Journal: Communications Biology

Article Title: Age-dependent cerebral vasodilation induced by volatile anesthetics is mediated by NG2 + vascular mural cells

doi: 10.1038/s42003-024-07200-7

Figure Lengend Snippet: a Experimental design for spatially confined ablation of cortical NG2 + cells. NG2-Cre mice were injected with AAV2-FLEx-DTR-EGFP and AAV2-FLEx-EGFP (control) into the right and left cortex, respectively, on day −21. An i.p. injection of diphtheria toxin (DT) on day −7 ablated DTR-expressing NG2 + cells, and two-photon imaging on day 0 assessed changes in arteriole diameter upon SEVO 2.5%. b Images of the cortex stained for cleaved caspase-3. In the control cortex, EGFP + cells (green arrows) express low levels of cleaved caspase-3; in the ablation cortex, most DTR-EGFP + cells show high caspase-3 expression (white arrows), with a few exceptions (yellow star). c Viral transduction range measured by distribution of EGFP + fluorescence intensity (f.u., fluorescence unit) in the control (EGFP) and ablation (DTR-EGFP) cortex ( n = 3 mice per group; F (22, 88) = 1.278, P = 0.2096). ML, midline. d Expression of cleaved caspase-3 measured by fluorescent intensity in either side of the cortex (9 slices from three mice; from left to right, n = 107, 102, 123, and 118 cells; P = 0.0856, 0.2568, and < 0.0001 for others). e Immunofluorescence images of the cortex stained for virally transduced EGFP (green), antibody-labeled NG2 (red), and DAPI (blue) from the control and ablation cortex. f Density of NG2 + DAPI + cells in the control and ablation cortex ( n = 12 slices from three mice per group; P < 0.0001). g Two-photon images of cortical arterioles before and 15 min after SEVO exposure. Arrows indicate SEVO-induced dilation of arterioles in the control (EGFP) but not the ablation (DTR-EGFP) cortex. h Time course of SEVO-induced arteriole dilation in the control ( n = 64 segments from four mice) and ablation ( n = 58 segments from four mice) cortex of adult mice exposed to SEVO at time 0. BL, baseline under awake conditions. i Distribution of initial diameters of the sampled arterioles. There is no difference between the control and ablation cortex ( P = 0.9924). **** P < 0.0001; NS , not significant; by two-way ANOVA ( c ), two-tailed unpaired t- tests ( d ), two-tailed Mann-Whitney U test ( f ), two-tailed paired t -tests ( h ), or Kolmogorov-Smirnov test ( i ).

Article Snippet: To locally ablate NG2 + cells, adult NG2-Cre mice were injected with 0.2 μl of AAV encoding Cre-dependent diphtheria toxin receptor (DTR) (AAV2-FLEx-DTR-GFP; titer 1.3 × 10 13 gc ml −1 ; 124364; Addgene) into the right cortex, and a control reporter (AAV2-FLEx-GFP; titer 1.2 × 10 13 gc ml −1 ; 51502; Addgene) into the left cortex, at the coordinates described above.

Techniques: Injection, Control, Expressing, Imaging, Staining, Transduction, Fluorescence, Immunofluorescence, Labeling, Two Tailed Test, MANN-WHITNEY