We thank Marianne Karlsberg, Anne Mari Haapaniemi, P&#228;ivi Partanen and Anne Kankkunen for excellent technical support. This work was funded by the Academy of Finland, P&#228;ivikki and Sakari Sohlberg Foundation, Tampere Tuberculosis Foundation, Finnish Medical Foundation, Pirkanmaa Hospital District Research Foundation and the Tampere University Hospital Research Fund.

The protocol described here has been approved by the National Animal Ethics Committee of Finland (protocol number ESAVI/9520/2020 and ESAVI/6421/04.10.07/2017).
1. Experimental animals and mouse OIR model induction
NOTE: Use time-mated animals, e.g., commonly used C57BL/6J mice, to get pups born on the same day. Use fostering dams, e.g., 129 strain (129S1/SvImJ or 129S3/SvIM) lactating dams, to nurse the pups during and after the induction of hyperoxia. Alternatively, make sure that there are extra lactating dams available in case the nursing dams need to be replaced due to exhaustion. Restrict the litter size to 6-7 pups for each dam when using C57BL/6J mice/dams (if the litters are larger than that the pups tend to have restricted weight gain)16.
<ol>
	<li>Record the weight of the animals before and after hyperoxia induction, and at the time of sacrifice.</li>
	<li>Make sure that there is enough food on the bottom of the cage, so the dams have an easy access to food.</li>
	<li>Add soda lime with color indicator to the bottom of the chamber to absorb excess CO2 when a filtration system is not used.</li>
	<li>Monitor the humidity and temperature inside the chamber and keep the humidity between 40 to 65%. Increase the humidity of the chamber, if needed, by placing dishes with water on bottom of the chamber (e.g., Petri dishes).</li>
	<li>Calibrate the O2 sensor with 100% O2 and normal room air.</li>
	<li>Place the P7 mice into a chamber and set up the O2 level to 75%. Keep the mice in the chamber for 5 days, until P12. Avoid opening the chamber during the hyperoxia induction. Check the gas pressure of the O2 cylinder and replace the cylinder when needed. Monitor the animals during the induction.</li>
	<li>Take the mouse cages out of the chamber and weigh all the pups. Group the pups based on the weight so that each experimental group has similar weight distribution in pups.</li>
</ol>
2. Experimental animals and rat OIR model induction (using semi-closed system)
NOTE: Use time-mated animals to get the pups born on the same day. For rat OIR, use increased litter size, approximately 18 pups/dam, to obtain sufficient NV induction in the rat model. Pool pups from several litters to obtain enough pups to each litter.
<ol>
	<li>Record the weight of the animals before and after induction, and at the time of sacrifice.</li>
	<li>Make sure that there is enough food on the bottom of the cage, so the dams have an easy access to food.</li>
	<li>Add soda lime with color indicator to the bottom of the chamber to absorb excess CO2 when filtration system is not used.</li>
	<li>Monitor the humidity and temperature inside the chamber. Absorb extra humidity (generated from multiple number of rats) by adding silica gel on the bottom of chamber.</li>
	<li>Calibrate the O2 sensor with 100% N2 and normal room air.</li>
	<li>Place the rats into the chamber at P0 (few hours after the birth). Set the O2 level to 50% and connect O2 cylinder to the chamber for 24 h. After that, switch the settings to 10% O2 and connect nitrogen (N2) cylinder to the chamber for 24 h. Continue the 24 h cycling between 50% and 10% O2 levels for 14 days.</li>
	<li>Monitor the gas consumption and the wellbeing of the animals during the study. Open the chamber during the change between 50/10% O2 and add more food and water if needed. Change the cages of the animals to clean ones during the induction.</li>
	<li>Take the rat cages out of the chamber and weigh all the pups. Group the pups based on the weight so that each experimental group has similar weigh distribution in the pups.</li>
</ol>
3. Drug administration (optional)
