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Corneal Assay 131
131
From:
Methods in Molecular Medicine, Vol. 46: Angiogenesis Protocols
Edited by: J. C. Murray © Humana Press Inc., Totowa, NJ
10
Corneal Assay for Angiogenesis
Marina Ziche
1. Introduction
In order to develop angiogenic and antiangiogenic strategies, concerted
efforts have been made to provide animal models for more quantitative analy-
sis of in vivo angiogenesis. In vivo techniques consist of the cornea pocket and
iris implant in the eye, the rabbit ear chamber, the dorsal skinfold chamber, the
cranial window, the hamster cheek pouch window, the sponge implant assay,
the fibrin clots, the sodium alginate beads and the Matrigel plugs, the rat
mesenteric window, the chick embryo chorioallantoic membrane and the air
sac in mice and rats (1). In this chapter we will discuss the avascular cornea
assay, and the advantages and disadvantages of using this assay in different
species. The cornea assay is based on the placement of an angiogenic inducer
(tumor tissue, cell suspension, growth factor) into a corneal pocket in order to
evoke vascular outgrowth from the peripherally located limbal vasculature. In
comparison to other in vivo assays, this assay has the advantage of measuring
only new blood vessels, because the cornea is initially avascular.
2. Materials
1. Sodium pentothal or sodium pentobarbital.
2. Pliable iris spatula (1.5 mm wide).
3. Ethylene-vinyl-acetate copolymer (Elvax-40, DuPont de Nemours, Wilmington, DE).
4. Absolute alcohol.
5. Methylene chloride.
6. Hydroxyethyl-methacrylate polymer (Hydron, Interferon Science, New
Brunswick, NJ).
7. Sucrose aluminum sulfate (Sucralfate, BukhMeditec, Copenhagen, Denmark).
8. Slit-lamp stereo-microscope with ocular grid
132 Ziche
3. Methods
3.1. Rabbit Cornea Assay
This assay was first described by Gimbrone et al. (2) for use in New Zealand
white rabbits (see Note 1), owing partly to the absence of a vascular pattern,
and for the ease of manipulation and monitoring of neovascular growth. We
have used this technique extensively for several years and have substantially
modified it to fulfil different experimental requirements.
3.1.1. Sample Preparation
The material to be tested can be in the form of slow-release pellets incorpo-
rating recombinant growth factors, cell suspensions, or tissue samples (3,4).
Recombinant growth factors are prepared as slow-release pellets by incorpo-
rating the protein under test into an ethylene-vinyl-acetate copolymer (Elvax-
40; see Note 2). In order to avoid nonspecific reactions, Elvax-40 has to be
carefully prepared as follows:
1. Elvax-40 beads are extensively washed in absolute alcohol at 37°C (see Note 3).
2. Prepare a 10% casting stock solution in methylene chloride and test for
biocompatibility (5). (The casting solution can be used if none of the implants
performed with this preparation induces a histological reaction in the rabbit cornea.)
3. A predetermined volume of Elvax-40 casting solution is mixed with a given
amount of the compound to be tested on a flat surface and the polymer is allowed
to dry under a laminar flow hood.
4. After drying, cut the film sequestering the test compound into 1 mm × 1 mm ×
0.5 mm pieces.
3.1.2. Cell and Tissue Implants
Cell suspensions are obtained by trypsinization of confluent cell monolay-
ers. Five microliters containing ~2 × 10
5
cells in medium supplemented with
10% serum are introduced into the corneal micropocket. If the overexpression
of growth factors by stable transfection of specific cDNA is being studied, one
eye is implanted with transfected cells and the other with the wild-type cell
line. When tissue samples are tested, samples of 2–3 mg are obtained by cut-
ting the original fragments under sterile conditions. The angiogenic activity of
tumor samples should be compared with macroscopically normal tissue.
3.1.3. Surgical Technique
1. Anesthetize rabbit with sodium pentothal (30 mg/kg intravenously) (see Note 4).
2. Under aseptic conditions, surgically produce a micropocket (1.5 mm × 3 mm) in
the lower half of the cornea using a pliable iris spatula (see Notes 5, 6, 7).
