Sensitivity of the Dorsal-Central Retinal Pigment Epithelium to Sodium Iodate-Induced Damage Is Associated With Overlying M-Cone Photoreceptors in Mice

Abstract

Purpose: Retinal pigment epithelium (RPE) degeneration is a leading cause of blindness in retinal degenerative diseases, but the mechanism of RPE regional degeneration remains largely unknown. This study aims to investigate the sensitivity of RPE to sodium iodate (SI) injury in the dorsal and ventral visual fields in mice and analyze whether overlaying cone photoreceptors regulate the sensitivity of RPE to SI-induced damage.

Methods: SI was used to induce RPE degeneration in mice. Hematoxylin-eosin staining, immunostaining, and TUNEL assay were used to evaluate retinal degeneration along the dorsal-ventral axis. Flat-mounted and sectional retinal immunostaining were used to analyze the distribution of cones along the dorsoventral axis in C57BL/6, albino, and 129 mice. Electroretinography was used to examine the retinal function.

Results: Dorsal-central RPE was more sensitive to SI-mediated injury along the dorsal-ventral axis in C57BL/6 mice. Compared with the ventral RPE, the dorsal-central RPE was dominantly covered by M cone photoreceptors in these mice. Interestingly, M cone photoreceptor degeneration was followed by dorsal RPE degeneration under a low dose of SI. Furthermore, the sensitivity of dorsal RPE to a low dose of SI was reduced in both albino and 129 mouse strains with dominant mixed cones instead of M cones in the dorsal visual field.

Conclusions: These findings suggest that dorsal-central RPE is more sensitive to SI injury and that SI-induced RPE degeneration could be controlled by modifying the dominant overlying cone population in the mouse dorsal retina, thereby highlighting a potential role of M cones in RPE regional degeneration.

Figure 1.

The dorsal retina exhibits severe damage in C57BL6/J mice treated with a low dose of SI. (A) Schematic representation of the experimental procedure of SI injury. A two-month-old C57BL/6 mouse was injected with a low dose of SI (15 mg/kg) via the tail vein, and retinal degeneration analysis was performed on day 7 after the single injection. (B) Histological structure of retinas from mice after seven days of low-dose SI or saline solution injection. The solid black arrow indicates RPE swelling and abnormal release of melanin granules apically into the photoreceptor outer segment. Scale bar, 200 µm (upper panel), 50 µm (middle panel), and 20 µm (lower panel). (C) Quantification of the thickness of the outer nuclear layer from the mice injected with a low dose of SI or saline solution. n = 6. (D) TUNEL assay of the retinas from the C57BL/6 mice in control and experimental groups. Scale bar, 200 µm (upper panel) and 50 µm (lower panel). (E) Quantification of the number of TUNEL-positive cells per field in the retina in the control and experimental group. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± standard error of the mean and were compared using a Student t-test or one-way ANOVA when appropriate. GCL, ganglion cell layer; INL, inner nuclear layer; IS, photoreceptor inner segment; NC, Normal control; ONH, optic nerve head; ERG, electroretinography.

Figure 2.

M cone degeneration was severe in C57BL6/J mice treated with a low dose of SI. (A) Immunostaining of anti-ZO1 (a), anti-Rhodopsin and Opsin (b) in the retinas treated with SI at day 0, day 4 or day 7. The solid white arrows indicate the incorrect localization of Rhodopsin or Opsin in the damaged dorsal retinas. ERG traces of the (B) M-cone and (C) S-cone responses from C57BL/6 mice injected with saline or low dose SI were elicited by green light with strength of 0.75 cd-s/m2 and UV light with strength of 3.00 mW-s/m2, respectively. The right bar graph shows the quantification of the amplitude of the b-wave from the (B) M-cone response and (C) S-cone response. n = 5.***P < 0.001. Data are presented as the mean ± standard error of the mean and were compared using a Student t-test. IS, photoreceptor inner segment; NC, Normal control.

Figure 3.

High dose of SI treatment induces severe retinal degeneration in the dorsal visual field of C57BL/6 mice. (A) Histological images of H&E staining from two-month-old C57BL/6 retinas treated with saline solution (left panels) or high-dose SI (right panels) (25 mg/kg) after three days. Scale bar: 200 µm (upper panel), 50 µm (lower panel). (B) Quantification of the thickness of the ONL from C57BL/6 retinas treated with saline solution or a high dose of SI. (C) Images of TUNEL assays from the C57BL/6 retinas three days after intraocular injection of saline or a high dose of SI and (D) quantitative analysis of the cell death rate of photoreceptor cells. (E) RPE degeneration analysis based on (a) ZO1 staining, (b) OTX2 staining, and (c) TUNEL detection of C57BL/6 mice one day after treatment with a high dose of SI. (F, G) Quantification of the number of (D) OTX2 D) and (E) TUNEL positive cells in the RPE. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± standard error of the mean and were compared using a one-way ANOVA. GCL, ganglion cell layer; INL, inner nuclear layer; IS, photoreceptor inner segment; NC, Normal control; ONH, optic nerve head.

Figure 4.

