Establishment of Facial Nerve Injury Rat Model for Idiopathic Facial Paralysis Research

Summary

The present protocol establishes a facial nerve injury rat model using microscopy to investigate the diagnostic and therapeutic mechanisms of idiopathic facial paralysis.

Abstract

Idiopathic facial paralysis is the most common type of facial nerve injury, accounting for approximately 70% of peripheral facial paralysis cases. This disease can not only lead to a change in facial expression but also greatly impact the psychology of patients. In severe cases, it can affect the normal work and life of patients. Therefore, the research on facial nerve injury repair has important clinical significance. In order to study the mechanism of this disease, it is necessary to carry out relevant animal experiments, among which the most important task is to establish an animal model with the same pathogenesis as human disease. The compression of the facial nerve within the petrous bone, especially the nerve trunk at the junction of the distal end of the internal auditory canal and the labyrinthine segment, is the pathogenesis of idiopathic facial paralysis. In order to simulate this common disease, a compression injury model of the main extracranial segment of the facial nerve was established in this study. The neurological damage was evaluated by behavioral, neuroelectrophysiological, and histological examination. Finally, 50 g constant force and 90 s clamp injury were selected as the injury parameters to construct a stable idiopathic facial paralysis model.

Introduction

As a type of peripheral facial paralysis, idiopathic facial paralysis is characteristic of unknown etiology, acute onset, and self-limiting course^1,2. The etiology and pathogenesis of idiopathic facial paralysis are still uncertain³. At present, there are various treatment methods for facial paralysis⁴, and the diversity of treatments reflects the lack of optimal treatment options. Using cellular and molecular biology techniques to study the mechanism of facial nerve injury is the foundation for establishing effective treatment methods for facial paralysis. Therefore, a suitable and stable facial nerve injury model is particularly important.

At present, there is no standard method for establishing a facial nerve injury model. The current preparation methods include virus inoculation⁵, transection⁶, cold stimulation⁷, and compression⁸ methods. It is believed that viral infection, neurotrophoblastic vasospasm, autoimmune inflammation, etc., may all cause local ischemia, degeneration, and edema of the facial nerve^9,10,11. Moreover, all of the above factors can cause compression of the main trunk of the facial nerve in the narrow bony facial nerve canal^12,13. In addition, the most common peripheral nerve injuries identified during surgical procedures were compression and contusion¹⁴. Based on the above theories and clinical phenomena, we believe that preparing the facial nerve injury model through compression injury is more reasonable. However, most of the current methods for implementing compression injuries do not provide quantitative parameters of force and time. In this study, we quantified the force and duration of compression injury to improve the reproducibility of the established model.

Protocol

All the animal experiments were approved and supervised by the Animal Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (XHEC-F-2023-061). Sprague-Dawley male Rats, 200-300 g, were used for the present study. The animals were obtained from a commercial source (see Table of Materials). The rats were randomly divided into four groups (n = 10): Sham surgery group, 30-s injury group, 60-s injury group, and 90-s injury group.

1. Induction of anesthesia and animal preparation

1. Wear the following personal protective equipment (PPE): surgical mask, surgical gloves, disposable gown.
2. Weight the rats and anesthetize them with ketamine hydrochloride at a dose of 50 mg/kg by intraperitoneal (i.p.) injection. Administer meloxicam (5 mg/kg; i.p.) for perioperative analgesia. Confirm the depth of anesthesia using a toe pinch.
3. Apply ophthalmic ointment on both eyes to prevent drying.
4. After anesthesia, place the rats in the prone position and fix the head so that the left side of the face is up. Shave off the hair behind the left ear and disinfect the skin. Cover the rat with the sterile surgical drape.

2. Establishing a local crush injury model of the extracranial trunk of the facial nerve

NOTE: Sterilize all equipment before use. All operations were performed in the operating room.

1. Make a 2 cm long longitudinal incision behind the left ear, and dissect the skin and subcutaneous tissue to separate the natural gap between facial and cervical muscles.
2. Use micro tweezers and micro scissors to fully dissociate the facial nerve trunk between the stylomastoid foramen and the parotid gland, with an exposed length of approximately 1 cm.
3. Use peripheral nerve quantitative injury forceps (Table of Materials) to clamp the facial nerve trunk to cause injury. Locate the injury site 0.5 cm away from the stylomastoid foramen. Apply an injury intensity of 50 g and injury time of 30 s, 60 s, and 90 s, respectively.
4. Suture the subcutaneous and skin with silk thread. Disinfect the incision.
5. For rats in the sham-surgery control group, cut the skin and subcutaneous tissue after anesthesia and expose and separate the corresponding main trunk of the facial nerve. Next, sutured the incision immediately.
6. Monitor the animal's health, maintain sternal recumbency, and keep it in warm conditions.
7. Move the rat back to the housing cage after the rat is conscious.

