Two recent elegant studies have shown that in apolipoprotein-E– deficient mice, the lamina adventitia is a major site of arterial wall inflammation associated with lymphocyte infiltration into atherosclerotic arteries and with formation of adventitial lymphoid-like tissues.1,2 These results suggest that lymphocyte responses in the lamina adventitia may play a crucial role in atherosclerosis development.1,

Baker, Andrew H.

Brewer, James M.

Garside, Paul

Ialenti, Armando

Kennedy, Simon

Maffia, Pasquale

McInnes, Iain B.

Zinselmeyer, Bernd H.

English

Maffia, P.

Zinselmeyer, B.H.

Ialenti, A.

Kennedy, S.

Baker, A.H.

McInnes, I.

Brewer, J.M.

Garside, P.

Enlighten: Publications

Multiphoton microscopy for 3-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deficient mouse carotid artery

University of Strathclyde Institutional Repository

Strathprints Institutional RepositoryMaffia, P. and Zinselmeyer, B.H. and Ialenti, A. and Kennedy, S. and Baker, A.H. and McInnes,I.B. and Brewer, J.M. and Garside, P. (2007) Images in cardiovascular medicine : multiphotonmicroscopy for three-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deficientmouse carotid artery. Circulation, 115. E326-E328. ISSN 0009-7322Strathprints is designed to allow users to access the research output of the University of Strathclyde.Copyright c© and Moral Rights for the papers on this site are retained by the individual authorsand/or other copyright owners. You may not engage in further distribution of the material for anyprofitmaking activities or any commercial gain. You may freely distribute both the url (http://strathprints.strath.ac.uk/) and the content of this paper for research or study, educational, ornot-for-profit purposes without prior permission or charge.Any correspondence concerning this service should be sent to Strathprints administrator:mailto:strathprints@strath.ac.ukhttp://strathprints.strath.ac.uk/H. Baker, Iain B. McInnes, James M. Brewer and Paul GarsidePasquale Maffia, Bernd H. Zinselmeyer, Armando Ialenti, Simon Kennedy, AndrewDeficient Mouse Carotid Artery−Into Apolipoprotein-EMultiphoton Microscopy for 3-Dimensional Imaging of Lymphocyte RecruitmentISSN: 1524-4539 Copyright © 2007 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online72514Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TXdoi: 10.1161/CIRCULATIONAHA.106.6584922007, 115:e326-e328Circulation  http://circ.ahajournals.org/content/115/11/e326located on the World Wide Web at: The online version of this article, along with updated information and services, is http://circ.ahajournals.org/content/suppl/2008/02/25/115.11.e326.DC1.htmlData Supplement (unedited) at:  http://www.lww.com/reprintsReprints: Information about reprints can be found online at    journalpermissions@lww.com410-528-8550. E-mail: Fax:Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters   http://circ.ahajournals.org//subscriptions/Subscriptions: Information about subscribing to Circulation is online at  by guest on December 5, 2011http://circ.ahajournals.org/Downloaded from Multiphoton Microscopy for 3-Dimensional Imaging ofLymphocyte Recruitment Into Apolipoprotein-E–DeficientMouse Carotid ArteryPasquale Maffia, PhD; Bernd H. Zinselmeyer, PhD; Armando Ialenti, PhD; Simon Kennedy, PhD;Andrew H. Baker, PhD; Iain B. McInnes, MRCP, PhD; James M. Brewer, PhD; Paul Garside, PhDTwo recent elegant studies have shown that in apoli-poprotein-E– deficient mice, the lamina adventitia is amajor site of arterial wall inflammation associated withlymphocyte infiltration into atherosclerotic arteries andwith formation of adventitial lymphoid-like tissues.1,2These results suggest that lymphocyte responses in thelamina adventitia may play a crucial role in atherosclerosisdevelopment.1,2However, in both studies, the detection of immune cellswithin the artery was limited to conventional analysissystems (flow cytometry, immunohistochemistry), whichrequire the removal, disruption, and processing of tissue atdefined time points to give a “snapshot” of the immuneresponse to vascular damage in vivo. For evaluation ofatherosclerotic processes and their relationship to immuneactivation, imaging of 3-dimensional structures in intactvascular tissues and functional aspects of the diseasedartery (eg, details of the interactions of immune cell withvessel wall structures) are required. A potential solution tothis problem is offered by the development of multiphotonlaser-scanning microscopy,3 with the ability to penetratedirectly into tissues at sufficient depths to image tissues insitu in the absence of significant phototoxicity. By movingthe focal point axially, a stack of optical sections at variousdepths can subsequently be viewed as a movie. Recently,multiphoton imaging has been applied in atherosclerosis toimage murine vascular structures.4 However, homing ofcells to the vessel wall was not addressed. In our labora-tory, we have established multiphoton scanning micros-copy to allow imaging of lymphocytes in real time, in situ,in vivo.3 Here, we report the 3-dimensional imaging of theentire structure of an isolated intact apolipoprotein-E–deficient mouse carotid artery, identifying homed, fluores-cently tagged, adoptively transferred lymphocytes.Cell suspensions were prepared from peripheral andmesenteric lymph nodes of female C57BL/6 mice and werelabeled with Cell Tracker Red CMTPX (Molecular ProbesInc.) at 107 cells/mL of CO2-independent medium (GIBCOBRL) plus 0.5 L of 20 mmol/L CMTPX stock solution inDMSO for 45 minutes at 37°C. Cells were washed withcomplete CO2-independent medium, and 2 to 5106 lym-phocytes were injected intravenously into 8-week-old fe-male apolipoprotein-E– deficient mice or C57BL/6 recipi-ents. Twenty-four hours after transfer, excised intactmouse carotid arteries were imaged by 2-photon micros-copy, as described previously.3 The excised vessel