Mice with an induced mutation in collagen 8A2 develop larger eyes and are resistant to retinal ganglion cell damage in an experimental glaucoma model

PURPOSE: To study susceptibility to glaucoma injury as it may be affected by mutations in ocular connective tissue components. METHODS: Mice homozygous for an N-ethyl-N-nitrosourea induced G257D exchange (Gly to Asp) missense mutation (Aca23) in their collagen 8A2 gene were studied to measure intraocular pressure (IOP), axial length and width, number of retinal ganglion cells (RGC), and inflation responses. Three month old homozygous Aca23 mutant and wild type (WT) mice had 6 weeks exposure to elevated IOP induced by polystyrene microbead injection. Additional Aca23 and matched controls were studied at ages of 10 and 18 months. RESULTS: Aca23 mice had no significant difference from WT in IOP level, and in both strains IOP rose with age. In multivariable models, axial length and width were significantly larger in Aca23 than WT, became larger with age, and were larger after exposure to glaucoma (n=227 mice). From inflation test data, the estimates of scleral stress resultants in Aca23 mice were similar to age-matched and younger WT C57BL/6 (B6) mice, while the strain estimates for Aca23 were significantly less than those for either WT group in the mid-sclera and in some of the more anterior scleral measures (p<0.001; n=29, 22, 20 eyes in Aca23, older WT, younger WT, respectively). With chronic IOP elevation, Aca23 eyes increased 9% in length and 7% in width, compared to untreated fellow eyes (p<0.05, <0.01). With similar elevated IOP exposure, WT eyes enlarged proportionately twice as much as Aca23, increasing in length by 18% and in nasal-temporal width by 13% (both p<0.001, Mann-Whitney test). In 4 month old control optic nerves, mean RGC axon number was not different in Aca23 and WT (46,905±7,592, 43,628±11,162, respectively; p=0.43, Mann-Whitney test, n=37 and 29). With chronic glaucoma, Aca23 mice had a mean axon loss of only 0.57±17%, while WT mice lost 21±31% (median loss: 1% versus 10%, n=37, 29, respectively; p=0.001; multivariable model adjusting for positive integral IOP exposure). CONCLUSIONS: The Aca23 mutation in collagen 8α2 is the first gene defect found to alter susceptibility to experimental glaucoma, reducing RGC loss possibly due to differences in mechanical behavior of the sclera. Detailed study of the specific changes in scleral connective tissue composition and responses to chronic IOP elevation in this strain could produce new therapeutic targets for RGC neuroprotection

Losartan Treatment Protects Retinal Ganglion Cells and Alters Scleral Remodeling in Experimental Glaucoma

<div><p>Purpose</p><p>To determine if oral losartan treatment decreases the retinal ganglion cell (RGC) death caused by experimental intraocular pressure (IOP) elevation in mice.</p><p>Methods</p><p>We produced IOP increase in CD1 mice and performed unilateral optic nerve crush. Mice received oral losartan, spironolactone, enalapril, or no drug to test effects of inhibiting angiotensin receptors. IOP was monitored by Tonolab, and blood pressure was monitored by tail cuff device. RGC loss was measured in masked axon counts and RGC bodies by β-tubulin labeling. Scleral changes that could modulate RGC injury were measured including axial length, scleral thickness, and retinal layer thicknesses, pressure-strain behavior in inflation testing, and study of angiotensin receptors and pathways by reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry.</p><p>Results</p><p>Losartan treatment prevented significant RGC loss (median loss = 2.5%, p = 0.13), while median loss with water, spironolactone, and enalapril treatments were 26%, 28% and 43%; p < 0.0001). The lower RGC loss with losartan was significantly less than the loss with spironolactone or enalapril (regression model p = 0.001; drug treatment group term p = 0.01). Both losartan and enalapril significantly lowered blood pressure (p< 0.001), but losartan was protective, while enalapril led to worse than water-treated RGC loss. RGC loss after crush injury was unaffected by losartan treatment (difference from control p = 0.9). Survival of RGC in cell culture was not prolonged by sartan treatment. Axonal transport blockade after 3 day IOP elevations was less in losartan-treated than in control glaucoma eyes (p = 0.007). Losartan inhibited effects of glaucoma, including reduction in extracellular signal-related kinase activity and modification of glaucoma-related changes in scleral thickness and creep under controlled IOP.</p><p>Conclusions</p><p>The neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes in the sclera expressed at the optic nerve head.</p></div

Multivariable model for percent axon loss.

<p>^† Treatments that share a</p><p>^¶ are not significantly different at p ≤ 0.05;</p><p>* Tukey adjustment for multiple comparisons.</p><p>Multivariable model for percent axon loss.</p

Primary antibodies used for histological studies and Immunoblot.

<p>(W) Western blot, (S) Immunohistochemistry sections, (WM) Immunohistochemistry wholemount.</p><p>Primary antibodies used for histological studies and Immunoblot.</p

Axial length and width.

<p>Data are mean (standard deviation) in mm.</p><p>Axial length and width.</p

Axon loss by treatment group.

<p>* p = 0.0001, t test for difference from zero percent loss; SD = standard deviation; n = number of animals providing data per group.</p><p>Axon loss by treatment group.</p

The effect of losartan on creep rate in ramp—hold inflation studies.

<p>The creep rate during a 30 minute period at 30 mm Hg for losartan- and water-treated glaucoma eyes was compared to control eyes (represented by the horizontal line drawn at the ratio of one), along with losartan without glaucoma compared to control eyes as a ratio. Losartan treatment alone and losartan-glaucoma eyes had no difference in creep rate from controls, but water-treated glaucoma eyes had a greater creep rate than controls.</p

Average IOP after bead injection glaucoma.

<p>All differences among non-glaucoma eye groups and among glaucoma eye groups are statistically insignificant, with lowest p value in t tests = 0.14. Data are mm Hg with mean (standard deviation), including 7 measurements per eye, 40 eyes per group.</p><p>*Both eyes were controls in this treatment group.</p><p>Average IOP after bead injection glaucoma.</p

Pressure—strain estimates in load—unload protocol for losartan and control groups.

<p>Pressure—strain relationships in load—unload inflation tests show steeper (stiffer) curves for glaucoma eyes of losartan (triangle) and water-treated (diamond) compared to their respective fellow, control eyes (x and square, respectively). Y axis = inflation pressure in mm Hg.</p

Fixed Peripapillary Thickness.

<p>Data are mean (standard deviation), in microns.</p><p>^†Glaucoma minus Control.</p><p>* Both are control eyes in this treatment group.</p><p>Fixed Peripapillary Thickness.</p