GLIA 50:321–328 (2005)
Identification of Oligodendrocytes in Experimental Disease Models JENNIFER K. NESS, MARIO VALENTINO, SALLY R. MCIVER, AND MARK P. GOLDBERG* Department of Neurology, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri
KEY WORDS
oligodendrocytes; histology; immunocytochemical identification; new methods; gene transfer
ABSTRACT The ability to identify oligodendrocytes in culture, in fixed tissue, and in vivo using unique markers is a requisite step to understanding their responses in any damage, recovery, or developmental process. Their nuclei are readily seen in histological preparations of healthy white and gray matter, and their cell bodies can be reliably identified with a variety of immunocytochemical markers. However, there is little consensus regarding optimal methods to assess oligodendrocyte survival or morphology under experimental injury conditions. We review common approaches for histological and immunocytochemical identification of these cells. Transgenic and viral methods for cell type-selective transfer of genes encoding fluorescent proteins offer promising new approaches for manipulating and visualizing oligodendrocytes in models of health and disease. V 2005 Wiley-Liss, Inc. C
INTRODUCTION Oligodendrocytes produce and sustain central nervous system (CNS) myelin and regulate axon function. In brain and spinal cord injury, these cells are not merely passive observers but are themselves directly vulnerable in a growing list of acute and chronic conditions (Ness and Goldberg, 2005). Like neurons, oligodendrocytes are highly sensitive to injury mediated by oxidative stress, excitatory amino acids, trophic factor deprivation, and activation of apoptotic pathways. Hypoxic-ischemic damage to oligodendrocytes is a common feature in focal ischemia (stroke), global ischemia (cardiac arrest), cyanide intoxication, and vascular dementia (Brierley et al., 1977; Garcia et al., 1977; Caplan et al., 1978; Pantoni and Garcia, 1995; Volpe, 2001), and oligodendrocytes are damaged also in brain and spinal cord trauma, multiple sclerosis, and Alzheimer’s disease. During the perinatal period, damage to oligodendrocyte progenitor cells results in periventricular leukomalacia and long-term demyelination, a major etiology of cerebral palsy (Volpe, 2001). It is important to assess oligodendrocyte survival or death in experimental disease models. We review conventional histological and immunocytochemical methods commonly used to C 2005 V
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identify oligodendrocyte injury. In addition, we discuss possible advantages of gene transfer techniques that allow visualization of oligodendrocytes in living or fixed tissue.
OLIGODENDROCYTE RECOGNITION BY HISTOCHEMICAL METHODS AND EM Nuclei of oligodendrocytes and those of other neuroglial cells are easily visualized in routine histological preparations stained with basic dyes. Most oligodendrocytes can be identified by their small round or oval nuclei and typical locations: in white matter tracts, intrafascicular oligodendrocytes align in rows parallel to myelinated axons; in gray matter, satellite cells Grant sponsor: National Institute of Neurological Disorders and Stroke, National Institutes of Health (NINDS/NIH); Grant number: P01 NS032636; Grant number: R01 NS36265; Grant number: T32 NS07205; Grant sponsor: Juvenile Diabetes Research Foundation. *Correspondence to: Mark P. Goldberg, Department of Neurology, Campus Box 8111, 660 S. Euclid Avenue, St. Louis, MO 63110-1193. E-mail:
[email protected] Received 9 December 2004; Accepted 18 February 2005 DOI 10.1002/glia.20206 Published online 21 April 2005 in Wiley InterScience (www.interscience. wiley.com).