wastransferred into CO2-independent medium at room temper-ature. The vessel was bound (Vetbond, 3M) on a coverslip.The coverslip was adhered with grease to the bottom of theimaging chamber, which was continuously supplied withwarmed (37°C) and gassed (95% O2 and 5% CO2) medium(RPMI 1640 with 25 mmol/L HEPES) before and through-out the period of microscopy. The 2-photon excitationsource was an all-solid-state, tuneable, titanium sapphirelaser system (Chameleon, Coherent Laser Group). Thelaser beam was routed into a multiphoton excitation laserscanning system (Radiance 2000MP, Bio-Rad Laborato-ries). The output laser beam from the scan head wasaligned through an upright microscope (E600-FN, Nikon).The objective lens used for all imaging investigations wasa CFi-60 Nikon Fluor 40/0.8 NA water-dipping objectivelens. The sample was illuminated with 830 nm, approxi-mately 210 fs in pulse duration and 76 MHz in repetitionfrequency. The excited fluorescence passed through a laserblocking filter (E625SP, Chroma Technologies) and wasdetected with a multialkali cathode photomultiplier tube(S20 PMT) in an external or nondescanned configurationas part of the Radiance 2000MP system. A custom-builtfilter block was used to separate fluorescence at 500 to 550From the Department of Experimental Pharmacology, University of Naples Federico II, Naples, Italy (P.M., A.I.); Division of Immunology, Infection& Inflammation (P.M., I.B.M.) and BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom (A.H.B.); andCentre for Biophotonics (B.H.Z., J.M.B., P.G.) and Department of Physiology and Pharmacology (S.K.), Strathclyde Institute of Pharmacy andBiomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.The online-only Data Supplement, consisting of movies I and II, is available with this article at http://circ.ahajournals.org/cgi/content/full/115/11/e326/DC1.Correspondence to: Pasquale Maffia, Department of Experimental Pharmacology, University of Naples Federico II, via D. Montesano 49, 80131Naples, Italy (e-mail pamaffia@unina.it); or Paul Garside, Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences,University of Strathclyde, Glasgow G4 ONR, United Kingdom (email paul.garside@strath.ac.uk).(Circulation. 2007;115:e326-e328.)© 2007 American Heart Association, Inc.Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.658492e326Images in Cardiovascular Medicine by guest on December 5, 2011http://circ.ahajournals.org/Downloaded from nm (autofluorescence) from that above 600 nm (red label).The scans were acquired with 500 lines per second andbetween 256256 and 10241024 pixel boxes, for aframe rate of 1.95 frames per second.As shown in Figure 1 and in the accompanying online3-dimensional movies, this system enabled us to clearlyimage lymphocyte infiltration mainly within the adventitia ofan intact mouse carotid artery. Consequently, this systemcould be a powerful tool to study immune cell behavior inartery disease.In an unlabeled sample, the whole structure of the vessel, with adventitial connective tissue and elastin fibers in the media, was visibleby autofluorescence.4 A set of longitudinal sections of intact mouse carotid artery was imaged to demonstrate the large penetrationdepth and resulting potential of 2-photon imaging in visualization of 3-dimensional structural information in intact arteries. A, The firstimage (10 m below the outer surface of the intact artery) is of the outer layer of the adventitia with autofluorescent connective tis-sue. B, The next image (30 m below the outer surface) originates from somewhat deeper in the adventitia, revealing more fibers. C,D and E, Entering the media further, elastin bands became clearly discernible. At approximately 80 m below the surface (F), the emptylumen finally was reached. For panels A through F, bar62 m. G, Three-dimensional reconstruction of an intact mouse carotid artery.Three-dimensional reconstruction of whole-vessel wall structure (Movie I) is available as online supplemental data. At 24 hours aftertransfer, no lymphocytes were detectable in C57BL/6 mice carotid arteries (A–F). H–J, Several red-labeled lymphocytes were detect-able, mainly in adventitia (10 to 30 m below the outer surface) and within elastin fibers in media (K–M, 30 to 50 m below theouter surface), as shown by the set of longitudinal sections of carotid arteries from apolipoprotein-E–deficient mice. For panels Hthrough M, bar41 m. N, Three-dimensional reconstruction of lymphocyte infiltration in the entire mouse carotid artery, also availableas online supplemental data (Movie II). Each imaged volume consisted of 16 to 50 planes, 2.5 m apart.Maffia et al 3D Imaging of Lymphocytes in Mouse Carotid Wall e327 by guest on December 5, 2011http://circ.ahajournals.org/Downloaded from DisclosuresNone.References1. Moos MPW, John N, Grabner R, Nobmann S, Gunther B, Vollandt R,Funk CD, Kaiser B, Habenicht AJR. The lamina adventitia is the majorsite of immune cell accumulation in standard chow-fed apolipoproteinE-deficient mice. Arterioscler Thromb Vasc Biol. 2005;25:2386–2391.2. Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K.Lymphocyte recruitment into the aortic wall before and during devel-opment of atherosclerosis is partially L-selectin dependent. J Exp Med.2006;203:1273–1282.3. Zinselmeyer BH, Dempster J, Gurney AM, Wokosin D, Miller M, Ho H,Millington OR, Smith KM, Rush CM, Parker I, Cahalan M, Brewer JM,Garside P. In situ characterization of CD4 T cell behaviour in mucosaland systemic lymphoid tissues during the induction of oral priming andtolerance. J Exp Med. 2005;201:1815–1823.4. Boulesteix T, Pena AM, Pages N, Godeau G, Sauviat MP, Beaurepaire E,Schanne-Klein MC. Micrometer scale ex vivo multiphoton imaging ofunstained arterial wall structure. Cytometry A. 2006;69:20–26.e328 Circulation March 20, 2007 by guest on December 5, 2011http://circ.ahajournals.org/Downloaded from 

