We thank all the members from our lab and Ophthalmic Animal Laboratory of Zhongshan Ophthalmic Center for their technical assistance. We also thank Prof. Chunqiao Liu for experimental support. This work was supported by grants from the National Natural Science Foundation of China (NSFC: 81670872; Beijing, China), the Natural Science Foundation of Guangdong Province, China (Grant No.2019A1515011347), and High-level hospital construction project from State Key Laboratory of Ophthalmology at Zhongshan Ophthalmic Center (Grant No. 303020103; Guangzhou, Guangdong Province, China).

All procedures involving the use of mice were approved by the animal experimental ethics committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, China (authorized number: 2020-082), and in accordance with the approved guidelines of Animal Care and Use Committee of Zhongshan Ophthalmic Center and the Association Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Induction of mouse OIR model
<ol>
	<li>Use mice with a low...

The susceptibility of mice to OIR is affected by many factors. The pups of different genetic background and strains cannot be compared. In BALB/c albino mice, vessels regrow into the VO area rapidly with significant reduced neovascular tufts38, which bring some difficulties to the research. In C57BL/6 mice, there is increased photoreceptor damage when compared to BALB/cJ mouse strain39,40. The same goes for different types of transgenic mi...

Retinal neovascularization (RNV), which is defined as a state where new pathologic vessels originate from existing retinal veins, usually extends along the inner surface of the retina and grows into the vitreous (or subretinal space under some conditions)1. It is a hallmark and common feature of many ischemic retinopathies, including retinopathy of prematurity (ROP), retinal vein occlusion (RVO), and proliferative diabetic retinopathy (PDR)2.
Numerous clinical and experimental observations have indicated that ischemia is the main cause of retinal neovascularization3,4. In ROP, neonates are exposed to high-level oxygen in closed incubators to increase the survival rates, which is also an important driver for the arrest of vascular growth. After the treatment is done, the retinas of newborns experience a relatively hypoxic period5. Other situations are seen in the occlusion of central or branch retinal veins in RVO and damage of retinal capillaries is also observed which is caused by microangiopathy in PDR2. Hypoxia further increases the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) through the hypoxia-induced factor-1&#945; (HIF-1&#945;) signaling pathway which in turn guide vascular endothelial cells to grow into the hypoxic area and form new vessels6,7.
ROP is a kind of vascular proliferative retinopathy in preterm infants and a leading cause of childhood blindness8,9, which is characterized by retinal hypoxia, retinal neovascularization and fibrous hyperplasia10,11,12. In the 1950s, researchers found that high concentration of oxygen can significantly improve the respiratory symptoms of premature infants13,14. As a result, oxygen therapy was increasingly used in premature infants at that time15. However, concurrent with the widespread use of oxygen therapy in preterm infants, the incidence of ROP increased year by year. Since then, researchers have linked oxygen to ROP, exploring various animal models to understand the pathogenesis of ROP and RNV16.
In human, most retinal vasculature development is completed before birth while in rodents the retinal vasculature develops after birth, providing an accessible model system to study angiogenesis in the retinal vasculature2. With the continuous progress of the research, oxygen-induced retinopathy (OIR) models have become major models for mimicking pathological angiogenesis resulting from ischemia. There are no specific animal species in the study of the OIR model and the model has been developed in various animal species, including kitten17, rat18, mouse19, beagle puppy20, and zebrafish21. All of the models share the same mechanism by which they are exposed to hyperoxia during early retinal development and then returned to the normoxic environment. Smith et al. observed that exposing mouse pups to hyperoxia from P7 for 5 days induced an extreme form of vessel regression in the central retina and bringing them back to the room air at P12 gradually triggered neovascular tufts, which grew toward the vitreous body19. This was a standardized OIR mouse model also named as Smith model. Connor et al. further optimized the protocol and provided a universally applicable method to quantify the area of VO (vaso-obliteration) and NV (neovascularization) in 2009, which increased the acceptance and utilization of the model22. OIR mouse model is still the most widely used model now because of its small size, fast reproduction, clear genetic background, good repeatability, and high success rate.
In mice, retinal vascularization starts after birth with the ingrowth of vessels from the optic nerve head into the inner retina toward the ora serrata. During normal retinal development, the first retinal vessels sprout from the optic nerve head around birth, forming an expanding network (the primary plexus) that reaches the periphery around postnatal day 7(P7)23. Then the vessels start to grow into the retina to form a deep layer, penetrate the retina, and establish a laminar network around the inner nuclear layer (INL) as in human24. By the end of the third postnatal week (P21), deeper plexus development is almost completed. For the OIR mouse model, vascular occlusion always appears in the central retina because of the rapid degeneration of a large number of immature vascular networks in the central region during hyperoxia exposure. So, the growth of pathological neovascularization also occurs in the mid-peripheral retina, which is the boundary of the non-perfusion area and the vascular area. However, human retinal vessels have almost formed before birth. As for premature infants, the peripheral retina is not completely vascularized when exposed to hyperoxia25,26. So vascular occlusion and neovascularization mainly appear in the peripheral retina27,28. Despite these differences, the mouse OIR model closely recapitulates the pathologic events that occur during ischemia-induced neovascularization.
