Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models

Arsh Ketabforoush; Meifang Wang; W. David Arnold

doi:10.3791/66452

JoVE Journal > Neuroscience

Neuroscience

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models

Published: March 08, 2024

doi:

10.3791/66452

Arsh Ketabforoush¹, Meifang Wang¹, W. David Arnold^1,2,3,4

¹NextGen Precision Health,University of Missouri, ²Department of Physical Medicine and Rehabilitation,University of Missouri, ³Department of Neurology,University of Missouri, ⁴Department of Medical Pharmacology and Physiology,University of Missouri

Summary

In this study, we demonstrate a refined single fiber electromyography (SFEMG) protocol to allow in vivo measurement of neuromuscular junction (NMJ) transmission in rodent models. A step-by-step approach to the SFEMG technique is described to allow quantification of NMJ transmission variability and failure in rat gastrocnemius muscle.

Abstract

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. Single fiber electromyography (SFEMG) is a clinically relevant and sensitive technique that measures single muscle fiber action potential responses during voluntary contractions or nerve stimulations to assess NMJ transmission. The assessment and quantification of NMJ transmission involves two parameters: jitter and blocking. Jitter refers to the variability in timing (latency) between consecutive single-fiber action potentials (SFAPs). Blocking signifies the failure of NMJ transmission to initiate an SFAP response. Although SFEMG is a well-established and sensitive test in clinical settings, its application in preclinical research has been relatively infrequent. This report outlines the steps and criteria employed in performing stimulated SFEMG to quantify jitter and blocking in rodent models. This technique can be used in preclinical and clinical studies to gain insights into NMJ function in the context of health, aging, and disease.

Introduction

Single fiber electromyography (SFEMG) was initially developed by Stålberg and Ekstedt in the 1960s to identify and analyze action potentials from individual muscle fibers, primarily to study muscle fatigue¹. SFEMG is the most sensitive clinical technique for the assessment of neuromuscular junction (NMJ) transmission². SFEMG is conducted by selectively recording single fiber action potentials (SFAPs)³. NMJ transmission can be compromised due to factors like aging⁴^,⁵ and various neuromuscular disorders such as myasthenia gravis and amyotrophic lateral sclerosis⁶. Furthermore, conditions such as ischemia, fluctuations in temperature, and the use of neuromuscular blocking agents can result in deficiencies in NMJ transmission, manifested by increased NMJ transmission variability and occurrences of NMJ failure².

There are two approaches to recording SFEMG: stimulated and voluntary SFEMG. Voluntary SFEMG involves recording SFAPs from two NMJs supplied by the same motor axon using a concentric needle electrode inserted into the muscle being tested during voluntary activation⁷. Accordingly, voluntary SFEMG requires cooperation from the subject and can only assess low-threshold motor units (those activated during weak contractions)³. Stimulated SFEMG uses a pair of stimulating electrodes to stimulate motor axons while recording SFAPs with an SFEMG needle electrode inserted into the muscle being tested⁷.

In both voluntary and stimulated SFEMG, jitter and blocking are the two parameters used to assess and quantify NMJ transmission⁸. Jitter describes the variability in timing (latency) between consecutive SFAPs. During voluntary SFEMG, jitter is quantified by assessing the latency differences between a pair of SFAPs (supplied by the same motor axon) during 50 to 100 consecutive discharges. During stimulated SFEMG, jitter is quantified by assessing the latency differences between the stimulation timing and the onset of the SFAP during 50 to 100 consecutive discharges. Blocking indicates failure of NMJ transmission to trigger an SFAP response, and it can be quantified as the presence or absence of each pair of SFAPs during voluntary SFEMG or for each NMJ during stimulated SFEMG²^,⁷.

While an established and sensitive test in the clinical setting, SFEMG has only been infrequently applied in preclinical research⁴^,⁵^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸. In this report, we outline the approach to performing and analyzing SFEMG recordings in preclinical rodent models. Furthermore, we present representative data that highlights representative findings on SFEMG that indicate impairment of NMJ transmission following administration of a non-depolarizing neuromuscular blocking agent, rocuronium.