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NESS ET AL. TABLE 1. Antibodies to Oligodendrocyte Lineage Cells
Antibody A2B5 R24 LB1 O4 O1 Ranscht Anti-NG2 Anti-GST-pi Anti-carbonic anhydrase II Anti-MBP Anti-MAG Anti-MOG Rip CCI Anti-OSP Anti-CNPase Anti-CD9
Antigen
Maturation stage
Location
Ganglioside GD3 ganglioside GD3 ganglioside Prooligodendrocyte antigen, sulfatide Galactocerebroside Galactocerebroside NG2 chondroitin sulfate proteoglycan Glutathione-S-transferase isoenzyme pi Carbonic anhydrase II Myelin basic protein Myelin-associated glycoprotein Myelin oligodendrocyte glycoprotein Unknown APC (binds a nonspecific antigen in OLs) Oligodendrocyte-specific protein/claudin-11 20 ,30 -cyclic nucleotide 30 -phosphodiesterase CD9
Early OL progenitors Early OL progenitors Early OL progenitors Late OL progenitors, adult progenitors Immature oligodendrocytes Immature oligodendrocytes Early OL progenitors, adult progenitors Mature oligodendrocytes All stages Mature oligodendrocytes Mature oligodendrocytes Mature oligodendrocytes Mature oligodendrocytes Mature oligodendrocytes Mature oligodendrocytes All stages, strong in mature OLs All stages, does not label adult NG2þ cells
Cell surface Cell surface Cell surface Cell surface Cell surface Cell surface Cell surface Intracellular Intracellular Intracellular Cell surface Cell surface Intracellular Intracellular Cell surface Intracellular Intracellular
surround neuronal cell bodies. A third group of oligodendrocyte lineage cells, the adult oligodendrocyte progenitor cells (OPCs), are widely distributed throughout gray and white matter. The development of specialized metal impregnation techniques by Cajal and Rio del Hortega allowed recognition of oligodendrocytes as multipolar neuroglial cells with a range of morphological appearances (Kettenmann and Ransom, 2005). The in situ morphology of oligodendrocyte processes was confirmed using intracellular injection of the fluorescent dye, Lucifer yellow (Butt and Ransom, 1989). In electron micrographs, oligodendrocytes are identified by their round or oval nuclei with variably clumped chromatin, and scanty, electron-dense cytoplasm. Their perikarya and processes include mitochondria and endoplasmic reticulum, but not intermediate filaments or glycogen granules that are associated with astrocytes (Butt, 2005; Peters et al., 1991). Conventional histochemical methods with light and electron microscopy can reliably distinguish oligodendrocytes from other cell types in normal tissue. However, these methods are not always sufficient under conditions of injury, in which oligodendrocyte identification is clouded by alterations in cell morphology, loss of epitopes, and confusion with activated microglia and infiltrating cells.
IMMUNOCYTOCHEMICAL METHODS The most widely used method for labeling oligodendrocytes has been immunocytochemistry, for which a wide range of antibodies to oligodendrocyte surface and intracellular markers have been described. The identification of multiple developmental stage-specific cell surface markers has allowed extensive characterization of the progressive development of cultured oligodendrocytes, as well as stage-specific vulnerability to damage in vitro. Antibodies A2B5, O4, and O1
identify the early oligodendrocyte progenitor, the late oligodendrocyte progenitor, and the postmitotic immature oligodendrocyte, respectively. Other stage-specific surface marker antibodies are listed in Table 1. Cell surface markers have been particularly advantageous in studies of cultured oligodendrocytes since they allow the visualization of the cell body and its processes. However, surface antigens such as galactocerebrosides (O1), gangliosides (LB1, R24), and sulfatides (O4), are sensitive to detergents, can nonspecifically label myelin, and require special immunolabeling techniques for use in intact tissue. Furthermore, identification of individual oligodendrocyte cell bodies using cell surface markers in adult tissue is difficult because of the concurrent labeling of their extensive processes and myelin. The NG2 antibody, directed against the NG2 chondroitin sulfate proteoglycan, identifies early oligodendrocyte progenitors and adult oligodendrocyte progenitors. Antibodies to myelin basic protein (MBP) label mature oligodendrocytes including their cell body and myelin sheets. Antibodies to the minor myelin components, myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), which constitute less than 1% of myelin proteins, have been shown to label mature oligodendrocytes and myelin in vivo (Linington et al., 1984); MAG also stains oligodendrocytes in cell culture (Dubois-Dalcq et al., 1986). Although antibodies to MBP and MOG can be used to visualize myelin in tissue, these may not adequately distinguish individual mature oligodendrocyte cell bodies because of the large amount of myelin in the adult brain. Antibodies to intracellular markers that primarily label the cell bodies of mature oligodendrocytes include CC1, and antibodies to 20 ,30 cyclic nucleotide 30 -phosphodiesterase (CNPase). While CC1 is directed against APC, the adenomatous polyposis coli, a tumor suppressor protein, it also binds to a nonspecific antigen in oligodendrocytes (Bhat et al., 1996; Brakeman et al., 1999). CC1 is