NOTE: Commonly used drug administration route in OIR is by intravitreal treatment (ivt), at P12-P14 for mice and at P14 for rats. Determine the treatment day based on the experimental setup. When multiple litters of pups are used in experiments, divide the treatment groups to have animals from all the litters. Preferably, inject the drug to only one eye, and keep the contralateral eye as a control.
<ol>
	<li>Weigh the animals and make identification&#160;marks to the tail and/or ear.</li>
	<li>Anesthetize the animal either with injectable anesthesia (for example mixture of ketamine and medetomidine, 30 mg/kg and 0.4 mg/kg for mice) or with inhalation anesthesia (isoflurane at 2-3.5% isoflurane and 200-350 mL/min air flow). Check the depth of the anesthesia by pinching the toes. Keep the animal on a heating pad during the treatment.</li>
	<li>For local anesthesia, apply a drop of analgesic onto the eyelid. Open the eyelid carefully with forceps before performing the ivt, as mice and rats open their eyes around P14. Apply a drop of analgesic (e.g., oxybuprocaine hydrochloride) onto the cornea.</li>
	<li>Apply a drop of iodine before conducting the ivt injection.</li>
	<li>For the ivt injection use a glass syringe with a 33-34 G needle attached. Press the eyelids down and grap the eyeball with forceps. Make the injection posterior to the limbus, approximately in 45&#176; angle needle pointing towards optic nerve.</li>
	<li>Avoid injecting more than 1.0 &#181;L&#160;into the intravitreal space. Keep the needle in place for 30 s after injecting the drug to avoid reflux of the injected solution.</li>
	<li>Examine the eye (e.g., with an ophthalmoscope) for any complications, such as hemorrhages or retinal damage, after removing the needle. Apply antibiotic ointment on top of cornea after the injection. 
	NOTE: The ivt injection volume for mice should be 0.5 &#8211; 1.0 &#181;L.</li>
	<li>Reverse the anesthesia (for example with an &#945;2-antagonist for medetomidine (2.5 mg/kg) and return the pup to the cage. House the litter normally until the end of the study.</li>
</ol>
4. In vivo imaging and electroretinography (optional)
<ol>
	<li>If desired, conduct in vivo imaging on live animals during the follow-up period to record changes that develop in retina during the angiogenic responses. For example, perform fluorescein angiography (FA) or scanning laser confocal microscopy19 to visualize the vasculature (Figure 4). Use spectral domain optical coherence tomography (SD-OCT) to visualize retinal layers in vivo (Figure 4).</li>
	<li>If desired, investigate functional changes in different retinal cell populations after OIR induction by using electroretinography (ERG) (Figure 5).</li>
</ol>
5. Tissue collection and preparation of retinal flat mounts
NOTE: Collect the tissues according to the desired research hypothesis. For mice, collect the samples for example at P12 (to study vaso-obliteration after the hyperoxic phase) or at the hypoxic period (P13-P17). Collect the mouse OIR samples at P17, which is the most common time point for sampling, to detect the peak in NV amount. In rat OIR, collect the samples at P18-P21 to observe the highest amount of NV (Figure 3).
<ol>
	<li>Weigh the animals before sampling.</li>
	<li>To label the retinal vasculature, deeply anesthetized animals can be transcardially perfused with FITC-dextran. (Alternatively, stain the retinal flat mounts with Isolectin later).</li>
	<li>Sacrifice the animals using either overdose of anesthesia drugs (for example mixture of ketamine and medetomidine, 300 mg/kg and 4 mg/kg for mice) or CO2 inhalation.</li>
	<li>Collect the eyes of the animals by grabbing behind the eyeball with curved forceps, cut the tissue around the eyes and lift the eye out from the orbit.</li>
	<li>Incubate the eyeballs in freshly made, filtered 4% paraformaldehyde (in phosphate-buffered saline, PBS) for 1-4 h. Remove the fixative and wash the eyeballs 3 x 10 min with PBS. Dissect the retinas immediately or store them in PBS at +4 &#176;C. 
	CAUTION: Paraformaldehyde is toxic by inhalation, in contact with skin and if swallowed. Please read safety data sheet before working with it. 
