Microneurography as a minimally invasive method to assess target engagement during neuromodulation

Abstract

Objective.Peripheral neural signals recorded during neuromodulation therapies provide insights into local neural target engagement and serve as a sensitive biomarker of physiological effect. Although these applications make peripheral recordings important for furthering neuromodulation therapies, the invasive nature of conventional nerve cuffs and longitudinal intrafascicular electrodes (LIFEs) limit their clinical utility. Furthermore, cuff electrodes typically record clear asynchronous neural activity in small animal models but not in large animal models. Microneurography, a minimally invasive technique, is already used routinely in humans to record asynchronous neural activity in the periphery. However, the relative performance of microneurography microelectrodes compared to cuff and LIFE electrodes in measuring neural signals relevant to neuromodulation therapies is not well understood.Approach.To address this gap, we recorded cervical vagus nerve electrically evoked compound action potentials (ECAPs) and spontaneous activity in a human-scaled large animal model-the pig. Additionally, we recorded sensory evoked activity and both invasively and non-invasively evoked CAPs from the great auricular nerve. In aggregate, this study assesses the potential of microneurography electrodes to measure neural activity during neuromodulation therapies with statistically powered and pre-registered outcomes (https://osf.io/y9k6j).Main results.The cuff recorded the largest ECAP signal (p< 0.01) and had the lowest noise floor amongst the evaluated electrodes. Despite the lower signal to noise ratio, microneurography electrodes were able to detect the threshold for neural activation with similar sensitivity to cuff and LIFE electrodes once a dose-response curve was constructed. Furthermore, the microneurography electrodes recorded distinct sensory evoked neural activity.Significance.The results show that microneurography electrodes can measure neural signals relevant to neuromodulation therapies. Microneurography could further neuromodulation therapies by providing a real-time biomarker to guide electrode placement and stimulation parameter selection to optimize local neural fiber engagement and study mechanisms of action.

Keywords: ECAP; microneurography; minimally invasive; neuromodulation; peripheral neural recordings; target engagement; vagus nerve.

Figure 1.

Illustration of three types of recording electrodes, synchronous versus asynchronous neural activity, and effect of electrode-fiber distance on recorded signals. (A) Illustration showing the three different types of recording electrodes characterized for their performance in measuring neural signals from the peripheral nervous system. The longitudinal intrafascicular electrode (LIFE) has a larger area of exposed metal for recording than the microneurography microelectrode, and the LIFE causes more damage to tissue upon insertion, indicated by the red blood track. (B) Synchronous electrically evoked compound action potential (ECAP) vs. asynchronous neural activity illustrated by number of fibers activated and synchrony of neural activity. Both illustrate a neural signal as a moving dipole with a finite length. This dipole length increases with conduction velocity and fiber size. (C) Cartoon illustrating the relevance of scale in interpreting neural recording electrode performance. For example, in the cervical vagus nerve (cVN) for smaller mammals, there is (1) less distance between the neural fiber and the surface of the nerve, where a recording electrode is placed, and hence less falloff in electric field, and (2) there is a thinner layer of perineurium insulation (Pelot et al 2020), and hence less falloff in electric field. Electric field falloff from the neural source, approximated as a monopole, is proportional to ~1/r² , where r is the distance from the neural source to the recording electrode (Plonsey and Barr 1995).

Figure 2.

Experimental setup and surgical preparation. (A) Cervical vagus nerve (cVN) instrumented with bipolar stimulation cuff (contact 2 and 5) and, at least 4 cm caudal, three replicates each of three different recording electrodes: three longitudinal intrafascicular electrodes (LIFEs), three microneurography electrodes, and a three-contact cuff. All recording electrodes were referenced to fat (off-nerve local tissue reference). (B) Great auricular nerve (GAN) instrumented with bipolar stimulation cuff and two replicates each of two different recording electrodes: two microneurography electrodes, and either a two-contact cuff or two LIFE electrodes (alternated by subject). All recording electrodes were referenced to fat (off-nerve local tissue reference). (C) Histology of the cVN and the GAN under the stimulation cuff illustrating the relative difference in size and fascicular organization. The cVN is approximately 5x larger than the GAN and contains a greater number of fascicles. GAN histology from additional subjects is shown in supplementary material 2.

Figure 3.

