Methods for Assessing Lower Urinary Tract Function in Animal Models

EUF-855; No. of Pages 4 E U RO P E A N U R O L O GY F O C U S X X X ( 2 019 ) X X X– X X X

available at www.sciencedirect.com journal homepage: www.europeanurology.com/eufocus

Mini Review – Neuro-urology

Methods for Assessing Lower Urinary Tract Function in Animal Models Andrea M. Sartori a,b,*, Thomas M. Kessler b, Martin E. Schwab a a

Institute for Regenerative Medicine, University of Zürich, Zürich, Switzerland;

b

Department of Neuro-Urology, Balgrist University Hospital, University of

Zürich, Zürich, Switzerland

Article info

Abstract

Article history: Accepted December 10, 2019

Lower urinary tract dysfunction affects a multitude of patients. Current therapeutic approaches are limited and very little is known about the mechanisms in failure of bladder control. Thus, more basic research is clearly needed to elucidate the underlying pathological mechanisms and to develop novel treatment strategies in urology. Noninvasive tests such as the void-spot assay and the metabolic cage and more invasive urodynamics investigations are currently used to assess lower urinary tract function in animals, in particular rodents. The noninvasive tests give some insights into the functionality of the system, whereas urodynamics testing yields an objective evaluation that allows distinction of different pathologies and investigations of the underlying neuronal malfunctions. Patient summary: We briefly summarize methods currently used to assess impairments of bladder function in animal models. Both noninvasive and invasive methods are available and can be used to understand and improve human health. An accurate and detailed diagnosis is, however, possible only with urodynamics assessments. © 2019 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Associate Editor: Richard Lee Keywords: Animal models Neuro-urology Urodynamics Void-spot assay Metabolic cage

* Corresponding author. Institute for Regenerative Medicine, University of Zürich, Wagistrasse 12, 8952 Schlieren, Switzerland. E-mail address: [email protected] (A.M. Sartori).

1.

Introduction

Lower urinary tract dysfunction can manifest as inability to empty the bladder or to store urine appropriately or a combination of both. The etiology can differ between pathologies and can be either neurogenic or nonneurogenic. Nevertheless, in both cases, little is known about the underlying mechanisms, hindering the discovery of new therapeutic approaches, especially in neurourology [1,2]. Thus, animal models are of crucial importance in gaining a better understanding of lower urinary tract dysfunction and testing the efficacy and safety of promising treatment options. Lower urinary tract function has been investigated in a variety of animal models [3], mostly cats and sheep in the

past, whereas recent studies mainly focus on rodents owing to the advantages of well-defined genetic lineages, straightforward gene manipulation, short lifespan, and minimal holding space, all factors that increase the quality and reduce the overall costs of experiments. Lower urinary tract function in animals can be assessed using a variety of behavioral experiments. The methods used are summarized in Table 1.

2.

Noninvasive assays

Noninvasive, relatively easy tests can be used to assess voiding frequency, voided volume, and, in some cases, the flow rate.

https://doi.org/10.1016/j.euf.2019.12.004 2405-4569/© 2019 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Sartori AM, et al. Methods for Assessing Lower Urinary Tract Function in Animal Models. Eur Urol Focus (2020), https://doi.org/10.1016/j.euf.2019.12.004

EUF-855; No. of Pages 4 2

E U RO P E A N U RO L O GY F O C U S X X X ( 2 019 ) X X X– X X X

Table 1 – Summary of LUT assessments used in urology. LUT assessment

Advantages

Disadvantages

Outcomes measured

Noninvasive Inexpensive Easy to implement and to perform High throughput Noninvasive 24-h cycle

Pathophysiology unclear Stress response to new environment

Voided volume, temporal and spatial organization of micturitions, (voiding frequency)

Pathophysiology unclear Stress response to new environment Limited precision

Voided volume, voiding time, voiding frequency, flow rate

Baseline pressure, threshold pressure, IVPmax, voided volume, voiding time, voiding frequency, flow rate, bladder compliance, EUS activity Baseline pressure, threshold pressure, IVPmax, voided volume, voiding time, voiding frequency, flow rate, bladder compliance, EUS activity Baseline pressure, threshold pressure, IVPmax, voided volume, voiding time, voiding frequency, flow rate, bladder compliance, EUS activity

Noninvasive Void-spot assay

Metabolic cage

Invasive Urodynamics in anesthetized animals

Full LUT function assessment

Expensive Time-consuming Unphysiological owing to anesthetic use

Urodynamics in restrained animals

Full LUT function assessment

Expensive Time-consuming Stress response to the restrainer

Urodynamics in freely moving animals

Full LUT function assessment

Expensive Time-consuming Movement artifacts

LUT = lower urinary tract; IVPmax = intravesical pressure; EUS = external urethral sphincter.

2.1.

