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.J Clin Invest, 110,1615 1617.Washington RA, Becher B, Balabanov R, Antel J, Dore-Duffy P (1996).Expression of the activa-tion marker urokinase plasminogen-activator receptor in cultured human central nervous sys-tem microglia.J Neurosci Res, 45, 392 399.Yamagishi S, Yonekura H, Yamamoto Y (1999).Vascular endothelial growth factor acts as a peri-cyte mitogen under hypoxic conditions.Lab Invest, 79, 501 509.Zhang ZG, Zhang L, Croll SD, Chopp M (2002).Angiopoietin-1 reduces cerebral blood vesselleakage and ischemic lesion volume after focal cerebral embolic ischemia in mice.Neuroscience, 113, 683 687.Zimmermann KW (1923).Der feinere Bau der Blutkapillaren.Z Anat Entwicklungsgesch, 68,29 109. This page intentionally blank Imaging Microglia in the Central NervousSystem: Past, Present and FutureDimitrios Davalos and Katerina AkassoglouThe development of in vivo imaging technology in mice has been a powerful toolto study mechanisms of physiology and pathology in both the nervous and theimmune systems.Recent studies have revolutionized our understanding on howglial cells, T cells and neurons interact with each other and respond to damage inthe nervous system.This chapter aims to summarize the advances of in vivo imag-ing as they relate to microglial activation and present the challenges of utilizing thistechnology directly in animal models of neuroimmunologic disease.1 In vivo ImagingImaging approaches have always been implemented by biologists in their efforts todescribe cellular morphology and structure and analyze cellular functions and inter-actions.A combination of advances in both microscopy and surgery has allowed thein vivo imaging of individual cellular responses as well as complex biological proc-esses, as they occur in real time and within their natural microenvironment, in bothhealth and disease.In particular, the development of two photon-excited fluores-cence laser scanning microscopy (Denk et al., 1990) that uses low-energy light andpenetrates deep in the intact living tissue has allowed the high-resolution imagingof cells located several hundred microns below the surface of various organs of livinganimals, with minimal photobleaching and photodamage (Helmchen and Denk,2005; Svoboda and Yasuda, 2006).This has been facilitated tremendously by thegeneration of transgenic animals which express fluorescent proteins by specific celltypes (Feng et al., 2000; Tsien, 1998), a technology that made the direct observa-tion of cells in vivo possible in both physiological and pathological settings(Germain et al., 2006; Helmchen and Denk, 2005; Misgeld and Kerschensteiner,2006; Svoboda and Yasuda, 2006).Prior to the development of in vivo imaging, studies were performed by eitherstandard histopathological techniques or in vitro imaging of individual culturedcells or ex vivo preparations of tissues.Histopathological techniques have provideda wealth of information on the anatomical and structural features of the nervoussystem and have revealed several aspects of nervous system pathophysiology.T.E.Lane et al.(eds.), Central Nervous System Diseases and Inflammation.45© Springer 2008 46 D.Davalos and K.AkassoglouHowever, the analysis of a snapshot of a biological event might obscure theappreciation and understanding of ongoing cellular processes, responses to envi-ronmental stimuli and transient cell-cell interactions.In vitro imaging of individualcultured cells that allows their manipulation by transfection of exogenous genesand the controlled addition of stimulatory agents or inhibitors has elucidated indetail both cellular and molecular mechanisms.Studies of cells within their micro-environment have been conducted in the context of acutely extracted tissue.Forexample, imaging in brain slices has expanded our understanding of dendritic calciumdynamics and the biochemical signals regulated by neural activity, as well as theprotein-protein interactions in neuronal micro-compartments such as axons, den-drites and their spines (Svoboda et al., 1997; Yasuda et al., 2006).However, inseveral cases cell viability and behavior within the brain slices may depend uponthe preparation and preservation conditions of the tissue in vitro.2 Imaging of Microglia In vivo2.1 Microglial SubtypesRamon y Cajal was the first who introduced a cellular third element besides theneurons and neuroglia in the central nervous system (CNS) in 1913.Del RioHortega (1932) further identified oligodendroglia and microglia within that thirdelement and discussed their morphological and ontogenic differences for the firsttime (Kaur et al., 2001).Microglia are unique among other cells of the