Evaluating the voiding spot assay in mice: a simple method with complex environmental interactions

Abstract

The voiding spot assay (VSA) on filter paper is an increasingly popular method for studying lower urinary tract physiology in mice. However, the ways VSAs are performed differ significantly between laboratories, and many variables are introduced compared with the mouse's normal housing situation. Rodents are intelligent social animals, and it is increasingly understood that social and environmental stresses have significant effects on their physiology. Surprisingly, little is known about whether change of environment during VSA affects mouse voiding and what the best methodologies are for retaining "natural" micturition patterns. It is well known that stress-related neuropeptide corticotropin-releasing factor is significantly elevated and induces dramatic voiding changes when rodents encounter stresses. Therefore we hypothesized that changes in the environmental situation could potentially alter voiding during VSA. We have examined multiple factors to test whether they affect female mouse voiding patterns during VSA, including cage type, cage floor, water availability, water bottle location, single or group housing, and different handlers. Our results indicate that mice are surprisingly sensitive to changes in cage type and floor surface, water bottle location, and single/group housing, each of which induces significant changes in voiding patterns, indicative of a stress response. In contrast, neither changing handler nor 4 h of water deprivation affected voiding patterns. Our data indicate that VSA should be performed under conditions as close as possible to the mouse's normal housing. Optimizing VSA methodology will be useful in uncovering voiding alterations in both genetic and disease models of lower urinary dysfunctions.

Disruption of Cav1.2-mediated signaling is a pathway for ketamine-induced pathology

“…A volume:area standard curve on this paper determined that a 1 mm 2 voiding spot represents 0.283 μl of urine. Voiding spots with an area ≥80 mm 2 were considered primary voiding spots (PVS), based on voiding spot patterns from hundreds of mice 18,62 .…”

Targetable purinergic receptors P2Y12 and A2b antagonistically regulate bladder function

“…Spontaneous VSA. VSAs were performed as described previously (58)(59)(60). Individual mice were gently placed in a standard polycarbonate mouse cage with Blicks Cosmos Blotting Paper (catalog 10422-1005) placed in the bottom, for 4 hours.…”

“…A volume/area standard curve on this paper determined that 1 mm 2 is equal to 0.283 μL urine. Voiding spots that have an area ≥80 mm 2 are considered to be PVSs (59,60).…”

Non-invasive voiding assessment in conscious mice

“…It Non-invasive murine voiding assessment was also noticed that CMG-results are less consistent across laboratories than non-invasive methods such as VSOP [25]. Besides the variability resulting from slightly different application of a certain measurement technique, mouse strain, sex, age, environmental factors and husbandry practices as well as time point in the animals' circadian rhythm clearly affect physiologic results as well, which mandates that this kind of information is provided in experimental reports [20,[36][37][38].…”