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&#176;, 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 &#38; 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 in vivo 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 in vivo imaging</td>
		</tr>
		<tr>
			<td>Eye spears</td>
			<td>Beaver-Visitec International, Inc.</td>
			<td>0008685</td>
			<td>For intravitreal injection and in vivo 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 in vivo 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 in vivo 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 &#38; 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 in vivo 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 in vivo 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...

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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 ...

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8.1K 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

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

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. ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...