IDENTIFICATION OF OLIGODENDROCYTES
used primarily to identify mature oligodendrocyte cell bodies, but its stage-specific expression patterns have not been fully characterized. CNPase is another antigen used to immunolabel mature oligodendrocyte cell bodies, but some labeling is also associated with myelin (Sprinkle et al., 1983). Although CNPase is expressed early in oligodendrocyte development, its expression is greatly increased in mature, myelinating cells. Rip is a monoclonal antibody that labels mature oligodendrocytes and their processes. Cell bodies can be identified with Rip by reducing detergents to decrease myelin staining (Friedman et al., 1989). Antibodies to the C-isoenzyme of carbonic anhydrase bind, with reasonable specificity, to oligodendrocytes in vivo and in vitro (Sarlieve et al., 1981) but are little used in routine identification of these cells.
RECOGNITION OF OLIGODENDROCYTE INJURY Assessment of oligodendrocyte injury in disease and recovery paradigms requires techniques to label injured as well as dead cells. A variety of labeling techniques have been used to identify end-stage cell death of oligodendrocytes in vitro and in vivo. In cell culture, oligodendrocyte survival and plasma membrane integrity is easily established by exclusion of large molecules such as trypan blue or propidium iodide. In vivo, DNA-binding dyes such as Hoechst 33258 and DAPI are commonly used to identify condensed chromatin and apoptotic bodies in oligodendrocytes. Oligodendrocyte death can also be identified by TUNEL and ISEL, which label nuclei with DNA strand breaks. Cytoskeletal and morphological changes in injured oligodendrocytes have also been used in recognizing oligodendrocyte damage. Myelin vacuolation and sheath detachment from axons in situ has also been identified in oligodendrocyte injury during ischemia (Pantoni et al., 1996). Development of tau-1 immunoreactivity has been identified as a marker for injured oligodendrocytes following ischemia (Dewar and Dawson 1995; Valeriani et al., 2000). It is unclear whether tau immunoreactivity is associated with a certain mode of cell death or whether tau-positive cells have the potential for recovery. Like other cell types, oligodendrocytes follow apoptotic or necrotic pathways, depending on their maturation state and the type or intensity of the insult. Activation of the apoptotic caspase cascade is involved in oligodendrocyte cell death from a variety of insults (Gu et al., 1999; Shibata et al., 2000; McBride et al., 2003; Sanchez-Gomez et al., 2003). Other apoptosis and necrosis indicators such as extracellular annexin binding have been used successfully to identify dying oligodendrocytes (Hollensworth et al., 2000, Matysiak et al., 2002). Changes in up-
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stream mediators of apoptosis also indicate injury of oligodendrocyte lineage cells, and may identify injured cells that have the potential for recovery. For example, upregulation of the pro-apoptotic protein Bim occurs after ischemia in vivo (Shibata et al., 2002), while Bax translocation to mitochondria precedes death of late oligodendrocyte progenitors after treatment with glutamate (Ness et al., 2004). Loss of mitochondrial membrane potential identified by Mitotracker Red or other mitochondrial stains can also be used to label dying oligodendrocytes (Ness et al., 2004). Mitochondrial release of cytochrome c has also been associated with oligodendrocyte apoptosis, particularly in response to excitotoxicity (Sanchez-Gomez et al., 2003). Dissection of upstream events in death pathways and identification of early injury markers will help to target therapies designed to rescue oligodendrocytes after various insults to white matter. Markers of cell injury are amenable to co-immunolabeling with oligodendrocyte-specific antibodies. However, this is problematic if the oligodendrocyte epitopes are lost early in cellular injury, as is often the case.
