Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: Immune cells are recruited to the surface of the lens in response to active EAU and continue to associate with the lens matrix capsule at late stages of disease resolution. Whole eye cryosections of normal mice and mice with EAU at D18 and D35 post disease induction were co-immunolabeled for CD45 (green), lens capsule protein perlecan (red), and nuclei (DAPI, blue), and imaged by confocal microscopy. (A) Model of the regions of the lens capsule (red line) in which images in (B) were acquired. (B) Representative images of CD45-labeled immune cells along the anterior (b,c) and posterior (e,f) lens capsule in response to EAU. Immune cells are absent from the anterior (a) and posterior (d) lens capsule of normal mice. All images are 2.5 μm projections. Images shown are representative of at least 3 independent studies. Mag bar: 20 μm. AqH - aqueous humor, CB – ciliary body, CZ – ciliary zonules, VH - vitreous humor. Model created with BioRender ( https://www.biorender.com/ ).

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Immunolabeling, Confocal Microscopy, Labeling

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: Fibrinogen increases its association with the lens capsule surface in response to EAU. Whole eye cryosections from mice with EAU at D18 and D35 post disease induction and from normal mice were co-immunolabeled for fibrinogen (FNG, white), CD45 (green), perlecan (red), and nuclei (DAPI, blue). Images of the entire lens (A) or the anterior (B) and posterior (C) regions of the lens capsule were acquired by confocal microscopy. Overview images of the entire lens and higher magnification at D18 (Ab,Ca,Da) and D35 (Ac,Cc,Dc) post EAU induction were compared to control lens (Aa,Ba,Bb) using the same settings to reveal the greater association of fibrinogen with the lens capsule in response to EAU over time. 3D surface rendering of confocal Z-stacks captured in the anterior (Cb,d) and posterior (Db,d) regions of the lens capsule were created to examine the relationship between LCA-ICs and fibrinogen deposited in each region at different stages of EAU. Single asterisk denotes an immune cell at D18 (Cb,bi) that had started to remove fibrinogen underneath it as indicated by the absence of a small amount of fibrinogen where the cell is present, shown in the cell-free 3D construct (Cbii). During the late resolution phase, LCA-ICs on the anterior lens capsule that had become partially covered by fibrinogen (Cd) are revealed by rendering fibrinogen transparent in a 3D construct at D35 (Cdi, double asterisk and arrowhead that also are noted in Cd). This view is also shown with only fibrinogen and perlecan to demonstrate an example of immune cells having gone through the fibrinogen matrix and degraded the basement membrane perlecan as they invade the lens capsule over time (Cdii, double asterisk). The 3D surface renderings of the posterior lens capsule revealed LCA-ICs at D18 traveled across the fibrinogen matrix with (Db, orange arrow) or without (Db,bi, white arrow) fibrinogen deposited over the top of the cells, whereas at D35 many LCA-ICs in this region were encapsulated by fibrinogen (Dd, arrowhead). The immune cell marked by a white arrow in Db (D18) is also shown with nucleus removed and CD45 rendered transparent to reveal the largely intact fibrinogen underneath the cell, with a small amount already removed (Dbii). A zoomed-in view of the area marked by the arrowhead at D35 is also created with transparent fibrinogen to fully reveal the morphology of immune cells within the fibrinogen matrix (Ddi). (E) Quantification of the immunolabeling studies of fibrinogen association with the anterior and posterior lens capsule surfaces at D18 and D35 post induction of EAU. Images shown are representative of at least 3 independent studies. (Aa-c) are tiles; (Ba,b; Ca,c; Da,c) are single optical planes; (Cb,bi,bii,d,di,dii; Db,bi,bii,d,di) are 3D surface renderings. Mag bar: (Aa-c) = 200 μm; (Ba,b; Ca,c; Da,c) = 20 μm; (Cc,e) = 5 μm; (Dc,e) = 8 μm; (Cbi,bii,di,dii) = 3 μm; (Dbi,bii,di) = 2 μm. AqH - aqueous humor, FNG – fibrinogen, VH -vitreous humor. *, p < 0.05; ****, p < 0.0001.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Immunolabeling, Confocal Microscopy, Control, Construct, Membrane

