Research Technician at Med Associates/Catamount R&D looks on during a conscious rat cystometry session

Bladder Function Studies in Conscious Rats: Use of Tether System for Chronic Studies

Gerald M. Herrera, Ph.D.
Med Associates, Inc. and Catamount Research and Development, Inc.

Saint Albans, Vermont, USA

 

Introduction

Overactive Bladder (OAB) is the loss of bladder control and the inability to control urination.  More than 34 million Americans, including 1 in 5 adults age 40 or over, suffer from OAB symptoms.  Current treatments for OAB are limited to antimuscarinics, which have negative side effects such as constipation and dry mouth.  In order to develop better therapies for OAB, reliable animal models of bladder dysfunction are needed so that preclinical screens can be established.

Several labs have utilized urodynamic testing in rodents in order to examine in vivo bladder function in animal models of bladder dysfunction, such as cystitis, outflow obstruction, and spinal cord injury.  This technique requires that a catheter be surgically implanted into the urinary bladder for simultaneously infusing a solution to fill the bladder and recording bladder pressure.  Med Associates sells a turn-key system for conducting urodynamic studies in rats and mice (Small Animal Cystometry Lab System).  This system consists of an isolation cubicle that isolates the experimental subject from environmental disturbances, a cage with wire-bottom floor, an analytical balance for recording weight of voided urine, an infusion pump for bladder filling, a bladder pressure transducer, polyethylene bladder catheters, and computer software/hardware (Figure 1). 

The original configuration of the Small Animal Cystometry Lab System supported a bladder catheter that was kept coiled in a subcutaneous skin pouch and exteriorized on the day of study.  This approach had two distinct limitations, which we addressed in the present study.  First, if an animal was to be studied more than once, the catheter would have to be re-implanted into the skin pouch at the end of the particular test session.  This required aseptic technique in order to minimize the risk of infection.  The second limitation was that this original configuration left the bladder catheter completely exposed during the experiment, such that the animal could twist and kink the catheter, or even more problematic, bite through the catheter.  The goal of this study was to incorporate a leash-based harness system into the Med Associates Small Animal Cystometry System that would facilitate chronic urodynamic testing in a single animal, as well as keep the bladder catheter protected during experimentation so that the animal could not bite or otherwise damage the catheter.

Methods

Male Sprague Dawley rats were used for all experiments. All experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Med Associates. A bladder catheter (CYT-102, Med Associates) was implanted into the urinary bladder using aseptic technique during a survival surgical procedure. The free end of the bladder catheter was routed subcutaneously to the back of the neck, exteriorized, and connected to a Quick Connect Harness from Strategic Applications Incorporated (SAI; Med Associates Part Number PHM-119-1). The Quick Connect Harness was secured around the upper torso of the rat. The harness was connected to a Quick Connect Swivel (PHM-119-2) via a Quick Connect Tether (PHM-119-3A). The swivel was connected to a delivery line (PHM-122). Post-operative analgesics were provided during the recovery period as directed by our staff veterinarian.

Immediately following recovery from anesthesia, the rats were transferred to a home cage (PHM-125A) with a swivel arm assembly (PHM-110) that allowed the Quick Connect Swivel to be attached to an infusion pump while the animals were in their home cage. A Triple Infusion Pump with Automatic Cycling Timer (PHM-102) was used to infuse a small bolus of saline (0.9 %) into the urinary bladder every 99 minutes. This was found to keep the bladder catheters patent for up to 6 weeks. If we did not include periodic saline infusions, the bladder catheters closed due to urothelium sealing over in about 2 to 4 weeks.

One week after the catheter implant surgery, the rats were transferred from their home cage to a Cystometry holding cage (ENV-272T) in a Med Associates Small Animal Cystometry Lab Station. The wire-bottomed cage was situated above an analytical balance, such that the weight of voided urine could be recorded. The swivel was attached to an infusion pump and in-line pressure transducer.  Following a 20 to 30 minute equilibration lperiod, saline was infused into the bladder at a rate of 175 µl/min. Infused volume, voided volume, minimum pressure, maximum pressure, average pressure, and threshold pressure were recorded during urodynamic testing using Med Associates MED-CMG software. Cystometrograms (CMGs) were analyzed using Cystometry Analysis Software (SOF-552).

