Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

Summary

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

Abstract

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Introduction

Studying the flow dynamics of the blood cells in the retinal and choroidal circulation is imperative to understanding the pathogenesis of potentially vision-threatening ocular diseases and other ocular inflammatory conditions. However, the conventional angiography techniques, which involve the binding of fluorescent dyes to plasma proteins, do not provide any information regarding the dynamics of the erythrocytes or leukocytes¹. The erythrocyte retinal flow dynamics are important for studying metabolically efficient circulation in the retina, and the leukocyte flow dynamics, for understanding the cell migration, recognition, adhesion and destru....

Protocol

The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of SingHealth, Singapore and are in accordance to the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Labeling of Erythrocytes and Leukocytes with Fluorescent Dyes

1. Preparation of reagents
   1. Prepare ICG (1.5 mg/mL) by dissolving 3 mg of ICG in 1800 µL of sterilized distilled water. Add 200 µL of 10x phosphate buffered saline (PBS).
   2. Prepare 40% 1x PBS by adding 4 mL of 1x PBS to 6 mL of sterilized dist....

Results

Erythrocytes labeled with 1.5% ICG were visualized in the retinal circulation of C57BL/6J mice (wild type). Both 1% and 5% hematocrit of 1.5% ICG labeled erythrocytes were distinguishable in the retinal circulation. However, the individual labeled cells were more clearly visualized with 1% hematocrit of 1.5% ICG labeled erythrocytes (Figure 1). In 5% hematocrit, due to the large number of labeled cells in the retinal vessels, it was not possible to mark the i.......

Discussion

Studying the hemodynamics in the retinal and choroidal circulation is vital for understanding the pathophysiology of many ocular diseases. The blood flow dynamics in the retinal circulation can be studied by Fourier-domain optical coherence tomography (FD-OCT), laser speckle flowgraphy (LSFG) and retinal oximetry. Although these methods use different approaches to study the total blood flow in the retinal circulation^40,41,42

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cardiogreen polymethine dye (Indocyanine green)	Sigma Aldrich	12633-50MG
Fluorescein 100 mg/mL	Novartis	U1705A/H-1330292
10x Phosphate-buffered saline (PBS) Ultra Pure Grade	1st BASE	BUF-2040-10X1L
Bovine serum albumin	Sigma Aldrich	A7906-100G
Microtainer tubes with K2E (K2EDTA) - EDTA concentration - 1.8 mg/mL of blood	BD, USA	REF 365974
Histopaque 1077 solution	Sigma Aldrich	10771
Centrifuge 5810 R	Eppendorf	05-413-401
Microcentrifuge tubes 2 mL	Axygen	MCT-200-C-S
Vortex mixer	Insta BioAnalytik pte. ltd	FINE VORTEX
Shaker incubator	Lab Tech
Ceva Ketamine injection (Ketamine hydrochloride 100 mg/mL)	Ceva	KETALAB03
ILIUM XYLAZIL-20 (Xylazine hydrochloride 20 mg/Ml)	Troy Laboratories PTY. Limited	LI0605
1% Mydriacyl 15 mL (Tropicamide 1%)	Alcon Laboratories, Inc. USA	NDC 0998-0355-15
2.5% Mydfrin 5 mL (Phenylephrine hydrochloride 2.5%)	Alcon Laboratories, Inc. USA	NDC 0998-0342-05
Terumo syringe with needle 1 cc/mL Tuberculin	Terumo (Philippenes) Corporation, Philippines	SS-01T2613
Vidisic Gel 10 G	Dr. Gerhard Mann, Chem.-Pharm, Fabrik Gmbh, Berlin, Germany
Alcohol swabs	Assure medical disposables	7M-004-L-01
Confocal laser scanning angiography system (Heidelberg Retina Angiograph 2)	Heidelberg Engineering, GmbH, Heidelberg, Germany
Hiedelberg Spectralis Viewing Module software, v4.0	Heidelberg Engineering, GmbH, Heidelberg, Germany
Fluorescent microscope	ZEISS	Model: axio imager z1