Noninvasive electrodiagnostic and motor function assessment of the common fibular nerve regeneration in the rabbit hindlimb

Abstract

Background

Although widely used, the rat model remains poorly transferable to humans for peripheral nerve regeneration studies. The rabbit is a much better choice from an anatomical perspective. However, it remains little used due to the lack of available literature. The aim of this article is to demonstrate the feasibility and effectiveness of an electrophysiological protocol combined with a motor function assessment to analyze nerve repair.

Methods

Ten white New Zealand rabbits underwent a 4 cm transection of the fibular nerve. Autograft regeneration over 36 weeks was compared to non‐repaired controls. The compound muscle action potential (CMAP) was recorded in the tibialis anterior and the extensor digitorum brevis. An electromyogram (EMG) was obtained after needle insertion and resting muscle activity recording. The electrophysiological results were compared to the toe spread index (TSI), which assesses the motor functional recovery promoted by fibular nerve regeneration.

Results

The autograft group regeneration starts between weeks 18 and 21 and normal EMG was observed around the 30th week. These electrophysiological results were compared to the well‐defined toe spread reflex. This motor test showed a significant functional return of 59% at 36 weeks (p < 0.05). Rabbits regain nearly 80% of their muscle mass.

Conclusion

Nerve conduction allows detection of nerve regeneration of the muscle while electromyography indicates when muscle activity returns to normal. These studies are reliable and non‐invasive techniques to evaluate fibular nerve regeneration in the rabbit's hindlimb. Nonetheless, it is necessary to have qualified personnel, since inter‐manipulator variations have been observed.

The rabbit model is well‐suited for peripheral nerve repair studies, using electrodiagnostic testing as an effective and reliable technique for fibular nerve regeneration evaluation, combined with motor function assessment using the toe spread index.

1. INTRODUCTION

Peripheral nerve injuries (PNIs) have a significant impact on the patient's quality of life and a huge economic impact on society. ¹ PNIs represent approximately 2.8% of all traumas and lead to 50 000 nerve graft surgeries every year in the United States alone. ² , ³ Researchers around the world are developing and designing innovative techniques to repair large nerve deficits (>3 cm). The rat model is the first choice for in‐vivo testing of those new technologies. In fact, about 75% of published studies related to peripheral nerve repair use rats. The main advantages of using rats are the availability of several well‐characterized motor tests in the literature, as well as a fast motor function recovery (12 weeks). ⁴ Unfortunately, the use of rat data alone may lead to results that do not transfer well into humans and can poorly evaluate the patient's risks versus benefits, especially for longer gaps. ⁵ , ⁶ It is mainly due to the rat's high neurobiological regenerative profile and small nerve size (maximal repairable distance of sciatic nerve being 1.5 cm). Thus, there is a need to use larger animals, especially when studying a critical human gap length of more than 3 cm. Various animal species have been used to study peripheral nerve repair, such as pigs, dogs, sheep and monkeys, allowing gap lesions up to 6 cm long. However, these large animals are very expensive to use, necessitate specific animal facilities, raise ethical concerns and require long post‐surgical follow up. ⁷ The rabbit is the most accessible and frequently used large animal model for nerve regeneration studies, and remains affordable compared to other larger animals. ⁸ However, the main challenge in using rabbits is their skin fragility, which increases the risk of wound formation caused by excessive licking. In addition, if the study involves the transplantation of heterologous cells to repair the peripheral nerve, it is necessary to administer immunosuppressive treatment to the animal, unlike rats or mice for which immunodeficient strains are available. Immunosuppression of rabbits involves expensive daily treatment and significant risk of side effects. ⁹

On the other hand, sections up to 5 cm can be performed in sciatic or fibular rabbit nerves. ⁵ , ¹⁰ The nervous system anatomy of the hindlimb is relatively conserved between mammals and is particularly similar for humans and rabbits. ¹¹ The rabbit hindlimb nerve injury model is increasingly popular within experimental settings but, to our knowledge, current available literature is scarce for electrodiagnostic and functional testing to assess the regeneration of peripheral nerves in rabbits. However, these two tests are described as the most reliable and accurate methods for monitoring peripheral nerve recovery following injury. ¹² Protocols are widely standardized in humans with confidence data for each nerve. In the literature, there is a lack of well‐defined methods and reference ranges for rabbits, though several teams have undertaken nerve conduction studies. ⁸

Hotson et al. ¹³ have shown the feasibility of studying fibular nerve conduction in distal rabbit hindlimbs. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ The fibular nerve passes through the lateral head of the gastrocnemius and travels medially down the leg giving innervation to tibialis anterior, the extensor digitorum longus and to the four fibular muscles. ¹⁹ Fibular nerve stimulation at the fibula head can evoke a compound muscle action potential (CMAP) from the tibialis anterior that can be monitored.

