Method Article

Mouse Sciatic Nerve Transection/Resuture Procedure for Studying Nerve Repair and Functional Recovery

DOI:

10.3791/68724

⸱

January 16th, 2026

 , 
1Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, 2Department of Cell Systems and Anatomy, University of Texas Health Science Center at San Antonio, 3Department of Molecular Medicine, University of Texas Health Science Center at San Antonio

In This Article

Summary

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This protocol describes a sciatic nerve transection and resuture model in mice to study peripheral nerve regeneration using adult dorsal root ganglion neurons. It enables in vivo analysis of axon regrowth, muscle reinnervation, and functional recovery following nerve injury.

Abstract

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Nerve repair is crucial for restoring function following axonal injury. However, mature neurons in the central nervous system (CNS) have a limited ability to regenerate their axons after injury due to diminished intrinsic growth capacity and a hostile environment. In contrast, neurons in the peripheral nervous system (PNS) exhibit robust regenerative potential. Among them, adult dorsal root ganglion (DRG) neurons are particularly well-known for their ability to regenerate effectively following peripheral nerve injuries, making them an ideal model for studying the cellular and molecular mechanisms underlying nerve repair. Here, a modified microsuture technique, termed "loop-before-transection," is described for generating a sciatic nerve transection and resuture model in mice, followed by analysis of axon regeneration. Behavioral assessments and muscle reinnervation are also demonstrated to evaluate functional recovery. After sciatic nerve transection and resuture, animals initially exhibit behavioral deficits, but full recovery is typically observed by 30 days post-injury. Applying drugs that promote axonal fusion at the injury site significantly accelerates functional recovery. This model system offers a powerful tool for investigating the mechanisms governing mammalian nerve repair in vivo.

Introduction

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Axonal injury is a common and severe consequence of brain and spinal cord damage, often caused by traumatic injuries, neurotoxins, or neurological disorders that disrupt neural connectivity and impair axonal regeneration1,2. Not allneurons possess an intrinsic capacity for regeneration, which is crucial for the repair of severed axons.The rate of axonal regeneration is influenced by multiple factors, including the neuron's intrinsic growth potential, protein synthesis efficiency, cytoskeletal dynamics, clearance of myelin debris, and the availability of growth factors3,4.

In the adult mammalian central nervous system (CNS), axonal injury leads to a diminished regenerative capacity, primarily due to a lack of intrinsic growth-promoting factors and the presence of inhibitory extrinsic cues1,5. In contrast, axons in the peripheral nervous system (PNS) possess a robust ability to regenerate following injury, owing to the intrinsic self-repair mechanisms of peripheral neurons and the rapid reactivation of pro-regenerative growth programs. Upon injury, the distal segment of the axon becomes disconnected from the neuronal soma and undergoes Wallerian degeneration, characterized by axonal disintegration and fragmentation6,7. Efficient clearance of axonal and myelin debris by glial cells is essential for successful regeneration, as it prevents scar formation that could impede axonal growth3,8. This process, together with cytoskeletal remodeling of injured axons, supports the regrowth of the proximal stump and facilitates target reinnervation. These events occur in partially overlapping phases, during which injury-induced activation of transcription factors such as ATF3 and c-Jun promotes pro-regenerative gene expression programs4,9,10.

Regenerative axonal fusion represents an efficient mechanism for restoring neuronal connectivity following axonal injury11. This process was first described in the crayfish Procambarus clarkii in 196712 and has since been documented across multiple species13,14,15,16, including Caenorhabditis elegans17. Although direct evidence for axonal fusion in mammals remains limited, fusion-inducing strategies have been successfully employed to enhance nerve repair11. Notably, the chemical fusogen polyethylene glycol (PEG) promotes membrane resealing and has been shown to facilitate functional recovery following both peripheral nerve and spinal cord injuries in mammalian models18,19,20,21,22,23,24. Recent findings indicate that glutathione peroxidase (GPX) depletion is sufficient to promote axonal fusion in C. elegans25. Similarly, Gpx4 conditional knockout mice or those treated with the GPX inhibitor ML162 displayed accelerated functional recovery after sciatic nerve transection, potentially through enhanced axonal fusion25.

