Margaret A. Vizzard - US grants

Micturition is regulated by neural circuits in the brain and spinal cord that coordinate the activity of the smooth and striated muscles of the lower urinary circuit. Disruption of these voluntary controls, as often occurs with spinal cord injury above the lumbosacral spinal cord, leads to the re-emergence of involuntary (reflex) voiding and incontinence. Detailed information about bladder reflex mechanisms and the manner in which they are modulated within the CNS is essential for understanding the pathophysiology of bladder hyperactivity and incontinence and for developing new therapeutic approaches to treat this disorder. It is proposed that reorganization of spinal micturition circuitry occurs in response to degeneration of bulbospinal axons as well as changes in neuron to target organ interactions. The overall goal of this proposal is to examine the effects of SCI on the neurochemical and organizational properties of spinal neurons (interneurons and preganglionic parasympathetic neurons) involved in the micturition reflex pathway. Three specific aims are proposed: 1. To determine the organization of urinary bladder interneurons and parasympathetic neurons in the lumbosacral spinal cord (L6-S1) using a combination of transneuronal tracing with pseudorabies virus (PRV) and conventional retrograde dye tracing techniques (Fluorogold) in control and SCI animals. The chemical phenotype of PRV-labeled urinary bladder interneurons in the L6-S1 spinal cord will be determined with immunohistochemistry for neuroactive compounds in control and SCI animals. 2. To determine the chemical phenotype of urinary bladder interneurons specifically responding to bladder afferent information (non-noxious and noxious) in control and SCI animals. Fos protein expression, as an indicator of cellular activation will be combined with immunohistochemistry for neuroactive compounds. 3. Previous studies have demonstrated significant increases in urinary bladder neurotrophic factor mRNA following chronic SCI. Thus, the role of neurotrophic factors (NGF and BDNF) play in mediating neurochemical and organizational plasticity of bladder interneurons following chronic SCI will be examined with chronic administration of neurotrophic factors or neurotrophic factor neutralizing antibodies in vivo.

DESCRIPTION (provided by applicant): The mechanisms involved in the storage and periodic elimination of urine exhibit marked changes during prenatal and postnatal development. In the young fetus, prior to maturation of the nervous system, urine is presumably eliminated from the urinary bladder by non-neural mechanisms. At later stages of development, micturition is regulated by spinal reflex pathways. As the central nervous system continues to mature during the postnatal period, reflex voiding is brought under voluntary control involving higher brain centers. In adults, injuries or diseases of the nervous system can lead to the reemergence of primitive functions that were prominent early in development but then were suppressed during neural maturation. Therefore, developmental studies of micturition reflex pathways are likely to provide key insights into the mechanisms underlying neurogenic disorders of urinary bladder function in adults. This research proposal will focus on alterations in spinal reflex voiding mechanisms that underlie the maturation of voiding function during the early postnatal period. Aim I). To determine the organization of urinary bladder interneurons and parasympathetic preganglionic neurons in the lumbosacral spinal cord. A combination of transneuronal tracing with pseudorabies virus (Bartha strain) and conventional retrograde dye mapping techniques (Fluorogold) will be utilized. Changes in connectivity between the various spinal elements will be assessed in postnatal rats prior to (P1, P7, P14) and following (P21, P28, P36) the emergence of the spinobulbospinal micturition reflex using design-based stereological techniques. Aim 2). To examine changes in central processing of afferent (A-s and/or C-fiber) information from the lower urinary tract of postnatal rats prior to and following the emergence of the spinobulbospinal micturition reflex. A combination of axonal tracing techniques and Fos protein expression will be utilized. These experiments will determine if A-[unreadable] and/or C-fiber bladder afferents are active during early postnatal development and how afferent information is processed by different neurons in different regions of the spinal cord. Aim 3). To examine the termination pattern, area occupied and morphology of perineal afferent nerve fibers and bulbospinal projections in close apposition to interneurons and/or preganglionic neurons in the lumbosacral spinal cord in postnatal rats prior to and following the emergence of the spinobulbosninal micturition reflex. These studies will determine if perineal afferent projections (Dil or WGA-HRP-labeled) and/or bulbospinal (CRF-IR) projections and varicosities are prominent in the lumbosacral spinal cord and which neuronal populations are in close apposition to afferent or bulbospinal efferent projections. Anatomical studies will be combined with immunoassay for CRF in the lumbosacral spinal cord.

