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.
The purpose of this research was to develop a reliable and repeatable inflation protocol to measure the scleral inflation response of mouse eyes to elevations in intraocular pressure (IOP), comparing the inflation response exhibited by the sclera of younger and older C57BL/6 mice. Whole, enucleated eyes from younger (2 month) and older (11 month) C57BL/6 mice were mounted by the cornea on a custom fixture and inflated according to a load-unload, ramp-hold pressurization regimen via a cannula connected to a saline-filled programmable syringe pump. First, the tissue was submitted to three load-unload cycles from 6 mmHg to 15 mmHg at a rate of 0.25 mmHg/s with ten minutes of recovery between cycles. Next the tissue was submitted to a series of ramphold tests to measure the creep behavior at different pressure levels. For each ramp-hold test, the tissue was loaded from 6 mmHg to the set pressure at a rate of 0.25 mmHg/s and held for 30 minutes, and then the specimens were unloaded to 6mmHg for 10 minutes. This sequence was repeated for set pressures of: 10.5, 15, 22.5, 30, 37.5, and 45 mmHg. Scleral displacement was measured using digital image correlation (DIC), and fresh scleral thickness was measured optically for each specimen after testing. For comparison, scleral thickness was measured on untested fresh tissue and epoxy-fixed tissue from age-matched animals. Comparing the apex displacement of the different aged specimens, the sclera of older animals had a statistically significant stiffer response to pressurization than the sclera of younger animals. The stiffness of the pressure-displacement response of the apex measured in the small-strain (6-15 mmHg) and the large-strain (37.5-45 mmHg) regime, respectively, were 287 ± 100 mmHg/mm and 2381 ± 191 mmHg/mm for the older tissue and 193 ± 40 mmHg/mm and 1454 ± 93 mmHg/mm for the younger tissue (Student t-test, p < 0.05). The scleral thickness varied regionally, being thickest in the peripapillary region and thinnest at the equator. Fresh scleral thickness did not differ significantly by age in this group of animals. This study presents a reliable inflation test protocol to measure the mechanical properties of mouse sclera. The inflation methodology was sensitive enough to measure scleral response to changes in IOP elevations between younger and older C57BL/6 mice. Further, the specimen-specific scleral displacement profile and thickness measurements will enable future development of specimen-specific finite element models to analyze the inflation data for material properties.
The TonoLab accurately reflects IOP in both normal mice and in eyes of mice with experimental or spontaneous glaucoma, with no detectable effect of age.
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