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. 2018 Aug 1;59(10):3889-3896.
doi: 10.1167/iovs.18-24567.

A Novel Murine Model of Radiation Keratopathy

Deshea L Harris  1   2   3 , Takefumi Yamaguchi  1   2   4 , Pedram Hamrah  1   2   3   5   6
Affiliations

A Novel Murine Model of Radiation Keratopathy

Deshea L Harris et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Radiation therapy results in severe chronic keratopathy and dry eye disease. We developed a novel mouse model for radiation keratopathy to allow future mechanistic studies.

Methods: Six to 8-week-old BALB/c mice underwent sublethal irradiation to the head only from a Cesium-137 irradiator, 2 × 550 rad, 3-hours apart. Irradiated mice were clinically evaluated by corneal fluorescein staining (CFS) at 1, 2, and 3 months, after which corneas were excised and immunofluorescence histochemistry performed with anti-CD45, anti-MHC class II, and anti-β-tubulin antibodies.

Results: The survival rate after irradiation was 100%. Mice demonstrated significant CFS and hair loss around the eyes. Corneal nerve density decreased in the central and peripheral corneas (P < 0.01) at 2 and 3 months, respectively. CD45+ immune cell densities increased in the central and peripheral corneas (P < 0.005, P < 0.001) at 2 and 3 months, respectively. MHC class II, a sign of antigen presenting cell activation, significantly increased after irradiation in the central and peripheral corneas at 2 and 3 months (P = 0.02). A strong inverse correlation was noted between decreased corneal nerves and increase in CD45+ cells in the central cornea at 2 (P = 0.04, r = -0.89) and 3 months (P = 0.03, r = -0.91) after irradiation.

Conclusions: We present a model of radiation keratopathy and demonstrate significant nerve loss and increase in immune cell influx and activation within months. This model will enable future investigations to understand the effects of radiation therapy on the eye, and to study mechanisms of neuro-immune crosstalk in the cornea.

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Figures

Figure 1
Figure 1
Six- to 8-week-old adult BALB/c mice were anesthetized and placed upright in a 50 mL conical tube wrapped in 6.6 mm lead shielding with the heads exposed. The mice underwent sublethal irradiation from a Cesium-137 irradiator, 2 × 550 rad, 3 hours apart.
Figure 2
Figure 2
Clinical findings of irradiated mice at 1, 2, and 3 months. Alopecia areata occurs around the eye beginning at 2 months (A). CFS in 2 representative mice at 1 (B), 2 (C) and 3 (D) months showing diffuse staining of the epithelium (white arrows) indicating damage to the ocular surface.
Figure 3
Figure 3
Representative histologic images of central (A, C, E, G) and peripheral (B, D, F, H) corneas stained with neuron-specific anti-β III tubulin NL557-conjugated antibody in normal (A, B) mice, and at 1 (C, D), 2 (E, F), and 3 (G, H) months after irradiation. Graph results shown in (I). Scale bar: 100 μm. Mean ± SEM. **P < 0.01; ***P < 0.001.
Figure 4
Figure 4
Representative histologic images of central (A, C, E, G) and peripheral (B, D, F, H) cornea stained with anti-CD45 FITC-conjugated antibody in normal (A, B) mice, and at 1 (C, D), 2 (E, F), and 3 (G, H) months after irradiation. Graph results shown in (I). Scale bar: 100 μm. Mean ± SEM. **P < 0.01; ***P < 0.001.
Figure 5
Figure 5
Representative histologic images of central (A, C, E, G) and peripheral (B, D, F, H) cornea stained with anti-MHC class II FITC-conjugated antibody in normal (A, B) mice, and at 1 (C, D), 2 (E, F), and 3 (G, H) months after irradiation. Graph results shown in (I). Scale bar: 100 μm. Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6
Figure 6
There was a severe significant reverse correlation (r = −0.83) between CD45+ cell density and nerve density in the central cornea of irradiated mice (P < 0.001).
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