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

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.

Early and Late Transcriptional Changes in Blood, Neural, and Colon Tissues in Rat Models of Stress-Induced and Comorbid Pain Hypersensitivity Reveal Regulatory Roles in Neurological Disease

“…The PI3K /Akt pathway has been reported as a regulator of spinal plasticity in the rat visceral pain model ( 56 ) and associated with chronic pain ( 57 ). Previous studies described an increased production of VEGF by intestinal mucosa cells of patients with IBS and associated vascular endothelial growth factors with visceral hyperalgesia, abdominal discomfort, and/or pelvic pain ( 58 , 59 ). Likewise, the neurotrophin pathway promotes blood vessel growth ( 60 ); neurotrophins such as NT-3 control the sympathetic innervation of blood vessels ( 60 , 61 ).…”

Therapeutic Approaches for Urologic Chronic Pelvic Pain Syndrome; Management: Research Advances, Experimental Targets, and Future Directions

“…Despite these clinical findings, the sites, and mechanisms of VEGF action in the CNS centers controlling micturition and pelvic pain, and the effects on the excitability of peripheral and central neurons innervating the LUT are poorly understood. Preclinical animal models determined that activation of VEGF signaling in the urinary bladder enhanced innervation of the bladder wall (Cheppudira et al, 2008;de Rijk et al, 2022;Malykhina et al, 2012) and that intravesical instillations of VEGF in mice promoted sensory and motor nerve density, triggered detrusor overactivity, and worsened pelvic sensitivity (Malykhina et al, 2012;Xie et al, 2022). In animal studies, silencing of lumbosacral sensory neurons with intrathecal delivery of DREADDs (designer receptors exclusively activated by designer drugs) reversed VEGF-induced visceral hypersensitivity (Xie et al, 2022), and the use of anti-VEGF antibodies effectively reduced pelvic nociception, suggesting potential benefits of anti-VEGF therapy in UCPPS patients (Lai et al, 2017).…”

“…Preclinical animal models determined that activation of VEGF signaling in the urinary bladder enhanced innervation of the bladder wall (Cheppudira et al, 2008;de Rijk et al, 2022;Malykhina et al, 2012) and that intravesical instillations of VEGF in mice promoted sensory and motor nerve density, triggered detrusor overactivity, and worsened pelvic sensitivity (Malykhina et al, 2012;Xie et al, 2022). In animal studies, silencing of lumbosacral sensory neurons with intrathecal delivery of DREADDs (designer receptors exclusively activated by designer drugs) reversed VEGF-induced visceral hypersensitivity (Xie et al, 2022), and the use of anti-VEGF antibodies effectively reduced pelvic nociception, suggesting potential benefits of anti-VEGF therapy in UCPPS patients (Lai et al, 2017). A transcriptome study of bladder biopsy samples from IC/BPS patients with Hunner's lesions found overexpression of VEGF and B-cell activating factor in the urinary bladder.…”

Transient receptor potential ankyrin 1 channels in the bladder mediate low temperature elicited bladder overactivity in rats

“…In lower urinary tracts, the TRPA1 channel has been implicated in overactive bladder, 11 or bladder pain 12–14 through afferent nerves 8–12 . The TRPA1 channel expression level is increased in the urinary bladders or the dorsal root ganglion (DRG) L6‐S1 in diseases such as spinal cord injury 15 or bladder and prostate inflammation 16–18 . Furthermore, it is reported that TRPA1 channels expressed within capsaicin‐sensitive afferent nerve fibers in the rat bladders mediate the basal tone of bladder strip activity 19 .…”

“…[8][9][10][11][12] The TRPA1 channel expression level is increased in the urinary bladders or the dorsal root ganglion (DRG) L6-S1 in diseases such as spinal cord injury 15 or bladder and prostate inflammation. [16][17][18] Furthermore, it is reported that TRPA1 channels expressed within capsaicin-sensitive afferent nerve fibers in the rat bladders mediate the basal tone of bladder strip activity. 19 This study determined that the TRPA1 channels located in the urinary bladder could mediate the LT-elicited bladder overactivity in normal rats.…”