Splenic Nerve Neuromodulation Reduces Inflammation and Promotes Resolution in Chronically Implanted Pigs

Figures

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  Figure 1. Burst Stimulation (10 Hz; 0.5s on 4.5s off) Sustains SpN Activation. (A) Representative SpN eCAPs obtained when stimulating the SpN at 1, 10, 30 Hz or 10 Hz burst (5 pulses delivered at 10 Hz, with a 4.5 s delay between bursts). Each image shows 300 consecutive pulses overlapped. The stimulation artefact was removed for clarity. The dashed lines indicate the latency of the peak of the first (white line) and last (red line) recorded eCAP. The arrows indicate the amplitude of the peak of the first (white arrow) and last (red arrow) recorded eCAP. (B) Quantified eCAP amplitude (expressed as percentage of the max response) over 600 consecutive pulses with the different pattern of stimulations shown. Data are shown as mean (n = 3). Least-squares regression curves were fitted against the data. (C) Quantified eCAP latency (expressed as percentage of the first response) over 600 consecutive pulses with the different pattern of stimulations shown in (A). Data are shown as mean (n = 3). Least-squares regression curves were fitted against the latency data. (D) eCAP amplitude (expressed as percentage of the first response) over the first 5-10 pulses with the different pattern of stimulations.

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  Figure 2. Burst Stimulation (10 Hz; 0.5s on 4.5s off) Sustains NA Release While Reducing Cardiovascular Effects. (A) Changes in SpA BF and mABP during a 60 s stimulation of the SpN using a 5 µC stimulus delivered at 10 Hz, 1 Hz or 10 Hz burst stimulation. Data are from stimulations within the same representative animal (shown as empty circles in 2B). (B) Relative SpA BF and mABP changes recorded during a 60 s stimulation delivered with stimulation at 1 or 10 Hz continuous or 10 Hz burst. Values are expressed as percentage of the average maximum change obtained at 10 Hz continuous across all animals. Individual data points are shown (at least 2 replicates from each animal) and mean (n = 4) + s.d. * represent significant statistical difference from 10 Hz continuous. (C) Graph showing the concentration of NA at each time point during baseline and stimulation periods (2 min) at different amplitude (0-11 µC) or pattern (continuous or burst at 10Hz) as indicated. Data are shown as mean (n = 5) + s.e.m. (D, E) Graph showing the comparison of NA release during stimulation at 10 Hz continuous or 10 Hz burst at the same stimulation intensity, expressed as AUC (D) or as AUC normalized over the total…

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  Figure 3. Chronic Conscious Neuromodulation Study Methods and Refinement of the Minimally-Invasive Surgical Implantation Procedure. (A) Schematic of overall chronic conscious study design. Each phase is described in more detail in the methods and supplementary information. (B) Schematic of surgical implantation procedure showing trocar position and use, relative to the spleen. (C) Schematic of chronic neuromodulation and blood sampling procedure. Animals were implanted with a catheter in the jugular vein and a circumferential cuff electrode around the splenic neurovascular bundle (NVB) connected to an implantable pulse generator (IPG). The IPG was wirelessly controlled by a tablet to control stimulation parameters and measure impedance.

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  Figure 4. Chronic SpN Neuromodulation Evokes Anti-Inflammatory Effects During LPS-Induced Endotoxemia. (A) Graphs showing TNF-α concentration quantified in the peripheral plasma collected at different time points after i.v. injection of 0.025 µg/kg of LPS. The data are shown for non-surgical (NS) sham (grey), sham (black) and SpNS (red) animals. (B) Graph showing the quantification of the relevant TNF-α AUC (between 0.5-2 h post LPS) derived from (A). Data are shown and were analyzed using the expressed as Log10 normalized AUC values. The single data points with mean are shown (C, D) Graphs showing total white blood cell (C) or monocyte (D) cell counts from peripheral blood between 0 and 24 h post LPS injection. (E, F) Quantification of peripheral blood monocytes stained with antibodies against CD16 (E) or CD172a (F) over time. (G) Representative histograms showing the changes over time of CD14 expression on CD16+ monocytes in a sham and a SpNS representative pig. (H) Quantification of the median fluorescence intensity (MFI) of CD14 expression on CD16+ monocytes over time. (A, C–F, H) Data are expressed as mean ± s.e.m. (E, F, H) Data are expressed as relative change over the baseline…

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  Figure 5. Chronic SpN Neuromodulation reprograms peripheral blood lipid mediator profiles. Porcine splenic nerve was stimulated chronically for 8 days. Plasma was collected immediately prior to LPS challenge (A), 0.5 (B), 3 (C) and 24 (D) h post LPS challenge and lipid mediators were investigated using LC-MS/MS-based profiling. Results were interrogated using Partial Least Square Discriminant Analysis. (Left panels) Display Score Plots. Colored area represents the 95% interval confidence. (Right panels) Plots displaying the lipid mediators with the 10 highest VIP scores from component 1. n = 5 for Sham group and n = 6 for SpNS group. (E) Flux down each of the bioactive metabolomes was assessed in the samples collected 24h post LPS injection. Pathway analysis for the differential expression of mediators from the DHA and n-3 DPA (left panel) and EPA and AA (right panel) bioactive metabolomes in SpNS group when compared to Sham group. Results are expressed as the fold change. Green boxes indicate the SPMs upregulated in the SpNS group at 24h post LPS (vs Sham). n = 5 for Sham group and n = 6 for SpNS group.

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  Figure 6. Physiological and Histological Assessment Confirms Integrity of Electrode-Nerve-Spleen Circuitry at Termination. (A, C) Percentage changes (normalized to pre-stimulation baseline) in mABP (A) and SpA BF (C) obtained when stimulating the SpN with the implanted cuff at different intensities, using an external stimulator, up to 40 μC. The curves obtained with two different pulse widths (1 and 2 ms) are shown. (B, D) Graphs showing the maximum percentage changes in mABP and SpA BF achieved at termination at relevant IPG output (15 or 40 μC), delivered at 10 Hz continuously for 60 s. Data in A-D are shown as mean ± s.e.m. (E) Low power image of transverse section from the splenic artery from a representative animal. The SpA is surrounded by numerous fascicles with normal morphology. The white band encircling the artery and fascicles represents the former location of the implanted cuff. The black box highlights the region represented in (E-e; higher power image) and shows nerve fascicles with normal morphology. A band of mature fibrotic tissue is part of the fibrotic capsule surrounding the implant and represents an expected and acceptable response to the device. (F) Low power image of…