The authors thank Linnea Tankred and Diana Rydholm for animal husbandry.

BALB/c mouse pups of either sex were used in accordance with the statement for the Use of Animals in Ophthalmologic and Vision Research, and the protocols were approved by Stockholm&#39;s Committee for Ethical Animal Research. Mice were housed in litters, together with the nursing mother, with a 12 h day/night cycle, free access to food and water, and monitored daily.
NOTE: For the surgical procedure, mice were kept under anesthesia with volatile isoflurane. Ocular ointments are discouraged during ocular procedures, as they might interfere with treatments and substances used. If necessary, to prevent dry-eye, a drop of sterile normal saline solution can be applied.Though uveal punctures are self-healing , care was taken during uveal punctures to ensure sterility with surgical instruments. Post-surgical treatment included hydration with normal saline solution subcutaneously, and analgesia with ocular topical administration of tetracaine hydrochloride. The pups were allowed to recover to sternal recumbency on a heating pad before being returned to the nursing mother, in a clean cage. Litters were kept with the same nursing mother to avoid stress.
1. Anesthesia
<ol>
	<li>Prepare the anesthesia induction chamber, to administer isoflurane anesthetic and ensure that a small mouse mask is also coupled. 
	NOTE: Perform all animal experiments in agreement with all applicable ethical permits.</li>
	<li>Prefill the induction chamber with 3-4% volatile isoflurane in atmospheric air. 
	NOTE: Different anesthetic substances other than isoflurane may be used, according to ethical permits. Volatile anesthetic procedures should be carried on a ventilated bench or fume hood.</li>
	<li>Place a postnatal (P)12.5-day-old BALB/c mouse pup in the induction chamber. Confirm that the anesthesia apparatus is ready to deliver isoflurane to the induction chamber. 
	NOTE: Mouse pups are fragile and best handled by the scruff. Keep the pups together as a litter or with the nursing mother to avoid stress.</li>
	<li>Allow the pup to be fully anesthetized.</li>
	<li>Gently, pick up the mouse pup by the scruff. 
	NOTE: Keep the induction chamber closed at all times for safe and stable induction.</li>
	<li>Transfer the mouse to the rodent mask. Make sure to switch the anesthetic flow from the induction chamber to the mask when moving the mouse.</li>
	<li>Perform a toe pinch to confirm anesthesia.</li>
</ol>
2. Puncture Procedure under Surgical Stereoscope
<ol>
	<li>Position the mouse in lateral recumbency position. Confirm the mask is well positioned so the mouse eye is in the field of view of the stereoscope.</li>
	<li>Gently rotate the mouse head so the eye is facing up toward the stereoscope. 
	NOTE: Clear focus of the eye should be achieved under the stereoscope. For positioning, a magnification of 16X is recommended, while 40X should be used to perform the surgical procedure. If ethical permits allow it, trimming the tip of the whiskers can facilitate the visualization of the eye.</li>
	<li>Using small tying forceps, carefully protrude the pup&#39;s eye by applying downward pressure to the eyelids dorsal and ventral to the eye.</li>
	<li>Keep a gentle but firm hold of the pup&#39;s eye lid, with small forceps. 
	NOTE: It is recommended to perform the eye protrusion and lid holding with the non-dominant hand, as the dominant hand should perform the puncture procedure.</li>
	<li>Use the stereoscope to locate the corneal limbus. Albino BALB/c limbus can be easily identified by the circular vascular plexus posterior to the cornea.</li>
	<li>With a 0.25 mm diameter (30 G) beveled needle, perform a small uveal puncture, near the posterior limbal limit of the uvea. Use only the tip of the needle, and no more than half the bevel (equivalent to 0.5 mm), puncture the uvea. If executed correctly, the uveal punctures are self-sealing, yet intraocular injection of study substances can be administered through the same puncture wound. 
	NOTE: If experienced with mouse ocular anatomy, the uveal puncture is executed between the ciliary body and ora serrata. Care must be taken while performing the uveal puncture to avoid rupturing the lens.</li>
	<li>Perform the second uveal puncture in the opposite site of the eye from the first puncture site. Optimize the distance and position of the punctures; consider 12 and 6 o&#39;clock punctures, with 12 o&#39;clock being as dorsal as possible.</li>
	<li>Repeat the uveal punctures procedure every four days until P24.5. Before every successive uveal puncture, monitor animal status paying particular attention to presence of traumatic cataract due to lens damage during the uveal puncture, or obvious diminished ocular pressure. Aim for executing repeat punctures at the same positions as previous punctures for optimal effects.</li>
</ol>
3. Noninvasive in Vivo Monitoring
<ol>
	<li>Prior to the puncture or injections on each experimental day, anesthetize the mouse as performed above.</li>
	<li>With the mouse in lateral recumbency position, gently protrude the eye.</li>
	<li>Focus on iris vasculature with the surgical stereoscope.</li>
	<li>Take a picture of the iris vasculature with a camera fitted to the surgical stereoscope. 
	NOTE: Though not necessary, inducing meiosis can be helpful to normalize iris area per animal. Consider ethical permits when selecting a meiosis-inducing agent. Careful focus of the iris vasculature will facilitate further quantitative analysis. A magnitude of 40X is recommended.</li>
</ol>
4. Post-Operative Care
<ol>
