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Abstract
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Medicina

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

Published: July 03, 2017
doi:
10.3791/55495
, , , , ,
1National Healthcare Group Eye Institute,Tan Tock Seng Hospital, 2Singapore Eye Research Institute (SERI),Singapore National Eye Center, 3School of Material Science and Engineering,Nanyang Technological University, 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University Health Systems,National University of Singapore, 5Ophthalmology Academic Clinical Research Program,DUKE-NUS Graduate Medical School

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 destruction in various inflammatory conditions2. There are several fluorescent molecules used in the identification and characterization of various cell types3. The hemodynamics of the blood cells can be measured by staining them with the appropriate fluorescent dyes and applying the proper imaging techniques4.

The presence of inflammatory responses in intraocular diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) involve the accumulation of lymphocytes in the diseased area5,6. Tracking the immune cells in the tissues can help understand the complex events involved in the mechanism of disease pathogenesis. Radioactive isotopes like 51Cr and 125I were used as cell tracers in early studies. These dyes are toxic and affect the cell viability. Although the radioactive markers 3H and 14C are less toxic to the cells, due to their lower emission energies, it is difficult to detect their signals in the system7,8. A number of fluorochrome dyes were introduced to overcome the potential problems associated with radioactive markers and track lymphocyte migration in vitro using fluorescent microscopy and flow cytometry9,10. Hoechst 33342 and thiazole orange are DNA binding fluorescent dyes, which are used to track lymphocytes in vivo. Hoechst 33342 binds to AT-rich regions in the DNA, is membrane permeable, retains fluorescent signals for 2 – 4 days and is resistant to quenching9,10. The disadvantages of Hoechst 33342 and thiazole orange are the inhibition of lymphocyte proliferation11 and the short half-life, respectively9.

Calcein-AM, fluorescein diacetate (FDA), 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM), 5-(and-6)-carboxyfluorescein diacetate (CFDA), and 5-(and-6)-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) are the cytoplasmic fluorescent dyes used for lymphocyte migration studies. However, FDA, CFDA and CFDA-AM have lower retention in the cells9. BCECF-AM reduces the proliferative response and influences the chemotaxis and superoxide production9,12. Calcein-AM is a fluorescent dye and useful for short-term in vivo lymphocyte migration studies. It emits strong fluorescent signals, does not interfere with most of the cellular functions and retains fluorescent signals for up to 3 days12,13. Fluorescein isothiocyanate (FITC) and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) are covalent coupling fluorescent dyes, which are used for lymphocyte migration studies. FITC exhibits no effect on the cell viability and has a stronger affinity with B lymphocytes than T lymphocytes14,15. The CFDA-SE labeled lymphocytes can be tracked in vivo for more than 8 weeks and up to 8 cell divisions9,16. C18 DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiO (3,3'-dioctadecyloxacarbocyanine perchlorate), Paul Karl Horan (PKH)2, PKH3, and PKH26 are membrane-inserting fluorescent lipophilic carbocyanine dyes used to label leukocytes and erythrocytes. C18 Dil and DiO exhibit higher signals when incorporated into the cell membrane and are relatively non-toxic12,17. PKH2, PKH3 and PKH26 labeled cells exhibit a good retention of the fluorescent signals with less toxicity18,19,20,21,22. However, PKH2 down regulates the CD62L expression and reduces the lymphocyte viability23.

Most of the above-mentioned studies have been performed for tracking the lymphocyte migration and proliferation in the lymphatics and studying the labeled erythrocytes in the non-ocular circulation. There are very few studies applying the labeling techniques to study the blood cells in the ocular circulation. Application of scanning laser ophthalmoscopy (SLO) has a great advantage in studying the labeled cells in the retinal and choroidal circulation in vivo by fundus angiography24. There are several fluorescent dyes, such as ICG, acridine orange, FITC, sodium fluorescein, and CFDA that are used to study the leukocytes in the retinal circulation by SLO25,26,27,28,29,30,31,32,33,34. The phototoxicity and carcinogenicity of acridine orange26,27, the interference of FITC with the cellular activity, and the requirement of an intravascular contrast agent for the resolution of the retinal and choroidal blood vessels limits their application in in vivo animal experiments29. Sodium fluorescein and ICG are non-toxic, approved by the Food and Drug Administration, and safe for testing on humans32,35. Most of the flow dynamic studies are related to the labeling of the leukocytes or erythrocytes and its visualization in the retinal and choroidal blood vessels36,37,38,39. Here, we describe a standardized protocol of ICG labeling of the erythrocytes, sodium fluorescein labeling of the leukocytes, and tracking the visualized labeled cells in the mouse retinal circulation using SLO.

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 Preparation of reagents Prepare ICG (1.5 mg/mL) by dissolving 3 mg of ICG in 1800 µL of sterilized dist…

Representative 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 circulation40,41,42<sup…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

The research project was funded under New Investigator grant from the National Medical Research Council (NMRC), Singapore. The team will like to acknowledge the research training provided to Dr Agrawal at Institute of Ophthalmology (IoO), University College London (UCL) under National Medical Research Council (NMRC) Overseas Research Training Fellowship from November 2012 till October 2014 under mentorship of Prof. David Shima. Dr. Agrawal acquired the concept and skills for labelling the cells and live imaging in Dr. Shima's lab. The team will hence like to acknowledge supervision and guidance during the training fellowship from Prof. David Shima, Pro.f Kenith Meissner, Dr. Peter Lundh and Dr. Daiju Iwata.

Materials

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 2mL Axygen MCT-200-C-S
Vortex mixer Insta BioAnalytik pte. ltd FINE VORTEX
Shaker incubator Lab Tech
Ceva Ketamine injection (Ketamine hydrochloride 100mg/mL) Ceva KETALAB03
ILIUM XYLAZIL-20 (Xylazine hydrochloride 20mg/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 1cc/mL Tuberculin Terumo (Philippenes) Corporation, Philippines SS-01T2613
Vidisic Gel 10G 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
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Referencias

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Tags

ErythrocytesLeukocytesRetinal CirculationMouseFluorescent Dye LabelingICGSodium FluoresceinScanning Laser OphthalmoscopeBlood Cell Flow DynamicsRetinal Diseases
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Agrawal, R., Balne, P. K., Tun, S. B. B., Sia Wey, Y., Khandelwal, N., Barathi, V. A. Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation. J. Vis. Exp. (125), e55495, doi:10.3791/55495 (2017).
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