BERKELEY, CA (UroToday.com) - Reproducibility is one of the cardinal qualities of valid animal models. Whether it is between labs across the country or experimenters in the same lab, reproducibility, or a lack there of, will influence the data obtained from an animal model and its resulting translatability. In this article, we discuss anesthesia maintenance and body temperature monitoring, two experimental factors that greatly contribute to the reproducibility of many animal models. We studied these factors in the context of urinary bladder distension (UBD), a common model of visceral pain. As its name suggests, UBD entails blowing compressed air into the bladder of a lightly anesthetized animal. Throughout the procedure, electrical activity from the overlying abdominal muscle is recorded. This measure, known as the visceromotor response (VMR) is an indicator of bladder nociception. While performing pilot UBD experiments, my colleagues and I started to observe unplanned variation amongst VMRs so we designed a series of experiments to uncover the reasons for these discrepancies. This article reports the effects of anesthesia and body temperature maintenance on UBD-evoked VMRs. Overall, we found that the length of anesthesia induction has a significant effect on VMR reproducibility. Likewise, the animal’s body temperature also has a significant effect on VMR stability.
Choosing the appropriate anesthetic for UBD is imperative to the study’s success. The anesthetic needs to have a quick induction rate, low amount of tissue build-up, and the ability to be easily adjusted. For these reasons, we chose inhaled isoflurane. Aside from the requirements already listed, isoflurane is well-tolerated in mice, has a higher safety margin than halothane, and is less expensive than sevoflurane. Two methods of isoflurane induction are common among UBD users: a short method in which the isoflurane is dropped to testing levels immediately following catheterization and electrode implantation, and a long method in which isoflurane is gradually dropped to testing levels over 1.5 hours. Following both methods, we tested VMR stability over the course of 1.5 hours. We found that when the short method was used, VMR stability was only maintained for approximately 30 minutes. Alternatively, VMR stability was maintained for at least the 1.5 hour testing time when the long induction method was used. Therefore, we recommend use of the long induction method for all experiments lasting longer than 1 hour total. In order to circumvent some of the physiological effects associated with prolonged anesthesia, we also suggest the following measures: (1) vaporize the isoflurane in 100% O2 to account for depressed respiration and (2) employ the use of a nebulizer, or bubble the gas in a temperature controlled water bath, to reduce bronchial secretions.
Body temperature maintenance is an obvious, but poorly reported, physiological measure that also significantly affects VMRs. Due to their high surface area to body weight ratio, mice and other small rodents have difficulty maintaining homeostatic mechanisms like thermoregulation while under general anesthesia. Additionally, a number of well-accepted surgical practices (e.g. hair removal, surgery site disinfection, IV administration, etc.) decrease the animal’s body temperature even further. It is essential, therefore, to monitor and maintain animal body temperature throughout the entire UBD procedure. We found that as an animal’s body temperature decreases, VMRs decrease in a parallel fashion. Additionally, if an animal’s body temperature drops during a procedure, the VMRs will remain reduced even if the animal’s body temperature is returned to normal. Preventative measures can be taken to reduce the development of decreased body temperatures. To start, we suggest pre-warming the procedure area by turning on heating pads, overhead radiant heat sources, etc. Once the procedure begins, body temperature should be monitored through a subcutaneous probe; rectal probes are commonly used in conjunction with heating systems but in these experiments this placement may interfere with bladder nociception. Heating systems should be continuously adjusted throughout the experiment to prevent both hyper and hypothermia.
The overall goal of this publication was to dissect the finer details of a well-accepted visceral pain model in the hopes of increasing its reproducibility. We discovered both isoflurane induction method and body temperature to play a significant role in VMR stability. The findings of this paper are not only applicable to UBD, but to other behavioral and physiological techniques as well.
Katelyn E. Sadler and Benedict J. Kolber as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.
Department of Biological Sciences and Chronic Pain Research Consortium, Duquesne University, Pittsburgh, PA USA