Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Summary

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Abstract

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

Introduction

Filling cystometry (FC) is a diagnostic method that involves placing a catheter into the urinary bladder to record pressure during slow bladder filling. First introduced in 1927 as a clinical diagnostic method to evaluate lower urinary tract function, it has remained widely used.¹ In research applications, FC can be used to test bladder function in healthy and diseased animal models and to study the effects of pharmacological agents. Rodent animal models are commonly used to investigate lower urinary tract function.² In this group of mammals, FC was first developed for use in rats.³ Here, the meth....

Protocol

Animals were housed in the University of Vermont Animal Care Facility according to institutional guidelines. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals.

1. Intravesical Tube Implantation

1. Preparation of tubing and instruments for the surgical procedure
   1. Cut a 7-cm piece of PE10 tubing to make the catheter for implantation.
   2. Create a flare at one end of the PE10 tube by slowly advancing the end towards an open flame.
      NOTE: Quickly withdraw the tube as soon as the flare develops.

Results

There was no significant difference between the tubing materials and diameters in the consistency of pressure rise and fall within the system during tube occlusion. Bladder wall swelling post intravesical tube implantation was significant for both polyethylene (PE) and polyurethane (PU) materials. On day 2, severe submucosal swelling developed. It occupied half the cross-section of the bladder, leading to obstruction of the lumen. On day 5, the edema resolved completely, leaving the submu.......

Discussion

Optimal material and size of intravesical tubing

To determine the effect tubing diameter has on pressure recordings, we tested different microfluidic tubes; PE50 (0.58 mm ID), polyurethane PU027 (0.4 mm ID), PE25 (0.46 mm ID), and PE10 (0.28 mm ID). For each tube, pressure was recorded with the infusion pump running at 1 mL/h, while rapidly moving the tube vertically from 0 to 30 cm. Initial in vivo experiments attempted to use PE50 tubing, but were unsuccessful due to the size of tubing compared.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyethylene (PE) 10 tubing	Instech	BTPE-10	Fits 30G connectors/plugs
Polyethylene (PE) 50 tubing	Instech	BTPE-50	Fits 22G connectors/plugs
22 G single channel stainless steel swivel	Instech	375/22
High Carbon Steel Utility Extension Spring (9/64" OD)	Grainger	1NAH1	Protects PE50 tubing - Cut to length
22 G connector	Instech	SP22/12
Yutaoz Professional Hot Melt Adhesive Glue Gun	Yutaoz		Use low temperature setting (100 °C) - Any hot melt glue gun with an adjustable temperature range will work
Surebonder DT-2010 all purpose glue stick	Surebonder		Any all purpose hot glue will work
Dumont #5 curved microforceps	World Precision Instruments	500232
Dumont #7 curved microforceps	World Precision Instruments	14188
Mini dissecting scissors - straight	World Precision Instruments	503240
Micro mosquito forceps (12.5 cm)	World Precision Instruments	500451
Dissecting scissors - straight	World Precision Instruments	14393
Castroviejo Needle Holder	World Precision Instruments	503258
Isoflurane, USP	Phoenix		2%, 1 L/min flow rate
Buprenorphine			0.05 mg/kg
0.9% Sodium Chloride Irrigation, USP	Baxter
6-0 Ethilon black monofilament, non-absorbable suture	Ethicon		Bladder tie
6-0 Vicryl violet braided, absorbable suture	Ethicon		Muscle suture, running
6-0 Prolene blue monofilament, non-absorbable suture	Ethicon		Skin suture, vertical mattress, buried interrupted
KD Legato 210 infuse/withdraw pump	KD Scientific		1.5 mL/hr
Disposable pressure transducer	Digitimer	NL108T2
Pressure Amplifier	Digitimer	NL108A
Power1401-3 data acquisition interface	Digitimer
Spike2	Cambridge Electronic Design Limited		PC pressure recording software
Leica MZ6 surgical operating microscope (3.2 - 20X)	Leica Microsystems		Magnification