Kilohertz frequency nerve block enhances anti-inflammatory effects of vagus nerve stimulation

Figure 1. (A) Experiment setup and electrophysiological configuration. The left cVN was exposed and fitted with three cuff electrodes. ENG measurements were made from the cranial end of the exposed nerve. cVNS was delivered to the caudal end of the exposed nerve, with a KES delivering electrode located cranially. (B) Preparation used for to measure ENG from the GSN. (C) Experiment timeline. Nerve and electrode preparation were followed by a 10 minute stimulation (pre-stim) period, in which either cVNS or cVNS + KES were delivered to the nerve. Vehicle or LPS was injected through the lateral tail vein, followed by another 10 minute stimulation (post-stim) period. For nerve block experiments, KES was on for the entire 70 minutes. Blood was collected 50 minutes after the post-stim period for biochemical analysis.

Figure 2. cVNS activates the GSN in a synchronous and dose-dependent manner. (A–C) Increasing stimulus intensities are delivered to the intact left cVN. Simultaneous ENG measurements are made on the ipsilateral GSN. (D) The caudal end of the transected cVN is stimulated, activating efferent pathways alone. (E) A cVNx is performed and the cranial end of the cVN is stimulated, activating afferent pathways. Waveforms shown are stimulus-triggered averages from 1000 stimulation trials. The red arrow indicates stimulus artifact, and θ values are presented for each waveform.

Figure 3. TNF-α expression and ENG data for baseline cVNS conditions. (A) TNF-α levels from animals receiving no stimulation (LPS, n = 8), stimulation of the intact cVN (cVNS, n = 6), vagotomized efferent cVNS (cVNx + ecVNS, n = 6), and complete afferent KES nerve block with paired efferent cVNS (Complete Block + ecVNS, n = 6). Asterisks denote significance between bracketed groups (α = 0.05). (B) Representative recordings from the caudal end of the cVN pre-, during-, and post-KES delivery. Average measurements (yellow and orange) are superimposed upon individual runs (grey). (A and C) Component regions depict the windows used for quantifying nerve activation (θ) and block efficacy (θ) for (A and C) components in the ENG measurements. Post-KES averages are from 10 runs only. Red arrow indicates stimulus artifact.

Figure 4. KES nerve block thresholds and θ computations. (A) KES block thresholds were determined during experimental preparation. Post-hoc analysis of ENG measurements and quantification of θ led to sorting of block thresholds into complete and incomplete block groups. Experiments with trials containing θ greater than the RMS noise floor during KES delivery were categorized as incomplete block for both biochemical and electrophysiological analysis. (B–E) Mean and standard deviation of θ throughout experiments with both complete afferent KES nerve block ((B,C) n = 9 from all experiments) and incomplete afferent KES nerve block ((D,E) n = 5 from all experiments). Recordings from the 70 minute experiments were parsed into 210 trials, each represented by a stimulus-triggered average waveform. Colored traces represent the mean from all experiments in each group, with the grey traces representing ±1σ. Experiments with complete afferent KES nerve block met the θ criteria, while incomplete block experiments did not.

Figure 5. Incomplete afferent KES nerve block and KES nerve block alone are not sufficient for activating anti-inflammatory pathways. (A) TNF-α expression from control (LPS, n = 8), incomplete afferent KES nerve block and paired ecVNS (Incomplete Block + ecVNS, n = 5), and complete afferent KES nerve block only (Complete Block, n = 3). (B) Example ENG measurements from the caudal end of the cVN during incomplete afferent KES nerve block. Average waveforms (red) are superimposed upon individual runs (grey), with θ presented for each CAP component. Red arrow indicates stimulus artifact. This example is from an experiment in which the calibration period was successfully completed, however post-hoc analysis revealed that afferent KES nerve block was incomplete. The A fiber component is partially blocked, however not complete, and the C fiber component is unmodified compared to baseline measurements.