TRANSGENIC MODELS The ability to track oligodendroglia in development and injury models has been greatly assisted by the identification of functional promoter sequences for oligodendrocyte-specific genes, proteolipid protein (PLP), MBP, and CNP. In transgenic mice, expression of the lacZ gene reporter that encodes b-galactosidase under the control of MBP, PLP, and CNP reporters has provided key information about critical regulatory sequences and developmental regulation of expression of glial genes and has identified cells of the oligodendrocyte lineage throughout the period of brain development and myelination (Gow et al., 1992; Wight et al., 1993; Gravel et al., 1998). The b-galactosidase reporter provides clear cellular labeling even with low levels of enzyme expression, but requires tissue fixation and a histochemical processing step. Expression of GFP and other fluorescent proteins allows cellular identification in living or fixed tissue without additional processing, potentially allowing visualization in intact systems. GFP expression may be difficult to detect without strong promoters or immunocytochemical signal amplification. Several lines of transgenic mice have been generated with a mouse PLP promoter and regulatory sequence that drive the expression of enhanced green fluorescent protein (eGFP) or the red protein, dsRed (Fuss et al., 2000, 2002). The initial PLP promoter element found to direct b-galactosidase expression in transgenic mice (Wight et al., 1993) was insufficient for signal detection with fluorescent reporters. Its expression required several sequence modifications (Fuss et al., 2000) and was enhanced by insertion of the 30 UTR of PLP (Mallon et al., 2002). Transgenic mice that
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(Fig. 3). Transgenic mice that express eGFP under control of a mouse CNP promoter have been reported to show selective labeling of cells of oligodendrocyte and Schwann cell lineage at all stages of development, beginning as early as embryonic day 10 (Yuan et al., 2002). In the adult mouse brain both mature, myelinating oligodendrocytes and adult oligodendrocyte progenitors were labeled (Belachew et al., 2001). Transgenic expression of fluorescent proteins enables time lapse imaging of oligodendrocytes within intact brain preparations (Valentino et al., 2004) (Fig. 2).
VIRAL GENE TRANSFER
Fig. 1. Transgenic mice with oligodendrocyte expression of fluorescent proteins under the proteolipid protein (PLP) promoter. Confocal micrographs show oligodendrocytes in corpus callosum from PLP-DsRed (A: F. Kirchhoff lab; Fuss et al., 2002) or PLP-eGFP (B: W. Macklin lab; Fuss et al., 2000) transgenic mice in control fixed sections (16-mm thickness) at low magnification brighter in oligodendrocyte cell bodies and processes in mice expressing eGFP than dsRed, possibly due to aggregation of multimeric dsRed.
