Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography

Summary

Currently, fluorescein angiography (FA) is the preferred method for identifying leakage patterns in animal models of choroidal neovascularization (CNV). However, FA does not provide information about vascular morphology. This protocol outlines the use of indocyanine green angiography (ICGA) to characterize different lesion types of laser-induced CNV in mouse models.

Abstract

Age-related macular degeneration (AMD) is a leading cause of blindness among older individuals, and its prevalence is rapidly increasing due to the aging population. Choroidal neovascularization (CNV) or wet AMD, which accounts for 10%-20% of all AMD cases, is responsible for an alarming 80%-90% of AMD-related blindness. Current anti-VEGF therapies show suboptimal responses in approximately 50% of patients. Resistance to anti-VEGF treatment in CNV patients is often associated with arteriolar CNV, while responders tend to have capillary CNV. While fluorescein angiography (FA) is commonly used to assess leakage patterns in wet AMD patients and animal models, it does not provide information about CNV vascular morphology (arteriolar CNV vs. capillary CNV). This protocol introduces the use of indocyanine green angiography (ICGA) to characterize lesion types in laser-induced CNV mouse models. This method is crucial for investigating the mechanisms and treatment strategies for anti-VEGF resistance in wet AMD. It is recommended to incorporate ICGA alongside FA for comprehensive assessment of both leakage and vascular features of CNV in mechanistic and therapeutic studies.

Introduction

Age-related macular degeneration (AMD) is a prevalent condition that leads to severe vision loss in older individuals¹. In the United States alone, the number of AMD patients is projected to double, reaching nearly 22 million by 2050, compared to the current 11 million. Globally, the estimated number of AMD cases is expected to reach a staggering 288 million by 2040².

Choroidal neovascularization (CNV), also known as "wet" or neovascular AMD, can have devastating effects on vision due to the formation of abnormal blood vessels beneath the central retina. This leads to hemorrhaging, ret....

Protocol

The animal experiments conducted in this study received approval from the Institutional Animal Care and Use Committees (IACUC) at Baylor College of Medicine. All procedures were carried out in compliance with the guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Young (7-9 weeks) and old (12-16 months) C57BL/6J male and female mice were used for the present study. The animals were obtained from a commercial source (se.......

Discussion

This study demonstrated the use of indocyanine green angiography (ICGA) to identify the vascular morphology of arteriolar and capillary choroidal neovascularization (CNV) in mouse models with laser-induced CNV. The hemoglobin-bound and infrared light properties of indocyanine green (ICG) dye enabled the detection of CNV morphology, which is challenging to achieve using fluorescein angiography (FA), the current method employed by the research community.

The first critical step in the protocol i.......

Materials

Name	Company	Catalog Number	Comments
32-G Insulin Syringe	MHC Medical Products	NDC 08496-3015-01
Alexa Fluor 488 goat anti-rabbit secondary antibody	Invitrogen	A11008
Anti-α smooth muscle Actin antibody	Abcam	ab5694
Bovine Serum Albumin	Santa Cruz Biotechnology, Inc.	sc-2323
C57BL/6J mice (7-9 weeks)	The Jackson Laboratory	Strain #:000664
Fluorescein Sodium Salt	Sigma-Aldrich	MFCD00167039
Gaymar T Pump Heat Therapy System	Gaymar	TP-500	Water circulation heat pump for mouse recovery after imaging
GenTeal Gel	Genteal	NDC 58768-791-15	Clear lubricant eye gel
GS-IB4 Alexa-Flour 568 conjugate	Invitrogen	I21412
Heidelberg Eye Explorerer	Heidelberg Engineering, Germany	HEYEX2
Indocyanine Green	Pfaultz & Bauer	I01250
Ketamine	Vedco Inc.	NDC 50989-996-06
Paraformaldehyde	Acros Organics	416785000
Proparacaine Hydrochloride Ophthalmic Solution (0.5%)	Sandoz	NDC 61314-016-01
Spectralis Multi-Modality Imaging System Heidelberg Engineering, Germany SPECTRALIS HRA+OCT Tropicamide ophthalmic solution (1%) Bausch & Lomb NDC 24208-585-64 for dilation of pupils GenTeal Gel Genteal NDC 58768-791-15 clear lubricant eye gel Ketamine Vedco Inc NDC 50989-996-06 Xylazine Lloyd Laboratories NADA 139-236 Acepromazine Vedco Inc NDC 50989-160-11 32-G Needle Steriject PRE-32013 1-ml syringe BD 309659 Indocyanine Green Pfaltz & Bauer I01250	Heidelberg Engineering, Germany	SPECTRALIS HRA+OCT
Triton X-100	Sigma-Aldrich	X100-1L
Tropicamide ophthalmic solution (1%)	Bausch & Lomb	NDC 24208-585-64	For dilation of pupils
Xylazine	Lloyd Laboratories	NADA 139-236