Protocol

All protocols were approved and performed in accordance with the regulations set forth by the Institutional Animal Care and Use Committee at the University of Missouri.

1. Animal preparation and anesthesia administration

Put on appropriate personal protection equipment.
Prior to the procedure, measure the rat's weight to determine the appropriate dose for weight-based medications and ventilator settings.
Induce anesthesia with 3%-5% inhaled isoflurane. Once an adequate level of anesthesia is established, position the rat in a prone position and maintain anesthesia with 1%-3% inhaled isoflurane.
Verify the adequacy of the depth of anesthesia by gently pressing the hindlimb footpad with forceps to observe the absence of a withdrawal response.
Maintain body temperature at 37 °C.
Apply veterinarian-approved petroleum-based ointment to the eyes to prevent dryness. Monitor the depth of anesthesia by observing the respiratory rate and assessing for withdrawal responses when applying pressure to the footpad using forceps.
Shave the hindlimb to be assessed using clippers. After adequate hair removal, using adhesive tape, position the limb to be studied with the ankle fixed at approximately 90° dorsiflexion, the knee in extension, and the hip in abduction.
Monitor the respirations of the rat during the entire experiment.
Administer isoflurane to euthanize the rat, with a dose of 5% or greater, until breathing has ceased for at least 3 min. Confirm the euthanasia by decapitation.

2. Electrode placement and setup

NOTE: NMJ transmission of the sciatic nerve and gastrocnemius muscle are assessed using the electromyography (EMG) system. Refer to the Table of Materials.

Insert a pair of insulated 28 G monopolar needles for stimulating the sciatic nerve with the cathode into the region of the proximal hind limb, while the anode is more proximally within the subcutaneous tissue overlaying the sacrum.
Ensure the stimulating electrodes are not placed in immediate proximity to the sciatic nerve or excessively deep to avoid direct harm to the sciatic nerve or other adjacent structures.
Place a disposable ground electrode on the contralateral hindlimb or tail.
Carefully place a 27-gauge, 25 mm needle electrode specifically designed for single fiber EMG, featuring a recording surface made of platinum-iridium material into the right gastrocnemius muscle. Ensure that the SFEMG electrodes are autoclaved prior to each use to maintain sterility.
Insert the SFEMG needle electrode in parallel with the gastrocnemius muscle fibers to capture single-fiber action potentials (SFAPs).
Avoid muscle damage while inserting and maneuvering the SFEMG needle within the muscle.

3. Stimulated single-fiber electromyography (SFEMG) procedure

Apply constant current stimulation to the right sciatic nerve at 10 Hz frequency using an intensity range of 0.3-10 mA and a pulse duration of 0.1 ms.
Configure the filter settings within a low frequency filter of 1 kHz and high frequency filter of 10 kHz. Adjust the Gain from 200 µV to 1000 µV per division to facilitate the visualization of potentials. Set the sweep speed at 500 µs per division.
Adjust the stimulus intensity to trigger and isolate SFAPs for recording and subsequent analysis.
1. To identify and analyze a response as an SFAP, ensure that the specific criteria mentioned below are met: Ensure the rise time from the baseline peak to the negative phase is less than 500 µs, the minimum amplitude (baseline peak to negative) is at least 200 µV, and the response consistently exhibit an all-or-none behavior (stable size and shape between responses).
  NOTE: It is essential that the recurring responses exhibit consistent upward phases without any notches or inflection points. Note that the last criterion is crucial for distinguishing a quality signal from the summation of multiple signals.
Calculate Jitter, or variability of SFAP latency (time between stimulation and rising phase of negative peak of SFAP) between consecutive discharges, following at least 50 stimulations (50-100 stimulations), and assess and quantify blocking.
NOTE: Blocking can be assessed as the presence or absence at each synapse or as the percentage of stimulations that fail to trigger single fiber action potential generation. Jitter is calculated using the following equation⁷ (Jitter is typically automatically calculated by clinic electromyography systems):

MCD = Mean value of Consecutive Difference
IPI = Interpotential Interval
Repeat the process for additional SFAP responses. On average, assess 10 synapses from each animal to calculate jitter and subsequently determine the average values per animal.
Exclude SFAPs with jitter of less than 4 µs from the analyses to prevent the inclusion of potentials that might have been evoked by direct muscle stimulation¹¹.
When recording potentials displaying intermittent blocking, increase the stimulus intensity to verify that SFAP failure is not attributable to submaximal stimulation.

