GLUT1 is up-regulated in SIRT6-KO retina.

<p>a) GLUTl immunoreactivity in cross-section of WT and SIRT6-KO mice retina. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL) Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). GLUT1 protein (b) and mRNA levels (c) were determined by Western blot and RT-PCR respectively. β-actin was used as loading control. Data are mean ± SE (n = 6 eyes/group) **p<0.01</p

SIRT6 is active in the mouse retina.

<p>a) H3K56 acetylation is shown by immunofluoescence. b) Representative Western blot showing protein levels of SIRT6 and the acetylation levels of H3K56 and H3K9 in chromatin preparations from WT and KO mice retinas. Total H3 was used for normalization. c) Quantification of the intensity of bands was determined by using the ImageJ and is represented as arbitrary units. Data are mean ± SE (n = 6 eyes/group). **p<0.01, ***p<0.001</p

Grm6 is down-regulated in SIRT6-KO retinas.

<p>Whole retina mRNA from WT and KO mice was used to profile the expression of several key genes of glutamate receptors involved in the synaptic transmission in an Affymetrix Mouse Gene 2.1 ST DNA microarray. a) Heatmap representing the hierarchical cluster analysis shows the differential expressed mRNAs between WT and SIRT6 KO retinas. The graphic depicts the expression levels of ionotropic AMPA glutamate receptors (Gria1–4), Glutamate receptor, ionotropic kainate (Grik1-2-4-5), Glutamate [NMDA] receptors (Grin1-2a-c) and metabotropic glutamate receptors (Grm1–8). The expression data for the hierarchical clustering image has been row normalized to a range of zero to one with blue representing the row minimum and red representing the row maximum. b) RNA was purified from SIRT6 WT and KO retinas, and Grm6 levels analyzed by RT-PCR. c) immunofluorescence was performed in SIRT6 WT and KO retinas with the indicated antibodies. PKC-alpha was used as a marker for ON bipolar cells. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). Data are mean ± SE (n = 4) **p<0.01 d) Representative fluorescent images of TUNEL analysis performed in WT and SIRT6 KO retinal sections. Apoptotic nuclei (bright green dots) labeled with fluorescein-dUTP were visualized by fluorescence microscopy. Data are mean ± SE (n  = 3) **p<0.01</p

Retinal functional evaluation.

<p>Representative scotopic (A) and photopic (C) electroretinograms from WT and SIRT6-KO mice at different light intensities (dBs). Plots B and D depict average amplitudes of <i>a</i>-wave and <i>b</i>-wave. Note that the fold decrease of the scotopic <i>a</i>-wave amplitude (8) is greater than the fold decrease of the photopic <i>a</i>-wave amplitude (2,5). Data are mean ± SE (n =  4). **p<0.01, ***p<0.001.</p

ONH sections from a control or a CS-treated eye without or with ischemia pulses.

<p>(A) Healthy, intact control optic nerve. Note the homogeneity of the staining. In vehicle-injected eyes, individual axons were generally uniform in shape, rounded and packed together tightly to form the fibers of the healthy nerve. In CS-treated eye without ischemia pulses (B) a less stained area indicates a nerve alteration. Disease in individual axons was characterized by axonal distention and distortion that resulted in a departure from the circular morphology of normal axons. In contrast, a conserved structure of the ONH was observed in the CS-treated eye with ischemia pulses (C). Toluidine blue. (D) Number of axons in eyes injected with vehicle or CS without or with ischemia pulses. A significant decrease in the axon number was observed in CS- injected eyes without ischemia pulses as compared with vehicle-injected eyes (sham), whereas ischemia pulses significantly preserved this parameter. Scale bar: 10 µm. Data are mean ± SEM (n = 5 eyes/group) *p<0.05 vehicle injected eyes without ischemia pulses (sham), a: p<0.05 versus CS-injected eyes without ischemia pulses (sham), by Tukey's test.</p

Retinal histology examination after 10 weeks of ocular hypertension.

