A Thermal Injury Model by Monopolar Radiofrequency on Sciatic Nerve in Mice

Summary

This study applied radiofrequency systems to the sciatic nerves of mice, establishing a radiofrequency ablation model of thermal nerve injury. This approach resolves issues of controllability in mice peripheral nerve thermal injury models, providing a practical modeling method for research on nerve injury repair and neuropathic pain.

Abstract

Peripheral nerve injury often results in neuropathic pain and triggers nerve repair processes. Whilst controllable physical injury models are now well-established, existing thermal injury models remain exploratory and exhibit poor controllability. In clinical practice, radiofrequency ablation (RFA) systems are frequently employed to induce thermal injury to tissues and nerves. RFA is a therapeutic modality that has been demonstrated to effectively block pain transmission by inducing thermal damage to nerves. The extent of the ablation can be modified by adjusting specific parameters, as well as the degree of numbness and motor dysfunction in the affected nerve distribution area. This study used a monopolar radiofrequency system to create a stable, controllable nerve injury model in the sciatic nerve of mice by applying 75°C for 30 s. A significant increase in mechanical withdrawal threshold (MWT) was observed from days 7–14 post-surgery, with recovery occurring by day 28. This research addresses the current gap in RFA nerve injury models, replicates the nerve damage observed clinically after RFA procedures, and provides a methodological framework for subsequent studies on RFA-induced nerve injury.

Introduction

Neuropathic pain presents ongoing challenges in clinical management, as its pain mechanisms continue to be investigated. To study the molecular and cellular mechanisms of neuropathic pain, spinal nerve ligation (SNL)¹, chronic constriction injury (CCI)², partial sciatic nerve ligation (pSNL)³, and sciatic nerve transection (SNT)⁴, and spared nerve injury (SNI)⁵ have been successfully used in rodents to simulate peripheral physical nerve injury. To enhance the controllability and stability of the model, previous studies proposed improvements, such as pressure gauges and selective damage, which have been implemented.

Although radiofrequency technology has yielded favorable clinical outcomes, it is also associated with several side effects. For instance, radiofrequency ablation (RFA), which is used for hemostasis during surgery⁶, can cause thermal damage to surrounding tissues and pose a risk of thermal injury to peripheral nerves due to the high temperature of the instrument's tip⁷. Additionally, RFA for treating trigeminal neuralgia⁸, postherpetic neuralgia⁹, and other neuropathic pain conditions reduce primary pain after surgery but are accompanied by long-term postoperative numbness in the innervated area, superficial hyperalgesia, and other discomforts^10,11. Consequently, research on thermal injury to peripheral nerves has gradually gained attention. However, existing animal models lack the ability to precisely control the temperature, duration, and location of thermal injury, thereby limiting uniformity in injury severity.

Previous studies have used heat-conducting rods connected to a constant-temperature water bath¹² or a hot water pump to inject hot water into a rubber tube in direct contact with the nerve¹³, aiming to simulate the thermal effects of RFA on nerve injury. However, previous thermal injury models have induced only thermal damage, thereby eliminating the influence of radiofrequency current and its parameters on nerves. Whether the damage from RFA is solely due to thermal effects, or whether factors such as changes in radiofrequency parameters also exert distinct effects on nerves, remains to be explored. The current lack of animal models that directly employ radiofrequency thermal coagulation systems for nerve injury hinders the advancement of research on nerve damage associated with RFA. This study directly used a monopolar RFA system to simulate nerve injury associated with RFA procedures in clinical settings by inducing RFA injury to the sciatic nerves of mice. It provides a methodological reference for basic research on RFA injury to peripheral nerves.

Protocol

Animal experiments were approved by the Animal Ethics Committee of the Experimental Animal Center, Xuanwu Hospital, Capital Medical University, and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals¹⁴. Adult male C57BL/6 mice (8–10 weeks old, 20–25 g) were group-housed (4–5 per cage) under controlled environmental conditions (22 ± 2 °C, 50%–60% humidity, 12 h light/dark cycle with lights on at 07:00) with ad libitum access to standard rodent chow and water. All behavioral assessments were performed during the light phase between 08:00 and 12:00. Details of the reagents, software, and equipment are listed in the Table of Materials.

