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Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model
In This Article
Summary
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.
Abstract
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.
Introduction
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
Protocol
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
- Use mice with a low.......
Representative Results
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 (<.......
Discussion
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.......
Acknowledgements
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).
....Materials
| Name | Company | Catalog Number | Comments |
| 1 mL sterile syringe | Solarbio | YA0550 | For preparation of retinal flat mounts and intraperitoneal injection |
| 1× Phosphate buffered saline (PBS) | Transgen Biotech |  FG701-01 | For preparation of retinal flat mounts |
| 2 ml Microcentrifuge Tube | Corning | MCT-200-C | For preparation of retinal flat mounts |
| 48 Well Clear TC-Treated Multiple Well Plates | Corning | 3548 | For preparation of retinal flat mounts |
| Adhesive microscope slides | Various | For preparation of retinal flat mounts | |
| Adobe Photoshop CC 2019 | Adobe Inc. | For image analysis | |
| Carbon dioxide gas | Various | For sacrifice | |
| Cover slide | Various | For preparation of retinal flat mounts | |
| Curved forceps | World Precision Instruments | 14127 | For preparation of retinal flat mounts |
| DAPI staining solution | Abcam | ab228549 | For labeling nucleus on retinal flat mounts |
| Dissecting microscope | Olmpus | SZ61 | For preparation of retinal flat mounts |
| Fluorescein sodium | Sigma-Aldrich | F6377 | For in vivo imaging |
| Fluorescent Microscope | Â Zeiss | AxioImager.Z2 | For acquisition of fluorescence images of retinal flat mounts |
| Fluoromount-G Mounting media | SouthernBiotech | Â 0100-01 | For preparation of retinal flat mounts |
| Hydroxypropyl Methylcellulose | Maya | 89161 | For in vivo imaging |
| Isolectin B4 594 antibody | Invitrogen | I21413 | For labeling retinal vasculature on retinal flat mounts |
| Mice C57/BL6J | GemPharmatech of Jiangsu Province | For OIR model induction | |
| Micro dissecting scissors-straight blade | World Precision Instruments | 503242 | For preparation of retinal flat mounts |
| No.4 straight forceps | World Precision Instruments | Â 501978-6 | For preparation of retinal flat mounts |
| Normal donkey serum | Abcam | ab7475 | For preparation of retinal flat mounts |
| O2 sensor | Various | For monitoring the level of O2 | |
| OxyCycler | Biospherix | A84XOV | For OIR model induction |
| Paraformaldehyde (PFA) | Sigma | P6148-1KG | For tissue fixation |
| Pentobarbital sodium | Various | For anesthesia | |
| Soda lime | Various | For absorbing excess CO2 in the oxygen chamber | |
| SPECTRALIS HRA+OCT | Heidelberg | HC00500002 | For in vivo imaging |
| SPSS Statistics 22.0 | IBM | For statistical analysis | |
| Tansference decloring shaker | Kylin-Bell | ZD-2008 | For preparation of retinal flat mounts |
| Tissue culture dish (Low attachment) | Corning | 3261-20EA | For preparation of retinal flat mounts |
| Transfer pipettes | Various | For preparation of retinal flat mounts | |
| Triton X-100 | Sigma-Aldrich | Â SLBW6818 | For preparation of retinal flat mounts |
| Tropicamide | Various | For in vivo imaging | |
| ZEN Imaging Software | ZEISS | For image acquisition and export |
References
- Vavvas, D. G., Miller, J. W. Chapter 26 - Basic Mechanisms of Pathological Retinal and Choroidal Angiogenesis. Retina (Fifth Edition). 1, 562-578 (2013).
- Selvam, S., Kumar, T., Fruttiger, M. Retinal vasculature develop....
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