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.
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.
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 alone1,2,3,4,5,6,7,8,9,10.
Recently, many prospective studies have used porcine models to optimize the use of IONM technology and to establish reliable strategies for preventing intraoperative RLN injury11,12,13,14,15,16,17,18,19,20. 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.
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
2. Equipment Setting and Animal Operation (Figure 1D)
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 effects11,17. The IONM equipment can be classified as stimulation equipment or recording equipment (Figure 2A).
4. RLN injury study (Figure 5)
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 amplituderesponse11,17. The EMG amplitude plateaued at the maximal stimulation levels of 0.7 mA for VN stimulation, and 0.5 mA for RLN stimulation11.
Electrical stimulation (intensity, safety, and cardiopulmonary effect)
In the safety study, there is no unwanted effects on EMG signal or hemodynamic stability observed after continuous pulsatile VN and RLN stimulations in the setting of 1 mA to 30 mA. In addition, baseline EMG amplitudes and latencies of the VN or RLN were relatively unchanged after the nerves was stimulated by a high-current. Therefore, it was suggested that an intermittent high stimulus current during IONM was not harmful to the VN or RLN19.
Effects of anesthetics (muscle relaxants and their reversals)
Experimental comparisons of NMBAs of this animal model showed that different types and doses of muscle relaxants have different natural recovery profile. For example, recovery times for succinylcholine (1 mg/kg) and low-dose rocuronium (0.3 mg/kg) were significantly shorter than that for standard dose rocuronium (0.6 mg/kg). The experiments for NMBA reversals confirm that sugammadex (reversal of rocuronium) effectively and rapidly restores neuromuscular function suppressed by rocuronium20.
Stimulating electrodes (stimulation probes and dissecting stimulators)
Typically, IONM is performed with a commercially available ETT-based surface recording electrode system (i.e., a so-called EMG tube). However, a limitation of the clinical use of EMG tubes is the need to maintain constant contact between the electrodes and vocal cords during surgery to obtain a robust EMG signal. False IONM results can result from an EMG tube that is mispositioned during intubation (e.g., due to incorrect insertion depth, incorrect tube size, or rotation of the electrode) or from an EMG tube that is displaced during surgical manipulation or neck retraction (e.g., causing rotation or upward/downward displacement of the electrode).
Experimental comparisons of stimulating electrodes showed that the stimulation probes/dissectors evoked typical EMG waveforms from the EBSLN/RLN/VN with 1 mA current. The stimulating current correlated positively with the resultant EMG amplitude. In monopolar probes and stimulating dissectors, maximum EMG was elicited by <1 mA. In bipolar probes, maximum EMG required a higher current. In all groups, evoked EMG amplitudes decreased as the distance from the probe/dissector to the nerve increased. Evoked EMG amplitudes also decreased in stimulated nerves that had overlying fascia. Therefore, the animal model confirmed that both stimulation dissectors and conventional probes are effective to evoke EBSLN, RLN, and VN waveforms to monitor real-time nerve function during surgery17. Various stimulation probes/dissectors are now available in IONM system for specific stimulation requirements, surgical monitoring application and the preference of the users.
Recording electrodes (EMG tubes, needle electrodes, and pre-gelled skin electrodes)
The feasibility study confirmed that the EMG tube electrodes on the vocalis, the transcutaneous/percutaneous needle electrodes, and the transcutaneous/transcartilage pre-gelled electrodes were effective for recording typical evoked laryngeal EMG waveforms from the VN and RLN under 1 mA stimulation. Figure 6 shows that transcutaneous/transcartilage pre-gelled electrodes generally recorded lower EMG amplitudes compared to EMG tube and needle electrodes.
In the stability study, real-time EMG tracings were compared before and after tracheal displacement was experimentally induced. Figure 7 shows that the change in contact between EMG tube electrodes and vocal folds after tracheal displacement significantly changed the recorded EMG signals. However, tracheal displacement had no apparent effect on electrode contact quality or on EMG signal quality from the transcutaneous or transcartilage electrodes.
