Fig. 1: AAV-sR3 induced dLV regression in WT and APdE9 mice impairs CSF outflow into cLNs but improves CSF outflow into blood circulation.
Comparison of littermate AAV-Ctrl and AAV-sR3 treated WT and APdE9 mice at 6 (female) and 16 (male) months of age. COS, confluence of sinuses; dcLN, deep cervical lymph node; scLN, superficial cervical lymph node; SSS, superior sagittal sinus; TS, transverse sinus. a, Schedule indicating AAV administration and experimental analysis time points. b, Western blot showing mVEGFR3-Ig protein in serum after AAV injection. Control sample is from a mouse with no detectable protein expression (unsuccessful injection), which was omitted from the analysis. c, Simplified schematic illustration of dLVs (green) attached to the basal and dorsal cranium and spinal canal. d–g, Comparison of LYVE1+/PROX1+ dLVs in the dorsal skull at 6 months (n = 7, 7, 4 and 6) (d,f) and 16 months (n = 3, 3, 3 and 3) (e,g) of age. LYVE1 staining in white (d,e). Yellow arrowheads point to dLV branches visible only in mice injected with AAV-Ctrl. Pineal gland was excised from the middle of COS in (d,e) to visualize all dLVs. h, The schedule of CSF drainage analysis. i–n, Comparison of IgG–RPE tracer signal in dcLNs (n = 9, 9, 5 and 6) of 6-month-old mice (i,j) and in scLNs (n = 14, 16, 9 and 11) and dcLNs (n = 14,16,10,11) (k,n) of 16-month-old mice 180 min after i.c.m. injection. LN values in (j, m, n) represent an average of both sides; maximum one LN per side per mouse was used for quantification. o, Kinetic analysis of IgG–RPE tracer appearance in systemic blood (saphenous vein) at 30, 60, 120 and 180 min after i.c.m. injection into 16-month-old mice (n = 11, 15, 9 and 11) visualized in three different ways. Data shown are representative of at least two independent experiments using littermate mice. The data points represent individual mice. LN and blood IgG–RPE tracer signal values are normalized to the average of the WT-Ctrl group of every experiment set at the 3-h time point. P values were calculated using two-way ANOVA (f,g,j,m,n) and three-way repeated measures mixed-effects model with Tukey’s post hoc test for multiple comparisons (o). Data are presented as mean ± s.e.m. Scale bars, 400 µm (d,e,i,l) and 1 mm (k).
