Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model

Summary

This study aims to develop a standard protocol of intra-operative neural monitoring of thyroid surgery in a porcine model. Here, we present a protocol to demonstrate general anesthesia, to compare different types of electrodes, and to investigate the electrophysiological characteristics of the normal and injured recurrent laryngeal nerves.

Abstract

Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere with breathing. In recent years, intraoperative neural monitoring (IONM) has been widely adapted as an adjunct technique to localize the RLN, detect RLN injury, and predict vocal cord function during the operations. Many studies have also used animal models to investigate new applications of IONM technology and to develop reliable strategies for preventing intraoperative RLN injury. The aim of this article is to introduce a standard protocol for using a porcine model in IONM research. The article demonstrates the procedures for inducing general anesthesia, performing tracheal intubation, and experimental design to investigate the electrophysiological characteristics of RLN injuries. Applications of this protocol can improve overall efficacy in implementing the 3R principle (replacement, reduction and refinement) in porcine IONM studies.

Introduction

Although thyroidectomy is now a commonly performed procedure worldwide, postoperative voice dysfunction is still common. Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere with breathing. Additionally, injury to the external branch of the superior laryngeal nerve can cause a major voice change by affecting pitch and vocal projection.

Intraoperative neural monitoring (IONM) during thyroid operations has obtained wide popularity as an adjunct technique for mapping and confirming the RLN, the vagus nerve (VN), and the external branch of the superior laryngeal nerve (EBSLN). Because IONM is useful for confirming and elucidating mechanisms of RLN injury and for detecting anatomic variations in the RLN, it can be used to predict vocal cord function after thyroidectomy. Therefore, IONM adds a new functional dynamic in thyroid surgery and empowers surgeons with information that cannot be obtained by direct visualization alone^{1,2,3,4,5,6,7,8,9,10}.

Recently, many prospective studies have used porcine models to optimize the use of IONM technology and to establish reliable strategies for preventing intraoperative RLN injury^{11,12,13,14,15,16,17,18,19,20}. Porcine models have also been used to provide practitioners with essential education and training in clinical applications of IONM.

Therefore, the combination of animal models and IONM technology is a valuable tool for studying the pathophysiology of RLN injury²¹. The aim of this article was to demonstrate the use of a porcine model in IONM research. Specifically, the article demonstrates how to induce general anesthesia, perform tracheal intubation, and set up experiments for investigating the electrophysiological characteristics of various RLN injury types.

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Protocol

The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University, Taiwan (protocol no: IACUC-102046, 104063, 105158).

