Assessment and Characterization of Hyaloid Vessels in Mice

Summary

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

Abstract

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

Introduction

The blood supply in the eye is essential to ensure the normal development of the retina and surrounding ocular tissues and to equip a proper visual function. There are three vascular beds in the eye: the retinal vasculature, the choroid, and a transient embryonic circulatory network of hyaloid vessels. The development of the ocular vasculature requires spatial and temporal coordination throughout embryogenesis and tissue maturation. Among the three vascular beds, the hyaloid vasculature is the first functional blood supply system to provide nutrition and oxygen to the newly formed embryonic lens and the developing retina. Hyaloid vessels regress at the same time that ....

Protocol

All animals were treated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research for animal experiments, following the Guidelines of the National Institutes of Health (NIH) regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at Boston Children’s Hospital. Lrp5^-/- mice (stock no. 005823; Jackson Laboratory) and its wild-type (WT) control C57BL/6J mice (stock no. 000664; Jackson Laboratory) were used for this study.

1. Part I....

Results

In vivo imaging of hyaloid vessels in live mice
Figure 3A reveals cross-sectional views of OCT images for the retina and hyaloid tissues in 3-month-old WT and Lrp5^-/- mice, an animal model with persistent hyaloid. The WT eye shows the absence of hyaloid tissue, whereas the Lrp5^-/- eye shows two persistent hyaloid vessels derived from the optic nerve head. Figure 3B dis.......

Discussion

Techniques to assess and characterize hyaloid vessels are intuitive and necessary procedures to observe the hyaloid vessel regression in animal models, to allow studies on the mechanisms underlying the vascular regression during development. While the in vivo retinal imaging allows the longitudinal observation of hyaloid regression in the same animal, access to a rodent fundus imaging system for OCT and FFA may be a limiting factor. In addition, in vivo imaging in live mice is not feasible before they open their eyes. Th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AK-Fluor (fluorescein injection, USP)	Akorn	17478-253-10
Anti-CD31 antibody	Abcam	ab28364
Antifade mounting medium	Thermo Fisher	S2828
Antifade Mounting Medium with DAPI	Vector Laboratories	H-1200
Artificial tear eyedrop	Systane	N/A
Bovine serum albumin (BSA)	Sigma-Aldrich	A2058
C57BL/6J mice	The Jackson Laboratory	Stock NO: 000664
Calcium chloride (CaCl2)	Sigma-Aldrich	C1016
Cryostat	Leica	CM3050S
Cryostat	Leica	CM3050 S
Cyclopentolate hydrochloride and phenylephrine hydrochloride eyedrop	Cyclomydril	N/A
Gelatin	Sigma-Aldrich	G9382
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	ThermoFisher Scientific	A-11008
Heating board	Lab-Line Instruments Inc.	N/A
Isolectin GS-IB4, 594 conjugate	ThermoFisher Scientific	I21413
Ketamine hydrochloride injection	KetaVed	NDC 50989-996-06
Lrp5-/- mice	The Jackson Laboratory	Stock NO. 005823	Developed by Deltagen Inc., San Mateo, CA
Micron IV and OCT	Phoenix Research Labs	N/A	Imaging software: InSight
Microscope	Zeiss	discovery v8
Microsurgery forceps	Scanlan International	4004-05
Microsurgery scissors	Scanlan International	6006-44
Optimal cutting temperature compound	Tissue-Tek	4583
Optimal cutting temperature compound	Agar Scientific	AGR1180
Paraformaldehyde (16%)	Electron Microscopy Sciences	15710
Peel-A-Way disposable embedding molds (tissue molds)	Fisher Scientific	12-20
Phosphate-buffered saline (PBS) buffer (10X)	Teknova	P0496
Slide cover glass	Premiere	94-2222-10
Superfrost microscope slides	Fisherbrand	12-550-15
Triton X-100	Sigma-Aldrich	X100
Xylazine sterile solution	Akorn: AnaSed	NDC: 59399-110-20