	<li>Remove the BALB/c pup from the rodent mask, and gently transfer it to a heating pad layered with a surgical mat.</li>
	<li>Apply one drop of 1% tetracaine solution to punctured eyes. 
	NOTE: Other analgesic solutions can be used, according to ethical permits.</li>
	<li>Hydrate the animal with a subcutaneous injection of 200 &#181;L of sterile normal saline solution.</li>
	<li>Return the pup to the nursing mother in a clean mouse cage.</li>
	<li>Monitor the recovery process. The pup should be able to deambulate and return to the litter.</li>
</ol>
5. Eye Enucleation
<ol>
	<li>Euthanize the animal by cervical dislocation. Perform enucleation and dissection under a stereoscope for better visualization of the procedure. 
	NOTE: Different euthanasia procedures can be used. It is recommended to strictly adhere to ethical permits.</li>
	<li>Pull apart the eyelids to improve the exposure to the eye.</li>
	<li>Make a small cut (approximately 0.5 cm) near the canthus of the eye lids with curved Bonn eye scissors.</li>
	<li>Use the exposed periocular space to insert micro tying forceps behind (under) the globe in the orbit.</li>
	<li>Grab the tissue surrounding the eye and gently pull upwards. Avoid holding onto the eyeball, use the surrounding tissues closer to the eye socket.</li>
	<li>Insert the curved Bonn eye scissors behind the eye globe in the orbit.</li>
	<li>Cut through the surrounding tissues until the eye is released from the socket. 
	NOTE: This dissection technique maintains eye anatomy and benefits subsequent analysis.</li>
	<li>Proceed to the removal of extraneous tissue from the eye. The removal of extraneous tissue can be performed after fixing, if preferred. 
	NOTE: BALB/c mice are melanin deficient, therefore, to increase contrast, a dark background is recommended to facilitate dissection procedure.</li>
	<li>Briefly rinse the whole-eye in a Petri dish filled with sterile phosphate-buffered saline (PBS).</li>
	<li>Transfer the eye to a 2 mL tube containing 1.5 mL of 4% buffered-formalin solution.</li>
	<li>Fix the eye for 6 h at room temperature. 
	NOTE: Fixing confers the rigidity of the tissue and facilitates dissection of the iris. Fixing time should be tested and adjusted for different applications.</li>
</ol>
6. Iris Dissection Procedure under Stereoscope
<ol>
	<li>Remove the fixative solution from the tube with a 1.5 mL plastic Pasteur pipette (or similar), and rinse the eye three times with fresh PBS.</li>
	<li>Place the fixed eye in a smooth dry surface (dissection stand) with the anterior chamber facing upwards.</li>
	<li>With a beveled 30 G needle, perform an entry point posterior to the limbus.</li>
	<li>Holding the anterior segment with small tying forceps, insert one tip of Clayman-Vannas straight scissors into the opening previously created. 
	NOTE: It is recommended to control the forceps with the non-dominant hand, and the scissor with the dominant.</li>
	<li>While rotating the eye with the tying forceps, cut around the limbus to remove the posterior segment of the eye. 
	NOTE: Performing partial cuts will allow the inner tip of the scissors to continue in the tissue and greatly facilitates the process.</li>
	<li>Position the anterior segment facing downward, exposing the lens.</li>
	<li>Remove the lens carefully by grabbing it with small forceps and pulling upwards. 
	NOTE: A beveled needle can be used to puncture and pull the lens.</li>
	<li>Position the anterior segment with the cornea facing down and the cut perpendicular to the field of view.</li>
	<li>Gently grab the tissue posterior to the ciliary body with the small tying forceps.</li>
	<li>With Clayman-Vannas straight scissors, proceed to trim just anterior to the ciliary body to remove the trabecular meshwork and isolate the iris. Confirm that the trabecular meshwork is removed; the iris should move within the cornea.</li>
	<li>Carefully transfer the anterior eyecup with the small tying forceps, holding the cornea, to a 96-well plate containing 200 &#181;L of PBS.</li>
	<li>Continue holding the cornea in the well and dissect the iris by flushing with PBS with a pipette. 
	NOTE: If the iris remains adherent to the cornea, some trabecular meshwork might be present, and the bevel of a 30 G needle can be used to help displacing the iris.</li>
	<li>Remove the cornea from the 96-well with the small tying forceps, and keep the isolated iris in the well for further analysis.</li>
	<li>Store free-floating iris samples at 4 &#176;C. 
	NOTE: Despite being fixed, long-term storage of the iris free-floating samples is not recommended.</li>
</ol>
7. Possible Experimental Read-Outs
<ol>
	<li>To visualize the blood vessels in the dissected irises, use free-floating whole-mount immunohistochemistry staining methods for markers such as platelet endothelial cell adhesion molecule (PECAM)-1 or isolectin B4. Alternatively, use whole-body perfusion with vascular staining, such as fluorescent-labeled high molecular weight dextrans.</li>
	<li>Use in vivo noninvasive images to perform in silico flat-mounting and quantify full iris vasculature with appropriate imaging software. 
	NOTE: Vasculature can be quantified, both in vivo and in vitro, using densitometry or with vasculature analysis software9.</li>
	<li>Process the whole eyeball for nucleic acids and protein extraction, followed by PCR or immunoblotting methods, to perform molecular analysis of the punctured eyes. 
	NOTE: Molecular analysis of tissues is best performed with non-fixed tissue, but dissection of non-fixed iris is extremely challenging, and the use of the whole eye has rendered statistically significant results in previous studies8.</li>
</ol>