1. Animal Preparation and Anesthesia

1. Porcine animal model
   Note: This study applied the protocol described in the literature to establish a prospective porcine model of IONM^{11,12,13,14,15,16,17,18,19,22}.
   1. Use KHAPS Black or Duroc-Landrace pigs (3-4 months old; weighing 18-30 kg).
   2. Ensure that the experimental protocol is consistent with national/international regulations and guidelines for animal experiments, including the 3R principles (replacement, reduction, and refinement). Obtain ethical approval of the experimental protocol from the committee for care and use of experimental animals at the relevant institution.
2. Anesthesia induction
   1. Pre-anesthesia preparations
      1. Withhold food 8 hours before anesthesia and withhold water 2 hours before anesthesia.
      2. Pre-medicate with intramuscular azaperone (4 mg/kg) at 2 hours before anesthesia. Use a 500 mL saline bottle to fabricate a face mask for each piglet. Trim as needed to ensure a secure fit to the snout.
      3. Use the weighing function on the operating table to measure the net weight of each piglet (Figure 1A).
      4. Maintain body temperature with a circulating water mattress set to 40 °C.
   2. Induce general anesthesia (GA) with 2-4% sevoflurane at a fresh gas flow of 3 L/min via the face mask with the piglet in a prone position. GA can also be induced by intramuscular tiletamine and zloazepam. An adequate depth of anesthesia is usually achieved in 3-5 minutes. Confirm the depth of anesthesia by no severe movement to pain due to peripheral venous catheterization.
   3. Identify a superficial vein on the outer side of one ear and sterilize the selected region (about 6 x 6 cm²) with 75% alcohol. For maximum safety, use a 24-gauge peripheral intravenous catheter.
   4. Administer intravenous anesthetic such as propofol (1-2 mg/kg) or thiamylal (5-10 mg/kg) to alleviate noxious stimulation by direct laryngoscopy.
      Note: Use of neuromuscular blocking agent (NMBA) is not suggested. In subsequent experiments, NMBA may complicate intubation by depressing spontaneous breathing and may diminish electromyography (EMG) signals. Additionally, sevoflurane inhalation combined with a bolus of propofol or short-acting barbiturates is reportedly sufficient for facilitating tracheal intubation.
3. Tracheal intubation (Figure 1B)
   1. Prepare the equipment and materials required for EMG tube intubation: a size #6 EMG endotracheal tube, a face mask for assisted ventilation, two slings to hold the mouth open, one gauze strip to pull the tongue, a blunt tip suction catheter, a veterinary laryngoscope with 20cm straight blades, an elastic bougie, a 20-mL syringe, a stethoscope, and adhesive tape.
   2. Position the piglet in a prone position on the operating table. Align the head and body to ensure clear visualization of the upper airway.
   3. Direct the assistant to apply traction of the upper and lower jaw to maintain an adequate mouth opening and to avoid rotation or overextension of the head. Cover the tongue with gauze and pull the tongue out to optimize the visual field.
   4. Hold the laryngoscope and place it directly in the oral cavity to depress the tongue.
   5. Directly visualize the epiglottis and use the laryngoscope to press the epiglottis downward toward the tongue base.
   6. When the vocal cords are clearly identified, gently advance the elastic bougie into the trachea. Slight rotation of the elastic bougie may be required to overcome resistance. Next, advance the EMG tube at the mouth angle to a depth of 24 cm.
   7. Inflate the EMG tube cuff to a volume no larger than 3 mL. If ventilation by manual bagging reveals no obvious air leakage, in situ deflation of the EMG tube is feasible.
   8. When the EMG tube is placed at the proper depth, confirm the free passage of fresh gas by manual bagging. Further confirm the proper tracheal intubation by end-tidal carbon dioxide (etCO₂) monitoring (capnography) and chest auscultation for early identification of inadvertent esophageal or endobronchial intubation.
      Note: Capnography showed both the etCO₂ waveform and the digital value in mmHg. When esophageal intubation occurred, etCO₂ was absent or near zero after 6 breaths. When the EMG tube was in the correct place, the typical etCO₂ waveform and adequate value (usually >30 mmHg) was noted. Furthermore, the breathing sound of a bilateral lung filled is clear and symmetric as determined by chest auscultation.
   9. Use medical tape to fix the EMG tube at the mouth angle. Since the tube usually requires adjustment during IONM experiments, do not fasten the tube to the snout.
   10. Connect the EMG tube to the ventilator. Continuous capnography is mandatory for monitoring the etCO₂ value and curve throughout the experiment.
4. Anesthesia maintenance (Figure 1C)
   1. After the EMG tube is fixed, position the piglet on its back with the neck extended (Figure 1C). Maintain general anesthesia with 1-3% sevoflurane in oxygen at 2 L/min.
   2. Ventilate the lungs in volume-control mode at a tidal volume of 8-12 mL/kg, and set the respiratory rate to 12-14 breaths/min.
   3. Begin physiologic monitoring, including capnography, electrocardiography (ECG)and monitoring of oxygenation (SaO₂).

2. Equipment Setting and Animal Operation (Figure 1D)

1. Equipment Setup
   1. Connect the channel leads from the EMG tube to the monitoring system.
   2. Set the monitoring system to run a 50 ms time window. Set pulsed stimuli to 100 μs and 4 Hz. Set the event capture threshold to 100 μV.
2. Surgical procedure
   1. Wear sterile surgical gloves and use povidone iodine with cotton swabs to disinfect the neck surgical site.
   2. Make a transverse collar incision about 10-15 cm in length with a scalpel to expose the neck and the larynx.
   3. Raise the subplatysmal flap 1 cm cranially from the clavicle to the hyoid bone.
   4. Remove the strap muscles and visualize the tracheal rings and nerves. Use monopolar and bipolar electrocautery to assist the surgical dissection and hemostasis.
   5. Localize, identify, and carefully expose the EBSLN, RLN, and VN with a handheld stimulation probe.
   6. Position an automated periodic stimulation (APS) electrode on one side of VN for stimulating during continuous IONM (CIONM). Connect the APS electrode with the monitoring system. Set pulsed stimuli to 1 Hz, 100 µs, and 1 mA.
3. At end of experiments, euthanize all piglets by the veterinarian.

