Real-Time Void Spot Assay
Summary
This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day.
Abstract
Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the nervous system. To study voluntary voiding behavior in mouse models, researchers have developed the void spot assay (VSA), a method that measures the number and area of urine spots deposited on a filter paper lining the floor of an animal’s cage. Although technically simple and inexpensive, this assay has limitations when used as an end-point assay, including a lack of temporal resolution of voiding events and difficulties quantifying overlapping urine spots. To overcome these limitations, we developed a video-monitored VSA, which we call real-time VSA (RT-VSA), and which allows us to determine voiding frequency, assess voided volume and voiding patterns, and make measurements over 6 h time windows during both the dark and light phases of the day. The method described in this report can be applied to a wide variety of mouse-based studies that explore the physiological and neurobehavioral aspects of voluntary micturition in health and disease states.
Introduction
Urine storage and micturition are coordinated by a central circuitry (central nervous system) that receives information about the bladder filling status through the pelvic and hypogastric nerves. The urothelium, the epithelium that lines the urinary tract from the renal pelvis to the proximal urethra, forms a tight barrier to the metabolic waste products and pathogens present in urine. It is an integral component of a sensory web, which senses and communicates the filling state of the bladder to underlying tissues and afferent nerves1,2. Disruption of the urothelial barrier, or alterations in urothelial mechanotransduction pathways, can lead to voiding dysfunction along with lower urinary tract symptoms such as frequency, urgency, nocturia, and incontinence3,4,5,6,7. Likewise, aging, diabetes, lower urinary tract infections, interstitial cystitis, and other disease processes that affect the urinary bladder, or the associated circuitry that controls its function, are known to cause bladder dysfunction8,9,10,11,12,13,14,15,16,17,18,19. A better understanding of normal and abnormal voiding behavior depends on the development of methods that can reliably discriminate among different urination patterns.
Traditionally, the voluntary voiding behavior of mice has been studied using the void spot assay (VSA), developed by Desjardins and colleagues20, and broadly adopted due to its simplicity, low cost, and noninvasive approach8,21,22,23,24. This assay is typically performed as an endpoint assay, in which a mouse spends a defined amount of time in a cage lined by a filter paper, which is subsequently analyzed by counting the number and assessing the size of urine spots when the filter paper is placed under ultraviolet (UV) light (the urine spots fluoresce under these conditions)20. Despite these many advantages, the traditional VSA presents some major limitations. Because mice often urinate in the same areas, investigators have to restrict the duration of the assay to a relatively short period of time (≤4 h)25. Even when the VSA is performed over shorter time periods, it is almost impossible to resolve small void spots (SVSs) that fall over large void spots or, to discriminate SVSs from the carryover of urine adhered to tails or paws. It is also very difficult to distinguish if SVSs are a consequence of frequent but individual voiding events (a phenotype that is often observed in response to cystitis4,26), or due to post-micturition dribbling (a phenotype associated with bladder outlet obstruction27). Furthermore, the desire to complete the assay during working hours, coupled with difficulties accessing housing facilities when the lights are turned off, often limits these assays to the light period of the 24 h circadian cycle. Thus, these time constraints prevent the evaluation of mouse voiding behavior during their active night phase, lessening the ability to analyze specific genes or treatments that are governed by circadian rhythms.
To overcome some of these limitations, researchers have developed alternative methods to assess voiding behavior in real time26,28,29,30,31,32. Some of these approaches involve the use of expensive equipment such as metabolic cages26,28,29, or the use of thermal cameras30; however, these too have limitations. For example, in metabolic cages, urine tends to adhere to the wires of the mesh floor and to the walls of the funnel, reducing the amount of urine that is collected and measured. Thus, it can be difficult to accurately collect data about small voids. Moreover, metabolic cages do not provide information about the spatial distribution of the voiding events (i.e., urination in the corners vs. the center of the chamber). Given that long-wavelength infrared radiation used by the thermographic cameras does not penetrate solids, voiding activity assessed by video thermography must be performed in an open system, which can be challenging with active mice, as they can jump several inches in the air. Another system is the automated voided stain on paper (aVSOP) approach33, which consists of rolled filter paper that winds up at a constant speed below the wire mesh floor of a mouse cage. This approach prevents paper damage and the overlap of urine spots that occur in the classical VSA, and its implementation allows the investigator to perform experiments over several days. However, it does not provide the investigator with precise timing of the voiding events, and there is no ability to examine behavior and how it correlates with spotting. To obtain this information, researchers have incorporated video-monitoring to voiding assays, an approach that allows the simultaneous assessment of mouse activity and urination events31,32. One approach consists of placing a blue light emitting diode (LED) and a video-camera with a green fluorescence protein filter set under the experimental cage to visualize the voiding events, and an infrared LED and a video-camera above the cage to capture mouse position32. This setup has been used to monitor voiding behavior while performing fiber photometry; however, the brightly lit environment of this system required the investigators to treat their mice with a diuretic agent to stimulate voiding. In another experimental design, wide-angle cameras were placed above and below the experimental cage to visualize mouse motor activity and urination events, respectively. In this case, urine spots deposited on a filter paper lining the cage’s floor were revealed by illuminating the filter paper with UV lights placed under the cage31. This setup was used in short assays, 4 min in duration, during the light phase of the day to study the brainstem neurons involved in voluntary voiding behavior31. The suitability of this system for its use during the dark phase or for periods of time >4 min was not reported.
