(A) Normalized CBF from laser Doppler during CCAO (black bar) in the absence (aCSF, n = 7) or presence of 10 μM Ani9 (n = 7). (B) Normalized CBF during CCAO or reperfusion (average of last 5 minutes of traces in A) in 15 month-old mice (n = 7 for each, each point is 1 Doppler recording) (paired 2-tailed Wilcoxon’s test with continuity correction and Mann-Whitney test). (C) TTC-stained brain sections at 6 hours of reperfusion after CCAO in aged mice. (D) Infarction quantified from mean intensity of TTC-stained sections (Mann-Whitney test) (vehicle, n = 8; Ani9, n = 6). Confocal images of fixed cortical (E) and striatal (F) slices from aged mice that were injected with pimonidazole (Hypoxyprobe) in vivo at 70 minutes after CCAO. Mice underwent 6 hours of reperfusion. Bar graphs indicate Hypoxyprobe intensities in the cortex (E) and striatum (F) from mice treated with aCSF (cortex, n = 40; striatum, n = 13) or Ani9 (cortex, n = 30; striatum, n = 9) (Mann-Whitney test and unpaired 2-tailed Student’s t test). (G) Confocal image of layers II and III in a fixed cortical slice from a mouse undergoing 6 hours of reperfusion after CCAO. (H) Proportion of cells with a glial or neuronal morphology labeled with Hypoxyprobe in the cortex quantified from images, as in H. (I) Cortical NeuN fluorescence intensity at 6 hours reperfusion (aCSF, n = 16; Ani9, n = 12) (unpaired 2-tailed Student’s t test). (J) Schematic of mechanisms revealed. G_qPCR activation triggers the IP₃ pathway; the resulting [Ca²⁺]_i rise stimulates TMEM16A, cell depolarization, and Cav-mediated Ca²⁺ entry. In ischemia, low ATP slows Ca²⁺ pumping, leading to TMEM16A activation, pericyte contraction, and death. Neutrophils and platelets become trapped as pericytes contract and capillaries narrow, further lowering CBF. TMEM16A inhibition enhances capillary reflow and reduces tissue damage. Animal numbers are provided in Supplemental Table 2. Scale bar: 50 μm.