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: Immune cells associated with the lens capsule in EAU exhibit region-specific interactions with fibronectin. Whole eye cryosections of normal mice and mice with EAU at D18 and D35 post disease induction were co-immunolabeled for fibronectin (white), perlecan (red), CD45 (green) or F-actin (yellow) and nuclei (DAPI, blue), and imaged by confocal microscopy. Overview images of the entire lens were acquired in control mice (Aa) and mice with uveitis during active inflammation (Ab) and resolution (Ac) to demonstrate the changes in fibronectin expression over the course of disease. Fibronectin is associated with the surface of the normal lens as part of its extracapsular matrix compartment (Aa,Ba,Ca). A greater association of fibronectin was observed by D18 post EAU induction (Ab,Bb,Cb), which continued to increase by D35 (Ac,Bd,Cd). Differential interactions between fibronectin and immune cells associated with the anterior (Bc,e) versus the posterior (Cc, e) lens capsule were examined either by removing immune cells (Bci,ei) or rendering the fibronectin transparent (Cci,ei) in 3D constructs of confocal Z-stacks created by Imaris software. Single and double asterisks denote immune cells that had begun to integrate with the fibronectin matrix by digesting some of it directly underneath them. Arrow and arrowhead denote immune cells within the fibrinogen matrix. (D) Quantification of the immunolabeling studies of fibronectin association with the anterior and posterior lens capsule surfaces at D18 and D35 post induction of EAU compared to normal mouse lenses. (Aa-c) are tiles, (Ba,b,d; Ca,b,d) are single optical planes, (Bc,ci,e,ei; Cc,ci,e,ei) are 3D surface renderings. Images shown are representative of at least 3 independent studies. Mag bars: (Aa-c) = 200 μm; (Ba,b,d; Ca,b,d) = 20 μm; (Cc,e) = 10 μm; (Bc,ci,e,ei) = 5 μm. AqH - aqueous humor, FN – fibronectin, VH - vitreous humor. *, p < 0.05; ****, p < 0.0001.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Immunolabeling, Confocal Microscopy, Control, Expressing, Construct, Software

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: Fibronectin and fibrinogen that become associated with the ciliary zonules in response to EAU provide pathways for immune cell migration. (A) Co-immunolabeling of whole eye cryosections from normal mice (a,d) and EAU mice at D18 (b,e) and D35 (c,f) post disease induction with ciliary zonule component MAPG1 (purple) and fibronectin (a-c, white) or fibrinogen (d-f, white) revealed association of fibronectin and fibrinogen with ciliary zonule fibers in uveitic eyes, but not non-uveitic ones. DAPI (blue) marks cell nuclei. (B–C) Confocal images of whole eye cryosections from uveitic eyes at D18 (Ba,Ca) and D35 (Bb,Cb) post EAU induction co-immunolabeled for CD45 (green), perlecan (red), fibronectin (Ba,b, white) or fibrinogen (Ca,b, white) in addition to nuclear staining (DAPI, blue) showed localization of immune cells along the fibronectin/fibrinogen-rich fibrils leading to the lens equator. Zoomed-in views of the 3D surface structures rendered from these confocal Z-stacks (Bai,bi; Cai,bi) are also created to highlight the interactions between immune cells and these fibronectin/fibrinogen-rich fibrils. Arrowhead, arrow, asterisk, and double asterisk denote the same cell in (Ba,ai), (Bb,bi), (Ca,ai), (Cb,bi), respectively. Images shown are representative of at least 3 independent studies. (Aa-c) are 1 μm projections; (Ad-f; Ca) are 2.5 μm projections; (Ba,b; Cb) are single optical planes; (Bai,bi; Cai,bi) are 3D surface structures. Mag bar: (Aa-f; Ba,b; Ca,b) = 20 μm; (Bai,bi; Cai,bi) = 5 μm. AqH - aqueous humor, CB – ciliary body, CZ – ciliary zonules, FNG – fibrinogen, FN – fibronectin, VH - vitreous humor.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Migration, Immunolabeling, Staining

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: MAGP1-rich fibrils extend across the anterior and posterior lens surface in response to EAU. Whole eye cryosections from mice without EAU and with EAU at D18 and D35 post disease induction were co-immunolabeled for MAGP1 (white), perlecan (red), and co-labeled for F-actin (yellow) and nuclei (DAPI, blue). Confocal images were acquired in the anterior (A) and posterior (B) regions of the lens capsule and shown as single optical planes (Aa,b,d; Ba,b,d) or 3D surface renderings after reconstruction with Imaris software (Ac,ci,e,ei; Bc,e). While MAGP1 is not found associated with the anterior or posterior surfaces of the lens in normal eyes (Aa, Ba), its expression is induced in these regions in response to EAU (Ab-e; Bb-e). The 3D surface renderings of confocal Z-stacks captured in the anterior lens capsule are also created without cells to reveal the presence (Aci, double asterisk) and absence (Aei, arrowhead) of MAGP1 fibrils directly underneath immune cells recruited at D18 and D35, respectively. Single asterisk, arrowhead, white arrow, and yellow arrow denote the same cell in (Ab,c), (Ad,e), (Bb,c), (Bd,e), respectively. (C) Quantification of immunolabeling studies of the de novo extension of MAGP1 across the anterior and posterior lens capsule surfaces at D18 and D35 post induction of EAU. Images shown are representative of at least 3 independent studies. Mag bar: (Aa,b,d; Ba,b,d) = 20 μm; (Ac,e; Bc,e) = 10 μm, (Aci,ei) = 3 μm. AqH - aqueous humor, MAGP1 - Microfibril-Associated Glycoprotein-1, VH - vitreous humor. ns, non-significant; **, p < 0.01; ****, p < 0.0001.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Immunolabeling, Labeling, Software, Expressing

