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JR5558 mice are a reliable model to investigate subretinal fibrosis - PubMed

  • ️Mon Jan 01 2024

JR5558 mice are a reliable model to investigate subretinal fibrosis

Yashar Seyed-Razavi et al. Sci Rep. 2024.

Abstract

Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options.

Keywords: Extracellular matrix; Fibrosis; Gliosis; Lesions; Müller cells; Retina.

© 2024. Crown.

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Conflict of interest statement

The authors have stated explicitly that there are no conflicts of interest in connection with this article.

Figures

Figure 1
Figure 1

Subretinal lesions and vascular leak in the retina of JR5558 mice. Fundus photography, OCT imaging and fluorescein-angiography performed on JR5558 mice reveal subretinal yellow lesion-like areas contain hyperreflective subretinal material and correlate with vascular leak. Bright yellow lesion-like areas continued to grow between 4 and 12 weeks, and some remain static from 8 weeks onwards. 8–12 wk, 14–16 wk and 18–20 wk timeline images presented were taken from 3 different animals, and vascular leak was not noted in all lesions. Scale bar = 50 μm.

Figure 2
Figure 2

Altered morphology in the retina and subretinal space of JR5558 mice. Hematoxylin and Eosin (H&E) (A–B), Picro-Sirius Red (PSR) (C–D) and Immunostaining with antibodies against collagen-I (E–F) reveal structural changes in the inner and outer nuclear layers as well as collagen I expression in the outer retina of JR5558 mice, compared to naïve C57Bl/6J controls. Scale bars = 50 μm.

Figure 3
Figure 3

Extracellular matrix, ⍺SMA, GLAST and Müller glia marker expression in JR5558 mice. Immunofluorescence imaging of JR5558 retinas with antibodies against collagen-IV, ⍺-smooth muscle actin (⍺SMA), and glial fibrillary acidic protein (GFAP) reveal collagen IV (A–D, white arrowheads) and ⍺SMA expression (E–H, white arrowheads, magnified in insert) aligning with subretinal Müller cell gliosis (green arrowheads) in fibrovascular lesion areas. Immunofluorescence imaging JR5558 retinal sections with antibodies against connective tissue growth factor (CTGF) and glutamate-aspartate transporter (GLAST) reveal altered CTGF expression (red) and GLAST expression in the outer retina (I–K, red arrowhead: area lacking CTGF expression, white arrowhead: CTGF expression, green arrows: GLAST expression.), compared to compared to naïve C57Bl/6J controls (L). Panels A–C, E–G and I–K are images from JR5558 retinal sections, where panels C, G and K are composites. Panels D, H, and L are composites of retinal sections from age matched naïve C57Bl/6J mice. Inserts represent a magnification within the corresponding panels. Scale bars = 50 μm, inserts = 20 μm.

Figure 4
Figure 4

Fibronectin expression localise around neovessels in the subretinal space of JR5558 mice. Immunofluorescence imaging reveals fibronectin expression localised around CD31 stained neovessels, and increased over time, in the fibrovascular lesion areas of retinal sections from JR5558 mice compared to naïve C57Bl/6J control retinas. Panels A, D, G (composite) and J (magnification of panel G) are of 4-week-old JR5558 retinal sections, whilst panels B, E, H (composite) and K (magnification of panel G) are of 8-week-old retinal sections from JR5558 mice. Panels C, F, I (composite) and K (magnification of panel G) are stained retinal sections from age matched naïve C57Bl/6J mice. The outer retina and RPE area (dashed square G–I) are magnified in panels J-L. Arrowheads indicate areas of distinct fibronectin expression. Arrows indicate neovessels within the outer retina. Scale bars: A–I = 50 μm, J–L = 20 μm.

Figure 5
Figure 5

Penetrating neovessels in JR5558 retinas originate from both the retinal vascular plexus and choroid. Immunofluorescence imaging of outer retinal neovascularisation and gliosis in JR5558 mice reveal examples of neovessels originating from retinal vascular plexus are presented in (panels D–F represent magnification of panel A–C, arrowhead) as well as neovessels of choroidal origin that penetrate the RPE (panels J–L represent magnification of panel G–I, arrowhead). Subretinal vessels perpendicular to the section plane were noted in subretinal lesion areas of JR5558 sections (arrows). Panels H–I & K–L have bright-field image added to indicate pigmented cells. Scale bars: A–C & G–I = 50 μm; D–F & J–L 20 μm.

