Fig. 7

Ischemia induces pericyte contraction by multiple calcium influx pathways. a-f Intravitreal injection of calcium fluorophore, Fluo-4, to NG2-DsRed (a-b) or wild type (c-d) mice labeled only a small number of pericytes in non-ischemic retinae (c, e), whereas ischemia strikingly enhanced the Fluo-4 signal in pericytes (b, d-e) (non-ischemia: n = 7 retinae; ischemia: n = 3 retinae; P = 0.001, ANOVA and Dunnett’s test). Most of the intensely calcium signal labeled pericytes were colocalized with microvascular constrictions (arrows in d). Vessel contours in non-ischemic retinae were traced by following the week Fluo-4 labeling along the vessel wall. Intravitreal administration of gap junction blocker carbenoxolone (CBX) also reduced the number of pericytes labeled with Fluo-4 (e; ischemia: n = 3 retinae; CBX-treated ischemia: n = 4 retinae; P = 0.018, ANOVA and Dunnett’s test). f Ischemia-induced microvessel constrictions (n = 3 ischemic retinae) were prevented by pre-ischemic intravitreal administration of calcium antagonist amlodipine (n = 3 retinae; P = 0.001, ANOVA and Dunnett’s test), pericyte relaxant adenosine (n = 4 retinae; P = 0.01, ANOVA and Dunnett’s test), and gap junction blocker carbenoxolone (n = 4 retinae), whereas recanalized retinas had similar number of constrictions to the ischemic ones (3 recanalized retinae; P = 0.53, ANOVA and Dunnett’s test). g-i Intravitreal administration of CBX caused glia and their end-feet over the microvessels to become intensely Fluo-4-positive (inset and arrowheads in g), suggesting that blockade of gap junctions might have led to calcium rise within glia (h; non-ischemia: n = 7 retinae; ischemia: n = 3 retinae; CBX-treated ischemia: n = 4 retinae; P < 0.0001, ANOVA and Tukey’s test). i The specific connexin-43 blocking peptide also led to a massive increase of intracellular calcium of Müller cell-like structures and their end-feet during ischemia. Arrowheads and asterisks indicate pericyte soma and Muller end-feet surrounding capillaries, respectively. Scale bars in a-b = 10 μm; in c-d = 40 μm; in g = 20 μm; in i = 5 μm