express either PLP-eGFP or PLP-dsRed show strong localization within MBP-immunoreactive oligodendrocytes in cerebral white and gray matter. Fluorescence is highest within oligodendrocyte cell bodies and nuclei, with modest spread of the cytosolic fluorophore to distal processes and to myelin (Figs. 1 and 2). Developmental studies show high expression in all oligodendrocyte lineage cells, beginning in the early postnatal period (Mallon et al., 2002). A similar pattern of developmental expression is observed in cultured oligodendrocytes isolated from PLP-eGFP mice
The use of transgenic mice is effective for identification of cell type and for gene transfer but has some limitations. It is not generally possible to control the proportion or location of labeled cells, or the timing or intensity of gene expression. Testing of each construct requires generation of a new mouse. Some of these issues can be addressed by other forms of gene transfer. Oligodendrocytes can be transfected through electroporation, particle bombardment, and liposomemediated transfection agents (Guo et al., 1996; Krueger et al., 1998). Recent advances in viral gene transfer technology have identified several recombinant viral vectors capable of transducing glial cells within the CNS. These attenuated viruses are significantly diverse in their cell specificity, transduction efficiency and duration, toxicity, and package size. Adenovirus efficiently transduces oligodendrocytes in vitro and in vivo (Guo et al., 1996; Krueger et al., 1998, Masamura et al., 2001). However, the intrinsic cytotoxicity and immunogenicity of adenovirus strains, especially at high titers, may limit their usefulness in the study of the exogenous CNS injury (Franklin et al., 1999). Adeno-associated virus (AAV), containing GFP driven by the MBP promoter, has labeled oligodendrocytes in rat cultures and to a lesser extent in adult mice (Chen et al. 1998, 1999). Lentiviral vectors have promising applications for live-cell imaging and gene transfer studies due to their high transduction efficiency, ability to transduce dividing and nondividing cells, and long-term gene expression (Naldini et al., 1996; Blomer et al., 1997; Baekelandt et al., 2002). We evaluated the expression patterns of several lentiviral constructs in primary oligodendrocyte cultures derived from postnatal mouse forebrain. Cells were infected with VSV-Gpseudotyped HIV-derived viral vectors that deliver constructs that express either cytosolic eGFP or plasma membrane-targeted eGFP (Lck-eGFP; Benediktsson et al., 2004) under the control of either a ubiquitous or an oligodendrocyte-specific promoter (Macauley et al., 2004). For each lentiviral construct, GFP expression was detected as early as 24 h postinfection and peaked at 4 days post-infection. Constructs that express GFP under control of a ubiqui-
IDENTIFICATION OF OLIGODENDROCYTES
Fig. 2. Multi-photon microscopy allows sequential imaging of oligodendrocytes from PLP-eGFP live brain slices at high spatial resolution within their native environment. Time lapse imaging of acutely isolated brain slice (400-mm thickness) under normoxic con-
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trol conditions shows fine detail of the oligodendrocyte cell bodies and processes in corpus callosum. Oligodendrocyte morphology remained stable over the 3-h observation period (Valentino et al., 2004).
Fig. 3. Developmental expression in cultured oligodendroglia generated from PLP-eGFP transgenic mice. Late oligodendrocyte progenitors (A: grown in the presence of fibroblast growth factor) express GFP at a lower level than postmitotic oligodendrocytes (B: grown in the presence of T3). Mature oligodendrocytes (C,D: showing the identical field) cultured from PLP-eGFP transgenic mice were immunostained with anti-myelin basic protein. GFP labels the cell body and processes of the oligodendrocytes (C), but not the MBPþ membrane sheets (D).
tous promoter derived from cytomegalovirus (CMV) effectively transduced glial cells and resulted in bright cytoplasmic expression. CMV promoted very high transduction efficiency and expression level in glial cultures; however, it nonselectively transduced both astrocytes and oligodendrocytes. A 1.9-kb fragment of the MBP gene, which has been previously
shown to confer oligodendrocyte-specific expression of reporter genes (Gow et al. 1992), was used to target GFP expression exclusively to oligodendrocytes. The small size of the MBP promoter sequence makes it ideal for use in lentiviral gene delivery, with some residual capacity for further addition of other genes of interest. Although transduction efficiency and
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Fig. 4. Lentiviral infection of cultured oligodendrocytes yields high expression of GFP. Oligodendrocyte cultures were transduced with 5 infecting units/cell of cytosolic MBP-eGFP lentiviral construct (A) or the membrane-targeted MBP-Lck-eGFP lentiviral construct (B) and digital fluorescence micrographs were taken of live cultures (Macauley et al., 2004).
