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), ...