The Miniature Pig: A Large Animal Model for Cochlear Implant Research

Summary

Miniature pigs (mini-pigs) are an ideal large animal model for research into cochlear implants. Cochlear implantation surgery in mini-pigs can be utilized to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches in a living system similar to human beings.

Abstract

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, no standard model exists for investigating the electrophysiology and histopathology in patients or animal models with a CI or for evaluating new models of electrode arrays. A large animal model with cochlea characteristics similar to those of humans may provide a research and evaluation platform for advanced and modified arrays before their use in humans.

To this end, we established standard CI methods with Bama mini-pigs, whose inner ear anatomy is highly similar to that of humans. Arrays designed for human use were implanted into the mini pig cochlea through a round window membrane, and a surgical approach followed that was similar to that used for human CI recipients. Array insertion was followed by evoked compound action potential (ECAP) measurements to evaluate the function of the auditory nerve. This study describes the preparation of the animal, surgical steps, array insertion, and intraoperative electrophysiological measurements.

The results indicated that the same CI used for humans could be easily implanted in mini-pigs via a standardized surgical approach and yielded similar electrophysiological outcomes as measured in human CI recipients. Mini-pigs could be a valuable animal model to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches before applying them to human beings.

Introduction

According to the World Health Organization (WHO), over 1 billion people are at risk of hearing loss globally, and it is estimated that, by 2050, one out of four people will suffer hearing loss¹. Over the last 2 decades, CIs have been the most effective intervention for people with permanent severe and profound sensorineural hearing loss (SNHL). A CI converts physical signals of sound into bioelectrical signals that stimulate the spiral ganglion neurons (SGNs), bypassing hair cells. Over time, the indications for a CI have been broadened so that they now include people with residual hearing, unilateral hearing loss, and very old or young people^2,3,4. Meanwhile, totally implantable CIs and advanced arrays have been developed⁵. There is, however, no economically feasible large animal model for investigating the electrophysiology and histopathology of the inner ear with a CI. This lack of a large animal model limits research seeking to improve CIs and gain insights into the electrophysiological impact of CIs on the inner ear.

Several rodent animal models have been applied in CI research, such as mouse⁶, gerbil⁷, rat⁸, and guinea pig⁹; however, the characteristics of morphology and electrophysiological responses are different from that in humans. Cochlear structures of animal models traditionally used for CI studies, such as cats, guinea pigs, and other animals, differ greatly from those of human cochlear structures¹⁰. Although array insertion has been conducted on cats¹¹ and rabbits¹², because of their smaller cochleae, this was done with arrays that were not designed for use in humans. Several large animal models have also been explored for CI. Lambs are well suited as a training model for atraumatic cochlear implantation, but the smaller size of the cochlea makes full array insertion impossible¹³. Primates might be the most suitable animals for CI research because of their anatomical similarity to humans^14,15; however, the sexual maturity of monkeys is delayed (4-5 years), the gestation period is up to about 165 days, and each female usually produces only one offspring per year¹⁶. These reasons, and the expensive cost, hinder the extensive application of primates in CI research.

In contrast, pigs reach sexual maturity at 5-8 months and have a gestation period of ~114 days, making pigs more accessible for CI research as a large animal model¹⁶. Bama mini pigs (mini-pigs) originated from a small-sized pig species in China in 1985, whose genetic background is well understood. They are characterized by an inherent small size, early sexual maturity, rapid breeding, and ease of management¹⁷. The mini-pig is an ideal model for otology and audiology because of its similarity to humans in morphology and electrophysiology¹⁸. The scala tympani length of a Bama mini-pig is 38.58 mm, which is close to the 36 mm length in humans¹⁰. The mini-pig cochlea has 3.5 turns, which is similar to the 2.5-3 turns seen in humans¹⁰. In addition to morphology, the electrophysiology of Bama mini-pigs is also highly similar to that of humans¹⁸. Therefore, in the present study, we inserted arrays designed for human use into the mini-pig cochlea via the round window membrane and followed a similar surgical approach to that used in human CI recipients. Intraoperative ECAP measurements were applied to evaluate the procedure. The process we describe herein could be used both for preclinical translational research associated with CIs and as a platform for resident training.

Protocol

All procedures and animal surgeries were conducted according to the guidelines of the Ethics Committee of the PLA General Hospital and were approved.

1. Anesthesia and surgical preparation

1. Inject the pig (male, 2 months old, 5 kg) muscularly with tiletamine and zolazepam with a dosage of 10-15 mg/kg and intubate it with a 5.5-French endotracheal tube. Maintain anesthesia by ventilator-assisted respiration with isofluorane inhalation. Monitor the oxygen saturation (>90%), breathing (15-20/min), and heart rate (60-120 beats/min) using the pulse oximetry clamp of an ECG monitor, which is connected to the pig's tongue.
2. Put the mini-pig in a left lateral position (when the right side is to be implanted) on a thermostatically regulated heating pad to prevent hypothermia. Confirm the pig is adequately anesthetized using various stimuli. Ensure the absence of all responses (e.g., toe pinch reflex). Apply artificial tear ointment to the eyes of the minipig to keep the cornea from drying. Keep the eyes closed using a medical patch.
3. Shave the surgical area around the earlobe, keeping it 10 cm in diameter (Figure 1) and disinfect it with three alternating swabs of iodine and alcohol in a circular motion from the center toward the outside. Cover the surgical area with sterile surgical drapes.
4. Cover the microscope with a sterile plastic sleeve and remove the parts covering the eyepieces and objective.