3. Electrical Stimulation

Note: To apply the 3R principle in porcine IONM studies, always perform repeatable electrophysiology studies that do not cause nerve injury before performing experiments that may cause nerve injury. This can be used to study the intensity, safety, and cardiopulmonary effects^11,17. The IONM equipment can be classified as stimulation equipment or recording equipment (Figure 2A).

1. Evaluate the baseline EMG responses of the target nerves, including the EBSLN, RLN, and VN (Figures 2B, 2C).
   1. Start with an initial stimulation current of 0.1-mA current and increase stimulation in 0.1-mA increments until an EMG response is detected and recorded.
   2. Further increase the current until the maximal EMG response is obtained.
   3. Record the baseline amplitude, latency, and waveform of the EMG response.
   4. Define the minimal stimulus level as the lowest current (mA) that clearly evoked EMG activity of >100 µV. Define the maximal stimulus level as the lowest current that evoked the maximal EMG response.
2. Evaluate the Safety of electrical stimulation^11,19
   1. Apply a continuous 1-minute stimulus at the fifth tracheal ring level of the VN or RLN.
   2. Progressively increase the stimulus current from 1 mA to 30 mA.
   3. During VN stimulation, evaluate hemodynamic stability by monitoring of heart rate, ECG, and invasive arterial blood pressure.
   4. Finally, evaluate nerve function integrity by comparing EMG responses proximal to the nerve stimulation site before and after each level of stimulation is applied.
3. Effect of anesthetics (muscle relaxants and their reversals)^12,20
   Note: Improper use of NMBAs is a potential cause of unsuccessful IONM. The proposed animal model was used to compare recovery profiles among different depolarizing NMBAs (e.g., succinylcholine) and nondepolarizing NMBAs (e.g., rocuronium) at varying doses and to identify the optimal NMBA for use in IONM. The animal model can also be used to evaluate the effectiveness of NMBA reversal drugs (e.g., sugammadex) for rapidly restoring neuromuscular function suppressed by rocuronium.
   1. Firstly, apply C-IONM and use the automatically calibrated baseline latencies and amplitudes of EMG as control data.
   2. Administer a bolus injection of 0.3 mg/kg rocuronium in a volume of 10 mg/mL and observe the real-time EMG changes.
   3. Three minutes after injection, perform one injection of 2 mg/kg sugammadex in a volume of 100 mg/mL as a rapid bolus. Record the recovery profile of laryngeal EMG for 20 minutes.
4. Stimulation electrodes (Stimulation probes/dissectors) (Figure 3)¹⁷
   Note: There are different types of stimulation electrodes that can be used for nerve stimulation during IONM, e.g., monopolar probes (Figure 3A), bipolar probes (Figure 3B), and stimulation dissectors (Figure 3C).
   1. To mimic direct stimulation of nerves during surgery, apply 1 mA stimulation to the EBSLN, RLN, and VN without overlying fascia.
   2. To mimic indirect mapping and localizing of the nerve position before visual identification during surgery, apply 1 mA stimulation at a 1- and 2-mm distance away from the nerves at overlying fascia.
   3. Record and compare the EMG responses between different types of stimulation electrodes.
5. Recording electrodes (EMG tubes/needle electrodes/pre-gelled skin electrodes) (Figure 4)
   1. Use the animal model to evaluate how rotation or upward/downward displacement of the EMG tube electrode (Figure 4A) affects the stability of the EMG signal. Additionally, use the animal model to compare the EMG responses between different electrode types (e.g., needle electrodes and adhesive pre-gelled electrodes, Figure 4B) and different recording approaches (e.g., transcutaneous/percutaneous and transcartilage approaches, Figures 4C and 4D) in terms of feasibility, stability, and accuracy during IONM.
   2. For a feasibility study, apply a 1 mA stimulus current to bilateral EBSLNs, VNs and RLNs. Record and compare EMG responses evoked by each electrode tested (i.e., EMG tube, transcutaneous, percutaneous, and transcartilage electrodes).
   3. For a stability study, evaluate and compare EMG signal stability in C-IONM under experimentally induced cricoid/tracheal cartilage displacement.
   4. For an accuracy study, evaluate and compare the accuracy of the tested electrodes in C-IONM for identifying EMG signal degradation under RLN injury.

