Differential susceptibility to experimental glaucoma among 3 mouse strains using bead and viscoelastic injection
Introduction
Glaucoma is the second leading cause of vision loss worldwide (Quigley and Broman, 2006). To understand its pathogenesis and to improve glaucoma therapy, animal models of glaucoma were developed in monkeys (Gaasterland and Kupfer, 1974, Quigley and Addicks, 1980, Quigley and Hohman, 1983) and rats (Shareef et al., 1995, Morrison et al., 1997, Sawada and Neufeld, 1999, Mittag et al., 2000, Schori et al., 2001, Ueda et al., 1998, Moreno et al., 2004, Levkovitch-Verbin et al., 2002) often using laser treatment to the trabecular meshwork to increase intraocular pressure (IOP). Similar laser treatment to the mouse anterior segment has been used to increase IOP (Grozdanic et al., 2003, Aihara et al., 2003b, Gross et al., 2003, Nakazawa et al., 2006), as have occlusion of episcleral veins (Ruiz-Ederra and Verkman, 2006) and injection of hypertonic saline into episcleral vessels (McKinnon et al., 2009). Improvements in the reliability of induced damage and in the minimization of collateral ocular damage would facilitate glaucoma research. Spontaneous glaucoma occurs in certain mammals, including beagle dogs (Samuelson et al., 1989) and in 3 mouse strains (John et al., 1998, Aihara et al., 2003a, Zhou et al., 2008). However, inducible mouse glaucoma models permit study of the effect of experimental disease in existing and transgenic strains without time-consuming cross-breeding.
The optimal mouse glaucoma model would have the following features: 1) prompt, consistent increased IOP in most eyes; 2) lack of detrimental effects on the cornea and sclera; 3) death of RGC, but not of other retinal neurons; and 4) ease and low cost of implementation. Previous investigators have obstructed the outflow channels by injecting either latex spheres in monkeys (Weber and Zelenak, 2001), polystyrene beads in rats and mice (Sappington et al., 2010), and repeated injections of latex microspheres with hydroxypropylmethylcellulose into rats (Urcola et al., 2006). We modified this approach by a single injection of polystyrene beads followed immediately by a viscoelastic substance in mouse eyes, a model that satisfies the above criteria. The development of a non-invasive tonometer to measure IOP accurately in mice was an important facilitating step (Goldblum et al., 2002, Pease et al., 2006). Its accuracy in glaucomatous mice was confirmed in a companion report (M.E. Pease, personal communication, 2010).
Susceptibility to glaucoma injury in humans is related to several known risk factors, including older age, ethnicity, central corneal thickness, and axial length (Boland and Quigley, 2007). We tested the effect of our mouse glaucoma model on both younger and older mice of one pigmented strain (C57BL/6) and compared the results to that of young mice of two other strains, the pigmented DBA/2J and the albino CD1. Our purpose was to determine the generalizability of the glaucoma model, as well as to compare cell loss in the retinal ganglion cell (RGC) layer and optic nerve across strains to identify differences in susceptibility.
Section snippets
Animals
A total of 168 female mice were utilized in this study. Ninety-seven were 8 week old C57BL/6, 25 were 8 month old C57BL/6, 25 were 8 week old DBA/2J, and 21 were 8 week old CD1. Two mice died due to anesthesia complications and 9 were euthanized due to severe ocular enlargement with corneal exposure or ulceration, leaving a total of 157 mice that provided data. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, using
IOP data
Prior to determining the optimal protocol, we used 57 young C57BL/6 mice in trials of various permutations of the methods. IOP data for 9 of these preliminary animals, which were sacrificed 3 weeks after bead injection, are shown with the 4 groups of mice in which the optimal injection protocol was used (young and older C57BL/6, young DBA/2J, and young CD1; Table 1).
In normal, paired mouse eyes under anesthesia, the 97.5% confidence interval for IOP difference between eyes was ≥5 mm Hg. We used
Discussion
The 3 different strains of mice and 2 age groups of the C57BL/6 strain showed significant differences in response to chronic IOP elevation that may provide important clues in glaucoma pathogenesis. First, the axial length and width of the mouse eye are rapidly increased with chronic IOP elevation, as do the eyes of human infants, and to a lesser degree monkey and rat eyes with experimental glaucoma. This fixed deformation began within the first week and was not reversed in those eyes that
Acknowledgements
Supported in part by Public Health Service Research Grants EY02120 and EY01765 (Dr Quigley and Wilmer Institute), the Leonard Wagner Charitable Trust, William T. Forrester, and Alcon Laboratories, Ft. Worth TX. Don Zack, MD PhD provided advice and manuscript review. David Calkins, PhD and Martin Wax, MD PhD provided important ideas that initiated this investigation.
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