title: A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.
abstract: We have developed a method to label and image myeloid cells infiltrating the mouse retina and choroid in vivo, using a single depot injection of indocyanine green dye (ICG). This was demonstrated using the following ocular models of inflammation and angiogenesis: endotoxin-induced uveitis, experimental autoimmune uveoretinitis and laser-induced choroidal neovascularization model. A near-infrared scanning ophthalmoscope was used for in vivo imaging of the eye, and flow cytometry was used on blood and spleen to assess the number and phenotype of labelled cells. ICG was administered 72 h before the induction of inflammation to ensure clearance from the systemic circulation. We found that in vivo intravenous administration failed to label any leukocytes, whereas depot injection, either intraperitoneal or subcutaneous, was successful in labelling leukocytes infiltrating into the retina. Progression of inflammation in the retina could be traced over a period of 14 days following a single depot injection of ICG. Additionally, bright-field microscopy, spectrophotometry and flow cytometric analysis suggest that the predominant population of cells stained by ICG are circulating myeloid cells. The translation of this approach into clinical practice would enable visualization of immune cells in situ. This will not only provide a greater understanding of pathogenesis, monitoring and assessment of therapy in many human ocular diseases but might also open the ability to image immunity live for neurodegenerative disorders, cardiovascular disease and systemic immune-mediated disorders.
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK University College London, Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: Dawn A
authors.initials: DA
authors.lastname: Sim
authors.affiliation: University College London, Institute of Ophthalmology, London EC1V 9EL, UK Academic Unit of Ophthalmology, School of Clinical Sciences, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Chu
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK University College London, Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: Senthil
authors.initials: S
authors.lastname: Selvam
authors.affiliation: University College London, Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Powner
authors.affiliation: University College London, Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: Sidath
authors.initials: S
authors.lastname: Liyanage
authors.affiliation: Academic Unit of Ophthalmology, School of Clinical Sciences, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: David A
authors.initials: DA
authors.lastname: Copland
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK University College London, Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: Pearse A
authors.initials: PA
authors.lastname: Keane
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK University College London, Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: Adnan
authors.initials: A
authors.lastname: Tufail
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Egan
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK University College London, Institute of Ophthalmology, London EC1V 9EL, UK.
authors.forename: James W B
authors.initials: JW
authors.lastname: Bainbridge
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK Academic Unit of Ophthalmology, School of Clinical Sciences, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Lee
authors.affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK Academic Unit of Ophthalmology, School of Clinical Sciences, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Dick
authors.affiliation: University College London, Institute of Ophthalmology, London EC1V 9EL, UK m.fruttiger@ucl.ac.uk.
authors.forename: Marcus
authors.initials: M
authors.lastname: Fruttiger
mesh_terms: D000818:Animals; D001327:Autoimmune Diseases / Q000209:etiology / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D058948:Cell Tracking / Q000379:methods*; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte; D002829:Choroid / Q000098:blood supply / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D020256:Choroidal Neovascularization / Q000209:etiology / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D004195:Disease Models, Animal; D018450:Disease Progression; D004731:Endotoxins; D005260:Female; D005451:Fluorescein Angiography; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000493:pharmacokinetics*; D005620:Freund's Adjuvant; D006801:Humans; D007208:Indocyanine Green / Q000008:administration & dosage / Q000493:pharmacokinetics*; D007274:Injections, Intraperitoneal; D007275:Injections, Intravenous; D007279:Injections, Subcutaneous; D007834:Lasers; D007962:Leukocytes / Q000276:immunology / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis; D009887:Ophthalmoscopy; D012160:Retina / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D012173:Retinitis / Q000209:etiology / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D013050:Spectrometry, Fluorescence; D014605:Uveitis / Q000139:chemically induced / Q000276:immunology / Q000378:metabolism* / Q000473:pathology
chemical_list: D004731:Endotoxins; D005456:Fluorescent Dyes; D005620:Freund's Adjuvant; D007208:Indocyanine Green
keywords: In vivo imaging; Indocyanine green; Inflammation