nervoussystem due to (a) their origin, (b) the multiple microglia subtypes present in thehealthy brain and (c) their differentiation upon injury that is accompanied by dra-matic changes in both cellular morphology and gene expression.Regarding their origin, microglia have been the subject of numerous studies bymany investigators using several experimental systems and animal models, yet theyremain among the least understood of all the cell types in the mammalian brain.Their exact origin and subclassification are matters of active debate, due to the lackof microglia-specific markers, their morphological polymorphism and the antigenicplasticity of microglial populations (Gonzalez-Scarano and Baltuch, 1999).It iscurrently thought that pial macrophages and peripheral monocytes infiltrate thebrain during the early developmental stages (Kaur et al., 2001), where they differ-entiate into a precursor form, the amoeboid microglia.Besides the amoeboid microglia, there are three additional microglial subtypes.The parenchymal or ramified microglia, represent the resting state of the cells andare found throughout the healthy CNS.They bear long processes with manybranches that define each cell s territory in a non-overlapping manner.The reactivemicroglia, are rounded cells without processes, present around various types oftraumatic injury.Finally the perivascular microglia are in close association with thevasculature, and are believed to be important for communicating with components Imaging Microglia in the Central Nervous System: Past Present and Future 47of the BBB and with peripheral immune cells in the blood circulation (Grossmannet al., 2002).Although these are distinct microglial subtypes, it is widely believedthat they can all arise by interconversion within a common set of cells (Stenceet al., 2001) depending on the presence of activating signals from the surroundingtissue and the severity of the tissue injury.It is well established that following braindamage microglia become activated (Gonzalez-Scarano and Baltuch, 1999;Kreutzberg, 1996; Raivich et al., 1999; Thomas, 1992).Their activation triggers astereotypical series of both morphological and molecular alterations which arebelieved to be responsible for the functional differences of the emerging microglialsubtypes (Davis et al [ Pobierz caÅ‚ość w formacie PDF ]
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.J Clin Invest, 110,1615 1617.Washington RA, Becher B, Balabanov R, Antel J, Dore-Duffy P (1996).Expression of the activa-tion marker urokinase plasminogen-activator receptor in cultured human central nervous sys-tem microglia.J Neurosci Res, 45, 392 399.Yamagishi S, Yonekura H, Yamamoto Y (1999).Vascular endothelial growth factor acts as a peri-cyte mitogen under hypoxic conditions.Lab Invest, 79, 501 509.Zhang ZG, Zhang L, Croll SD, Chopp M (2002).Angiopoietin-1 reduces cerebral blood vesselleakage and ischemic lesion volume after focal cerebral embolic ischemia in mice.Neuroscience, 113, 683 687.Zimmermann KW (1923).Der feinere Bau der Blutkapillaren.Z Anat Entwicklungsgesch, 68,29 109. This page intentionally blank Imaging Microglia in the Central NervousSystem: Past, Present and FutureDimitrios Davalos and Katerina AkassoglouThe development of in vivo imaging technology in mice has been a powerful toolto study mechanisms of physiology and pathology in both the nervous and theimmune systems.Recent studies have revolutionized our understanding on howglial cells, T cells and neurons interact with each other and respond to damage inthe nervous system.This chapter aims to summarize the advances of in vivo imag-ing as they relate to microglial activation and present the challenges of utilizing thistechnology directly in animal models of neuroimmunologic disease.1 In vivo ImagingImaging approaches have always been implemented by biologists in their efforts todescribe cellular morphology and structure and analyze cellular functions and inter-actions.A combination of advances in both microscopy and surgery has allowed thein vivo imaging of individual cellular responses as well as complex biological proc-esses, as they occur in real time and within their natural microenvironment, in bothhealth and disease.In particular, the development of two photon-excited fluores-cence laser scanning microscopy (Denk et al., 1990) that uses low-energy light andpenetrates deep in the intact living tissue has allowed the high-resolution imagingof cells located several hundred microns below the surface of various organs of livinganimals, with minimal photobleaching and photodamage (Helmchen and Denk,2005; Svoboda and Yasuda, 2006).This has been facilitated tremendously by