Different levels of pigmentation between the dorsal and ventral RPE. (A) Representative H&E staining image of sectional retina from two-month-old C57BL/6 mice kept under normal conditions (n = 8). Scale bars, 200 µm (upper panel) and 50 µm (lower panel). (B) Melanosomes in the dorsal and ventral RPE cells were revealed by TEM. The black arrows indicated the melanosomes. Scale bars: 2 µm. (C) Quantification of melanosomes in the RPE cells based on the TEM analysis (n = 6). ***P < 0.001 by Student's t test. (D, E) Images of the flat-mounted RPE of the C57BL/6 mice at postnatal day 2 (D) and the quantification of melanin content of the dorsal and ventral RPE (E) (n = 4). Scale bars: 500 µm (flat-mounted RPE) and 20 µm (right enlarged images). (F) Representative images of immunostaining of dorsal and ventral RPE from two-month-old C57BL/6 mice detected by anti-PEML17 and anti-MITF antibodies (n = 5). Data are presented as the mean ± standard error of the mean, and were compared using Student's t-test. *P < 0.05, ***P < 0.001. INL, inner nuclear layer; D, dorsal; V, ventral; N, nasal; T, temporal.

Figure 5.

Deficiency of melanin granules does not accelerate SI-induced RPE degeneration. (A) Histological images of H&E staining from two-month-old albino mice three days after a single injection of saline solution or a high dose of SI (25 mg/kg). Scale bar: 200 µm (upper panel) and 50 µm (lower panel). (B) Quantification of the nuclei in the RPE and (C) the thickness of ONL from the albino mice under the indicated conditions. (D) Retinal degeneration analysis of the albino mice injected with high-dose SI by double staining of anti-Rhodopsin and Opsin. (E) Images of TUNEL assays from the two-month-old albino retinas treated with high-dose SI (25 mg/kg) for one or three days. Scale bar: 200 µm (upper panel), 50 µm (lower pane). (F) Quantitative analysis of the cell death rate in RPE (left bars) one day after injection of high dose SI and in the ONL (right bars) three days after injection of high dose SI (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± standard error of the mean and were compared using a one-way ANOVA. NC, normal control; GCL, ganglion cell layer; INL, inner nuclear layer.

Figure 6.

RPE degeneration is prevented in the dorsal retina of albino mice under low dose of SI treatment. (A) Histological images of H&E staining from two-month-old C57BL/6 (left panels) and albino (right panels) mice seven days after a single injection of saline solution or low dose of SI (15 mg/kg) (n = 6). The black arrows point to the mislocalization of melanin granules in the OS of dorsal retinas from the C57BL/6 mice. Scale bar: 200 µm (upper panel) and 50 µm (lower panel). (B) Immunostaining images of M and S opsins across the dorsoventral retinas of the C57BL/6 (left panels) and albino (right panels) mice by retinal sections (n = 6). INL, inner nuclear layer; IS, photoreceptor inner segment. Scale bar: 200 µm (upper panel) and 50 µm (lower panel).

Figure 7.

The dorsal retina is rich in mixed cones instead of M cones in 129 mice. (A, B) Immunodetection of M and S wavelength-sensitive opsins in (A) dorsal and ventral retinas from two-month-old C57BL/6 (left panels) and 129 (right panels) mice by retinal sections and (B) flat-mounted retinas. (C) The white square in the retinal scheme shows the analyzed dorsal and ventral regions for detecting M and S opsins, and the pie graphs show the percentage of cones manually classified as M+S− (green), S+M− (red), and M+S+ (mixed, pink) based on the opsin expression in the dorsal and ventral retinas from these two strain mice (n = 4). D, dorsal visual field; V, ventral visual field. M, M-opsin; S, S-opsin.

Figure 8.

RPE degeneration in the dorsal region is prevented in 129 mice treated with a low dose of SI treatment. (A) Histological images of H&E staining from two-month-old C57BL/6 (left panels) and 129 (right panels) mice seven days after a single injection of saline solution or low dose of SI (15 mg/kg) (n = 6). The black arrows point to the mislocalization of melanin granules in the OS of dorsal retinas from the C57BL/6 and 129 mice. Scale bar: 200 µm (upper panel) and 50 µm (lower panel). (B) Immunodetection of OTX2 in the RPE of C57BL/6 (left panels) and 129 (right panels) mice under the indicated conditions (n = 6). The white arrows point to the abnormal accumulation of RPE cells in the dorsal subretinal cavity of C57BL/6 mice under low dose of SI treatment. Scale bar: 200 µm (upper panel) and 50 µm (lower panel). INL, inner nuclear layer; IS, photoreceptor inner segment.

Figure 9.

A potential mechanism of selective dorsal RPE degeneration in C57BL/6 mice under low dose SI treatment. The dorsal RPE is hypopigmented and is predominantly covered by M cones characterized by expression of M opsin, and the ventral RPE is hyperpigmented and predominantly covered by S cones characterized by expression of S opsin. M opsin is thought to be more unstable than S opsin under a cyclic light condition, thus M cones are much more sensitive to oxidative stress than S cones, such as that caused by SI injury. Under low dose SI treatment, M opsin was notably degraded in the dorsal M cones, and the damaged M cones likely induced the dorsal RPE degeneration.