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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: Retinal ischemia induces α-SMA-mediated capillary pericyte contraction coincident with perivascular glycogen depletion

Fig. 2

Ischemia-induced microvascular constrictions are caused by pericyte contraction mediated by α-SMA. a and b Ex vivo fluorescence imaging of vasculature filled with FITC–dextran-70S on whole-mounted retinae from non-ischemic and recanalized eyes confirms incomplete filling of microcirculation after recanalization (b) compared to the well-visualized microvessels in a non-ischemic retina (a). c-e Ex vivo co-labeling of the retinae with anti-claudin-5 and anti-NG2 antibodies (c and e) or lectin and anti-PDGFRβ antibody (d) shows that several capillary segments (green) under the pericyte soma or processes (red, in c and d) were constricted (a focal narrowing to < 1/2 diameter, arrows) in retinae subjected to 1 h of central retinal artery occlusion and 1 h of recanalisation. Note that, in addition to the constricted segments under the junctional pericytes, constrictions were also present near the pericytes with helical processes located at the straight parts of capillaries (e, see also the green channel separately in Additional file 1: Figure S1). f The number of microvessel (< 9 μm) constrictions (measured within < 10 μm away from or under pericyte soma) in a whole-mount retina that colocalized with NG2+ pericytes increased significantly with ischemia (non-ischemia: n = 5 retinae; ischemia: n = 3 retinae; P = 0.004, ANOVA and Dunnett’s test) and did not recover after recanalisation, (recanalisation: n = 5 retinae; P = 0.002 compared to non-ischemia, ANOVA and Dunnett’s test). g The diameter of the microvessels was also reduced after ischemia (red; n = 4268 microvessels in three retinae) and recanalisation (green; n = 4533 microvessels in three retinae) compared to the non-ischemic retina (blue; n = 5444 microvessels in three retinae). The diameter reduction was more evident in small microvessels. h and i Knocking down α-SMA expression prevented 1 h-ischemia-induced constrictions. h Illustrates microvessels from an ischemic retina pre-treated with α-SMA siRNA 24 h before induction of ischemia. α-SMA was immunostained red and vessels were labeled green with lectin. There were no constrictions at capillary segments where α-SMA expression was knockdown (only green labeled, arrowheads), whereas constrictions continued (arrows) at segments where α-SMA expression was preserved (red). Note that the capillary on the right bottom had a smaller radius at the proximal part where α-SMA was still expressed whereas its distal segment had a larger diameter and no constrictions (inset on the right). A similar pattern is also seen in the left upper capillary (inset on the left). i Graph illustrates the number of constrictions in non-ischemic, scrambled siRNA-pretreated ischemic and α-SMA siRNA-pretreated ischemic retinae (n = 3 retinae per group; *P = 0.007, ANOVA and Tukey’s test). Scale bar in a-b = 50 μm; in c-d = 10 μm; in e = 5 μm; in h = 5 μm (left), 25 μm (middle), 5 μm (right)

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