Fig. 5

The effect of LIPUS stimulation on EAM mice with splenic denervation surgery. A Timeline of a LIPUS treatment experiment on EAM mice with splenic denervation surgery. B Photographs of surgical exposure and alcohol application for denervation operation. C Representative images of whole spleen sections of EAM mice or denervated EAM mice 6 days after surgery. Red, tyrosine hydroxylase (TH) staining. Blue, nuclear staining. In statistical analysis, quantification revealed a significant decrease in tyrosine hydroxylase immunoreactivity in denervated spleens relative to spleens from mice subjected to EAM (n = 3). ^* Represents EAM vs. EAM + SDN. D HE and MASSON staining for the degree of inflammatory cell infiltration and collagen deposition of EAM hearts. Scale: 500 μm (top) and 50 μm (bottom). Representative HE and MASSON staining images of indicated groups. Bar graphs on the right show quantitation of data (n = 6). E The BW change, SW/BW and HW/BW of EAM mice in different experimental groups (n = 6). F, G Representative FACS quantification of Treg and macrophage percentage of splenic (F) or heart (G) isolated immune cells from EAM mice with additional LIPUS stimulation at 21 days (n = 6). ^* Represents EAM vs. LIPUS-0.35 MPa; ^# represents LIPUS-0.35 MPa vs. LIPUS + SDN; ⁺ represents EAM vs. LIPUS + SDN. (Data were shown as the mean ± SD, Student’s t-test for two-group comparison and one-way analysis of variance, followed by Tukey’s multiple comparison tests for comparison of multiple groups; ^*P or ^#P < 0.05, ^**P or ^##P < 0.01, ^***P or ^###P < 0.001.) EAM: experimental autoimmune myocarditis; LIPUS: low-intensity pulsed ultrasound. SDN: splenic denervation; TH: tyrosine hydroxylase; HE: hematoxylin and eosin; BW: body weight; SW/BW: spleen weight to body weight; HW/BW: heart weight to body weight; FACS: fluorescence activated cell sorting