Alsmadi et al. ²⁰ used high‐speed photography and standardized equipment to objectively measure the degree of toe abduction and showed that the toe‐spread index, the distance between the first and fourth toes, allowed them to statistically discriminate between experimental groups after fibular nerve gap repair with different nerve tube compositions. The reflex is noninvasively obtained, can be easily repeated and does not need preconditioning. At the moment, it is one of the only reported way to test the resulting functional repair of the peripheral nervous system in a rabbit fibular nerve defect model.

This study aimed to show the feasibility of evaluating rabbit fibular nerve regeneration after a large nerve gap defect by (1) recording the CMAP for the tibialis anterior and extensor digitorum brevis (EDB) muscles and (2) conducting needle electromyography (EMG) of the tibialis anterior and EDB muscle. We also compared these electrophysiological data to the toe‐spread index functional assessment to describe an effective evaluation methodology for rabbit peripheral nerve regeneration that takes both nerve growth and muscle function into consideration.

2. MATERIALS AND METHODS

2.1. Animals

All animal experiments were approved by the CHU de Quebec‐Universite Laval Animal Care Committee (# 2020–577) following the guidelines of the Canadian Council on Animal Care. The rabbits were part of another research protocol, whose objective was to assess nerve recovery with different experimental nerve conduits. For that protocol, rabbits had to be immunosuppressed with a daily oral intake of 1 mg/kg of Tacrolimus (FK‐506, Sandoz, Boucherville, QC, Canada). ⁹ A total of 10 female New Zealand white rabbits (weight 2.0–2.5 kg; Charles River, QC, Canada) were tested throughout this study. The cages were ventilated in a temperature‐controlled room (21°C ± 1°C) with a cycle of 12 h of light and 12 h of darkness with water and hay ad libitum. Rabbits receive a controlled amount of food once a day (pellets and vegetables).

2.2. Surgery

Rabbits were anesthetized by intubation with inhalation of isoflurane (3%–4%, oxygen 0.8–1.5 L/min, Forane®) with injection of 1 mg/kg acepromazine (Boehringer Ingelheim Animal HEALT, Canada). Pain control was provided with subcutaneous preoperative injections of 0.12 mg/kg sustained‐release buprenorphine and postoperative injection of 2.2 mg/kg carprofen twice a day for 2 days. The sciatic nerve was exposed with a muscle splitting incision of 5 cm along the femoral axis. In all rabbits the right fibular nerve was surgically isolated in the common trunk of the thigh and a 4 cm length was transected. For the autograft group, the cut nerve was rotated 180 degrees and then stitched at each end with 8–0 Ethilon (Ethicon, Johnson & Johnson company, San Lorenzo, Puerto Rico) (n = 5). For the non‐repaired group, the nerve was removed, and the nerve ends sutured to the underlying muscle fascia to create a gap of 4 cm (n = 5). The left contralateral limb was left untreated and used as a comparison. At the end of the study, the animals were euthanized under deep isoflurane anesthesia with an overdose of barbiturate.

2.3. Electrophysiology methods

2.3.1. Animal preparation

All the rabbits were evaluated on the experimental limb every 3 weeks following surgery for 36 weeks. When rabbits began to show electrodiagnostic signs of reinnervation, they also had concurrent assessments of the uninjured control limb every 3 weeks until the end of the study. The rabbits received premedication by injection of acepromazine 0.5 mg/kg subcutaneously 30 min before the procedure. The rabbits received pre‐oxygenation with 100% oxygen for 5 min followed by sedation with isoflurane (2%–3%, oxygen 0.8–1.5 L/min, Forane®) delivered by mask. Anesthetic monitoring was carried out by an animal health technician with continuous monitoring of vital signs and temperature during the procedure and until a fully conscious recovery. The leg was shaved with an electric clipper to give easy access to the underlying muscles. The anesthetized rabbits were kept warm using a heating pad to maintain a constant physiological core body temperature and limb temperature was assessed with an infrared thermometer (Maximum®). The assessed limb was disinfected with a 70% alcohol–0.5% chlorhexidine solution prior to nerve conduction study measurements.

2.3.2. Nerve conduction study

The electrophysiological measurements were recorded using a clinical EMG system (Natus® UltraProS100). In the nerve conduction study, the CMAPs were recorded with subdermal needle electrodes (019–477 000, Natus®). The nerves were stimulated with a surface probe (9031E0172, Natus®) with pediatric tips (1.5 cm spacing between the anode and the cathode). A subdermal needle ground electrode was implanted in the ipsilateral leg (Figure 1).

FIGURE 1.

