Adaptation to cortical hypoperfusion is impaired in the absence of microglia. (a) In vivo two-photon imaging reveals increased microglial process motility (arrowheads) to repeated (3×) CCAo in CX3CR1^tdTomato mice (1st, first-order capillary). n = 6 mice; ****, P < 0.0001, Mann–Whitney U test. Scale bar, 20 μm. (b) Automated morphological analysis demonstrates reduced number of branching and ending nodes of microglial processes ipsilaterally in CX3CR1^GFP/+ mice 24 h after 3× CCAo compared with the contralateral side (contra) and sham animals in the cerebral cortex. Branching/ending nodes of n = 386–388 sham, n = 197 contralateral (contra), and n = 134 ipsilateral (ipsi) cells from n = 3 sham and n = 3 CCAo mice; ***, P = 0.0008, Kruskal–Wallis test followed by Dunn’s multiple comparisons test (branching nodes: ***, P = 0.0008, sham versus ipsi; **, P = 0.005, contra versus ipsi; ending nodes: ***, P = 0.0007, sham versus ipsi; **, P = 0.0083, contra versus ipsi). (c) Representative perfusion (first and third rows), and difference LSCI images (second and fourth rows) showing cortical perfusion changes in response to 3× CCAo (occl.) in control and microglia-depleted mice. Dashed lines indicate the area of quantification in both the ipsilateral (white arrowheads) and contralateral (empty arrowheads) hemisphere as shown in d. Venous sinuses were excluded from the analysis. Scale bar, 1 mm. reperf., reperfusion. (d) CBF responses to 3× CCAo are shown as the percentage of baseline. A significant CBF reduction is seen in the absence of microglia in both hemispheres. n = 9 control and n = 12 depleted mice; ****, P < 0.0001, two-way ANOVA followed by Sidak’s multiple comparison test (ipsilateral second reperfusion [rep.], **, P = 0.0099; third occl., *, P = 0.0270; third rep., ****, P < 0.0001 control versus depleted; contralateral second occl., *, P = 0.0233; second rep., **, P = 0.0052; third occl., ***, P = 0.0001; third rep., ****, P < 0.0001 control versus depleted). (e) ICV clodronate administration resulted in the depletion of CD206-positive PVMs but did not affect microglial cells (P2Y12R labeling, green). Blood vessels were visualized using the endothelial marker, tomato lectin (blue). Scale bar, 20 µm. Quantification of the number of PVMs after ICV clodronate liposomes or PBS injection. n = 5–5 mice control versus clodronate injected; ****, P < 0.0001, unpaired t test with Welch’s correction. (f) Quantification of the number of P2Y12-positive microglia cells after ICV clodronate liposomes or PBS injection. n = 5–5 mice control versus clodronate injected, unpaired t test with Welch’s correction. (g) PVMs were eliminated from the brain by ICV liposomal clodronate injection before LSCI measurements. (h) No difference in CBF is seen between clodronate-treated and control mice after 3× CCAo. n = 5 and 5 mice control versus clodronate injected, two-way ANOVA followed by Sidak’s multiple comparison test. Data are expressed as mean ± SEM. LSCI data have been pooled from two to three independent experiments.