2. Surgical procedure

1. Locate the surface projection site of the cochlea 1 cm behind the posterior auricular sulcus at the level of the earlobe. Make a postauricular incision about 5 cm long with the projection site as the center using a #15 scalpel. Divide the subcutaneous tissue, parotid gland, and sternocleidomastoid muscle with micro-scissors to expose the surface of the mastoid bone (Figure 2A). Use bipolar cautery when necessary to minimize bleeding.
2. Cortical mastoidectomy
   1. Drill the mastoid at the surface projection of the cochlea on the mastoid bone (Figure 2B) to the external auditory canal (EAC), which is dense and pale blue (Figure 2C). Be careful not to damage the pale or reddish vertical segment of the facial nerve dorsal to the EAC to avoid bleeding (Figure 2C).
      NOTE: If the facial nerve is damaged, bipolar cautery is a good choice to stop bleeding.
3. Expose the tympanum by drilling the bone surrounding the posterior bony EAC (Figure 2D). Separate the skin of the EAC and the facial nerve with a hypodermic needle to avoid damaging the facial nerve. Carefully push the skin of the EAC away to expose the tympanum (including the ossicular chain) and round window niche (Figure 3A).
4. Expose the round window membrane. Remove the round window niche with a small diamond burr, and keep continuous suction-irrigation to expose the basal turn of the cochlea and round window membrane (Figure 3B).
5. Fix the receiver package. Separate the cranial parietal muscle to form a pocket just large enough for the receiver. Place the internal receiver package in the muscular pocket and fix it with a fixation suture.
6. Insert the electrode array, which is connected to a receiver fixed in a muscular pocket, by opening the round window membrane with a sharp microsurgical knife and inserting the array using micro forceps slowly, steadily, and continuously in relationship to the modiolus of the cochlea (Figure 3C). Suture the surgical incision with an absorbable suture 2-0.
7. ECAP measurements
   NOTE: The setup is composed of a PC with MAESTRO Software connected to the patient's cochlear implant (CI) through the stimulation device (MAX Programming Interface) and the CI coil.
   1. Magnetically connect the CI coil to the CI receiver through the skin. Confirm the integrity of the CI and check the electrode impedance for all channels before ECAP measurements using the telemetry function of the CI, which is conducted by MAESTRO Software automatically (Figure 4A,B).
   2. Conduct the ECAP measurements as described previously¹⁹. Select the ECAP module, choose all 12 electrodes for stimulation, and wait for the ECAP tests of the electrodes to be completed. See the Table of Materials for the software and stimulation device used to measure ECAP responses. Stimulate all 12 electrodes for ECAP measurements using biphasic stimuli of 30 µs phase duration, with an alternating-polarity paradigm, averaging over 25 iterations, and a stimulation rate of 45.1 pulses/s.

3. Postoperative care

1. Keep monitoring the mini-pig to avoid harm due to unconscious movement until it has regained sufficient consciousness to maintain sternal recumbency. Put the minipig on the thermostatically regulated heating pad to prevent hypothermia.
2. Place the mini-pig back into its home cage alone.
3. Inject antibiotics to prevent postoperative infection for 7 days.
4. Check the mini-pig for symptoms of vestibular injury such as nystagmus, circling, or rolling over.

4. Postoperative CT scan

1.  Administer intramuscular injection of 3% sodium pentobarbital 1 ml/kg, and 0.1 ml/kg of Sulforaphane to minipig to induce anesthesia. Use a 37℃ warming plate to keep it warm. After 3 or 5 minutes, a CT scan can be conducted. 
2. To confirm the correct position of the electrode array, narcotize the mini-pig with tiletamine and zolazepam 1 week after the operation. Perform the CT scan and 3D reconstruction²⁰ using the 3D slicer image computing platform (see the Table of Materials). Import DICOM data of the CT and conduct the Volume rendering module to achieve 3D images of the CI.

Results

The integrity (Figure 4A) and impedances (Figure 4B) of the CI were confirmed by MAESTRO Software. ECAP results showed that all 12 electrodes demonstrated good neural responses (Figure 4C), meaning the electrode array was well attached to the cochlear axis and stimulated the auditory nerve. Figure 5 demonstrates postoperative 3D reconstructed electrode coils in the right cochlea. The array did not ...

Discussion

Around 15% of the world's population have some degree of hearing loss, and over 5% have disabling hearing loss²¹. CI provision is the most efficient treatment for both adult and pediatric patients with severe and profound sensorineural hearing loss. As the first successful implantable cranial nerve stimulator, over the past 2 decades, CIs have offered thousands of people with hearing loss the opportunity to return to the world of sound and (re)integrate into mainstream society. Even though CIs are...

Materials

Name	Company	Catalog Number	Comments
0.5 mm diamond burr
1 mm diamond burr
5 mm diamond burr
2-0 suture silk
3D Slicer image computing platform			3D reconstruction of CT image
Alcohol
Bipolar cautery
Bipolar electrocoagulation			Stop bleeding
CI designed for human use (CONCERTO FLEX28)	MED-EL	Concerto F28
Dressing forceps
ECG monitor
Iodine tincture
Isoflurane			3.6 mL/h
Laryngoscope
MAESTRO Software	MED-EL		Measure ECAP responses
Micro forceps
Micro spatula
Mosquito forceps
Needle holder
Needle probe
Negative pressure suction device
Otological surgical instruments
Respiratory Anesthesia Machine
Scalpel with blade No. 15
Scissors
Shaver
Stimulation device (MAX Programming Interface)	MED-EL		Measure ECAP responses
Surgery microscope	Leica
Surgical drill
Surgical Power Device
Tiletamine and zolazepan			10-15 mg/kg
Tissue forceps
Trachea cannula