UBD, baseline micturition frequency, and micturition reflexes in CMG tests of adult rats, (2) the structure of the bladder, and the density and spinal distribution of bladder afferents in studies of histopathology using protein gene product 9.5 (PGP 9.5), wheat germ agglutinin horse-radish peroxidase (WGA-HRP), substance P, CGRP, and TRPV1 immunhistochemisty, and (3) responses of C- and A-5 bladder afferents to phasic UBD in adult rats. We believe these systematic studies, based on an innovative hypothesis, will lay the groundwork for potential novel therapeutic modalities for the treatment of urinary bladder pain by identifying the substrates for the development of bladder hypersensitivity. Translation to the treatment of painful bladder syndromes like 1C would be highly probable.

DESCRIPTION (provided by applicant): Our previous research in rats has shown that exposure to bladder inflammation during the neonatal period of development markedly enhances the degree of bladder hyperalgesia that occurs in response to a second exposure to bladder inflammation as an adult. Neonatal bladder inflammation also significantly increases micturition frequency and significantly decreases the threshold for micturition reflexes in adulthood. These outcomes are consistent with the primary symptoms of painful bladder syndrome (PBS) / interstitial cystitis (IC) and suggest that neonatal bladder inflammation may be a contributing factor to bladder disorders. The general hypothesis of the present proposal is that neonatal bladder inflammation predisposes an organism to adult bladder pain by impairing the development and/or function of an opioid inhibitory system that normally serves to suppress bladder pain. The specific hypotheses that refine and develop this general hypothesis are tested in the following specific aims: Specific Aim 1 will test the hypothesis that the estrous cycle influences the magnitude of the endogenous opioid inhibitory response evoked by bladder inflammation in the adult rat. Specific Aim 2 will test the hypothesis that neonatal bladder inflammation predisposes an organism to adult bladder pain by impairing a descending inhibitory system originating from neurons located in the rostroventral medulla (RVM). Specific Aim 3 will test the hypothesis that neonatal bladder inflammation predisposes an organism to adult bladder pain by altering <-, :- and/or 4-opioid receptor expression in the spinal cord. Specific Aim 4 will test the hypothesis that neonatal bladder inflammation predisposes an organism to adult bladder pain by altering spinal cord opioid peptide content. Specific Aim 5 will test the hypothesis that neonatal bladder inflammation predisposes an organism to adult bladder pain by reducing opioid inhibition of spinal dorsal horn neurons We believe these systematic studies, based on an innovative hypothesis, will lay the groundwork for potential novel therapeutic modalities for the treatment of urinary bladder pain by identifying opposing substrates underlying the development of bladder pain. Translation to the treatment of painful bladder syndromes would be highly probable because we will be able to identify key loci for targeted interventions. PUBLIC HEALTH RELEVANCE: We believe these systematic studies, based on an innovative hypothesis, will lay the groundwork for potential novel therapeutic modalities for the treatment of urinary bladder pain associated with painful bladder syndrome (PBS) / interstitial cystitis (IC) by identifying opposing substrates underlying the development of bladder pain. Translation to the treatment of PBS/IC would be highly probable because we will be able to identify key developmental factors that give rise to the disorders, underlying mechanisms responsible for the disorders, and loci for targeted interventions.