Accelerated onset of the vesicovesical reflex in postnatal NGF-OE mice and the role of neuropeptides

Abstract

The mechanisms underlying the postnatal maturation of micturition from a somatovesical to a vesicovesical reflex are not known but may involve neuropeptides in the lower urinary tract. A transgenic mouse model with chronic urothelial overexpression (OE) of NGF exhibited increased voiding frequency, increased number of non-voiding contractions, altered morphology and hyperinnervation of the urinary bladder by peptidergic (e.g., Sub P and CGRP) nerve fibers in the adult. In early postnatal and adult NGF-OE mice we have now examined: (1) micturition onset using filter paper void assays and open-outlet, continuous fill, conscious cystometry; (2) innervation and neurochemical coding of the suburothelial plexus of the urinary bladder using immunohistochemistry and semi-quantitative image analyses; (3) neuropeptide protein and transcript expression in urinary bladder of postnatal and adult NGF-OE mice using Q-PCR and ELISAs and (4) the effects of intravesical instillation of a neurokinin (NK)-1 receptor antagonist on bladder function in postnatal and adult NGF-OE mice using conscious cystometry. Postnatal NGF-OE mice exhibit age-dependent (R2= 0.996–0.998; p ≤ 0.01) increases in Sub and CGRP expression in the urothelium and significantly (p ≤ 0.01) increased peptidergic hyperinnervation of the suburothelial nerve plexus. By as early as P7, NGF-OE mice exhibit a vesicovesical reflex in response to intravesical instillation of saline whereas littermate WT mice require perigenital stimulation to elicit a micturition reflex until P13 when vesicovesical reflexes are first observed. Intravesical instillation of a NK-1 receptor antagonist, netupitant (0.1 μg/ml), significantly (p ≤ 0.01) increased void volume and the interval between micturition events with no effects on bladder pressure (baseline, threshold, peak) in postnatal NGF-OE mice; effects on WT mice were few. NGF-induced pleiotropic effects on neuropeptide (e.g., Sub P) expression in the urinary bladder contribute to the maturation of the micturition reflex and are excitatory to the micturition reflex in postnatal NGF-OE mice. These studies provide insight into the mechanisms that contribute to the postnatal development of the micturition reflex.

Rhythmic Calcium Events in the Lamina Propria Network of the Urinary Bladder of Rat Pups

“…The spinobulbospinal micturition reflex is triggered by tension receptor afferents in the bladder and begins to elicit voiding in the rat between P16-18 (Capek and Jelinek, 1956 ; Girard et al, 2016 ) and continues to mature during the postnatal weeks 3–6 (Capek and Jelinek, 1956 ; Ng et al, 2007 ). As the CNS matures during the postnatal period in humans, reflex voiding becomes voluntary (2–5 years) and originates in the higher brain centers (de Groat et al, 1998 ; de Groat and Araki, 1999 ; Zvarova and Zvara, 2012 ).…”

Effects of CYP-Induced Cystitis on Growth Factors and Associated Receptor Expression in Micturition Pathways in Mice with Chronic Overexpression of NGF in Urothelium

“…Consistent with the current studies demonstrating differential changes in growth factors and receptors transcripts in urothelium and detrusor of NGF-OE mice with CYP-induced cystitis, neuropeptides and associated receptors also exhibited changes in urothelium transcript expression in NGF-OE mice following CYP-induced cystitis (Girard et al, 2012). As a follow-up to the demonstration that PAC1 receptor transcript expression was significantly increased in urothelium of NGF-OE mice in the presence or absence of CYP-induced cystitis, we evaluated the contribution of PACAP/PAC1 receptor signaling to increased urinary bladder frequency and somatic sensitivity in NGF-OE mice (Girard et al, submitted). Intravesical instillation of the PAC1 receptor antagonist, PACAP(6-38) (300 nM) significantly reduced urinary frequency, the presence of non-voiding bladder contractions during the filling phase and pelvic sensitivity as determined with von Frey monofilament testing (Girard et al, submitted).…”

“…As a follow-up to the demonstration that PAC1 receptor transcript expression was significantly increased in urothelium of NGF-OE mice in the presence or absence of CYP-induced cystitis, we evaluated the contribution of PACAP/PAC1 receptor signaling to increased urinary bladder frequency and somatic sensitivity in NGF-OE mice (Girard et al, submitted). Intravesical instillation of the PAC1 receptor antagonist, PACAP(6-38) (300 nM) significantly reduced urinary frequency, the presence of non-voiding bladder contractions during the filling phase and pelvic sensitivity as determined with von Frey monofilament testing (Girard et al, submitted). Similar functional studies evaluating the contributions of NGF/P75 NTR , BDNF/TrkB and VEGF-VEGF receptor systems to urinary bladder dysfunction and increased somatic sensitization will be performed now that potential contributors have been identified in the present study.…”

Expression and Function of Chemokines CXCL9-11 in Micturition Pathways in Cyclophosphamide (CYP)-Induced Cystitis and Somatic Sensitivity in Mice

“…Quantitative RT-PCR (Girard et al, 2008 , 2010 , 2013 , 2016 ; Gonzalez et al, 2013 ) was used to determine CXCL9, CXCL10, and CXCL11 mRNA transcript levels in the urothelium and detrusor muscle as previously described. Total RNA was extracted using STAT-60 total RNA/mRNA isolation (Tel-Test “B”, Friendswood, TX, USA).…”

“…Complementary DNA templates, diluted 10-fold to minimize the inhibitory effects of the reverse transcription reaction components, were assayed using HotStart-IT SYBR Green qPCR Master Mix (USB, Cleveland, OH, USA) and 300 nM of each primer in a final 25-μL reaction volume. Real-time quantitative PCR was performed on an Applied Biosystems 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) as previously described (Girard et al, 2008 , 2010 , 2013 , 2016 ; Gonzalez et al, 2013 ). Oligonucleotide primer sequences were: CXCL9: upper primer (5′-GTGGAGTTCGAGGAACCCTAG-3′); lower primer (5′-ATTGGGGCTTGGGGCAAAC-3′); CXCL10: upper primer (5′-GTGGGACTCAAGGGATCCCTC-3′); lower primer (5′-CAGGATAGGCTCGCAGGGATG-3′); CXCL11: upper primer (5′-GCTCAAGGCTTCCTTATGTTCAAAC-3′); lower primer (5′-CTTTGTCGCAGCCGTTACTCG-3′).…”