Fabry Disease Rat Model Develops Age- and Sex-Dependent Anterior Segment Ocular Abnormalities

Abstract

Purpose: Fabry disease is an X-linked lysosomal storage disorder that results in multi-systemic renal, cardiovascular, and neuropathological damage, including in the eyes. We evaluated anterior segment ocular abnormalities based on age, sex (male and female), and genotype (wild-type, knockout [KO] male, heterozygous [HET] female, and KO female) in a rat model of Fabry disease.

Methods: The α-Gal A KO and WT rats were divided into young (6-24 weeks), adult (25-60 weeks), and aged (61+ weeks) groups. Intraocular pressure (IOP) was measured. Eyes were clinically scored for corneal and lens opacity as well as evaluated for corneal epithelial integrity and tear break-up time (TBUT). Anterior chamber depth (ACD) and central corneal thickness (CCT) using anterior segment-optical coherence tomography (AS-OCT).

Results: The Fabry rats showed an age-dependent increase in IOP, predominantly in the male genotype. TBUT was decreased in both male and female groups with aging. Epithelial integrity was defective in KO males and HET females with age. However, it was highly compromised in KO females irrespective of age. Corneal and lens opacities were severely affected irrespective of sex or genotype in the aging Fabry rats. AS-OCT quantification of CCT and ACD also demonstrated age-dependent increases but were more pronounced in Fabry versus WT genotypes.

Conclusions: Epithelial integrity, corneal, and lens opacities worsened in Fabry rats, whereas IOP and TBUT changes were age-dependent. Similarly, CCT and ACD were age-related but more pronounced in Fabry rats, providing newer insights into the anterior segment ocular abnormalities with age, sex, and genotype in a rat model of Fabry disease.

Figure 1.

Age-, sex-, and genotype-dependent changes in intraocular pressure (IOP) levels in a rat model of Fabry disease. Mean IOP was obtained in Fabry rats using a Tonolab rebound tonometer. All readings averaged the 6 values recorded between 9 and 11 AM to avoid diurnal variation during the day. (A) There was a significant increase in IOP levels in an age-dependent manner. (B) The aged male Fabry rats were observed to have higher IOP levels than the adult rats group. Similarly, the adult male rats showed a significant increase in IOP compared to the young male rats. (C) When the Fabry rats were divided based on genotypes, IOP levels showed an increasing trend in KO male rats, heterozygous (HET) female rats, and KO female rats but were not significant versus wild-type (WT). *, P < 0.05.

Figure 2.

Age-, sex-, and genotype-dependent changes in tear break up time (TBUT) levels in a rat model of Fabry disease. Fabry rats showed a significant decrease in TBUT in the adult rats group compared to the young rats group (A). The bar graph represents the average mean values of TBUT for different age groups. When Fabry rats were compared based on sex irrespective of gender, the female rats in the adult rats group showed significantly low TBUT values compared to the young rats group. Further analysis comparing genotypes in Fabry rats, adult and aged KO male rats depict significantly decreased TBUT compared to the young KO male rats (C). Similarly, heterozygous (HET) female rats in the adult rats group showed significantly lower TBUT values than the young HET female rats. Data are represented as mean ± SE. *, P < 0.05; ****, P < 0.0001.

Figure 3.

Age-, sex-, and genotype-dependent changes in corneal epithelial integrity in a rat model of Fabry disease. Fabry rats were administered 1% sodium fluorescein dye topically to the eye. The images were randomized and scored by at least three investigators in a blinded manner using cornea scoring rubrics described previously. Based on the reported literature, the scoring was given on a scale from 0 to 5. (A) Present the representative images of rat cornea of male and female Fabry rats in different age groups and genotypes based on the epithelial defects; the arrows point at the damage to the cornea indicated by the deposition of fluorescein stain. (B) The aged rats developed more severe corneal defects and attained a higher stain score than the young Fabry rats. (C) Sex-related differences were observed only in the aged male Fabry rats, depicting a significant increase in epithelial damage compared to the young male group. (D) The genotypic separation of Fabry rats suggests a significant increase in the KO male Fabry rats in the adult group compared to the young rats group. Similarly, the KO male rats in the aged group showed increased corneal damage compared to the adult group. On the other hand, the heterozygous (HET) adult female rats showed significantly increased corneal stain scores compared to their HET counterparts in the young group. *, P < 0.05; **, P < 0.01.