Fig. 5. Oligodendrocyte-specific GFP expression using lentiviral vectors in vivo. Stereotaxic injection of MBP-eGFP to the corpus callosum of adult mice brightly labels oligodendrocyte cell bodies (A), while MBP-Lck-eGFP specifically labels their myelinating processes (B) (McIver et al., 2004).
expression levels were relatively lower with the MBP promoter, mature oligodendrocytes expressed GFP at levels sufficient for live imaging by conventional fluorescence and confocal microscopy. Compared with cytosolic eGFP, constructs expressing a plasma membrane-targeted Lck-eGFP were found to enhance visualization of oligodendroglial morphology (Fig. 4). Similar expression patterns were found when the same lentiviral constructs were tested in vivo (McIver et al., 2004). Stereotaxic injections into the corpus callosum of adult mice resulted in GFP expression detectable as early as 3 days post-injection (dpi) and remained stable for at least 28 dpi. Use of the CMV promoter produced bright GFP expression in both
astrocytes and oligodendrocytes, which was almost exclusively confined to the white matter tract. The MBP promoter specifically targeted GFP to oligodendrocytes throughout the corpus callosum. MBP-driven expression of cytosolic eGFP resulted in brightly labeled cell bodies (Fig. 5A), while expression of membrane-targeted Lck-eGFP very effectively labeled the myelinating processes (Fig. 5B). It remains to be determined whether this label extends to compact myelin as well as the surface of oligodendrocyte processes. Viral gene transfer with Lck-eGFP yielded excellent visualization of the variety of oligodendrocyte morphologies which previously were seen with intracellular dye injections and special immunocytochemical methods.
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IDENTIFICATION OF OLIGODENDROCYTES
CONCLUSIONS
REFERENCES
Standard histological stains offer a simple method for identification of oligodendroglia in tissue sections. However, interpretation of results can be limited during damage, if oligodendroglia cells are difficult to distinguish due to changes in cell morphology and similarity to infiltrating immune cells. Electron microscopy is labor intensive but is the gold standard for identifying cell types and the mode of cell death, and may be the method of choice if other options are inadequate. The choice of antibody for immunocytochemistry depends on whether the cells are cultured or in situ, since cell surface markers that work well for cultured cells may prove inadequate for tissue sections. Other considerations include the efficient labeling of oligodendrocyte cell bodies and whether colabeling with cell death markers can be used. Many of these problems can be reduced with the use of transgenic animals that carry oligodendrocytespecific reporter genes. Unless labeling of the reporter gene product is lost because of cell death, there should be no question about the proper identification of oligodendrocytes during injury. The greatest advance offered by fluorescent mice is the opportunity to conduct live imaging experiments in vivo and in vitro. Viral techniques provide the possibility for spatially and temporally localized gene expression, rapid and flexible construct formation, and an opportunity for transfer of additional genes of interest in addition to the reporters. Generation of viral vectors takes considerably less time than generation of transgenic mice lines, and permits testing of several different constructs prior to mass production of the virus of interest. However, it will be necessary to use conventional cell identification methods to verify that transfer of fluorescent proteins or viral infection does not itself alter cellular vulnerability to subsequent insults. Stereotaxic microinjection of viruses allows spatial and temporal restriction of gene expression, and some level of control of transduction numbers. Thus, low cellular transduction efficiency for visualization of fluorescent reporters is beneficial in many imaging applications because individual cells and their processes are easily distinguished. The ability to visualize oligodendrocytes in living brain provides new opportunities to understand cell-cell interactions in diseases of the myelinating unit.
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ACKNOWLEDGMENTS The authors are grateful for the transgenic mice graciously provided by Frank Kirchhoff (PLP-dsRed) and Wendy Macklin (PLP-eGFP). This research was supported by NIH NINDS grants P01 NS032636 (to M.P.G.), R01 NS36265 (to M.P.G.), T32 NS07205 (to J.K.N.), and a grant from the Juvenile Diabetes Research Foundation (to M.P.G.).
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