Neural bridging achieves transcutaneous regulation of vagus nerve function and treatment of depressive disorders

Figures

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  Fig. 1. Schematic of end-to-side neurorrhaphy and other surgery. a, Schematic of end-to-side neurorrhaphy (ventral view) with a magnified inset in the lower-right corner showing the completed anastomosis site. GAN: great auricular nerve. TN: transverse cervical nerve. C3: the third cervical nerve. C3R: the third cervical nerve root. VN: vagus nerve. b, Top: Normal anatomical relationship between the left C3 nerve and vagus nerve in a mouse, with the vagus nerve elevated by a probe. Bottom: The C3 nerve and vagus nerve after end-to-side neurorrhaphy. Both images are shown in a ventral view. Scale bar ​= ​1 ​mm. c, Overview of the control groups and their experimental significance. ETE: end-to-end neurorrhaphy. s-ETS: spiral end-to-side neurorrhaphy. n-ETS: non-windowed end-to-side neurorrhaphy. d, Schematic diagrams of surgical procedures for control groups, each with a magnified inset of the completed anastomosis site.

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  Fig. 2. Neurorrhaphy facilitates axonal regeneration. a, e, Transmission electron microscopy (TEM) images of C3 nerve cross-sections at 5000× and 10,000× magnifications, 8 weeks post-surgery, in the Sham, ETE, Mod, and Cut C3 groups (a) and in the s-ETS and n-ETS groups (e). Scale bar: 5 ​μm (5000×), 2 ​μm (10,000×). b, f, Distribution of myelin sheath thickness across myelinated nerve fibers with varying diameters. c, g, Mean g-ratio values of myelinated fibers in different groups. d, h, Average number of myelinated fibers per field at 2000× magnification. i, Summary of mean g-ratio and myelinated fiber density values. b-c, f–g: 150 randomly selected myelinated fibers per group. d, h: For each mouse, three random fields were selected and the average was taken. n ​= ​10 per group. Statistical significance: ∗∗∗p ​< ​0.001; n.s., no significant difference.

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  Fig. 3. Neurorrhaphy preserved vagus nerve integrity. a, Left: Schematic of the sampling sites for the VN in Mod group mice. Right: Gross observation showing the VN upstream and downstream of the anastomosis site, with sampling locations marked, 8 weeks post-surgery. b, Cross-sectional electron microscopy images of the VN upstream (PVN) and downstream (DVN) of the anastomosis site in the Mod group and the normal VN (NVN) in the Sham group, captured at 2000×, 5000×, and 10,000× magnifications. Scale bars: 10 ​μm (2000×), 5 ​μm (5000×), 2 ​μm (10,000×). c, Distribution of myelin sheath thickness across myelinated nerve fibers of varying diameters in the VN. d, Comparison of mean g-ratio values among the three groups. e, Average number of myelinated fibers per field at 2000× magnification. f, Summary of mean g-ratio and myelinated fiber density values. g, Immunofluorescence co-labeling of NF200, ChAT, and DAPI in the VN across the three groups. Scale bar ​= ​100 ​μm ​h, Fluorescence intensity analysis of NF200 protein. i, Fluorescence intensity analysis of ChAT protein. c, d: 150 randomly selected myelinated fibers per group. e, h, i: For each mouse, three random fields were selected and…

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  Fig. 4. Neurorrhaphy establishes new projections. a, Schematic diagram of the viral retrograde tracing experiment. b, Horizontal sections of the Nucleus of the Solitary Tract (NTS) region (outlined by a white frame). Scale bar ​= ​1 ​mm. Directions: r, rostral; C, caudal; L, left; R, right. c, Enlarged view of the framed region in panel b. Scale bar ​= ​500 ​μm ​d, e, Further magnified views of the framed region in panel c, highlighting the Solitary Tract (ST), Nucleus of the Solitary Tract (NTS), and Fourth Ventricle (4V). Scale bar ​= ​100 ​μm. f, Fluorescence intensity analysis of EGFP in the left NTS region. g, Fluorescence intensity analysis of tdTomato in the left NTS region. h, Colocalization analysis of EGFP and tdTomato in the left ST and NTS regions of the Mod group. Analysis was performed along the black line shown in the left panel. Scale bar ​= ​100 ​μm. i, Schematic diagram of the viral anterograde tracing experiment. Regions a, b, and c correspond to nerve sampling sites after viral injection, representing the proximal vagus nerve (PVN), distal vagus nerve (DVN), and ipsilateral C3 nerve, respectively. j, Longitudinal sections of different nerve segments after viral…

