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, including the pudendal and peroneal nerves, are being studied^3,4,5,6.

Although neuromodulation has been used in the treatment of bladder and bowel disease for several decades, its mechanism of action is not fully understood. As stated in the multidisciplinary expert group review, currently used stimulation parameters were mostly chosen by manufacturers based on a trial-and-error approach⁷. It is therefore important to continue using animal models to elucidate underlying pathways and optimize stimulation parameters to improve therapeutic effect. Several preclinical experiments have been conducted, using awake and anesthetized animals, including mice, rats, sheep, dogs, and cats⁴. With ethical guidelines and research policies worldwide encouraging the use of less complex, smaller species, rodent models have been employed most frequently in recent years.

SNS in rat models of bladder dysfunction has predominantly employed two methodological approaches. The first, described by Zvara et al., involves implanting a stimulating electrode into the sacral foramen using an angiocatheter or a spinal needle⁸. The second approach requires the exposure of the L6 nerve root and positioning stimulating electrodes under the L6 nerves, with silicone glue used to secure the contact between the electrode and the nerve⁹. The technique involving electrode placement in sacral foramen mirrors the clinical approach. The method using exposure of the L6 root, while technically challenging, provides precise localization and more consistent access to the nerve.

Rat animal models mimicking the posterior tibial nerve stimulation have been described in the literature. Most frequently used method involves surgical exposure of the tibial nerve on the medial aspect of the hindlimb above the ankle^{10,11,12,13,14,15,16}. Another method consists of percutaneous insertion of needle electrodes near the tibial nerve¹⁷. This article aims to provide a detailed description of tibial nerve stimulation at its origin, between the gluteus maximus and biceps femoris muscles, where the sciatic nerve splits into three branches: the tibial, common peroneal, and sural nerves. In addition, this study introduces peroneal nerve stimulation as a novel animal model for investigating a new neuromodulation method. The translational relevance of this rat model is supported by the recent clinical introduction of peroneal nerve stimulation for bladder neuromodulation^18,19.

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 were used²⁰. The following steps provide a brief description of electrode design and construction.

1. Cut two equal-length 125 µm polytetrafluoroethylene (PTFE)-coated silver wires and twist them together.
2. Strip 4–5 mm of PTFE from one end of the wires, trim the exposed ends evenly, and bend the tips upwards over a 23 G needle to form two parallel hooks spaced 2 mm apart.
3. Secure the electrode to a plate and position the hooks facing upward and extending over the edge of the plate.
4. Mix the two-component silicon glue for 5 s. Apply a drop 1–2 mm from the hooks and allow it to dry for approximately 5 min.
5. Strip a 1–2 cm long section of the PTFE coating from the opposite end of the electrode to allow connection to the stimulator.

2. Animal preparation

NOTE: Under sterile conditions, PE-50 catheters, with an end flared by heat, were implanted into the bladder dome of female Sprague-Dawley rats (250–300 g) and secured with a purse string suture. The distal end of the tubing was externalized at the animal's back and coiled in a subcutaneous pouch²¹. Five days were allowed for the bladder to recover after the surgery.

1. Anesthetize the animal with urethane (1.5 g/kg, intraperitoneally) (following institutionally approved protocols). Administer urethane in three divided doses at 15-min intervals, approximately 2 h before the start of the experiment.
2. Transfer the animal onto a heating pad and place it in a prone position.
3. For tibial and peroneal nerve stimulation, shave the rat’s hindlimb around the thigh region as well as the lower half of the back. Place the tail and a 5 mL syringe under the left hindlimb of the rat and fix the tail and the leg as shown in Figure 1A.
4. For SNS, shave the rat’s back from the tail to the thoracic spine and place a 50 mL centrifuge tube under the abdomen to elevate the lumbar and sacral region.
   NOTE: All neurostimulation experiments represent non-survival procedures. Use clean surgical techniques.

3. Dissection of the peroneal and tibial nerve and implantation of the electrodes

1. Use a scalpel to make a 3–3.5 cm incision in the center between the knee joint and the ischial tuberosity (Figure 1B).
2. Separate the skin from the muscle by sliding the scissors under the skin and cutting the connective tissue.
3. Spread the skin open using retractors and expose the fascia connecting the gluteus maximus to the biceps femoris muscles (Figure 1C).
4. Use straight scissors to cut the fascia and dissect the space between the gluteus maximus and the biceps femoris until the sciatic nerve is visualized (Figure 1D,E).
5. Reposition the retractors on the biceps femoris to achieve good exposure of the sciatic nerve and its branches - the tibial, peroneal, and sural nerves (Figure 2A). In this context, “peroneal nerve” refers to the common peroneal nerve prior to its division into deep and superficial peroneal nerves. The tibial nerve is the largest branch; the sural nerve is the smallest and the peroneal has an intermediate diameter.
6. Separate the peroneal nerve from the surrounding connective tissue using curved forceps and micro scissors. Then use microforceps to carefully spread the tissue on both sides and beneath it to isolate a 5 mm-long section of the nerve. Repeat the same procedure with the tibial nerve for tibial nerve stimulation (Figure 2B).
   NOTE: Avoid crushing the nerve by picking it up with the forceps and reduce pulling on the nerve to a minimum.
7. Make a 5 mm-long incision on the lower half of the back using a scalpel and slide a hemostat under the skin through the incision to create a subcutaneous channel. Advance the hemostat carefully until the tip emerges at the hindlimb incision (Figure 2C).
8. Using the hemostat, grasp the unhooked end of the electrode and slowly withdraw the instrument through the incision at the lower half of the back. Using two forceps, bend the other end of the electrode (hooked end) to a 90° angle with respect to the subcutaneous plane and ensure that the hooks of the electrode are positioned adjacent and perpendicular to the nerve (peroneal or tibial) (Figure 2D).
   NOTE: Proper positioning of the electrode is important to avoid stretching the nerve.
9. Use curved micro forceps to lift the nerve and slide the hooks of the electrode under the nerve. Mix the two-component silicon glue for 5 s, use a Q-tip to dry the nerve, and apply enough glue to cover the hooks and the area adjacent to the nerve (Figure 2E–G).
   NOTE: Before applying glue, connect the electrodes to the stimulator, and stimulate the nerve at the level of the motor threshold to confirm proper placement of the electrode. The motor threshold was determined as the minimum voltage required to provoke consistent phasic foot movement. Stimulation of the peroneal nerve generates a phasic dorsal flexion of the foot, whereas tibial nerve stimulation generates a plantar flexion. In the absence of the motor response to stimulation, check the connection between the stimulator and the electrode.
10. Place a 5-0 nylon stay suture in the connective tissue at the back incision, leaving both suture ends sufficiently long. These ends are used to secure the electrode in place, ensuring stable positioning.
11. Close the skin at the hindlimb incision using a 4-0 Vicryl suture.

