Fig. 1
Cervical lymph node activation in rats after stroke: a Evans Blue dye (2%, 10 µL) was injected into lateral ventricles in normal male Sprague Dawley (SD) rats. Within 30 min, Evans Blue fluorescence was detected in deep and superficial cervical lymph nodes (CLNs) but not in axillary lymph nodes (ALNs) or inguinal lymph nodes (ILNs). Scale: 500 nm. b–d Male SD rats were subjected to 100 min of transient focal cerebral ischemia. Deep and superficial CLNs were collected and analyzed by immunohistochemistry. We did not count LYVE-1 or Ki67 single-positive cells to exclude potential signal from the macrophage population. b Proliferation of lymphatic endothelial cells were also observed in deep CLNs, but it was not statistically significant. c LYVE-1 positive lymphatic endothelial cells in the area of subcapsular sinus in superficial CLNs rapidly proliferated as early as 3 h until 24 h after focal ischemia (n = 4 biologically independent animals). d At the same time, ILNs were collected as a negative control. Note: Some of Ki67 could be co-expressed with immune cells including macrophages and T cells58. *P < 0.05, **P < 0.01 vs Sham, one-way ANOVA followed by Fisher’s LSD test. Scale: 100 nm. e, f Western blot, following immunoprecipitation, demonstrated that VEGFR3 was phosphorylated in superficial CLNs at 24 h after transient focal ischemia (n = 3). *P < 0.05, unpaired t-test. g PBS (5 µL) or MAZ51 (50 ng/5 µL) was injected into lateral ventricles immediately after reperfusion and FACS analysis was performed at 24 h. h FACS analysis indicated that focal ischemia promoted the proliferation of VEGFR3 positive cells in the superficial CLNs. Intracerebroventricular injection of MAZ51 significantly suppressed the proliferation of VEGFR3-positive cells in CLNs (Sham; n = 3, MCAO; n = 11, MCAO + MAZ51; n = 7 biologically independent animals). *P < 0.05, **P < 0.01, one-way ANOVA followed by Fisher’s LSD test. i Immunohistochemistry demonstrated that MAZ51 treatment decreased the proliferation of LYVE-1 positive lymphatic endothelium that was observed after ischemia (n = 4). Scale: 100 nm. **P < 0.01, unpaired t-test. All values are mean +/− SD.
