Fig. 2: Vagus nerve stimulation harnesses choline acetyltransferase-expressing T lymphocytes in the spleen to stimulate circulating platelets via α7nAChR.

a C57BL6/J mice underwent splenectomy or sham-splenectomy followed by vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. Sham SPX + Sham VNS (n = 7), Sham SPX + VNS (n = 6), SPX + Sham VNS (n = 9), SPX + VNS (n = 8) mice per group. **p = 0.009 vs. sham in sham-splenectomy group. p = 0.15 VNS vs. sham after SPX. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. b Wild-type BALB/c or T lymphocyte deficient (Foxn1^nu) mice received vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. WT + Sham VNS (n = 5), WT + VNS (n = 4), Foxn1^nu + Sham VNS (n = 9), Foxn1^nu + VNS (n = 10) mice per group. *p = 0.03 vs. sham in WT mice. p = 0.60 VNS vs. sham in Foxn1^nu mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. c T lymphocyte deficient (Foxn1^nu) mice were reconstituted with ChAT-eGFP⁺ or ChAT-eGFP^- cells followed by vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. ChAT-eGFP⁻ + Sham VNS (n = 8), ChAT-eGFP⁻ + VNS (n = 7), ChAT-eGFP⁺ + Sham VNS (n = 5), ChAT-eGFP⁺ + VNS (n = 6) mice per group. **p = 0.005 vs. sham in ChAT-eGFP⁺ cells. p = 0.85 VNS vs. sham in ChAT-eGFP^- cells. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. d Representative confocal microscopy image (10X) of spleen from ChAT-TdTomato mouse with immunostaining of CD41⁺ platelets (green), CD4⁺ T lymphocytes (purple), and ChAT-eGFP⁺ cells (red) throughout spleen but mostly in the peripheral white pulp (arrows). Scale bar = 200 µm. Experiments were performed independently at least three times with similar results. e Representative merged confocal microscopy image (left, 63X) of spleen from ChAT-TdTomato mouse showing CD4⁺ ChAT-eGFP⁺ T lymphocyte (red and pink, center) in direct contact with CD41⁺ platelets (green). Scale bar = 10 µm. Representative confocal microscopy of individual color channels (right, 63X) including CD41⁺ platelets (green), CD4⁺ T lymphocytes (purple), ChAT-eGFP⁺ cell (red), and DAPI (blue). Scale bars = 10 µm. Experiments were performed independently at least three times with similar results. f Wild-type or α7nAChR-deficient (α7KO) mice received vagus nerve stimulation or sham stimulation before tail transection. Data were presented as mean ± s.e.m. WT + Sham VNS (n = 7), WT + VNS (n = 7), α7KO + Sham VNS (n = 9), α7KO + VNS (n = 9) mice per group. *p = 0.025 vs. sham in WT mice. p = 0.87 VNS vs. sham in α7KO mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. g Wild-type or α7nAChR-deficient (α7KO) mice received vagus nerve stimulation or sham stimulation before carotid artery injury. Data were presented as mean ± s.e.m. WT + Sham VNS (n = 5), WT + VNS (n = 5), α7KO + Sham VNS (n = 5), α7KO + VNS (n = 5) mice per group. *p = 0.046 vs. sham in WT mice. p = 0.82 VNS vs. sham in α7KO mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. h Wild-type or α7nAChR-deficient (α7KO) mice received nicotine or vehicle before tail transection. Data were presented as mean ± s.e.m. WT + Vehicle (n = 4), WT + Nicotine (n = 5), α7KO + Vehicle (n = 8), α7KO + Nicotine (n = 7). **p = 0.0037 vs. vehicle in WT mice. p = 0.88 Nic vs. vehicle in α7KO mice. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. i α7nAChR-deficient (α7KO) mice were reconstituted with platelets from wild-type or α7KO mice, followed by treatment with nicotine or vehicle before tail transection. Data are presented as mean ± s.e.m. WT platelets + Vehicle (n = 6), WT Platelets + Nicotine (n = 7), α7KO Platelets + Vehicle (n = 5), α7KO Platelets + Nicotine (n = 5). *p = 0.029 vs. vehicle with WT platelets. p = 0.52 Nic vs. vehicle with α7KO platelets. Statistical significance was determined by unpaired two-tailed Student’s t-test. The figure represents pooled results from two experiments performed independently. Source data are provided as a Source Data File.