Schematic of the distal rabbit hind limb and the experimental setup. Placement of nerve conduction electrodes and the transcutaneous stimulator at site number 1, fibular head, and site number 2, sciatic notch. Ground electrode (green), recording electrode (black) and reference electrode (red).

First, CMAPs were recorded at the extensor digitorum brevis muscle with the needle electrodes implanted subcutaneously at a spacing of 3 cm following the belly‐tendon montage of the muscle to the dorsum of the foot. Three sites were then stimulated: (1) the deep fibular nerve at the ankle 4 cm proximal to the active‐recording electrode and medial to the midline of the leg, (2) the common fibular nerve at the fibular head, lateral to the popliteal fossa about 8 cm proximal to the previous stimulation site, and (3) the sciatic nerve at the sciatic notch, as proximal as possible on the rabbit, about 4 cm proximal to the previous site of stimulation.

Subsequently, CMAPs were recorded in the tibialis anterior muscle. The subdermal needle electrode placement followed the muscle in a belly‐tendon montage and were spaced 3 cm apart (Figure 1). The common fibular nerve was stimulated at the fibular head lateral to the popliteal fossa 4 cm proximal to the active‐recording electrode; then the sciatic nerve was stimulated at the sciatic notch, approximately 4 cm proximal to the previous stimulation site (Figure 1).

At the end of the study, in situ compound muscle action potential (in situ CMAP) was recorded once at the tibialis anterior. Before the necropsy, the rabbits were put on general anesthesia and the fibular nerve was exposed. The subdermal needle electrode placement followed the muscle in a belly‐tendon montage and the electrodes were spaced 3 cm apart. The fibular nerve was directly stimulated proximally (in situ) and distal to the graft and the evoked CMAPs were recorded at the tibialis anterior muscle.

For CMAPs, the stimulus was a 1 ms square wave current pulse with a supramaximal intensity of 20%. The amplitudes (milliamperes, mA) were measured from the beginning of the negative deflection to its peak. The latency (milliseconds, ms) was measured from the stimulation until the start of the negative deflection.

2.3.3. Electromyogram

The electrophysiological measurements were recorded with EMG system (Natus® UltraProS100). An electromyography needle (disposable concentric needle S53153, Natus®) was used to assess the EMG. The needle was inserted into the tibialis anterior muscle (Figure 2A). The evaluation was performed with an observation of insertional activity and resting activity. The fibrillation potential present in the muscle was graded on a conventional scale from 0 (Figure 2B) to 4. The +4 pattern is only seen when nearly all muscle fibers are denervated simultaneously. In nerve trauma, the screen is completely filled, and it is not possible to distinguish individual fibrillation (Figure 2C). ²¹ The electromyogram measures the muscle quantity and quality of electrical activity and is an indirect measure of nerve recovery (Figure 2D).

FIGURE 2.

Scoring system for fibrillation potential as conventionally graded. ²¹ Electromyogram of tibialis anterior to evaluate muscle activity at the needle insertion site (A). (B–D) Examples of waveform results in a healthy muscle (B), a denervated muscle (C), and a muscle in the process of re‐innervation (D). *Decreased fibrillation may also be a sign of muscle death from total denervation. The result must be analyzed in conjunction with the physiological data.

2.4. Wet tibialis anterior muscle weight

The tibial anterior muscle of the injured and the contralateral control leg were removed under deep anesthesia followed by euthanasia. The wet muscle mass has been shown to be a reliable measurement of reinnervation status. ²² A muscle mass ratio was obtained between the operated leg and the contralateral healthy leg. ²³ The whole muscle was then fixed overnight in 3.7% phosphate buffered formaldehyde (Chaptec inc, Montreal, QC, Canada). Thin muscle slices were then embedded in hot paraffin. Five‐micrometer thick sections were stained with Harris' Hematoxylin (Inter Medico, Markham, ON, Canada) and 0.25% Eosin stain (Fisher Scientific, Ottawa, On, Canada). The slides were observed, and photos taken under light microscopy using Zeiss Axio Imager (Carl Zeiss Canada Ltd., Toronto, ON, Canada) with Zen software. The muscle fiber area and Feret diameter were measured from muscle sections using Image J software (NIH, Bethesda, MD, USA).

2.5. Toe spread reflex

The rabbit is placed in a harness attached to a spring and held high by a pin (Figure 3A). When the pin is removed, the rabbit falls suddenly 20–30 cm and evokes a startled toe spread reflex in preparation for contact with the floor, which is avoided due to a strong spring that engages and safely slows the fall, stopping any contact with the ground (Figure 3B). The fall was recorded by 180 image bursts in 3 seconds. 5 falls were recorded every 3 weeks. To facilitate the recording of the toe position, a red toenail polish is applied to the claws. The toe spread index (TSI) is obtained by measuring the distance between the first and the fourth toe at the bottom of the fall. The TSI represents the ratio between the toe distance of the operated leg and the contralateral leg (Figure 3C). The TSI before the fibular nerve lesion gives a ratio of approximately 1.