Void-spot assay

Similar to a human bladder diary, voiding behaviors are used to characterize lower urinary tract dysfunction in animals placed singly in a special cage with a filter paper covering the floor for a defined time period (Fig.1A) [4]. Illumination of the filter paper with ultraviolet light reveals the voiding pattern, which can be analyzed for spot size (proportional to voided volume) and disposition. Furthermore, a real-time camera located below the cage can be used to determine voiding frequency. Although this noninvasive test can describe some general aspects of the voiding pattern of the animal, there are some limitations. Without a simultaneous camera recording system, overlapping micturitions can result in a biased analysis. In addition, it is very difficult to distinguish between different types of lower urinary tract dysfunction. For instance, if the void-spot assay shows a multitude of small micturitions, without invasive measurements the experimenter is unable to discriminate urinary incontinence caused by detrusor overactivity or by a weak sphincter muscle. 2.2.

Flow studies in a metabolic cage

The animals are single-housed in cages with a grid floor for 12–24 h (Fig. 1B) with food and water provided ad libitum [5,6]. The advantage of 24-h measurements is that both active and inactive phases of the animal are recorded. Below the grid, a digital balance collects the urine, allowing determination of voided volume, voiding time, flow rate, and voiding frequency. As for the void-spot assay, it is difficult to make a precise diagnosis of the underlying pathophysiological conditions. Precision is also limited for the very small volumes (adsorption to the grid, drying). In addition, the animals are placed in a new, empty environment for several hours that can cause stress and thus might have an influence on voiding behaviors.

3.

Invasive assays

Urodynamics is the only method that can objectively assess lower urinary tract function. Three main types of urodynamics measurements are currently performed. 3.1.

Urodynamics in anesthetized animals

Most commonly, urodynamics investigations are performed under anesthesia (Fig. 1C). A transurethral or implanted catheter is inserted into the bladder to fill it with a physiological solution for simultaneous recording of the intravesical pressure. A variety of parameters can be obtained via urodynamics (Table 1), allowing accurate differentiation of various forms of lower urinary tract dysfunction. Although the use of anesthesia can help to reduce artifacts due to the animal’s movements, it has been shown that anesthetics have an impact on lower urinary tract function [7,8]. 3.2.

Urodynamics in restrained animals

Performing urodynamics investigations in awake animals (Fig. 1D) requires implantation of a chronic catheter into the bladder, as well as electrodes for concomitant external urethral sphincter (EUS) electromyography [8–10]. During a urodynamics session, the awake, previously tamed animal is placed into a restrainer for 1–3 h and micturitions are induced by filling the bladder via the catheter. Although restraint of the animal reduces movement artifacts, it is of crucial importance to acclimatize and habituate the animal to the restrainer to reduce stress-related changes in voiding behaviors. 3.3.

Urodynamics in freely moving animals

As for restrained urodynamics, a bladder catheter and EUS electrodes need to be implanted into freely moving animals

Please cite this article in press as: Sartori AM, et al. Methods for Assessing Lower Urinary Tract Function in Animal Models. Eur Urol Focus (2020), https://doi.org/10.1016/j.euf.2019.12.004

EUF-855; No. of Pages 4 E U R O P E A N U R O L O GY F O C U S X X X ( 2 019 ) X X X– X X X

3

Fig. 1 – Schematic representations of lower urinary tract assessments used in urology. (A) Void-spot assay. (B) Metabolic cage. (C) Urodynamics in anesthetized animals. (D) Urodynamics in restrained animals. (E) Urodynamics in freely moving animals.

undergoing urodynamics. The unrestrained conditions resemble the everyday life of the animals and are assumed to best reflect physiological lower urinary tract function [11]. Animals are kept in a metabolic cage to collect the voided urine. 4.

Important considerations

The overall goal of basic research using animal models is to improve human health by gaining a better understanding of the mechanisms underlying certain pathologies. For practical and ethical reasons (minimal numbers of animals used), most studies do not perform experiments in both sexes or multiple strains, a fact that might increase the risk of gender- and strain-specific outcomes [12]. Genderspecific differences in anatomy and, for example, hormonal status can influence lower urinary tract function [13], whereas a number of basic parameters such as vesical pressure, detrusor distention, sphincter EMG, and

neuronal wiring will be largely identical in many species and both sexes. Lower urinary tract function assessed via urodynamics can be influenced by the presence and type of catheter used. A transurethral catheter requires the animal to be sedated and may partly obstruct the urethra and activate nociceptive reflexes, thus altering normal voiding behavior [14]. A suprapubic catheter is implanted in the bladder and has the advantages of keeping the urethra free of obstacles and being suitable for use in awake animals. Nevertheless, bladder morphology can change after implantation, and urodynamics studies performed a few days after implantation show an altered voiding behavior [15].

5.

