The effect of triamcinolone acetonide on laser-induced choroidal neovascularization in mice using a hypoxia visualization bio-imaging probe

Abstract

Hypoxic stress is a risk factor of ocular neovascularization. Hypoxia visualization may provide clues regarding the underlying cause of angiogenesis. Recently, we developed a hypoxia-specific probe, protein transduction domain-oxygen-dependent degradation domain-HaloTag-Rhodamine (POH-Rhodamine). In this study, we observed the localization of HIF-1α proteins by immunohistochemistry and the fluorescence of POH-Rhodamine on RPE-choroid flat mounts. Moreover, we compared the localization of POH-Rhodamine with pimonidazole which is a standard reagent for detecting hypoxia. Next, we investigated the effects of triamcinolone acetonide (TAAC) against visual function that was evaluated by recording electroretinogram (ERG) and choroidal neovascularization (CNV) development. Mice were given laser-induced CNV using a diode laser and treated with intravitreal injection of TAAC. Finally, we investigated POH-Rhodamine on CNV treated with TAAC. In this study, the fluorescence of POH-Rhodamine and HIF-1α were co-localized in laser-irradiated sites, and both the POH-Rhodamine and pimonidazole fluorescent areas were almost the same. Intravitreal injection of TAAC restored the reduced ERG b-wave but not the a-wave and decreased the mean CNV area. Furthermore, the area of the POH-Rhodamine-positive cells decreased. These findings indicate that POH-Rhodamine is useful for evaluating tissue hypoxia in a laser-induced CNV model, suggesting that TAAC suppressed CNV through tissue hypoxia improvement.

Figure 1. Localization of HIF-1α and POH-Rhodamine in RPE-choroid-sclera flat mounts and localization of POH-Rhodamine and pimonidazole stain.

Fluorescent staining of FITC-dextran (green) and POH-Rhodamine (red), and immunohistochemical analyses of HIF-1α (blue) and pimonidazole (blue) in RPE-choroid-sclera flat mounts on day 3 after laser photocoagulation. The images were shown that the localization of POH-Rhodamine and HIF1α (A), HIF1α negative control (B), the localization of POH-Rhodamine and pimonidazole (C). The localization of POH-Rhodamine-, FITC-dextran- and Hoechst33342-stained cells in CNV are demonstrated in 300 × magnifications (D). The color dotted lines represent the edge of the area of CNV lesions or the stained area. (A) (B) (C) Scale bar = 100 μm, (D) scale bar = 50 μm (left) and 10 μm (right).

Figure 2. Measurement of dark-adapted ERG amplitudes 15 d after laser treatment in the retina of laser-induced CNV model mice.

(A) Representative ERG tracing from normal, or laser plus vehicle- and laser plus TAAC-treated groups. Amplitudes of (B) a-waves and (C) b-waves from the laser photocoagulation plus vehicle-treated group vs. the laser photocoagulation plus TAAC-treated group. Data are presented as means ± SE (n = 7 to 10). ## p < 0.01 vs. normal. * p < 0.05, ** p < 0.01 vs. laser photocoagulation plus vehicle-treated group (Student's t-test).

Figure 3. TAAC-suppressed CNV formation in laser-induced CNV model mice.

(A) Representative micrographs of CNV lesions in the RPE-choroid flat mounts from vehicle and TAAC-treated mice 3, 7, and 14 d after laser photocoagulation. CNV is indicated by green fluorescence (FITC-dextran) angiography. Scale bar = 100 μm. (B) The color dotted lines represent the edge of area of CNV lesions or stained area. Analysis included the total areas within the dotted lines. Data are presented as means ± SE (n = 5 to 8). * p < 0.05, ** p < 0.01 vs. vehicle-treated group (Student's t-test).

Figure 4. TAAC decreased POH-Rhodamine stained area around laser injury sites.

(A) Representative micrographs of CNV lesions in the RPE-choroid flat mounts 3 d after laser photocoagulation. POH-Rhodamine-stained area indicated HIF-1α positive cells. (B) Measurement of the POH-Rhodamine-stained area around the laser injury sites. The color dotted lines represent the edge of area of CNV lesions or stained area. Analysis included the total areas within the dotted lines. Data are presented as means ± SE (n = 8). * p < 0.05 vs. vehicle-treated group (Student's t-test). Scale bar = 100 μm.

Figure 5. TAAC decreased the level of HIF-1α and the fluorescent intensity of POH-Rhodamine in ARPE-19 cells under hypoxic conditions.

(A) Time-dependent change of HIF-1α levels. ARPE-19 cells were exposed to normoxia or hypoxia for the indicated times. The individual protein levels were semiquantified and normalized by β-actin levels. Data are shown as means ± SE (n = 4 or 5). * p < 0.05 vs. normoxia (Student's t-test). (B) Western blot analysis of HIF-1α levels. Vehicle or TAAC (1 μg/ml) were added to ARPE-19 cells and incubated under hypoxic condition for 3 h. Data are shown as means ± SE (n = 4). ## p < 0.01 vs. normal, * p < 0.05 vs. vehicle-treated group (Student’s t-test). (C) Measurement of the fluorescent intensity with an Ex/Em wavelength of 550 nm/573 nm after incubation of ARPE-19 cells with POH-Rhodamine under hypoxic condition for 3 h. Data are shown as means. ## p < 0.01 vs. normal, ** p < 0.01 vs. vehicle-treated group (Student’s t-test). The cropped blots are used in this Figure and the full-length blots are presented in Supplementary Figure S4.