FIGURE 3.

Toe spread reflex method. (A) The rabbit is secured in a harness which is attached to a support using a spring and held up high by a pin. (B) Before the intervention, the rabbit toe reflex gives a TSI of approximately 1 (C) After the graft, there is a loss of reflex, and the leg is droopy. The ratio is calculated based on the distance between the outer toes of each leg (red line).

2.6. Statistical analysis

The amplitude and the latency of the evoked CMAP at the tibialis anterior were compiled. The muscle mass and the fiber area were analyzed using Students t test. The TSI was analyzed with a two‐way ANOVA with Šídák's multiple comparisons test. The probability level was considered significant at p < 0.05. All statistical analyses were carried out in GraphPad Prism 8 software and values were all expressed as the mean ± standard deviation.

3. RESULTS

Motor function recovery in 10 rabbits, after a 4 cm fibular nerve transection, (5 non‐repaired control nerves and 5 autografts) was followed over 36 weeks. The CMAP's to tibialis anterior and EDB muscles were assessed by needle electromyography. These results were then linked to the analysis of motor function recovery using the toe‐spread index functional assessment. We have highlighted the relationship between nerve conduction analysis and muscle functionality.

It is important to note that one of the five rabbits in the autograft group showed no sign of reinnervation. At necropsy, the nerve autograft of this rabbit was not in continuity as a result of a microsuture dehiscence, which explained the absence of reinnervation. It was removed from the analysis. One of five rabbits in the non‐repaired group was sacrificed due to autotomy. Therefore, only 8 rabbits were kept for analysis, 4 in each group (n = 4). Additionally, the fact that this analysis was derived from a larger study of 30 rabbits (the maximum number of rabbits that could be housed in our animal facility), consisting of 6 different groups, limited the number of rabbits to 5 per group.

3.1. The compound muscle action potential of the tibialis anterior

CMAP was obtained at the tibialis anterior muscle by stimulation of the fibular nerve and two parameters were assessed: the amplitude and the latency. The stimulation was done at two different nerve sites: the fibular head (Figure 4A,C) and the sciatic notch (Figure 4B,D). The two sites gave a similar wave pattern. The evoked CMAP on the untreated limb typically showed a rapidly ascending negative waveform, with a notch at the maximal peak of the curve followed by a slower positive descent. A second waveform was commonly seen at the end of the first one, positive then negative, and corresponds to a stimulus artifact of the adjacent tibial nerve co‐stimulation. Fibular nerve stimulation evoked a CMAP for every healthy contralateral leg and autograft leg. As expected, there was no CMAP in the non‐repaired group (Figure 4A–D).

FIGURE 4.

Compound muscle action potential of the tibialis anterior to the fibular head or the sciatic notch over 36 weeks. (A, C) Latency curve (A) and amplitude curve (C) at the fibular head site. (B, D) Latency curve (B) and amplitude curve (D) at the sciatic notch site. The mean and the standard deviation are represented (n = 4 individual biological replicates). Solid and dotted lines: Mean and standard deviation for the reference of the contralateral paw (n = 3 individual biological replicates).

The beginning of nerve regeneration for the autograft group was observed between weeks 18 and 21, with an ill‐defined waveform and low amplitude. From the 24th week onward, the amplitude gradually increases and reaches a plateau. Compared to the contralateral leg, a waveform of longer duration can be seen, owing to the heterogeneity of the nerve fiber's remyelination within the autograft. The maximum amplitude of the waveform remained the same until the end of the study but could be associated with the reappearance of the distinctive notch at the negative peak. The mean CMAP amplitudes on the contralateral limb and autograft limbs at the end of the experiment were, respectively, 21.44 ± 5.74 mA and 9.9 ± 4.56 mA; thus the ratio of recuperation was 54% nine months after the surgery (Figure 4C,D). Intra‐ and inter‐observer variation was observed for the measurement of this parameter and required precise manipulation to avoid co‐stimulation of the tibial nerve. As it will be discussed below, this was more pronounced when testing was performed at the EDB muscle.

3.2. In situ CMAP of the tibialis anterior

At the end of the study, the fibular nerve was stimulated in situ, directly on the graft, proximal and distal to the graft sutures to record the CMAP at the tibialis anterior (Figure 5). The evoked CMAP on the untreated limb typically showed a rapidly ascending negative waveform, a flat head, followed by a rapid positive descent (Figure 5C). In the autograft group, the latency was longer and the amplitude smaller (Figure 5B). In situ CMAPs were recorded in every healthy contralateral limb and experimental autograft limb. No CMAP was recorded in the non‐repaired group (Figure 5A).

