Introduction
Objective electrophysiological assessment of facial nerve function plays an important role in clinical evaluation of facial nerve disorders, including nerve injury and regeneration [1]. In experimental research, particularly in rodent models, facial nerve function is most commonly assessed by recording compound muscle action potentials (CMAPs) from vibrissal muscles, which provide a quantitative surrogate of facial nerve integrity and neuromuscular transmission. In many previously reported rodent studies [2,3], CMAPs have been obtained using indirect stimulation of distal facial nerve branches or under closed-skin conditions, approaches that may be susceptible to variability in electrode positioning, stimulus spread, and signal stability. These factors can limit reproducibility and complicate interpretation of electrophysiological changes following nerve injury.
Methods employing direct stimulation of the facial nerve trunk under an open surgical field have been described and offer improved control over stimulation site and consistency of CMAP acquisition. However, such protocols have often been implemented using specialized electrophysiological systems that may not be readily available in many laboratories [4,5].
In this brief communication, we describe a practical CMAP recording method using a widely available BIOPAC electrophysiological system, adapted for direct stimulation of the exposed facial nerve trunk and recorded from vibrissal muscles in rats. This report focuses on the experimental setup and recording parameters required to obtain stable and reproducible CMAP waveforms in a facial nerve crush injury model.
Materials and Methods
Animals
Male Sprague–Dawley (SD; Narabio) rats were used for facial nerve CMAP recordings. A total of seven male SD rats (200–250 g) were included in the study and were kept in a 12-hour day/night animal facility supplemented with food and water ad libitum. All animals underwent baseline CMAP recordings from both facial nerves to establish normal reference values.
After baseline measurements, a unilateral facial nerve damage model was created on the left side, and follow-up CMAP recordings were performed at predefined postoperative time points to evaluate electrophysiological changes during nerve regeneration.
All experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Dankook University (approval number: IACUC No. DKU-21-060).
Anesthesia
All surgical and electrophysiological procedures were performed under general inhalational anesthesia using isoflurane (Fig. 1A). Anesthesia was induced in an acrylic chamber using an animal inhalation anesthesia system (JD-C107A, Jeungdo B&P). Isoflurane was delivered in oxygen at a flow rate of 0.8–1.0 L/min with the vaporizer set to 3%–5% until loss of the righting reflex was confirmed.
Following induction, animals were positioned on a stereotaxic apparatus (JD-SI-02, Jeungdo B&P) (Fig. 1B), and anesthesia was maintained via a nose mask connected to a Bain breathing circuit. During maintenance, the isoflurane concentration was adjusted to 2.0%–2.5% with an oxygen flow rate of 0.4–0.8 L/min. Ophthalmic ointment was applied to prevent corneal drying, and adequate depth of anesthesia was confirmed by the absence of withdrawal responses to paw pinch.
After completion of the procedure, isoflurane administration was discontinued while oxygen delivery was maintained. Animals were returned to their cages after spontaneous movement was observed and allowed to recover fully.
Facial nerve damage model
Following induction of anesthesia, a skin flap was elevated through a left preauricular incision to expose the facial nerve (Fig. 1C and D). Under an operating microscope, the main trunk of the left facial nerve was identified at its emergence from the stylomastoid foramen and carefully dissected free from surrounding connective tissue (Fig. 1E).
A standardized crush injury was induced by tightly clamping a 2-mm segment of the facial nerve trunk using a 12.5-cm mosquito forceps applied perpendicular to the nerve axis. The nerve was compressed for 2 minutes to produce a consistent crush injury (Fig. 1F). After completion of the crush injury, the nerve trunk was transected at the center of the crushed segment (Fig. 1G), and immediate end-to-end repair was performed using 10-0 nylon epineurial sutures under microscopic visualization (Fig. 1H). Nerve continuity and alignment were carefully restored.
The surgical field was irrigated with sterile saline, and the incision was closed in layers using 5-0 nylon sutures. Successful induction of facial nerve damage was confirmed postoperatively by ipsilateral vibrissal immobility. Animals were monitored daily for general condition and wound integrity.
CMAP recording
Facial nerve electrophysiological evaluation was performed by recording CMAPs from the vibrissal muscles using a BIOPAC data acquisition system (MP160, BIOPAC Systems Inc.) with AcqKnowledge software. A schematic overview of the CMAP recording setup is shown in Fig. 2, and detailed materials are listed in Table 1.
Stimulation
Electrical stimulation was delivered to the main trunk of the left facial nerve using a constant-current stimulator module (STM100C) coupled with an isolator (STMISOC, BIOPAC Systems Inc.).
For nerve stimulation, a commercially available unipolar needle electrode (12 mm, 28 G; EL452, BIOPAC Systems Inc.) was manually bent to form a hook-shaped electrode. Unmodified electrodes of the same type were used for electromyography (EMG) recording.
Following surgical exposure, the facial nerve trunk was gently lifted, and the hook-shaped stimulation electrode was applied directly to the nerve (Fig. 2A). Electrical stimulation was delivered as rectangular current pulses with the following parameters: pulse duration of 100 μs, stimulation delay of 100 ms, inter-stimulus interval of 1 s, and delivered in trains of five stimuli. Stimulation intensity was set at a supramaximal level of 2 mA to ensure consistent activation of the facial nerve.
Recording
CMAPs were recorded from the ipsilateral vibrissal muscles using a unipolar needle electrode (12 mm, 28 G) inserted into the mid-portion of the vibrissal muscle pad. A reference (ground) needle electrode was placed subcutaneously in the postauricular region.
All electrodes used in the study were disposable, uncoated stainless-steel needle electrodes.
