1Laboratory of Experimental Urology, Department of Development and Regeneration, KU Leuven, Belgium, 2Laboratory for Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Belgium, 3TRP Research Platform Leuven (TRPLe), KU Leuven, Belgium
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Uvin, P., Everaerts, W., Pinto, S., Alpízar, Y. A., Boudes, M., Gevaert, T., et al. The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation. J. Vis. Exp. (66), e3869, doi:10.3791/3869 (2012).
The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.
1. Laboratory Animals
3. Surgical Procedure - Bladder Catheter Implantation
4. Setup and Cystometry
5. Representative Results
Figure 1. Laparotomy overview. A) Place the rat in the supine position. B) Shave and antiseptically prepare the surgical site. C) Incision of the skin. D) Incision of the abdominal muscles, and bladder exposure.
Figure 2. A purse-string suture.
Figure 3. Catheter implantation.
Figure 4. Tunnelling of the catheter.
Examples of pressure measurements obtained during intravesical perfusion of saline in a conscious rat and an anesthetized mouse are shown in Figure 5. Multiple parameters can be extracted from the pressure signal (e.g. the intercontractile interval, the baseline pressure and the threshold pressure). For comprehensive descriptions of these parameters, please see Andersson et al. (ref 4) and Yoshiyama et al. (ref 10).
We have recently used cystometry to identify the molecular targets of mustard oil (MO), a highly reactive compound that has been long used in experimental models of inflammation and hyperalgesia of visceral organs such as the urinary bladder11,12. Intravesical infusion of 10 mM MO induced a strong increase in the voiding frequency (decrease in the intercontractile interval) in wild type mice (Figure 6A, B) and a decrease of the voided volume6. Interestingly, MO induced similar changes in mice deficient of the MO receptor TRPA1. In contrast, MO induced much weaker changes in cystometric parameters in Trpv1 KO mice than in WT mice and was without any effect in Trpa1/Trpv1 KO mice. Together with measurements of the release of the Calcitonin Gene Related Peptide (CGRP)6, these data indicate demonstrate that TRPV1 may play a key role in visceral irritation induced by MO.
Figure 5. Representative traces of intravesical pressure recorded in a conscious female rat (A) and in an anesthetized female mouse (B). The lowest pressure is defined as the "baseline pressure" (red arrows). The pressure at the end of the filling phase is marked with blue arrows. The volume of fluid infused between these points, divided by the pressure difference, allows calculation of the compliance of the bladder wall (compliance = infused volume/(threshold pressure - baseline pressure). The "intercontractile interval" (ICI) is the time between two voiding contractions.
Figure 6. Effects of intravesical application of mustard oil on the cystometry pattern in wild type and Trpa1, Trpv1 and Trpa1/Trpv1 knockout mice. (A) Representative examples of intravesical pressure changes recorded in WT, Trpa1 KO, Trpv1 KO and Trpa1/Trpv1 KO mice in response to infusion of saline and 10 mM MO. (B) Time course of the average instantaneous voiding frequency before and during intravesical infusion of MO. For all mice, the data were normalized to the average frequency obtained during saline infusion. These data is adapted from Everaerts et al. (ref 6), with permission from Elsevier.
The cystometry technique presented here allows performing in vivo measurements of bladder function in animal models. Rats are probably the most used animal model. Mice are more difficult to handle, but offer the advantage of using genetically manipulated animals. Because of the technical difficulty of using conscious mice, which tend to be very active resulting in loosening of the implanted catheter and changes in the intra-abdominal pressures that may influence the intravesical pressure, we advise to keep them anesthetized with urethane during the full cystometry protocol. Of course, the benefits of having sedated mice, should be weighed against the effects of the anesthetics. As such, urethane can have a significant influence on post-voiding residual volume7,8. We therefore wait until we have a stable degree of anesthesia, before we start our recordings and interventions.
Researchers should consider the differences between male and female rodent micturition13. Also, the age of the animals is of great importance. We perform cystometry in animals that are 10 - 12 weeks old. All the experiments should be performed in a calm environment, especially when using conscious animals. A number of infusion rates have been described for our animal models. We typically use infusion rates of 20 μl/min for mice and 100 μl/min for rats.
As mentioned above, micturition in humans and rodents differ significantly. For example, ATP is a major contributor to the detrusor contraction in mice and rats, whereas in humans, the bladder contraction, under physiological conditions is mainly mediated by acetylcholine4. However, the possibilities to perform invasive measurements that are generally prohibitive in the clinical context and the use of genetically modified animals, allow exploring the current boundaries of bladder pathophysiology and pharmacology. In this respect, significant advances have been recently obtained in elucidating the role of muscarinic14, prostaglandin15 and adrenergic receptors16, neuropeptides17, Ca2+-activated K+ channels18 and TRP channels3,5,6.
No conflicts of interest declared.
This work was supported by grants from the Belgian Federal Government (IUAP P6/28), the Research Foundation-Flanders (F.W.O.) (G.0565.07 and G.0686.09), the Astellas European Foundation Award 2009 and the Research Council of the KU Leuven (GOA 2009/07, EF/95/010 and PFV/10/006). P.U. and W.E. are doctoral fellows of the Research Foundation-Flanders (FWO). M.B. is a Marie Curie fellow. D.D.R. a fundamental-clinical fellow of the FWO.
|urethane||Urethane, Sigma-Aldrich||315419||group 2B carcinogen|
|isoflurane||Isoba, Schering-Plough Animal Health|
|polyethylene catheter||Intramedic Polyethylene tubing PE50, Becton Dickinson||427411|
|surgical microscope||Op-Mi 6, Carl Zeiss||Op-Mi 6|
|purse-string suture||Prolene 6/0, Ethicon||8610H|
|fascia and skin suture||Ethilon 4/0 or 5/0, Ethicon||662G or 661G|
|postoperative analgesics||Temgesic, Schering-Plough Animal Health||dosage for rats: 0.05 mg/kg|
|amplifier||78534c monitor, Hewlett Packard|
|analytical balances and balance data acquisition software||FZ 300i, A&D||FZ-300i|
|infusion pumps||pump 33, Harvard apparatus||HA33|
|cystometry recording system||Dataq instruments, DI-730 series and Windaq/Lite||DI-730-USB Windaq/Lite|
|temperature registration||Fluke 52 KJ thermometer||52 KJ|
|pressure transducers||Edwards Lifesciences, pressure monitoring set||T322247A|