DOCSLIB.ORG

Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury This information is current as Masaki Honda, Takayuki Takeichi, Shintaro Hashimoto, of September 29, 2021. Daiki Yoshii, Kaori Isono, Shintaro Hayashida, Yuki Ohya, Hidekazu Yamamoto, Yasuhiko Sugawara and Yukihiro Inomata J Immunol 2017; 198:1718-1728; Prepublished online 6 January 2017; Downloaded from doi: 10.4049/jimmunol.1601773 http://www.jimmunol.org/content/198/4/1718 Supplementary http://www.jimmunol.org/content/suppl/2017/01/06/jimmunol.160177 http://www.jimmunol.org/ Material 3.DCSupplemental References This article cites 37 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/198/4/1718.full#ref-list-1 Why The JI? Submit online. by guest on September 29, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury Masaki Honda, Takayuki Takeichi, Shintaro Hashimoto, Daiki Yoshii, Kaori Isono, Shintaro Hayashida, Yuki Ohya, Hidekazu Yamamoto, Yasuhiko Sugawara, and Yukihiro Inomata Neutrophils are considered responsible for the pathophysiological changes resulting from hepatic ischemia-reperfusion (I/R) injury, which is a complication of trauma, shock, liver resection, and transplantation. Recently, evidence is accumulating that formyl- peptide receptor (FPR) signaling constitutes an important danger signal that guides neutrophils to sites of inflammation. This study aimed to investigate dynamic neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 Downloaded from blockade during hepatic I/R. LysM-eGFP mice were subjected to partial warm hepatic I/R. They were pretreated with an FPR1 antagonist, cyclosporine H (CsH), or formyl peptide, fMLF. Liver was imaged after hepatic laser irradiation or I/R using the TPLSM technique. CsH treatment alleviated hepatic I/R injury, as evidenced by decreased serum transaminase levels, reduced hepatocyte necrosis/apoptosis, and diminished inflammatory cytokine, chemokine, and oxidative stress. In contrast, systemic ad- ministration of fMLF showed few effects. Time-lapse TPLSM showed that FPR1 blockade inhibited the accumulation of neutrophils in the necrotic area induced by laser irradiation in vivo. In the CsH-treated I/R group, the number and crawling velocity of neu- http://www.jimmunol.org/ trophils in the nonperfused area were lower than those in the control group. Meanwhile, FPR1 blockade did not affect monocyte/ macrophage recruitment. Hepatic I/R promoted the retention of neutrophils and their active behavior in the spleen, whereas CsH treatment prevented their changes. Intravital TPLSM revealed that formyl-peptide–FPR1 signaling is responsible for regulating neutrophil chemotaxis to allow migration into the necrotic area in hepatic I/R. Our findings suggest effective approaches for elucidating the mechanisms of immune cell responses in hepatic I/R. The Journal of Immunology, 2017, 198: 1718–1728. epatic ischemia-reperfusion (I/R) injury is a complication to play critical roles in hepatic I/R through a specific pattern of trauma, shock, liver resection, and transplantation (1, recognition receptor (5–8). In addition, cellular injury also re- by guest on September 29, 2021 H 2). In the ischemic liver, an imbalance of the metabolic leases mitochondrial DAMPs, including formyl peptide and mito- supply and demand results in severe tissue hypoxia, mitochondrial chondrial DNA, which are recognized by formyl-peptide receptor dysfunction, and the generation of reactive oxygen species (ROS). 1 (FPR1) and TLR9, respectively (3). Endogenous formyl peptides Subsequent reperfusion causes the activation of innate and adap- are released secondarily from the N terminus of mitochondrial tive immune responses and cell death programs, leading to liver NADH dehydrogenase and cytochrome c oxidase in dead cells, dysfunction and failure. and mainly exert a chemotactic function by promoting neutrophil Cellular injury can release endogenous damage-associated Ca2+ flux and MAPKs (9). Signals through FPR1 in neutrophils molecular patterns (DAMPs), which activate innate immunity (3, overcome signals hierarchically through CXCR2, which is known 4). Several DAMPs have been identified in which intranuclear as a key chemokine receptor, allowing neutrophils to migrate to- high-mobility group box 1, DNA, and histones have been shown ward end-target chemoattractants (10). We therefore hypothesized that hepatic I/R releases mitochondrial DAMPs, thereby inducing Department of Transplantation and Pediatric Surgery, Postgraduate School of Med- the accumulation of neutrophils in the ischemic liver in an FPR1- ical Sciences, Kumamoto University, Kumamoto 860-8556, Japan dependent manner. Recent studies have shown the therapeutic ORCIDs: 0000-0002-6102-3201 (T.T.); 0000-0002-7477-9614 (Y.I.). potential of FPR1 blockade in acetaminophen-induced acute liver Received for publication October 17, 2016. Accepted for publication December 5, failure and smoking-induced lung emphysema (11, 12); however, 2016. the impact of the formyl-peptide–FPR1 interaction on hepatic I/R This work was supported by the Ministry of Education, Culture, Sports, Sciences and injury is unknown. Technology of Japan (Grants KAKENHI 22591410 and KAKENHI 25461954). Advances in intravital microscopy have enabled the visualization Address correspondence and reprint requests to Dr. Yukihiro Inomata, Department of Transplantation and Pediatric Surgery, Postgraduate School of Medical Sciences, and quantification of real-time biological processes in situ (13, 14). Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan. E-mail Above all, the monitoring of immune cell recruitment has an es- address: [email protected] pecially broad range of applications. We recently showed a novel The online version of this article contains supplemental material. method for examining in vivo real-time neutrophil recruitment Abbreviations used in this article: ALT, alanine aminotransferase; AST, aspartate during hepatic I/R using two-photon laser-scanning microscopy aminotransferase; CsH, cyclosporine H; DAMP, damage-associated molecular pat- tern; FPR, formyl-peptide receptor; I/R, ischemia-reperfusion; KC, keratinocyte che- (TPLSM) (15). TPLSM is one of the most progressive develop- mokine; 8-OHdG, 8-hydroxy-29-deoxyguanosine; ROS, reactive oxygen species; ments in imaging technology and has numerous advantages, in- TPLSM, two-photon laser-scanning microscopy; TRITC, tetramethylrhodamine cluding high-resolution deep-site imaging, less phototoxicity, and isothiocyanate. less photobleaching in comparison with conventional confocal Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 laser-scanning microscopy. These major advantages are especially www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601773 The Journal of Immunology 1719 suited to imaging live tissues and organs in their native envi- Olympus UMPLFLN 103 W (numerical aperture 0.30) objective lens. For ronment (16, 17). Using this method, we could investigate the high-magnification imaging, images were recorded using an Olympus 3 structure of hepatic lobes, circulation of the sinusoids, cell death, XLPLN 25 WMP (water-immersion, numerical aperture 1.05) objective lens with x-y planes spanning 508 3 508 mm. In some experiments, mice and changes in the number, velocity, and morphology of recruited were placed in the right lateral recumbent position, and the spleen was neutrophils. Because these findings provided valuable new in- exteriorized by a left flank incision. Spleen was fixed and imaged using sights into the functional-anatomical changes that occur during TPLSM as described. hepatic I/R, we applied the technique to test the dynamic effect of Induction of selective hepatic necrosis FPR1 blockade. By combining the information of intravital TPLSM imaging Selective hepatic necrosis was induced by high-power laser irradiation using and conventional biological parameters, we provided visual and TPLSM. Time-lapse imaging at low-magnification was zoomed up by six quantitative evidence of the therapeutic effects of FPR1 blockade in times, and the laser transmissivity was increased to 100% (it was ordinarily locked at 25% during low-magnification imaging). After 3 min of irradi- hepatic I/R injury. FPR1 blockade inhibited the chemotaxis of ation, the imaging conditions were returned to normal. Necrotic area was neutrophils into the necrotic area and subsequent innate immune- blacked out with x-y planes spanning 210 3 210 mm. Twenty-minute mediated inflammation. FPR1 blockade also
Load more

Recommended publications
  • When Two Is Better Than One: Elements of Intravital Microscopy David W
    Open access, freely available online Primer When Two Is Better Than One: Elements of Intravital Microscopy David W. Piston ver the last 20 years, many cell biological studies have moved from the single-cell level to the tissue level, Oand even to whole animals. This progress has been led by developments in fl uorescence microscopy that permit molecular observations from single cells within intact tissue or animals. Concurrent developments in fl uorescent probes, especially the cloning of the Green Fluorescent Protein and its use in transgenic animals, have also fueled this movement. The key instrumental technology for this work is optical sectioning microscopy; in this technique, instead of fi xing DOI: 10.1371/journal.pbio.0030207.g001 and physically sectioning a sample, the investigator obtains a 3-D dataset from an intact (and more importantly, live) Figure 1. Images of a Shark Choroid Plexus Stained with Fluorescein specimen. The most common optical sectioning technique (A) and (B) were collected 70 µm into the sample, and (C) is confocal microscopy, where fl uorescence is created was collected 140 µm into the sample. The contrast of the confocal image (A) is signifi cantly degraded at this depth, while throughout the sample and a confocal pinhole is placed in two-photon excitation at the same focal plane (B) allows the front of the detector so that only the in-focus fl uorescence is collection of an image with excellent intensity contrast. Further, recorded. For live samples, whose cells can be killed by the using two-photon excitation to image deeper into the sample (C) excitation light (via photo-toxicity, particularly of ultraviolet does not signifi cantly degrade the image contrast.