FIGURE 5.

In situ CMAP of the tibialis anterior 36 weeks post‐surgery. (A–C) Examples of the waveform results for the grafts with no nerve conduction (A), partial nerve conduction (B), and a normal nerve conduction (C).

The mean in situ CMAP amplitudes on the contralateral limb and autograft limb were 26.2 ± 10.1 mA and 15.1 ± 8.0 mA, respectively (Table 1). The ratio of reinnervation was 61%. The evoked in situ CMAP was concordant with the CMAP obtained during the non‐invasive evaluation.

TABLE 1.

In situ stimulation of the fibular nerve (proximal and distal to the nerve graft) in autografts and their contralateral control.

Leg	Latency (ms)			Amplitude (mV)
	Proximal	Median	Distal	Proximal	Median	Distal
Autograft	1.8 ± 0.3	1.7 ± 0.2	1.9 ± 0.1	11.7 ± 6.1	8.6 ± 2.2	15.1 ± 8.0
Contralateral	1.5 ± 0.1	1.8 ± 0.1	1.8 ± 0.2	23.2 ± 9.2	25.9 ± 8.9	26.2 ± 10.1
Ratio	1.2	1.0	1.1	0.5	0.3	0.6

3.3. The compound muscle action potential for the extensor digitorum brevis

The evoked CMAP at the EDB was obtained with stimulation of the fibular nerve at three different nerve sites: medial leg, fibular head and sciatic notch. Three parameters were assessed: the amplitude, the latency and the conduction velocity. In the healthy limb, the stimulation elicited a negative triangular low‐amplitude waveform (about 3–4 mV), followed by a smaller shallow positive peak (Figure S2).

However, significant variations were observed in the results when measuring evoked CMAP and EDB, both on healthy and injured nerves. This was associated with inter‐ and intra‐manipulator variations. When performed on the injured nerve, the variations showed a significant increase. It was difficult to obtain reproducible results over weeks. Due to this inconsistency, EDB was not considered a reliable site to use and was removed from the analyses. The results obtained were not consistent over weeks; therefore, only analyses of the tibialis anterior muscle were retained and fully exploited. Furthermore, insertion of the needle into the extensor digitorum minor caused repeated bleeding, unlike the tibialis anterior muscle, which was problematic from an ethical point of view.

3.4. Electromyography

The EMG represents the motor unit potential of the tibialis anterior muscle, which gives valuable information about the innervation integrity, but also indexes the condition of the neuromuscular junctions and myofibers. ²⁴

During the EMG, we specifically evaluated the muscle activity at the needle insertion time point and at rest. On the untreated limb of every rabbit, normal insertional and rest activity was seen during the needle electromyography testing of the tibialis anterior and EDB muscles. In the non‐repaired group, both muscles showed complete denervation at the first post‐operative EMG testing and until the completion of the study (Table 2). For the first months, strong fibrillation and positive sharp waves were seen on the waveform, which transformed into weaker fibrillation at the end of the study. In the autograft group, tibialis anterior and EDB muscles initially showed complete denervation with fibrillation and positive sharp waves. Then, the tibialis anterior muscle fibrillations became significantly weaker around the 18th week post‐surgery, and the waveform gradually returned to a near normal insertional and rest activity level at around the 30th week (Table S2). Rare polyfasciculations were noted in the rest activity of 1 rabbit. Reinnervation was not observed in the EDB muscles for any rabbits, independent of group. No sign of reinnervation was found in the non‐repaired group.

TABLE 2.

Electromyogram of tibialis anterior to evaluate muscle activity at rest at the site of needle insertion over a 36‐week period following grafting.

Rabbit	Time to reach onset regeneration (weeks)	Time to reach rest activity (weeks)	Delay between onset and rest (weeks)
Autograft 1	24	33	9
Autograft 2	24	30	6
Autograft 3	21	36	15
Autograft 4	18	27	9
EMG score mean	21.8 ± 2.9	31.5 ± 3.9	9.7 ± 3.8

3.5. Muscle functional analysis

Rabbits were euthanized after a 36‐week follow‐up and tibialis anterior muscles from both limbs (operated and healthy contralateral leg) were removed. A ratio was calculated by dividing the muscle weight of the operated side by the contralateral side. As expected, the muscle weight of the autograft repair leg was significantly higher than for the non‐repaired group (p = 0.0467) (Figure 6F). Three out of the four autograft rabbits had muscles which had nearly completely recovered from atrophy and gave a muscle mass ratio approaching 1 (mean = 0.78 ± 0.18). The remaining rabbit still showed signs of muscle atrophy. All rabbits in the non‐repaired group showed obvious signs of muscle atrophy (mean = 0.3 ± 0.03). Muscle fiber area, obtained from histological cross‐sections (Figure 6A–D), is also a good index of muscle atrophy, indirectly expressing the level of muscle reinnervation (Figure 6G–J). Both the muscle Feret diameter (Figure 6I,J) and area (Figure 6G,H) of the autograft group were higher compared to the non‐repaired group.

