Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Summary

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Abstract

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Introduction

Chronic pain is officially defined as pain that persists for approximately three months, or for longer than the normal tissue healing time¹. This type of pain is one of the main reasons that people are driven to seek medical attention² and can cost up to $635 billion dollars annually³. Current chronic pain coping strategies are considered archaic; after decades of medical advancements, NSAIDs (nonsteroidal anti-inflammatory drugs) and opioids are still the primary treatments prescribed by physicians. However these treatments target all different types of pain in the same way, providing analgesic effects all over the body as opposed to ....

Protocol

The following protocol has been approved by the Institutional Animal Care and Use Committee at Duquesne University and is consistent with guidelines from the National Institutes of Health.

1. Surgical Implantation of Wire Electrodes and Urinary Bladder Catheter Using Sterile Techniques (total time 5-10 min)

1. The following items should be prepared prior to starting surgery:
   1. Actively monitor and adjust body temperature to 37.5±0.5 °C using temperature monitoring system with a battery-operated heating pad and animal body temperature probe (Materials List) Connect automatic system to a low-wattage heat lamp....

Results

An illustration of the overall UBD-VMR setup can be seen in Figure 1A. Increasing pressure steps induce an increase in the raw VMR (i.e. EMG) (Figure 1B.) The raw VMR, pressure, and stimulus marker signal that should be observed during recording using digitizer software can be seen in Figure 2. The top channel seen in Figure 2 is the EMG trace (in V). The background signal of the EMG trace should be steady and low amplitude (<0.2 V). If backg.......

Discussion

In humans, interstitial cystitis/bladder pain syndrome (IC/BPS) represents a significant clinical problem because patients have debilitating pain that is often unresponsive to regular pain treatment¹⁵. One of the major challenges in understanding and ultimately treating chronic bladder pain is to understand the normal neural processes that are involved in the response to noxious bladder distention. In order to overcome this challenge, an animal model of bladder pain is necessary. This model should be reproduci.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Infrared heating blanket and monitoring system	Kent Scientific	Right-Temp System	This system is set up to monitor two separate temperatures.  This should include the animal and the heating blanket.  In addition, the system can automatically adjust the temperature to maintain a set temp.  However, this automatic function produces electrical interference during EMG recording and must be turned off.  Kent can provide a battery pack for the heating pad for use during the riEMG recording part of the experiment.
Isoflurane vaporizer	Draeger	Vapor 19.1	Any isoflurane vaporizer will work, but it is helpful to have one that has multiple notches between 0-2% isoflurane.
Isoflurane	multiple sources	n/a
100% Oxygen air tank	multiple sources	n/a	For ventilation of animal
Air breathing grade	multiple sources	n/a	For bladder distention
24 G 0.56 in IV catheter	BD Biosciences	381411	For bladder catheterization
Surgilube (sterile)	Savage Laboratories	0281-0205-02	Any surgical grade lubricate would work fine.
Mineral oil (sterile)	multiple sources	n/a
Saline (sterile)	multiple sources	n/a
AG8W Silver Wire, 2 m, 0.20 mm (.008 in) D, L, No Insulation	Warner Instruments	W4 64-1318	Any silver wire with these specifications will work.  Wire does not need to be "chlorinated."
Ophthalmic ointment	multiple sources	n/a
Small surgical scissors	multiple sources	n/a
Sharp forceps	multiple sources	n/a
21 G Needle	multiple sources	n/a
Grass amplifier P511 with 3-lead input cable	Grass Instruments	P511 (F-P5IC3/REV1)	This is the "amplifier" used in the protocol.  Amplifier with the following settings: Calibrator = 1 mV; Lo freq = 300 Hz; Amplification = 20; Hi freq = 10 kHz; Line filter = in.
Cambridge Electronic Design (CED) 1401 Plus (or equivalent)	Cambridge Electronic Design	1401 Plus	This is the "digitizer" used in the protocol.  Other digitizer systems from WINDAQ or other companies would work fine; Need inputs for pressure signal, EMG, and stimulus signal.
CED Spike2 software	Cambridge Electronic Design	Spike 2	This is the the "digitizer software" used in the protocol.  Should be from same manufacturer as digitizer.  Program should be setup with 3 channels for pressure (0-100 mmHg scale), EMG signal (typically -5 to +5 V range), and stimulus marker (0-2 V) range.
Flow chart from air tank to bladder catheter	n/a	n/a	Sequence of connections from pressurized air tank to animal bladder:  Air tank to 1/4 in tubing to Gilmont flowmeter to y connector.  Branch 1 of y connector to to sphygmomanometer.  Branch 2 to a single input on the 4-way gang valve to 4-way valve output to the timed pressure regulator to 3/32 tubing from timed pressure regulator to 2nd y connector (branch 1 to sphygmomanometer) with branch 2 to 3/32 tubing  to a 3rd y connector.  Branch 1 of y connector to a 3rd sphygmomanometer and branch 2 to animal bladder catheter.
Gilmont Flowmeter	Gilmont	GF8321-1401	Multiple brands of flowmeters will work.  For bladder distention air, flow meter should be able to handle high pressures (such as this Gilmont meter).  For breathing air, flow rate should be adjustable down to 100 ml/min (typical mouse rate is 500-1,000 ml/min).
4-way Gang valve	Elite		This is a specific piece of equipment.  The Elite gang valve is designed for fish tanks at low air pressures.  In the bladder distention setup, this valve acts as a safety valves in case the pressure spikes.  Too high pressure during initial turning on of the tank will ruin the pressure transducer in the Timed pressure regulator and/or the sphygnometers. In addition, by providing a small amount of leak in the system, this valve makes it easier to adjust the pressure between 10-80 mmHg.
1/4 in Tubing	multiple sources	n/a
3/32 inch Tubing	multiple sources	n/a
"Y" connectors (1/4 and 3/32 in)	multiple sources	n/a
Sphygmomanometer	CVS		Any analog sphygmomanometer from a drug store will work for this application.
Timed Pressure Regulator	custom		This is a custom built machine (Washington University in St. Louis Machine shop) that allows for automated pressure delivery including digitization of air pressure, control of trial length, inter-trial interval automation, control of trial number, and stimulus onset digital signal.  However, the basic components of the system (pressure on and off for a given trial period) could be controlled with a simple on/off in-line switch.   Such analog control of a trial would necessitate additional analysis parameters (see Protocol 4).  In addition, one would have to manually assign the pressure based on the analog sphygmomanometer during data analysis.
IGOR Pro	Wavemetrics	n/a	For analysis of EMG signal. Many different types of software can be used for data analysis in these experiments.