The accuracy study evaluated the accuracy of real-time signals in reflecting adverse EMG degradation during RLN stress experimentally induced by continuous VN stimulation with the APS electrode. When RLN traction stress was experimentally induced, the EMG tube electrodes on the vocalis muscle and the transcartilage/percutaneous/transcutaneous electrodes recorded similar patterns of progressive degradation in EMG amplitude (Figure 8).
RLN injury study
Traction injury
Typical real-time EMG changes during RLN traction revealed a progressive amplitude decrease combined with a latency increase (the so-called "combined event"). In addition, the EMG signals gradually recovered after release of traction (Figure 9A). The histopathology study showed that morphological changes occurred mostly in outer nerve structures such as the epi- and peri-neurium. Structures in the endoneurium remained relatively intact13,16.
Clamping injury
All RLNs showed an immediate LOS (within less than 1 s) after acute mechanical injury was experimentally induced. In addition, no gradually EMG recovery can be observed in a short period of time after the injury (Figure 9B). The histopathology study showed that distortion of the epineurium and perineurium was greater in the clamping injury group compared to the traction injury group13,16.
Thermal injury
During the thermal injury study, the real-time EMG reveals a combined event, which then rapidly degrades to LOS (Figure 9C). The reaction time before LOS and the severity of electrophysiologic injury may be related to the dose of thermal stress14. Studies of EBDs reveal that the safe activation distance to the RLN and the cooling time vary by EBD type. For example, the safe activation distances and cooling times are 5 mm and 1 second for monopolar electrocautery (15 watts), 3 mm and 1 second for bipolar electrocautery (30 watts), 2 mm and 3 to 10 seconds for the Harmonic scalpel, and 2 mm and 2 to 5 seconds for the Ligasure system, respectively. Notably, the Harmonic scalpel should be cooled for more than 10 seconds or cooled by a quick (2 seconds) muscle touch maneuver before it touches the RLN. The Ligasure system should be cooled for more than 2 seconds or cooled by a quick muscle touch maneuver before it touches the RLN15,18. The histopathological examination of the thermal injured nerves showed relatively severe damage to the inner endoneurium with less distortion of the outer nerve structure16.
Figure 1. Preparation and anesthesia of KHAPS Black/Duroc-Landrace Pigs for IONM research. (A) Net weight of each piglet was measured before anesthesia. (B) An assistant maintained an adequate mouth opening while traction was applied to the upper and lower jaw. A laryngoscope was then used to press the epiglottis downward toward the base of the tongue. When the vocal cords were clearly identified, the elastic bougie was gently advanced into the trachea. The EMG tube was then inserted to a depth of 24 cm at the appropriate mouth angle. (C) The piglet was placed on its back with the neck extended. The channel leads from the recording electrodes were connected to the monitoring system. Physiologic monitoring was performed during the study. (D) The neck and the larynx were exposed for experiments. Please click here to view a larger version of this figure.
Figure 2. The multifaceted electronic equipment and principle of the IONM system. (A) The basic equipment included the neural stimulating electrodes (stimulator) and the recording electrodes (connected to the ETT). (B) The stimulating electrodes can be used to determine the location and functional status of the EBSLN, RLN, and VN during IONM. (C) The evoked EMG response is displayed on an LCD screen. Please click here to view a larger version of this figure.
Figure 3. The various stimulation electrodes available for use in IONM. (A) Monopolar probes (B) bipolar probes, and (C) stimulation probes/dissectors. The selection of stimulation probes/dissectors used for IONM depends on the specific stimulation requirements, the specific application desired and the preference of the surgeon. Please click here to view a larger version of this figure.
Figure 4. Various recording electrode types are available for use in IONM. (A) The EMG ETT electrodes include (1a) Trivantage (1b) Contact Reinforced (1c)-Standard Reinforced, and (1d)- FLEX EMG Tubes); (B) (2)-adhesive pre-gelled electrodes and (3)-needle electrodes. (C and D) The EMG tube is designed to touch the vocal fold through intubation (I), and the adhesive pre-gelled or needle electrodes can be used in transcutaneous (II), percutaneous (III), or transcartilage (IV) approach for EMG recording during IONM. Please click here to view a larger version of this figure.