4. Exposure of the L6 nerve and implantation of the electrode

1. Use a scalpel to make a 4–5 cm midline incision over the lumbar region and sacrum. Place retractors to open the incision, expose the underlying back muscles, and the tips of the vertebral spinous processes (Figure 3A).
2. Palpate the iliac crest and use it as a landmark to locate the spinous processes of L6. It can be identified as the first spinous process located just below the iliac crest (Figure 3B).
3. Use scissors to remove the fascia on top of the vertebrae from L5 to S2. Then carefully isolate and detach the paravertebral muscles bilaterally to expose the spinous processes, laminae, and articular processes. Remove the remaining connective tissue and muscle with micro scissors until the bony structures are clearly visible (Figure 4A,B).
   NOTE: This creates an anatomical window that exposes landmarks and provides access to the L6 nerve trunk. Identify the posterior facet joint of L6; the L6 nerve trunk runs caudal to this joint. Also, identify the sacrum and the sacroiliac joints. The L6 nerve trunk runs caudal and medial to this joint (Figure 4B).
4. Use a rongeur to carefully remove the spinous process of S1 and gradually break and remove small fragments of the sacral bone in the region between the S1 and S2 vertebrae to expose the L6 nerve trunk (Figure 4C).
5. Separate the L6 nerve from the surrounding tissue using curved forceps and micro scissors. Hook the nerve onto a bipolar PTFE-coated silver wire electrode and isolate it from the surrounding tissue using biocompatible silicone glue, as described in step 3.9 (Figure 4D).
   NOTE: Stimulation of the L6 nerve generates pelvic floor muscle contraction and tail twitch.
6. Repeat the same procedure on the opposite side for bilateral nerve stimulation.
7. Close the skin with a 4-0 Vicryl suture.

5. Insertion of a stimulating electrode into the sacral foramen

1. Use a scalpel to make a midline incision over the sacrum and use retractors to open the incision and expose the underlying back muscles.
   NOTE: Palpate the iliac crest as mentioned in step 4.2 to locate the L6/S1 landmarks.
2. Separate the paravertebral muscles from the spinous processes using blunt dissection. Reposition the retractors on the muscles to obtain exposure of the spinous processes and facilitate access to the S1 sacral foramen (Figure 5A).
3. Identify the location of the S1 foramen, which lies caudal and lateral to the S1 spinous process. Probe the S1 sacral foramen with an 18-gauge needle preloaded with the monopolar electrode (Figure 5B).
   NOTE: A 180 µm PTFE-coated stainless steel wire is used, with 5 mm of insulation stripped from both ends.
4. Position the needle tip at the S1 foramen, keeping it parallel to the spine and angled approximately 30° relative to the animal’s back. Gently advance the wire along the nerve trajectory while slowly withdrawing the needle. Repeat the procedure on the contralateral S1 foramen for bilateral stimulation.
   NOTE: To confirm the correct positioning of the electrode, connect the distal end of the wire to the stimulator. Electrical stimulation generates a tail twitch.
5. Secure the wires to the adjacent spinous process (L6) using a 5-0 Vicryl suture (Figure 5C).
6. Make a 1 cm incision at the back of the neck using a scalpel and create a subcutaneous tunnel using a hemostat from the incision at the back of the neck to the incision at the sacral level.
7. Pass the distal end of the wire through the tunnel and exteriorize it at the back of the neck (Figure 5D).
8. Close the fascia and the skin at the sacral level, as well as the skin at the back of the neck, using 4-0 Vicryl suture.

6. Cystometry recording

1. After 5 days, prior to performing the stimulation experiments, externalize the PE-50 tubing and connect it to the pressure transducer and infusion pump.
2. Infuse the bladder continuously at a rate of 3 mL/h for 2 h to allow for stabilization of bladder parameters.
3. Evaluate cystometry parameters, including baseline pressure, threshold pressure, bladder compliance, micturition pressure, and bladder capacity before, during, and after nerve stimulation using a single micturition cycle intravesical infusion²¹.
4. At the conclusion of each experiment, in compliance with ethical standards, euthanize animals by anesthetic overdose.

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