Void spot assay: recommendations on the use of a simple micturition assay for mice

Abstract

Investigators have for decades used mouse voiding patterns as end points for studying behavioral biology. It is only recently that mouse voiding patterns were adopted for study of lower urinary tract physiology. The spontaneous void spot assay (VSA), a popular micturition assessment tool, involves placing a mouse in an enclosure lined by filter paper and quantifying the resulting urine spot pattern. The VSA has advantages of being inexpensive and noninvasive, but some investigators challenge its ability to distinguish lower urinary tract function from behavioral voiding. A consensus group of investigators who regularly use the VSA was established by the National Institutes of Health in 2015 to address the strengths and weaknesses of the assay, determine whether it can be standardized across laboratories, and determine whether it can be used as a surrogate for evaluating urinary function. Here we leverage experience from the consensus group to review the history of the VSA and its uses, summarize experiments to optimize assay design for urinary physiology assessment, and make best practice recommendations for performing the assay and analyzing its results.

Keywords: urinary dysfunction; void spot assay; voiding behavior.

Fig. 1.

Prototypical void spot assay (VSA) assay designs. Enclosure size and shape vary across laboratories but the basic design is the same: the enclosure is lined with filter/chromatography paper and the mouse is introduced in direct contact with the paper or elevated above it on a wire mesh cage bottom. The mouse is maintained in the cage for a fixed time interval, the paper is removed, dried, imaged and spot number, size and distribution are determined using custom methods or existing image analysis software (46).

Fig. 2.

Evidence of urine residue on wire grid elevated above the enclosure bottom. A single 12-wk-old female C57BL/6 mouse was housed for 4 h in an enclosure containing a wire grid elevated over filter paper. A: the grid was illuminated with UV light and imaged. Note multiple white spots that indicate urine residue on the grid. B: the filter paper was illuminated by UV light and imaged. The grid and filter paper images are shown in the same orientation (i.e., left side of the grid corresponds to the left side of filter paper).