Figure 5. Continuous IONM was performed via APS of the VN (*) to investigate real-time EMG changes in the RLN during (A) traction and (B) thermal injury. (C). Throughout the experiment, the C-IONM system displayed and continuously recorded the induced EMG changes and sequential recoveries in real time. Please click here to view a larger version of this figure.
Figure 6. Comparison of evoked EMG responses between four different types of recording electrodes. The feasibility studies indicated that all electrode types (i.e., EMG tube, transcutaneous, percutaneous, and transcartilage electrodes) accurately recorded typical evoked laryngeal EMG waveforms from the RLN under 1 mA stimulation. Please click here to view a larger version of this figure.
Figure 7. Comparison of real-time EMG tracings before and after experimental tracheal displacement. For stability study, tracheal displacement was experimentally induced. Changes in contact between the EMG tube electrodes and vocal folds caused significant variation in recorded EMG signals. (A) Electrodes in the normal position recorded strong EMG signals. (B) Electrodes with slight upward displacement (1 cm) recorded relatively weaker EMG signals. (C) Electrodes with moderate to severe upward displacement (2 cm) showed an EMG LOS. Please click here to view a larger version of this figure.
Figure 8. Comparison of real-time EMG tracings during experimental RLN experimental RLN traction injuries between four different types of recording electrodes. The accuracy studies showed that, when RLN traction stress was experimentally induced, all electrode types (i.e., EMG tube, transcutaneous, percutaneous, and transcartilage electrodes) recorded similar patterns of progressively degrading EMG amplitude. Please click here to view a larger version of this figure.
Figure 9. Comparison of real-time EMG changes and sequential recoveries after different RLN injury types. (A) In traction injury, the EMG signals gradually degraded under nerve stress and gradually recovered after release of traction. (B) In clamping injury, the EMG signals showed an immediate LOS and no recovery. (C) In thermal injury, the EMG signals revealed a combined event and then rapidly gradually degraded to LOS with no recovery. Please click here to view a larger version of this figure.
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 models are necessary to these clinical issues.
Recently, plenty of animal experimental studies have tried to overcome pitfalls of IONM and to investigate new applications. Most of these studies have used medium-sized animals such as canine/dog23,24,25 and porcine/swine/mini-pig11,12,13,14,15,16,17,18,19,22,26,27,28,29. Canine models of the RLN and laryngeal function are well-established and highly mimic human anatomy, size and physiology. The porcine model is the oldest animal applied in RLN research30,31. The first experiments in live pigs performed by Galen in the second century A.D. demonstrated functional alterations in a transected RLN. Currently, the porcine model is most commonly used for IONM research because its anatomy and physiology are very similar to those in humans. Experimental pigs have a medium size that enables easy handling and are widely available at a relatively low cost21.
This article demonstrates standard protocols for using the porcine model in IONM research, including protocols for general anesthesia and tracheal intubation. The 3R principle is implemented in the design of experiments for investigating electrophysiological characteristics of RLN injuries. Key issues in the use of the proposed porcine model include(1) EMG parameter characteristics and safety considerations when applying electrical stimulation11,17,19, (2) the use of muscle relaxants and reversals12,20,32, (3) stimulating and recording electrodes17, and most importantly (4) models of RLN injuries13,14,15,16,18 that cannot be accurately quantified in humans. The protocols were set up to induce different severity and types of RLN injuries. Recorded real-time EMG data were correlated with postoperative vocal cord function and histopathology examinations. Although some data from experimental studies are inapplicable to clinical practice, our porcine model provides a valuable research platform not merely in understanding technology of IONM, but also in guiding future experiments to improve surgical strategies for lesser RLN injuries during thyroid surgery.
The authors have nothing to disclose.
This study was supported by grants from Kaohsiung Medical University Hospital, Kaohsiung Medical University (KMUH106-6R49) and from Ministry of Science and Technology (MOST 106-2314-B-037-042-MY2.), Taiwan
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 |