DESCRIPTION (provided by applicant): Interstitial cystitis (IC) is a chronic inflammatory bladder disease syndrome characterized by urinary frequency, urgency, suprapubic and pelvic pain. The working hypothesis for this proposal is that urinary bladder hyperreflexia and altered sensation that accompany IC are due to an alteration in the primary afferent limb of the micturition reflex and, in part, to an alteration in interneuronal mechanisms in the spinal cord. Urinary bladder hyperreflexia after cystitis may also be due to an alteration in the efferent limb of the micturition reflex. The combination of these changes facilitates a spinal reflex pathway to the urinary bladder by altering various types of synaptic transmission at the interneuronal level in the spinal cord and by altering synaptic transmission to the pelvic ganglia. The following aims address these hypotheses: 1). Previous studies have demonstrated significant increases in urinary bladder neurotrophic factor (NTF) mRNA/protein after chronic cyclophosphamide (CYP)-induced cystitis. Thus, the role that NTFs (eq., nerve growth factor, NGF; brain derived neurotrophic factor, BDNF) play in mediating, functional, neurochemical and organizational plasticity of bladder reflexes after cystitis will be examined by chronic administration of NTFs in vivo. The companion studies will examine the effects of administration of NTF neutralizing antibodies on this plasticity after cystitis. 2). Previous studies have demonstrated dramatic upregulation of the peptide, pituitary adenylate cyciase-activating polypeptide, (PACAP) in bladder afferent and spinal pathways after cystitis. We will extend these observations by examining the functional significance of this upregulation. Thus, the influence of PACAP antagonists on bladder reflexes will be determined. Furthermore, the role of PACAP in cystitis-induced bladder hyperreflexia will be examined in PACAP knockout mice. 3). In the current proposal, we will take a new approach by examining changes in the properties of an efferent component of micturition reflexes. Thus, the electrical and pharmacological properties of bladder postganglionic neurons after CYP-induced cystitis will be examined. These studies will involve, in vitro, intracellular recordings from the cell body of the postganglionic neuron located in the major pelvic ganglion. Axons of these neurons terminate in the inflamed urinary bladder and these cells are also likely to be influenced by the inflammatory milieu.

DESCRIPTION (provided by applicant): Interstitial cystitis (IC) is a chronic inflammatory bladder disease syndrome characterized by urinary frequency, urgency, suprapubic and pelvic pain. Although the etiology and pathogenesis of IC are unknown, numerous theories including; infection, autoimmune disorder, toxic urinary agents, deficiency in bladder wall lining and neurogenic causes have been proposed. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts diverse and prevalent roles in the lower urinary tract (LUT). Sensory fibers expressing PACAP have been identified in the bladder wall and in suburothelial plexuses, PACAP expression in the micturition reflex pathway is upregulated following chronic cystitis and pharmacological agents that block PACAP receptor function reduce bladder overactivity after cystitis. PACAP expression can be regulated by neurotrophins; conversely, recent studies have also suggested that PACAP may regulate neurotrophin receptor tyrosine kinase expression and activation. Cystitis markedly alters the profile of neurotrophin expression in bladder tissues. Thus, the resulting changes in target organ growth factor levels may drive the neurochemical and functional plasticity in the micturition pathway with cystitis. The overall hypothesis for our work is that pain and micturition dysfunction in IC involves an alteration in bladder smooth muscle, urothelium and sensory physiology. The central hypothesis is that the VIPIPACAP system is a prominent modulator of bladder sensation and function and the inflammation-induced changes in neurotrophic factors and/or neural activity arising in the bladder alter PACAP/PACAP receptor expression in LUT to mediate altered micturition function in IC. The following three aims test these hypotheses. 1). To characterize PACAP and PACAP receptor expression in urothelium, bladder smooth muscle and bladder afferent cells in the lumbosacral DRG; 2). To establish the functional relationships between PACAP and neurotrophin systems in the normal micturition reflex pathway and after cystitis.; 3). To evaluate the physiological roles of PACAP and neurotrophins in the micturition reflex pathway using PACAP knockout mice.