Representative Results

To demonstrate increased jitter and blocking in the context of NMJ transmission failure, stimulated SFEMG was performed with and without intravenous administration of rocuronium. Rocuronium is an intermediate-acting, non-depolarizing neuromuscular blocking agent widely used in clinical settings to induce muscle paralysis during surgeries or medical procedures. It operates by competitively binding to nicotinic acetylcholine receptors at the NMJ¹⁹. Prior to the administration of rocuronium, the adult wild-type rat (Sprague Dawley) was prepared and anesthetized as described in section 1. Due to paralysis induced by rocuronium, invasive ventilatory support was used to obtain representative data (contrasting to the typical approach for anesthesia during SFEMG recordings described above in section 1). An endotracheal tube was placed, and ventilation was maintained at 60 respirations/min using a pressure-controlled mode with a pressure of 17 mmHg and positive end-expiratory pressure of 3 cm H₂O while delivering 2%-3% isoflurane. Once ventilatory support and anesthesia were achieved, single fiber action potentials were recorded with and without treatment with rocuronium (bolus of 0.05 mg/kg rocuronium bromide intravenous). Figure 1 demonstrates three representative single-fiber action potential recordings from the same NMJ in an adult rat. Figure 1A demonstrates the typical morphology of a single fiber action potential (after a single stimulation) with a rise time of less than 500 µs from the baseline to the negative peak and a minimum amplitude (peak to peak) of at least 200 µV. Figure 1B demonstrates features of increased jitter and intermittent blocking following injection of rocuronium. Figure 1C demonstrates the normalization of jitter and blocking from the same synapse after the resolution of the NMJ block. For viewing simplicity, Figure 1B,C only show responses following 9 consecutive stimulations. Moreover, for better comprehension, Figure 2 shows an additional example of three superimposed single fiber action potentials observed after 10 consecutive stimulations, revealing heightened jitter and blocking.

To quantify the impact of rocuronium on NMJ transmission using stimulated SFEMG, recordings were made from 5 NMJs without any treatment and from 5 NMJ following delivery of rocuronium and were analyzed for jitter and blocking (Figure 3). Following rocuronium, jitter (Figure 3A) and blocking (Figure 3B) were significantly increased.

Figure 1: Three representative single-fiber electromyography recordings from the same neuromuscular junction (NMJ). (A) Example of a single fiber action potential following a single nerve stimulation showing criteria for amplitude (baseline to negative peak amplitude of at least 200 µV) and rise time (baseline to negative peak less than 500 µs) criteria for acceptance as single fiber potential. (B) Example of superimposed single fiber action potentials following 9 consecutive stimulations showing increased jitter and blocking indicating impaired NMJ transmission following rocuronium administration (jitter: 68.35 µs). (C) Example of superimposed single fiber action potentials following 9 consecutive stimulations showing reduced jitter and no blocking, indicating normalizing NMJ transmission after resolution of rocuronium treatment (jitter: 21.7 µs). Representative SFEMG traces reveal single muscle fiber action potentials recorded from a healthy synapse with no blocking and normal jitter in superimposed view. Please click here to view a larger version of this figure.

Figure 2: Three superimposed single fiber action potentials (SFAPs) recorded after consecutive stimulations during rocuronium administration. Three superimposed single fiber action potentials (SFAPs) were recorded after 10 consecutive stimulations during rocuronium administration, indicating increased jitter and blocking. The jitter values for A, B, and C were 24.3 µs, 46.9 µs, and 88.7 µs, respectively. Please click here to view a larger version of this figure.

