Visualization of Mouse Choroidal and Retinal Vasculature Using Fluorescent Tomato Lectin Perfusion

Abstract

Purpose: To develop a reliable and simplified method to assess choroid and retinal vasculature on whole mount and cross sections in mice using tomato lectin (TL; Lycopersicon esculentum).

Methods: Albino mice (n = 27) received 1 mg/mL of TL (conjugated to Dylight-594) intravascularly through the tail vein, jugular vein, or cardiac left ventricle. Whole mounts of the retina and choroid were evaluated using fluorescence microscopy. Perfusion with GSL-IB4 conjugated to Dylight-594 and fluorescein isothiocyanate was performed to compare against labeling with TL. Co-labeling of choroidal endothelial cells with perfused TL on cross-sections with antibodies directed against the choriocapillaris-restricted endothelial cell marker CA4 was performed. The percentage of perfused choroidal and retinal vessels was assessed semiquantitatively. One mouse was subjected to thermal laser damage before perfusion to cause retinal and choroidal vasculature ablation.

Results: Intravascular injection of TL led to consistent, robust labeling of retinal and choroidal vascular walls. On cross-sections, choriocapillaris was co-labeled with CA4 and TL. On flat mount, TL perfusion resulted in better labeling of choroidal vessels using tail/jugular vein injection compared with cardiac perfusion (P < .01). More consistent labeling of the choroidal and retinal vascular trees was observed with TL than with GSL-IB4. Vascular damage caused by laser ablation was detected readily using this method.

Conclusions: TL injection intravascularly can reliably label normal and ablated choroid and retinal vasculature in mouse in a quick, simple manner.

Translational relevance: These data will help to facilitate modeling in rodents for diseases such as age-related macular degeneration, diabetes, and other ischemic/angiogenic processes that can also be used for treatment evaluation.

Keywords: choriocapillaris; lectins; retinal vasculature.

Figure 1.

Choroid flat mounts of mice (B6[CG]-TyrC-2J/J) perfused with Dy594-conjugated TL (in red). (A) Low magnification image (collected with a 2× objective lens) of normal albino mouse with optic nerve in the center. (B) Higher magnification image collected with a 10× objective lens showing detailed view of normal choroid and choriocapillaris. (C) High magnification microscopic image showing well-demarcated area of focal, central ablation of choroid and choriocapillaris corresponding to the lasered area surrounded by relatively preserved vessels.

Figure 2.

Choroid and retina flat mounts in mouse (BALB/CJ) perfused simultaneously with 100 µL of TL and 100 µL of GSL-IB4 conjugated to different fluorescent antibodies. (A–D) Choroid flat mount images of (A) TL conjugated to Dy594 showing clear delineation of choroidal and choriocapillaris vessels, (B) GSL-IB4 conjugated to FITC with choriocapillaris details obscured, (C) TL conjugated to FITC showing clear delineation of vessels, and (D) GSL-IB4 conjugated to Dy594 demonstrating relatively nonuniform staining of vessels. (E–H) Retinal flat mount images of (E) TL conjugated to Dy594, (F) GSL-IB4 conjugated to FITC, (G) TL conjugated to FITC, and (H) GSL-IB4 conjugated to Dy594. Note the increased signal intensity of GSL-IB4 at vascular branching points (arrows) in B, D, F, and H with relative absence of staining in the smaller vessels compared with more uniform staining of all vessels using TL perfusion in A, C, E, and G.

Figure 3.

Immunofluorescent-labeled cross-sections with the choriocapillaris enriched CA4 (in green) of mouse (B6[CG]-TyrC-2J/J) choroid perfused with Dy594-conjugated TL (in red). (A) Labeling of choriocapillaris by CA4 immunofluorescent marker. (B) Labeling of choriocapillaris and larger choroid vessels by TL perfusion. (C) Merged image of perfused TL showing co-labeling with CA4 in the choriocapillaris. (D) Merged image with no primary antibody control demonstrating no positive staining of the choriocapillaris with secondary antibody (Cy2 anti-goat) alone. CA4, carbonic anhydrase IV; DAPI, 4′,6-diamidino-2-phenylindole; CH, choroid

Figure 4.

Retinal flat mounts of mouse (B6[CG]-TyrC-2J/J) perfused with Dy594-conjugated TL (in red). (A) Low magnification fluorescence microscopic image of normal albino mouse with optic nerve in the center. (B) High magnification fluorescence microscopic image showing detailed view of normal retinal vessels, some not in focus owing to limitations of the microscope. (C) High magnification confocal projection yielding better visualization of all retinal vessels. (D) High magnification microscopic image showing well-demarcated area of focal, central ablation of the retina corresponding with lasered area surrounded by relatively preserved vessels.