	NOTE: Do not apply pressure to the eyeball during the sampling or any phase of the tissue processing in order to avoid retinal detachment, if cross-sections from whole eyeballs are done.</li>
	<li>Prepare retinal flat mounts to quantify the amount of NV and the size of AVAs. Alternatively, process the eyeballs/retinas for histology, or RNA or protein analysis. Dissect the retina under a stereo microscope using micro scissors and forceps.
	<ol>
		<li>Place the eyeball in PBS to keep it moist and puncture the eyeball at limbus with a needle (23G) and cut around limbus with curved micro scissors to remove iris and the cornea.</li>
		<li>Carefully place the tip of the scissors between sclera and retina and cut sclera towards the optic nerve. Do the same to the other side of the eyeball, and carefully cut/tear the sclera until the retinal cup is exposed. Pull the lens out from the retinal cup and add PBS to the cup.</li>
		<li>Remove all the hyaloid vessels, vitreous and debris without damaging the retina. Wash the retina by adding PBS to the retinal cup. Perform four incisions (at 12, 3, 6 and 9 o&#8217;clock) to the retina with straight micro scissors to make a flower-like structure. Optionally, make the cuts with surgical blade prior mounting the samples. Lift the retina using a soft paintbrush to a well-plate for staining.</li>
	</ol>
	</li>
	<li>Label the retinal vasculature using Isolectin B4 which stains the surface of endothelial cells (if the animals were not perfused with FITC-dextran). Incubate the retinas in blocking buffer (10% NGS + 0.5% Triton in TBS) for 1 h and wash with 1% NGS + 0.1% Triton in TBS for 10 min. Incubate the retinas with fluorescent dye conjugated Isolectin B4 (5-10&#181;g/ml) in 1% NGS + 0.1% Triton in TBS overnight at +4 &#176;C while protected from the light. 
	NOTE: If desired, label other cells such as inflammatory cells and pericytes using specific antibodies.</li>
	<li>Wash the retinas 3x for 10 min with 1% NGS + 0.1% Triton in TBS and lift retinas on a microscopic slide, inner retina facing upwards. Carefully spread out the retina using soft paintbrush and remove any remaining hyaloid vessels or debris. Add mounting medium to a cover slip and place it on top of the retina. Store retinas at +4 &#176;C and protect from light.</li>
</ol>
6. Analysis of the flat mounts
<ol>
	<li>Take images of the retinal flat mounts using a fluorescence microscope with 10x objective. Focus to the superficial vascular plexus and to the preretinal neovascularization. Make a tile scan image to capture the whole retina and merge the tile scans</li>
	<li>Quantify the images by measuring the AVAs, area of NV and total retinal area using an image processing program (see Table of Materials).
	<ol>
		<li>Draw the AVAs and total retinal area using a free hand drawing tool and select the neovascular areas using a selection tool. The software measures the regions of interest in pixels, and the AVA and NV areas (expressed in pixels) can be used to calculate their percentage in relation to the total retinal area. Also, some software tools are available for quantifying NV. 
		NOTE: Recently, an open-source, fully automated programs&#160;for the quantification of key values of OIR images using deep learning neural networks have been introduced and provide a reliable tool for reproducible quantification of retinal AVA and NV (e.g., https://github.com/uw-biomedical-ml/oir/tree/bf75f9346064f1425b8b9408ab792b1531a86c64)20,21.</li>
	</ol>
	</li>
	<li>If using antibodies for immunohistochemical detection of individual cell populations, quantify the number of stained cells (such as microglia, Figure 2B) from the retinal flat mounts by hand or by automated image analysis systems if desired.</li>
</ol>
7. Statistics
<ol>
	<li>Analyze normally distributed data by Student&#8217;s t test or One-Way ANOVA followed by Dunnett&#8217;s or Tukey&#8217;s multiple comparisons test, as appropriate. Use nonparametric tests like Mann-Whitney U test or Kruskal Wallis test for non-normally distributed data. Consider differences statistically significant at the P &#60; 0.05 level.</li>
</ol>

