Evaluation of voiding assays in mice: impact of genetic strains and sex.

Void spot assays (VSA) and cystometry are two of the most common tests performed in mice to assess lower urinary tract function. Assay protocols and methodology vary greatly among laboratories, and little is known about reproducibility of results generated by different laboratories. We performed VSA in four mouse strains, comparing males with females and comparing results between two independent laboratories. Unique aspects of the current study include direct comparison of results of VSA performed in a similar manner in two locations and comparison of cystometry performed using two different rates of infusion in these two laboratories. Both assays were performed in male and female 129S1/SvImJ, C57BL/6J, NOD/ShiLtJ, and CAST/EiJ mice, and cystometry was performed under urethane anesthesia (10/group). Assays were performed and results analyzed as previously described. Results obtained in female mice were compared with previously reported values. Results of lower urinary tract function testing in mice vary in a consistent manner with strain and sex. Variables in husbandry, testing techniques, and analysis of results can significantly affect conclusions, particularly those obtained by cystometry. Although VSA results were remarkably similar between the two laboratories, consistent methods for performing lower urinary tract function testing in mice are required to compare results among studies with confidence.

Caffeine enhances micturition through neuronal activation in micturition centers.

Caffeine may promote incontinence through its diuretic effect, particularly in individuals with underlying detrusor overactivity, in addition to increasing muscle contraction of the bladder smooth muscle. Caffeine may also affect bladder function via central micturition centers, including the medial preoptic area, ventrolateral periaqueductal gray, and pontine micturition center. However, the biochemical mechanisms of caffeine in central micturition centers affecting bladder function remain unclear. In the present study, the effects of caffeine on the central micturition reflex were investigated by measuring the degree of neuronal activation, and by quantifying nerve growth factor (NGF) expression in rats. Following caffeine administration for 14 days, a urodynamic study was performed to assess the changes to bladder function. Subsequently, immunohistochemical staining to identify the expression of c­Fos and NGF in the central micturition areas was performed. Ingestion of caffeine increased bladder smooth muscle contraction pressure and time as determined by cystometry. Expression levels of c­Fos and NGF in all central micturition areas were significantly increased following the administration of caffeine. The effects on contraction pressure and time were the most potent and expression levels of c­Fos and NGF were greatest at the lowest dose of caffeine. These results suggest that caffeine facilitates bladder instability through enhancing neuronal activation in the central micturition areas.

Spontaneous voiding by mice reveals strain-specific lower urinary tract function to be a quantitative genetic trait.

Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18-24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations.

Characterization of bladder function in a cannabinoid receptor type 2 knockout mouse in vivo and in vitro.

AIMS: The contribution of individual CB receptors (CB1 R and CB2 R) to normal micturition has not been clearly defined. Our goal was to study if differences in urodynamic parameters or in vitro bladder contractility can be demonstrated between CB2 R knockout (CB2 RKO) and C57BL/6J control (wild type, WT) mice. METHODS: Female WT and CB2 RKO mice underwent bladder catheterization and cystometry was performed after 2 and 3 days. Cystometric evaluations were performed in awake animals without drug administration, and WT were also given HU-308 (CB2 R agonist) followed by AM630 (CB2 R antagonist). Bladders were removed for in vitro assessment of contractile responses to carbachol and electrical field stimulation (EFS). RESULTS: CB2 RKO mice had significantly higher intercontraction intervals (ICIs), bladder capacity (BC) and compliance (Bcom) than WT controls (P < 0.05). In WT mice, BC and ICI were increased from baseline by HU-308 exposure, and then returned to baseline levels after AM630 administration (P < 0.05). There were no differences in contractility after carbachol or EFS between the groups. CONCLUSIONS: Lack of CB2 R was associated with longer ICI and higher BC and Bcom than its presence (WT controls). This was unexpected since in WT, an increase in BC and ICI from baseline was observed after CB2 R agonist administration, and this action was reversed by a CB2 R antagonist. Since there were no differences in the in vitro responses to carbachol and EFS in bladder strips, it may be speculated that the urodynamic differences are caused by a change in the central nervous micturition control in CB2 RKO animals. Neurourol. Urodynam. 33:566-570, 2014. © 2013 Wiley Periodicals, Inc.