1. Anesthesia and mice preparation

1. Anesthetize mice via intraperitoneal injection of a 20 mg/mL tribromoethanol solution at a dose of 0.2 mL per 10 g of body weight.
2. Cover both eyes with an ophthalmic ointment using a cotton wool applicator.
3. Place the mice in the left lateral position. Remove the hair on the right side of the body (1 cm × 1 cm area) with an electric razor (Figure 1).
   NOTE: This step increases the contact area between the torso and the negative plate of the circuit to facilitate reducing circuit resistance.
4. Place part of the mouse torso in contact with the metal negative electrode plate in a right lateral orientation. Apply several drops of ionic liquid (0.9% NaCl or modified Ringer’s solution; see Table of Materials for composition) to the contact area between the torso and the plate to enhance electrical conductivity.
   NOTE: Ensure that the head and face remain off the negative plate to prevent unintended current exposure to the eyes or other organs.
5. Secure the left hind limb in an upward-facing position with the knee flexed using adhesive tape on the negative electrode plate. Remove hair from the left thigh region and disinfect the surgical site three times with 75% ethanol.
6. Confirm adequate depth of anesthesia before proceeding by verifying the absence of a withdrawal response to a pinch stimulus applied to the hind limb or tail.
   NOTE: Avoid administering local anesthetics (e.g., lidocaine) or nonsteroidal anti-inflammatory drugs (e.g., acetaminophen) during the perioperative period, as these agents may influence postoperative neurobehavioral assessments.

2. Radiofrequency ablation surgery

1. Use the femur as the reference. Use micro scissors to cut a 1 cm skin incision in the direction of the long axis of the femur, with a 0.5 cm lateral translation to the caudal side (Figure 2).
2. Bluntly dissect the biceps femoris muscle (BFM) along the skin incision and search for the main sciatic nerve trunk using micro forceps. The sciatic nerve bifurcates near the knee joint (Figure 3).
3. Use micro forceps to dissect the sciatic nerve trunk from the surrounding tissue approximately 2 mm above the bifurcation of the sciatic nerve, to a length of approximately 5 mm. Avoid injury to blood vessels during blunt separation.
   NOTE: In case of accidental vascular injury, use a sterile cotton wool swab to absorb the blood and apply appropriate pressure to stop bleeding. Continue the operation after there is no subsequent blood seepage.
4. Connect the RFA coagulation needle to the radiofrequency treatment device. Place the needle tip flat on the muscle tissue to test the resistance. If the resistance is higher than 1 kΩ, add ionic liquid between the mice body and the negative plate using a dropper until the resistance falls below 1 kΩ.
5. Gently lift the separated sciatic nerve with a glass minute hand. Place the RFA needle below the sciatic nerve, with the exposed metal tip positioned 3 mm from the sciatic nerve bifurcation (Figure 4).
6. Set the radiofrequency therapeutic instrument parameters as follows: thermal coagulation mode, 75 °C, 30 s, 2 Hz frequency, and 1 ms pulse width. Activate RFA energy delivery.
7. Withdraw the RFA needle gently. Reposition the sciatic nerve using tweezers to lift the muscle tissue. Close the muscle with 6-0 absorbable polyglycolic acid (PGA) suture. Close the skin incision with 4-0 absorbable PGA suture.
8. After the surgery, place the mice on a circulating warm water blanket (38 °C) or a thermostatically controlled heating pad (37–38 °C) covered with toilet paper or a towel to allow the hair to dry and prevent direct heat-induced damage.
9. Return the mice to the cage and wait for recovery from anesthesia. Routinely house and check daily for incision integrity, and monitor food intake, water consumption, and general body condition.

3. Experimental group

1. RFA group: Perform the complete RFA surgery protocol as described in steps 2.1–2.9.
2. Sham group: Perform identical procedures as the RFA group (steps 2.1–2.5 and 2.7–2.9), but do not activate the RFA system delivery in step 2.6. Maintain the RFA needle in position beneath the sciatic nerve for 30 s without current activation, then proceed to step 2.7.
   NOTE: The Sham group controls for surgical trauma, anesthesia, nerve manipulation, and needle placement independent of thermal injury.