The induction of the OIR model can be divided into two phases29: in phase 1 (hyperoxia phase), retinal vascular development is arrested or retarded with occlusion and regression of blood vessels as a result of the decline in VEGF and the apoptosis of endothelial cells24,30; in phase 2 (hypoxia phase), the retinal oxygen supply will become insufficient under room air conditions29, which is essential for neural development and homeostasis19,31. This ischemic situation usually results in unregulated, abnormal neovascularization.
Currently, the commonly used modeling method is alternating high/low oxygen exposure: Mothers and their pups are exposed to 75% oxygen for 5 days at P7 followed by 5 days in room air till P17 demonstrated comparable results22, which is the endpoint of OIR mouse model induction. (Figure 1). In addition to simulating ROP, this ischemia-mediated pathological neovascularization can also be used to study other ischemic retinal diseases. The main measurements of this model include quantifying the area of VO and NV, which are analyzed from retinal flat mounts by immunofluorescence staining or FITC-dextran perfusion. Each mouse can be studied only once because of the lethal operation. At present, there are few methods to observe dynamic changes of retinal vasculature continuously during the process of vascular regression and pathologic angiogenesis32. In this paper, we provide a detailed protocol of OIR model induction, analysis of retinal flat mounts as well as a workflow of fluorescein fundus angiography (FFA) on mice which would be helpful to gain a more comprehensive understanding of vascular dynamic changes during two phases of the OIR mouse model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL sterile syringe</td>
			<td>Solarbio</td>
			<td>YA0550</td>
			<td>For preparation of retinal flat mounts and intraperitoneal injection</td>
		</tr>
		<tr>
			<td>1&#215; Phosphate buffered saline (PBS)</td>
			<td>Transgen Biotech</td>
			<td>&#160;FG701-01</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>2 ml Microcentrifuge Tube</td>
			<td>Corning</td>
			<td>MCT-200-C</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>48 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Adhesive microscope slides</td>
			<td>Various</td>
			<td></td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Adobe Photoshop CC 2019</td>
			<td>Adobe Inc.</td>
			<td></td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>Carbon dioxide gas</td>
			<td>Various</td>
			<td></td>
			<td>For sacrifice</td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Various</td>
			<td></td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>World Precision Instruments</td>
			<td>14127</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>DAPI staining solution</td>
			<td>Abcam</td>
			<td>ab228549</td>
			<td>For labeling nucleus on retinal flat mounts</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olmpus</td>
			<td>SZ61</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Fluorescein sodium</td>
			<td>Sigma-Aldrich</td>
			<td>F6377</td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>&#160;Zeiss</td>
			<td>AxioImager.Z2</td>
			<td>For acquisition of fluorescence images of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting media</td>
			<td>SouthernBiotech</td>
			<td>&#160;0100-01</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Hydroxypropyl Methylcellulose</td>
			<td>Maya</td>
			<td>89161</td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>Isolectin B4 594 antibody</td>
			<td>Invitrogen</td>
			<td>I21413</td>
			<td>For labeling retinal vasculature on retinal flat mounts</td>
		</tr>
		<tr>
			<td>Mice C57/BL6J</td>
			<td>GemPharmatech of Jiangsu Province</td>
			<td></td>
			<td>For OIR model induction</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors-straight blade</td>
			<td>World Precision Instruments</td>
			<td>503242</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>No.4 straight forceps</td>
			<td>World Precision Instruments</td>
			<td>&#160;501978-6</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Abcam</td>
			<td>ab7475</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>O2 sensor</td>
			<td>Various</td>
			<td></td>
			<td>For monitoring the level of O2</td>
		</tr>
		<tr>
			<td>OxyCycler</td>
			<td>Biospherix</td>
			<td>A84XOV</td>
			<td>For OIR model induction</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148-1KG</td>
			<td>For tissue fixation</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Various</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Soda lime</td>
			<td>Various</td>
			<td></td>
			<td>For absorbing excess CO2 in the oxygen chamber</td>
		</tr>
		<tr>
			<td>SPECTRALIS HRA+OCT</td>
			<td>Heidelberg</td>
			<td>HC00500002</td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>SPSS Statistics 22.0</td>
			<td>IBM</td>
			<td></td>
			<td>For statistical analysis</td>
		</tr>
		<tr>
			<td>Tansference decloring shaker</td>
			<td>Kylin-Bell</td>
			<td>ZD-2008</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Tissue culture dish (Low attachment)</td>
			<td>Corning</td>
			<td>3261-20EA</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Various</td>
			<td></td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;SLBW6818</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Tropicamide</td>
			<td>Various</td>
			<td></td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>ZEN Imaging Software</td>
			<td>ZEISS</td>
			<td></td>
			<td>For image acquisition and export</td>
		</tr>
	</tbody>
</table>

