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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 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 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 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.

Protocol

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.

  1. Record the weight of the animals before and after hyperoxia induction, and at the time of sacrifice.
  2. Make sure that there is enough food on the bottom of the cage, so the dams have an easy access to food.
  3. Add soda lime with color indicator to the bottom of the chamber to absorb excess CO2 when a filtration system is not used.
  4. 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).
  5. Calibrate the O2 sensor with 100% O2 and normal room air.
  6. 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.
  7. 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.

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.

  1. Record the weight of the animals before and after induction, and at the time of sacrifice.
  2. Make sure that there is enough food on the bottom of the cage, so the dams have an easy access to food.
  3. Add soda lime with color indicator to the bottom of the chamber to absorb excess CO2 when filtration system is not used.
  4. 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.
  5. Calibrate the O2 sensor with 100% N2 and normal room air.
  6. 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.
  7. 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.
  8. 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.

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.

  1. Weigh the animals and make identification marks to the tail and/or ear.
  2. 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.
  3. 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.
  4. Apply a drop of iodine before conducting the ivt injection.
  5. 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° angle needle pointing towards optic nerve.
  6. Avoid injecting more than 1.0 µL into the intravitreal space. Keep the needle in place for 30 s after injecting the drug to avoid reflux of the injected solution.
  7. 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 – 1.0 µL.
  8. Reverse the anesthesia (for example with an α2-antagonist for medetomidine (2.5 mg/kg) and return the pup to the cage. House the litter normally until the end of the study.

4. In vivo imaging and electroretinography (optional)

  1. 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).
  2. If desired, investigate functional changes in different retinal cell populations after OIR induction by using electroretinography (ERG) (Figure 5).

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).

  1. Weigh the animals before sampling.
  2. To label the retinal vasculature, deeply anesthetized animals can be transcardially perfused with FITC-dextran. (Alternatively, stain the retinal flat mounts with Isolectin later).
  3. 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.
  4. 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.
  5. 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 °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.
  6. 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.
    1. 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.
    2. 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.
    3. 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’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.
  7. 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 (5-10µg/ml) in 1% NGS + 0.1% Triton in TBS overnight at +4 °C while protected from the light.
    NOTE: If desired, label other cells such as inflammatory cells and pericytes using specific antibodies.
  8. 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 °C and protect from light.

6. Analysis of the flat mounts

  1. 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
  2. Quantify the images by measuring the AVAs, area of NV and total retinal area using an image processing program (see Table of Materials).
    1. 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 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.
  3. 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.

7. Statistics

  1. Analyze normally distributed data by Student’s t test or One-Way ANOVA followed by Dunnett’s or Tukey’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 < 0.05 level.

Results

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...

Discussion

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...

Disclosures

The authors Maria Vähätupa, PhD, Niina Jääskeläinen, 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 Järvinen, M.D., PhD, and Hannele Uusitalo-Järvinen, M.D., PhD, have nothing to disclose.

Acknowledgements

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

Materials

NameCompanyCatalog NumberComments
33 gauge, Small Hub RN NeedleHamilton Company7803-05, 10mm, 25°, PS4For intravitreal injection
Adobe PhotoshopAdobe Inc.For image analysis
Air pump air100Eheim GmbH & Co. KG.143207For inhalation anaesthesia
Anaesthesia unit 410 APUniventor Ltd.2360309For inhalation anaesthesia
AnalaR NORMAPUR Soda limeVWR International Ltd22666.362For CO2 control during model induction
Attane Vet 1000 mg/gVET MEDIC ANIMAL HEALTH OYvnr 17 05 79For inhalation anaesthesia
BrushFor preparation of flat mounts
Carbon dioxide gasFor sacrifice
Celeris D430 ERG systemDiagnosys LLC121For in vivo ERG
Cell culture dishesGreiner Bio-One International GmbH664 160For preparation of flat mounts
Cepetor Vet 1 mg/mLVET MEDIC ANIMAL HEALTH OYvnr 08 78 96For anaesthesia
Cover slipsThermo Fisher Scientific15165452For preparation of flat mounts
O2 Controlled InVivo Cabinet, Aninal Filtrarion System and DehumidifierCoy Laboratory ProductsClosed system for disease model induction, optional for semi-closed system
E702 O2 sensorBioSphenix, Ltd.E207, 1801901For oxygen level measurement
Envisu R2200 Spectral Domain Optical Coherence Tomograph (SD-OCT)Bioptigen, Inc.BPN000668For in vivo imaging
Eye spearsBeaver-Visitec International, Inc.0008685For intravitreal injection and in vivo imaging
Flexilux 600LL Cold light sourceMikron11140For intravitreal injection or tissue collection
Fluorescein sodium saltMerck KGaAF6377-100GFor in vivo imaging
Gas Exhaust unit (+Double 3-way valve, mouse and rat face masks, UNOsorb filter)UNO Roestvaststaal BVGEX 17015249For inhalation anaesthesia
Glass syringe, Model 65 RNHamilton Company7633-01For intravitreal injection
HRA2 Retina angiograph (FA)Heidelberg Engineering GmbHSpec-KT-05488For in vivo imaging
Isolectin GS-IB4, Alexa Fluor 488 ConjugateThermo Fisher ScientificI21411For labeling retinal vasculature on flat mounts
Ketaminol Vet 50 mg/mLIntervet International B.V.vnr 51 14 85For anaesthesia
Medicinal Oxygen gasFor disease model induction
Mice C57BL/6JRjJanvier LabsAlso other strains possible
Microscope slidesThermo Fisher ScientificJ1800AMNZFor preparation of flat mounts
Minims Povidone Iodine 5% (unit)Bausch & Lomb U.K Limitedvnr 24 11 304For intravitreal injection
Nitrogen gasFor disease model induction (rat)
Oftan Chlora 10 mg/gSanten Pharmaceutical Co., Ltd.vnr 55 01 11For intravitreal injection
Oftan Metaoksedrin 100 mg/mlSanten Pharmaceutical Co., Ltd.vnr 55 03 43For in vivo ERG
Oftan Obucain 4 mg/mlSanten Pharmaceutical Co., Ltd.vnr 55 03 50For intravitreal injection
Oftan Tropicamid 5 mg/mlSanten Pharmaceutical Co., Ltd.vnr 04 12 36For in vivo imaging
ProOx Model 110 O2 controller and animal chamberBioSphenix, Ltd.803For disease model induction, semi-closed system, optional for closed system
ProOx Model P360 O2 controller and animal chamberBioSphenix, Ltd.538For disease model induction, semi-closed system, optional for closed system
Rats CD(SD)Charles River LaboratoriesAlso other strains possible
Revertor 5 mg/mLVET MEDIC ANIMAL HEALTH OYvnr 13 04 97For anaesthesia reversal
Silica gelFor humidity control during model induction
Systane Ultra 10mlAlconTamro 2050250For hydration of the eye
Systane Ultra unit 0.7mlAlconTamro 2064871For hydration of the eye
Transfer pipetteThermo Fisher Scientific1343-9108For preparation of flat mounts
VENTI-Line VL 180 PRIME Drying ovenVWRVL180S 170301For drying silica gel
VisiScope SZT350 StereomicroscopeVWR481067For intravitreal injection or tissue collection

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Oxygen induced RetinopathyIschemic Retinal DiseasesNeovascular Retinal DiseasesPathological AngiogenesisHypoxia ModelPreclinical StudiesRetinopathy Of PrematurityDiabetic RetinopathyAge related Macular DegenerationAnimal ModelP7 MiceOxygen Level CalibrationIVT InjectionRetinal ImagingSpectral Domain Optical Coherence Tomography

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