Rodent models for urodynamic investigation.

Rodents, most commonly rats, mice, and guinea pigs are widely used to investigate urinary storage and voiding functions, both in normal animals and in models of disease. An often used methodology is cystometry. Micturitions in rodents and humans differ significantly and this must be considered when cystometry is used to interpret voiding in rodent models. Cystometry in humans requires active participation of the investigated patient (subject), and this can for obvious reasons not be achieved in the animals. Cystometric parameters in rodents are often poorly defined and do not correspond to those used in humans. This means that it is important that the terminology used for description of what is measured should be defined, and that the specific terminology used in human cystometry should be avoided. Available disease models in rodents have limited translational value, but despite many limitations, rodent cystometry may give important information on bladder physiology and pharmacology. The present review discusses the principles of urodynamics in rodents, techniques, and terminology, as well as some commonly used disease models, and their translational value.

Vesico-ureteric reflux: using mouse models to understand a common congenital urinary tract defect.

Vesico-ureteric reflux (VUR) is a common congenital urinary tract defect in which urine flows retrogradely from the bladder to the kidneys because of an abnormally formed uretero-vesical junction. It is associated with recurrent urinary tract infections, renal hypo/dysplasia, reflux nephropathy, hypertension, and end-stage renal disease. In humans, VUR is genetically and phenotypically heterogeneous, encompassing diverse renal and urinary tract phenotypes. To understand the significance of these phenotypes, we and others have used the mouse as a model organism and this has led to the identification of new candidate genes. Through careful phenotypic analysis of these models, a new understanding of the genetics and biology of VUR is now underway.

The voltage-gated sodium channel Nav1.9 is required for inflammation-based urinary bladder dysfunction.

Tetrodotoxin (TTX)-resistant sodium channels are found in small diameter primary sensory neurons and are thought to be important in the maintenance of inflammatory pain. Here we examined bladder urodynamics of Nav1.9 voltage-gated sodium channel knock out (KO) mice, and the contribution of Nav1.9 to the development of inflammation-based bladder dysfunction. Basal urodynamics were not different between wildtype (WT) mice and those lacking Nav1.9. Peripheral nerve recordings from pelvic afferents in Nav1.9 KO mice revealed a lack of sensitization to intravesicularly applied prostaglandin E2 (PGE2). Consistent with this, cyclophosphamide treatment in vivo, which is associated with an enhancement of PGE2 production, evoked a reduction in bladder capacity of WT, but not Nav1.9 KO mice. We conclude that the Nav1.9 sodium channel provides an important link between inflammatory processes and changes in urodynamic properties that occur during urinary bladder inflammation.

The molecular basis of urgency: regional difference of vanilloid receptor expression in the human urinary bladder.

AIM: Treatments targeting vanilloid receptor TRPV1 are effective in some bladder disorders. Our aim was to determine the expression profiles of TRPV1 in regions of human bladder and test the hypothesis that there would be an upregulation of TRPV1 in mucosa of patients with bladder hypersensitivity but not idiopathic detrusor overactivity (IDO). MATERIALS AND METHODS: Women with sensory urgency (SU), interstitial cystitis (IC), and IDO were investigated by videourodynamics and cystoscopy. Control biopsies were used for comparison. Biopsies were dissected into mucosa and muscle, and evaluated for TRPV1 mRNA expression using quantitative competitive RT-PCR (QC-RT-PCR). RESULTS: TRPV1 mRNA from SU trigonal mucosa was significantly higher than control trigonal mucosa or SU bladder body mucosa. In contrast, in IDO patients, there was no difference between trigonal mucosa and body mucosa. In IC biopsies, RNA quality was substandard and unable to be used for analysis. The most striking finding was that TRPV1 mRNA expressed in SU trigonal mucosa was significantly inversely correlated with the bladder volume at first sensation of filling during cystometry. No such relationship was seen for IDO trigonal mucosa. No difference was seen in bladder body mucosa from any disease groups compared with age-matched control. CONCLUSIONS: The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.