3. Behavioral testing

NOTE: The facial nerve function of the rats was evaluated before surgery and 48 h after surgery (Figure 1). The scores of blink reflex, palp movement, and nasal tip position were calculated¹⁵. The higher the total score, the more severe the degree of facial nerve injury (Table 1).

1. Blink reflex (BR):
   1. Attach an 18 G needle to a 2 mL syringe and blow air into the rat's eye from a distance of 2 cm. Observe the eyelid movement and closure.
   2. Score per the following criteria: No significant difference on both sides: 0 points; Delayed closure of the affected side compared to the healthy side: 1 point; Inability to close the affected eyelid: 2 points.
2. Vibrissae movement (VM):
   1. Count the bilateral tentacle movements of rats within 30 s.
   2. Score per the following criteria: No significant difference in bilateral tentacle movement: 0 points; The movement of the affected side's whiskers is weaker than that of the healthy side: 1 point; Loss of whisker movement on the affected side: 2 points.
3. Nasal tip position.
   1. Center nose tip: 0 points; Nose tip leaning towards the healthy side: 1 point.
      ​NOTE: A total score of 0 points indicates normal, 1-2 points indicate mild facial paralysis (paresis), 3-4 points indicate obvious facial paralysis (paresis), and 5 points indicate complete facial paralysis¹⁵.

4. Neuroelectrophysiological detection

NOTE: Facial electrography (ENoG) was performed before the injury, immediately after surgery, and 48 h after surgery (Figure 2, Table 2, Table 3, and Table 4).

1. Place the grounding electrode under the skin of the left lower limb.
2. Insert the recording electrode (bipolar concentric needle electrode) into the injured side of the tentacle muscle, with a penetration depth of 5 mm.
3. Place the stimulation electrode (concentric needle electrode) on the facial nerve membrane. Stimulate the proximal and distal ends of the injured facial nerve separately.
4. Use a square wave pulse current with a frequency of 1 Hz, a wave width of 0.1 ms, and a filtering range of 10-3000 Hz.
5. Use stimuli of 2 mA, 5 mA, 10 mA, 15 mA, and 20 mA to induce the generation of compound muscle action potential.
6. Record the latency (Lm) and peak amplitude (Am) of the M-wave.
   NOTE: The M-wave refers to the first and most obvious waveform recorded. The point where the waveform leaves the baseline is the starting point of the waveform. The distance from the starting point of the baseline to the starting point of the waveform is Lm. The distance between the highest and lowest peaks of the waveform is Am.
7. Allow a 5-min interval between each stimulation to ensure nerve recovery.

5. Histological examination

1. After completing the electrophysiological tests, use micro tweezers to lift the nerve and micro scissors to cut the nerve specimen. The specimen includes the facial nerve trunk from the injury point to the parotid gland, which is the nerve fiber at the distal end of the injury point, with a total length of about 0.5 cm.
2. Fix the nerve specimen in 4% paraformaldehyde for 24 h and prepare paraffin-embedded sections¹⁶.
3. Stain the sections with the hematoxylin-eosin staining (H&E) method¹⁶ and acquire images at 100x and 400x magnification using an optical photographic microscope (Figure 3).
   NOTE: After removing the nerve specimen and suturing the skin under anesthesia, the rats were euthanized by pentobarbital sodium (150 mg/kg; i.p.).

Results

Behavioral testing
Before surgery, the scores of blink reflex, palp movement, and nasal tip position were 0 points in all experimental rats, indicating that all rats had intact facial nerve function. In the facial nerve function assessment 48 h after surgery, it was found that the individual scores of rats in each injury group were increased. Moreover, the total score increased gradually with the prolongation of facial nerve injury time (Table 1).

The fa...

Discussion

It is necessary to study the repair mechanism of facial nerve injury in patients with idiopathic facial paralysis¹⁷. The degree of facial nerve injury model should meet the following requirements. Firstly, the degree of facial nerve injury should not be too mild, such as Sunderland Grade 1^st degree¹⁸, which can completely self-repair without drug intervention. Secondly, it should not be too severe, such as Sunderland 5th degree, which requires surgical interventi...

Materials

Name	Company	Catalog Number	Comments
4% paraformaldehyde fixing solution	Beyotime Biotechnology	P0099
Clean bench	Airtech
Electronic balance	Shanghai Precision Instrument Factory	AS909
Freezing microtome	Leica	CM1900
Hematoxylin eosin (HE) staining kit	Beyotime Biotechnology	C0105S
Ketamine	Sigma	57074-21-2
Optical photographic microscope	Olympus	IX90
Pentobarbital sodium	ChemSrc	57-33-0
Quantitative peripheral nerve injury forceps	In-house		Patent number: CN20082015530.3
Sprague-Dawley rats	Jiangsu Jicui Yaokang Biotechnology Co., Ltd
Surgical operating microscope	OPMI 1FR proergo	ZEISS