4. RLN injury study (Figure 5)

1. In accordance with the 3R principle, perform RLN injury experiments in the porcine model after all repeatable electrophysiology studies are completed. Perform tests of nerve segments from proximal nerve segments to distal nerve segments (i.e., proceed from the caudal part of the RLN to the cranial part of the RLN).
2. Use C-IONM to confirm and compare patterns of real-time changes in evoked laryngeal EMG signals during and after acute RLN injuries with different injury mechanisms (e.g., traction, clamping, transection, or thermal injuries) (Figures 5A and 5B). Use C-IONM for continuous real-time display and recordation of EMG changes and sequential recoveries throughout the experiment (Figure 5C).
3. Collect injured RLN segments for histopathological analysis of morphological alterations caused by the nerve injury experiments.
4. Traction compression/stretch injury
   Note: Traction compression or stretch injuries are the most common intraoperative RLN injuries. Experimentally induce traction stress and observe the resulting electrophysiological EMG changes and histopathological changes.
   1. Traction compression injury¹³
      1. Wrap a thin plastic loop (e.g., a vascular loop 1.3-mm wide) around the RLN and use a force gauge to apply retraction with 50 g of tension (Figure 5A). This scheme mimics an RLN trapped against a dense, fibrous band or a crossing artery at the region of Berry’s ligament during medial traction of the thyroid lobe.
   2. Traction stretch injury¹⁶
      1. Wrap the RLN with a wider elastic material (e.g., a 10-mm wide silicone Penrose drain), and use a force gauge to retract the RLN with 50 g of tension) This scheme mimics an RLN adhered to or encased in the goiter capsule and stretched forward during medial traction.
5. Clamping injury
   Note: Intraoperative mechanical trauma to the RLN usually results from poor exposure or visual misidentification of the RLN. ^13,16
   1. After the traction compression RLN injury experiment, pinch the distal segment of the RLN with hemostatic forceps for one second. This scheme mimics the nerve being inadvertently clamped owing to visual misidentification as a vessel during the operation. Record the accompanying EMG signal change for comparison with further histopathological findings of the nerve specimen.
6. Thermal injury
   Note: Most intraoperative RLN thermal injuries result from thermal spread when electrocautery devices and various energy-based devices (EBDs) are used to induce hemostasis near the RLN. Like traction injury, thermal injury is rarely visible to the naked eye. Therefore, perform animal IONM experiments to determine the best model for evaluating the pathophysiology of RLN thermal injury and to test the thermal tolerance¹⁴ and the safety of EBDs^15,18.
   1. Use C-IONM to register the EMG changes continuously throughout the experiment.
   2. For the activation study, investigate how energy-based devices (EBD) can be safely applied for hemostasis and dissection near the RLN during surgery (Figure 5B).
      1. Activate the EBD (electrothermal bipolar vessel sealing system, set power at level 2, and the energy discontinues automatically by 2 to 4 seconds) at a 5-mm distance away from the RLN.
      2. If EMG signals remain stable after several tests, perform a further test at the narrower distance (e.g., 2-mm, and followed by 1mm distance).
      3. If any substantial EMG change occurs after any test the experiment is complete and followed by continuous real-time EMG recording for at least 20 minutes.
   3. For the cooling study, evaluate the cooling time to determine post-activation optimal EBD cooling parameters.
      1. Contact the activated EBD on the RLN directly after a 5 second cooling time.
      2. If the EMG signals remain stable after three tests, test the shorter cooling time (e.g., 2 seconds, and followed by 1 second).
      3. If the EMG remains stable after repeated tests, confirm the safety of the EBD by touching the RLN immediately after activation.

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Results

Electrophysiology study
Baseline EMG data, minimal/maximal stimulus level, and the stimulus-response curves
Using a standard monopolar stimulating probe, the obtained minimal stimulation level for VN and RLN stimulation ranges from 0.1 to 0.3 mA, respectively. In general, the stimulus current correlated positively with the resulting EMG amplituderesponse^11,17. The EMG amplitude plateaued at the maximal stimulation...

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Discussion

Injury to the RLN and EBSLN remains a significant source of morbidity caused by thyroid surgery. Until recently, nerve injury could only be identified by direct visualization of trauma. The use of IONM now enables further functional identification of the RLN by applying stimulation and recording the contraction of the target muscles. Currently, however, both conventional intermittent and continuous IONM systems have some technical limitations in false-positive and false-negative interpretations. Hence, suitable animal mo...