<ol><li>Morrison, J. C., Van Buskirk, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anterior+collateral+circulation+in+the+primate+eye%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">Anterior collateral circulation in the primate eye.</a> Ophthalmology. 90 (6), 707-715 (1983).</li>
<li>Dreyfuss, J. L., Giordano, R. J., Regatieri, C. V. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600930/">Ocular Angiogenesis.</a> J Ophthalmol. 2015, 892043(2015).</li>
<li>Breuss, J. M., Uhrin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VEGF-initiated+angiogenesis+and+the+uPA%2FuPAR+system%5BTitle%5D)+AND+%22Cell+Adh+Migr%22%5BJournal%5D)">VEGF-initiated angiogenesis and the uPA/uPAR system.</a> Cell Adh Migr. 6 (6), 535-615 (2012).</li>
<li>Rodrigues, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neovascular+glaucoma%3A+a+review%5BTitle%5D)+AND+%22Int+J+Retina+Vitreous%22%5BJournal%5D)">Neovascular glaucoma: a review.</a> Int J Retina Vitreous. 2, 26(2016).</li>
<li>Stahl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mouse+retina+as+an+angiogenesis+model%5BTitle%5D)+AND+%22Invest+Ophth+Vis+Sci%22%5BJournal%5D)">The mouse retina as an angiogenesis model.</a> Invest Ophth Vis Sci. 51 (6), 2813-2826 (2010).</li>
<li>DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenesis+and+wound+repair%3A+when+enough+is+enough%5BTitle%5D)+AND+%22J+Leukocy+Biol%22%5BJournal%5D)">Angiogenesis and wound repair: when enough is enough.</a> J Leukocy Biol. 100 (5), 979-984 (2016).</li>
<li>Eming, S., Brachvogel, B., Odorisio, T., Koch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+angiogenesis%3A+Wound+healing+as+a+model%5BTitle%5D)+AND+%22Prog+Histochem+Cytoc%22%5BJournal%5D)">Regulation of angiogenesis: Wound healing as a model.</a> Prog Histochem Cytoc. 42 (3), 115-170 (2007).</li>
<li>Beaujean, O., Locri, F., Aronsson, M., Kvanta, A., André, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+in+vivo+model+of+puncture-induced+iris+neovascularization%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">A novel in vivo model of puncture-induced iris neovascularization.</a> PloS One. 12 (6), e0180235(2017).</li>
<li>Zudaire, E., Gambardella, L., Kurcz, C., Vermeren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Computational+Tool+for+Quantitative+Analysis+of+Vascular+Networks%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">A Computational Tool for Quantitative Analysis of Vascular Networks.</a> PloS One. 6 (11), (2011).</li>
<li>Dorrell, M., Uusitalo-Jarvinen, H., Aguilar, E., Friedlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+neovascularization%3A+basic+mechanisms+and+therapeutic+advances%5BTitle%5D)+AND+%22Surv+Ophthalmol%22%5BJournal%5D)">Ocular neovascularization: basic mechanisms and therapeutic advances.</a> Surv Ophthalmol. 52 Suppl 1, S3-S19 (2007).</li>
<li>Kvanta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+angiogenesis%3A+the+role+of+growth+factors%5BTitle%5D)+AND+%22Acta+Ophthalmol%22%5BJournal%5D)">Ocular angiogenesis: the role of growth factors.</a> Acta Ophthalmol. 84 (3), 282-288 (2006).</li>