DESCRIPTION (provided by applicant): There are a number of childhood voiding dysfunctions that may be attributable to abnormal postnatal maturation of voiding reflexes. Recent studies have shown a strong correlation between childhood lower urinary tract (LUT) dysfunction and adult overactive bladder. Thus, understanding the factors that regulate the neural control of voiding function during the postnatal period may provide insight into childhood and adult voiding dysfunction. Furthermore, injuries or diseases of the adult nervous system can lead to the reemergence of primitive functions that were prominent early in development but then were suppressed during neural maturation. Therefore, developmental studies of micturition reflex pathways are likely to provide key insights into the mechanisms underlying neurogenic disorders of urinary bladder function in adults. The central hypothesis of this proposal is that changes in both the central nervous system (CNS) and peripheral nervous system (PNS) drive micturition reflex maturation. Marked changes in the neural control of voiding function that occur during early postnatal development may be mediated by multiple factors, including upregulation of mature supraspinal mechanisms (corticotropin releasing factor, CRF/CRF receptor system) and changes in neuron and target organ (postganglionic neuronyenurinary bladder) interactions. In this competitive renewal application, we propose aims that will provide further mechanistic insight into developmental-induced changes in the neural control of micturition reflexes using a multidisciplinary experimental approach involving a rat model as well as a novel, chronic overexpressing nerve growth factor (NGF) mouse line (UPII-NGFv2). The aims of this proposal are: (1) to characterize the CRF/CRF receptor system in the lumbosacral spinal cord and to define the function of the CRF/CRF receptor system in LUT pathways as a function of development;(2) to determine the mechanisms underlying an accelerated development of mature voiding function in UPII-NGFv2 mice;(3) to define the functional properties of bladder postganglionic cells in the major pelvic ganglion (MPG) during development, the underlying channel properties that contribute to the diversity of electrophysiological properties in the adult and the contribution of urinary bladder NGF to these properties. PUBLIC HEALTH RELEVANCE: Project Narrative There are a number of childhood voiding dysfunctions that may be attributable to abnormal postnatal maturation of voiding reflexes. Recent studies have shown a strong correlation between childhood lower urinary tract dysfunction and adult overactive bladder. Thus, understanding the factors that regulate the neural control of voiding function during the postnatal period may provide insight into childhood and adult voiding dysfunction. Furthermore, injuries or diseases of the adult nervous system can lead to the reemergence of primitive functions that were prominent early in development but then were suppressed during neural maturation. Therefore, developmental studies of micturition reflex pathways are likely to provide key insights into the mechanisms underlying neurogenic disorders of urinary bladder function in adults. These studies will focus on the development of the prominent neurotransmitter/receptor system in descending spinal pathways and changes and mechanisms involved in neuron and target organ (i.e., postganglionic neuronyenurinary bladder) interactions that underlie maturation of voiding function. It is anticipated that these studies will provide insights into potential targets and therapeutic interventions for childhood and adult voiding dysfunctions.

DESCRIPTION (provided by applicant): Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain syndrome characterized by pain, pressure or discomfort perceived to be bladder related with at least one urinary symptom. The impact of BPS/IC on quality of life and economic burden are enormous. Over the life of this grant, we have hypothesized that pain associated with BPS/IC involves an alteration of visceral sensation/bladder sensory physiology. Changes in visceral sensation may be mediated, in part, by inflammatory changes in the urinary bladder including nerve growth factor (NGF). Monoclonal antibody treatment that specifically inhibits NGF in patients with BPS/IC demonstrates proof of concept; however, clinical trials have halted enrollment due to severe side effects. The need for additional targets beyond NGF is clear. With this competitive renewal application, we propose aims that will provide mechanistic insight into additional NGF-mediated pleiotropic changes that contribute to urinary bladder hyperreflexia and pelvic hypersensitivity in a novel transgenic mouse model of chronic NGF overexpression (NGF-OE) using the urothelium-specific uroplakin II promoter that was characterized during the last funding cycle. The working hypothesis is that increases in urinary frequency and altered sensation that accompany BPS/IC are due to an alteration in the expression, function and interactions of neurochemical mediators and the sensory transducer, transient receptor potential (TRPV) family member TRPV4, in the sensory limb of the urinary bladder reflex. These studies examine the contributions of and interactions between the neuropeptide, PACAP, and receptor PAC1 and TRPV4 to increased voiding frequency and somatic sensitivity in NGF-OE mice. Aim 1: We hypothesize that NGF overexpression exhibited in urothelium and lumbosacral dorsal root ganglia (DRG) of the NGF-OE mouse model induces upregulation of the sensory transducer, TRPV4, in bladder afferent cells in DRG and in urothelial cells of the urinary bladder. We hypothesize that tissue-specific expression of TRPV4, in sensory components (urothelium, DRG) of the micturition reflex contributes to urinary bladder hyperreflexia and pelvic hypersensitivity in NGF-OE mice. Aim 2: We hypothesize that interactions between TRPV4 and PACAP/PAC1 may represent a novel mechanism by which PACAP/PAC1 signaling activates phospholipase C and inositol triphosphate to sensitize TRPV4-mediated changes in voiding behavior and painful sensation. Furthermore, we hypothesize that PAC1 and TRPV4 are co-expressed in bladder afferent DRG and urothelial cells. We will use a multidisciplinary approach including anatomical, biochemical, molecular, electrophysiological, and functional methodologies and a novel ex vivo peripheral nerve recording approach to address these aims. Results will provide key insights into new targets with therapeutic potential to improve urinary bladder function and visceral sensation.

