Journal: Frontiers in Neuroscience

Article Title: Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma

doi: 10.3389/fnins.2019.01139

Figure Lengend Snippet: Elevated IOP decreases expression of total and α1 Na/K-ATPase in retina. (A) Box plots of fold change (log scale; left) and percent change (right) in expression of the Atp1 gene family between retina from saline- and microbead-injected mice, as determined by RNA sequencing. n (saline) = 5 retinas, n (microbead) = 5; ∗ p < 0.005. (B) Representative confocal micrographs of total Na/K-ATPase (green) and β-Tubulin III (β-Tub; red) immunolabeling in retinal sections from naïve, saline-, and microbead-injected mice. Bottom panels of each image are a zoom of the ganglion cell layer (GCL). Scale bar = 40 μM. (C) Mean intensity of total Na/K-ATPase immunolabeling in retina from naïve, saline-injected, and microbead-injected mice. Asterisk indicates p < 0.05 and error bars represent SEM. n (naïve) = 8 retinal sections, n (saline) = 9, n (microbead) = 9 (from n = 4 eyes/condition). (D) Representative confocal micrographs of α1 Na/K-ATPase (green) and β-Tubulin III (β-Tub; red) immunolabeling in retinal sections from naïve, saline-, and microbead-injected mice. Bottom panels of each image are a zoom of the GCL. Scale bar = 40 μM. (E) Mean intensity of α1 Na/K-ATPase immunolabeling in retina sections from naïve, saline-injected, and microbead-injected mice. ∗ p < 0.05 and error bars represent SEM. n (naïve) = 4 retinal sections, n (saline) = 8, n (microbead) = 5 (from n = 4 eyes/condition). ONL, outer nuclear layer, OPL, outer plexiform layer, INL, inner nuclear layer, IPL, inner plexiform layer, GCL, ganglion cell layer.

Article Snippet: Samples were then incubated overnight at 4°C in primary antibody solution (3% NHS and 0.1% Triton X-100 diluted in 1 × PBS) containing rabbit anti-Total Na/K-ATPase (2 μg/ml, Cat# ab58475, Abcam, Cambridge, United Kingdom), rabbit anti-α1 Na/K-ATPase (2.85 μg/ml, Cat# ANP-001, Alomone, Jerusalem, Israel); and mouse anti-β-Tubulin III (2 μg/ml, Cat# 801201, BioLegend, London, United Kingdom).

Techniques: Expressing, Injection, RNA Sequencing Assay, Immunolabeling

Journal: Frontiers in Neuroscience

Article Title: Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma

doi: 10.3389/fnins.2019.01139

Figure Lengend Snippet: Elevated pressure decreases expression of total and α1 Na/K-ATPase in RGCs in vitro . (A) Representative fluorescent micrographs of total and α1 Na/K-ATPase (green) and β-Tubulin (red) immunolabeling in primary cultures of purified RGCs exposed to ambient or elevated pressure for 48 h. Scale bar = 40 μm. (B) Representative fluorescent micrographs of total and α1 Na/K-ATPase (green) and β-Tubulin (red) immunolabeling in primary cultures of purified RGCs exposed to ambient or elevated pressure for 4 h. Scale bar = 40 μm. (C) Mean intensity (arbitrary units; AU) of total (left) and α1 (right) Na/K-ATPase staining in RGC cultures following a 48-h exposure to ambient or elevated hydrostatic pressure. ∗ p < 0.05. Total: n (AMB48) = 10 cells, n (ELEV48) = 9; α1: n (AMB48) = 13, n (ELEV48) = 11. (D) Mean intensity (arbitrary units; AU) of total (left) and α1 (right) Na/K-ATPase staining in RGC cultures following a 4 h exposure to ambient or elevated hydrostatic pressure. ∗ p < 0.05. Total: n (AMB4) = 14 cells, n (ELEV4) = 9; α1: n (AMB4) = 10, and n (ELEV4) = 10.

Article Snippet: Samples were then incubated overnight at 4°C in primary antibody solution (3% NHS and 0.1% Triton X-100 diluted in 1 × PBS) containing rabbit anti-Total Na/K-ATPase (2 μg/ml, Cat# ab58475, Abcam, Cambridge, United Kingdom), rabbit anti-α1 Na/K-ATPase (2.85 μg/ml, Cat# ANP-001, Alomone, Jerusalem, Israel); and mouse anti-β-Tubulin III (2 μg/ml, Cat# 801201, BioLegend, London, United Kingdom).

Techniques: Expressing, In Vitro, Immunolabeling, Purification, Staining

Journal: Frontiers in Neuroscience

Article Title: Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma

doi: 10.3389/fnins.2019.01139

Figure Lengend Snippet: Inhibition of endocytosis and degradation pathways preserves Na/K-ATPase expression in RGCs following short-term pressure exposure. (A) Representative confocal micrographs of total Na/K-ATPase (green) and β-Tubulin III (red) immunolabeling in primary, purified RGCs treated with vehicle, 10 μM bisindolylmaleimide, or 20 μM MG-132 and exposed to ambient or elevated pressure for 4 h. Scale bar = 40 μM. (B) Mean intensity (arbitrary units; AU) of total Na/K-ATPase staining per cell in each drug and pressure condition. ∗ p < 0.05. n (AMB) = 14 cells, n (ELEV) = 9; n (AMB + BIS) = 11, n (ELEV + BIS) = 10; n (AMB + MG) = 13, and n (ELEV + MG) = 14.

