Reflections on the void: the art of micturition analysis

Many men and women of all ages experience lower urinary tract symptoms (LUTS) such as overactive bladder, voiding obstructions, and urinary tract infections. In addition to significantly decreasing quality of life, LUTS are often lifelong problems that add substantially to medical costs. Given that LUTS often worsen with age and that our aged population is growing, we must identify new means to treat these afflictions, which will require a more detailed understanding of the underlying mechanisms.

Mice are a useful model to study LUTS because many of these symptoms, such as urinary tract infections, can be experimentally induced (4). Additionally, investigators can examine the effect of variables such as obesity, diet, sex hormones, drugs, and genetics on urinary function. One method to assess such effects is cystometry, in which a catheter is placed into the bladder of a mouse and used to measure bladder capacity, peak bladder pressure, and spontaneous contractility (3). However, although this assay can be done with and without anesthesia, it is difficult, invasive, and time-consuming.

Some researchers have instead begun employing an assay long used by those studying mouse behavior, the void spot assay (VSA). In this assay, the investigator measures the number, size, and spatial distribution of urine spots on filter paper over a defined period of time. The VSA requires no specialized equipment and is easy to perform, reproducible, inexpensive, noninvasive, rapid, and can be repeated on the same mouse several times. Studies have shown that there are many different factors that can have an effect on voiding behaviors. Spontaneous urination patterns can be used to evaluate how changes in environment, age, and genetic background impact urinary function (5). This assay has provided researchers with a noninvasive tool to further study the effects of stimuli on LUTS. Despite these major advantages, there have remained concerns regarding whether this assay can be successfully used to distinguish alterations in urinary physiology.

To address this important question, the National Institute of Diabetes, Digestive and Kidney Diseases tasked a consensus group with evaluating the methodologies, interpretations, and findings of VSAs. In this issue of American Journal of Physiology-Renal Physiology, Hill et al. (3) provide a comprehensive report on the outcome of this group's work, describing numerous research questions that have been addressed with the VSA, pointing out parameters that should be considered and reported on for VSAs. We highlight these suggested parameters and optimal approach for conducting a VSA in Fig. 1.

Fig. 1.

Schematic depicting best practices for the design and analysis of void spotting assays.

Historically, cystometry, a clinical diagnostic procedure that measures the contractility of the bladder, has been used to evaluate bladder capacity, peak bladder pressure, and spontaneous contractility (3). There have been some divergent findings using the two assays that suggest that VSA and cystometry may not be necessarily interchangeable. While some groups have been able to confirm VSA results using cystometry (and vice-versa) (6), others have shown contrasting data between the two methods (7). Based on these and other findings, the consensus of the group is that cystometry and VSA both provide information about physiological changes due to LUTS, albeit providing different types of measurements.

Hill et al. (3) outline how investigators in the field could use the VSA to gain new insights into mechanisms underlying disorders that affect the lower urinary tract. The relationship between the voided volume and the urine spot area has been extensively studied (9). In general, the data collected from VSAs include: the total number of spots, the total voided volume, the average volume voided, and the spatial distribution of the spots. Depending on the investigator’s interest, the changes in these measurements may be interpreted differently. Spatial distribution of spots may be particularly interesting due to mice engaging in “wall seeking” behavior. This is thought to be related to anxiety, fear, and learning, and it is an inherited but modifiable property. Because of this, mice will often urinate near the walls/outer edges of the cage. Therefore, changes in the ratio of edge voiding to center voiding are an indicator of aberrant void behavior. Interestingly, these behaviors occur independent of enclosure shape.

Furthermore, researchers could use the VSA to assess the effects of genetic differences on susceptibility to urinary tract infections. For example, it has been shown that C3H/HeN mice are more susceptible to urinary tract infections than C57BL/6J mice (8). Sex and age are important considerations as women are disproportionally affected by urinary tract infections, and postmenopausal women are highly susceptible to recurrent urinary tract infections (2). Studies using VSA to examine voiding behaviors in different mouse strains at different ages, with and without infection, may provide valuable insights into urinary tract infection pathogenesis, incidence of reinfections, and efficacy of current and new therapies.

One major disadvantage of VSA that Hill et al. (3) address is that small differences in procedure can lead to variable results. Some of the biggest variations can potentially come from the use of a grid, the type of filter paper used, and most notably, the data analysis method. Furthermore, there is no consensus on how each experiment should be planned. In this review, Hill et al. (3) provide a valuable list of VSA “best practices,” which provides insight as to how to conduct experiments and report results. When similar protocols were used, different laboratories in different geographical locations produced consistent results, further supporting the reproducibility of VSA (1). Additionally, by archiving VSA filter images, future work can be done to compare results between experimental conditions and labs. The review also highlights an open source software called “Void Whizzard” (http://sites.imagej.net/VezinaLab) that was recently developed by Vezina and colleagues to facilitate rapid and objective analysis of VSA data (10). This software package may ultimately enable VSA to be a standard tool in the analysis of LUTS. Thus, when done correctly and consistently, void spot assays could provide a unique tool that can be used to further understand the physiology of the lower urinary tract.

GRANTS

B. Fashemi is supported by NIH Training Grant T32-AI 007172. I. U. Mysorekar is supported by NIH Grants R01 AG052494, R01 DK1000644, and P20-DK119840.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

B.E.F. and I.U.M. prepared figure; B.E.F. and I.U.M. drafted manuscript; B.E.F. and I.U.M. edited and revised manuscript; B.E.F. and I.U.M. approved final version of manuscript.