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Materials

Name	Company	Catalog Number	Comments
Criticare systems	nGenuity	8100E	physiologic monitoring, including capnography, electrocardiography (ECG) and monitoring of oxygenation (SaO2)
Intraoperative NIM nerve monitoring systems	Medtronic	NIM-Response 3.0	monitor EMG activity from multiple muscles. If there is a change in nerve function, the NIM system may provide audible and visual warnings to help reduce the risk of nerve damage.
NIM TriVantage EMG Tube	Medtronic	8229706	6 mm ID, 8.2 mm OD. The NIM TriVantage EMG Tube is a standard size, non-reinforced, DEHP-free PVC tube that features smooth, conductive silver ink electrodes and a cross-band to guide placement. It has reduced sensitivity to rotation and movement while offering increased EMG responses that facilitate improved nerve dissection.
NIM Contact Reinforced EMG Endotracheal Tube	Medtronic	8229506	6 mm ID, 9 mm OD. The NIM Contact EMG Tube continuously monitors electromyography (EMG) activity during surgery. An innovative design allows the tube to maintain contact, even upon rotation. Vocal cords are more easily visible against the white band. Recording electrode leads are twisted pair. Packaged sterile with one green and one white subdermal needle. Single use.
NIM Standard Reinforced EMG Endotracheal Tube	Medtronic	8229306	6 mm ID, 8.8 mm OD. The NIM Standard EMG Tube continuously monitors electromyography (EMG) activity during surgery. Recording electrode leads are twisted pair. Packaged sterile with one green and one white subdermal needle. Single use.
NIM Flex EMG Endotracheal Tube	Medtronic	8229960	6 mm. The NIM Flex EMG Tube monitors vocal cord and recurrent laryngeal nerve EMG activity during surgery. An updated, dual-channel design allows the tube to maintain contact with the vocal cords, even upon rotation. Recording electrode leads are twisted pair. Packaged sterile with one green and one white subdermal needle. Single use.
Standard Prass Flush-Tip Monopolar Stimulator Probe	Medtronic	8225101	Tips and Handles. For locating and mapping cranial nerves in the surgical field, the single-use Standard Prass Monopolar Stimulating Probe features a flush 0.5 mm tip diameter. The probe is insulated to the tip to prevent current shunting. Individually sterile packaged.
Ball-Tip Monopolar Stimulator Probe	Medtronic	8225275/ 8225276	Tip and Handle, 1.0 mm/ 2.3mm. Featuring a flexible ball tip and flexible shaft, the single-use Ball-Tip Monopolar Stimulating Probe allows greater access to neural structures. The 1.0 mm tip diameter allows atraumatic contact to larger neural structures. The probe is insulated to the tip to prevent current shunting. Individually sterile packaged.
Yingling Flex Tip Monopolar Stimulator Probe	Medtronic	8225251	Tips and Handles. The highly flexible single-use Yingling Monopolar Stimulating Probe allows stimulation in areas outside the surgeon’s field of view. The platinum-iridium wire of the probe is fully insulated to the ball tip to prevent current shunting. Individually sterile packaged with one green subdermal electrode.
Prass Bipolar Stimulator Probe	Medtronic	8225451	The single-use Prass Bipolar Stimulating Probe features a slim, flexible tip that allows greater access to neural structures. The probe tip is 0.5 mm in distance between cathode and anode for minimal shunting. Individually sterile packaged.
Concentric Bipolar Stimulator Probe	Medtronic	8225351	The single-use Concentric Bipolar Stimulating Probe features a 360° contact area. Insulation is complete to the active tip; cables and handles are polarized. Individually sterile packaged.
Side-by-Side Bipolar Stimulator Probe	Medtronic	8225401	The single-use Side-by-Side Bipolar Stimulating Probe features probe tips that are 1.3 mm apart, allowing neural structures to be stimulated between the tips. Insulation is complete to the active tip; cables and handles are polarized. Individually sterile packaged.
APS (Automatic Periodic Stimulation) Electrode*	Medtronic	8228052 / 8228053	2 mm/ 3mm. The APS Electrode offers continuous, real-time monitoring. The electrode is placed on the nerve and can provide early warning of a change in nerve function.
Neotrode ECG Electrodes	ConMed	1741C-003	The electrode is made of a clear tape material, which allows for continuous observation of the patient's skin during monitoring.
LigaSure Small Jaw	Medtronic	LF1212	A FDA-approved electrothermal bipolar vessel sealing system for surgery