Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

After its development, the oxygen-induced retinopathy model has become widely used in preclinical studies of neovascular retinal diseases. It has been used as a model to study pathological angiogenesis in response to hypoxia. The main advantage of this model is that it induces a robust neurovascular response that is reproducible and easily quantifiable.

This model can be used as a preclinical model for studying the effects of possible therapeutic candidates. It is considered as a relevant model for several human diseases, such as retinopathy of prematurity, diabetic retinopathy, and wet age-related macular degeneration. Record the weight of the animals before and after hypoxia induction and at the time of sacrifice.

Make sure that there is enough food on the bottom of the cage and add soda lime with color indicator to the bottom of the chamber to absorb excess carbon dioxide when a filtration system is not used. Monitor the humidity and temperature inside the chamber, keeping the humidity between 40 and 65%If needed, increase the humidity of the chamber by placing dishes with water on the bottom. Calibrate the oxygen sensor with normal room air and 100%oxygen.

Then place the P7 mice into a chamber and set the oxygen level to 75%Keep the mice in the chamber for five days until P12, monitoring the animals during the induction. When finished, remove the mice from the chamber. Weigh the animals and make identification marks on the tail or ear.

After the animal is anesthetized, perform a toe pinch. A small reflex is acceptable. Keep the animal on a heating pad during treatment.

Use a glass syringe with a 33 to 34 gauge needle for the IVT injection. Apply a drop of iodine, then press the eyelid down and grab the eyeball with forceps. Make the injection posterior to the limbus, approximately at a 45 degree angle pointing towards the optic nerve.

Keep the needle in place for 30 seconds after injecting the drug to avoid reflux of the injected solution. Examine the eye for any complications such as hemorrhages or retinal damage after removing the needle. Apply antibiotic ointment on top of the eye after the injection.

If desired, conduct in vivo imaging on live animals during the followup period to record changes that develop in the retina during the angiogenic responses. Use spectral domain optical coherence tomography to visualize retinal layers in vivo. Collect the eyes of the animals by cutting the tissue around the eye, grabbing behind the eyeball with curved forceps and lifting the eye out from the orbit.

Incubate the eyeballs in freshly made filtered 4%paraformaldehyde for one to four hours. Remove the fixative and wash the eyeballs three times with PBS for 10 minutes per wash. Dissect the retinas immediately or store them in PBS at four degrees Celsius.

Prepare retinal flat mounts to quantify the amount of neovascularization and the size of avascular areas or AVAs. Dissect the retina under a stereo microscope using micro scissors and forceps. Place the eyeball in PBS to keep it moist and puncture it at the limbus with a 23 gauge needle, then cut around the limbus with curved micro scissors to remove the iris and the cornea.

Carefully place the tip of the scissors between RPE and neural retina and cut the RPE choroid sclera complex towards the optic nerve. Do the same to the other side of the eyeball. Then gently cut or tear the tissue until the retinal cup is exposed.

Pull the lens out from the retinal cup and add PBS to the cup. Remove all of the hyaloid vessels, vitreous, and debris without damaging the retina. Make four incisions to the retina with straight micro scissors to create a flower-like structure.

Use a soft paintbrush or a transfer pipette to transfer it to a well plate for staining. Label the retinal vasculature with isolectin B4 which stains the surface of endothelial cells. Incubate the retinas in blocking buffer for one hour and wash them with 1%NGS and 0.1%Triton in TBS for 10 minutes.

Incubate the retinas with 5 to 10 micrograms per milliliter fluorescent dye conjugated isolectin B4 overnight at four degrees Celsius while protected from light. On the next day, wash the retinas three times with 1%NGS and 0.1%Triton in TBS for 10 minutes per wash. Place the retinas on a microscope slide inner retina facing upwards.

Then carefully spread out the retina using a soft paintbrush and remove any remaining hyaloid vessels or debris. Add mounting medium on top of the sample and place a coverslip on top of the slide. Store the retinas at four degrees Celsius protected from light.

Image the retinal flat mounts using a fluorescence microscope with a 10X objective. Focus on the superficial vascular plexus and the pre-retinal neovascularization. Make a tile scan image to capture the whole retina and merge the tile scans.

Quantify the images by measuring the AVAs, area of neovascularization, and total retinal area using an image processing program. Draw the AVAs and total retinal area using a freehand drawing tool and select the neovascular areas using a selection tool. In the mouse oxygen-induced retinopathy model, the vaso-obliteration occurs in the central retina.

While in the rat model, it develops in the periphery similar to human ROP. Pre-retinal neovascularization develops near the avascular areas, specifically the central retina in the mouse and periphery in rats. Histological analysis using either cross-sections or flat mounts can be done to evaluate morphological changes in OIR retinas or the presence of cell types of interest, for example, inflammatory cells.

Optionally, non-invasive in vivo imaging can be conducted during the OIR followup period. Retinal and hyaloid vasculature can be visualized with fluorescein angiography and spectral domain optical coherence tomography can be used to evaluate structural changes in the retina. Functional changes can be measured by electroretinography.

Aflibercept, a soluble VEGF-Trap, inhibits both neovascular and physiological revascularization in OIR. OIR eyes injected at P14 with a high dose of aflibercept had even bigger retinal AVAs than untreated eyes, suggesting that aflibercept also blocks physiological retinal revascularization driven by hypoxia. When attempting this protocol, remember to consider the proper litter size for your experiment in advance and monitor the wellbeing of the dams and the pups in the oxygen chamber.

Take into account that postnatal weight gain affects the outcome in the model. Retinal samples obtained from this model can be used for analytical methods, such as immunohistochemistry, gene or protein expression analysis, and to measure the effects of potential therapeutic candidates on the retina at the molecular level.