We thank Kristin Johnson for the graphic work.

All protocols involving animals are presented to and approved by the Institutional Animal Care and Use Committee at Children&#39;s Hospital Boston and are conducted in accordance with the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Be sure to use sterile instrumentation and aseptic techniqure while performing the procedure.&#160;
1. Preparation of Pellets
<ol>
	<li>Weigh out 10 mg of sucralfate and 60 mg of hydron with the sterile unbent spatula.&#160; Place in separate microcentrifuge tubes.</li>
	<li>Place a 1 cm square piece of mesh into a sterile 10 cm dish.&#160; Invert so mesh rests on shallow lid.&#160; Set aside.</li>
	<li>Add 500 &#956;l of ethanol to the hydron and vortex for at least 10 min.</li>
	<li>Add the appropriate amount of growth factor (standard: 20 &#956;g for bFGF, 50 &#956;g for VEGF) to the sucralfate and vortex briefly.&#160; Store growth factor in a -80 &#176;C freezer at a concentration of 1 mg/ml.&#160; Liquid volumes added should be a minimum of 20 &#956;l to ensure the sucralfate is completely moistened but should not exceed 50 &#956;l.&#160; Greater volume makes the evaporation of the liquid in the next step difficult.</li>
	<li>Place the sucralfate mixture into the centrifugal evaporator set on low until the mixture is completely dry, normally 30-50 min depending on the volume of liquid used.
	<ol>
		<li>Check the mixture for residual moisture with the tapered end of a sterile spatula.&#160; The mixture should feel &#34;crunchy&#34;.</li>
	</ol>
	</li>
	<li>Use the tapered end of the spatula to break up the sucralfate mixture within the tube.</li>
	<li>Add 10 &#956;l of the hydron to the sucralfate.
	<ol>
		<li>Due to the viscous nature of hydron, cut the end from a 200 &#956;l pipet tip and then draw and release the 10 &#956;l before pipetting up again and adding to sucralfate.</li>
	</ol>
	</li>
	<li>Quickly use bent spatula to mix the hydron and sucralfate and remove from tube.
	<ol>
		<li>Gather the mixture along the bottom side of the tip of the bent spatula and then rotate the tube while drawing the spatula out along the wall of the tube.&#160; The mixture will dry rapidly so this step must be done efficiently.</li>
	</ol>
	</li>
	<li>Spread the mixture onto the prepared mesh using forceps to hold it in place.&#160; The mixture should be an even layer and fill into the holes of the mesh.</li>
	<li>Immerse the bent spatula into the tube containing hydron and use it coat both sides of the mesh.</li>
	<li>Place the coated mesh leaning on side against edge of dish and allow drying at RT for 30-45 min.</li>
	<li>Place the mesh in -20 &#176;C freezer or proceed immediately to next step.&#160; Keep mesh inside dish.</li>
	<li>Place the 35 mm dish inside of the larger petri dish, lid off.&#160; Use the pair of forceps to carefully pull the fibers of the mesh apart over the 35 mm dish.&#160; Stray pellets can be collected from the larger dish at the end.
	<ol>
		<li>Check pellets for uniformity under scope.&#160; The process yields approximately 250 pellets thus the standard dose per pellet for bFGF and VEGF is 80 ng and 200 ng, respectively.</li>
	</ol>
	</li>
	<li>Store pellets in 35 mm dish in -20 &#176;C freezer for up to 3 months.</li>
</ol>
2. Surgical Implantation of Pellets
<ol>
	<li>Set up a surgical area under operating microscope.</li>
	<li>Anesthetize the mouse with avertin (400-500 mg/kg, intraperitoneal) and position the mouse under microscope so eye is visible.&#160; Perform toe pinch to ensure sufficient level of anesthesia.</li>
	<li>Anesthetize the mouse eye by placing 1 drop of proparacaine solution atop eyeball.&#160; Wait 20-30 sec and dab with a gauze pad.</li>
	<li>Proptose the mouse eye with the dulled #1 jewelers forceps being certain to leave skin between forceps and eye.&#160; It is important to judge the pressure applied correctly.&#160; Holding the eye too firmly will cause it to become red and irritated, while too loose a hold will allow eye to move during surgery.</li>
