Supplementary Figure 7: Control studies showing that diphtheria toxin (DT) does not alter pericyte coverage, cerebral blood flow response, cerebrovascular integrity, neuronal numbers, neurofilament density, and behavior in iDTR mice treated with TAM and DT, or in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle.
From: Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss
(a,b) Confocal microscopy images (a) and quantification (b) of CD13+ pericyte coverage in the primary somatosensory cortex (Ctx) in iDTR animals treated with tamoxifen (TAM, 40 mg/kg i.p. daily for 7 consecutive days) and DT (0.1 µg per day for 10 consecutive days), and in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle, using the same protocol as in the main Figs. 2 and 3. The analyses were performed 3 days post-DT or vehicle treatment. Bar = 20 μm. Mean ± S.E.M.; n = 5 mice/group. (c) Cerebral blood flow (CBF) response to an electrical hindlimb stimulus by laser doppler flowmetry in iDTR animals treated with TAM + DT, and in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle 3 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group; one way ANOVA followed by Bonferroni posthoc test. (d,e) Confocal images (d; Bar = 20 μm) and quantification (e) of extravascular fibrinogen and IgG deposits in the primary somatosensory cortex of iDTR animals treated with TAM + DT, and Cre mice treated with TAM and vehicle at 3 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group. (f,g) Confocal images (f; Bar = 20 μm) and quantification of ZO-1 tight junction protein length (g) in the Ctx of iDTR animals treated with TAM + DT, and Cre mice treated with TAM and vehicle 3 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group. (h-j) Confocal images (h) and quantification of NeuN+ neurons (i) and SMI312+ neurofilaments (j) in the Ctx and Hipp of iDTR animals treated with TAM + DT, and Cre mice treated with TAM and vehicle 15 days post-DT or vehicle treatment. Bar = 50 μm. Mean ± S.E.M.; n = 5 mice/group. (k,l) Novel object location (k) and fear conditioning (l) in iDTR animals treated with TAM + DT, and in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle at 15 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group. In b, e, g, i-l, significance by two-tailed Student’s t-test. (Supplementary Figure 3 and Fig. 4e-k).