3. Drain a small amount of the aqueous humor from the anterior chamber to reduce
corneal tension (see Note 8).
Corneal Assay 133
4. Position the implant 2.5–3 mm from the limbus to avoid false positives owing to
mechanical procedure. This also allows diffusion of test substances and the sub-
sequent formation of a gradient for the endothelial cells of the limbal vessels
(Fig. 1 A, B).
5. Implants sequestering test materials, and appropriate controls (empty pellets of
Elvax-40 can be used as controls), should be coded and implanted in a double-
blinded manner.
3.1.4. Quantification
Subsequent daily observation of the implants is made with a slit lamp stereo-
microscope without anesthesia. Angiogenesis, edema, and cellular infiltrate
are recorded daily with the aid of an ocular grid by an independent operator
who does not perform the surgery. An angiogenic response is scored positive
when budding of vessels from the limbal plexus occurs after 3–4 d and capil-
Fig. 1. Schematic representation of the corneal micropocket assay. A micropocket
is surgically produced in the corneal stroma with a pliable spatula (A) and the test
substance is inserted into the micropocket (B). The newly formed vessels start from
the limbal vasculature (C, t = 0) and progress toward the implanted stimulus (D, t = 7 d).
134 Ziche
laries reach the implanted pellet in 7–10 d (Fig. 1 C,D). Implants that fail to
produce neovascular growth within 10 d are considered negative, while
implants showing an inflammatory reaction are discarded (6). The ratio of posi-
tive implants to total implants performed is determined during each observa-
tion. The potency of angiogenic activity is evaluated on the basis of the number
and growth rate of newly formed capillaries, and an angiogenic score is calcu-
lated by the formula (vessel density) × (distance from limbus) (7–9).
A density value of 1 corresponds to 0–25 vessels per cornea, 2 from 25–50,
3 from 50–75, 4 from 75–100, and 5 for more than 100 vessels. The distance
from the limbus (in mm) is graded with the aid of an ocular grid.
3.1.5. Histological Examination
Corneas are removed at the end of the experiment as well as at defined inter-
vals after surgery and/or treatment and fixed in formalin for histological
examination. Newly formed vessels and the presence of inflammatory cells are
detected by hematoxylin & eosin staining or specific immunohistochemical
procedures (e.g., antirabbit macrophage [RAM11], antiCD-31 for endothelium)
(8). Double staining (i.e., antiCD-31 for vascular endothelium and specific
markers for tumor cells) could be useful to label newly formed vessels of the
host and proliferating tumor cells implanted in the cornea.
3.2. Mouse Cornea Micropocket Assay
The mouse cornea micropocket assay was first described by Muthuk-
karuppan and Auerbach (10). After the animals are anesthetized with meth-
oxyflurane, corneal micropockets are made in both eyes, reaching within 1 mm
of the limbus, and pellets containing substances to be tested coated with Hydron
are implanted. Hydron is used as a casting solution of 12% (w/v) prepared by
dissolving the polymer in absolute alcohol at 37°C (5). When peptides are
tested, sucralfate is added to stabilize the molecule and to slow its release from
Hydron (11,12). The vascular response, measured as the maximal vessel length
and number of clock hours of neovascularization, is scored at fixed time (usu-
ally at d 5 and 7 postoperation). For this purpose, the eyes are examined by slit-
lamp biomicroscopy and photographed. To quantify the section of the cornea
in which new vessels are sprouting from the preexisting limbal vessels, the
circumference of the cornea is divided into the equivalent of 12 clock hours.
The measure of the number of clock hours of neovascularization for each eye
is performed during each observation.
3.3. Rat Corneal Assay
Purified growth factors are combined 1/1 with Hydron, as described by
Polverini and Leibovich (13). Pellets are implanted 1–1.5 mm from the limbus
Corneal Assay 135
of the cornea of anesthetized rats (sodium pentobarbital, 30 mg/kg, ip).
Neovascularization is assessed at fixed days (usually d 3, 5, and 7). To photo-
graph responses, animals are perfused with colloidal carbon solution to label
vessels, and eyes are enucleated and fixed in 10% neutral-buffered formalin
overnight. The following day, corneas are excised, flattened, and photographed.
