Oxysterol Compounds in Mouse Mutant αA- and αB-Crystallin Lenses Can Improve the Optical Properties of the Lens

Abstract

Purpose: To investigate how cataract-linked mutations affect the gradient refractive index (GRIN) and lens opacification in mouse lenses and whether there is any effect on the optics of the lens from treatment with an oxysterol compound.

Methods: A total of 35 mice including wild-type and knock-in mutants (Cryaa-R49C and Cryab-R120G) were used in these experiments: 26 mice were treated with topical VP1-001, an oxysterol, in one eye and vehicle in the other, and nine mice were untreated controls. Slit lamp biomicroscopy was used to analyze the lens in live animals and to provide apparent cataract grades. Refractive index in the lenses of 64 unfixed whole mouse eyes was calculated from measurements with X-ray phase tomography based on X-ray Talbot interferometry with a synchrotron radiation source.

Results: Heterozygous Cryaa-R49C lenses had slightly irregularly shaped contours in the center of the GRIN and distinct disturbances of the gradient index at the anterior and posterior poles. Contours near the lens surface were denser in homozygous Cryab-R120G lenses. Treatment with topical VP1-001, an oxysterol, showed an improvement in refractive index profiles in 61% of lenses and this was supported by a reduction in apparent lens opacity grade by 1.0 in 46% of live mice.

Conclusions: These results indicate that α-crystallin mutations alter the refractive index gradient of mouse lenses in distinct ways and suggest that topical treatment with VP1-001 may improve lens transparency and refractive index contours in some lenses with mutations.

Figure 1.

Slit lamp images of eyes from mice of different genotypes. Representative slit lamp images show the extent of lens opacity in WT lenses aged (a) 255 days, (b) 493 days, and (c) 738 days with apparent cataract gradings of 1.0, 4.0, and 3.0, respectively. The 493-day-old WT lens is compared in d with 416-day-old Cryaa-R49C-Het lenses (e and f); both had lower apparent cataract gradings than the WT 493-day-old lens. The 493-day-old WT lens is compared in g with 402-day-old Cryab-R120G-Het lenses (h and i) and in j with 449-day-old Cryab-R120G-Hom lenses (k and l). The apparent cataract gradings for the Cryab-R120G-Het lenses were lower than in the WT lens; the Cryab-R120G-Hom lenses were very opacified and had the same apparent grading as the WT lens. Images shown are of OS (a, b, d, g, h, i, k, j) and OD (c, e, f, l) lenses. The nasal side is on the left side of the image in OS lenses and on the right side of the image in OD lenses.

Figure 2.

Comparison of refractive index profiles in WT lenses with age. Refractive index distributions in the mid-sagittal cross-sectional plane of each WT mouse lens shown both as contour plots (first row) and as three-dimensional GRIN profiles (second row) for lenses aged 255 days (a, d) [ID 1378-3 Supplementary Table S1], 493 days (b, e) [ID 1078-4 Supplementary Table S1] and 738 days (c, f) [ID 1170-5 Supplementary Table S1]. Magnitudes of refractive index are indicated using color bar on the right side of each image. Slight disturbances at the poles of the 255-day-old WT lens, shown by arrows (a) are likely to be capsular damage. The arrows in b show irregularities in peripheral contours in the polar regions of the 493-day-old lens; the arrows in c show perturbed contours near the anterior pole with slighter perturbation at the posterior pole. The maximum refractive index found in the innermost contour is 1.54; adjacent contours differ in refractive index by a value of 0.01. WT = wild type, d = day, F = female, M = male, a = anterior pole, p=posterior pole, LD = lens diameter, ND = diameter of the innermost contour.

Figure 3.

Comparison of refractive index profiles between WT lens and Cryaa-R49C-Het lenses. Refractive index distributions in the mid-sagittal cross-sectional plane of each mouse lens shown both as contour plots (first row) and as 3-dimensional GRIN profiles (second row) for a 493-day-old WT lens (a, d) [ID 1078-4 Supplementary Table S1], and two 416-day-old Cryaa-R49C-Het lenses (b, e [ID 1310-2 Supplementary Table S1] and (c, f) [ID 1311-5 Supplementary Table S1]. Magnitudes of refractive index are indicated by the color bar on the right side of each image. Slight disturbances, shown by arrows, are seen at the poles of the WT lens (a) and could be a manifestation of age-related changes. Arrows in b and c indicate significant disturbances in the refractive index gradient in the polar regions. WT = wild type, d = day, F = female, M = male, a = anterior pole, p = posterior pole.

Figure 4.

Comparison of refractive index profiles between WT lens and Cryab-R120G-Het lenses. Refractive index distributions in the mid-sagittal cross-sectional plane of each mouse lens shown both as contour plots (first row) and as three-dimensional GRIN profiles (second row) for a 493-day-old WT lens (a, d) [ID 1078-4 Supplementary Table S1], and two 402-day-old Cryab-R120G-Het lenses (b, e) [ID 1145-1 Supplementary Table S1], and (c, f) [ID 1144-6 Supplementary Table S1]. Magnitudes of refractive index are indicated using color bar on the right side of each image. Slight disturbances, shown by arrows, are seen at the poles of the WT lens (a) and could be a manifestation of age-related changes. Arrows in b and c indicate greater disturbances of refractive index gradient in the anterior polar regions than seen in the WT lens (a). This is particularly evident in c. WT = wild type, d = day, F = female, M = male, a = anterior pole, p = posterior pole.

