Extended Data Fig. 4: Restructuring of the PNS in the vicinity of adventitia NICIs.

a, Approach on tracing in tissue-cleared intact abdominal aorta of young (12 weeks) WT mice. 3D segmentation and manual tracing of NF200⁺ nerves/axon bundles along their entire paths (800–1200 µm thickness) shows connectivities of the sympathetic CG (yellow) and SycGs (light yellow) with the adventitia. An example of a traced axon bundles (red) contacting the adventitia; approximations of diameters of different sizes of nerves/axon bundle connecting the ganglia are indicated. Arrowheads, beginning and ending of the traced axon bundle from adventitia to CG; arrow, direction of tracing. Scale bars, 0.7 mm. b, Light sheet 3D reconstruction images of NF200⁺ nerves and axons in the intact abdominal aorta and periaortic tissues (autofluorescence, magenta) of aged WT (n = 3) and Apoe^−/− (n = 2) mice showing NF200⁺ neuronal structures (green) in the 600 µm-thick abdominal aortic segments (4 µm per z-step; arrow, CG; arrowhead, SycGs). Scale bars, 0.5 mm. c, Light sheet 3D reconstruction in the intact abdominal aorta of aged Apoe^−/− mice showing varicosities (arrows) in axons adjacent to plaque. Inset, single plan image of the 3D projected whole stack (z = 1.2 mm; arrow, varicosities in axons; arrowhead, nerve) (c). Dashed line, media; yellow line, plaque. Scale bars, 500 µm (left), 50 µm (rihgt). d,e, 3D reconstruction and segmentation views of an abdominal aorta region showing NF200⁺ nerves and axons and their spatial relationship to the intact abdominal aorta (z = 320 µm; 4 µm per z-step) in aged Apoe^−/− mice (n = 2). Nerves and axons are evident in the vicinity of the aorta; video 11. Scale bars, 40 µm. f, 3D reconstruction of TDE-cleared whole mount abdominal aorta (z = 80 µm; 1 µm per z-step; n = 3) showing NF200⁺ nerves and axons in the adventitia. White, second harmonic generation (SHG) from collagen in the adventitia. Arrow, axon; double arrowhead, nerve; asterisk, axon neogenesis in ATLO. Scale bars, 70 µm. g, NF200⁺ axons (arrow, arrowhead) in T cell (T) and B cell (B) areas in ATLO, and their quantification in ATLO and Apoe^−/− paraaortic lymph node (pLN). n = 7 ATLOs, 4 pLNs. Scale bar, 30 µm. h, Single neurofilament L (NFL)⁺ immature newly formed axons and double NFL⁺/neurofilament H (NFH)⁺ mature axons in aged WT adventitia vs ATLOs (n = 3 WT, 3 Apoe^−/−). Scale bars, 10 µm. Data are means ± s.e.m. n represents biologically independent animals. Two-sided unpaired Student´s t-test (g); two-way ANOVA with Bonferroni post hoc test (h)

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