<li>Salman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrasilicone+Bevacizumab+Injection+for+Iris+Neovascularization+after+Vitrectomy+for+Proliferative+Diabetic+Retinopathy%5BTitle%5D)+AND+%22Ophthalmic+Res%22%5BJournal%5D)">Intrasilicone Bevacizumab Injection for Iris Neovascularization after Vitrectomy for Proliferative Diabetic Retinopathy.</a> Ophthalmic Res. 49 (1), 20-24 (2013).</li>
<li>Jeong, Y. C., Hwang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Etiology+and+Features+of+Eyes+with+Rubeosis+Iridis+among+Korean+Patients%3A+A+Population-Based+Single+Center+Study%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Etiology and Features of Eyes with Rubeosis Iridis among Korean Patients: A Population-Based Single Center Study.</a> PloS One. 11 (8), e0160662(2016).</li>
<li>Speier, S., Nyqvist, D., Köhler, M., Caicedo, A., Leibiger, I. B., Berggren, P. -O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+high-resolution+in+vivo+imaging+of+cell+biology+in+the+anterior+chamber+of+the+mouse+eye%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye.</a> Nat Protoc. 3 (8), 1278-1286 (2008).</li>
<li>Stefansson, E., Landers, M. B., Wolbarsht, M. L., Klintworth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neovascularization+of+the+Iris+-+an+Experimental-Model+in+Cats%5BTitle%5D)+AND+%22Invest+Ophth+Vis+Sci%22%5BJournal%5D)">Neovascularization of the Iris - an Experimental-Model in Cats.</a> Invest Ophth Vis Sci. 25 (3), 361-364 (1984).</li>
<li>Nork, T. M., Tso, M., Duvall, J., Hayreh, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+Mechanisms+of+Iris+Neovascularization+Secondary+to+Retinal+Vein+Occlusion%5BTitle%5D)+AND+%22Arch+Ophthalmol-Chic%22%5BJournal%5D)">Cellular Mechanisms of Iris Neovascularization Secondary to Retinal Vein Occlusion.</a> Arch Ophthalmol-Chic. 107 (4), 581-586 (1989).</li>
<li>Hjelmeland, L. M., Stewart, M. W., Li, J. W., Toth, C. A., Burns, M. S., Landers, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Experimental-Model+of+Ectropion+Uveae+and+Iris+Neovascularization+in+the+Cat%5BTitle%5D)+AND+%22Invest+Ophth+Vis+Sci%22%5BJournal%5D)">An Experimental-Model of Ectropion Uveae and Iris Neovascularization in the Cat.</a> Invest Ophth Vis Sci. 33 (5), 1796-1803 (1992).</li>
<li>Tolentino, M. J., Miller, J. W., Gragoudas, E. S., Chatzistefanou, K., Ferrara, N., Adamis, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+endothelial+growth+factor+is+sufficient+to+produce+iris+neovascularization+and+neovascular+glaucoma+in+a+nonhuman+primate%5BTitle%5D)+AND+%22Arch+Ophthalmol-Chic%22%5BJournal%5D)">Vascular endothelial growth factor is sufficient to produce iris neovascularization and neovascular glaucoma in a nonhuman primate.</a> Arch Ophthalmol-Chic. 114 (8), 964-970 (1996).</li>
<li>Paula, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rabbit+Rubeosis+Iridis+Induced+by+Intravitreal+Latex-derived+Angiogenic+Fraction%5BTitle%5D)+AND+%22Curr+Eye+Res%22%5BJournal%5D)">Rabbit Rubeosis Iridis Induced by Intravitreal Latex-derived Angiogenic Fraction.</a> Curr Eye Res. 36 (9), 857-859 (2011).</li>
<li>Takei, A., Ekström, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+Transfer+of+Prolyl+Hydroxylase+Domain+2+Inhibits+Hypoxia-inducible+Angiogenesis+in+a+Model+of+Choroidal+Neovascularization%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization.</a> Sci Rep. 7, 42546(2017).</li>
</ol>