    [Show full text]
  • Intravital Microscopy in Tissue Engineering
    fbioe-09-627462 February 11, 2021 Time: 18:1 # 1 REVIEW published: 17 February 2021 doi: 10.3389/fbioe.2021.627462 Actually Seeing What Is Going on – Intravital Microscopy in Tissue Engineering Ravikumar Vaghela, Andreas Arkudas, Raymund E. Horch and Maximilian Hessenauer* Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich–Alexander University Erlangen–Nürnberg (FAU), Erlangen, Germany Intravital microscopy (IVM) study approach offers several advantages over in vitro, ex vivo, and 3D models. IVM provides real-time imaging of cellular events, which provides us a comprehensive picture of dynamic processes. Rapid improvement in microscopy techniques has permitted deep tissue imaging at a higher resolution. Advances in fluorescence tagging methods enable tracking of specific cell types. Moreover, IVM can serve as an important tool to study different stages of tissue regeneration processes. Furthermore, the compatibility of different tissue engineered Edited by: Jetze Visser, constructs can be analyzed. IVM is also a promising approach to investigate host Radboud University Medical Center, reactions on implanted biomaterials. IVM can provide instant feedback for improvising Netherlands tissue engineering strategies. In this review, we aim to provide an overview of the Reviewed by: requirements and applications of different IVM approaches. First, we will discuss the Roberto Weigert, National Institute of Dental history of IVM development, and then we will provide an overview of available optical and Craniofacial Research (NIDCR), modalities including the pros and cons. Later, we will summarize different fluorescence United States Lia Rimondini, labeling methods. In the final section, we will discuss well-established chronic and acute University of Eastern Piedmont, Italy IVM models for different organs.
    [Show full text]
  • Chronic Two-Photon Imaging of Neuronal Activity in Alzheimer's
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 Chronic two-photon imaging of neuronal activity in alzheimer’s disease animal models Bhat, Annapoorna Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-72425 Dissertation Published Version Originally published at: Bhat, Annapoorna. Chronic two-photon imaging of neuronal activity in alzheimer’s disease animal mod- els. 2012, University of Zurich, Faculty of Science. Chronic Two-photon Imaging of Neuronal Activity in Alzheimer’s Disease Animal Models Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Annapoorna Bhat aus Indien Promotionskomitee Prof. Dr. Roger M. Nitsch (Vorsitz, Leitung der Dissertation) Prof. Dr. Fritjof Helmchen (Leitung der Dissertation) Prof. Dr. Markus Rudin Prof. Dr. Esther Stöckli Zürich, 2012 1 2 “It is good to have an end to journey toward; but it is the journey that matters, in the end.” ― Ernest Hemingway 3 4 Table of Contents 1. Abbreviations ...........................................................................................................7 2. Summary/Zusammenfassung ...............................................................................11 3. Introduction............................................................................................................15 3.1 Alzheimer’s Disease .....................................................................................................17
    [Show full text]
  • Intravital Microscopy of Capillary Hemodynamics in Sickle Cell Disease
    Intravital microscopy of capillary hemodynamics in sickle cell disease. H H Lipowsky, … , N U Sheikh, D M Katz J Clin Invest. 1987;80(1):117-127. https://doi.org/10.1172/JCI113036. Research Article Direct intravital microscopic examinations were made in nailfold capillaries in subjects with homozygous sickle cell disease (HbSS red cells). In the resting state, capillary red cell (rbc) flux exhibited greater intermittence compared with normal subjects, which increased with painful crisis. In crisis-free HbSS subjects, capillary occlusion and red cell sequestration occurred in only 8.2% of all capillaries and diminished to 5.8% during crisis, possibly due to sequestration of less deformable rbcs in other organs. Velocities of rbc's (Vrbc) were measured by video techniques under resting conditions and during postocclusive reactive hyperemia (PORH) induced by a pressure cuff around the finger. Resting Vrbc was normal in crisis-free HbSS subjects, averaging 0.7 mm/s. In contrast, Vrbc was significantly elevated during crisis, to 0.98 mm/s, apparently due to compensatory arteriolar dilation. Crisis subjects exhibited a significantly depressed PORH with the ratio of peak red cell velocity to resting values reduced by 15% due to a loss of vasodilatory reserve, whereas crisis-free subjects exhibited a normal response. A 55% increase in the time to attain peak Vrbc was attributed to resistance increases, possibly resulting from red cell and leukocyte-to-endothelium adhesion during the induced ischemia. Find the latest version: https://jci.me/113036/pdf Intravital Microscopy of Capillary Hemodynamics in Sickle Cell Disease Herbert H. Lipowsky, Naeem U. Sheikh, and Donald M.