Figure 3: Single Fiber Electromyography assessment of neuromuscular junction transmission (NMJ) following treatment with a non-depolarizing NMJ blocking agent. (A) During rocuronium administration (0.05 mg/kg), SFEMG showed increased variability of transmission (jitter) versus healthy condition (untreated: 12.9 µs, 95% CI [7.2-16.9 µs] versus rocuronium: 40.70 µs, 95% CI [34.7-70.7 µs]) (Mann Whitney test: p = 0.0079). (B) Percentage of stimulations with NMJ blocking from each synapse on SFEMG was increased versus healthy condition (untreated: 0%, 95% CI [0-0%] versus rocuronium: 31%, 95% CI [14.0-59.0%]) (Mann Whitney test: p= 0.0079). All measurements (jitter and percentages of stimulations with NMJ blocking) were obtained from a single rat, n = 5 synapses untreated and n = 5 synapses following rocuronium treatment. Data shown as a median ± 95% confidence interval. Please click here to view a larger version of this figure.

Discussion

SFEMG is commonly used for diagnostic testing in patients with suspected autoimmune, acquired, and genetic forms of NMJ disease. SFEMG is considered the most sensitive test for the diagnosis of the NMJ disorder, myasthenia gravis²⁰^,²¹. Repetitive nerve stimulation (RNS) is another method that is more commonly used in clinical diagnostic testing and involves stimulating a peripheral nerve with a train of stimuli and quantifying the summated compound muscle action potential response of the innervated muscle⁵^,²². RNS has consistently shown to have significantly lower sensitivity as compared with SFEMG²³^,²⁴. The advantage of SFEMG lies in the ability to selectively evaluate individual muscle fibers and quantify not only failure of NMJ transmission (blocking) but also variability (jitter)²^,²⁵^,²⁶. While an established and sensitive test in the clinical setting, SFEMG has only been infrequently applied in preclinical research⁴^,⁵^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶. Animal models of neuromuscular diseases play a pivotal role in determining the underlying mechanisms of human diseases and developing therapeutic interventions. The capacity to apply outcome measures and biomarkers, such as SFEMG, across different species can streamline and expedite the transition of promising preclinical discoveries into human clinical studies²⁷. For instance, a multicenter study in Japan focused on establishing reference values for SFEMG using concentric needles in healthy human subjects. The study indicated that the recommended cut-off values for individual MCD were 58.8 µs for stimulated SFEMG in the extensor digitorum communis (EDC) muscle²⁸. It is noteworthy that the Gastrocnemius muscle is not commonly subjected to SFEMG, with EDC being the closest muscle studied in this context. Consequently, the jitter values we obtained from rats were observed to be lower than those reported in clinical studies. These discrepancies suggest that the distribution of the safety factor varies depending on the muscle and the species under consideration.

In this report, we present a stepwise approach to applying stimulated SFEMG in rodents including steps for animal preparation, electrode placement, single fiber action potential recording, and analyses. Additionally, we present representative data in an induced model of NMJ failure (rocuronium administration) to help clearly demonstrate the concepts of increased jitter and blocking as well as the all-or-none concept of a single fiber action potential in SFEMG recording.Previously, in an experimental study, a rat without the blocking agent showed a minor MCD change (around 10 µs) with no blocking in the first 3000 s. In contrast, a rat injected with 0.08 mg/Kg vecuronium exhibited increased jitter after 200 s, leading to blocking around 500 s, with an initial MCD of about 50 µs, consistent with our results¹⁷. Establishing the correct dosage of rocuronium in the rats was essential to attain the targeted level of neuromuscular blockade while mitigating potential complications. While various studies employed dosages ranging from 0.1 mg/kg to 5 mg/kg for inducing muscle paralysis¹⁷^,²⁹^,³⁰, we opted for a dosage of 0.05 mg/kg. This choice aligns with the recognition that NMJ impairment initiates before the onset of muscle paralysis.