The authors have nothing to disclose.

In the present protocol, a novel method for induction of iris vascular response by uveal puncture is presented. The puncture triggers wound healing mechanisms and promotes vascular responses in the iris10,11. This is in agreement with ocular pathologies, such as PDR and NVG, where exacerbated angiogenic responses from the retina in the posterior segment of the eye culminate in the clinical condition of rubeosis iridis, an increased vascularization of the iris<sup...

The iris, together with the ciliary body and the choroid, comprises the uvea, which is the most vascularized tissue of the eye. Iris vasculature is essential in maintaining homeostasis in the anterior chamber of the eye. As a result of abundant anastomotic connections between arteries and veins, iris blood vessels provide nutrients and supply of oxygen not only to the iris itself, but to the entire anterior segment of the eye1.
The formation of new blood vessels, or angiogenesis from pre-existing ones, is fundamental in physiological processes, such as development and wound healing2. Angiogenesis is finely regulated by a multitude of canonical factors, such as vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor (PAI), as well as multiple inflammatory factors, and an imbalance of these factors can lead to pathologic angiogenesis3.
In the eye, neovascularization is the cause of sight-threatening diseases, such as proliferative diabetic retinopathy (PDR) and neovascular glaucoma (NVG). In these ocular diseases, the focal neovascularization is commonly located in retinal tissues, yet the imbalance in inflammatory and angiogenic factors in both the posterior and anterior ocular chambers of the eye has been associated with rubeosis iridis, the clinical term for iris pathological neoangiogenesis4. These pathologies indicate the capability of the adult iris to undergo angiogenesis. In mice, ocular vasculature is immature after birth and continues maturation postpartum. This peculiarity of development is exploited in the mouse model of oxygen-induced retinopathy, a model that closely mimics the clinical condition of retinopathy of prematurity5. In addition, angiogenesis and inflammation play a pivotal role in wound healing mechanisms6, and wound healing itself has been associated with angiogenesis models7.
In this study, we describe a model of puncture-induced iris neovascularization. Uveal punctures are performed near the outer limit of the limbus, which induce iris neovascularization by triggering the wound healing system. Due to the transparency of the cornea, iris vasculature can be analyzed easily in vivo by noninvasive methods. Punctured eyes present an increase of vascular bed in the iris, which has been associated with an increase of plasminogen activating and inflammatory markers8. The presented model has great potential as a new tool to study angiogenesis and screening angiogenic compounds, and allows direct in vivo visualization of the angiogenic processes.