    [Show full text]
  • Research Techniques Made Simple: Two-Photon Intravital Imaging of the Skin Peyman Obeidy1, Philip L
    RESEARCH TECHNIQUES MADE SIMPLE Research Techniques Made Simple: Two-Photon Intravital Imaging of the Skin Peyman Obeidy1, Philip L. Tong1,2,3 and Wolfgang Weninger1,2,3 Over the last few years, intravital two-photon microscopy has matured into a powerful technology helping basic and clinical researchers obtain quantifiable details of complex biological mechanisms in live and intact tissues. Two-photon microscopy provides high spatial and temporal resolution in vivo with little phototoxicity that is unattainable by other optical tools like confocal microscopy. Using ultrashort laser pulses, two-photon microscopy allows the visualization of molecules, cells, and extracellular structures up to depths of 1 mm within tissues. Consequently, real-time imaging of the individual skin layers under both physiological and pathological conditions has revolutionized our understanding of cutaneous homeostasis, immunity, and tumor biology. This review provides an overview to two-photon microscopy of the skin by covering the basic concepts and current applications in diverse preclinical and clinical settings. Journal of Investigative Dermatology (2018) 138, 720e725; doi:10.1016/j.jid.2018.01.017 CME Activity Dates: 21 March 2018 CME Accreditation and Credit Designation: This activity has Expiration Date: 20 March 2019 been planned and implemented in accordance with the Estimated Time to Complete: 1 hour accreditation requirements and policies of the Accreditation Council for Continuing Medical Education through the joint Planning Committee/Speaker Disclosure: All authors, providership of Beaumont Health and the Society for Inves- planning committee members, CME committee members tigative Dermatology. Beaumont Health is accredited by the and staff involved with this activity as content validation fi ACCME to provide continuing medical education for physi- reviewers have no nancial relationship(s) with commercial cians.
    [Show full text]
  • Visualizing the Dynamics of Immune Surveillance in Brain Tumors by Intravital Multiphoton Microscopy
    The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2017 VISUALIZING THE DYNAMICS OF IMMUNE SURVEILLANCE IN BRAIN TUMORS BY INTRAVITAL MULTIPHOTON MICROSCOPY Felix Nwajei Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Medicine and Health Sciences Commons Recommended Citation Nwajei, Felix, "VISUALIZING THE DYNAMICS OF IMMUNE SURVEILLANCE IN BRAIN TUMORS BY INTRAVITAL MULTIPHOTON MICROSCOPY" (2017). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 735. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/735 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. Visualizing the dynamics of immune surveillance in brain tumors by intravital multiphoton microscopy by Felix I. Nwajei, MD APPROVED: ______________________________
    [Show full text]
  • Label-Free Multiphoton Microscopy: Much More Than Fancy Images
    International Journal of Molecular Sciences Review Label-Free Multiphoton Microscopy: Much More than Fancy Images Giulia Borile 1,2,*,†, Deborah Sandrin 2,3,†, Andrea Filippi 2, Kurt I. Anderson 4 and Filippo Romanato 1,2,3 1 Laboratory of Optics and Bioimaging, Institute of Pediatric Research Città della Speranza, 35127 Padua, Italy; fi[email protected] 2 Department of Physics and Astronomy “G. Galilei”, University of Padua, 35131 Padua, Italy; [email protected] (D.S.); andrea.fi[email protected] (A.F.) 3 L.I.F.E.L.A.B. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, 35128 Padua, Italy 4 Crick Advanced Light Microscopy Facility (CALM), The Francis Crick Institute, London NW1 1AT, UK; [email protected] * Correspondence: [email protected] † These authors contributed equally. Abstract: Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment.