The authors Maria V&#228;h&#228;tupa, PhD, Niina J&#228;&#228;skel&#228;inen, Marc Cerrada-Gimenez, PhD, and Rubina Thapa are employees of Experimentica Ltd.
The author Giedrius Kalesnykas, PhD, is an employee (President and Chief Executive Officer) and shareholder of Experimentica Ltd. that offers contract research services employing preclinical OIR models used in this Article.
Tero J&#228;rvinen, M.D., PhD, and Hannele Uusitalo-J&#228;rvinen, M.D., PhD, have nothing to disclose.

The severity of disease phenotype is dependent on both the strain and even vendor in both mouse and rat OIR models23. This suggests that there is a wide genotypic variability in the pathology development. In general, pigmented rodents develop more severe phenotype than the albino ones. For example, the retinal vasculature of albino BALB/c revascularizes rapidly after hyperoxia and does not develop NV at all24. Similarly, in rats, pigmented Brown Norway rats show more severe...

Reliable and reproducible experimental models are needed to study the pathology behind angiogenic eye diseases and to develop novel therapeutics to these devastating diseases. Pathological angiogenesis is the hallmark for wet age-related macular degeneration (AMD) and for many ischemic retinal diseases among them retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO)1,2,3,4. Human and rodent retinas follow a similar pattern of development, as both human and rodent retina are among the last tissues that are vascularized. Before the retinal vasculature has completely developed, retina receives its nutrient supply from hyaloid vasculature, which, in turn, regresses when the retinal vasculature starts to develop1,2. In human, retinal vascular development is completed before birth, whereas in rodents the growth of retinal vasculature occurs after birth. Since the retinal vascular development occurs postnatally in rodents, it provides an ideal model system to study the angiogenesis2,3. The newborn rodents have an avascular retina that develops gradually until complete vascular retina development is achieved by the end of third postnatal week4. The growing blood vessels of neonatal mouse are plastic, and they undergo regression during hyperoxia stimulus5.
ROP is the leading cause for childhood blindness in Western countries, as it affects almost 70% of the premature infants with birthweight under 1,250 g6,7. ROP occurs in premature infants who are born before retinal vessels complete&#160;their normal growth. ROP progresses in two phases: in Phase I, preterm birth delays the retinal vascular growth where after in phase II, the unfinished vascularization of the developing retina causes hypoxia, which induces the expression of angiogenic growth factors that stimulate new and abnormal blood vessel growth8. The OIR model has been a widely used model to study the pathophysiology of ROP and other ischemic retinopathies as well as to test novel drug candidates2,3,9. It is widely considered as a reproducible model for carrying out proof-of-concept studies for potential antiangiogenic drugs for ocular as well as non-ocular diseases. The two rodent models i.e., mouse and rat OIR differ in their model induction and disease phenotype. The rat model mimics ROP phenotype more accurately, but the mouse model provides a more robust, fast and reproducible model for retinal neovascularization (NV). In the mouse model, NV develops to the central retina. This pathological read-out is important in pharmacologic efficacy studies for many ischemic retinopathies, such as PDR, RV and exudative AMD as well as for non-ocular, angiogenic diseases such as cancer. Moreover, availability of genetically manipulated (transgenic and knockout) mice makes the mouse OIR model a more popular option. However, neither mouse nor rat OIR model creates retinal fibrosis, which is typical in human diseases.
The understanding that high oxygen levels contribute to the development of ROP in 1950s10,11 led to the development of animal models. The first studies about the effect of oxygen on&#160;retinal vasculature were done in 195012,13,14 and until the 1990s there were many refinements to the OIR model. The research by Smith et al. in 1994 set a standard for the&#160;current mouse OIR model that separates hyaloidopathy from retinopathy15. A wide adoption of the method to quantify vaso-obliteration and pathological NV by Connor et al. (2009) further increased its popularity16. In this model, mice are placed at 75% oxygen (O2) for 5 days at P7, followed by 5 days in normoxic conditions. Hyperoxia from P7 to P12 causes retinal vasculature to regress in central retina. Upon return to normoxic conditions, avascular retina becomes hypoxic (Figure 1A). Due to the hypoxic stimuli of the avascular central retina, some of the retinal blood vessels sprout towards the vitreous, forming preretinal NV, called preretinal tufts2,3. These tufts are immature, and hyperpermeable. The amount of NV peaks at P17, after which it regresses. The retina is fully revascularized and NV is fully regressed by P23 - P25 (Figure 2A)2,3.
The rat OIR model (using varying levels of O2) was first described in the 1990s showing that varying O2 levels at 80% and 40% cause more pronounced NV than under 80% O2 constant exposure17. Later it was discovered that the intermittent hypoxia model, where O2 is cycled from hyperoxia (50%) to hypoxia (10-12 %), causes even more NV than the 80/40% O2 model18. In the 50/10% model, rat pups are exposed to 50% for 24 hours, followed by 24 hours in 10% O2. These cycles are continued until P14, when the rat pups are returned to normoxic conditions (Figure 1B). As in human ROP patients, in the rat model the avascular areas develop to the periphery of retina because of immature retinal vascular plexus (Figure 3).
In both models, the main parameters that are usually quantified are the size of AVA and NV. These parameters are typically analyzed from retinal flat mounts where the endothelial cells are labeled4,16. Previously the amount of preretinal NV was evaluated from retinal cross sections by counting blood vessel or vascular cell nuclei extending to vitreous above the inner limiting membrane. The major limitation of this approach is that it is not possible to quantify the AVAs.