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.

In the OIR mouse model, the most important and basic result is the quantification of the VO and NV area. After living in the hyperoxia environment for 5 days from P7, the central retina of the pups showed the largest non-perfusion area. Under the stimulation of hypoxia in another 5 days, retinal neovascularization was gradually produced which fluoresced more intensely than surrounding normal vessels. After P17, the fluorescence signal of pathological neovascularization regressed rapidly as the remodeling of the retina (<...

Watch this Scientific Journal Video about Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model at JoVE.com

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

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), ...

This protocol can help researchers easily separate the retinas of mice and observe the dynamic changes in the retinal vasculature of the OIR mouse model. The main advantages of this method are preservation of the integrity of the separated retinas and allowing each mouse to serve as its own control, which makes the results more comparable. Researchers trying this method for the first time may struggle while taking the five orientation imagings because it is a bit difficult to place pups in the correct position.To collect the tissue for retinal whole mounting, use curved scissors to disconnect the eyeballs from the orbital tissue of a euthanized C57-Black/6 pup. Then, insert curved forceps into the posterior part of the eyeball, clamping the optic nerve and quickly lifting the eye out from the orbit. Place the eyeballs in pre-cooled PBS to remove any hair and blood from the surface.When all of the eyeballs have been collected, transfer the cleaned eyeballs to a two-milliliter microcentrifuge tube containing 4%paraformaldehyde, and incubate for 15 minutes at room temperature and 12 to 15 revolutions per minute. At the end of the incubation, add a drop of PBS to the center of a culture dish under a dissecting microscope, and place one eyeball into the drop. Holding the eyeball with a pair of forceps, use a one-milliliter syringe needle to carefully puncture the cornea at the corneal limbus.Insert the tip of a pair of scissors into the puncture, and cut the cornea along the corneal limbus, being careful not to cut the retina. Use forceps to remove the iris and lens, and place the remaining eyecup in 4%paraformaldehyde for a 45-minute incubation at room temperature on the shaker. At the end of the incubation, place the fixed eyecup in a fresh drop of PBS at the center of a new culture dish.Holding the eyecup with one pair of forceps, use a second pair of forceps to gently separate the retina and sclera layers. Place the tip of the scissors between the retina and sclera layers, and cut the sclera toward the optic nerve. Peel the sclera off of the retina to obtain the retinal cup.Using forceps, release the connection between the radial hyaloid vessels and peripheral retina. Use a two-milliliter pipette with the tip cut off to transfer the retinal cup into one well of a 48-well plate for three five-minute washes in room temperature PBS on the shaker. After the last wash, incubate the cup in 1%Triton X-100 and 5%normal donkey serum in PBS overnight at four degrees Celsius.To label the retinal vasculature with isolectin B4, incubate the retina in one well of a 48-well plate in 400 microliters 0.1%normal donkey serum supplemented with isolectin B4-594 overnight at 4 degrees Celsius on a shaker. If necessary, another primary antibody can be added at the same time. On the following day, wash the retina with three 20-minute washes in 0.1%PBST on the shaker.After the last wash, incubate the retina with an appropriate high-affinity secondary antibody for one hour at room temperature before labeling the sample with DAPI for 20 to 25 minutes. At the end of the incubation, wash the retina with three 30-minute washes in 0.1%PBST at room temperature with shaking. After the last wash, transfer the retinal cup to a clean slide with the