IOP in eyes injected with vehicle or CS with or without RON.

<p>TonoPen measurements of IOP from eyes bilaterally injected with vehicle or CS and submitted to a sham procedure or RON performed at 3 (left panel) or 6 (right panel) weeks of treatment with vehicle or CS. At all time points examined, CS significantly increased IOP as compared with vehicle-injected eyes. RON did not modify this parameter in vehicle or CS-injected eyes at any time point. Data are the mean ± SEM (n = 10 eyes per group). **p<0.01 versus vehicle-injected eyes with sham procedure, by Tukey's test.</p

Flash VEPs in eyes injected with vehicle or CS with or without RON.

<p>Animals were weekly injected with vehicle or CS for 10 weeks and submitted to a sham operation or RON at 3 (upper panel) or 6 (lower panel) weeks of intracameral injections. A significant reduction in flash VEP N2-P2 amplitude component was observed in eyes injected with CS with a sham procedure. RON significantly abrogated the effect of ocular hypertension. No changes between vehicle- injected eyes with or without RON were observed. Representative VEPs traces are shown on the right side. Data are mean ± SEM (n = 10 eyes per group). **p<0.01 versus vehicle-injected eyes without RON (sham), b: p<0.01 versus CS-injected eyes with sham procedure (sham), by Tukey's test.</p

Retinal histology examination after 10 weeks of ocular hypertension.

<p>A–E: Representative photomicrographs of retinal sections stained with hematoxylin and eosin from a vehicle-injected sham operated eye at 3 weeks of intracameral treatment (A), a vehicle-injected eye submitted to RON at 3 weeks of intracameral treatment (B) and a hypertensive eye without (C) or with RON performed at 3 (D) or 6 (E) weeks of treatment with CS. Note the diminution of GCL cells in the eye injected with CS without RON. RON preserved this parameter. The other retinal layers showed a normal appearance in all groups. Immunohistochemical detection of Thy-1 (F–J), Brn3a (K–O) or NeuN (P–T)-positive cells in the GCL from a vehicle-injected eye submitted to a sham procedure or RON, a hypertensive eye without or with RON performed at 3 or 6 weeks of treatment. The presences of all these markers were confined to the GCL in all experimental groups. A decrease in GCL cell number was observed in CS- injected eyes with sham procedure as compared with vehicle-injected eyes (sham or RON), whereas RON, which showed no effect in vehicle-injected eyes, preserved GCL cell count in CS-injected eyes. No differences were observed between CS-injected eyes submitted to a sham operation at 3 and 6 weeks of treatment (not shown). Scale bar: 100 µm. A representative (out of five per group) photograph of retina is shown. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.</p

ON from a vehicle- or a CS-treated eye with or without RON.

<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 with sham procedure (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 ON was observed in the CS-treated eye with RON at 3 (C), or 6 weeks (D) of ocular hypertension. Toluidine blue. Number of axons in eyes injected with vehicle or CS with sham procedure or RON at 3 (E) or 6 (F) weeks of treatment. A significant decrease in the axon number was observed in CS- injected eyes without RON as compared with vehicle-injected eyes (sham), whereas RON significantly preserved this parameter. Scale bar: 10 µm. Data are mean ± SEM (n = 5 eyes/group). **p<0.01 versus vehicle injected eyes with sham procedure (sham), a: p<0.05 versus CS-injected eyes without RON, by Tukey's test.</p

Electroretinographic preservation in hypertensive eyes induced by RON.

<p>ERGs were registered after 10 weeks of treatment with vehicle or CS in eyes submitted to sham operation or RON at 3 (left panel) or 6 weeks (right panel) of treatment. In sham operated eyes, CS induced a significant decrease in ERG a- and b-wave amplitude, as compared with vehicle-injected eyes. In hypertensive eyes submitted to RON at 3 or 6 weeks of ocular treatment with CS, a significant reduction of these alterations was observed. The lower panel shows representative scotopic ERG traces from eyes injected with vehicle or CS with or without RON. Data are the mean ± SEM (n = 10 eyes per group). **p<0.01 versus vehicle injected eyes with sham operation (sham); a: p<0.05 versus CS-injected eyes with sham operation, by Tukey's test.</p

Effect of RON on GCL cell count in vehicle or CS-injected eyes.

<p>Cell count in the GCL/100 µm was evaluated by H&E staining and Thy-1, NeuN and Brn3a immunostaining after 10 weeks of treatment with vehicle or CS in eyes submitted to sham operation or RON at 3 (upper panel) or 6 (lower panel) weeks of treatment. In sham operated eyes, CS induced a significant decrease in the GCL cell and in Thy-1, NeuN and Brn3a positive ganglion cell number, whereas in hypertensive eyes submitted to RON at 3 or 6 weeks, a significant preservation of GCL cells was observed. Data are the mean ± SEM (n = 5 retinas per group);</p>*<p>p<0.05,</p>**<p>p<0.01 versus vehicle- injected eyes with sham operation (sham);</p>a<p>: p<0.05,</p>b<p>: p<0.01 versus CS-injected eyes with sham operation, by Tukey's test.</p

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

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

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