<table><tbody><tr><td>33 gauge, Small Hub RN Needle</td><td>Hamilton Company</td><td>7803-05, 10mm, 25&amp;deg;, PS4</td><td>For intravitreal injection</td></tr><tr><td>Adobe Photoshop</td><td>Adobe Inc.</td><td></td><td>For image analysis</td></tr><tr><td>Air pump air100</td><td>Eheim GmbH &amp;amp; Co. KG.</td><td>143207</td><td>For inhalation anaesthesia</td></tr><tr><td>Anaesthesia unit 410 AP</td><td>Univentor Ltd.</td><td>2360309</td><td>For inhalation anaesthesia</td></tr><tr><td>AnalaR NORMAPUR Soda lime</td><td>VWR International Ltd</td><td>22666.362</td><td>For CO2 control during model induction</td></tr><tr><td>Attane Vet 1000 mg/g</td><td>VET MEDIC ANIMAL HEALTH OY</td><td>vnr 17 05 79</td><td>For inhalation anaesthesia</td></tr><tr><td>Brush</td><td></td><td></td><td>For preparation of flat mounts</td></tr><tr><td>Carbon dioxide gas</td><td></td><td></td><td>For sacrifice</td></tr><tr><td>Celeris D430 ERG system</td><td>Diagnosys LLC</td><td>121</td><td>For &lt;em&gt;in vivo&lt;/em&gt; ERG</td></tr><tr><td>Cell culture dishes</td><td>Greiner Bio-One International GmbH</td><td>664 160</td><td>For preparation of flat mounts</td></tr><tr><td>Cepetor Vet 1 mg/mL</td><td>VET MEDIC ANIMAL HEALTH OY</td><td>vnr 08 78 96</td><td>For anaesthesia</td></tr><tr><td>Cover slips</td><td>Thermo Fisher Scientific</td><td>15165452</td><td>For preparation of flat mounts</td></tr><tr><td>O2 Controlled InVivo Cabinet, Aninal Filtrarion System and Dehumidifier</td><td>Coy Laboratory Products</td><td></td><td>Closed system for disease model induction, optional for semi-closed system</td></tr><tr><td>E702 O2 sensor</td><td>BioSphenix, Ltd.</td><td>E207, 1801901</td><td>For oxygen level measurement</td></tr><tr><td>Envisu R2200 Spectral Domain Optical Coherence Tomograph (SD-OCT)</td><td>Bioptigen, Inc.</td><td>BPN000668</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Eye spears</td><td>Beaver-Visitec International, Inc.</td><td>0008685</td><td>For intravitreal injection and &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Flexilux 600LL Cold light source</td><td>Mikron</td><td>11140</td><td>For intravitreal injection or tissue collection</td></tr><tr><td>Fluorescein sodium salt</td><td>Merck KGaA</td><td>F6377-100G</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Gas Exhaust unit (+Double 3-way valve, mouse and rat face masks, UNOsorb filter)</td><td>UNO Roestvaststaal BV</td><td>GEX 17015249</td><td>For inhalation anaesthesia</td></tr><tr><td>Glass syringe, Model 65 RN</td><td>Hamilton Company</td><td>7633-01</td><td>For intravitreal injection</td></tr><tr><td>HRA2 Retina angiograph (FA)</td><td>Heidelberg Engineering GmbH</td><td>Spec-KT-05488</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Isolectin GS-IB4, Alexa Fluor 488 Conjugate</td><td>Thermo Fisher Scientific</td><td>I21411</td><td>For labeling retinal vasculature on flat mounts</td></tr><tr><td>Ketaminol Vet 50 mg/mL</td><td>Intervet International B.V.</td><td>vnr 51 14 85</td><td>For anaesthesia</td></tr><tr><td>Medicinal Oxygen gas</td><td></td><td></td><td>For disease model induction</td></tr><tr><td>Mice C57BL/6JRj</td><td>Janvier Labs</td><td></td><td>Also other strains possible</td></tr><tr><td>Microscope slides</td><td>Thermo Fisher Scientific</td><td>J1800AMNZ</td><td>For preparation of flat mounts</td></tr><tr><td>Minims Povidone Iodine 5% (unit)</td><td>Bausch &amp;amp; Lomb U.K Limited</td><td>vnr 24 11 304</td><td>For intravitreal injection</td></tr><tr><td>Nitrogen gas</td><td></td><td></td><td>For disease model induction (rat)</td></tr><tr><td>Oftan Chlora 10 mg/g</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 55 01 11</td><td>For intravitreal injection</td></tr><tr><td>Oftan Metaoksedrin 100 mg/ml</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 55 03 43</td><td>For &lt;em&gt;in vivo&lt;/em&gt; ERG</td></tr><tr><td>Oftan Obucain 4 mg/ml</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 55 03 50</td><td>For intravitreal injection</td></tr><tr><td>Oftan Tropicamid 5 mg/ml</td><td>Santen Pharmaceutical Co., Ltd.