Results & Discussion

In order to determine if the Quick Connect harness/tether system introduces pressure artifacts in the CMG recordings, we first obtained a series of CMG recordings from rats implanted with standard bladder catheters that directly attached to a pressure transducer. Figure 2 shows a CMG recorded from a rat in which the bladder catheter was attached directly to a pressure transducer, with no swivel or connection harness in the fluid path. Note that bladder pressure remains low during filling (~10 mmHg), and increases sharply when bladder capacity is reached and voiding occurs. Our goal in selecting a swivel/harness/tethering system was to choose a system that resulted in no distortion of the pressure waveform recorded during cystometry. We tested several devices that caused unacceptably large increases in resistance to flow, which resulted in high baseline pressures and oscillating pressure artifacts during bladder filling. We eventually settled on the Quick Connect Luer system from SAI Infusion Products, which Med Associates now distributes. The Quick Connect system did not alter bladder pressure waveforms.

A CMG recorded from a rat using the Quick Connect tethering system is shown in Figure 3.  Notice that baseline (minimum) pressure during filling is relatively low (~10 mmHg).  Also, we observed no unusual pressure fluctuations when using the Quick Connect system compared to rats implanted with a catheter that attached directly to the pressure transducer. 

Figure 4 shows summary data comparing urodynamic parameters recorded from rats in which their bladder catheters were connected directly to the pressure transducer (Control) to those obtained from rats in which the bladder catheter was connected to a Quick Connect luered tethering system.  No significant differences were found in any of the parameters measured between control animals and the Quick Connect rats.  Thus, we felt confident that the Quick Connect luered tethering system from SAI did not introduce recording artifacts or otherwise adversely affect urodynamic recordings.

A major advantage of the Quick Connect system is that it facilitates the use of one animal for more than a single test session.  In our case, we simply left animals tethered chronically in their home cages, which they tolerate quite well.  Alternatively, the tether can easily be detached from the harness, and a luer lock plug can be placed over the opening on the harness while the animals are in their home cage.  Keeping the animals chronically tethered allows the intermittent infusion of saline into the bladder while the animals are in their home cages.  We found this to be important.  If catheters were not regularly flushed, the urothelium would seal over and occlude the catheter after about 2 to 4 weeks.  Using intermittent home cage catheter flushing, we recorded CMGs from rats for up to 6 weeks (total duration of this study).  It is likely that animals could even be used longer than 6 weeks when home cage catheter flushing is performed.

The scientific power of using a single animal for more than one test session is that it allows for the use of within-subjects experimental designs, in which each animal serves as its own control.  To demonstrate the utility of studying a single animal in multiple test sessions, we examined an acute model of bladder overactivity: acetic acid-induced cystitis.  We obtained urodynamic recordings from a rat equipped with a Quick Connect tether system.  As soon as three consistent CMGs were obtained under baseline conditions, acetic acid (0.1%) was introduced into the saline infused into the bladder (Figure 5).  Acetic acid caused bladder overactivity, as indicated by elevated bladder pressure and shortening the interval between consecutive voids.  This decrease in intermicturition interval (IMI) has been interpreted as an increase in the sensitivity of, or recruitment of normally quiescent, bladder afferents mediating the micturition reflex.  Most studies characterizing the properties of acetic acid-induced cystitis have utilized between subjects designs, in which the acetic acid-induced bladder overactivity is compared between separate groups of animals receiving different treatments.  In the present study, we used a within-subjects design.  Immediately following conclusion of the first cystometry session (after acetic acid was instilled), fresh saline was infused into the bladder to eliminate residual acetic acid.  At this point, animals were injected with resiniferatoxin (0.3 mg/kg, s.c.), which has been shown to prevent/reverse acetic acid-induced bladder overactivity.  24 hrs post resiniferatoxin treatment, cystometry was repeated, and three successive micturition cycles were recorded under baseline conditions.  The intermicturition interval was significantly prolonged after resiniferatoxin treatment (Figure 5), consistent with desensitization of bladder afferents.  After baseline recordings, acetic acid was again infused into the bladder.  Acetic acid did not decrease the intermicturition interval post-resiniferatoxin treatment (Figure 5). 

In conclusion, the use of a Quick Connect luered tethering system (PHM-119A) represents a major refinement in the Med Associates Small Animal Cystometry Lab System.  We offer a standard cage for rats (ENV-272T) that is tall enough to accommodate the tether and securely hold the swivel in a counter-balanced lever arm.  The addition of a tether prevents the animal from chewing or clawing at its catheter, which can be a significant problem with un-tethered animals.  Also, the Quick Connect system allows a single animal to be easily studied on more than one test session, facilitating within-subjects experimental designs.  Figure 6 shows a schematic of a rat in the ENV-272T cystometry cage, attached to a PHM-119A Quick Connect system.