<p>Upper panel: Representative photomicrographs of retinal sections stained with hematoxylin and eosin from a vehicle-injected eye, and a hypertensive eye without or with pulses of ischemia. Note the diminution of GCL cells in the eye injected with CS without ischemia pulses. The application of ischemia pulses preserved this parameter. The other retinal layers showed a normal appearance in all groups. Middle panel: Immunohistochemical detection of NeuN-positive neurons in the GCL from a vehicle-injected eye, a hypertensive eye without or with ischemia pulses. A strong NeuN-immunostaining (red) was confined to ganglion cells in the GCL. The number of NeuN positive ganglion cells was lower in hypertensive eyes without ischemia pulses than in vehicle- injected eyes, whereas the application of ischemia pulses in CS-injected eyes increased NeuN-immunostaining. A similar profile was observed for cell nuclei counterstained with DAPI (blue). Lower Panel: cell count in the GCL evaluated by H&E staining, NeuN immunostaining, and DAPI labeling. By all these methods, a significant decrease of the number of cells in the GCL was observed in CS- injected eyes without ischemia pulses as compared with vehicle-injected eyes (sham), whereas ischemia pulses significantly preserved this parameter in CS-injected eyes. Scale bar: Upper panel  =  50 µm; Middle panel  =  50 µm. Data are the mean ± SEM (n = 5 animals per group). * p<0.05, ** p<0.01 vehicle injected eyes without ischemia pulses (sham), a: p<0.05, b: p<0.01 versus CS-injected eyes without ischemia pulses (sham), by Tukey's test. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.</p

Effect of ischemia pulses on IOP in vehicle and CS-injected eyes.

<p>TonoPen measurements of IOP from eyes bilaterally injected with vehicle or CS and submitted to ischemia pulses or sham procedure. IOP was assessed at 6, 7, 8, 9, and 10 weeks of weekly intracameral injections. At all time points examined, CS significantly increased IOP as compared with vehicle-injected eyes. Ischemia pulses did not modify this parameter in vehicle or CS-injected eyes at any time point. Data are the mean ± SEM (n = 10 animals per group).</p><p>**p<0.01 versus vehicle-injected eyes without ischemia pulses (sham), by Tukey's test.</p

Electroretinographic preservation in hypertensive eyes induced by the application of brief ischemia pulses.

<p>ERGs were registered after 10 weeks of treatment with vehicle or CS. CS induced a significant decrease in ERG a- and b-wave amplitude, as compared with vehicle-injected eyes. In hypertensive eyes submitted to ischemia pulses, a significant reversion of these alterations was observed. The lower panel shows representative scotopic ERG traces from eyes injected with vehicle or CS without or with ischemia pulses. Data are the mean ± SEM (n = 10 animals per group); **p<0.01 versus vehicle injected eyes without ischemia pulses (sham); a: p<0.05, versus CS-injected eyes without ischemia pulses (sham), by Tukey's test.</p

Flash VEPs in eyes injected with vehicle or CS without or with ischemia pulses.

<p>Animals were weekly injected with vehicle or CS for 10 weeks. Ischemia pulses were applied in one eye, while the contralateral eye was submitted to a sham procedure. Left panel shows average amplitudes of VEP N2-P2 component amplitude and right panel shows representative VEP traces. A significant reduction in flash VEP N2-P2 amplitude component was observed in eyes injected with CS for 10 weeks without ischemia pulses. The application of weekly ischemia pulses significantly abrogated the effect of ocular hypertension. No changes between vehicle injected eyes without or with ischemia pulses were observed. Data are mean ± SEM (n = 10 eyes/group), **p<0.01 versus vehicle injected eyes without ischemia pulses (sham), a: p<0.05 versus CS-injected eyes without ischemia pulses (sham), by Tukey's test.</p

Retinal TBARS levels in animals injected with CS or vehicle, without o with ischemia pulses.

<p>This parameter was significantly higher in eyes injected with CS without ischemia pulses than in those injected with vehicle. The application of brief ischemia pulses in CS-injected significantly reversed the increase in retinal lipid peroxidation. Data are mean ± SEM (n = 12 eyes/group), * p<0.05, **p<0.01 vs. vehicle injected eyes without ischemia pulses (sham), a: p<0.05 vs. CS-injected eyes submitted to a sham procedure, by Tukey's test.</p