Optimizing the sodium iodate model: Effects of dose, gender, and age

Abstract

Sodium iodate (NaIO₃) is a commonly used model for age-related macular degeneration (AMD), but its rapid and severe induction of retinal pigment epithelial (RPE) and photoreceptor degeneration can lead to the premature dismissal of potentially effective therapeutics. Additionally, little is known about how sex and age affect the retinal response to NaIO₃. This study aims to establish a less severe yet reproducible regimen by testing low doses of NaIO₃ while considering age- and sex-related effects, enabling a broader range of therapeutic evaluations. In this study, young (3-5 months) and old (18-24 months) male and female C57Bl/6J mice were given an intraperitoneal (IP) injection of 15, 20, or 25 mg/kg NaIO₃. Damage assessment one week post-injection included in vivo imaging, histological examination, and qRT-PCR analysis. The results revealed that young mice showed no damage at 15 mg/kg IP NaIO₃, with varying degrees of damage observed at 20 mg/kg. At 25 mg/kg, most young mice displayed widespread retinal damage, with females exhibiting less retinal thinning than males. In contrast, older mice at 20 and 25 mg/kg displayed a more patchy degeneration pattern, outer retinal undulations, and greater variability in degeneration than the young mice. The most effective model for minimizing damage while maintaining consistency utilizes young female mice injected with 25 mg/kg NaIO₃. The observed sex- and age-related differences underscore the importance of considering these variables in research, aligning with the National Institutes of Health's guidance. While the model does not fully replicate the complexity of AMD, these findings enhance its utility as a valuable tool for testing RPE/photoreceptor protective or replacement therapies.

Keywords: Age differences; Oxidative stress; Retinal degeneration; Sex differences; Sodium iodate.

Figure 1.. Damage caused by 15, 20, and 25 mg/kg NaIO₃ in young mice.

(A) Quantification of the area of damage, defined as speckled autofluorescence, induced by 15 (n=10), 20 (n=86), and 25 (n=35) mg/kg NaIO₃ in young male mice, measured by percent of 102° field of view cSLO image containing damage. Each dot represents one mouse; mean ± SEM is reported. (B) Comparison of damage seen in cSLO images of young male (n = 35) and female (n = 26) mice injected with 25 mg/kg NaIO₃. Each dot represents one mouse; mean ± SEM is reported. (C) Proportions of the different categories of damage induced by 15 (male n=10), 20 (male n=86), and 25 (male n=35; female n=26) mg/kg NaIO₃. (D) cSLO images (blue autofluorescence (BAF), top; infrared autofluorescence (IRAF), middle) and vertical OCT images (bottom; left side of each image is inferior, right side is superior) of the four categories induced by low doses of NaIO₃ and saline-injected age-matched male control. All images except for the saline control are from male mice given 20 mg/kg NaIO₃. The set of images in each column come from the same mouse. The cSLO images cover a larger area than the OCT images, and the OCT image length is shown as the red line in the top left cSLO image.

Figure 2.. Damage caused by 20 and 25 mg/kg NaIO₃ in old mice.

(A) Quantification of the area of damage induced by 20 (male n=10, female n=7) and 25 (male n=14, female n=13) mg/kg NaIO₃, as measured by percent of 102° field of view cSLO image containing damage. Each dot represents one mouse; mean ± SEM is reported. (B) Proportions of the different categories of damage induced by 20 and 25 mg/kg NaIO₃ (same n as A). (C) cSLO images (BAF, top; IRAF, middle) and vertical OCT images (bottom) of the four categories induced by low doses of NaIO₃: none, sparse, patchy, and widespread. The representative “none” and “sparse” images are from male mice at 25 mg/kg NaIO₃, the “patchy” and “widespread” images are from female mice at 25 mg/kg NaIO₃, and the “saline control” images are from a saline-injected 24-month male mouse. The set of images in each column come from the same mouse. cSLO images cover a larger area than OCT images, with the OCT image length indicated by the red line in the top-left cSLO image.

Figure 3.. OCT analysis of both sexes of young and old mice injected with 25 mg/kg NaIO₃.

(A) Representative horizontal and vertical OCT images (OCT-H and OCT-V respectively) of saline-injected 24-month-old male mouse and young male, young female, old male, and old female mice injected with 25 mg/kg NaIO₃, taken one week after injection. Each pair of images (horizontal and vertical) was captured from the same eye. White arrow shows an example of an undulation. (B) cSLO image showing the range of the OCT images. The white circle shows how far the central images in A extend. Peripheral images (Fig. S3) extended this range for measurements in C. Blue line: horizontal plane; red line: vertical plane. (C) Quantification of OCT images, measuring from the top of the INL to Bruch’s membrane, as shown in the brackets on the rightmost images in A. Measurements were taken every 50 pixels (approximately 109 μm). Mean ± SD is reported with the following distribution: young untreated males (n = 5), young male (n = 16), young female (n = 20), old untreated male (n = 5), old male (n = 14), and old female (n = 13). All locations were averaged together to get one data point per mouse for statistical analysis: Pairwise comparisons showed young control males, young males, and young females to be significantly different. Old male and female mice lacked significant distinction, but old females differed from untreated controls. Because the retinal thickness of untreated females is statistically equivalent to that of untreated males, only the data from untreated males is presented.

Figure 4.. Retinal plastic sections of mice injected with 25 mg/kg NaIO₃.

(A–D) Representative images of plastic sections of (A) young male, (B) young female, (C) old male, and (D) old female mice injected with 25 mg/kg NaIO₃; mice were euthanized one week after injection. Scale bars represent 100 μm. (E) Quantification of the thickness of the ONL, measured by nuclei count. Nuclei were counted at 250 μm intervals, and the results were averaged. Each dot represents one mouse. Mean ± SEM is reported. (F) Quantification of the number of undulations present in each plastic section image. Each dot represents one image from different mice. (G) Undulations seen in IRAF cSLO image of an old female mouse injected with 25 mg/kg NaIO₃, imaged one week after injection. White box shows a magnified region with many curvilinear black lines representing undulations.

Figure 5.. qRT-PCR analysis of retinal tissue.

(A-B) Fold change analysis of mRNA levels in the neural retina of (A) rhodopsin (Rho) and (B) heme oxygenase 1 (Hmox1). The letters above each group signify significant differences. Groups sharing the same letter indicate statistical similarity. Mean ± SEM is reported and each dot represents one mouse. Only one eye from each mouse was studied by qPCR.