Microglia modulation does not change blood gases but impacts on cGMP levels in the cerebral vasculature. (a) In vivo two-photon imaging was performed with resonant scanning (32 Hz) in the somatosensory cortex of CX3CR1^GFP/+ × P2Y12R KO and CX3CR1^GFP/+ (P2Y12R-competent) mice following intravenous Rhodamine B-Dextran (Rhod.-dextran) administration to visualize blood vessels. After recording 60 s of baseline, vasodilation was induced by inhalation of 10% CO₂ in air for 120 s (61–180 s) under normoxic conditions, followed by 60 s of posthypercapnia recording, using a protocol identical to that shown in Fig. 5 d. Scale bar, 10 µm. (b) Arterial pCO₂, pO₂, and pH measurements under ketamine-medetomidine anesthesia after the administration of atipamezole performed before and after hypercapnic challenge. Blood samples were taken from the femoral artery. No significant difference was observed between control and microglia-depleted mice. n = 10 control and n = 8 depleted mice, two-way ANOVA followed by Sidak’s multiple comparison test. (c and d) Both intracellular (c) and extracellular (d) pH markedly decreases within a few minutes after exposing cells to 15% CO₂/85% air gas mixture, as a model of hypercapnia. Extracellular pH was determined by Phenol Red absorbance measurements, and intracellular pH was measured as changes in pHrodo Green AM dye fluorescence in glial cells. n = 4 parallels per group; ***, P = 0.0001, 0 min versus 5 min, paired t test (c); n = 10 parallels per group; ****, P < 0.0001, 0 min versus 5 min, paired t test (d). (e) Hypoxia Green AM loaded cells exhibit significant increase in fluorescent intensity within 10 min after placing microglia cultures to hypoxic environment (1% O₂/5% CO₂/94% N₂). The reagent begins to fluoresce when oxygen levels drop below 5%. n = 50 parallels per group; **, P = 0.0019 0 min versus 10 min, Mann–Whitney U test. Scale bar, 30 µm. Data are shown as mean ± SEM (b–e). (f) Single image planes for CLSM imaging show small blood vessel segments from second and third layer of the neocortex in acute brain slices. Lectin (blue) outlines the vessels, CD13 labels contractile elements (pericytes and smooth muscle cells), microglial P2Y12R is orange, and cGMP signal can be seen in green (arrows). Note that PSB0739 treatment has no effect on SNP-induced cGMP. (g) CLSM imaging shows small blood vessel segments from the second and third layer of the neocortex in perfusion-fixed brain sections. Hypercapnia was induced in vivo and maintained in anesthetized mice until sacrifice. Lectin (blue) outlines the vessels, CD13 labels contractile elements (pericytes and smooth muscle cells), microglial P2Y12R is orange, and cGMP signal can be seen in green (arrows).