A positive neovascularization response is recorded only if sustained directional
ingrowth of capillary sprouts and hairpin loops toward the implant are
observed. A negative response is recorded when either no growth is observed
or when only an occasional sprout or hairpin loop showing no evidence of
sustained growth is detected.
3.4. Features of the Corneal Assay for Angiogenesis
Species: Rabbit cornea has been found to be avascular in all strains exam-
ined so far. In some strains of rat the presence of preexisting vessels within the
cornea and the development of keratitis are serious disadvantages.
Animal size: Rabbits are more docile and amenable to handling and experi-
mentation than mice or rats. The size of a rabbit (2–3 kg) allows easy manipu-
lation of the whole animal, and the eye to be easily extruded from its location
and surgically manipulated. General anesthesia is required only for surgery, while
daily examinations occasionally need local anesthesia. Conversely, the small
size of rats and mice allows the use of small amounts of drugs given systemically.
Measurements: In mice and rats it is possible to obtain time-point results.
Following the evolution of the angiogenic response in only one animal is not
recommended because each time the cornea is observed the animal has to be
anesthetized. Experiments are made with a large number of animals, and ves-
sel growth over time can be visualized by perfusion of individual animals with
colloidal carbon. Multiple observations are however possible with rabbits. The
use of a slit lamp stereomicroscope to observe unanesthetized animals allows
the observation of newly formed vessels for periods up to 1–2 mo.
Monitoring of inflammation: Inflammatory reactions are easily detectable
by stereomicroscopic examination of rabbits as corneal opacity.
Different experimental procedures: In the wide area of rabbit eye, stimuli
can be placed in different forms. In particular, the activity of specific growth
factors can be studied in the form of slow-release pellets (3,14–17) (Fig. 2),
and of tumor or nontumor cell lines stably transfected for the overexpression
of angiogenic factors (8,18–22) (Fig. 3). The modulation of the angiogenic
responses by different stimuli can be assessed in the rabbit cornea assay through
the implantation and/or removal of multiple pellets placed in parallel
micropockets produced in the same cornea (6,7) (Fig. 4). Implanting tumor
samples from different locations can be performed both in corneal
micropockets and in the anterior chamber of the eye to monitor angiogenesis
136 Ziche
Fig. 2. Representative pictures of a negative implant (A) and an implant producing
neovascular growth (B). In A, a control pellet of Elvax alone has been inserted into the
micropocket. In B arrows indicate the newly formed vessels that progress into the
corneal stroma toward a pellet of FGF-2 (200 ng). Photographs were taken at d 10
after surgical implantation, through a slit stereomicroscope (×18).
Corneal Assay 137
Fig. 3. Representative pictures of corneal angiogenesis induced by wild type MCF-7
cells (A) and by MCF-7 stably transfected with VEGF
121
(B). Wild-type MCF-7
cells induce the growth of few slowly progressing capillaries from preexisting limbal
vessels, while VEGF transfectants induce a strong angiogenic response. Photographs
were taken at d 18 after surgical implantation, through a slit stereomicroscope (×18).
138 Ziche
produced by hormone-dependent tissues or tumors (e.g., human breast or ova-
rian carcinoma in female rabbits). This also allows the detection of neovascular
growth in both the iris and the cornea (4).
The use of nude mice allows the study of angiogenesis in response to effec-
tors produced and released by tumors, or tumor cell lines, of human origin
growing subcutaneously (11). Treatment of mice with antiangiogenic or anti-
tumor drugs allows the simultaneous measurement of tumor growth and
metastasis, and corneal angiogenesis.
3.5. Morphological Analysis and Quantification
of Neovascularization
In the past angiogenesis has been described qualitatively (strong, weak,
absent) or by measuring the length of the most advanced vascular sprout at
daily intervals. Moreover, flat-mount preparations of cornea have been used to
obtain more quantitative assessments using imaging techniques and vector
Fig. 4. Representative pictures of corneal angiogenesis induced by implanting in
the same eye two pellets containing two different angiogenic triggers at suboptimal
doses. Photograph taken at d 10 after surgical implantation, through a slit stereomicro-
scope (×18).