In-vivo application of low frequency alternating currents on porcine cervical vagus nerve evokes reversible nerve conduction block

Fig. 1. Electrode placement on the isolated, left cervical vagus of a swine for the LFAC experiments. The CE, a bipolar cuff, was used for vagal stimulation. The LFAC conditioning waveform was presented at the RE. A tripolar recording electrode for ENG recordings was placed most rostral on the vagus nerve and secured with suture. A ligature was placed caudal to all three electrodes to eliminate cardiac effects due to stimulation of the left vagus nerve

Fig. 2. LFAC waveform at 1 Hz and sync pulses generated. The sync pulses were phased to trigger at the peaks of the sinusoidal waveform

Fig. 3. Example of a raw respiration data set during experiment. Vagal stimulation alone occurred at approximately 140 seconds, shown as the shaded region. In this period, there is an evident increase in inter-breath interval and a slight decrease in amplitude. This indicates that the HB reflex was adequately activated

Fig. 4. Temporal raster plots of the continuous breathing rate (Top), and normalized breathing rate (Middle) records as a function of stimulation case (Bottom) for all experimental runs across all animals (N=30). Times were aligned to the start of the ’Stim Only’ epoch to show the transition between the breathing rate during LFAC + vagal stim and vagal stimulation only, which shows a drop in breathing rate when LFAC is removed. Top panel’s breathing rate is the instantaneous rate calculated using the instantaneous interval between inspirations. The middle panel shows the instantaneous breathing rate normalized to the average ’Pre’ epoch breathing rate. Middle panel shows the experimental normalized breathing rate percentage with respect to the averaged breathing rate in ‘pre’ epoch. Bottom panel shows the times when of each stimulation cases were applied

Fig. 5. Breathing rate during each epoch of the experimental paradigm. The percent block is shown in the legend on the bottom left (± standard deviation). The ’Pre’ epoch was normalized to 100% block and the ’Stim Only’ epoch was normalized to 0% block. The overshoot seen in the first few seconds of the ‘Post’ epoch is likely due to sympathetic rebound before returning to baseline

Fig. 6. BRrate during a control case in which the electrode connections were swapped. Vagal stimulation was delivered via the CE and the LFAC waveform was presented at the RE. In this control case, the percent block (± SD) during the ’LFAC+Stim’ epoch was calculated to be -22%. A negative percent block indicates that the breathing rate during ’LFAC+Stim’ was lower than the breathing rate of the ’Stim Only’ epoch

Fig. 7. Test case of Swine ID 21435. In this unconventional case, the test sequence was applied out of order. Following a baseline reading, the HB reflex was established. The amplitude of the LFAC waveform was then slowly ramped up until block was achieved. The LFAC waveform was then discontinued which resulted in the activation of the HB reflex almost instantly. The left panel shows the unconventional test sequence order. To quantify the percent block (± SD), 4 epochs were isolated, shown on the right panel: Pre, LFAC+Stim, Stim Only, and Post. The ’LFAC+Stim’ epoch had a max amplitude of 1mAp applied at 1 Hz, thus the window of ’LFAC+Stim’ was determined based on the location of the max amplitude

Fig. 8. ENG recordings during an LFAC test case. In the ’Stim Only’ epoch (left panel) there are 2 clear CNAPs. The LFAC waveform during ’LFAC+Stim’ slow these CNAPs down and cause a decrease in amplitude. The right panel shows the breathing rate and percent block (± SD) during ’Stim Only’ and ’LFAC+Stim’

Fig. 9. ENG recordings during an LFAC control experiment. During the ’Stim Only’ epoch (left panel) there are 2 clear CNAPs. Conversely to what occurs in the test case, the LFAC waveform in the ’LFAC+Stim’ epoch does not affect the 2 CNAPs that are visible. The CNAPs visible in the ’LFAC+Stim’ epoch are unaffected and superimposed on those seen during the ’Stim Only’ epoch, which supports the hypothesis that LFAC block does not occur due to an electrode or waveform interaction. The right panel shows the breathing rate and percent block (± SD) during ’Stim Only’ and ’LFAC+Stim’ epochs in the control case