The sciatic nerve is a mixed nerve composed of sensory, somatic motor, and autonomic motor axons26. It arises from the L4 and L5 spinal segments in rats27, and from the L3 and L4 segments in mice28. Due to its accessibility and anatomical consistency, sciatic nerve injury has become one of the most widely used models for studying peripheral nerve regeneration29,30. These models enable the investigation of both intrinsic neuronal mechanisms and extrinsic environmental factors that influence axonal growth and functional recovery3,4,9. The use of sciatic nerve crush as an experimental paradigm in neuroscience dates back to the late 19th century29. In addition to nerve crush models, nerve transection is widely used to study axon regeneration and functional recovery. However, the classical sciatic nerve transection followed by suture in mice is often less reproducible and reliable than in rats, due to the nerve's small size and fragile texture31.

Using a modified sciatic nerve transection and resuture model, termed "loop-before-transection", reproducible sciatic nerve transection and resuture models were successfully generated25. This approach eliminates the need for post-transection nerve handling by pre-inserting the suture prior to cutting, thereby reducing mechanical stress and minimizing variability due to surgical technique. Moderate levels of GPX modulation were found to contribute to functional recovery after complete transection of the mouse sciatic nerve25. Co-treatment of the low concentrations ML162 (0.25 µM)32, GPX inhibitor, and polyethylene glycol (PEG, 500 mM)18,19,20,21,22,23,24 on the sciatic nerve transection (SNT) / resuture lesion successfully enhanced the behavior score assessed by pinprick, foot fault asymmetry, and toe spreading test25,33,34. Amputated distal axon fragments of mammalian dorsal root ganglion (DRG) neurons degenerate within 3 days, resulting in disruption of neuromuscular junctions (NMJ)6,35, and regenerative DRG neurons will reinnervate muscles to recover their functions. SNT/resuture-ML162/PEG co-treatment group showed innervated NMJs and enhanced behavior scores at the early time point (3 days post-operation), indicating early recovery of neuromuscular function25.

Here, the "loop-before-transection" microsuture method and drug administration that enabled the above observations in mice are described. A previously established PEG-fusion protocol, which enables rapid repair of severed rat nerves, was modified18. Polyethylene glycol (PEG) was initially reported to promote exogenous reconnection of cut crayfish medial giant axons (MGA) in vitro36. PEG-fusion protocols improved behavioral outcomes in 25%-30% of trials involving crush-severance injuries of the rat sciatic nerve, though functional recovery was not observed at early time points34. Subsequent refinements incorporated the concept of endogenous membrane sealing37. After nerve injury, rapid plasmalemmal repair is neuroprotective and prevents neurodegeneration. Ca2+-induced accumulation of membrane-bound vesicles facilitates this sealing process38. Complete nerve transection disrupts the axolemma as well as the endoneurial, perineurial, and epineurial sheaths at the lesion site. Microsuturing of the epineurial sheath aligns the proximal and distal nerve stumps. During the suture step, the use of Ca2+-free hypotonic saline reduces the formation of membrane-bound vesicles, keeping the endoneurial and perineurial sheaths open and allowing them to be closely approximated by the epineurial sutures. Following this, the application of Ca2+-containing saline induces vesicle accumulation to seal remaining plasmalemmal disruption18.

In this protocol, Adult FVB mice (8-10 weeks old) and C57BL/6 mice were used, and no strain-specific difference was observed. The sciatic nerve in mice is located approximately 1-2 mm beneath the gluteal muscle at mid-thigh level and is notably thinner and more fragile than in rats, necessitating greater surgical precision during dissection and suturing. Care must be taken to avoid nerve overstretching or excessive manipulation that may compromise functional recovery. This approach involves fully severing the nerve, resulting in a physical gap between the proximal and distal stumps. Because this gap inhibits direct axonal regeneration, successful recovery typically requires microsurgical intervention or nerve grafting.

