Genetic modification of glaucoma associated phenotypes between AKXD-28/Ty and DBA/2J mice

Abstract

Background: Glaucoma is a common disease but its molecular etiology is poorly understood. It involves retinal ganglion cell death and optic nerve damage that is often associated with elevated intraocular pressure. Identifying genes that modify glaucoma associated phenotypes is likely to provide insights to mechanisms of glaucoma. We previously reported glaucoma in DBA/2J mice caused by recessive alleles at two loci, isa and ipd, that cause iris stromal atrophy and iris pigment dispersion, respectively. A approach for identifying modifier genes is to study the effects of specific mutations in different mouse strains. When the phenotypic effect of a mutation is modified upon its introduction into a new strain, crosses between the parental strains can be used to identify modifier genes. The purpose of this study was to determine if the effects of the DBA/2J derived isa and ipd loci are modified in strain AKXD-28/Ty.

Results: AKXD-28/Ty mice develop glaucoma characterized by intraocular pressure elevation, retinal ganglion loss, and optic nerve excavation. In AKXD-28/Ty, isa causes an iris stromal atrophy phenotype as in DBA/2J. However, the iris pigment dispersion phenotype associated with ipd in DBA/2J does not occur in AKXD-28/Ty. Additionally, a greater severity and speed of retinal and optic nerve damage following intraocular pressure elevation in AKXD-28/Ty compared to DBA/2J mice suggests that AKXD-28/Ty is more susceptible to pressure-induced cell death.

Conclusions: The consequences of the ipd and isa mutations are modified in the AKXD-28/Ty background. These strains provide a resource for the identification of modifier genes that modulate pigment dispersion and susceptibility to pressure-induced cell death.

Figure 1

AKXD28 develop ISA but not IPD. Representative iris phenotypes in aging AKXD28 (a-c and g, h) and D2 (d-f and i) mice are shown. Age and disease severity increase from left to right. (a, d) Two month old AKXD28 and D2 mice have a normal, complex iris morphology with clearly evident iris details including crypts, small central pupil, prominent peripupillary sphincter muscle, and pupillary ruff. (b, e) At 12 months AKXD28 (b) have iris stromal atrophy characterized by loss of iris detail, thinning of the iris stroma with exposure of the sphincter muscle (arrowhead in b), and mild transillumination defects (arrow). Dispersed pigment is not prominent in the anterior chamber. The eyes of similarly aged D2 mice (e) are more severely affected with both stromal atrophy and iris pigment dispersion. The stromal atrophy is characterized by abnormally shaped pupils, exposure of the sphincter, and loss of iris detail while the pigment dispersion is evidenced by the prominent accumulation of pigment on the front of the iris and lens (arrowheads in e). (c, f) 26 month old AKXD28 and D2 mice have marked stromal atrophy with enlarged irregular pupils, iris holes, and increased transillumination. In D2, severe loss of both iris stromal and iris pigment epithelium pigmentation due to the ISA and IPD phenotypes results in large transparent iris regions. In contrast, the AKXD28 iris remains generally pigmented due to the lack of IPD and retention of much of the pigment epithelium. Histologic analysis confirms the presence of ISA and lack of IPD in AKXD28 mice. (g) In a young AKXD28 mouse the iris has a robust stroma (S) and pigment epithelium (P) that are separated by the dilator muscle (masked by pigment in this section but located at the level of the arrows). (h) Although severity varies locally, the stroma of old AKXD28 mice is severely atrophied and almost non-existent in many places (arrow). The iris pigment epithelium (arrowhead) of old mice has a flattened morphology but remains remarkably intact considering the overall condition of the iris. (i) In old D2 mice, both the iris stroma and iris pigment epithelium are severely atrophic.

Figure 2

Anterior synechiae and elevated IOP in AKXD28. (a) Young mice have normal, unobstructed iridocorneal angles. The angle has a well developed trabecular meshwork (TM, arrowhead) and open Schlemm's Canal (SC). (b) A large anterior synechia (arrow) has occluded the TM and SC in a 14 month old mouse. As shown in c (and see Table 1), the formation of synechiae is followed by IOP elevation. Two standard deviations (2 SD) above the mean IOP for 7-10 month mice is indicated by the dashed line (females) and a full line (males). Ciliary body atrophy occurs in some mice and its incidence increases with age. Although histologic analysis and IOP measurement were not performed on the same eyes, it seems likely that ciliary body atrophy explains the drop in female IOP at 19 to 21 months. Original magnifications 400X.

Figure 3

Severe retinal and optic nerve damage in AKXD28 mice. The panels are arranged to display the progressive increase in severity from left to right. All images are from strain AKXD28 mice except for f, (D2) and g,h (AKXD28B6F1 X AKXD28, backcross N2). (a) Young AKXD28 retinas have normal morphology. The retinal ganglion cell layer (G) is continuous and 1-2 cells thick. The inner nuclear layer is approximately 5 to 6 cells thick (flanked by arrowheads). (b) Moderately affected AKXD28 retinas contain fewer retinal ganglion cells while the inner nuclear layer has some cell loss but remains relatively normal. (c) Severely affected AKXD28 retinas have very few retinal ganglion cells, the inner nuclear layer (arrow) is only 1-2 cells thick, and the total thickness of the retina is greatly reduced. Focal loss of photoreceptors is also present. The image represents the severe phenotype attained by all old AKXD28 eyes; some old eyes have even more cell loss with severe photoreceptor depletion and the remnants of retina are very thin. This severe atrophy does not occur in D2 mice (see f for a typical severe D2 retina). (d) Normal optic nerve head of a young mouse characterized by a thick nerve fiber layer entering the optic nerve (arrows), a central vessel (V), and well organized pial septae (P). (e) Advanced optic nerve excavation (arrowheads) with atrophy extending to a level external to the choroid (C). There is severe peripapillary atrophy with thinning of most retinal layers near the nerve. Although not prominent in this image, gliosis was frequently observed in severely damaged nerves. (f) Representative retina from a D2 mouse exhibiting advanced end stage retinal disease typical for that strain. Note that the inner nuclear layer is relatively unaffected and overall retinal thickness is maintained. (g) Normal fundus. (h) Glaucomatous fundus with an asymmetric and severely excavated optic nerve head (arrowhead). Peripapillary chorioretinal atrophy is also distinctly recognizable in this eye. These fundi are from backcross mice since all old AKXD28 mice had severe cataracts that made photography very difficult. The appearance of these backcross fundi closely resembles those of age matched AKXD28. Original magnifications 400X (a,b,c,f,) and 200X (d,e).

Figure 4

Optic nerve damage in AKXD28 mice. Representative nerve sections scored as (a) mild, (b) moderate, and (c) severe are shown. (d) Nerve damage is first evident in females. At 19 to 20 months all females are severely affected whereas approximately half of the males are. Original magnifications 630X.