Mutant human myocilin induces strain specific differences in ocular hypertension and optic nerve damage in mice - PubMed (original) (raw)

Mutant human myocilin induces strain specific differences in ocular hypertension and optic nerve damage in mice

Colleen M McDowell et al. Exp Eye Res. 2012 Jul.

Abstract

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12-15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.

Copyright © 2012 Elsevier Ltd. All rights reserved.

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Figures

Figure 1

Figure 1. Myocilin is overexpressed in the trabecular meshwork after adenovirus transduction

All mice were injected in one eye with 5×107 pfu Ad5.MYOC.Y437H. The contralateral uninjected eye served as a control. All samples were collected 7 days post-injection. The first two columns are labeled with anti-myocilin antibody and myocilin expression is represented by the brown labeling (A–B, E–F, I–J, M–N; n= 5 mice per strain). Myocilin expression in the TM is indicated by black arrows (B, F, J, N). The last two columns are sections from the same eye used in the first two columns and are stained with hematoxylin. The stars represent an open iridiocorneal angle (C–D, G–H, K–L, O–P; n=5 mice per strain).

Figure 2

Figure 2. Ad5.MYOC.Y437H induces strain dependent ocular hypertension in mice

All mice were injected in one eye with 5×107 pfu Ad5.MYOC.Y437H and IOP was measured for 8 weeks post injection. The contralateral uninjected eye served as a control. (A) A/J mice had significant IOP elevation beginning at 5 days post-injection (p=0.0002). The IOP elevation remained significant for at least 56 days (p<0.0001, Days 7–56). (B) BALB/cJ mice had significant IOP elevation beginning at 5 days post-injection (p<0.0001). The IOP elevation remained significant for at least 56 days (p<0.0001, Days 7–56). (C) C57BL/6J mice had significant IOP elevation beginning at 5 days post-injection (p=0.0393). The IOP elevation remained significant for at least 56 days (p<0.0001, Days 7–56). (D) C3H/HeJ mice had significant IOP elevation at Day 5 (p=0.0018), Day 7 (p=0.0042) and Day 14 (p=0.0104). At all other time points there was no significant elevation in IOP. Ad5.MYOC.Y437H injected eyes represented in grey, uninjected control eye represented in black. Data reported as mean +/− SD.

Figure 3

Figure 3. Ad5.MYOC.WT has no effect on IOP

BALB/cJ mice were injected in one eye with 5×107 pfu Ad5.MYOC.WT and IOP was measured for 8 weeks post injection. The contralateral uninjected eye served as a control. There was no statistical difference in IOP between the injected and uninjected eye at any time point throughout the 8-week time period. Ad5.MYOC.WT injected eyes represented in grey, uninjected control eye represented in black. Data reported as mean +/− SD.

Figure 4

Figure 4. Ad5.MYOC.Y437H damages the optic nerve in A/J mice at 8 weeks post injection

Optic nerve damage was assessed by PPD stain and quantified by using the optic nerve damage score (ONDS) system, which clinically grades optic nerve damage from 1 (no damage) to 5 (severe damage). At 8 weeks post-injection there was a significant difference in ONDS in A/J mice (injected eye ONDS=2.64 +/− 0.19, n=18; uninjected eye ONDS=1.40 +/− 0.14, n=18, p<0.001). There was no significant difference in ONDS in BALB/cJ, C57BL/6J or C3H/HeJ mice at 8 weeks. Data represented as mean +/− SEM.

Figure 5

Figure 5. Ad5.MYOC.Y437H has no effect on RGC loss or SC damage at 8 weeks post injection

(A) Nissl stained retina flat mounts were used to quantify RGC loss 8 weeks after Ad5.MYOC.Y437H injection. At 8 weeks post-injection there was no significant difference in RGC number in A/J (n=8), BALB/cJ (n=10), C57BL/6J (n=10), or C3H/HeJ (n=10) mice. Two images (400X) were taken from each quadrant of the retina flat mount (8 images total) and four regions within each image were counted, representing about 2% of total RGC’s. (B) Nissl stained superior colliculus sections were used to quantitate damage 8 weeks after Ad5.MYOC.Y437H injection. At 8 weeks post-injection there was no significant difference in cell number in the superior colliculus in A/J (n=10), BALB/cJ (n=10), C57BL/6J (n=10), or C3H/HeJ (n=8) mice. Data represented as mean +/− SD.

Figure 6

Figure 6. Mutant myocilin is expressed in the TM 8 weeks post-injection in A/J mice

All mice were injected in one eye with 5×107 pfu Ad5.MYOC.Y437H. The contralateral uninjected eye served as a control. All samples were collected 8 weeks post-injection. The left column is labeled with anti-myocilin antibody and myocilin expression is represented by the brown labeling (A, C, E, G; n= 5 mice per strain). Myocilin expression in the TM is indicated by black arrows. The right column is sections from the same eye used in the left column and are stained with hematoxylin. The stars represent open iridiocorneal angles (B, D, F, H; n=5 mice per strain).

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