Pharmacogenetic inhibition of lumbosacral sensory neurons alleviates visceral hypersensitivity in a mouse model of chronic pelvic pain.

Alison Xiaoqiao Xie,Taylor C Clarkson,Nao Iguchi,Anna P Malykhina,Praveen Thumbikat

doi:10.1371/journal.pone.0262769

Abstract

The study investigated the cellular and molecular mechanisms in the peripheral nervous system (PNS) underlying the symptoms of urologic chronic pelvic pain syndrome (UCPPS) in mice. This work also aimed to test the feasibility of reversing peripheral sensitization in vivo in alleviating UCPPS symptoms. Intravesical instillation of vascular endothelial growth factor A (VEGFA) was used to induce UCPPS-like symptoms in mice. Spontaneous voiding spot assays and manual Von Frey tests were used to evaluate the severity of lower urinary tract symptoms (LUTS) and visceral hypersensitivity in VEGFA-instilled mice. Bladder smooth muscle strip contractility recordings (BSMSC) were used to identify the potential changes in myogenic and neurogenic detrusor muscle contractility at the tissue-level. Quantitative real-time PCR (qPCR) and fluorescent immunohistochemistry were performed to compare the expression levels of VEGF receptors and nociceptors in lumbosacral dorsal root ganglia (DRG) between VEGFA-instilled mice and saline-instilled controls. To manipulate primary afferent activity, Gi-coupled Designer Receptors Exclusively Activated by Designer Drugs (Gi-DREADD) were expressed in lumbosacral DRG neurons of TRPV1-Cre-ZGreen mice via targeted adeno-associated viral vector (AAVs) injections. A small molecule agonist of Gi-DREADD, clozapine-N-oxide (CNO), was injected into the peritoneum (i. p.) in awake animals to silence TRPV1 expressing sensory neurons in vivo during physiological and behavioral recordings of bladder function. Intravesical instillation of VEGFA in the urinary bladders increased visceral mechanical sensitivity and enhanced RTX-sensitive detrusor contractility. Sex differences were identified in the baseline detrusor contractility responses and VEGF-induced visceral hypersensitivity. VEGFA instillations in the urinary bladder led to significant increases in the mRNA and protein expression of transient receptor potential cation channel subfamily A member 1 (TRPA1) in lumbosacral DRG, whereas the expression levels of transient receptor potential cation channel subfamily V member 1 (TRPV1) and VEGF receptors (VEGFR1 and VEGFR2) remained unchanged when compared to saline-instilled animals. Importantly, the VEGFA-induced visceral hypersensitivity was reversed by Gi-DREADD-mediated neuronal silencing in lumbosacral sensory neurons. Activation of bladder VEGF signaling causes sensory neural plasticity and visceral hypersensitivity in mice, confirming its role of an UCPPS biomarker as identified by the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) research studies. Pharmacogenetic inhibition of lumbosacral sensory neurons in vivo completely reversed VEGFA-induced pelvic hypersensitivity in mice, suggesting the strong therapeutic potential for decreasing primary afferent activity in the treatment of pain severity in UCPPS patients.

Highlights

Urologic chronic pelvic pain syndrome (UCPPS) is characterized by chronic pain in the pelvic region or genitalia that is often accompanied by urinary frequency and urgency [1]
We demonstrated that 1) intravesical instillations of vascular endothelial growth factor A (VEGFA) did not affect the baseline nor maximum detrusor muscle contractility tested in vitro; nor did they change spontaneous voiding patterns in vivo, suggesting that VEGFA exposure mostly targeted bladder innervation with minimal impact on the detrusor muscle itself; 2) bladder VEGFA instillations enhanced nerve-mediated RTX-sensitive detrusor contractions, suggesting a predominant contribution of sensory neural pathways to neurogenic contractions; 3) activation of VEGFA signaling in the urinary bladder increased gene and protein expression of TRPA1 channel in lumbosacral dorsal root ganglia (DRG), and induced visceral mechanical hypersensitivity; 4) silencing the activity of lumbosacral sensory neurons with Gi-Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) abolished the VEGFA-induced visceral mechanical hypersensitivity, suggesting a potential therapeutic strategy for alleviating visceral pain
Our data suggest that Vascular endothelial growth factor (VEGF) signaling in the urinary bladder differentially impacts pelvic sensitivity and voiding function in this mouse model of UCPPS, while predominantly affecting sensory nerve-mediated bladder contractions and pelvic mechanical sensitivity

Summary

Introduction

Urologic chronic pelvic pain syndrome (UCPPS) is characterized by chronic pain in the pelvic region or genitalia that is often accompanied by urinary frequency and urgency [1]. Patients with UCPPS reported higher sensitivity to pressure pain, which correlated with a lower likelihood of symptom improvement using current treatment strategies [3]. Many patients complained of widespread systemic pain, which correlated with more severe UCPPS symptoms than those with pelvic pain only [4]. Longitudinal clinical changes in UCPPS were associated with structural and functional disturbances in the sensorimotor systems of the brain representing pelvic area [5]. These data suggested that increased pain sensitivity significantly contributes to UCPPS symptoms [1]

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