4. Von Frey assessment for MWT

1. Before each test, place the mice randomly into the von Frey test platform. Allow acclimation for 15 min until exploratory behavior ceases.
   NOTE: The platform consists of a metal mesh with 0.6 cm × 0.6 cm holes at the bottom and several red transparent containers for the mice, which are placed in different containers without interfering with each other.
2. Perform the von Frey assessment using the up-down method¹⁵. Apply the von Frey fiber wire to stimulate the middle of the left hind paw sole with sufficient force to cause slight bending. Hold for 3 s or until a withdrawal response occurs¹⁶.
   NOTE: Begin testing with the 0.4 g filament. Apply the next higher filament following a negative response, or the next lower filament following a positive response. Continue for four additional applications after the first direction change. In practice, the 2 g filament was set as the upper limit in this study because at this force, the von Frey fiber lifted the forepaw off the ground without displaying escape or withdrawal responses.
3. Record responses and calculate the 50% MWT using the up-down method calculator (Up-down method for von Frey experiments, https://bioapps.shinyapps.io/von_frey_app/).
4. Conduct von Frey testing at baseline (preoperative) and on postoperative days 7, 14, 21, 28, and 35.

5. DRG tissue collection and immunofluorescence staining

1. At postoperative day 7, collect L4–L6 DRG from the RFA and Sham groups.
2. Fix the tissues in 10% sucrose-paraformaldehyde at 4 °C for 6–12 h.
3. Cryoprotect the tissues in 30% sucrose-PBS at 4 °C overnight.
4. Embed the tissues in optimal cutting temperature compound¹⁷.
5. Cut 13 µm cryosections and permeabilize the sections with a non-ionic surfactant.
6. Block using a blocking reagent and incubate overnight with the polyclonal antibody at 4 °C.
7. After washing, incubate with the secondary antibody for 2 h at room temperature.
8. Mount with antifade medium.

6. Statistical analysis

1. Present normally distributed continuous data as mean ± standard error of the mean (SEM).
2. Perform between-group comparisons using the independent samples t-test.
3. Analyze within-group comparisons across different time points using repeated-measures analysis of variance (ANOVA).
4. Apply Bonferroni correction for post-hoc pairwise comparisons.
5. Process all data using a statistical software and set statistical significance at P < 0.05.

Results

Successful model establishment was confirmed by both intraoperative and postoperative indicators. At the completion of RFA (75 °C, 30 s), mild adhesion between the RFA needle tip and the sciatic nerve was observed upon withdrawal. This adhesion results from localized protein denaturation and coagulation at the contact site and suggests appropriate thermal energy delivery along with direct nerve contact. Absence of adhesion may indicate needle displacement during the procedure, leading to inconsistent or suboptimal nerve ...

Discussion

With the rapid development of minimally invasive surgery, radiofrequency technology has become increasingly widespread^6,7,18. As radiofrequency technology has expanded in clinical practice, related research has facilitated its broader adoption. However, the effects of radiofrequency current on tissue require further investigation, especially after radiofrequency technology is applied to the treatment of neuropathic pain, and the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-0 suture	Jinhuan Medical	C412	Close the skin
6-0 suture	Jinhuan Medical	KCR631	Close the muscular layer
Alexa Fluor 488 secondary antibody	Invitrogen	A-11008	Secondary antibody
ATF3 rabbit polyclonal antibody	Novus Biologicals	NBP1-85816	Polyclonal antibody
Curved fine forceps	Beyotime Biotechnology	FS031	Cut muscle and fascia
DAPI antifade mounting medium	Beyotime Biotechnology	P0131	Antifade medium
Glass minute hand	Yuyan Instruments	BLFZ10	Lift up the nerve
GraphPad Prism	Graphpad Software, LLC	version 9.0	Output data images
Micro Scissors	Beyotime Biotechnology	FS217	Cut skin incision
Modified Ringer’s solution	Self-prepared	N/A	Sodium lactate 3.10 g/L; NaCl 6.00 g/L; KCl 0.30 g/L; CaCl2·2H2O 0.20 g/L
Needle Holder	Yuyan Instruments	Y32010	Hold needles
QuickBlock blocking solution	Beyotime Biotechnology	P0260	Blocking reagent for immunofluorescence
Radiofrequency therapeutic device	Cosman Medical	RFG-1A	Radiofrequency system
radiofrequency thermo-coagulation electrode trocar	Cosman Medical	TCD-10	Insulated RF needle
Sodium Lactate Ringer's Injection	Sichuan Kelun Pharmaceutical Co., Ltd	86902180001566	ionic liquid to reduce the resitance
SPSS Statistics	IBM Corp	version 27.0	Statistical analysis software
Straight fine forceps	Beyotime Biotechnology	FS027	Cut muscle and fascia
Von Frey fiber wire	Danmic Global	Aesthesio Semmes-Weinstein	Behaviour test