Article Snippet: Samples were then incubated overnight at 4°C in primary antibody solution (3% NHS and 0.1% Triton X-100 diluted in 1 × PBS) containing rabbit anti-Total Na/K-ATPase (2 μg/ml, Cat# ab58475, Abcam, Cambridge, United Kingdom), rabbit anti-α1 Na/K-ATPase (2.85 μg/ml, Cat# ANP-001, Alomone, Jerusalem, Israel); and mouse anti-β-Tubulin III (2 μg/ml, Cat# 801201, BioLegend, London, United Kingdom).

Techniques: Inhibition, Expressing, Immunolabeling, Purification, Staining

Journal: Frontiers in Neuroscience

Article Title: Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma

doi: 10.3389/fnins.2019.01139

Figure Lengend Snippet: Inhibition of Na/K-ATPase endocytosis and degradation prevents pressure-induced reduction of inward cation flux. (A) Representative heat maps showing the fluorescent signal of Thallos dye in RGCs exposed to ambient and elevated pressure for 4 h plus treatment with vehicle, 10 μM bisindolylmaleimide, or 20 μM MG-132. Images were taken after the addition of thallium. Scale bar = 100 μM. (B) Line graph displaying the mean fluorescence intensity of Thallos dyes over time for RGCs exposed to ambient or elevated pressure for 4 h plus treatment with vehicle or bisindolylmaleimide. ∗ p < 0.05 and error bars represent SEM. n (AMB) = 30 cells, n (ELEV) = 27, n (AMB + BIS) = 7, and n (ELEV + BIS) = 16. (C) Line graph displaying the mean fluorescence intensity of Thallos dyes over time for RGCs exposed to ambient or elevated pressure for 4 h plus treatment with vehicle or MG-132. ∗ p < 0.05 and error bars represent SEM. n (AMB) = 30 cells, n (ELEV) = 27, n (AMB + MG-132) = 3, and n (ELEV + MG-132) = 13.

Article Snippet: Samples were then incubated overnight at 4°C in primary antibody solution (3% NHS and 0.1% Triton X-100 diluted in 1 × PBS) containing rabbit anti-Total Na/K-ATPase (2 μg/ml, Cat# ab58475, Abcam, Cambridge, United Kingdom), rabbit anti-α1 Na/K-ATPase (2.85 μg/ml, Cat# ANP-001, Alomone, Jerusalem, Israel); and mouse anti-β-Tubulin III (2 μg/ml, Cat# 801201, BioLegend, London, United Kingdom).

Techniques: Inhibition, Fluorescence

Journal: Frontiers in Neuroscience

Article Title: Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma

doi: 10.3389/fnins.2019.01139

Figure Lengend Snippet: Na/K-ATPase inhibition reproduces cation dyshomeostasis and repolarization deficits. (A) Representative heat maps depicting the fluorescent signal of Thallos dye in RGCs exposed to ambient and elevated pressure for 4 h in the presence of vehicle or 20 μm ouabain or vehicle. Scale bar = 100 μM. (B) Line graph displaying the mean fluorescence intensity of Thallos dyes over time for RGCs exposed to ambient or elevated pressure for 4 h in the presence of vehicle or 20 μm ouabain. ∗ p < 0.05 and error bars represent SEM. n (AMB) = 30, n (ELEV) = 27, n (AMB + OUA) = 5, and n (ELEV + OUA) = 6. (C–F) Whole-cell patch-clamp physiology in RGCs from retina of saline-injected eyes following puff administration of vehicle, 10, or 20 μM ouabain. ∗ p < 0.05. (C) Frequency of spontaneous spiking depicted as mean event frequency (Hz) across cells for each condition. n (vehicle) = 12 cells, n (10) = 9, n (20) = 9 (from n = 3 retina/condition). (D) Peak amplitude of spontaneous spiking depicted as mean peak amplitude (mV) across cells for each condition. n (vehicle) = 11 cells, n (10) = 10, n (20) = 11 (from n = 3 retina/condition). (E) Max rise slope of spontaneous spiking depicted as mean max rise slope (mV/ms) across cells for each condition. n (vehicle) = 14 cells, n (10) = 10, n (20) = 11 (from n = 3 retina/condition). (F) Max decay slope of spontaneous spiking depicted as mean max decay slope (mV/ms) across cells for each condition. n (vehicle) = 10 cells, n (10) = 7, n (20) = 11 (from n = 3 retina/condition).

Article Snippet: Samples were then incubated overnight at 4°C in primary antibody solution (3% NHS and 0.1% Triton X-100 diluted in 1 × PBS) containing rabbit anti-Total Na/K-ATPase (2 μg/ml, Cat# ab58475, Abcam, Cambridge, United Kingdom), rabbit anti-α1 Na/K-ATPase (2.85 μg/ml, Cat# ANP-001, Alomone, Jerusalem, Israel); and mouse anti-β-Tubulin III (2 μg/ml, Cat# 801201, BioLegend, London, United Kingdom).

Techniques: Inhibition, Fluorescence, Patch Clamp, Injection