    [Show full text]
  • Imaging Windows for Long- Term Intravital Imaging: General Overview and Technical Insights
    Imaging windows for long- term intravital imaging: General overview and technical insights The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Alieva, Maria, Laila Ritsma, Randy J Giedt, Ralph Weissleder, and Jacco van Rheenen. 2014. “Imaging windows for long-term intravital imaging: General overview and technical insights.” IntraVital 3 (2): e29917. doi:10.4161/intv.29917. http://dx.doi.org/10.4161/intv.29917. Published Version doi:10.4161/intv.29917 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:31731691 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA METHODS ARTICLE RESOURCE ARTICLE IntraVital 3, e29917; August 2014; © 2014 Landes Bioscience Imaging windows for long-term intravital imaging General overview and technical insights Maria Alieva1,†, Laila Ritsma2,3,†, Randy J Giedt4, Ralph Weissleder4,5, and Jacco van Rheenen1,* 1Cancer Genomics Netherlands; Hubrecht Institute-KNAW and University Medical Centre Utrecht; CT Utrecht, The Netherlands; 2Center for Cancer Research and Center for Regenerative Medicine; Massachusetts General Hospital; Richard B. Simches Research Center; Harvard Medical School; Boston, MA USA; 3Broad Institute of Harvard and Massachusetts Institute for Technology; Cambridge, MA USA; 4Center for Systems Biology; Massachusetts General Hospital; Richard B. Simches Research Center; Harvard Medical School; Boston, MA USA; 5Department of Systems Biology; Harvard Medical School; Boston, MA USA †These authors contributed equally to this work.
    [Show full text]
  • 2 Annual IMMUNE IMAGING SYMPOSIUM
    2nd Annual IMMUNE IMAGING SYMPOSIUM Hosted by: THE PROGRAM FOR ADVANCED IMMUNE BIOIMAGING & UNIVERSITY OF ROCHESTER Saturday, November 5th, 2016 Saunders Research Building University of Rochester 8:00 a.m. – 6:30 p.m. About our program: PROGRAM FOR ADVANCE IMMUNE BIOIMAGING Deborah Fowell, Minsoo Kim, Jim Miller and David Topham Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester, Rochester, NY Pathogen control ultimately requires the recruitment and activation of innate and adaptive immune effectors to specific infected tissue microenvironments. While we have gained much insight into effector T cell generation in lymphoid tissues there exists a significant knowledge gap on the fate of effector T cells once they leave the lymph node. The ability of T cells to sense and interpret different inflammatory environments in infected or damaged tissues is poorly understood. Yet it is within the inflamed tissue milieu that T cells must mediate their effector functions, including cytokine secretion and cytolysis, to clear infection. The central premise of this program is that the specific tissue and the local inflammatory milieu will shape T cell recruitment and effector function. Such tissue-control is likely to impact the magnitude and functional diversity of the immune response. Optimizing T cell function in tissues is critical for pathogen clearance and the avoidance of collateral damage. The goal of this program is to define the checkpoints and identify molecular interactions that guide successful immunity at sites of inflammation. The objective is to bring together scientific expertise in migration, effector function and tissue structure to address fundamental effector T cell processes in infected tissues using cutting- edge intra-vital imaging approaches.
    [Show full text]
  • Application of GFP Imaging in Cancer Robert M Hoffman
    Laboratory Investigation (2015) 95, 432–452 & 2015 USCAP, Inc All rights reserved 0023-6837/15 $32.00 PATHOBIOLOGY IN FOCUS Application of GFP imaging in cancer Robert M Hoffman Multicolored proteins have allowed the color-coding of cancer cells growing in vivo and enabled the distinction of host from tumor with single-cell resolution. Non-invasive imaging with fluorescent proteins enabled the dynamics of metastatic cancer to be followed in real time in individual animals. Non-invasive imaging of cancer cells expressing fluorescent proteins has allowed the real-time determination of efficacy of candidate antitumor and antimetastatic agents in mouse models. The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm can visualize the nuclear–cytoplasmic dynamics of cancer cells in vivo including: mitosis, apoptosis, cell-cycle position, and differential behavior of nucleus and cytoplasm that occurs during cancer-cell deformation and extravasation. Recent applications of the technology described here include linking fluorescent proteins with cell-cycle-specific proteins such that the cells change color from red to green as they transit from G1 to S phases. With the macro- and micro-imaging technologies described here, essentially any in vivo process can be imaged, giving rise to the new field of in vivo cell biology using fluorescent proteins. Laboratory Investigation (2015) 95, 432–452; doi:10.1038/labinvest.2014.154; published online 16 February 2015 ‘...during periods of revolution when the fundamental tenets cancer cells of a specific genotype or phenotype. For example, of a field are once more at issue, doubts are repeatedly the behavior of cancer stem cells labeled with GFP and non- expressed about the possibility of continued progress if one stem cells labeled with red fluorescent protein (RFP) can be or another of the opposed paradigms is adopted.’ simultaneously compared in vivo.3 The stroma and the cancer Thomas S.