In situ characterization of CD4  T cell behaviour in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance.

In situ characterization of CD4 T cell behaviour in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance.

Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent.

Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.

The lamina adventitia is the major site of immune cell accumulation in standard chow-fed apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol.

Images in cardiovascular medicine : multiphoton microscopy for three-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deficient mouse carotid artery

Enlighten

McInnes, I.B.

Name not available

In this study we report the 3-dimensional imaging by 2-photon microscopy of the entire structure of an isolated intact apolipoprotein-E–deficient mouse carotid artery, identifying homed, fluorescently tagged, adoptively transferred lymphocytes

MAFFIA, PASQUALE

IALENTI, ARMANDO

ZINSELMEYER BH

KENNEDY S

BAKER AH

MCINNES IB

BREWER JM

GARSIDE P.

Archivio della ricerca - Università degli studi di Napoli Federico II

Images in cardiovascular medicine. Multiphoton microscopy for 3-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deficient mouse carotid artery.

Pasquale Maffia

Bernd H. Zinselmeyer

Armando Ialenti

Simon Kennedy

Andrew H. Baker

Iain B. McInnes

James M. Brewer

Paul Garside

Crossref

Multiphoton Microscopy for 3-Dimensional Imaging of Lymphocyte Recruitment Into Apolipoprotein-E–Deficient Mouse Carotid Artery

http://dx.doi.org/10.1161/CIRCULATIONAHA.106.658492

Images in cardiovascular medicine : multiphoton microscopy for three-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deficient mouse carotid artery

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