FIGURE 6.

Motor function analysis over 36 weeks of nerve regeneration. (A–D) Hematoxylin and Eosin histological staining with a macroscopic image insert of the tibialis anterior muscle for the contralateral (A), the autograft (B), the non‐repaired group (C) and for the non‐responder autograft (D) 36 weeks after graft. (E) The return of motor function was determined by the toe spread index test, with follow‐up every 3 weeks. (F) The tibialis anterior muscle index was determined by a calculation of the ratio of the tibialis anterior muscle weight between the injured and healthy legs of each rabbit. (G–J) The average and ratio of muscle fiber area (G and H, respectively) and Feret diameters (I and J) were compared between groups. Two‐way ANOVA with Šídák's multiple comparisons test was used for the functional analysis in graph E and Students t test was used for the muscle analysis in graphs F–J (n = 4 individual biological replicates). Comparison between the non‐repaired group and the autograft (*p < 0.05). Scale bar in A (applies to panels A–D): 100 μm; Scale bar in insert in A (applies to panel inserts in A–D): 1 cm.

The toe spread index allows evaluation of the motor function recovery of the fibular nerve. The index was assessed every 3 weeks. Three out of the four autograft rabbits showed a significant amelioration over the weeks (p = 0.04) while one rabbit did not have motor functional improvement over the 36 week follow‐up (mean = 0.7 ± 0.08). The TSI curves (Figure 6E) showed a return to function beginning at week 12 followed by a plateau from week 15 to week 30. A second phase of improvement began in week 33. The end of the experiment in week 36 probably prevented us from seeing any further functional improvement. No rabbits in the non‐repaired group demonstrated any sign of motor function recovery (mean = 0.5 ± 0.03).

3.6. Complications

The only adverse effects related to the electrodiagnostic testing were minor. The most frequent complications were infracentimetric wounds associated with electric clipper shaving and small subcutaneous hematomas in the dorsum of the foot. These were all either self‐healing or cured with conservative wound care. No infections were noted. The weight of the animals was monitored daily and evolved normally without variation associated with the electrodiagnostic testing. The only side effects observed were caused by the repetitive anesthesia. Decreasing the acepromazine dose (1.0–0.5 mg/kg) and keeping the analysis under 15 min greatly improved the recovery of the rabbits.

4. DISCUSSION

Our long‐term results have shown the feasibility of repeated non‐invasive electrodiagnostic evaluation in the assessment of rabbit peripheral nerve regeneration. Our model allows quantifiable observations to measure the success of nerve regeneration. Sacrificing the rabbit fibular nerve in an experimental setting gives little functional or sensory deficit, greatly limiting pain and post‐operative complications, while its innervation still allows assessment of motor and sensory recovery. ²⁵

Motor nerve conduction studies form the majority of electrodiagnostic tests in animal studies on peripheral nervous system regeneration. Regenerated axons mature with an increase in size and myelination that helps to restore normal conduction properties, ²⁶ which can be tested using a variety of parameters. The most common measures are CMAP amplitudes and latencies.

The CMAP amplitude evoked at the tibialis anterior muscle seems to be the most reliable parameter of nerve regeneration. It accurately represents the number of regenerated axons and is the least likely to be affected by technical issues. Latency and conduction velocity are inherent in the measurements and were easy to analyze but, in our experience, it is technically challenging to get consistent results. Owing to the small size of the hindlimb, the slightest change in the position of the surface stimulator causes relatively large changes in latency, while the amplitude gives a much more repeatable measure.

In the last decade, researchers studying fibular nerve regeneration following nerve gap repair with nerve autograft, compared to experimental conduits, have shown that the CMAP amplitude is a promising parameter for finding statistically significant differences between groups. ²⁰ , ²⁷ , ²⁸ , ²⁹ Our results also showed that we can identify the early signs of regeneration and then serially evaluate the process of reinnervation using a minimally invasive technique. It obviates the necessity of having multiple groups that are periodically sacrificed to assess the success of regeneration. The CMAP amplitude was the first sign of electrical activity during the nerve conduction study that was consistent over the weeks, gradually increasing until it reached a plateau at around week 27. Three weeks after the surgery, needle electromyography showed denervation with strong fibrillation potentials and positive sharp waves. At around the 18th–21st week, the first signs of reinnervation appeared, progressing into the diminution of fibrillation and appearance of fasciculation. The return of normal insertional and rest activity associated with CMAP confirmed the success of reinnervation.

