An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Summary

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Abstract

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

Introduction

Choroidal angiogenesis dysregulation is associated with neovascular age-related macular degeneration (AMD)¹. The choroid is a microvascular bed present underneath the retinal pigment epithelium (RPE). It has been shown that reduced blood flow in the choroid is associated with progression of AMD². The intricate relationship between vascular endothelium, RPE, macrophages, pericytes and other cells is responsible for the homeostasis of the tissue^3,4,5. Therefore, a reproducible assay modeling choroidal microenvironment is critical ....

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Protocol

All animal experiments described were approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital (ARCH protocol number 19-04-3913R).

1. Preparation

1. Add 5 mL of Penicillin/Streptomycin (10000 U/mL) and 5 mL and 10 mL of commercially available supplements to 500 mL of complete classic medium with serum. Aliquot 50 mL of the medium initially.
   NOTE: Do not return any medium back to the stock to avoid contamination.
2. Put an aliquot of complete classic medium on ice.
3. Use 70% ethanol to clean the dissecting microscope, forceps, and scissors.
4. Prepare two cell cultu....

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Results

Comparison of choroid sprouting growth per day

We dissected the choroid with sclera, embedded in BME and cultured them for 6 days (Figure 1). The choroid sprouting in C57BL/6J mice from day 3 to day 6 were examined with a microscope and quantified with SWIFT-Choroid a semi-automated quantification method in ImageJ. In a representative case, the choroidal sprouting area (the vessels extending from the explant, excluding the explant itself) was 0.38.......

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Discussion

The choroidal sprouting assay aids research in neovascular AMD^{9,10,18,19,20}. Choroid explants can be isolated from mice as well as rats and humans^17,21. The choroid explant includes ECs, macrophages, and pericytes¹⁷. In this assay the interaction between choroidal ECs and adjace.......

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Materials

Name	Company	Catalog Number	Comments
AnaSed (Xylazine)	AKORN	59339-110-20
Basal membrane extract (BME) Matrigel	BD Biosciences	354230
Cell culture dish	NEST	704001	10cm
Complete classic medium with serum and CultureBoost	Cell systems	4Z0-500
Ethyl alcohol 200 Proof	Pharmco	111000200	use for 70%
Kimwipes	Kimberly-Clark	06-666
Microscope	ZEISS	Axio Observer Z1
Penicillin/Streptomycin	GIBCO	15140	10000 U/mL
Tissue culture plate (24-well)	Olympus	25-107
VetaKet CIII (Ketamine)	AKORN	59399-114-10