Characterization of neural damage and neuroinflammation in Pax6 small-eye mice

Abstract

Aniridia is a panocular condition characterized by a partial or complete loss of the iris. It manifests various developmental deficits in both the anterior and posterior segments of the eye, leading to a progressive vision loss. The homeobox gene PAX6 plays an important role in ocular development and mutations of PAX6 have been the main causative factors for aniridia. In this study, we assessed how Pax6-haploinsufficiency affects retinal morphology and vision of Pax6^Sey mice using in vivo and ex vivo metrics. We used mice of C57BL/6 and 129S1/Svlmj genetic backgrounds to examine the variable severity of symptoms as reflected in human aniridia patients. Elevated intraocular pressure (IOP) was observed in Pax6^Sey mice starting from post-natal day 20 (P20). Correspondingly, visual acuity showed a steady age-dependent decline in Pax6^Sey mice, though these phenotypes were less severe in the 129S1/Svlmj mice. Local retinal damage with layer disorganization was assessed at P30 and P80 in the Pax6^Sey mice. Interestingly, we also observed a greater number of activated Iba1+ microglia and GFAP + astrocytes in the Pax6^Sey mice than in littermate controls, suggesting a possible neuroinflammatory response to Pax6 deficiencies.

Figure 1.. Pax1. Pax6^Sey phenotype results in smaller retinas.

<p/>A) Pax1^Sey in both 129S1/Svlmj and C57BL/6 exhibit small-eye phenotypes. Yellow dashed lines highlight the ocular anatomy. B) Example of whole-mounted retina stained with DAPI showing the contour shape for retinal area quantification (S: superior, T: temporal, I: inferior, N: nasal). C) Pax6^Sey mice in both backgrounds show significantly smaller whole-mount area compared to their WT counterparts. 129S1/Svlmj is labeled as 129S and C57BL/6 as C57; same for all figures below. *: P < 0.05 in one-way ANOVA post-hoc Tukey’s test.

Figure 2.. Intraocular pressure (IOP) increases, and visual acuity decreases significantly with age in Pax6^Sey mice.

A) Pax6^Sey C57BL/6 mice show a significant increase of IOP by P30; Pax6^Sey 129S1/Svlmj mice show a significant IOP increase by P60. B) Visual acuity was significantly decreased in Pax6^Sey of both C57BL/6 and 129S1/Svlmj backgrounds at P20. C) Plots of acuity versus IOP. *: P < 0.05, ***: P < 0.001; ****: P < 0.0001 in one-way ANOVA post-hoc Tukey’s test.

Figure 3.. Abnormal layer organization in Pax6^Sey mouse retinas.

A) Layer disorganization was present in Pax6^Sey mice of both genetic backgrounds. P90 full sagittal retina sections are shown as well as high magnification of normal regions and hotspots, the localized damages. B) Quantification of total retinal thickness of the hotspots in WT, Pax6^Sey normal area and Pax6^Sey hotspots (hs). *: P < 0.05 in one-way ANOVA post-hoc Tukey’s test. Sample numbers are shown within the bars.

Figure 4.. Retinal subpopulations show varied densities in Pax6^Sey mice.

Confocal imaging of retinal cell-type markers, Rbpms, ChAT, and AP-2 in C57BL/6 (A) and 129S1/Svlmj (B) P90 retina sections. The somas labeled Rbpms, ChAT, and AP-2 showed varied distributions in Pax6^Sey mice.

Figure 5.. C57BL/6 Pax6^Sey mice exhibit increased density of activated (Iba1+) microglial cells.

A) Confocal imaging of a whole-mounted P80 retina immuno-stained with Iba1 (red) and DAPI (blue). S: superior, T: temporal, I: inferior, N: nasal. B) Enlarged images of Iba1 staining and quantification of Iba1+ staining at P25 (dark red) and P80 (light red) in C57BL/6 background (W = wildtype, P = Pax6^Sey). C) Enlarged images of Iba1 staining and quantification of Iba1+ staining at P25 (dark red) and P80 (light red) in 129S1/Svlmj background (W = wildtype, P = Pax6^Sey). Each point represents one eye for each mouse *: P < 0.05, ***: P < 0.001 in one-way ANOVA post-hoc Tukey’s test.

Figure 6.. C57BL/6 Pax6^Sey mice also exhibit an increased density of astrocytes.

A) Confocal imaging of whole-mounted P80 retina immuno-stained with GFAP (green), a marker for astrocytes in the superficial layer of the retina. S: superior, T: temporal, I: inferior, N: nasal. B) Two magnified images that have been stained with GFAP (green) and DAPI (blue) with an area of 30,000 μm² and have been used for cell counting. C) Quantification of cells with GFAP+ staining above the GCL at P25 (dark blue) and P80 (light blue) (W = wildtype, P = Pax6^Sey). Each point represents one eye for each mouse. **: P < 0.01 in one-way ANOVA post-hoc Tukey’s test.