An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 for the study of neovascular AMD.

Ex vivo angiogenesis assays and in vitro endothelial cell cultures can complement studies of microvascular behavior in vivo, for testing new drugs and for studies of pathogenesis. Endothelial cells such as human retinal microvascular endothelial cells (HRMECs), Human Umbilical Vein Endothelial Cells (HUVEC), isolated primary animal brain or retinal ECs are often used in in vitro studies for ocular angiogenesis research^6,7,8. HRMECs in particular have been widely used as a model of in vitro choroidal neovascularization (CNV)⁹ by assessing endothelial proliferation, migration, tubular formation, and vascular leakage to evaluate interventions^6,10. However, ECs in culture are limited as a model of CNV because of the lack of interactions with other cell types found in the choroid and because most EC used in these assays do not originate from choroid. Mouse choroidal ECs are difficult to isolate and maintain in culture.

The aortic ring assay is widely used as a model of macro vascular proliferation. Vascular sprouts from aortic explants include ECs, pericytes and macrophages¹¹. The aortic ring assay models large vessel angiogenesis well^12,13,14. However, it has limitations as a model of choroidal neovascularization as aortic rings are a macrovascular tissue lacking the characteristic choroidal microvascular environment, and sprouts from large vessels may differ from sprouts from capillary networks involved in microvascular pathology. Recently a group published an ex-vivo retinal assay^15,16. Although, it is suitable for retinal neovascular disease, it is not as appropriate for choroidal neovascularization as seen in AMD.

The choroidal sprouting assay using mouse RPE, choroid, and scleral explanted tissue was developed to better model CNV. The tissue can easily be isolated from mouse (or other species) eyes¹⁷. This assay allows reproducible evaluation of pro- and anti-angiogenic potential of pharmacologic compounds and evaluation of the role of specific pathways in choroidal neovascularization using tissue from genetically modified mice and controls¹⁸. This choroidal sprouting assay has been referenced in many subsequent publications^{9,10,18,19,20}. Here, the method involved in the use of this assay are demonstrated.

Protokół

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 culture dishes (10 cm), one on the dissection microscope, one on ice; put 10 mL of complete classic medium in each dish.

2. Experimental steps (Figure 1)

1. Sacrifice C57BL/6J mice around postnatal (P) 20 using 75-100 mg/kg ketamine and 7.5 -10 mg/kg xylazine injected intraperitoneally. Keep the eyes in complete classic medium on ice before dissection.
2. Remove the connective tissue (muscle and fatty tissue) and optic nerve on the eye.
3. Use a micro-scissor to circumferentially cut 0.5 mm posterior to the corneal limbus. Remove the cornea/iris complex, vitreous and the lens.
4. Make a 1 mm incision perpendicular to the cut edge towards the optic nerve and cut a circumferential band of 1 mm width. Separate the central and peripheral regions of the complex. Use forceps to peel off the retina from RPE/choroid/sclera complex. 
5. Keep the peripheral choroid band in complete classic medium on ice; isolate the other eye and repeat the process to cut a second band.
6. Cut the circular band into 6 ~equal square pieces (~1 mm x 1 mm).
   NOTE: Never touch any edge.
7. Thaw the basal membrane extract (BME) per manufacture’s instruction. Add 30 µL/well of BME into the center of each well of a 24 well tissue culture plate. Make sure the droplet of BME forms a convex dome at the bottom of the plate without touching the edges.
   NOTE: Thaw the BME overnight in a refrigerator. BME should be on the ice any time after thawing.
8. Place the tissue in the middle of the BME.
   NOTE: Do not flatten the choroid explant; generally, let the tissue expand within the BME. The orientation of the tissue (scleral side up or down) does not impact the experimental outcome.
9. Incubate the plate at 37 °C for 10 min to let the gel solidify.
10. Add 500 µL of the complete classic medium/well.
11. Change the classic medium every other day (500 µL). Choroid sprouting can be observed after 3 days with a microscope.
    NOTE: For growth factor treatment, starve the tissue for 4 h. Dilute a trial compound in growth factor-reduced medium (1:200 boost instead of 1:50).

3. SWIFT-Choroid computerized quantification method¹⁷ (Figure 2)

NOTE: A computerized method to measure the area covered by growing vessels was used. A macro plugin to ImageJ software is needed prior to quantification (see Supplemental Information for further detail).

1. Open the choroid sprouting image with ImageJ and check “Image | Type| 8-bit” with gray scale.
2. Go to “Image | Adjust | Brightness/Contrast (Ctrl/shift/C)” and optimize the contrast.
3. Use the magic wand function to outline and remove from the image the choroid tissue which are present in the center of the sprouts (using shortcut key “F1”) (Figure 2A,B).
   NOTE: Set the tolerance rate of the magic wand to 20-30%.
4. Remove the background of the image with the free selection tools (Figure 2C). Go to “Image | Adjust | Threshold (Ctrl/shift/T)”. Use the threshold function to define the microvascular sprouts against the background and periphery (Figure 2D).
5. Click “F2” and a summary will appear. Save an image of the selected area by clicking “Save”. Save in the same folder as the original image for future reference.
6. After a group of samples is measured, copy the recorded for data analysis.
   NOTE: It is also possible to measure the area (µm²) by “Analyze | Set Scale” using images with scale bars.

Wyniki

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...

Dyskusje

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...

Materiały

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