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 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.
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.
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'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
2. Puncture Procedure under Surgical Stereoscope
3. Noninvasive in Vivo Monitoring
4. Post-Operative Care
5. Eye Enucleation
6. Iris Dissection Procedure under Stereoscope
7. Possible Experimental Read-Outs
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 triggering the wound healing system (Figure 1). Due to transparency of the cornea and melanin-deficient BALB/c mice, increased iris vasculature is readily visible from experimental day 4 (P16.5) and intensifies throughout the duration of the protocol. Immunohistochemistry images for PECAM-1 denote an increase in overall vasculature in irises of punctured eyes compared to controls (Figure 2).
Figure 1: Noninvasive monitoring of puncture-induced iris angiogenesis. Representative images of day 0, 4, 8, 12, and 15 of control and punctured eyes. Iris vascular response is evident from day 4 onwards in the punctured eyes. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 2: Iris vascular response in puncture-induced eyes. Illustrative images of PECAM-1 staining from day 15 irises, displayed as full view and magnified area (square), from control and punctured eyes. Scale bar = 400 µm (top); 200 µm (bottom). Note the increase of vascular branches in punctured irises. Please click here to view a larger version of this figure.
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 iris12,13.
The cornea is avascular and possesses a peculiar collagen structure resulting in transparency. In line with this, angiography of the anterior segment of the eye for visualization of iris vasculature is achievable by direct visualization of iris blood vessels with in vivo noninvasive methods13,14. Previous animal models of iris neovascularization relied on large-eyed animals where complex surgical interventions were performed15,16,17, or by intraocular injection of specific angiogenic substances18,19. In this method, the use of pigment deficient BALB/c mice enables direct observation of the iris vasculature in vivo, and allows noninvasive quantification of the iris neovascularization in mice, thus providing a new tool to study in vivo angiogenesis.
Furthermore, the presented method for mouse iris dissection allows analysis of puncture-induced iris neovascularization with vascular specific markers. Here, iris vessels were illustratively stained with PECAM-1 antibodies and visualized by fluorescence microscopy. Free-floating, whole-mount immunostaining protocols are easily achieved and fairly routine. In addition, perfusion methods for blood vessels can be performed in this model. Vascular specific staining grants assessment of iris blood vessel structure and thus quantification, by user-based or software analysis, of the iris neovasculature9. As previously described8,20, the illustration of iris neovasculature was assessed at day 15 post-puncture-induction. Nevertheless, effects on iris vascularization can be observed as early as day 4 post-puncture-induction, suggesting that evaluation of the puncture-induced iris neovascularization model could be carried out at different time points, based on specific experimental requirements.
It is noteworthy to state that this protocol presents some challenges, particularly related to the size of a mice eyes relative to other animal model. Extreme care should be taken while executing the uveal puncture procedure, to avoid lens touches or damage to the eye. Observation of ocular condition is paramount, and animals displaying experimental-related cataracts or drop in ocular pressure should be excluded and appropriately euthanized. Mouse irises are extremely fragile and somewhat tacky. Proper dissection and isolation of the tissue presents another critical step for the protocol. A correct separation from the trabecular meshwork is necessary for displacement of the iris from the anterior chamber. Moreover, due to fragility, iris isolation requires fixation of the tissue, which impairs downstream molecular analysis. Though, a molecular analysis of an unfixed whole-eye has been successfully applied to the presented method8.
In summary, the described protocol presents a novel puncture-induced iris neovascularization mouse model. This model has the advantage of allowing in vivo noninvasive direct visualization of angiogenesis. In addition, the use of small rodents renders the presented model attractive for studies of rubeosis iridis, as it avoids the use of large-eyed animals and accompanied ethical restriction. Furthermore, the procedure can be combined with injection of pro- or anti-angiogenic substances through the self-sealing puncture, enhancing its usefulness as a new model of in vivo angiogenesis.
The authors have nothing to disclose.
The authors thank Linnea Tankred and Diana Rydholm for animal husbandry.
Bonn eye scissors | Bausch & Lomb | 23060 | |
Clayman-Vannas curved scissors | Bausch & Lomb | E3383 C | |
Clayman-Vannas straight scissors | Bausch & Lomb | E3383 S | |
Objective adapter for camera | Handcrafted | N/A | Or any system that allows adapting a camera to the microscope |
Heating Pad 100-110 watts | Non Applicable | N/A | Available in pet/veterinarian stores |
Hypodermic 30g beveled needle | KDM GmBH germany | 911914 | |
Iphone 4S | Apple | Non Applicable | Or other high resolution image acquistion device |
Isoflurane | Baxter | KDG 9623 | |
McPherson tying forceps | Bausch & Lomb | E1815 S | |
Micro tying forceps | Bausch & Lomb | 63140 | |
Minims tetracaine hydrochloride | Bausch & Lomb | N/A | 1 % (w/v) Eye Drops |
Neutral-buffered formalin | Bioreagens | 0018-40 | |
Normal saline solution | Fresenius Kabi | 210352 | 0.9 % (w/v) NaCl in injectable water |
Phosphate-buffered saline | ThermoFisher Scientific | 10010023 | Balanced and buffered PBS pH 7.4 |
Petri dish 10 cm | Starstedt | 83.3902 | |
Petri dish 3 cm | Starstedt | 83.3900 | |
Safe Seal Tube 2.0 mL | Starstedt | 72.685.200 | Or any eppendorf style tubes |
TC plate 96-well | Starstedt | 83.3924 | |
Transfer pipette 3.5 mL | Starstedt | 86.1171 | Or any other Pasteur pipette style |
Univentor 400 anesthesia unit | Univentor Limited | N/A | Or equivalent flow regulator with induction chamber and mask for volatile anesthesia |
Wild M650 surgical microscope | Wild Heerbrugg | N/A | Or other surgical or magnifying stereoscope |