Fig 2: Effect of caspase-6 or -8 inhibition on caspase-3 activation after cerebral ischemia. (a) Two millimeter-thick coronal sections of rat brain stained with 2, 3, 5-triphenyletrazolium chloride (TTC) solution. Individual schematic representations of each group showing the location of tissue sampling (black square) for western blot analysis: control (DMSO; n=4), caspase-6 inhibitor (Z-VEID-FMK; n=4), and caspase-8 inhibitor (Z-IETD-FMK; n=4); (b and c) western blot analysis of brain samples (b) and whole retina (c) following control, caspase-6 inhibitor, or caspase-8 inhibitor delivery. The bands corresponding to the cleaved caspase-6 p10 subunit, cleaved caspase-8 p18 subunit and cleaved caspase-3 p17 are shown, with the corresponding GAPDH loading control at the bottom. For each treatment group, two lanes were loaded with lysate from the same tissue sample (Z-VEID, Z-IETD, or control DMSO); (d–g) quantification of cleaved caspase-6 (p10) subunit, cleaved caspase-8 (p18) subunit and cleaved caspase-3 (p17) levels at 48 h after embolization. Band intensity was normalized to the amount of GAPDH in each sample. Results are expressed as the mean of three separate brains±S.E.M. Z-VEID and Z-IETD treatment significantly reduced the amount of activated caspase-6 (p10), caspase-8 (p18), and caspase-3 (p17) at 48 h following thromboembolic ischemic injury; (h and i) Caspase activity assay showing CASP3 and CASP6 activity, based on cleavage of a colorimetric substrate in brain samples (h) and whole retinas (i). CASP3 and CASP6 activity was significantly decreased in rats treated with Z-VEID-FMK or Z-IETD-FMK compared with control (n=4 per group, *P<0.001)

Fig 3: Effect of caspase-6 or caspase-8 inhibition on the number of proliferating cells after MCAO. Intravenous administration of a caspase-6 or caspase-8 inhibitor increased the percentage of Ki-67 positive cells after MCAO. (a–l) Fluorescence micrographs of peri-infarct area of the injured hemisphere. Columns (left to right) show Ki-67 immunoreactivity (proliferating cells), DAPI labeling, a merge of the two preceding images, and a higher magnification view of the inset white box in the merged images. Control samples showed scarce Ki-67 positive cells (a–d), whereas samples treated with a caspase-6 inhibitor Z-VEID-FMK (e–h) or caspase-8 inhibitor Z-IETD-FMK (i–l) had an increased percentage of Ki-67 positive cells after MCAO. Arrow in d, h, and l show Ki-67 positive proliferating cells in the peri-infarct region of the infarcted hemisphere; (m) quantification of the mean percentage of Ki-67 positive cells (±S.E.M.) following MCAO. Z-VEID-FMK or Z-IETD-FMK significantly increased the percentage of Ki-67 positive cells (*P<0.01) at 7 days after stroke. Scale bar, 50 µm. n=6 each group

Fig 4: Caspase inhibition promotes RGC survival after ophthalmic artery ligation. (a–i) Epifluorescence micrographs of flat-mounted retinas showing Fluorogold-labeled RGCs at 14 days following ophthalmic artery ligation and various treatments (a–c) control retinas (n=6) had few surviving RGCs; (d–f) caspase-6 inhibition (Z-VEID-FMK; n=6) and caspase-8 inhibition (Z-IETD-FMK; n=6; g–i) increased RGC survival after retinal ischemia; (j) schematic of retinal flat-mounts, showing the three eccentric areas of RGC quantification (inner, middle, outer); (k) quantification of the density (cells/mm2) of surviving RGCs (±S.E.M.) at 14 days following ophthalmic artery ligation and treatment with caspase inhibitors. Z-VEID (caspase-6 inhibitor) or Z-IETD (caspase-8 inhibitor) significantly increased RGC survival (*P<0.001) after retinal ischemia. Scale bar, 50 µm

Fig 5: Caspase-6 and caspase-8 siRNAs promote RGC survival after retinal ischemia. (a–l) Epifluorescence micrographs of flat-mounted retinas showing RNA-binding protein with multiple splicing (RBPMS)-labeled RGCs at 14 days following ophthalmic artery ligation and various treatments. Images were taken in the inner, mid-periphery (middle), or periphery (outer) of the retina. Images in the right-hand column show magnified portions of the boxed regions in the third column (a–d) control retinas (n=6); (e–h) caspase-6 siRNA1 (CASP6 siRNA1; n=6) or caspase-8 siRNA1 (CASP8 siRNA1; n=6; i–l) increased RGC survival after retinal ischemia; (m) quantification of the density (cells/mm2) of surviving RGCs (±S.E.M.) at 14 days following ophthalmic artery ligation and treatment with caspase siRNAs. CASP6 siRNA1 and siRNA2 or CASP8 siRNA1 and siRNA2 significantly increased RGC survival (*P<0.001) after retinal ischemia; scale bar, 50 µm