	<li>Use the 30&#176; microknife to make an incision into the cornea approximately 1 mm from the limbus.&#160; The incision should be 1-2 mm in length.&#160; Incision should be deep enough to penetrate beyond the epithelial layer into midstroma but not so deep to rupture the eye.</li>
	<li>Use the von Graef knife to make a pocket perpendicular to the incision.&#160;
	<ol>
		<li>Slide the knife under the corneal layer at the incision site and gently work knife in and move it along the incision to enlarge the space, thus forming the pocket.&#160; It helps to move the mouse as well to be able to work with the curvature of the eye.&#160; Be careful not to push downward with the tip to avoid rupturing the eye.</li>
	</ol>
	</li>
	<li>Wet the #5 jeweler&#39;s forceps with proparacaine and pick a pellet up out of dish.&#160; Place a pellet on the eye.</li>
	<li>Moisten the von Graef knife with proparacaine and transfer some of liquid onto pellet to make it rubbery and pliable.&#160; Remove excess moisture with gauze pad or cotton swab.</li>
	<li>Insert the pellet into the pocket using the von Graef knife to push it inside beneath the cornea layer.&#160; Once the whole pellet is inside, run the flat side of von Graef knife over site to check that the pellet is secure.</li>
	<li>Coat the eye with triple antibiotic ointment.</li>
	<li>Turn the mouse over and repeat surgery in the opposite eye.</li>
</ol>
3. Quantification of Corneal Neovascularization.
Leave animals to develop vessels over a set period of time.&#160; The day of vessel grading depends on growth factor used. Grade bFGF on day 5 and the weaker VEGF on day 6.&#160; The day of implantation is day 0.
<ol>
	<li>Anesthetize the mouse with avertin (400-500 mg/kg, intraperitoneal).</li>
	<li>Use the slit lamp microscope for grading.&#160; One ocular has a reticule, lines within the eyepiece used as a measuring aid to quantify vessel length and distance around the eye&#39;s circumference that the vessels have sprouted.
	<ol>
		<li>Hold the mouse in front of scope, positioning mouse so eye is seen in ocular with pellet directly ahead.&#160; Stabilize the head between thumb and forefinger so skin is tightened on face and eye is slightly proptosed.</li>
	</ol>
	</li>
	<li>Place the y-axis of the reticule along the limbal vessel directly beneath pellet.&#160; Measure the length of vessels branching upward toward pellets.&#160; Record this measurement in tenths of millimeters and designate vessel length (VL) (Figure 1A).</li>
	<li>Turn either the mouse or the reticule so that the distance around the eye that these vessels have sprouted becomes clear.&#160; It is easiest to think of the eye as a clock face with intervals of 1 through 12.&#160; Designate this number clock hour (CH) (Figure 1B).&#160; It can be a whole number or fractions of 0.25 (e.g., 2, 2.25, 2.5 or 2.75). Note: CH is subjective.&#160; For example, some may measure the distance around the eye to include all vessels sprouted from the limbus while others may stop measuring at the point where vessel length is half maximal.&#160; The important factor is to be consistent with how one chooses to grade.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51375/51375fig1highres.jpg" src="/files/ftp_upload/51375/51375fig1.jpg" /> 
Figure 1. Quantification of corneal neovascularization. &#160;(A) 80 ng bFGF pellet implanted in C57BL/6J mouse showing slit lamp reticule oriented to measure vessel length. VL = 0.9 mm. (B) Same eye showing slit lamp reticule rotated to measure clock hour.&#160; CH = 3.25.&#160; Calculated vessel area = 1.84 mm2.&#160; These images have been digitally enhanced to allow for the addition of reticule schematic. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51375/51375fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<ol start="5">
	<li>Calculate the vessel area (VA) using the formula below, which is based on the area of an oval (Note, it is helpful to keep this in mind when grading.)&#160; Express VA in millimeters squared.&#160; The formula is as follows: 
	Vessel length x Clock hour x 0.2 &#960; = Vessel area</li>
</ol>