</td><td>vnr 04 12 36</td><td>For &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>ProOx Model 110 O2 controller and animal chamber</td><td>BioSphenix, Ltd.</td><td>803</td><td>For disease model induction, semi-closed system, optional for closed system</td></tr><tr><td>ProOx Model P360 O2 controller and animal chamber</td><td>BioSphenix, Ltd.</td><td>538</td><td>For disease model induction, semi-closed system, optional for closed system</td></tr><tr><td>Rats CD(SD)</td><td>Charles River Laboratories</td><td></td><td>Also other strains possible</td></tr><tr><td>Revertor 5 mg/mL</td><td>VET MEDIC ANIMAL HEALTH OY</td><td>vnr 13 04 97</td><td>For anaesthesia reversal</td></tr><tr><td>Silica gel</td><td></td><td></td><td>For humidity control during model induction</td></tr><tr><td>Systane Ultra 10ml</td><td>Alcon</td><td>Tamro 2050250</td><td>For hydration of the eye</td></tr><tr><td>Systane Ultra unit 0.7ml</td><td>Alcon</td><td>Tamro 2064871</td><td>For hydration of the eye</td></tr><tr><td>Transfer pipette</td><td>Thermo Fisher Scientific</td><td>1343-9108</td><td>For preparation of flat mounts</td></tr><tr><td>VENTI-Line VL 180 PRIME Drying oven</td><td>VWR</td><td>VL180S 170301</td><td>For drying silica gel</td></tr><tr><td>VisiScope SZT350 Stereomicroscope</td><td>VWR</td><td>481067</td><td>For intravitreal injection or tissue collection</td></tr></tbody></table>

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.

The main outcome of the model is the vascular phenotype: the size of AVAs and the amount of NV. In the mouse OIR model, the vaso-obliteration occurs in the central retina (Figure 2A), while in the rat model it develops in the periphery, i.e., similar to human ROP22 (Figure 3A). This is because the superficial vascular plexus has already developed when mice are exposed to hyperoxia, whereas in the rat model the retina is avascular at the t...

Watch this Scientific Journal Video about Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents at JoVE.com

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

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 ...

oxygen-induced-retinopathy-model-for-ischemic-retinal-diseases

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8.3K Views.  Tampere University & Tampere University Hospital. 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.

Video: Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Neuroscience

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

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, ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

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 ...

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, ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

Biology

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, ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

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Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

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

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

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

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

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