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 du...

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B., 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=Neovascular+glaucoma:+a+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+retina+as+an+angiogenesis+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+wound+repair:+when+enough+is+enough.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+angiogenesis:+Wound+healing+as+a+model.">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., Andr&#233;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vivo+model+of+puncture-induced+iris+neovascularization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Computational+Tool+for+Quantitative+Analysis+of+Vascular+Networks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+neovascularization:+basic+mechanisms+and+therapeutic+advances.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+angiogenesis:+the+role+of+growth+factors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasilicone+Bevacizumab+Injection+for+Iris+Neovascularization+after+Vitrectomy+for+Proliferative+Diabetic+Retinopathy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiology+and+Features+of+Eyes+with+Rubeosis+Iridis+among+Korean+Patients:+A+Population-Based+Single+Center+Study.">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., K&#246;hler, M., Caicedo, A., Leibiger, I. B., Berggren, P. -. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+high-resolution+in+vivo+imaging+of+cell+biology+in+the+anterior+chamber+of+the+mouse+eye.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neovascularization+of+the+Iris+-+an+Experimental-Model+in+Cats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Mechanisms+of+Iris+Neovascularization+Secondary+to+Retinal+Vein+Occlusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Experimental-Model+of+Ectropion+Uveae+and+Iris+Neovascularization+in+the+Cat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+is+sufficient+to+produce+iris+neovascularization+and+neovascular+glaucoma+in+a+nonhuman+primate.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+Rubeosis+Iridis+Induced+by+Intravitreal+Latex-derived+Angiogenic+Fraction.">Rabbit Rubeosis Iridis Induced by Intravitreal Latex-derived Angiogenic Fraction.</a> Curr Eye Res. 36 (9), 857-859 (2011).</li><li>Takei, A., Ekstr&#246;m, M., 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=Gene+Transfer+of+Prolyl+Hydroxylase+Domain+2+Inhibits+Hypoxia-inducible+Angiogenesis+in+a+Model+of+Choroidal+Neovascularization.">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 &#38; Lomb</td>
			<td>23060</td>
			<td></td>
		</tr>
		<tr>
			<td>Clayman-Vannas curved scissors</td>
			<td>Bausch &#38; Lomb</td>
			<td>E3383 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Clayman-Vannas straight scissors</td>
			<td>Bausch &#38; 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 &#38; Lomb</td>
			<td>E1815 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tying forceps</td>
			<td>Bausch &#38; Lomb</td>
			<td>63140</td>
			<td></td>
		</tr>
		<tr>
			<td>Minims tetracaine hydrochloride</td>
			<td>Bausch &#38; 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...

Watch this Scientific Journal Video about Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis at JoVE.com

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 ...

The overall goal of this procedure is to induce iris vasculature, which allows direct in-vivo visualization of angiogenic processes. This method can help answer key questions in the ophthalmology field, particularly in neovascular diseases. The main advantage of this technique is that it utilizes albino mice to allow for directing vivo visualization of the blood vessels.Through this method can give insight into ocular neovascular disease. It can also be applied as a general model of angiogenesis, such as testing for new angiogenic therapies. Visual demonstration of this method is critical, as the iris dissection is challenging because of the small size of the mouse eye.With practice, however, it can be mastered. After anesthetizing a P12.5 mouse and preparing it for the procedure, put it in the lateral recumbancy position under a stereoscope and confirm the mask is well positioned. The eye should fill the field of view.Clear focus of the eye is imperative. Next, using small tying forceps in the non-dominant hand, carefully protrude the eye by applying downward pressure to the eyelids, dorsal and ventral to the eye. Keeping a firm hold on the eyelid helps.Before proceeding, photo document the vasculature at 40X. Next, use the stereoscope to locate the corneal limbus. The limbus of albino BALB/c mice can be easily identified as the circular vascular plexus posterior to the cornea.Now, in the dominant hand, hold a 30 gauge beveled needle and use it to perform a small uveal puncture near the posterior limbal limit of the uvea between the ciliary body and the ora serrata. Make the puncture using only the tip of the needle and no more than half the bevel. Avoid rupturing the lens.The puncture should be self-sealing but still allow for intra-ocular injection of study substances through the wound. Next, perform a second uveal puncture in the opposite site of the eye. Optimize the punctures to be at the most dorsal and most ventral positions.Repeat the uveal punctures at the same two positions every four days until P24.5. And prior to repeating the puncture, always check for traumatic cataract. After enucleating the eye and fixing it as described in the text protocol, remove the fixative solution and rinse the eye three times with fresh PBS.Then, place the eye on a smooth, dry surface with the anterior chamber upwards. Next, insert a beveled 30 gauge needle posterior to the limbus to make an entry point. Then, secure the anterior segment with small tying forceps and use Clayman-Vannas straight scissors to cut around the limbus while rotating the eye.Using partial cuts can make the process easier. When completed, the posterior segment of the eye should release. Next, position the anterior segment downward to expose the lens.Remove the lens carefully by grabbing it with small forceps and pulling upwards. Now, position the anterior segment with the cornea facing down and with the cut perpendicular to the field of view. Then, gently grab the tissue posterior to the ciliary body using small tying forceps in the non-dominant hand.Clayman-Vannas straight scissors to trim just anterior to the ciliary body with the goal of removing the trabecular meshwork and isolating the iris. The iris should then move within the cornea. Next, use small tying forceps to grip the cornea and carefully transfer the anterior eye cup to a 96-well plate containing 200 microliters of PBS.Maintain the grip on the cornea and continue dissecting the iris by flushing the tissue with PBS. If needed, a 30 gauge needle can be used to help displace the iris. Now, remove the cornea using the small tying forceps.The isolated iris can then be stored in PBS at four degrees Celsius temporarily until it is experimented upon. Albino BALB/c mice at P12.5 were subjected to uveal punctures repeated every fourth day until P24.5, as described. Due to the transparency of the cornea and the melanin deficiency of BALB/c mice, iris vasculature is readily visible in P16.5 pups and intensifies throughout the duration of the protocol.At P27.5, mice were euthanized and their irises were carefully dissected. The punctures induced a vascular response from the iris by triggering the wound healing system. Immunohistochemistry was performed against PECAM1.The resulting stain showed an increase in overall vasculature in irises of punctured eyes compared to controls. After watching this video, you should have a good understanding of how to induce iris angiogenesis and successfully isolate the mouse iris. Following this procedure, other methods like QBCR can be performed in order to answer additional molecular and cellular questions such as testing the efficacy of pro-angiogenic or anti-angiogenic substances.After its development, this technique has paved the way for researchers to explore neovascular diseases by in-vivo, non-invasive methods using small rodents as a model.

puncture-induced-iris-neovascularization-as-mouse-model-rubeosis

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8.7K Views.  Karolinska Institutet. The overall goal of this procedure is to induce iris vasculature, which allows direct in-vivo visualization of angiogenic processes. This method can help answer key questions in the ophthalmology field, particularly in neovascular diseases. The main advantage of this technique is that it utilizes albino mice to allow for directing vivo visualization of the blood vessels.Through this method can give insight into ocular neovascular disease. It can also be applied as a general model of an...