Novel telemetry recordings from the urinary bladder and external urethral sphincter in awake freely mobile mice

Ikeda Y1, Zabbarova I1, Kozlowski M1, Kanai A1

Research Type

Basic Science / Translational

Abstract Category

Research Methods / Techniques

Abstract 20
Novel Techniques and Approaches in Basic Science
Scientific Podium Short Oral Session 3
Wednesday 4th September 2019
09:07 - 09:15
Hall G3
Animal Study Basic Science Pre-Clinical testing
1.University of Pittsburgh
Presenter
Y

Youko Ikeda

Links

Abstract

Hypothesis / aims of study
There have been a variety of methodologies developed to obtain functional recordings from the lower urinary tract (LUT) of various animal models.  These include intravesical pressure recordings from the bladder and electromyograms from the external urethral sphincter (EUS).  Simultaneous recordings from both structures are essential to obtaining a clear indication of voiding and storage functions for effective comparison with clinical data.  However, in order to achieve this in animal models, most methodologies use anaesthesia which dampens central nervous system (CNS) reflexes.  Other methods such as whole-body restraints which are accompanied by motion artefact due to struggling and can cause stress to the animal. Decerebration cystometry does not require anaesthesia but removes CNS influence and could impact interpretation of pain-induced responses.  Telemetry bladder pressure recordings have been achieved in a rat model [1] however, to our knowledge, simultaneous bladder and EUS activity by radiotelemetry has not been recorded in awake rodents.  Therefore, we have developed a technique to record bladder and EUS activity in freely mobile unanaesthetised mice utilizing implantable telemeters combined with a metabolic cage system with custom load cells fitted with filter paper to measure urine void volumes, flow rates and spot patterns.
Study design, materials and methods
Telemetry implantation: Adult (8-12 weeks) female and male C57Bl/6 mice were anesthetized with 1.5-2% isoflurane and, under sterile conditions, a lower midline incision was made to expose the urinary bladder and proximal/mid urethra.  An HDX-11 telemeter (Figure 1A; Data Sciences International) was adapted to allow for implantation of a pressure catheter via the bladder dome and insertion of recording electrodes (50-micron stainless steel wire) into the EUS muscle (Figure 1B).  Telemeter sending units were placed subcutaneously on the flank of the animal and mice were allowed to recover with prophylactic analgesic and antibiotics.
Metabolic cage recordings: Customized metabolic cages (Columbus Instruments) were designed to accommodate mice for up to 72-hour periods whilst recording food and water consumption in a sound-proofed, climate-controlled cabinet (Figure 1C).  Each metabolic cage was equipped with a high sensitivity load cell balance set to acquire at 10 Hz frequency to record the time, volume and the speed of voids as well as urine spot patterns.  Load cells were lined with Whatman paper before animals were placed in cages to record urine spot patterns.  The data from the telemeters and metabolic cage load cells were recorded using LabChart 8 (ADInstruments).  Metabolic cage recordings were taken from each mouse before telemeter implantation, 1, 7, 14 and 28 days post implantation (DPI).   Voided volumes (based on load cell recordings), bladder pressure, EUS-EMG recordings and time between voiding events were measured for each session.  Data were expressed as mean ± standard deviation; Student's t-test was used to evaluate group differences between non-implanted and implanted data sets.
Results
Bladder pressure and EUS electrical activity were recorded from both female and male mice (N=4 each) for up to three months in recording periods lasting up to six hours; telemeter batteries last up to 30 days but are only activated during recording session being switched on/off using an external magnet.  Micturition contractions recorded through telemetry indicated by a sharp rise in bladder pressure, were correlated with decreased tonic firing of the EUS indicating relaxation of the sphincter muscle (Figure 2A and 2B).  These events were followed by the measurement of urine flow rates and volumes, after a 0.5-1 second time delay for the urine to reach the load cells (Figure 2C).  Voiding patterns were measured using filter paper mounted on the load cells and corresponded to the number of recorded voids (Figure 2D). Before telemeter implantation, female and male mice voided 263 ± 164 and 569 ± 83 µl, respectively.  At 1 DPI, there was a sharp and significant decline in voided volumes in both sexes (females - 28.3 ± 12.7 µl, males - 55.8 ± 32.5 µl; p<0.05 versus controls without implants; Figure 2E) which were recovered by 14 DPI.   The decline in voided volumes correlated with decreases in the time between voiding events (Figure 2F) which also returned to control levels by 14 DPI.  There was no significant change in baseline, threshold and maximal voiding pressure values as a result of implantation surgery and between the sexes (not shown).
Interpretation of results
We have successfully recorded simultaneous bladder and EUS activity using telemetry implants in awake freely moving mice.  The implantation surgery initially result in a decrease of voided volumes and increased frequency of voids, which were normalized by 14-28 DPI.  The presented methodology has the added advantage of giving an accurate measurements of voided volumes, rates of voiding and voiding patterns.
Concluding message
Bladder and EUS activity can be drastically different in recordings from awake freely moving animals compared to other cytometric methodologies, making it difficult to correlate findings from animal studies to the human situation.  This may be particularly important in studying neurogenic bladder dysfunction which often is associated with discoordination of the bladder and urethral activity.  Our telemetry technique has the potential for long-term monitoring of LUT function in animal models to better characterize pathologies and determine the efficacy of treatments.
Figure 1 Figure 1. Images of the HDX-11 telemeter, implanted catheter and EMG electrodes and metabolic cage setup.
Figure 2 Figure 2. Example telemetry recordings of A) bladder pressure, B) EUS-EMG, C) load cell and resulting D) urine spots from a male mouse. E) change in voided volume and F) time between voids following telemeter implantation.
References
  1. Monjotin N et al., Neurourol Urodyn., 36(2):308-315, 2017.
Disclosures
<span class="text-strong">Funding</span> Awards from NIH/NIDDK; R01 DK071085 (Kanai), R01 DK098361 (Kanai and Drake), P01 DK093424 (Kanai) and Department of defense SC170171 (Kanai and Ikeda) <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> mouse <span class="text-strong">Ethics Committee</span> University of Pittsburgh Institutional Animal Care and Use Committee