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	<li>Gospodarowicz, D., Moran, J., Braun, D., Birdwell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+growth+of+bovine+vascular+endothelial+cells:+fibroblast+growth+factor+as+a+survival+agent.">Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent.</a> Proc. Natl. Acad. Sci. USA. 73, 4120-4124 (1976).</li><li>Glaser, B. M., D'Amore, P. A., Seppa, H., Seppa, S., Schiffmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+tissues+contain+chemoattractants+for+vascular+endothelial+cells.">Adult tissues contain chemoattractants for vascular endothelial cells.</a> Nature. 288, 483-484 (1980).</li><li>Kubota, Y., Kleinman, H. K., Martin, G. R., Lawley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> J. Cell Biol. 107, 1589-1598 (1988).</li><li>Montesano, R., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-promoting+phorbol+esters+induce+angiogenesis+in+vitro.">Tumor-promoting phorbol esters induce angiogenesis in vitro.</a> Cell. 42, 469-477 (1985).</li><li>Korff, T., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+endothelial+cells+in+multicellular+spheroids+prevents+apoptosis+and+induces+differentiation.">Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation.</a> J. Cell Biol. 143, 1341-1352 (1998).</li><li>Nicosia, R. F., Tchao, R., Leighton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histotypic+angiogenesis+in+vitro:+light+microscopic,+ultrastructural,+and+radioautographic+studies.">Histotypic angiogenesis in vitro: light microscopic, ultrastructural, and radioautographic studies.</a> In Vitro. 18, 538-549 (1982).</li><li>Wong, K. H., Chan, J. M., Kamm, R. D., Tien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+models+of+vascular+functions.">Microfluidic models of vascular functions.</a> Annu Rev Biomed Eng. 14, 205-230 (2012).</li><li>Sandison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+microscopic+study+of+living+growing+tissues+by+the+introduction+of+a+transparent+chamber+in+the+rabbit's+ear.">A new method for the microscopic study of living growing tissues by the introduction of a transparent chamber in the rabbit's ear.</a> Anat. Rec. 28, 281-287 (1924).</li><li>Jain, R. K., Schlenger, K., Hockel, M., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+angiogenesis+assays:+progress+and+problems.">Quantitative angiogenesis assays: progress and problems.</a> Nat. Med. 3, 1203-1208 (1997).</li><li>Rogers, M. S., Birsner, A. E., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+micropocket+angiogenesis+assay.">The mouse cornea micropocket angiogenesis assay.</a> Nat. Protoc. 2 (10), 2545-2550 (2007).</li><li>Gimbrone, M. A., Leapman, S. B., Cotran, R. S., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+dormancy+in+vivo+by+prevention+of+neovascularization.">Tumor dormancy in vivo by prevention of neovascularization.</a> J. Exp. Med. 136, 261-276 (1972).</li><li>Gimbrone, M. A., Cotran, R. S., Leapman, S. B., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+growth+and+neovascularization:+an+experimental+model+using+the+rabbit+cornea.">Tumor growth and neovascularization: an experimental model using the rabbit cornea.</a> J. Natl. Cancer Inst. 52, 413-427 (1974).</li><li>Rohan, R. M., Fernandez, A., Udagawa, T., Yuan, J., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+heterogeneity+of+angiogenesis+in+mice.">Genetic heterogeneity of angiogenesis in mice.</a> FASEB J. 14 (7), 871-876 (2000).</li><li>Chang, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+response+of+FGF-2+for+lymphangiogenesis.">Dose-dependent response of FGF-2 for lymphangiogenesis.</a> Proc Natl Acad Sci U S A. 101 (32), 11658-11663 (2004).</li><li>Udagawa, T., Birsner, A. E., Wood, M., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+suppression+of+angiogenesis+following+radiation+exposure+is+independent+of+hematopoietic+reconstitution.">Chronic suppression of angiogenesis following radiation exposure is independent of hematopoietic reconstitution.</a> Cancer Res. 67 (5), 2040-2045 (2007).</li><li>Turner, P. V., Albassam, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+rats+to+corneal+lesions+after+injectable+anesthesia.">Susceptibility of rats to corneal lesions after injectable anesthesia.</a> Med Comp. 55 (2), 175-182 (2005).</li><li>Calderone, L., Grimes, P., Shalev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+reversible+cataract+induced+by+xylazine+and+by+ketamine-xylazine+anesthesia+in+rats+and+mice.">Acute reversible cataract induced by xylazine and by ketamine-xylazine anesthesia in rats and mice.</a> Exp. Eye Res. 42, 331-337 (1986).</li></ol>