Given that the mouse sciatic nerve is thinner and more delicate than that of the rat, greater surgical precision is required. Although microsuturing is commonly used, it may inadvertently cause additional trauma due to excessive manipulation or repeated needle passes39. These limitations can introduce variability and potentially confound the evaluation of novel treatment strategies. Therefore, achieving consistent surgical outcomes while minimizing suture-induced nerve damage is essential for reliably assessing functional recovery in nerve transection models. Using this "loop-before-transection" method, reproducible sciatic nerve transection and resuture models were generated, and the effects of dual treatment with PEG and ML162 on functional recovery were evaluated. Overall, despite its widespread use, the mouse sciatic nerve transection model poses technical challenges due to the nerve's small size and fragility. This modified resuture method described here addresses a critical need for a reproducible, standardized microsurgical protocol that minimizes mechanical damage.

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Protocol

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All animal procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Health Science Center at San Antonio (Protocol Number: 20200082AR). Adult male and female FVB mice (8-10 weeks old, 20-25 g) were used for the JoVE experimental procedures described here. C57BL/6 mice (8-10 weeks old, 20-25 g) were used only in preliminary practice surgeries conducted prior to the formal experiments to optimize handling and suturing precision. These practice sessions confirmed that the "loop-before-transection" procedure produced comparable outcomes in both strains; therefore, all formal data presented in this manuscript were obtained from FVB mice. A total of ten FVB mice (five males and five females) were initially included for sciatic nerve transection followed by ML162 + PEG treatment. One animal exhibited a congenital anatomical variation in which the sciatic nerve bifurcated into two main branches before the transection site, resulting in incomplete surgery and exclusion from analysis. Therefore, behavioral and histological analyses were conducted on nine animals (n = 9). Both sexes showed comparable regenerative and behavioral recovery, and no sex-specific differences were observed. An additional mouse underwent double suture penetration without nerve transection (sham control) to assess the potential impact of the suture procedure itself on behavioral outcomes. No noticeable differences in regenerative response were observed between sexes. Animals were housed under standard conditions with a 12-h light/dark cycle, at 75 °F and 40%-60% relative humidity, with ad libitum access to food and water. Both male and female mice were used, and no noticeable difference was observed between male and female animals. Surgical procedures were performed under isoflurane anesthesia of the SomnoSuite low-flow anesthesia system. Behavioral tests were conducted beginning on post-operative day 2, and repeated on days 4, 7, 10, 14, 18, 22, and 27, as indicated in the corresponding figures. The reagents and the equipment used are listed in the Table of Materials.