    [Show full text]
  • The Amyloid-Beta Rich CNS Environment Alters Myeloid Cell Functionality Independent of Their Origin
    www.nature.com/scientificreports OPEN The Amyloid-beta rich CNS environment alters myeloid cell functionality independent of their origin Natalia Drost1,14, Judith Houtman1,2,14, Zoltán Cseresnyés 3,4, Raluca Niesner3,5, Jan-Leo Rinnenthal1,6, Kelly R. Miller1,7, Stefan Prokop1,8,9,10,15 & Frank L. Heppner 1,11,12,13,15 ✉ Microglia, the innate immune cells of the central nervous system (CNS) survey their surroundings with their cytoplasmic processes, phagocytose debris and rapidly respond to injury. These functions are afected by the presence of beta-Amyloid (Aβ) deposits, hallmark lesions of Alzheimer’s disease (AD). We recently demonstrated that exchanging functionally altered endogenous microglia with peripheral myeloid cells did not change Aβ-burden in a mouse model mimicking aspects of AD at baseline, and only mildly reduced Aβ plaques upon stimulation. To better characterize these diferent myeloid cell populations, we used long-term in vivo 2-photon microscopy to compare morphology and basic functional parameters of brain populating peripherally-derived myeloid cells and endogenous microglia. While peripherally-derived myeloid cells exhibited increased process movement in the non- diseased brain, the Aβ rich environment in an AD-like mouse model, which induced an alteration of surveillance functions in endogenous microglia, also restricted functional characteristics and response to CNS injury of newly recruited peripherally-derived myeloid cells. Our data demonstrate that the Aβ rich brain environment alters the functional characteristics of endogenous microglia as well as newly recruited peripheral myeloid cells, which has implications for the role of myeloid cells in disease and the utilization of these cells in Alzheimer’s disease therapy.
    [Show full text]
  • High-Pulse-Energy Multiphoton Imaging of Neurons and Oligodendrocytes in Deep Murine Brain with a Fiber Laser
    www.nature.com/scientificreports OPEN High‑pulse‑energy multiphoton imaging of neurons and oligodendrocytes in deep murine brain with a fber laser Michael J. Redlich1,2, Brad Prall3, Edesly Canto‑Said3, Yevgeniy Busarov1, Lilit Shirinyan‑Tuka1, Arafat Meah1 & Hyungsik Lim1,2* Here we demonstrate high‑pulse‑energy multiphoton microscopy (MPM) for intravital imaging of neurons and oligodendrocytes in the murine brain. Pulses with an order of magnitude higher energy (~ 10 nJ) were employed from a ytterbium doped fber laser source at a 1‑MHz repetition rate, as compared to the standard 80‑MHz Ti:Sapphire laser. Intravital imaging was performed on mice expressing common fuorescent proteins, including green (GFP) and yellow fuorescent proteins (YFP), and TagRFPt. One ffth of the average power could be used for superior depths of MPM imaging, as compared to the Ti:Sapphire laser: A depth of ~ 860 µm was obtained by imaging the Thy1‑YFP brain in vivo with 6.5 mW, and cortical myelin as deep as 400 µm ex vivo by intrinsic third‑harmonic generation using 50 mW. The substantially higher pulse energy enables novel regimes of photophysics to be exploited for microscopic imaging. The limitation from higher order phototoxicity is also discussed. Two-photon excitation microscopy (2PM) with descanned detection has a superior depth of imaging, far exceeding that of one-photon excitation microscopy1–3. Similar gain can be attained in general for multiphoton microscopy (MPM), expanding biological investigations by light microscopy from cultured cells to fresh thick tissues. By virtue of the safety of the near-infrared (NIR) excitation4, MPM has been the primary tool for intra- vital microscopy leading to breakthrough discoveries in broad biomedical felds.
    [Show full text]