PROJECT SUMMARY/ABSTRACT Loss of bladder control is one of the most challenging outcomes facing spinal cord injured patients, with no drug treatments available at the present time. NGF has long been implicated in the development of bladder dysfunction after spinal cord injury (SCI). After SCI as well as in overactive bladder and interstitial cystitis/painful bladder syndromes, an increase in NGF levels is detected in the urine. As the increase in urinary NGF is implicated in bladder hypersensitivity, many have tried to neutralize NGF, but with mixed results. As in the CNS after injury or seizure, we have discovered that proNGF, and not mature NGF, is rapidly released into the urine after SCI in rodents as well as in humans. These results suggest that selective release of proNGF right after SCI may be a common feature in mammals, playing an analogous role. Our study in mice revealed that proNGF acts both in the CNS and in the bladder: Blocking proNGF binding to p75 systemically with a small molecule, LM11A-31, that crosses the blood-brain/spinal cord barrier efficiently, resulted in dramatic improvement in reflex voiding. The hyperreflexia was attenuated with normal bladder pressure, acquiring spontaneous voiding weeks earlier than the control. The improvement was accompanied by preservation of the bladder luminal surface, which normally undergoes massive cell loss followed by hyperplasia and detrusor hypertrophy after SCI. On the other hand, when proNGF binding to p75 was blocked locally by conditionally deleting p75 in urothelial cells, bladder function worsened after SCI, although umbrella cell loss was completely prevented. Since our data indicate that the death of umbrella cells is entirely due to urinary proNGF activating p75 on the luminal surface, these results suggest that the loss of umbrella cells and subsequent urothelial turnover influence voiding function positively. We thus hypothesize that that proNGF-p75 signaling plays a role in bladder function after SCI both in the bladder circuit and in the periphery. Under the hypothesis, we propose to determine the mechanism by which p75 induces the turnover of the urothelium after SCI, urothelial p75 influences voiding, and where in the bladder circuitry that proNGF-p75 signaling acts to influences bladder function after SCI.

Project Summary/Abstract Bladder Pain Syndrome (BPS)/Interstitial Cystitis (IC) is a chronic pelvic pain disorder with at least one urinary symptom and the perception that the pain originates from the bladder. Stress exacerbates symptoms of BPS/IC. Despite intense research, we lack understanding of how structural and functional changes in the micturition reflex are linked to BPS/IC and how stress exacerbates symptoms, thus impeding effective therapies. Addressing these challenges requires, in part: (1) a novel hypothesis involving NGF/TrkA/MAPK signaling for downstream transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4)/Ca2+ activation in the sensory components of the micturition reflex; (2) innovative and multidisciplinary approaches; and (3) animal models that recapitulate the clinical signs/symptoms of BPS/IC including symptom exacerbation (flares) precipitated by psychological stress. Our laboratory is unique in the complementary use of several relevant models to reinforce our studies including (e.g., cyclophosphamide (CYP)-induced cystitis, transgenic mice with chronic, urothelial overexpression of NGF (NGF-OE), repeated, low dose CYP (alone insufficient to produce significant symptoms) coupled with repeated variate stress (RVS) to assess how stress can exacerbate disease. Our overall hypothesis is that increases in urinary frequency and pelvic sensation that accompany BPS/IC reflect increased expression, function and interactions of neurochemical mediators and the sensory transducer, TRPV4, in the sensory components of the micturition reflex that favor a pro-excitatory state. Building from our previous work, the maladaptive role(s) of NGF/TrkA/p75NTR signaling and downstream activation of TRPV4/Ca2+ in the sensory components of the micturition reflex will be assessed as contributory mechanisms to BPS/IC. Aim 1: Determine if interrupting NGF/TrkA/p75NTR signaling pathways reduces voiding frequency and pelvic pain by: reducing (1) urothelial Ca2+ events; (2) urothelial ATP release and (3) bladder afferent activity. Further interrupting NGF/p75NTR signaling reduces voiding frequency by: (1) reducing urothelial cell apoptosis; (2) promoting urothelial cell recovery and (3) maintaining transepithelial resistance. Aim 2: Determine if disruption of NGF signaling in the micturition pathway has short- and long-term consequences on TRPV4/Ca2+ function in BPS/IC-like symptoms. The acute NGF-mediated TRPV4/Ca2+ BPS/IC-related responses include heightened urothelial Ca2+ signaling, urothelial ATP secretion and bladder afferent nerve activity. Maladaptive, long-term NGF signaling promotes BPS/IC by increasing TRPV4 transcript and protein expression. Using three models with BPS/IC-like symptoms and a multidisciplinary, cell-tissue- systems experimental approach, we will determine: (1) underlying structural and functional changes contributory to BPS/IC-like symptoms; (2) the influence of psychological stress on bladder function and pain and (3) novel treatments.