Figure 6
Figure 6

Altered RPE structure and cell morphology in JR5558 mice. Immunofluorescence imaging of JR5558 retinal sections with antibodies against GFAP (green) and RPE65 (red) revealed subretinal Müller cell gliosis and altered RPE structure around the lesion area (A), corresponding to the altered outer retina and RPE structure noted in OCT image (B). Arrow and arrowhead highlighting subretinal gliosis and altered RPE, respectively. Phalloidin staining of JR5558 retinal flatmounts revealed the normal morphology of RPE cells at 4 (C) and 8 weeks (D) is changed from 12 weeks onwards (E–F). Dashed box represents area imaged in panel A. Scale bars: 50 μm.

Figure 7
Figure 7

Increased microglia distribution in the outer retina of JR5558 mice. Immunofluorescence imaging of JR5558 retinal sections with antibodies against Iba-1 reveal increased ramified microglia in the inner and outer plexiform layers, as well as amoeboid microglia in the outer retain, compared to naïve C57Bl/6J controls (A–B). Flatmount staining of JR5558 eye cups confirmed microglia in the outer retina (C), located sub-ONL (D). Panel D is the orthagonal view of panel C, rendered for clarity. Scale bars: 50 μm.

Figure 8
Figure 8

JR5558 retinas have altered protein expression in extracellular matrix and fibrotic markers. Western blot analysis of whole posterior eye cups revealed significant or near significant increases in fibronectin (p = 0.080), GFAP (p = 0.078), and MMP9 (p = 0.012) in JR5558 mice compared to 9-week-old naïve C57Bl/6J controls (A). Western blot analysis of lysed neural retinas revealed increased fibronectin expression at 4- (p < 0.001), 8- (p < 0.001) and 12-week (p = 0.025) JR5558 mice (B), whereas α-SMA expression significantly increased later at 12- (p = 0.009) and 20-weeks (p < 0.001) (C). Insert: α-SMA expression of neural retinas from 4-week-old JR5558 mice compared to 4-week-old naïve C57Bl/6J retinas. MMP-2 expression increased in the neural retina to reach significance at 20 weeks (p = 0.001) (D), whereas MMP-9 expression was elevated at 4- (p = 0.011), 8- (p = 0.043), 12- (p = 0.082), and 20-week-old (p = 0.46) (E). GFAP expression elevated at 8- (p < 0.001), 12- (p < 0.001), and 20-week-old (p = 0.15) animals (Fig. 8F). Insert: GFAP expression at 4-weeks compared to 4-week-old naïve C57BL/6J retinas (p = 0.014). Panels A, C insert and F insert: t-test, *p < 0.05; Panels B–F: One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 9
Figure 9

Lesions in fundus and OCT align with gliosis in immunohistochemical stained sections and retinal flatmounts Brightly yellow subretinal lesions noted in fundus (A) and OCT images (B) aligned with subretinal areas of gliosis in fixed retinal sections of the same retina stained with antibodies against GFAP and α-SMA (also highlighting vasculature) (C). GFAP positive Müller cell gliosis (green arrowheads) was not present in the subretinal space of all subretinal lesions (Fig. 9A–C, middle red arrow highlights subretinal lesion lacking gliosis). Scale bars: A = 200 μm, B = 100 μm and C = 50 μm.

Figure 10
Figure 10

Subretinal gliosis in retinal flatmounts align with retinal OCT images. Immunofluorescence imaging of JR5558 retinal sections with antibodies against GFAP (green) and CD31 (red) revealed neovessels (arrowheads) but not gliosis (arrow) at 4 weeks (A), whereas both gliosis and neovessels are significant at 8 week (B) and older JR5558 retinas compared to 4wk JR5558 and naïve C57BL/6J controls (C). Gliosis in the photoreceptor layer (sub-ONL) of retinal flatmounts stained with antibodies against GFAP (green, D–G). Utilising CD31-positive retinal vasculature as a guide, areas of subretinal gliosis were aligned with fundus images taken from the same animal (G–I). Dashed boxes represent the same area across panels. Numbered areas 1–3 represent the same area in panels G–H, and coincide with panels D–F, respectively. Scale bars: A–F = 50 μm, G–H = 200 μm.

Figure 11
Figure 11

Vascular leak and lesion area quantification following intravitreal Aflibercept treatment. Fundus photography, OCT imaging and fluorescein-angiography on JR5558 mice before (A) and 2 weeks following intravitreal injection of Aflibercept (B) reveal a reduction in vascular leak and subretinal lesions, with no growth in lesion area observed compared to untreated controls (C) (n = 4 per group).

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