The CMAP amplitude evoked at the EDB was not identified as a reliable test for fibular nerve regeneration. It was difficult to find the perfect spot that maximizes stimulation of the fibular nerve in the rabbit leg without recording tibial nerve co‐stimulation, even in the healthy contralateral limb (Figure S2B). Rabbits have relatively small nerve and muscular anatomy in the distal limb compared to the size of the pediatric stimulator used. Additionally, rabbits have very thin and mobile skin on the anterior part of the leg, which adds to the technical difficulty of precisely stimulating the fibular nerve. An effort was made to replace the surface stimulator with a subcutaneous needle to stimulate the nerve, but this was also suboptimal given the rabbit morphology and size. On the dorsum of the foot, the recording electrode (30 gauge subcutaneous needle) was also too large to precisely measure the evoked CMAP at the EDB muscle. Unlike the autografted rabbits, which gave consistent tibialis anterior muscle responses during fibular nerve stimulation, the presence or absence of evoked CMAP at the EDB muscle does not appear to correlate with reinnervation. We encountered favorable CMAP readings at the EDB muscle in the experimental rabbits on several occasions. However, we were unable to consistently replicate the results across testing sessions. Those difficulties could also be explained by the later regeneration of this distal muscle.

To minimize inter‐individual variability during CMAP measurements, it is essential that operators practice on the animals before surgery to obtain similar CMAP values over several attempts. They must use a template that will allow them to always place the stimulating electrode at the same distance from the recording electrode. In addition, the use of a smaller surface probe electrode for the stimulation of the EDB muscle should help to prevent tibial nerve co‐stimulation.

The in situ CMAPs evoked at necropsy, which are the gold standard of electrodiagnostic testing on animals, confirmed the results we had with the non‐invasive electrodiagnostic testing. Considering the two nerve anastomoses at each end of the graft and the latency before the start of regeneration, the first sign of regeneration at 18–21 weeks coincides with a reinnervation speed of approximately 2 mm/d following the initial latency period, which Gutmann has also observed. ³⁰ , ³¹

Some limitations should be noted. Our testing and results do not necessarily provide the ultimate healthy baseline values for nerve conduction studies because only the left side (healthy side) was analyzed, and side dominance has been demonstrated in rabbits as asymmetry in bone weight and in isometric tetanic muscle force tests. ³² , ³³ We did not measure the baseline level before transection; thus, our result does not allow direct comparison of the same limb before and after repair. The absence of pre‐injury baseline electrophysiological measurements is a limitation of this study, and such analysis would be necessary as a complement to the data obtained from the healthy leg, which accounts for the animals' growth over time. Moreover, the 3‐week assessment interval was based on a human evaluation paradigm, and we may have missed data during this interval.

Two conduction curve rejection criteria were followed. The curve was considered inappropriate if (1) the curve starts too quickly (latency <1 ms) or, (2) the curve is inverted, meaning that there is co‐stimulation (Figure S2). Particular caution was required to avoid co‐stimulation of the tibial nerve and direct stimulation of the tibialis anterior muscle, especially in the early phase of the reinnervation process. The rabbit limb has a small dimension when compared to the surface stimulator and thus the tibial nerve or the tibialis anterior muscle can easily be erroneously stimulated, especially at the fibular neck. The easiest way to detect the presence of tibial nerve co‐stimulation was an inversion of the usual waveform with a strong positive peak followed by a strong negative peak. The direct stimulation of the tibialis anterior muscle evoked a waveform with a significantly shorter latency (Figure S1).

The only convincing functional motor test in published rabbit studies is the toe‐spread reflex. ²⁰ , ²⁵ , ³⁴ , ³⁵ This assesses the function of the fibular nerve, which innervates the muscles responsible for spreading the toes on the hind legs. As already described by Schmitz and Beer, the toe spread reflex is a reliable indicator of the onset of motor recovery in rabbit peripheral nerve injury models. ²⁵ The huge advantage of this technique compared to other motor analyses is that the test is non‐invasive, requires no prior training and can be repeated as often as desired. At the moment, it is one of the only reported ways to test the end functionality of the peripheral nervous system in a rabbit nerve defect model. Alsmadi et al. ²⁰ used high‐speed photography and standardized equipment to objectively measure the degree of toe abduction and showed that the toe‐spread index allowed statistical discrimination between experimental groups after fibular nerve gap repair with different nerve tube compositions. In our experiment, the TSI effectively differentiated the two groups and accurately determined the time of the motor function return. In the nerve conduction study, regeneration in autografts began between weeks 18 and 21, which resulted in a return of motor function at around weeks 30–33.