opening facing up, and use the microscope to cut the retina radially at the three, six, nine, and 12 o'clock positions from the periphery toward the center, up to one to 1.5 millimeters from the optic nerve head.Rinse the retina three times with a few drops of PBS per wash before using air-laid paper to dry and flatten the retina. Add a drop of mounting medium to the center of the coverslip until the diameter of the droplet covers half of the coverslip. Then quickly place the coverslip mounting medium side down onto the outspread retina.Once all of the retinal flat mounts have been prepared, the samples can be imaged by confocal fluorescence microscopy. For in vivo imaging, add 20 microliters of mydriatic eye drops to achieve long-lasting pupil dilation. After five minutes, place the pups in a stable position on a small heating pad in front of the imaging device.To maintain moisture in the corneas, regularly add artificial tears throughout the imaging procedure. Next, click Infrared Fundus Imaging on the device to adjust the optic nerve head to the center of the screen. Intraperitoneally inject 150 microliters of 0.5%fluorescein sodium salt solution, and immediately click the FA and Injection buttons on the touch panel of the imaging device to start the timer.After three minutes, when the blood circulation of the retina has entered the venous phase, acquire the first image of the central retina. Move the lens of the imaging device horizontally to the nasal side of the eye until the optic nerve head is located at the midpoint of one side of the image acquisition area, and take the second image. Continue to move the lens and acquire images of the temporal, superior, and inferior retina in the same manner.To process the retinal images in an appropriate imaging processing program, open the File menu and click New to create a new canvas with a black background. Open the image of the central retina. Then click File to add a second image.Adjust the opacity of the second image to 60%and move and resize the image until the two images overlap. Click Switch Between Free Transform and Warp Modes, and make subtle adjustments to the vessels if necessary. Then set the opacity of the second image back to 100%Select both images, and click Auto-Blend Layers.Check Panorama as the blend method, and also check the boxes for Seamless Tones and Colors and Content Aware Fill Transparent Areas. Click OK to complete the stitching of the first two images. Next, add the third image, and stitch this image to the first two images as demonstrated until all five of the images from a single retina have been stitched.When all of the images from the analysis have been processed, use the Crop Tool to crop the FFA images to a uniform size to facilitate observation of the changes in the retinal vasculature dynamics at different developmental time points. After five days of exposure to hyperoxia, the central retinas of postnatal day 12 pups demonstrate a large non-perfusion area. Upon transfer to five days of hypoxic conditions, the retinas gradually neovascularize, exhibiting an increase in fluorescence signal stronger than that observed in the surrounding normal vessels.After postnatal day 17, the pathological neovascularization regresses rapidly and the retina demonstrates a completely normal appearance by postnatal day 25. A good repeatability and stability of oxygen-induced retinopathy model can be achieved by controlling the litter size and postnatal weight gain of the pups. As observed, the vaso-obliteration typically peaks at postnatal day 12 and disappears at postnatal day 25 while the neovascularization peaks at postnatal day 17 and regresses by postnatal day 25.In postnatal day 15 to 25 oxygen-induced retinopathy mice, the retinal blood vessel diameter increases and becomes highly torturous compared to that observed for the retinas of normal mice. The forceps in the left hand mainly play in a supporting role. Take care not to squeeze the eyeball, which can deform or rupture it.

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

Abstract