Signal acquisition
EMG signals were amplified using an electromyogram amplifier (EMG100C, BIOPAC Systems Inc.) with a gain of 1,000×. Signals were band-pass filtered between 10 Hz and 5 kHz and digitized at a sampling rate of 50 kHz. A 50/60 Hz notch filter was applied as needed to minimize electrical noise. CMAP waveforms were displayed and analyzed using AcqKnowledge v5.0 software.
Data analysis
CMAP amplitude was quantified as the single-peak amplitude, defined as the voltage difference between baseline and the maximal positive or negative deflection of the evoked waveform, depending on polarity. For each recording session, five consecutive CMAP responses evoked by a supramaximal stimulus train were obtained, and the amplitudes from individual traces were averaged to reduce variability. All pertinent results were represented as a mean with standard deviation based on the number of animals tested at a particular time point.
Results
CMAP waveform characteristics and stimulation–response relationship
Direct electrical stimulation of the facial nerve trunk reliably evoked CMAPs from the ipsilateral vibrissal muscles in intact rats (Fig. 3). As stimulation intensity increased from 0.1 to 2.0 mA, CMAP amplitudes increased in a graded manner (Fig. 3A). Low-intensity stimulation (0.1–0.2 mA) produced small or barely detectable responses, whereas higher intensities (0.5–1.0 mA) elicited progressively larger CMAPs. Maximal and stable responses were consistently observed at 2.0 mA, indicating supramaximal stimulation.
To assess waveform reproducibility, a stimulus train consisting of five identical supramaximal stimuli (2.0 mA, 1-s inter-stimulus interval) was applied. Five consecutive CMAP waveforms demonstrated minimal variability in both amplitude and morphology (Fig. 3B). Averaging these five responses resulted in a representative CMAP waveform with an improved signal-to-noise ratio (Fig. 3C), which was used for visualization purposes.
Changes in CMAP amplitude following unilateral facial nerve injury
Baseline CMAP recordings obtained prior to nerve injury showed comparable amplitudes between the right and left sides, with a left/right ratio close to 100% (Table 2). One week after unilateral facial nerve crush injury followed by transection and epineurial repair, CMAP responses from the injured (left) side were absent in all animals examined, whereas responses from the contralateral (right) side remained detectable. Consequently, the left/right amplitude ratio was 0% at this time point.
At 2 weeks post-injury, small but detectable CMAP responses reappeared on the injured side, although amplitudes remained markedly reduced compared with the contralateral side. This resulted in a low left/right ratio, indicating limited functional recovery. By 4 weeks post-injury, CMAP amplitudes on the injured side increased further, accompanied by a corresponding increase in the left/right ratio, suggesting partial electrophysiological recovery of the facial nerve.
Overall, these results demonstrate that vibrissal CMAP recordings using direct facial nerve trunk stimulation provide a sensitive and reproducible method for tracking functional changes following facial nerve injury and repair.
Discussion
In the present study, we established a practical and reproducible protocol for vibrissal CMAP recording following direct facial nerve trunk stimulation in rats and evaluated longitudinal changes after unilateral facial nerve injury. The baseline vibrissal CMAP amplitude observed in intact rats (mean single-peak amplitude≈5.5 mV) was comparable to previously reported values. Takeuchi, et al. [4] reported a mean CMAP amplitude of 4.12±2.13 mV in intact rats using buccal branch stimulation and vibrissal muscle recordings, supporting the validity of our stimulation and recording setup.
CMAP amplitude is influenced by multiple experimental factors, including depth of anesthesia, body temperature, elec-trode impedance, and subtle variations in electrode placement [6,7]. In addition, the marked decrease in absolute CMAP amplitude observed on the intact (right) side at 1 week suggests that procedural factors inherent to longitudinal experimental designs may also contribute to electrophysiological variability. Repeated surgical re-exposure and direct manipulation of the facial nerve trunk, including repeated electrode contact, may induce transient physiological stress or minor mechanical and microvascular effects, even in the absence of intentional nerve injury. Importantly, this observation further supports the necessity of ratio-based normalization. In clinical practice, facial nerve function is commonly evaluated using side-to-side amplitude ratios rather than absolute values [1]. Consistent with this principle, we assessed functional recovery using the left/right CMAP amplitude ratio. In our study, absolute CMAP amplitudes of the intact facial nerve varied across baseline and follow-up time points despite the absence of nerve injury, whereas the left/right ratio showed a clear reduction after unilateral injury and gradual recovery over time, providing a more robust and biologically meaningful measure of asymmetric facial nerve dysfunction and recovery.
We also observed variability in CMAP waveform morphology, including biphasic and triphasic patterns. Similar waveform heterogeneity has been reported in experimental EMG and CMAP recordings and is thought to reflect differences in motor unit recruitment, spatial summation, and recording geometry rather than pathological conduction abnormalities alone [6]. In small animal models, minor changes in electrode position or muscle fiber orientation can substantially alter CMAP shape without indicating true changes in nerve integrity.
Given this waveform complexity, CMAP amplitude was quantified using single-peak amplitude from baseline to the maximal positive or negative deflection, rather than peak-topeak amplitude. In our experimental setup, the stimulation and recording electrodes were positioned in close proximity, and in some recordings, stimulus-related artifacts or exaggerated negative deflections were observed. Under these conditions, peak-to-peak measurements could be disproportionately influenced by stimulus artifacts, leading to inconsistent amplitude estimation. Single-peak amplitude provided a more stable and interpretable index of net muscle activation and was therefore considered more suitable for longitudinal comparisons.
Together, these findings support the use of direct facial nerve trunk stimulation combined with single-peak CMAP amplitude and side-to-side ratio analysis as robust outcome measures for evaluating facial nerve dysfunction and recovery in rodent models.
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