This comparative study between electromyography and muscular evaluation in rabbits undergoing fibular nerve repair revealed significant observations. In all the individuals studied, a normalization of the resting potential was observed around the 30th week following nerve repair. Moreover, all four subjects exhibited conclusive conduction across the nerve graft. However, a striking inconsistency was observed during the muscle biopsy of one rabbit, namely pronounced muscular atrophy associated with the presence of adipose tissue (Figure 6D). These findings underscore the necessity of corroborating electrophysiological data with physiological findings, as decreased fibrillation could also indicate muscular degeneration due to total denervation. Thus, a careful analysis of results should integrate physiological parameters for a more accurate interpretation.

Nerve conduction was generally recorded earlier after transplantation, between 18 and 21 weeks, consistent with progressive axonal regeneration of the muscle, while the return to normal for EMG occurred later, between 27 and 36 weeks (Table S1). Interestingly, functional recovery assessed by TSI also reached a plateau around week 30, correlating with the return of functional motor units in the muscle. However, TSI is controlled by the EDB muscle, which is further from the graft than the tibialis anterior muscle, and should therefore be innervated with a few days of delay. This delay is probably compensated by the latency due to the formation of neuromuscular junctions after muscle reinnervation. However, variability may be observed between electrophysiological reconnection and functional recovery data due to partial dehiscence of the graft anastomosis, or interindividual variability.

Please note that the plateau reached by EMG and TSI results between the 26th and 36th week was confirmed for the autograft group in another study to be published soon (data not shown).

In this study, because some groups received human cell grafts, all animals were immunosuppressed with tacrolimus. Tacrolimus blocks the synthesis of interleukin‐2 (IL‐2) by T cells, thus preventing acute graft rejection, but does not alter macrophage functionality, which favors nerve regeneration. ⁹ However, tacrolimus has been shown to improve axonal regeneration. ³⁶ , ³⁷ , ³⁸ Since no non‐immunosuppressed controls were included, its specific impact remains unclear and should be considered when interpreting the results.

Electrodiagnostic testing is a fast, objective, and reliable technique to evaluate the regeneration of the fibular nerve in the hindlimb of the rabbit. It is a non‐invasive method and associated with very few minor complications.

The rabbit is an effective model for evaluating the potential of a new peripheral nerve repair strategy prior to transferal to the clinic. Indeed, its anatomical and physiological characteristics are much closer to those of humans than those of rats, allowing for a more predictive assessment of graft performance and regeneration strategies. Additionally, the size of the rabbit nerve allows for surgical techniques and functional assessments that are directly relevant to clinical scenarios, thereby bridging the gap between preclinical results and human applications. The ability to monitor nerve regeneration in muscle through non‐invasive electrodiagnostics represents another major advantage.

AUTHOR CONTRIBUTIONS

Olivier Larrivée: Conceptualization; data curation; formal analysis; investigation; methodology; validation; writing – original draft; writing – review and editing. Alexane Thibodeau: Conceptualization; data curation; formal analysis; investigation; methodology; validation; writing – original draft; writing – review and editing. Rosemarie Rinfret‐Paquet: Data curation; formal analysis; investigation. Todd Galbraith: Data curation; formal analysis; investigation; methodology; writing – review and editing. Oumayma Hayouni: Data curation; formal analysis; investigation. Hélène T. Khuong: Conceptualization; funding acquisition; investigation; methodology; supervision; validation; visualization; writing – review and editing. François Berthod: Conceptualization; data curation; funding acquisition; methodology; project administration; resources; supervision; validation; visualization; writing – original draft; writing – review and editing.

FUNDING INFORMATION

This study was supported by the Canadian Institutes of Health Research (CIHR grant PJT‐175015), the Fonds de recherche du Québec (FRQ) through the research centre grant for the CHU de Québec‐Université Laval Research Center (reference: 30641), and the Quebec Cell, Tissue and Gene Therapy Network – ThéCell, a thematic network supported by the FRQ. Alexane Thibodeau received a scholarship award from the FRQ, the Fondation du CHU de Québec‐Université Laval and from NeuroQuébec.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interests.

ETHICS STATEMENT

All animal experiments were approved by the CHU de Quebec‐Laval University Animal Care Committee (#2020‐577) following the guidelines of the Canadian Council on Animal Care.

Supporting information

Figure S1.

Larrivée O, Thibodeau A, Rinfret‐Paquet R, et al. Noninvasive electrodiagnostic and motor function assessment of the common fibular nerve regeneration in the rabbit hindlimb. Anim Models Exp Med. 2025;8:2080‐2090. doi: 10.1002/ame2.70085

DATA AVAILABILITY STATEMENT

The data generated are available from the corresponding author upon reasonable request.