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  Fig. 5. Postoperative auricle stimulation activates ipsilateral NTS neurons. a, Schematic of the c-Fos immunostaining groups and workflow. b, Left: Location of the NTS in the coronal plane of the Allen Mouse Brain Atlas (black frame and ∗ mark). Right: Corresponding location of the NTS in a mouse brain section (white frame and ∗ mark). Scale bar ​= ​500 ​μm. c, Representative grayscale images of c-Fos expression in the left NTS region for different groups (Left) and the corresponding pseudo-colored 3D visualizations (Right). Scale bar ​= ​100 ​μm ​d, Quantitative analysis of c-Fos+ cells in the left NTS region in the Sham, Con, and Mod groups. e, Quantitative analysis of c-Fos+ cells in the Mod group at different time points post-surgery, shown as the ratio of Mod group cell counts to Sham group cell counts. A 1:1 reference line is marked on the graph. f, Spike potentials and local field potentials (LFPs) recorded from the left NTS. Stim: Time of stimulation. g, Power spectral analysis of LFPs from 2 ​s before to 2 ​s after stimulation. h-j, Changes in average power spectral density (PSD) (h), average delta band power (i), and average Spike firing frequency (j) before and after…

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  Fig. 6. End-to-side neurorrhaphy significantly improves depressive behavior. a, Experimental group design and treatment workflow. b, Schematic diagram of the stimulation area, showing the ear clip electrode and stimulation parameters. The red area indicates the anterior auricular region, and the blue area represents the posterior auricular region. ES: Electrical Stimulation. c, Immobility time in the TST. d, Representative movement trajectories in the EPM. e, Representative movement trajectories in the OFT. f, Time spent in the closed arms (left) and open arms (right) of the EPM. g, Comparison of behavioral metrics in the OFT. n ​= ​8 per group. Statistical significance: comparison with the Sham group, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001; n.s.: no significant difference.

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  Fig. 7. Sleep disruption in CUMS mice is improved. a, Implantation sites for EEG and EMG electrodes. In the lower diagram, blue lines represent EEG electrodes, and red lines represent EMG electrodes. b, Schematic diagram of the sleep monitoring system. c, Representative EEG and EMG traces during different sleep phases (WAKE, NREM, and REM). d, 24-h EEG and EMG traces with corresponding PSD images. e, Time-course changes in NREM sleep duration across the four groups over 24 ​h f, Average hourly NREM sleep duration during different phase. g, Average duration of individual NREM sleep bouts. h, Average frequency of NREM sleep bouts per hour. i-k, Relative PSD of NREM sleep during the 24-h period (i), the 12-h light phase (j), and the 12-h dark phase (k). n ​= ​6 per group. Statistical significance: ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001; n.s.: no significant difference.

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  Fig. 8. Treatment normalizes inflammation in the hippocampus. a, Number of DEGs in the hippocampus across comparisons. b, Transcriptomic changes in the hippocampus across different comparisons. c, Heatmap of DEG expression, showing clustering patterns among groups. d, Top 20 biological processes identified by GO enrichment analysis, with the number and proportion of upregulated and downregulated genes. e, Results of KEGG enrichment analysis of DEGs. f, GSEA reveals changes of multiple inflammation-related pathways. g, Detailed GSEA results for inflammation-related pathways from panel f. h, Venn diagram of overlapping DEGs across comparisons. i, KEGG enrichment analysis of the most significant overlapping DEGs and mapped pathways. j, Expression levels of DEGs in the TNF signaling pathway. n ​= ​5 per group. Statistical significance: comparison between CUMS and Sham groups: ∗p ​< ​0.05, ∗∗∗p ​< ​0.001; comparison between Mod and CUMS groups: #p ​< ​0.05, ##p ​< ​0.01, ###p ​< ​0.001.