The acquisition of single fiber action potential responses using SFEMG involves several crucial steps. Notably, obtaining selective recordings requires practice to perform several tasks concurrently, including adequately delivering nerve stimulation while acquiring adequate responses. During recordings, the SFEMG recording electrode is carefully inserted and adjusted with small, steady movements to minimize muscle trauma and improve yield. Once an action potential has been identified, small adjustments of stimulation and recording electrode position should be made to optimize single fiber action potential amplitude and "sharpness" (short rise time). Once a potential is identified and amplitude and rise time are optimized, movements of electrodes must be minimized to ensure stability of the all-or-none responses. Steady positioning of electrodes is crucial as even minor shifts in the recording needle position can result in fluctuations in action potential characteristics, including shape, amplitude, and latency. It is important to ensure that the action potentials are stable and in compliance with all-or-none appearance. Variability of responses can be related to electrode movement or superimposition of multiple single fiber action potentials from different NMJs. Together, these factors play key roles in achieving reproducibility and stable measurement of the action potential²^,²⁶. Moreover, excluding SFAPs with jitter less than 4 µs from the analyses is a prudent approach to ensure the reliability of results. This stringent criterion helps avoid the inclusion of potentials that could potentially be artifacts induced by direct muscle stimulation, thereby enhancing the accuracy of the assessment¹¹. Another important factor in stimulation jitter studies is subliminal stimulation, particularly in pathological conditions, where many spikes have apparently increased jitter. It is essential to rely on the operator to ensure that stimulus intensity exceeds the threshold for all such spikes, as this determination cannot be made retrospectively³¹.

The stimulated SFEMG technique outlined in this manuscript utilizes a specialized SFEMG electrode rather than a standard concentric electromyography needle electrode. Our SFEMG electrode features a recording surface of 0.0005 mm², in contrast to the approximately 0.019 mm² recording surface of a standard pediatric-sized concentric electromyography needle electrode. This distinction is crucial, as larger concentric needle electrodes, with less selectivity due to their larger recording surfaces, may be suitable for voluntary SFEMG to capture apparent single fiber action potential pairs. However, in stimulation SFEMG, where multiple axons or motor units are often stimulated simultaneously, employing larger concentric needle electrodes becomes more challenging⁷.

A few limitations should be considered. It is also important to point out that SFEMG cannot ascertain the mechanism underlying a defect in neuromuscular transmission as may be achieved with intracellular recordings performed ex vivo²⁷. Another essential consideration regarding SFEMG is its limitation in selectively recording from specific muscle fiber types. Consequently, SFEMG recordings are likely to represent a variable mixture of different muscle fiber types⁵.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to thank Dr. Martin Brandhøj Skov from NMD Pharma for his valuable advice on rocuronium dosing and Arash Karimi from the Biomedical Engineering Department of Stony Brook University for his assistance in calculations. This study was supported in part by funding from NIH to WDA (R01AG067758 and R01AG078129).

Materials

27 G Reusable Single Fiber Needle Electrode	Technomed	202860-000	singlefiber recording electrode
2 mL Glass Syringe	Kent Scientific Corporation	SOMNO-2ML
Detachable Cable	Technomed	202845-0000	to connect the recorder electrode to the electrodiagnostic machine
Disposable 2" x 2" disc electrode with leads	Cadwell	302290-000	ground electrode
disposable monopolar needles 28 G	Technomed	202270-000	cathode and anode stimulating electrodes
EMG needle cable (Amp/stim switch box)	Cadwell	190266-200	to connect monopolar electrodes to electrodiagnostic stimulator
Helping Hands alligator clip with iron base	Radio Shack	64-079	Maintaining recording electrode placement
Isoflurane (250 mL bottle)	Piramal Healthcare	NA
monoject curved tip irrigating syringe	Covidien	81412012	utilized for application of electrode gel
PhysioSuite Physiological Monitoring System with RightTemp Homeothermic Warming	Kent Scientific Corporation	PS-RT	Includes infrared warming pad, rectal probe, and pad temperature probe
Pro trimmer Pet Grooming Kit	Oster	078577-010-003	clippers for hair removal
Rat Endotracheal Tubes (16 G)	Kent Scientific Corporation
Rocoronium Bromide	Sigma	PHR2397-500MG	neuromuscular blocker agent
Sierra Summit EMG system	Cadwell Industries, Inc., Kennewick, WA	NA	portable electrodiagnostic system
SomnoSuite Low-Flow Digital Anesthesia System	Kent Scientific Corporation	SOMNO	Includes anti-spill, anti-vapor bottle top adapter; Y adapter tubing; charcoal scavenging filter
Veterinarian petroleum-based ophthalmic ointment	Puralube	26870	applied during anesthesia to avoid corneal injury

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