Void spot assay visualization optimization for use of Void Whizzard in rats (Rattus norvegicus)

Void spot assay (VSA) noninvasively evaluates urination. VSA is often not performed in rats due to difficulty analyzing larger papers compared with mouse. This study optimizes VSA for rats by comparing post-assay visualization techniques: bright field light (BF), ultraviolet light (UV), and ninhydrin spray (N). Male rats were placed in cages lined by filter paper for 4 hours. Water and food was provided ad lib. After the assay all papers were dried overnight. BF images were photographed (digital camera). UV images were captured after cutting papers in half using a Darkroom ultraviolet imaging cabinet. Papers were sprayed with ninhydrin and photographed (digital camera). All images were converted to black and white for analysis with Void Whizzard. UV vs. BF showed significant differences in area. All three groups had significant differences in overall spot count and in the smallest sized spots (0-0.1 cm2). UV vs. N and UV vs. BF showed significant differences in 0.1-0.25 cm2 spots, UV vs. N in 0.25-0.5 cm2, and N vs. BF in spots 0.5-1 cm2. Overall BF visualization proved difficult. N provided an ideal way to highlight urine and image with a digital camera. Human fingerprints from pre-assay handling of paper interfered with analysis of the smallest sized spots however there were no differences in detection of larger spots, spot distribution, or overall spot area. This study contributes to the development of a standardized VSA protocol for assessing bladder function in both mouse and rat models.