thegeneration of transgenic animals which express fluorescent proteins by specific celltypes (Feng et al., 2000; Tsien, 1998), a technology that made the direct observa-tion of cells in vivo possible in both physiological and pathological settings(Germain et al., 2006; Helmchen and Denk, 2005; Misgeld and Kerschensteiner,2006; Svoboda and Yasuda, 2006).Prior to the development of in vivo imaging, studies were performed by eitherstandard histopathological techniques or in vitro imaging of individual culturedcells or ex vivo preparations of tissues.Histopathological techniques have provideda wealth of information on the anatomical and structural features of the nervoussystem and have revealed several aspects of nervous system pathophysiology.T.E.Lane et al.(eds.), Central Nervous System Diseases and Inflammation.45© Springer 2008 46 D.Davalos and K.AkassoglouHowever, the analysis of a snapshot of a biological event might obscure theappreciation and understanding of ongoing cellular processes, responses to envi-ronmental stimuli and transient cell-cell interactions.In vitro imaging of individualcultured cells that allows their manipulation by transfection of exogenous genesand the controlled addition of stimulatory agents or inhibitors has elucidated indetail both cellular and molecular mechanisms.Studies of cells within their micro-environment have been conducted in the context of acutely extracted tissue.Forexample, imaging in brain slices has expanded our understanding of dendritic calciumdynamics and the biochemical signals regulated by neural activity, as well as theprotein-protein interactions in neuronal micro-compartments such as axons, den-drites and their spines (Svoboda et al., 1997; Yasuda et al., 2006).However, inseveral cases cell viability and behavior within the brain slices may depend uponthe preparation and preservation conditions of the tissue in vitro.2 Imaging of Microglia In vivo2.1 Microglial SubtypesRamon y Cajal was the first who introduced a cellular third element besides theneurons and neuroglia in the central nervous system (CNS) in 1913.Del RioHortega (1932) further identified oligodendroglia and microglia within that thirdelement and discussed their morphological and ontogenic differences for the firsttime (Kaur et al., 2001).Microglia are unique among other cells of the nervoussystem due to (a) their origin, (b) the multiple microglia subtypes present in thehealthy brain and (c) their differentiation upon injury that is accompanied by dra-matic changes in both cellular morphology and gene expression.Regarding their origin, microglia have been the subject of numerous studies bymany investigators using several experimental systems and animal models, yet theyremain among the least understood of all the cell types in the mammalian brain.Their exact origin and subclassification are matters of active debate, due to the lackof microglia-specific markers, their morphological polymorphism and the antigenicplasticity of microglial populations (Gonzalez-Scarano and Baltuch, 1999).It iscurrently thought that pial macrophages and peripheral monocytes infiltrate thebrain during the early developmental stages (Kaur et al., 2001), where they differ-entiate into a precursor form, the amoeboid microglia.Besides the amoeboid microglia, there are three additional microglial subtypes.The parenchymal or ramified microglia, represent the resting state of the cells andare found throughout the healthy CNS.They bear long processes with manybranches that define each cell s territory in a non-overlapping manner.The reactivemicroglia, are rounded cells without processes, present around various types oftraumatic injury.Finally the perivascular microglia are in close association with thevasculature, and are believed to be important for communicating with components Imaging Microglia in the Central Nervous System: Past Present and Future 47of the BBB and with peripheral immune cells in the blood circulation (Grossmannet al., 2002).Although these are distinct microglial subtypes, it is widely believedthat they can all arise by interconversion within a common set of cells (Stenceet al., 2001) depending on the presence of activating signals from the surroundingtissue and the severity of the tissue injury.It is well established that following braindamage microglia become activated (Gonzalez-Scarano and Baltuch, 1999;Kreutzberg, 1996; Raivich et al., 1999; Thomas, 1992).Their activation triggers astereotypical series of both morphological and molecular alterations which arebelieved to be responsible for the functional differences of the emerging microglialsubtypes (Davis et al [ Pobierz caÅ‚ość w formacie PDF ]