Angiopoietin-1 Knockout Mice as a Genetic Model of Open-Angle Glaucoma

Figures

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  Figure 1.. Angpt1 knockout mice exhibit elevated IOP and vision loss. (A) Compared to controls, whole-body Angpt1 knockout mice induced at mid gestation exhibit chronic IOP elevation when measured using rebound tonometry. Wild-type (WT): n = 34–54 (20 days to 4 months), n = 18 (6 months), and n = 12 (8–12 months); Angpt1ΔWB: n = 38–52 (20 days to 4 months), n = 24 (6 months), and n = 10 (8–12 months). (B) Similarly, progressive loss of visual acuity was detected in Angpt1ΔWB mice by optomotor response testing. WT: n = 25 (20 days), n = 9–14 (1–4 months), n = 21 (6 months), n = 8 (8 months), and n = 10 (12 months); Angpt1ΔWB: n = 10–15 (20 days to 2 months), n = 26 (4 months), and n = 8–10 (6–12 months). (C) Globe enlargement is apparent by 12 weeks of age, quantified in (D), and buphthalmos is visible at 6 months. Scale bars: 1 mm. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by two-way ANOVA followed by Bonferroni's method for multiple comparisons (A, B) or Student's two-tailed t-test (D). Average axial length from each animal is plotted as a single data point in (D).

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  Figure 2.. Retinal ganglion cell loss in Angpt1 knockout mice. Compared to controls, 12-week-old Angpt1ΔWB mice exhibit reduced density of (A) BRN3A-, (B) BRN3B-, and (C) TUBB3-positive retinal ganglion cells when quantified in retina flat mounts. Scale bars: 50 µm; 20× fields represent an area of 65,413 µm2. *P < 0.05, **P < 0.01, ***P < 0.001, as determined using two-way ANOVA followed by Bonferroni's method for multiple comparisons (A, B) or Student's two-tailed t-test (C). Average density obtained from each retina is shown as a single data point.

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  Figure 3.. Optic nerve dysfunction in Angpt1 knockout mice. Plastic sections (1-µm) stained with toluidine blue revealed reduced axon density and gliosis (red arrows) in 6-month-old mutant mice. **P < 0.01 as determined by Student's two-tailed t-test. Each data point represents the average value obtained from a single animal.

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  Figure 4.. Angpt1 knockout retinas have normal morphology. (A, B) The 4′,6-diamidino-2-phenylindole-stained cryosections from 12-week-old mice show normal retinal morphology, with no significant difference in ONL thickness between control and Angpt1ΔWB mice in either central (red squares) or peripheral (blue squares) retina (control n = 14, Angpt1ΔWB n = 14; P = 0.8). (C) Calbindin, choline acetyltransferase (ChAT), PROX1, and calretinin show no difference in horizontal or amacrine cell morphology in Angpt1ΔWB mice. (D) Likewise, no changes in PKCα-stained rod bipolar cells were observed. Scale bars: 500 µm and 50 µm (A) and 50 µm (C, D). Not significant P > 0.05 as determined by two-way ANOVA followed by Bonferroni's method for multiple comparisons.

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  Figure 5.. Scotopic ERG response in Angpt1 knockout mice. (A) Representation of light stimulation in scotopic recording. Before stimulation, mice were dark adapted for 12 hours to maximize retinal sensitivity. (B) Example waveform for scotopic response. The negative a-wave is mainly derived from rods, and the b-wave is a positive-going potential primarily derived from bipolar cells. (C) The STR waveform consists of pSTR and nSTR components that represent RGCs and AII amacrine cells, respectively. (D) Examples of scotopic ERG response traces from control and Angpt1ΔWB mice in response to flash intensities ranging from –4.8 to 1.4 lg cd·s/m2 (–52 to 10 dB). (E) Intensity response curves from 4- to 6-month-old control and Angpt1 knockout mice. Compared to controls, mutant animals exhibited similar a- and b-wave amplitudes with only a slight reduction observed at high flash intensity (control n = 9, Angpt1ΔWB n = 7). (F) Latency of a- and b-waves was largely unchanged in Angpt1ΔWB mice. (G) Scotopic threshold measurements recorded at –4 lg cd·s/m2 in control and Angpt1ΔWB mice revealed an age-related loss of RGC-derived positive current (pSTR) in mutant animals (control, 1–2 months, n = 4;…

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  Figure 6.. Angpt1 knockout mice have normal retinal vasculature. (A) Retinal flat mounts of P5 mice reveal normal angiogenesis and sprouting front progression in Angpt1 knockout retinas. (B) In adult eyes, color fundus microscopy revealed normal vascular patterning in Angpt1ΔWB mice at 3 months of age. (C) This finding was confirmed in retinal flat mounts, where no differences in vascular patterning or CD31-positive (pos.) area were observed in the superficial (sup.) layer of Angpt1ΔWB retinas. (D) Likewise, normal patterning was observed in the intermediate (int.) and deep vascular layers. Scale bars: 500 µm (A, C), 400 µm (B), and 100 µm (D). Lack of significance in (B) was confirmed using a two-tailed Student's t-test.