Figure 5.

Comparison of refractive index profiles between WT lens and Cryab-R120G-Hom lenses. Refractive index distributions in the mid-sagittal cross-sectional plane of each mouse lens shown both as contour plots (first row) and as three-dimensional GRIN profiles (second row) for a 493-day-old WT lens (a, d) [ID 1078-4 Supplementary Table S1], and two 449-day-old Cryab-R120G-Hom lenses (b, e) [ID 905-3 Supplementary Table S1] and (c, f) [ID 906-8 Supplementary Table S1]. Magnitudes of refractive index are indicated using color bar on the right side of each image. Slight disturbances, shown by arrows, are seen at the poles of the WT lens (a) and could be a manifestation of age-related changes. The refractive index contours in the old Cryab-R120G-Hom lenses are narrow, not concentric and misshapen (b and c). Arrows show disturbances in refractive index indicative of localized high protein concentration (b,e and c,f). The three-dimensional profiles (e and f) are narrow and steep with ridges (shown by arrows) on the surface. WT = wild type, d = day, F = female, M = male, a = anterior pole, p = posterior pole.

Figure 6.

Slit lamp images of eyes from mice of different genotypes. Representative slit lamp images show the comparison between vehicle-treated (left) lenses and VP1-001-treated (right) lenses in (A) WT lens pair aged 270 days, (B) Cryaa-R49C-Het lens pair aged 186 days, (C) Cryab-R120G-Het lens pair aged 402 days, and (D) Cryab-R120G-Hom lens pair aged 185 days. (The nasal side is on the left side of the image in OS [vehicle] lenses and on the right side of the image in OD [VP1-001] lenses).

Figure 7.

Comparison of refractive index profiles between control and treated eyes of similar ages. Refractive index distributions shown as contour plots in the mid-sagittal cross-sectional plane and as three-dimensional GRIN profiles in control lenses from mice that had not been treated with either vehicle or VP1-001 aged (a) 255 days [ID 1378-3 Supplementary Table S1] and (c) 415 days [ID 938-1 Supplementary Table S1] and in lenses from mice treated with vehicle in the OS eye and with VP1-001 in the OD eye aged (b) 270 days [ID 1210-3 Supplementary Table S1] and (d) 493 days [ID 1077-3 Supplementary Table S1]. The contour plots and three-dimensional profiles are generally indicative of normal optical function with show slight disturbances (marked by arrows) in the polar regions of the 255-day-old WT lens (a) that could be a manifestation of capsular damage. There is a slight disturbance to the refractive index in the anterior pole of the 415-day-old OD lens as marked by the arrows in the contour plot and the three-dimensional profile (c). The treated pairs b and d show some small irregularities in the polar regions (marked by arrows on the contour plots) in the vehicle treated lenses; these irregularities are not apparent in the lenses treated with VP1-001 (b and d). a = anterior pole, p = posterior pole.

Figure 8.

Refractive index profiles in Cryaa-R49C-Het eyes and Cryab-R120G-Het lenses. Refractive index distributions shown as contour plots in the mid-sagittal cross-sectional plane and as three-dimensional profiles in lenses from mice treated with vehicle in the OS eye and with VP1-001 in the OD eye: (a) Cryaa-R49C-Het mouse aged 186-days [ID 1237-7 Supplementary Table S1] and (b) Cryab-R120G-Het aged 402-days [ID 1144-6 Supplementary Table S1]. The polar regions of both vehicle treated lenses have small disturbances to the refractive index profile (indicated by arrows); these are slightly reduced in the VP1-001–treated lenses (indicated by arrows). a = anterior pole, p = posterior pole.

Figure 9.

Refractive index profiles in Cryab-R120G-Hom lenses. Refractive index distributions shown as contour plots in the mid-sagittal cross-sectional plane and as 3-dimensional profiles in lenses in Cryab-R120G-Hom mouse treated with vehicle in the OS eye and with VP1-001 in the OD eye at (a, c, d, e) 260 days old [a: ID 1216-4, Supplementary Table S1; c: ID 1213-3 Supplementary Table S1; d:ID 1212-1 Supplementary Table S1], (b) 185 days old [ID 1231-4 Supplementary Table S1], and (f) 380 days old [ID 1150-4 Supplementary Table S1]. In the 260-day-old lenses (a, c, e) the refractive index profiles are skewed, and there is little if any difference between vehicle or VP1-001–treated lenses. Some small differences between lenses in the pair seen in (d) are apparent with irregularities in the refractive index contours in vehicle treated lens (indicated by the arrows) which are not present in the corresponding VP1-001–treated lens. There are greater differences evident in the 185-day-old (b) and 380-day-old lens pairs (f). In b the vehicle-treated 185-day-old lens has a narrow and asymmetric profile with a relatively sharp peak, an indentation in the contours and in the GRIN profile in the posterior polar region (indicated by arrows), protruding contours and slight distortion in the GRIN profile in the anterior polar region (indicated by arrows). These are not seen in the VP1-001–treated lens (b) which manifests a profile closer in breadth to normal lenses, more regular contours. The 380-day-old lens in f shows localized peaks in refractive index indicative of protein aggregation in the vehicle-treated lens (indicated by arrows) that are not seen in the VP1-001–treated lens. a = anterior pole, p = posterior pole.