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In This Article

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

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

  1. 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

NameCompanyCatalog NumberComments
1 mL sterile syringeSolarbioYA0550For preparation of retinal flat mounts and intraperitoneal injection
1× Phosphate buffered saline (PBS)Transgen Biotech FG701-01For preparation of retinal flat mounts
2 ml Microcentrifuge TubeCorningMCT-200-CFor preparation of retinal flat mounts
48 Well Clear TC-Treated Multiple Well PlatesCorning3548For preparation of retinal flat mounts
Adhesive microscope slidesVariousFor preparation of retinal flat mounts
Adobe Photoshop CC 2019Adobe Inc.For image analysis
Carbon dioxide gasVariousFor sacrifice
Cover slideVariousFor preparation of retinal flat mounts
Curved forcepsWorld Precision Instruments14127For preparation of retinal flat mounts
DAPI staining solutionAbcamab228549For labeling nucleus on retinal flat mounts
Dissecting microscopeOlmpusSZ61For preparation of retinal flat mounts
Fluorescein sodiumSigma-AldrichF6377For in vivo imaging
Fluorescent Microscope ZeissAxioImager.Z2For acquisition of fluorescence images of retinal flat mounts
Fluoromount-G Mounting mediaSouthernBiotech 0100-01For preparation of retinal flat mounts
Hydroxypropyl MethylcelluloseMaya89161For in vivo imaging
Isolectin B4 594 antibodyInvitrogenI21413For labeling retinal vasculature on retinal flat mounts
Mice C57/BL6JGemPharmatech of Jiangsu ProvinceFor OIR model induction
Micro dissecting scissors-straight bladeWorld Precision Instruments503242For preparation of retinal flat mounts
No.4 straight forcepsWorld Precision Instruments 501978-6For preparation of retinal flat mounts
Normal donkey serumAbcamab7475For preparation of retinal flat mounts
O2 sensorVariousFor monitoring the level of O2
OxyCyclerBiospherixA84XOVFor OIR model induction
Paraformaldehyde (PFA)SigmaP6148-1KGFor tissue fixation
Pentobarbital sodiumVariousFor anesthesia
Soda limeVariousFor absorbing excess CO2 in the oxygen chamber
SPECTRALIS HRA+OCTHeidelbergHC00500002For in vivo imaging
SPSS Statistics 22.0IBMFor statistical analysis
Tansference decloring shakerKylin-BellZD-2008For preparation of retinal flat mounts
Tissue culture dish (Low attachment)Corning3261-20EAFor preparation of retinal flat mounts
Transfer pipettesVariousFor preparation of retinal flat mounts
Triton X-100Sigma-Aldrich SLBW6818For preparation of retinal flat mounts
TropicamideVariousFor in vivo imaging
ZEN Imaging SoftwareZEISSFor image acquisition and export

References

  1. Vavvas, D. G., Miller, J. W. Chapter 26 - Basic Mechanisms of Pathological Retinal and Choroidal Angiogenesis. Retina (Fifth Edition). 1, 562-578 (2013).
  2. Selvam, S., Kumar, T., Fruttiger, M. Retinal vasculature develop....

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