Renal Nerve Activity and Arterial Depressor Responses Induced by Neuromodulation of the Deep Peroneal Nerve in Spontaneously Hypertensive Rats

Figures

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  FIGURE 1. Experimental set-up for DPNS to modulate MAP. Adult SRH rats were anesthetized. Phase I animals received assisted ventilation (n = 5), and phase II did not (n = 8), and phase III the MAP was measured by telemetry and DPNS with a wireless system (n = 11). The effect of DPNS on RSNA and MAP was evaluated, (A) Animal setup: a stimulator system with a pulse generator for short (0.15 ms) or long (1 ms) pulses was connected to a hook electrode placed on the DPN (i). A second electrode was placed on the renal nerve to record neural activity (ii). A catheter was implanted in the femoral artery and connected to a pressure transducer to monitor MAP (iii). Dotted-line inserts (i and ii) show the site where pictures in panels (B–D) were obtained. Photographs of the DPN before (B) and after (C) electrode implantation are displayed. Insert in panel B is magnified in panel C after electrode placement. (D) Identification of renal nerve. SHR, spontaneous hypertensive rats; RSNA, renal sympathetic nerve activity; DPNS, Deep peroneal nerve stimulation; DPN, deep peroneal nerve: MAP, mean arterial pressure; TAM, tibialis anterior muscle.

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  FIGURE 2. Reduction in MAP by DPNS. (A) Stimulation of the DPN at 2 Hz (arrows) evokes a reduction in MAP and increases the SRNA (red dotted lines). (B) 1 ms pulse duration and (C) 1 mA pulse amplitude induced more effective MAP depressor responses (unpaired t-test; *p < 0.05; **p < 0.008, n = 4–5). (D) Reproducible MAP reduction evoked by two sequential DPNS trains. (E) MAP reduction evoked by three consecutive trains of DPNS in five individual SHR animals. (F,G) 1 ms pulses induced a reduction in MAP in each animal and a significant reduction in MAP compared to baseline (unpaired t-test; **p < 0.02, n = 8). ES, electrical stimulation, MAP, mean arterial pressure; HR, heart rate; BPM, beats per minute; RSNA, renal sympathetic nerve activity; DPN, deep peroneal nerve.

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  FIGURE 3. DPNS neuromodulation of RSN activity. (A) Spontaneous RSNA (green) increased in amplitude and frequency during the 10 s DPNS. Concurrently, the MAP decreases (blue). (B,B’) The onset of RSNA was observed at approximately 0.2 s after the first DPNS stimulation pulse. Arrows point before and after RSNA, to highlight the decrease in amplitude. (C) Nicotinic receptor blocker hexamethonium (60 mg/kg) confirmed RSNA activity. (D) Power spectral analysis in two different SHR animals after 10 and 20 s of DPNS showed the RSNA band at 10–50 Hz; arrows point signals and stimulus artifact. (E) Compared to baseline (Bl), the mean SSD showed a significant increase in the RSNA range (ANOVA *p < 0.05, n = 3), but not 10 s after stimulation. (F) Raster plots of two waveforms from RSNA, one was evoked during the stimulation and the second was present before stimulation and reduced thereafter. (G) 1 mA stimulation pulses increased the amplitude of the evoked RSNA (**p < 0.004, paired t-test, n = 5). ES, electrical stimulation; DPN, deep peroneal nerve; DPNS, deep peroneal nerve stimulation; RSNA, renal sympathetic nerve activity.

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  FIGURE 4. w-μCE for sub-chronic DPNS. (A) Picture of the w-μCE top view. (A’) Side view of the insertion z-shaped channel and electrode chamber. (B) Schematic and picture of the w-μCE implanted onto the DPN. (C) Voltage output from the w-μCE as a function of power from the external antenna (n = 3). (D) Illustration of the device implanted in the DPN and evoked dorsiflexion. (E) Placement of the external antenna, 3–4 cm from the w-μCE; the hind-limb was stained to facilitate dynamic pixel tracking during videotaping. (F) Pixels tracked from the video recorded during DPNS sessions are displayed in an X/Y graph. The evoked movement was directly proportional to the RF power (0–50%). w-μCE, wireless multichannel electrode; DPNS, deep peroneal nerve stimulation; RF, radio frequency.

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  FIGURE 5. Effect of sub-chronic DPNS on mean arterial pressure in SHR animals. (A) Experimental setup; mean arterial pressure was measured with a telemetric device from the femoral artery. The external RF antenna delivered pulses to the w-mCE on the DPN. (B) Mean arterial pressure modulation by DPN stimulation, at day 21 of implantation (n = 6 SHR animals treated, n = 3 SHR sham). At baseline, all animals showed MAP values of (150 ± 14 mmHg; n = 9); the shadowed square represents the period of stimulation. SHR treated showed a decrease from 159 to 128 mmHg (n = 6), while the sham group maintained a MAP of 150 ± 13 mmHg (n = 3). (C) Individual datapoints of SHR with an active w-μCE (n = 6) with their means obtained every 2 min. The depressor response progressively returned to baseline levels at minute 20 from the start of the stimulation (164 ± 26 mmHg). (D) Effect of DPN stimulation at minute 8 significantly reduced the MAP (**p < 0.004 one-way ANOVA followed by Dunnett’s test) in the DPNS-treated group (135 ± 8 mm Hg; n = 6), while MAP values were similar in sham animals (150 ± 13 mm Hg; n = 3). DPN, deep peroneal nerve; DPNS, DPN stimulation; RF, radio frequency; SHR, spontaneous…

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  FIGURE 6. Effect of sub-chronic DPNS on blood pressure in SHR awake animals. (A) Experimental setup; blood pressure was measured with a tail cuff in restrained and fully awake animals. The external antenna delivered pulses to the w-mCE on the DPN. (B,C) Multiple systolic and diastolic pressure measurements were taken during baseline (i.e., pre-implant and 10 days post-implant) and 15 days after daily 10-min DPNS treatment, at 1 and 2 h post-DPNS (8–19 measurements in two animals, *p < 0.05, **p < 0.001, and ****p < 0.0001). DPN, deep peroneal nerve; DPNS, DPN stimulation; SHR, spontaneous hypertense rats; w-mCE, wireless micro-channel electrode.

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  FIGURE 7. Nerve histology after sub-chronic wMCE implantation. (a) Picture of DPN in the device 30 days after implantation. (b) Nerve segment after device removal, arrows point out the area where the electrode was implanted. Cross-section histology of control (c–d’), and wMCE implanted DPN (e–f’), d’ and f’ are magnifications of the dotted line squares in panel c,d and e,f, respectively. Fluorescent labels: b-tubulin (axons), P0 (myelin), ED-1 (activated macrophages).