Infravesical obstruction in aromatase over expressing transgenic male mice with increased ratio of serum estrogen-to-androgen concentration.

PURPOSE: The potential role of estrogen in the development of infravesical obstruction is still unresolved. Aromatase over expressing transgenic mice provide a novel instrument for investigating the consequences of prolonged systemic or local increases in endogenous estrogen concentrations. Two aromatase over expressing transgenic mouse strains with different prostatic phenotypes (reduced and normal size, respectively) were compared in urodynamic studies with each other and with the wild-type strain. MATERIALS AND METHODS: The bladder and urethra were exposed in adult male wild-type or transgenic mice. High frequency oscillations of intraluminal bladder pressure and flow rate from the distal urethra were simultaneously recorded with the mice under anesthesia. RESULTS: No changes were observed in voiding in MMTV-arom+ mice. These mice are known to have only slightly elevated estradiol concentrations in serum, suggesting a localized increase in estrogen production. In AROM+ mice the aromatase gene was detected in several organs, including the testis and bladder. These mice are known to have markedly increased estrogen and decreased serum androgen concentrations, and reduced prostate size. Compared with wild-type mice AROM+ mice showed higher mean maximal bladder pressure plus or minus standard deviation (33.1 +/- 6.4 versus 25.6 +/- 4.8 mm. Hg, p = 0.046) and decreased mean maximal flow rate (3.1 +/- 1.6 versus 17.7 +/- 5.4 ml. per minute, p <0.0001), consistent with the presence of the infravesical obstruction. Morphologically the proximal rhabdosphincter in AROM+ mice showed atrophy (relative mean thickness 0.005 +/- 0.015 versus 0.013 +/- 0.002 mm., p <0.0001). CONCLUSIONS: Activation of the aromatase gene during an earlier developmental stage under the ubiquitin C promoter and highly elevated serum estrogen concentrations may explain the differences in voiding and prostate size in the AROM+ mouse strain.

Mitochondrial DNA deletion of the human detrusor after partial bladder outlet obstruction-correlation with urodynamic analysis.

OBJECTIVES: To investigate mitochondrial DNA (mtDNA) mutations in human detrusor after partial bladder outlet obstruction (BOO) and correlate the findings with the results of urodynamic studies. METHODS: Sixty-two male patients with and without BOO were recruited and assessed by the International Prostate Symptom Score, a quality-of-life assessment index, and sonography. The severity of partial BOO was determined by pressure-flow study with an International Continence Society (ICS) nomogram. Random detrusor biopsies obtained cystoscopically were analyzed by polymerase chain reaction (PCR) techniques to detect possible mtDNA deletions. Primer-shift PCR and DNA sequencing were then performed to characterize specific mtDNA deletions. A semiquantitative PCR method was used to determine the proportion of the deleted mtDNA in detrusor. Finally, the mtDNA deletion and the urodynamic results were compared statistically. RESULTS: A 4977-bp mtDNA deletion was identified in the human detrusor. Its incidence and proportion were found to increase after partial BOO (P = 0.005 and 0.012, respectively). The incidence of the mtDNA deletion was 4.2% (1 of 24) in the unobstructed group, 27.8% (5 of 18) in the equivocal group, and 40% (8 of 20) in the obstructed group. The mean proportion of the 4977-bp deleted mtDNA was 23.7 and 12.7 times higher in the obstructed and equivocal groups, respectively, compared with that of the unobstructed group. CONCLUSIONS: We found mtDNA with the 4977-bp deletion in human detrusor and an increase of this deletion after partial BOO. This molecular change might account for the previous observations of mitochondrial functional impairment and voiding dysfunction after partial BOO.