<table><tbody><tr><td>Bonn eye scissors</td><td>Bausch &amp;amp; Lomb</td><td>23060</td><td></td></tr><tr><td>Clayman-Vannas curved scissors</td><td>Bausch &amp;amp; Lomb</td><td>E3383 C</td><td></td></tr><tr><td>Clayman-Vannas straight scissors</td><td>Bausch &amp;amp; Lomb</td><td>E3383 S</td><td></td></tr><tr><td>Objective adapter for camera</td><td>Handcrafted</td><td>N/A</td><td>Or any system that allows adapting a camera to the microscope</td></tr><tr><td>Heating Pad 100-110 watts</td><td>Non Applicable</td><td>N/A</td><td>Available in pet/veterinarian stores</td></tr><tr><td>Hypodermic 30g beveled needle</td><td>KDM GmBH germany</td><td>911914</td><td></td></tr><tr><td>Iphone 4S</td><td>Apple</td><td>Non Applicable</td><td>Or other high resolution image acquistion device</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>KDG 9623</td><td></td></tr><tr><td>McPherson tying forceps</td><td>Bausch &amp;amp; Lomb</td><td>E1815 S</td><td></td></tr><tr><td>Micro tying forceps</td><td>Bausch &amp;amp; Lomb</td><td>63140</td><td></td></tr><tr><td>Minims tetracaine hydrochloride</td><td>Bausch &amp;amp; Lomb</td><td>N/A</td><td>1 % (w/v) Eye Drops</td></tr><tr><td>Neutral-buffered formalin</td><td>Bioreagens</td><td>0018-40</td><td></td></tr><tr><td>Normal saline solution</td><td>Fresenius Kabi</td><td>210352</td><td>0.9 % (w/v) NaCl in injectable water</td></tr><tr><td>Phosphate-buffered saline</td><td>ThermoFisher Scientific</td><td>10010023</td><td>Balanced and buffered PBS pH 7.4</td></tr><tr><td>Petri dish 10 cm</td><td>Starstedt</td><td>83.3902</td><td></td></tr><tr><td>Petri dish 3 cm</td><td>Starstedt</td><td>83.3900</td><td></td></tr><tr><td>Safe Seal Tube 2.0 mL</td><td>Starstedt</td><td>72.685.200</td><td>Or any eppendorf style tubes</td></tr><tr><td>TC plate 96-well</td><td>Starstedt</td><td>83.3924</td><td></td></tr><tr><td>Transfer pipette 3.5 mL</td><td>Starstedt</td><td>86.1171</td><td>Or any other Pasteur pipette style</td></tr><tr><td>Univentor 400 anesthesia unit</td><td>Univentor Limited</td><td>N/A</td><td>Or equivalent flow regulator with induction chamber and mask for volatile anesthesia</td></tr><tr><td>Wild M650 surgical microscope</td><td>Wild Heerbrugg</td><td>N/A</td><td>Or other surgical or magnifying stereoscope</td></tr></tbody></table>

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.

Albino BALB/c mouse pups at P12.5 were subjected to uveal punctures, repeated every fourth day (experimental day 0, 4, 8, 12), until P24.5. At P27.5, mice were euthanized and irises carefully dissected (experimental day 15). Pictures of mouse eyes were taken with a camera attached to a surgical stereoscope before every puncture series in each experimental day to assess noninvasive evaluation of the iris vascular response. Uveal punctures induce a vascular response from the iris by trigger...

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

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 ...

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8.9K Views.  Karolinska Institutet. 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.

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

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) ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Neuroscience

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Mouse Model for Retinal Ischemia-Reperfusion Injury to Study Retinal Pathologies

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, and other vaso-occlusive conditions. This manuscript presents a method for inducing ischemia-reperfusion injury in a mouse model. The method utilized anterior chamber cannulation attached to a saline reservoir, generating hydrostatic pressure to raise the intraocular pressure to 90-100 mmHg. This method effectively caused constriction of retinal capillaries to induce retinal ischemia. At the end of the ischemic period (60 min), the intraocular pressure was normalized (&#8804;20 mmHg) before removing the cannula from the anterior chamber to initiate reperfusion. Days after the ischemia/reperfusion procedure, the eyes were collected and sectioned for histological staining. The histopathology of the retinal sections was scored by evaluating eight parameters of retinal injury: folds, hemorrhage, deformation, cell loss in the ganglion cell, inner nuclear, outer nuclear, and photoreceptor layers, and damage to retinal pigment epithelial cells. This method provided a reproducible model to study the mechanisms and pathology of retinal ischemia/reperfusion injury. In addition, this model can facilitate the discovery of potential therapeutic targets to treat retinal ischemia/reperfusion injury, advancing the study of retinal pathologies and improving patient outcomes.

This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, ...

Biology

 Induction of Elevated Intraocular Pressure in a Rat Model

<a href='https://app.jove.com/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)</a>This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison.

Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)This ...

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

Summary

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