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: MAGP1 fibrils extending across the anterior and posterior lens capsule surfaces in response to EAU occasionally co-localize with accumulating fibrinogen. Whole eye cryosections from mice at D35 post induction of EAU were co-immunolabeled for MAGP1 (green), fibrinogen (red), and perlecan (white), with nuclei detected by DAPI (blue). Confocal Z-stack images were acquired along the anterior (A) and posterior (B) lens capsule and visualized in 3D view as overlay (Aa,d; Ba, d) or single channel (Ab,c; Bb,c). Arrows denote colocalization of MAGP1 fibrils and fibrinogen. Images shown are representative of at least 3 independent studies. Mag bar = 10 μm. AqH - aqueous humor, FNG – fibrinogen, LC – lens capsule, MAGP1 - Microfibril-Associated Glycoprotein-1, VH - vitreous humor.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Immunolabeling

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: Versican and vitronectin, known PEX materials, become associated specifically with the anterior surface of the lens capsule in EAU. Whole eye cryosections from mice without EAU and with EAU at D18 and D35 post disease induction were co-immunolabeled for versican (A) or vitronectin (B) (white), basement membrane component perlecan (A) or laminin (B) (red), and co-labeled for F-actin (yellow) and nuclei (DAPI, blue). Confocal images were acquired in the anterior region of the lens capsule and shown as single optical planes (Aa,b,d; Ba,b,d,f) or 3D surface renderings created with Imaris software (Ac,e; Bc,e,g). While versican and vitronectin were normally undetected on the anterior surface of the lens (Aa,Ba), patches of these matrix proteins were occasionally found accumulating in this region of the lens capsule during both active inflammation and resolution of EAU (Ab-e; Bb,c,f,g). Single asterisk and double asterisk denote the same cell in (Bd,e) and (Bf,g), respectively. (C) Quantification of the immunolabeling studies of versican and vitronectin association with the anterior and posterior lens capsule surfaces at D18 and D35 post induction of EAU. Images shown are representative of at least 3 independent studies. Mag bar: (Aa,b,d; Ba,b,d,f) = 20 μm; (Ac,e; Bc,e,g) = 10 μm. AqH - aqueous humor, LMN - laminin, VCAN - versican, VH - vitreous humor, VTN - vitronectin. ns, non-significant; *, p < 0.05.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Immunolabeling, Membrane, Labeling, Software

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: The iris adheres to the anterior surface of the lens in EAU, mimicking a phenotype of uveitic glaucoma. (A) Whole eye cryosections from mice with EAU at D35 post disease induction were co-labeled for fibrinogen (red), F-actin (white) and nuclei (DAPI, blue). Confocal fluorescence images were acquired in combination with differential interference contrast (DIC) in region of the anterior lens capsule. Panels ai and bi show a higher magnification of the boxed regions in a and b, respectively. (B) Whole mouse eye cryosections of normal mice (a) and mice with EAU at D18 (b) and D35 (c) after induction of uveitis were stained for hematoxylin and eosin and imaged using a Nikon SMZ800 microscope (Melville, NY) equipped with a Nikon DS-Fi1 camera. Images are representative of at least three independent studies. Mag bar: (Aa,b) = 100 μm; (Aai,bi) = 10 μm; (Ba-c) = 500 μm. AqH - aqueous humor, FN – fibrinogen, LC – lens capsule.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Labeling, Fluorescence, Staining, Microscopy

Journal: Experimental eye research

Article Title: Matrix molecules associated with both regulation of inflammation and pathogenic outcomes associated with the surface of the lens in the eyes of EAU mice

doi: 10.1016/j.exer.2025.110529

Figure Lengend Snippet: Accumulation of matrix proteins on the lens capsule during active EAU correlates with increased lens capsule rigidity. Whole mouse eye cryosections of normal mice and mice with EAU at D18 and D35 after induction of uveitis were examined by atomic force microscopy. The Young’s modulus of the anterior (A) and posterior (B) lens capsule was measured for control eyes, uveitic eyes at both D18 and D35. Each data point represents the mean value of Young’s moduli recorded across each field of view (FOV) per lens capsule region (n = 5–6 FOV per group). ns, non-significant; *, p < 0.05; **, p < 0.01.

Article Snippet: For histological studies, whole eye cryosections were stained with hematoxylin and eosin (Vector Laboratories, Newark, CA, USA).

Techniques: Microscopy, Control