Noninvasive Vagus Nerve Electrical Stimulation for Immune Modulation in Sepsis Therapy

Abstract

Sepsis presents a significant medical challenge due to its intense inflammatory response to infection, often resulting in high mortality rates. A promising therapeutic strategy targets the cholinergic anti-inflammatory pathway (CAIP), which regulates immune responses. This study investigates the ingestion of piezoelectric particles that adhere to the stomach lining, specifically targeting TRPV1 receptors. In a mouse model of sepsis, these particles, when activated by low-intensity pulsed ultrasound, generate mild electrical pulses. These pulses stimulate vagal afferent fibers, transmitting signals to the brain and modulating the neural-immune network via the CAIP. Consequently, this leads to a reduction in systemic inflammation, mitigating weight loss, alleviating multiple tissue injuries, and preventing death by modulating immune cells in the spleen. This approach addresses the critical need for noninvasive sepsis therapies, potentially improving patient outcomes. Utilizing portable ultrasound equipment with minimal thermal effects, this technique offers a safe and convenient treatment option, even for home use.

Figure 1

A noninvasive vagus nerve electrical stimulation system for immune modulation and its operating mechanism. (a) Synthesis and composition of BTO@Cap particles. (b) Upon oral administration, BTO@Cap particles adhere to the stomach lining, specifically targeting TRPV1 receptors. When exposed to low-intensity pulsed US, these particles generate mild electric pulses. These pulses stimulate vagal afferent fibers, which then transmit signals to the brain. This interaction modulates the neural-immune network via the CAIP, reducing systemic inflammation, multiple organ injuries, and mortality rates. As a result, this process significantly improves outcomes in mice with LPS-induced sepsis. Images were created with Biorender.com.

Figure 2

Characteristics of BTO@Cap particles. (a) SEM images of BTO particles and BTO@Cap particles before and after treatment in SGF or SIF under US stimulation. (b) Raman spectra of BTO and BTO@Cap particles. (c) COMSOL simulation of a single BTO particle under US irradiation at a power intensity of 0.3 W/cm². (d) PFM images showing the topography, amplitude, and phase of BTO and BTO@Cap particles. (e) Piezoresponse amplitude–voltage and phase–voltage curves of BTO and BTO@Cap particles. (f) Photographs of the system with an LED bulb, demonstrating the illumination of the bulb when connected to an electromechanical device with BTO or BTO@Cap particles under US irradiation. (g) Measured current values of electrical outputs from the electromechanical device, comparing outputs without particles and with BTO or BTO@Cap particles at different power intensities. (h) Local levels of H₂O₂, ·OH, and ·O₂^– detected in PBS containing BTO@Cap particles with or without US stimulation over various time periods. (i) TGA curves of BTO particles, BTO–OH particles, Cap, and BTO@Cap particles before and after treatment in SGF or SIF under US stimulation. Each dot represents one observed data point. n.s., not significant (P > 0.05).

Figure 3

In vitro safety and potential of using BTO@Cap/+US for activating VNS. (a) Cytotoxicity assessment of BTO@Cap particles at various concentrations with and without US irradiation. (b) Assessment of cytotoxicity for BTO@Cap particles at a concentration of 4 mg/mL under varying power intensities of single-dose US irradiation. (c) Schematic representation of the US treatment schedule for different doses. (d) Evaluation of cytotoxicity for BTO@Cap particles at a concentration of 4 mg/mL under various doses of US irradiation at a power intensity of 0.3 W/cm². (e) Schematic representation of the transwell model (created with Biorender.com). (f) Representative microscope images of Caco-2 cells illustrating the adsorption of BTO or BTO@Cap particles onto the cell membranes, indicated by white arrowheads, rather than internalization. (g) Fluorescence images depicting Ca²⁺ influx in SH-SY5Y cells following various treatments. (h) Quantification of mean fluorescence intensity representing intracellular Ca²⁺ signal through analysis of fluorescence images. Each dot represents one observed data point. *(P < 0.05), **(P < 0.01), and ***(P < 0.001); n.s., not significant (P > 0.05).

Figure 4

In vivo biodistribution and safety profile of orally ingested BTO@Cap particles. (a) Ex vivo NIR-II images depicting the biodistribution of ICG-labeled BTO and BTO@Cap particles on gastric surfaces at specific time intervals following oral administration, with CPZ pretreatment used as a TRPV1 inhibitor. The average fluorescence intensity on the gastric surface is quantified and presented below each image. (b) Serum levels of AST, ALT, CRE, and BUN in healthy mice and in mice treated with BTO@Cap/+US. (c) H&E staining images of major organs harvested from healthy mice and from mice treated with BTO@Cap/+US. Each dot represents a single data point. n.s., not significant (P > 0.05).

Figure 5

Therapeutic efficacy on survival rate and body weight. (a) Schematic timeline and treatment protocol for studying survival rate and body weight in LPS-induced mice (created with Biorender.com). (b) Survival rate and (c) body weight of healthy mice and LPS-induced septic mice after different treatments. ***(P < 0.001).

Figure 6

Therapeutic efficacy in mitigating cytokine storm and multiple organ injuries. (a) Schematic timeline and treatment protocol for studying pro-inflammatory cytokines and multiple organ injuries in LPS-induced septic mice (created with Biorender.com). (b) Serum levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, and IL-17A) and (c) serum levels of AST, ALT, CRE, and BUN collected from healthy mice and septic mice after different treatments. (d) H&E staining images of liver, kidney, lung, and stomach tissues collected from healthy mice and septic mice after various treatments. Inflamed regions are outlined with red dashed lines, and necrotic regions are outlined with black dashed lines. Each dot represents one data point. *(P < 0.05), **(P < 0.01), and ***(P < 0.001); n.s.: not significant (P > 0.05).

Figure 7

Brain-specific mechanism underlying the anti-inflammatory effects. (a) Schematic diagrams illustrating the location of the NTS in the brainstem (created with Biorender.com). (b) Immunofluorescence staining images depicting c-Fos expression in the area defined as the NTS in septic mice, without or with vagotomy, before and after treatment with BTO@Cap/+US. (c) Illustration of the selected brain regions for analyzing rs-fMRI data. (d) Results of fALFF analysis in the selected brain regions of the two mouse groups. (e) Functional connectivity between the left insular and left amygdala with other selected brain regions, indicated by Pearson’s correlation coefficient (r). mPFC, medial prefrontal cortex; ACA, anterior cingulate area; RSP, retrosplenial area; CPu, caudate putamen; Hip, hippocampus; DG, dentate gyrus; Thal, thalamus; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; M1, primary motor cortex; M2, secondary motor cortex; Ins, insular cortex; TeA, temporal association area; Amyg, amygdala; HT, hypothalamus. Each dot represents one observed data point. *(P < 0.05), and **(P < 0.01).

Figure 8

Spleen-specific mechanism underlying the anti-inflammatory effect. (a) Schematic timeline illustrating the treatment protocol for studying the role of spleen in CAIP in the LPS-induced septic mice (created with Biorender.com). (b) Serum levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, and IL-17A) collected from healthy mice and septic mice that underwent splenectomy after various treatments. (c) Schematic timeline illustrating the treatment protocol for studying the CAIP in the spleen in the LPS-induced septic mice (created with Biorender.com). (d) Splenic NE, (e) Ach, and (f) serum TNF-α levels in healthy mice and septic mice before and after BTO@Cap/+US treatment. Reserpine pretreatment was used to deplete catecholamines. *(P < 0.05), and **(P < 0.01); n.s., not significant (P > 0.05).