In this article, a method is described that enhances the traditional VSA by allowing for long-term video monitoring of mouse voiding behavior. This cost-effective approach provides temporal, spatial, and volumetric information about voiding events for extended periods of time during the light and dark phases of the day, along with details related to mouse behavior3,4,34. Detailed information for the construction of the voiding chambers, the implementation of a real-time VSA (RT-VSA), and the analysis of the data is provided. The RT-VSA is valuable for researchers seeking to understand the physiological mechanisms that control the function of the urinary system, to develop pharmacological approaches to control micturition, and to define the molecular basis of disease processes that affect the lower urinary tract.
Protocol
Representative Results
Discussion
The incorporation of video-monitoring is a cost-effective modification that presents several advantages over the classical VSA. In the classical VSA, which is typically used as an end-point assay, it is difficult to distinguish overlapping void spots. This is not a trivial concern, as mice tend to urinate multiple times in the same area when the assay is prolonged for several hours, typically in the corners of their cage. Thus, the first advantage of RT-VSA is that the investigator can readily identify individual spots t…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by an NIH grant R01DK119183 (to G.A. and M.D.C.), a pilot project award through P30DK079307 (to M.G.D.), an American Urology Association Career Development award and a Winters Foundation grant (to N.M.), and by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307).
Materials
| 1.00” X 1.00” T-Slotted Profile – Four Open T-Slots – cut to 10 inches | 80/20 | 1010 | Amount: 20 |
| 1.00” X 1.00” T-Slotted Profile – Four Open T-Slots – cut to 12 inches | 80/20 | 1010 | Amount: 6 |
| 1.00” X 1.00” T-Slotted Profile – Four Open T-Slots – cut to 40 inches | 80/20 | 1010 | Amount: 4 |
| 1.00” X 1.00” T-Slotted Profile – Four Open T-Slots – cut to 14.75 inches | 80/20 | 1010 | Amount: 12 |
| 1.00” X 1.00” T-Slotted Profile – Four Open T-Slots – cut to 32 inches | 80/20 | 1010 | Amount: 5 |
| 1/4-20 Double Slide-in Economy T-Nut | 80/20 | 3280 | Amount: 16 |
| 1/4-20 Triple Slide-in Economy T-Nut | 80/20 | 3287 | Amount: 18 |
| 10 & 25 Series 2 Hole – 18mm Slotted Inside Corner Bracket with Dual Support | 80/20 | 14061 | Amount: 6 |
| 10 Series 3 Hole – Straight Flat Plate | 80/20 | 4118 | Amount: 8 |
| 10 Series 5 Hole – "L" Flat Plate | 80/20 | 4081 | Amount: 8 |
| 10 Series 5 Hole – "T" Flat Plate | 80/20 | 4080 | Amount: 8 |
| 10 Series 5 Hole – Tee Flat Plate | 80/20 | 4140 | Amount: 2 |
| 10 Series Standard Lift-Off Hinge – Right Hand Assembly | 80/20 | 2064 | Amount: 2 |
| 10 to 15 Series 2 Hole – Lite Transition Inside Corner Bracket | 80/20 | 4509 | Amount: 6 |
| 24”-long UV tube lights | ADJ Products LLC | T8-F20BLB24 | Amount: 2 20W bulb – 24” Wavelength: 365nm |
| Acrylic Mirror Sheet | Profesional Plastics | Amount: 1 82.5 cm x 26.5 cm |
|
| Acrylic Mirror Sheet | Profesional Plastics | Amount: 2 26.5 cm X 30.5 cm |
|
| Acrylic Mirror Sheet | Profesional Plastics | Amount: 2 82.5 cm x 30.5 cm |
|
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 2 4.5mm Thick, Clear, 38.5 cm x 26.5 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear, 38.5 cm x 21.5 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear, 26.5 cm x 21.5 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear 37.5 cm x 23.9 cm |
| AR polycarbonate (UV resistance) | 80/20 | 65-2641 | Amount: 4 4.5mm Thick, Clear , 24.4 cm x 23.9 cm |
| Chromatography paper (thin paper) | Thermo Fisher Scientific | 57144 | |
| Cosmos blotting paper (thick paper) | Blick Art Materials | 10422-1005 | |
| Excel | Microsoft Corporation | ||
| GraphPad Prism | GraphPad Software | Version 9.4.0 | graphing and statistics software |
| ImageJ FIJI | NIH | ||
| Parafilm | Merck | transparent film | |
| Quick Time Player 10.5 software | Apple | multimedia player | |
| Security spy | Ben software | video surveillance software system | |
| Standard End Fastener, 1/4-20 | 80/20 | 3381 | Amount: 80 |
| UV transmitting acrylic | Spartech | Polycast Solacryl SUVT | Amount: 2 38.5 cm x 26.5 cm |
| Water gel: HydroGel | ClearH2O | 70-01-5022 | (https://www.clearh2o.com/product/hydrogel/) |
| Webcam | Logitech | C930e | Amount: 4 |
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