Future directions

In the ideal case, lower urinary tract function would be continuously assessed in animals in their home cage, since

Please cite this article in press as: Sartori AM, et al. Methods for Assessing Lower Urinary Tract Function in Animal Models. Eur Urol Focus (2020), https://doi.org/10.1016/j.euf.2019.12.004

EUF-855; No. of Pages 4 4

E U RO P E A N U RO L O GY F O C U S X X X ( 2 019 ) X X X– X X X

invasive urodynamics requires catheterization and filling of the bladder and may thus have an impact on voiding patterns. To overcome the use of a bladder catheter constantly linked to the urodynamics set-up, efforts are being made to develop wireless pressure sensors that could measure bladder pressure for multiple days in the home cage. As an example, Mickle et al [16] developed a sensor that can be wrapped around the bladder for measuring changes in resistance during bladder filling and emptying over a long time period [17].

[2] Groen J, Pannek J, Castro Diaz D, et al. Summary of European Association of Urology (EAU) guidelines on neuro-urology. Eur Urol 2016;69:324–33. [3] de Groat WC, Griffiths D, Yoshimura N. Neural control of the lower urinary tract. Compr Physiol 2015;5:327–96. [4] Hill WG, Zeidel ML, Bjorling DE, Vezina CM. Void spot assay: recommendations on the use of a simple micturition assay for mice. Am J Physiol Renal Physiol 2018;315:F1422–9. [5] Kurien BT, Everds NE, Scofield RH. Experimental animal urine collection: a review. Lab Anim 2004;38:333–61. [6] Sidler M, Aitken KJ, Forward S, Vitkin A, Bagli DJ. Non-invasive voiding assessment in conscious mice. Bladder 2018;5:e33.

6.

Conclusions

A variety of animal models, currently mostly rodents, are available for testing the functionality of the lower urinary tract in healthy animals and disease conditions, which can ultimately help in improving human health. Noninvasive tests can be easily implemented in many laboratories and allow measurements for a considerable number of animals in a relatively short time period. Conversely, more invasive urodynamics investigations are more time-consuming and require a sophisticated set-up, but give much more precise readouts, including the activity of relevant muscles. Urodynamics is currently the only method that allows different forms of lower urinary tract dysfunction, such as detrusorsphincter dyssynergia, detrusor overactivity, and detrusor underactivity, to be distinguished.

[7] Smith PP, Kuchel GA. Continuous uroflow cystometry in the urethane-anesthetized mouse. Neurourol Urodyn 2010;29:1344–9. [8] Schneider MP, Hughes Jr FM, Engmann AK, et al. A novel urodynamic model for lower urinary tract assessment in awake rats. BJU Int 2015;115(Suppl 6):8–15. [9] Foditsch EE, Roider K, Sartori AM, et al. Cystometric and external urethral sphincter measurements in awake rats with implanted catheter and electrodes allowing for repeated measurements. J Vis Exp 2018;131:e56506. [10] Schneider MP, Sartori AM, Tampe J, et al. Urodynamic measurements reflect physiological bladder function in rats. Neurourol Urodyn 2018;37:1266–71. [11] LaPallo BK, Wolpaw JR, Chen XY, Carp JS. Long-term recording of external urethral sphincter EMG activity in unanesthetized, unrestrained rats. Am J Physiol Renal Physiol 2014;307:F485–97. [12] Ito H, Pickering AE, Igawa Y, Kanai AJ, Fry CH, Drake MJ. Muroneuro-urodynamics; a review of the functional assessment of mouse lower urinary tract function. Front Physiol 2017;8:49. [13] Robinson D, Toozs-Hobson P, Cardozo L. The effect of hormones on the lower urinary tract. Menopause Int 2013;19:155–62.

Conflicts of interest: The authors have nothing to disclose.

[14] Smith PP, Hurtado E, Smith CP, Boone TB, Somogyi GT. Comparison of cystometric methods in female rats. Neurourol Urodyn 2008;27: 324–9. [15] Andersson KE, Soler R, Fullhase C. Rodent models for urodynamic

References

investigation. Neurourol Urodyn 2011;30:636–46. [16] Mickle AD, Won SM, Noh KN, et al. A wireless closed-loop system for

[1] Panicker JN, Fowler CJ, Kessler TM. Lower urinary tract dysfunction

optogenetic peripheral neuromodulation. Nature 2019;565:361–5.

in the neurological patient: clinical assessment and management.

[17] Kessler TM, Birder LA, Gomery P. Neuromodulation of urinary tract

Lancet Neurol 2015;14:720–32.

function. N Engl J Med 2019;380:2067–9.

Please cite this article in press as: Sartori AM, et al. Methods for Assessing Lower Urinary Tract Function in Animal Models. Eur Urol Focus (2020), https://doi.org/10.1016/j.euf.2019.12.004