Figure 4.

Age-, sex-, and genotype-dependent changes in corneal opacity in a rat model of Fabry disease. The slit lamp images were captured, and corneal opacity was scored on a scale of 1 to 5 based on the severity of corneal deposits using a cornea scoring rubric described previously. Briefly, a score of 1 represents no or few scattered deposits, a score of 2 represents < 25% of the honeycomb network, a score of 3 represents 25% to 50% of the honeycomb network, a score of 4 represents >50% of the honeycomb network, and a score of 5 represents confluent deposits and plaques. (A) The representative images of rat cornea slit images of male and female Fabry rats in different age groups based on the severity of corneal deposits; the arrows point at the deposits similar to cornea verticillata observed in the Fabry cornea. (B) The Fabry rats in the aged rats group developed significantly higher corneal opacities than the adult and young rats groups. (C) When compared between the sexes, the aged male rats showed significantly higher corneal scores compared to the young male rats. (D) When the Fabry rats were defined based on their genotype, the group most affected was the KO male rats, which showed significantly high corneal scores in the adult rats group and a further increase in the aged rats group. The KO female rats group also showed elevated corneal scores in the adult versus the young female rats. *, P < 0.05; **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001.

Figure 5.

Age-, sex-, and genotype-dependent changes in central corneal thickness (CCT) in a rat model of Fabry disease. (A) Representative optical coherence tomography (OCT) images were used to measure the central corneal thickness (CCT) in Fabry rats. (B) An age-dependent increase in CCT was observed in the Fabry rats. (C) Compared among the sexes, Fabry rats displayed significantly higher CCT values in the adult rats group and the aged rats group than the young rats group. (D) When Fabry rats were compared based on genotypes, the wild-type (WT) and KO male rats had an age-dependent increase in CCT. *, P < 0.05; **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001.

Figure 6.

Age-, sex-, and genotype-dependent changes in the anterior chamber depth (ACD) in a rat model of Fabry disease. (A) Representative optical coherence tomography (OCT) images were used to measure the anterior chamber depth (ACD) in Fabry rats. (B) An age-dependent increase in ACD was observed in the Fabry rats. (C) Compared between the sexes, the aged male and female rats displayed significantly higher ACD values in the adult and aged groups than the young rats group (adult group; and n = 11 male rats and n = 18 female rats to the aged rats group). (D) When Fabry rats were compared based on genotypes, the wild-type (WT), KO male rats, heterozygous (HET) female rats, and KO female rats, all had an age-dependent increase in ACD values. *, P < 0.05; **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001.

Figure 7.

Age-, sex-, and genotype-dependent changes on lenticular opacity in a rat model of Fabry disease. (A) Representative images of the lens examined using the slit lamp showed varying levels of lenticular opacities in the Fabry rat lens. The arrows indicate lenticular opacities, scored and assessed in a rubric based on the severity described previously. A lens score of 1 represents none or developmental suture, a lens score of 2 represents some puncta, a lens score of 3 means central focal cataract, and a lens score of 4 represents nuclear cataract and halo pattern. (B) A significant increase in the lens score was observed in an age-dependent manner in the Fabry rats. (C) When analyzed based on sex, the aged male rats showed a significant increase in lens score compared to the adult and young rats groups. (D) There were significant differences in the lens score in various genotypes of the Fabry rats. Wild-type (WT) Fabry rats showed increased lenticular opacity based on age. Similarly, there was an increased lens score in the KO males in an age-dependent manner. On the other hand, KO female rats showed high lenticular opacities overall, regardless of age. The mean score of individual age groups was compared using 1-way ANOVA, and 2-way ANOVA was performed comparing the gender versus age and genotype versus age datasets. *, P < 0.05; **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001.