The authors have nothing to disclose.

There are several critical steps in performing a successful corneal assay.&#160; The first is making uniform pellets capable of being implanted and stimulating vessels.&#160; The most important parts of pellet preparation are 1) using carrier-free growth factor; 2) ensuring a good mixture of growth factor with the sucralfate and hydron and 3) moving the final mixture swiftly but carefully from the eppendorf tube to the mesh where the pellets are cast.&#160; It is recommended that &#34;dummy&#34; pellets be made without g...

The process of angiogenesis, the formation of new blood vessels form preexisting ones, is highly complex and is regulated by several endogenous factors that control different steps of vessel sprouting and morphogenesis.&#160; Angiogenesis is triggered due to a shift in balance between pro- and anti-angiogenic factors, a balance that normally maintains the vasculature in a quiescent state.&#160; Angiogenesis in adults occurs in certain physiological conditions such as during the female ovarian cycle or in repair processes such as wound healing and tissue regeneration. &#160;However, it is also a hallmark of several pathologies including malignancies, autoimmune conditions and inflammatory diseases. &#160;The involvement of angiogenesis in these physiological and pathological conditions makes it an important subject for research and an attractive target for therapy.
Due to the complexity of angiogenesis and the involvement of several cells and factors in the process, including endothelial cells, pericytes, circulating cells and stromal cells, in vitro models remain limited and cannot recapitulate the unique in vivo microenvironment.&#160; &#160;The main in vitro assays of angiogenesis are largely focused on observing direct effects on endothelial cells and measuring certain steps in angiogenic process under controlled conditions. These assays include the quantification of endothelial cell proliferation1, migration2, network formation3, tube formation4 and sprouting from spheroids5. &#160;Ex vivo models, unlike the in vitro ones, are more complex and incorporate multiple tissue cell types, an example being the aortic ring assay6.&#160; Nevertheless, like other systems, it cannot capture the contribution of circulating cells and the natural stroma of endothelial cells as exists in vivo.&#160; Attempts to study angiogenesis under flow to mimic the in vivo setting are performed using microfluidic systems7, however even these assays, although much improved, are still unable to account for all the compartments existing in vivo.
Due to the limitations of the in vitro and ex vivo angiogenesis models, in vivo models remain the more reliable choices for angiogenesis studies.&#160; Examples of these models include the implantation of transparent chambers, &#34;windows&#34;, that allow the visualization of growing blood vessels under microscope8, injectable subcutaneous implants such as matrigel and vessel formation in normal tissues such as mouse ear and the chicken chorioallantoic membrane (CAM).&#160; However, one of the most acceptable and quantitative in vivo angiogenesis models is the corneal micropocket neovascularization assay described here, which exploits the naturally avascular cornea as a &#34;screen&#34; to visualize and assess new angiogenic growth9.
Here we describe the corneal micropocket assay as developed in mice.&#160; Initially the model was used to measure nonspecific angiogenic stimuli in rabbit corneas. This was done by introducing tumor pieces into the aqueous humor of the anterior chamber of the rabbit eye and measured tumor-induced neovascularization11.
&#160;However, the assay was later evolved to study effects of specific growth factors10 to better specify and standardize the angiogenic effect.&#160;&#160; In order to release the growth factor in the eye, slow-release pellets containing known quantities of angiogenic growth factors were used instead of tissues.&#160; The availability of purified recombinant angiogenic proteins such as bFGF or VEGF enabled their use as specific targets of angiogenesis modulaters12.&#160; Initially, the assay was largely used in rabbits, which are easier to work with due to their size but later the model was translated into mice; a smaller and less expensive animal model.&#160; Shifting from rabbit to mice provided an important advantage of being able to use genetically manipulated animals, thereby creating a new area of research into the genetic components impacting angiogenesis13.&#160; In addition to the more acceptable usage of cornea assay in studying angiogenesis, other biological processes can also being investigated using modified assays.&#160; For example, studies of lymphangiogenesis were made possible through the implantation of low dose bFGF pellets which allowed the visualization of lymphatic vessels through specific molecular markers14.&#160; In addition, this assay has provided a means to evaluate the effects of radiation on angiogenesis15.
In summation, the corneal micropocket angiogenesis assay is a quantitative, reproducible, flexible assessment of angiogenesis in vivo.&#160; A major advantage of this assay is that the measurement of background vessels is unnecessary because the vessels grow on a naturally avascular tissue.&#160; We describe here the protocol of this assay in details and discuss different scenarios which can occur. The assay consists of 3 discrete segments.&#160; Here we will describe the preparation of growth factor-inclusive pellets, the subsequent surgical implantation and finally the method used to quantify the resulting neovascular growth.

