Rat Animal Models for Evaluation of the Effects of Sacral and Peripheral Nerve Stimulation on Bladder Function

Summary

This protocol describes detailed surgical techniques for stimulating the sacral, tibial, and peroneal nerves in anesthetized rats. These models enable investigation of neuromodulation effects on bladder function, aid in determining optimal stimulation parameters, and allow for the study of the mechanisms of action of sacral and peripheral nerve stimulation therapies.

Abstract

Neuromodulation, a therapeutic approach to the dysfunction of various organs, has proven effective in treating lower urinary tract dysfunctions. Despite its clinical success, the mechanisms underlying neuromodulation remain incompletely understood, emphasizing the need for standardized and reproducible animal models. Preclinical models of urinary bladder neuromodulation using sacral and peripheral nerve stimulation have been established for more than two decades; however, the existing literature provides limited methodological detail regarding the surgical procedures. This article offers a detailed description and visual representation of well-established techniques for sacral nerve stimulation. It also describes a modified approach to tibial nerve stimulation and introduces a novel bladder neuromodulation method using peroneal nerve stimulation in a rat model. The modified tibial nerve stimulation approach involves exposing and stimulating the tibial nerve at its origin, as a branch of the sciatic nerve, rather than at the medial ankle, as described in previous studies. Given the recent introduction of peroneal nerve stimulation for bladder neuromodulation in clinical practice, this article proposes a unique rat model to investigate its mechanisms in the control of lower urinary tract function. Step-by-step guidance for all three neuromodulation methods is outlined for nerve exposure, electrode placement, and verification of successful electrode placement based on characteristic motor responses. A representative example of the effect of peroneal nerve stimulation on bladder function in a rat model of acetic acid–induced bladder overactivity is included to demonstrate its applicability. This article presents a standardized, technically accessible protocol for studying neuromodulation mechanisms, including alterations in bladder sensory signaling, spinal and supraspinal regulatory pathways, optimization of stimulation parameters, and comparison of the efficacy of different stimulation targets.

Introduction

Neuromodulation uses low-amplitude electrical stimulation to alter the function of the end organ¹. In the context of lower urinary tract dysfunction, such as overactive bladder syndrome (OAB), non-obstructive urinary retention, neurogenic bladder, and bladder pain syndrome, neuromodulation is employed as a third-line treatment for patients who failed behavioral therapy and pharmacotherapy². For neuromodulation in patients with OAB, posterior tibial nerve stimulation and sacral nerve stimulation (SNS) have been widely used in clinical practice. Experimental neuromodulation approaches targeting additional peripheral nerves....

Protocol

The animals were housed at the University of Southern Denmark Animal Care Facility following 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. The Ethics Committee of the Danish Animal Experiments Inspectorate approved the study procedures (Protocol No. 2022-15-0201-01158). The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of the electrodes

NOTE: For tibial, peroneal, and L6 nerve stimulation, bipolar hook-shaped electrodes we....

Results

To demonstrate the experimental design for assessing the effect of neuromodulation on the lower urinary tract function, a study is presented in which peroneal nerve stimulation was applied in a rat model of bladder overactivity induced by intravesical infusion of 0.5% acetic acid. Bladder function was recorded using a single micturition cycle cystometry before, during, and after stimulation (Figure 6 A,B). The experimental setup included two different durations of peroneal n.......

Discussion

This article provides a detailed description of the surgical methods for implantation of electrodes for sacral, tibial, and peroneal nerve stimulation in an anesthetized rat.

Implantation of a lead electrode into the sacral foramen for continuous low-amplitude stimulation of the S3 sacral nerve is a broadly accepted treatment of lower urinary tract (LUT) dysfunction and fecal incontinence. In the patients, the electrode is typically placed in the S3 foramen and targets a mix of myelinated and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
18 G disposable injection needle	KRUUSE	121282
Dumont forceps style 5TM	DUMONT	1708-5TM-PO	Curved
Dumont forceps style 7XL	DUMONT	0508-7XL-PO	Curved
Grass SD9 square pulse stimulator	Somatco	1077/183
Hemostat	Fine Science Tools/Teleflex	13018-14/PO181059	≥ 14 cm, fine tip
Iris scissors	World Precision Instruments	14218	Straight scissors
Kwik-Sil silicone Elastomer	World Precision Instruments	KWIK-SIL	Two-component glue
Micro Adson forceps	World Precision Instruments	501245
Microscissors	S&T	SAC-15 R-8	curved tip ≤ 10mm long
NaCl 0.9% 100 mL	B.Braun	N/A
PE-50 tubing	Instech	BTPE-50
Prolene 5-0 suture	Ethicon	EH7257
Rongeur	George Tiemann & Co	160-424-08	Fine curved tip
Surgical Scalpel	Swann-Morton	0505
Urethane	Sigma-Aldrich	U2500
Vicryl 4-0 suture	Ethicon	V451
Vicryl 5-0 suture	Ethicon	V303
Wire Silver Teflon-coated 0.125 mm	World Precision Instruments	AGT0525
Wire Stainless Steel Teflon-coated 0.18 mm	World Precision Instruments	SST30407-50