Comparison of electrodes in recording ECAPs from larger myelinated fibers. (A) Representative ECAP traces from concurrent cuff, microneurography electrode, and longitudinal intrafascicular electrode (LIFE) recordings at 1.5 mA of stimulation on the cervical vagus nerve (cVN). Contact one of three was selected for each electrode type. Stimulation artifact shaded in orange and Aβ-fiber ECAP shaded in yellow. (B) Bar plot summarizing magnitude of Aβ-fiber ECAP recorded with three different electrodes on cVN showing significantly larger recording on cuff compared to both the microneurography electrode and LIFE electrode. (C) (top left) Dose-response curves of Aβ-fiber ECAPs for three replicates each of the three recording electrode types characterized. From top left, in an anti-clockwise direction, shows the steps of fitting dose-response curves with logistic growth functions: (1) averaging of replicate contacts, (2) normalization to ECAP amplitude at 5 mA of stimulation, and (3) fitting to logistic growth functions and rescaling y-range to be from zero to one. (D) Bar plot summarizing sensitivity of the three recording electrodes across subjects. Sensitivity was measured as EC10 stimulation currents extracted from the dose-response curve logistic growth function fits in (C).

Figure 4.

Noise floor of recording electrodes. Noise measurements in electrophysiology recordings showing largest noise levels on the microneurography electrode. Note the different y-axis scales in the three plots as the noise level after filtering and averaging is reduced by orders of magnitude. (A) Unfiltered time series electrophysiology. (B) Filtered time series electrophysiology. (C) Filtered averaged ECAPs (median of n = 750).

Figure 5.

Comparison of electrodes in recording ECAPs from smaller myelinated fibers and correlation to evoked bradycardia. (A) Authenticity of B-fiber recordings was confirmed by signal propagation delay across recording electrodes in the range of 3–15 m s⁻¹ (Erlanger and Gasser 1937, Manzano et al 2008). An artifact (e.g. EMG, motion) could occur in a similar time window but would not show up with a signal propagation delay across spatially separated recording electrodes. Stimulation artifact shaded in orange, Aβ-fiber ECAP shaded in yellow, and B-fiber ECAP shaded in green. Stimulation was delivered at 10 mA. A similar plot is shown for the LIFE and microneurography electrodes in supplementary material 10. (B) B-fiber dose-response curves for all three recording electrodes on a single subject showing the largest ECAP magnitude recorded with the cuff electrode. The cuff electrode also recorded ECAPs at the lowest stimulation current. The ECAP detection threshold for each electrode is boxed; a clear threshold was not apparent on the microneurography electrode. The dash line from (C) indicates the bradycardia onset threshold. (C) Evoked heart rate decrease (bradycardia) dose-response curve in the same subject as (B). The bradycardia stimulation current threshold is lower than the B-fiber ECAP detection thresholds in (B).

Figure 6.

Effect of referencing strategy on stimulation artifact and ECAP. (A) Representative data showing ECAP traces with three different referencing strategies at 2.3 mA of stimulation: (left) reference in ‘local tissue’, (center) bipolar ‘on-nerve’ reference, and (right) tripolar on-nerve reference. Orange highlights overlap the stimulation artifact, an example of common mode noise, which is substantially reduced in the on-nerve referencing strategies. The Aβ-fiber ECAP (~3–4 ms) is also smaller in the on-nerve referencing strategies. In the illustration of the recording setup in each plot, recording electrodes are in blue and reference electrodes are in gray. (B) Zoomed out plot of local tissue referenced recording from (A) to show larger y-axis range.

Figure 7.

Comparison of electrodes in recording asynchronous sensory evoked neural activity. (A) Using a toothbrush to gently stroke the ear at off-target (red, not innervated by GAN) and on-target (green, innervated by GAN) areas corresponding to green and red color coding in (B). (B) Representative electrophysiology recordings: first 30 s with no stroking, middle 30 s with on-target stroking, and last 30 s with off-target stroking to control for motion artifacts from stroking. Only the microneurography electrode showed a robust response specific to on-target stroking. LIFE and cuff recordings were selected to illustrate artifacts, which appeared neural, but were not authenticated by appropriate controls. The gray line in each plot is the spike detection voltage threshold and the red dots indicate detected ‘spikes’. Note, the three recordings shown are not from the same subject. (C) Spike shapes for electrophysiology recordings in (B) indicating multi-unit hash recordings on the microneurography electrode, tremor or cardiac motion artifact on the LIFE electrode, and ventilator artifact on the cuff electrode. (D) Bar plot summarizing spike count recorded by the three electrodes across all subjects showing the microneurography electrode records significantly more spikes than both the cuff and LIFE electrodes.