1. Sciatic nerve transection/resuture and drug treatment

  1. Place the animal on a heated pad where the sciatic nerve could be observed under a stereo dissecting microscope (Figure 1A).
  2. Anesthetize the mouse using the SomnoSuite low-flow anesthesia system.
    NOTE: Confirm the depth of anesthesia by the absence of reflex responses, such as reaction to a toe pinch, before initiating surgery. Apply vet ophthalmic ointment to eyes to prevent corneal dryness.
  3. Shave the hair on the thigh and around.
    NOTE: Remove the shaved and dropped hairs as cleanly as possible. Otherwise, the hairs will stick to the 10-0 nylon suture and contaminate the sciatic nerve. Residual hairs can be gently removed using laboratory tape or scotch tape after shaving to ensure a clean surgical field. Disinfect the surgical site by sequentially wiping the shaved area with 70% ethanol, betadine, and a final wipe with 70% ethanol using autoclaved cotton balls.
  4. Locate the femur bone and make a small skin incision of approximately 1.0 cm (range 0.8-1.2 cm) at the mid-thigh level, about ~1 mm posterior and in parallel to the femur bone using fine surgical scissors.
    1. Gently retract the gluteus maximus to expose the underlying biceps femoris. Then, use the tips of the scissors to gently separate the anterior and posterior heads of the biceps femoris, without cutting, to expose the sciatic nerve beneath the sciatic notch. These steps are performed under direct visual observation, not under the microscope.
  5. Pull the sciatic nerve out of the body using micro-dissecting forceps to facilitate surgery on the sciatic nerve (Figure 1A).
    NOTE: Secure the forceps with tape to prevent them from spreading beyond a certain level to minimize damage to the sciatic nerve. Place the tip of the forceps under the sciatic nerve and lift the sciatic nerve upward using the mouse thigh as a fulcrum. Take care to lift the nerve only as much as needed to facilitate suturing, and avoid applying tension or pulling it outward, as excessive stretch can damage the axons or surrounding structures.
  6. Focus the microscope on the exposed sciatic nerve. From this step, proceed through Step 15 using a microscope (Figure 1A).
  7. Penetrate twice the sciatic nerve with 10-0 nylon suture (Figure 1B,C). Go to Step 1-8 for uncut control. Go to Step 1-11 for the nerve transection.
    NOTE: Two penetrations were made approximately one nerve width apart (~0.7 mm), positioned symmetrically on either side of the intended transection site. This spacing ensured proper alignment of the proximal and distal stumps while minimizing mechanical stress at the lesion. Make penetration through the center of the sciatic nerve with the suture, ensuring that the two penetrations made by the suture are precisely parallel. 10-0 sutures are small and must be controlled with super fine tweezers. Non-absorbable 10-0 nylon was used because it provides excellent tensile stability and minimal tissue reactivity during the 30-day observation period. This material is widely used in microsurgical nerve repair models in mice and rats18,31. Absorbable sutures were not used because gradual degradation could compromise alignment of the transected nerve stumps before functional recovery is complete. To minimize operator-dependent variability, all surgical procedures were performed by a single trained surgeon after extensive microsurgical practice under a stereomicroscope. The "loop-before-transection" method itself further reduces variability by eliminating the need for post-transection nerve manipulation, thereby ensuring consistent alignment and tension across animals. This requires considerable practice and a high level of control skills. Before performing animal surgeries, beginners may find it helpful to practice the suturing motion using fine synthetic threads such as dental floss to develop dexterity and control under a stereomicroscope.
  8. Carefully tie a knot with the nylon suture.
  9. Place the exposed sciatic nerve under the muscle layer and close the skin using 4-0 nylon sutures.
  10. Determine that double penetration of the sciatic nerve with a 10-0 nylon suture does not affect mouse behavior.
    NOTE: To confirm this, mice that underwent double suture penetration without transection were subjected to the same behavioral tests used in the experimental groups, including pinprick, foot fault asymmetry, and toe spreading. These mice consistently achieved full scores equivalent to unoperated wild-type controls, indicating that the suture procedure alone did not impair sciatic nerve function.
  11. Cut the sciatic nerve midway between the two 10-0 nylon sutures using a scalpel (Figure 1D).
    NOTE: To minimize variability, all surgeries were performed by the same trained experimenter using a standardized procedure. In addition, the "loop-before-transection" design inherently minimizes variability by avoiding post-transection nerve handling, ensuring consistent suture placement and tension across all animals. The position of the sutures was verified under a dissecting microscope to ensure uniformity across animals.
  12. Put the forceps away.
  13. Wash the cut nerve with Ca2+-free phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4/NaH2PO4, pH 7.4), prepared without calcium chloride.
    NOTE: Rinse the severed nerve stump with calcium-free PBS for approximately 10-20 s to clear blood and prevent premature sealing. Resuturing should be completed promptly within 1-2 min to maintain alignment and tissue viability.
  14. Remove the Ca2+-free PBS by absorbing it with a tissue.
  15. Carefully tie a knot with the nylon suture (Figure 1E). Proceed to Step 20 if drug treatment is not planned or required.
    NOTE: A standard square knot was used for microsuturing, consisting of two opposite throws gently tightened with fine forceps under a stereomicroscope. The two severed axons connected by the suture return to a shape close to their original shape. The reconnection of a severed blood vessel can be a visual indicator that the proximal and distal nerve stumps are properly aligned. During the procedure, the tissue should appear fresh and moist, without pallor or compression. Avoid tying the suture too tightly, as this may collapse blood vessels or deform the nerve. Before tying the final knot, ensure that the nerve ends are approximated without tension. If needed, gently reposition the limb to facilitate natural alignment rather than pulling on the nerve, as excessive tension may increase the risk of neuroma or fibrosis.
  16. Place 0.25 µM of ML162 in DMSO diluted in PBS onto the sutured sciatic nerve using a syringe. Allow the sutured sciatic nerve to be immersed in the drug for 1 min.
  17. Remove the ML162 by absorbing it with a sterile cotton swab.
  18. Place 500 mM of PEG in distilled water, pre-warmed to 37 °C onto the sutured sciatic nerve using a syringe. Allow the sutured sciatic nerve to be immersed in the drug for 1 min.
    NOTE: 500 mM of PEG is a dense solution that may become solid at operating room temperature. Place the syringe on a heated pad during operation to ensure the PEG remains liquid. PEG is thought to promote membrane fusion by removing water molecules from between adjacent lipid bilayers, thereby enabling close membrane contact and spontaneous fusion. Although the precise molecular mechanism remains under investigation, this approach has been shown to facilitate axonal continuity and enhance functional recovery in peripheral nerve injury models.
  19. Remove the PEG by absorbing it with a tissue.
  20. Wash the sutured nerve with Ca2+-containing PBS (PBS supplemented with 1 mM CaCl2; 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH7.4).
  21. Remove the Ca2+-containing PBS by absorbing it with a tissue.
  22. Place the exposed sciatic nerve under the muscle layer and close the skin using 4-0 nylon sutures.
  23. Once the animal has recovered from the anesthesia, return it to its cage.
    NOTE: After skin closure, the surgical site was treated with a topical antibiotic ointment containing pain reliever. Each animal was monitored on a heating pad until it regained sufficient consciousness to maintain sternal recumbency. Animals were then housed individually and returned to their group only after full recovery of motor function. Although the surgical environment was not maintained under a laminar flow hood, all surgical instruments were sterilized, and procedures were performed using aseptic technique with gloved hands and sterile tools.