<table><tbody><tr><td>Section 1: Pellet preparation</td><td></td><td></td><td></td></tr><tr><td>Sucralfate</td><td>Sigma</td><td>#S0652</td><td></td></tr><tr><td>Hydron (aka Poly(2-Hema))</td><td>Sigma</td><td>#P3932-10G</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco Products Inc</td><td>#111000200CSGL</td><td></td></tr><tr><td>Growth factors:</td><td></td><td></td><td>Must be carrier-free (no bovine serum albumin(BSA))</td></tr><tr><td>&amp;nbsp;Fibroblast growth factor (FGF)</td><td>PeproTech</td><td>#AF-100-18B</td><td></td></tr><tr><td>Vascular endothelial growth factor (VEGF)</td><td>R D Systems</td><td>#293-VE-050/CF</td><td></td></tr><tr><td>35 mm dish</td><td>Becton-Dickson</td><td>#353001</td><td>Used for storage of pellets</td></tr><tr><td>10 cm petri dish</td><td>VWR</td><td>#25384-342</td><td>Used as work surface for preparing pellets</td></tr><tr><td>Mesh</td><td>Sefar America</td><td>#03--300/51</td><td>300 um nitex nylon, cut into cm square pieces and sterilzed in autoclave</td></tr><tr><td>Spatulas</td><td>Fisher Scientific</td><td>#21-401-10</td><td>Use tapered end of one to break up pellet mixture. Bend tapered end of other to help remove mixture from microcentrifuge tube.</td></tr><tr><td>Microcentrifuge tubes</td><td>Fisher Scientific</td><td>#05-408-146</td><td>One for hydron, one for sucralfate</td></tr><tr><td>Jewelers forceps, #5</td><td>Ambler Surgical</td><td>#2315E</td><td>Need 2 for pulling mesh apart</td></tr><tr><td>Centrifugal evaporator</td><td>ThermoSavant</td><td>DNA110 SpeedVac</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Section 2: Surgical implantation of pellets</td><td></td><td></td><td></td></tr><tr><td>Operating microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>2.5% Avertin</td><td></td><td></td><td>General anesthetic</td></tr><tr><td>Proparacaine hydrochloride ophthalmic solution 0.5%</td><td>Falcon</td><td>NDC# 6131401601</td><td>Eye anesthetic</td></tr><tr><td>Triple Antibiotic Ophthalmic Ointment</td><td>Bausch Lomb</td><td>NDC# 2420878055</td><td>Contains neomycin, polymixin and bacitracin</td></tr><tr><td>Ophthalmic microknife, 5 mm</td><td>Surgistar</td><td>#924501</td><td>30 degree angle</td></tr><tr><td>von Graef knife</td><td>Ambler Surgical</td><td>#3401E</td><td></td></tr><tr><td>Jewelers forceps, #1</td><td>Ambler Surgical</td><td>#2301E</td><td>Must be blunted with sharpening stone for proptosing eye</td></tr><tr><td>Jewelers forceps, #5</td><td>Ambler Surgical</td><td>#2305E</td><td>For picking up pellets and placing on eye</td></tr><tr><td>Small curved scissors</td><td>Ambler Surgical</td><td>#5636E</td><td>For trimming whiskers</td></tr><tr><td>Gauze</td><td></td><td></td><td>For blotting eye after proparacaine</td></tr><tr><td>Section 3: Grading of Corneal Neovascularization</td><td></td><td></td><td></td></tr><tr><td>2.5% Avertin</td><td></td><td></td><td>General anesthetic</td></tr><tr><td>Slit lamp</td><td>Nikon</td><td>FS-2</td><td>Needs an ocular with a reticule to assist in measuring</td></tr></tbody></table>

the corneal micropocket assay: a model of angiogenesis in the mouse eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

Typical results for bFGF and VEGF pellets in the normal low angiogenic C57BL/6J mice are shown in Figure 2A and B, respectively.&#160; Figure 2E shows the normal distribution of vessel area (VA) in the C57BL/6J strain with varying doses of these growth factors.&#160; 80 ng bFGF pellets normally result in a VA of approximately 2.0 mm2, though values in a range of 1.8-2.4 mm2 are acceptable.&#160; 200 ng VEGF pellets typically cause a response of appr...

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The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

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16.6K Views.  Boston Children's Hospital. The protocol describes the corneal micropocket assay as developed in mice.

Video: The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Medicine

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biology

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

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.

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.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

Alkali Burn-Induced Corneal Neovascularization in Mice

A Mouse Model for Corneal Neovascularization by Alkali Burn

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus&#160;and adversely&#160;affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

Author Spotlight: Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies 

This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization.

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

Summary

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Chapters in this video

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

A Mouse Model for Laser-induced Choroidal Neovascularization

Rat Mesentery Angiogenesis Assay

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

A Mouse Model for Corneal Neovascularization by Alkali Burn