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

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 microL of 6 mum diameter, polystyrene beads followed by 3 microL 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 >or=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 <or= 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.

Figure 1

Difference between bead-injected eyes and fellow control eyes expressed as mean ± standard error IOP (mmHg) for C57BL/6 younger, C57BL/6 older, DBA/2J younger, and CD1 younger mice throughout study. Linear regression of mean values for each time point included in each of 4 graphs.

Figure 2

A: Anterior segment of control, non-injected, C57BL/6 younger mouse. B: Anterior segment of C57BL/6 younger mouse 6 weeks after bead injection. Beads are seen as clear spheres due to extraction of polystyrene during epoxy embedding. Beads are found in the iris stroma, in the uveoscleral pathway between ciliary body and sclera (US) and in outflow channels equivalent to Schlemm’s canal (S). Anterior chamber (AC), Retina (R). (1% toluidine blue; scale bars = 70μm).”

Figure 3

CD1 mouse retinas 12 weeks after bead injection. A: Normal mouse retina. B: Bead-injected mouse retina showing thinning of nerve fiber and RGC layer, but normal thickness of inner nuclear and outer nuclear layers (1% toluidine blue stain; scale bar = 30μm).

Figure 4

Optic nerve cross-sections from CD1 mice. A: Normal mouse optic nerve. B: Nerve from bead-injected eye 12 weeks after injection, showing major loss of normal axon profiles, as well as clumps of myelin debris and macrophages filled with clear vacuoles (epoxy-embedded, 1 μm section, 1% toluidine blue, scale bars = 20 μm).

Figure 5

Retinal whole mounts in confocal micrographs of CD1 mice 12 weeks after bead injection. A and C: control retinas. B: shows significant cell loss in the RGC layer after bead injection. D: one of the rare examples of a excluded retinal whole mount showing increased RGC layer cells due to an inflammatory infiltrate (superior retina, DAPI stain, scale bars = 50 μm).