2. Behavior test - Pinprick

NOTE: To assess the high-threshold mechanical sensitivity responsiveness after injury33.

  1. Place the mouse in a wire mesh grid and allow it to roam freely for 30 min to habituate to the new environment.
  2. The most lateral part of the plantar surface of the hind paw was divided into 5 areas (A to E). Gently apply the Austerlitz (size 000) to the plantar surface of the ipsilateral paw. The pinprick was applied twice from the most lateral toe (area A) to the heel (area E).
    NOTE: Do not penetrate the skin.
    NOTE: A response was considered positive when the animal briskly removed its paw from the two pinprick stimuli. The mouse was graded 1 for this area and then tested for the next one (Maximum 5). If none of the applications elicited a positive response, the overall grade was 0. Use the saphenous territory of the same ipsilateral side as a positive control. It will always elicit a positive response.

3. Behavior test - Foot fault (FF) asymmetry

NOTE: To measure how well sciatic axons innervate more proximal muscles34.

  1. Allow the animals to roam freely on a wire mesh grid elevated on the cage to habituate the environment. One trial consists of 50 total steps per hindlimb.
  2. Score when the animals misstep. The scoring criteria are as follows.
    Score 1: Partial fault. Pulled back before the leg entirely fell out to the thigh.
    Score 2: Full fault. Completely fell off to the crotch.
  3. Calculate the composite foot fault score from all animals' injured and uninjured hindlimbs at each post-operative time.
    Composite foot fault score = (# partial faults x 1) + (# full faults x 2)
  4. Divide the composite foot fault score by 50 (total steps/limb) to obtain the percentage of fault for each hindlimb.
     % foot fault = Composite foot fault score/50 (total steps) x 100%
  5. Subtract the foot faults in the injured hindlimb from the proportion of foot faults in the intact hindlimb to yield an asymmetry score for each animal at a given post-operative time.
    Foot fault (FF) asymmetry score = %FF (intact limb) - %FF (injured limb)

4. Behavior test - Toe spreading

NOTE: To assess motor recovery after nerve injury. According to a previous study, the toe spreading motor test is more sensitive than the gait analysis to detect the recovery of motor function after sciatic nerve injury30.

