Increased levels of extracellular ATP in glaucomatous retinas: Possible role of the vesicular nucleotide transporter during the development of the pathology

Abstract

Purpose: To study retinal extracellular ATP levels and to assess the changes in the vesicular nucleotide transporter (VNUT) expression in a murine model of glaucoma during the development of the disease.

Methods: Retinas were obtained from glaucomatous DBA/2J mice at 3, 9, 15, and 22 months together with C57BL/6J mice used as age-matched controls. To study retinal nucleotide release, the retinas were dissected and prepared as flattened whole mounts and stimulated in Ringer buffer with or without 59 mM KCl. To investigate VNUT expression, sections of the mouse retinas were evaluated with immunohistochemistry and western blot analysis using newly developed antibodies against VNUT. All images were examined and photographed under confocal microscopy. Electroretinogram (ERG) recordings were performed on the C57BL/6J and DBA/2J mice to analyze the changes in the electrophysiological response; a decrease in the scotopic threshold response was observed in the 15-month-old DBA/2J mice.

Results: In the 15-month-old control and glaucomatous mice, electrophysiological changes of 42% were observed. In addition, 50% increases in the intraocular pressure (IOP) were observed when the pathology was fully established. The responses in the retinal ATP net release as the pathology progressed varied from 0.32±0.04 pmol/retina (3 months) to 1.10±0.06 pmol/retina (15 months; threefold increase). Concomitantly, VNUT expression was significantly increased during glaucoma progression in the DBA/2J mice (58%) according to the immunohistochemical and western blot analysis.

Conclusions: These results may indicate a possible correlation between retinal dysfunction and increased levels of extracellular ATP and nucleotide transporter. These data support an excitotoxicity role for ATP via P2X7R in glaucoma. This modified cellular environment could contribute to explaining the functional and biochemical alterations observed during the development of the pathology.

Figure 1

Changes in the intraocular pressure and electroretinogram recordings in glaucomatous and control mice. A: Assessment of intraocular pressure (IOP) measurements in the glaucomatous (DBA/2J) and control (C57) mice as a function of age. Animals were measured at 3, 9, and 15 months of age. A significant increase was observed in DBA/2J mice at 12 months when compared with the control mice (*p<0.05). B: Scotopic threshold responses from control C57BL/6J or DBA/2J glaucomatous mice as a function of age. Superposed averaged positive scotopic threshold response (pSTR) amplitudes were measured from the electroretinogram (ERG) flash response as a function of stimulus light intensity recorded from C57BL/6J (triangles) and DBA/2J (circles) mice at 3 (white symbols), 9 (gray symbols), and 15 months (black symbols). Plot data correspond to mean values ± standard deviation (SD) (n=8). A significant reduction in the STR amplitudes in the DBA/2J mice between 3 and 15 months is observed for pSTR amplitudes (***p<0.001) and when compared with the control mice at 15 months (^$p<0.001).

Figure 2

Study of the ATP release in control (C57BL/6J) and glaucomatous (DBA/2J) mice. A: Basal and KCl stimulated ATP release from whole mount retinas obtained from the C57BL/6J mice. The left panel represents the mean of the ATP amounts at the given mice ages either under basal (solid symbol) or stimulated conditions (open symbol). The middle and right panels are representative high-pressure liquid chromatography (HPLC) elution profiles of basal (middle panel) and stimulated conditions (right panel). B: Basal and KCl stimulated ATP release from whole mount retinas obtained from glaucomatous DBA/2J mice. The left panel shows the mean of the ATP amounts at the given mice ages either under basal (solid symbol) or stimulated conditions (open symbol). The middle and right panels are representative HPLC elution profiles of the basal (middle panel) and stimulated conditions (right panel). C: Net ATP release in the glaucomatous DBA/2J mice (open circles) and the control C57BL/6J mice (open squares) followed from 3 to 22 months. D: Total ATP content in the retinas of the control and glaucomatous mice at the different ages under study (n=8). *p<0.05 between 9 and 15 months of age in the DBA/2J animals. #p<0.05 C57BL/6J mice versus DBA/2J mice at 15 months and ##p<0.01 C57BL/6J versus DBA/2J mice at 22 months.

Figure 3

Correlation between electroretinogram recordings, ATP and vesicular nucleotide transporter expression. Relationship between ERG amplitude and ATP levels (A) and ERG amplitude and VNUT expression (B). Correlation was performed in glaucomatous animals comparing 3 versus 15 months of age.

Figure 4

Immunohistochemistry for VNUT in C57BL/6J and DBA/2J retinas. Vertical sections at 3, 9, 15, and 22 months of retinas from glaucomatous mice (B, C, E, F) and control mice at 3 and 15 months (A, D). Vesicular nucleotide transporter (VNUT)-stained retinas (green) showed a significant increase at 15 months in the DBA/2J mice (E) compared to that observed in the DBA/2J and C57BL/6J mice at 3 months (A, B) and 23 months (F). G–H: High magnification of the photoreceptor layer, showing an increase in VNUT labeling in the DBA/2J mice at 15 months (G) and VNUT staining found in ganglion cells (arrows; H). Nuclei were stained with propidium iodide (red). All images were collected from the central area of the retina. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 μm.

Figure 5

VNUT expression in the glaucomatous and control retinas at different ages (3, 9, 15, and 22 months). A: Detection of vesicular nucleotide transporter (VNUT) (68 kDa) levels in the C57BL/6J mice at 3, 9, and 15 months and in the DBA/2J mice at 3, 9, 15, and 22 months by western blotting. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 37 kDa) levels are shown as a loading control. B: Western blot analysis of the VNUT at 3, 9, 15, and 22 months of age. Values are the mean ± standard deviation (SD) of six independent animal experiments (***p<0.001).