Retinal responses.

<p>A, B) ERGs recorded from 4 weeks old WT RA, WT OIR, and A2−/− OIR mice at intensities of 7 cd/m² (candela per square meter) and 13 cd/m² respectively. The data show averages of responses over both eyes, in 9, 6, and 16 WT RA, WT OIR, and A2−/− OIR mice, respectively. C) Mean b-wave amplitudes for WT RA, WT OIR, and A2−/− OIR mice are plotted against intensity. Responses are represented as mean ± SEM of the b-wave amplitudes, measured from the trough of the a-wave to the peak of the b-wave. Asterisks indicate significant differences between the two WT and A2−/− OIR groups (P<0.05, t-test). WT RA responses were significantly larger compared to both OIR groups at all intensities studied. Sample size varies from 6–16. * A2−/− OIR vs WT OIR (P<0.05).</p

Signaling mechanism involved in A2-deficiency mediated neuro-glial protection in OIR retina.

<p>A) Western blot analysis showing the levels of p53, and cleaved caspase 9 in WT RA, WT OIR and A2−/− OIR at stages P12, P14 and P17 of the hypoxic OIR phase. B) Western blots showing changes in the expression of apoptotic markers Bim, BCLxl and cytochrome C in WT RA, WT OIR and A2−/− OIR at P14. N varies from 3–5, and representative images are shown.</p

Arginase activity in retinal samples.

<p>Arginase activity was analyzed in WT RA and WT OIR retinal samples at P12, P14 and P17 and is shown as nmol/mg protein/min. A minimum of 3 animals were used in each group and data are presented as mean ± SEM.</p

Preservation of bipolar cells in A2−/− OIR retina.

<p>A) Immunostaining of WT RA, WT OIR and A2−/−OIR retinal sections at P17 using rod bipolar cell marker PKCα. Scale bar 100 µm B) High magnification images of rod bipolar cells demonstrating the neurodegeneration of rod bipolar cells in WT OIR retina. Scale bar 25 µm. C) Quantification of PKCα positive cells showing significantly increased number in A2−/−OIR retina compared to WT OIR. Data presented as mean±SD. §WT RA vs WT OIR (P<0.01), * A2−/− OIR vs WT OIR (P<0.05), N varies from 3–4. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.</p

Decreased apoptosis in A2−/− OIR retina.

<p><b>A</b>) TUNEL assay of retinal sections showing the presence of numerous apoptotic cells (colocalized with DAPI) in WT OIR compared to room air (RA) controls and A2−/− OIR at P14. B) Quantification of TUNEL positive cells showing significantly increased number in WT OIR at all stages. Numbers of apoptotic cells were significantly reduced in A2−/− OIR compared to WT OIR. Data presented as mean±SD. C) Characterization of TUNEL positive cells on WT OIR retinal cryostat sections at P14, by double immunolabelling using NeuN (ganglion and amacrine cells), CRALBP (Muller glia), PKCα (bipolar cells) and recoverin (photoreceptors) antibodies. Insets show magnified images of TUNEL colocalization with respective cellular markers. N varies from 4–6. §WT RA vs WT OIR (P<0.01), * A2−/− OIR vs WT OIR (P<0.05). Scale bar 100 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.</p

Expression of arginase 2 during OIR.

<p>A) Western blot analysis showing changes in the expression of A2 during OIR. Increased expression in WT OIR was observed at P12. B) Immunohistochemistry of retinal cryostat sections at P12 showing A2 (green) expression in horizontal cells. Calbindin (red) is used as a marker for horizontal cells. Scale bar 100 µm. C) Low magnification confocal images of retinal flat mounts (periphery is outlined) from WT RA, WT OIR and A2 −/− OIR at P12 immunostained for A2 showing enhanced levels in the horizontal cells in WT OIR. White boxes represent area for high magnification images in panel D. Scale bar 100 µm. D) High magnification images of WT RA and WT OIR retinal flatmounts at P12 immunostained for A2, demonstrating increased A2 expression in the horizontal cells of WT OIR compared with WT RA. Scale bar 50 µm. A minimum of three animals was used for each experiment and representative images are shown. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.</p

Decreased glial activation in A2−/− OIR retina.

<p>A) Immunohistochemistry of retinal cryostat sections from WT RA, WT OIR and A2−/− OIR using anti- GFAP antibody at different stages of hypoxic phase. B) Western blot analysis using retinal homogenates showing the changes in GFAP expression in WT OIR retina compared to RA and A2−/− OIR during different stages. C) Quantification of GFAP expression levels in WT RA, WT OIR and A2−/− OIR at P12, P14 and P17. Data presented as mean±SD. §WT RA vs WT OIR (P<0.01), *A2−/− OIR vs WT OIR (P<0.01), N varies from 4–6. Scale bar 100 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.</p

Diabetes-induced increase in tyrosine nitration is blocked in NOX2^-/- chimeras.

<p><b>A</b>. Double labeling of frozen sections with the vascular marker islolectin B₄ (red) and anti-nitrotyrosine antibody (green) shows high levels of nitrotyrosine immunoreactivity in the ganglion cell and inner nuclear layers and within and around the retinal vessels (arrows) in the diabetic WT→WT chimeras. Nitrotyrosine immunoreactivity is weak or absent within the retinas of the control WT→WT chimeras and in the retinal parenchyma of both NOX2^-/-→WT, WT→NOX2^-/- diabetic chimeras. However blood cells within the vessels are nitrotyrosine positive in the diabetic WT→NOX2^-/- chimeras. Scale bar = 40 μm, GCL = ganglion cell layer, INL = inner nuclear layer. <b>B</b>. High power images of the boxed areas in A show nitrotyrosine-positive blood cells (green) within the lectin-labeled blood vessels (red). Scale bar = 25 μm.</p

Diabetes-induced increases in ROS are suppressed in NOX2^-/- chimeras.

<p><b>A</b>. Real time DHE imaging of superoxide formation in retinas from diabetic WT→WT chimeras mice show a strong reaction to DHE as compared with control WT→WT chimeras, the reaction is almost completely abolished in diabetic NOX2^-/-→WT and WT→NOX2^-/- chimeras. Diabetes-induced increase in superoxide production is also abolished by pretreatment with SOD (400U/mL) or apocynin (1 mM). GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer. <b>B</b>. Quantitative analysis of fluorescence intensity in DHE images shows a significant increase in superoxide formation in diabetic WT→WT chimeras. Superoxide production is significantly reduced in diabetic WT→NOX2^-/- and NOX2^-/-→WT chimeras as compared with the diabetic WT→WT chimeras. Pretreatment with apocynin or SOD blocked the DHE reaction. Data represent mean <u>+</u> S.E., *=P<0.001 vs. WT→WT control mice. #=P<0.001 vs WT→WT diabetic mice, n=4, scale bar = 20 μm.</p

Donor genotype determines the genotype of blood cells in recipient mice.

<p><b>A</b>. Genotype of tissue samples from wild type (240 bp) and NOX2^-/- mice (195 bp) before transplant. <b>B</b>. Genotype of blood samples from recipient wild type (240 bp) and NOX2^-/- mice (195 bp) after bone marrow cell transplantation show a band corresponding to the donor genotype. <b>C</b>. Genotype of tissue samples from recipient mice after bone marrow transplant show no changes.</p