Elsevier

Experimental Eye Research

Volume 91, Issue 3, September 2010, Pages 415-424
Experimental Eye Research

Differential susceptibility to experimental glaucoma among 3 mouse strains using bead and viscoelastic injection

https://doi.org/10.1016/j.exer.2010.06.018Get rights and content

Abstract

The purpose of this experiment was to test the susceptibility to retinal ganglion cell (RGC) axon loss and RGC layer cell loss from experimental glaucoma among 3 mouse strains, and between younger and older mice. We obstructed the mouse aqueous outflow channels by injecting 2 μL of 6 μm diameter, polystyrene beads followed by 3 μL of viscoelastic solution into the anterior chamber with a glass micropipette. We evaluated intraocular pressure (IOP) and damage to RGC as measured by optic nerve axon counts and RGC layer neuron counts in 3 strains of young mice (2 month old C57BL/6, DBA/2J, and CD1) and 10 month C57BL/6 mice. Bead and viscoelastic injection produced IOP elevation at ≥1 time point in 94.1% of eyes (112/119), with mean IOP difference from fellow eyes of 4.4 ± 3.0 mmHg. By 6–12 weeks, injected eyes were 10.8% longer and 7.6% wider (p < 0.0001). Young DBA/2J and C57BL/6 eyes increased axial length significantly more than young CD1 or older C57BL/6 (all p ≤ 0.02). RGC layer and axon loss was greatest in CD1 mice, significantly more than the other groups (p from 0.04 to <0.0001). Young C57BL/6 eyes elongated more and lost more RGC layer cells than older C57BL/6 mice (p = 0.02 and 0.01, respectively). With this mouse glaucoma model, there was differential susceptibility to ocular elongation and RGC layer and axon damage among mouse strains and by age. Factors that determine sensitivity to RGC injury can be studied using transgenic mouse strains with inducible models.

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.

References (39)

  • M. Aihara et al.

    Ocular hypertension in mice with a targeted type 1 collagen mutation

    Invest. Ophthalmol. Vis. Sci.

    (2003)
  • M. Aihara et al.

    Experimental mouse ocular hypertension: establishment of the model

    Invest. Ophthalmol. Vis. Sci.

    (2003)
  • M. Blair et al.

    Effect of glatiramer acetate on primary and secondary degeneration of retinal ganglion cells in the rat

    Invest. Ophthalmol. Vis. Sci.

    (2005)
  • M.V. Boland et al.

    Risk factors and open-angle glaucoma: concepts and applications

    J. Glaucoma

    (2007)
  • Y. Dai et al.

    Outflow facility in mice with a targeted type I collagen mutation

    Invest. Ophthalmol. Vis. Sci.

    (2009)
  • D. Gaasterland et al.

    Experimental glaucoma in the rhesus monkey

    Invest. Ophthalmol.

    (1974)
  • D. Goldblum et al.

    Non-invasive determination of intraocular pressure in the rat eye. Comparison of an electronic tonometer (TonoPen), and a rebound (impact probe) tonometer

    (2002)
  • R.L. Gross et al.

    A mouse model of elevated intraocular pressure: retina and optic nerve findings

    Trans. Am. Ophthalmol. Soc.

    (2003)
  • S.D. Grozdanic et al.

    Laser-induced mouse model of chronic ocular hypertension

    Invest. Ophthalmol. Vis. Sci.

    (2003)
  • Cited by (139)

    View all citing articles on Scopus
    View full text