  1. Gently cover the animal with a piece of cloth and lift the tail for clear observation of the hind paws.
    NOTE: Alternative: Gently grab the mouse's nape, fix the tail, turn it over, and make the mouse squirm to observe the toe movement.
  2. Award the toe spreading score as follows:
    Score 2: Full spreading. Define as a complete, wide, and sustained spreading of all toes for at least 2 s.
    Score 1: Intermediate spreading. Any movement that does not reach a full spreading score. Tremble slightly, or partial movement.
    Score 0: No spreading. No movement at all.
    NOTE: All behavioral assessments (pinprick, foot fault, and toe spreading) were double checked by an experimenter blinded to treatment groups. Animals were randomly assigned to treatment groups at the time of surgery in accordance with the design used in our prior study (Ko et al.25), which included four experimental conditions (Resuture only, PEG, ML162, ML162 + PEG). The current article presents data from the ML162 + PEG group, analyzed independently while maintaining the original experimental rigor.

5. Tissue perfusion and processing for histology

  1. Deeply anesthetize the animal and perform transcardial perfusion with phosphate-buffered saline (PBS) via the left ventricle.
  2. Incise the right atrium to allow complete blood drainage.
  3. Immerse the collected tissues in 4% paraformaldehyde (PFA) at 4 °C overnight for fixation.
  4. Wash the fixed tissues three times with PBS, then incubate them in PBS at 4 °C overnight.
  5. Cryoprotect the tissues by immersing them in 30% sucrose at 4 °C overnight.
  6. Embed the tissues in OCT compound, freeze them, and section at 12 µm thickness using a cryostat.
  7. Store tissue blocks at -80 °C and cryosections at -20 °C until use.
  8. For sciatic nerve collection alone, omit perfusion, as blood content does not significantly affect immunostaining quality in these peripheral tissues.

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Results

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The previous PEG-fusion protocol18 was modified to maintain consistent surgical results and minimize nerve damage caused by suturing25. By placing the suture to penetrate through the nerve before transection, damage caused by pinching with tweezers or pricking with a needle after transection was minimized. To test the effect of tying a suture penetrating a nerve on its function, the suture was passed through an intact nerve twice and knotted (Figure 2A<...

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Discussion

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Nerve regeneration studies using nerve transection models are widely performed on the mouse sciatic nerve31,39. However, due to its small size and soft texture, the mouse sciatic nerve is difficult to handle gently with surgical tools, and the tissue is prone to tearing during suturing. The microsuture process, often involving excessive manipulation, can lead to nerve deformation and trauma. Even when unnecessary instrument stimulation is minimized, excessive mov...

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Disclosures

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Authors have no conflicts of interest to disclose.

Acknowledgements

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This work was supported by NIA R01AG070214 to L.C., NIA R01AG071591 to L.C., and NIA 1R01AG085545 to L.C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
#55 ForcepsDumostar-Biology11295-51One in each hand. You will need two to grasp a 10-0 nylon suture.
10-0 nylon sutureDemeTECHNL761000,4F0P
4-0 nylon sutureMatrix Wizard0197
Anti-beta-tubulinR&D SystemsMAB1195
Anti-NF-200Millipore SigmaN4142
Austerlitz size 000For Pinprick test
CF488 conjugated-alpha-bungarotoxinBiotium00005
Dissecting scissorsKent Scientific 
Fine suturing scissorsKent Scientific 
Iris forcepsKent Scientific 
K&H Small Animal Heated PadK&H pet products
ML162Millipore SigmaSML2561
Mouse surgical kitKent Scientific 
Needle holderKent Scientific 
Polyethylene glycol (PEG)Millipore SigmaP5413
SCG10Novus BiologicalsNBP1-49461
SomnoSuiteKent Scientific For isoflurane anesthesia
Splitter forcepUsed to expose the sciatic nerve.

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Sciatic Nerve TransectionNerve RepairFunctional RecoveryAxon RegenerationPeripheral Nerve InjuryDorsal Root GanglionMicrosuture TechniqueMuscle ReinnervationAxonal FusionBehavioral Assessment
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