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Microglia Identification Methods
Microglia Identification Methods 849
Microglia Identification Methods E Raibon and T Mo¨ller, University of Washington, Seattle, WA, USA ã 2009 Elsevier Ltd. All rights reserved.
Brief History of Microglial Identification Utilizing the silver staining method developed by Golgi, Ramo´n y Cajal first described an uncharacterized ‘third element’ within the central nervous system in 1913. In his definition, this term applied to all cells morphologically different from either neurons (first element) or astrocytes (second element). As we know today, this term actually applies to two distinct cell groups, oligodendrocytes and microglia. Pio del Rio Hortega formally described ‘microglia’ as their own unique entity in 1921 and provided the first systematic investigation of microglial cells in 1932. He described microglia as cells that ‘‘possess liberal ramified expansions and display migratory and phagocytic activity.’’ Both Ramo´n y Cajal and del Rio Hortega used silver staining and relied on cell morphology for their assessments. Their method reveals resting microglia as cells with characteristically elongated, almost bipolar, cell bodies with spine-like processes having many short branches. The nucleus of microglial cells is large in comparison with the small perikaryon. While this early morphological description of microglial cells still holds true today, the staining methods of Ramo´n y Cajal and del Rio Hortega were neither specific nor reproducible. Consequently, for an extended period of time there was little progress in our understanding of microglial cells. This quest was further hampered by the fact that microglia show a remarkable morphological plasticity, which ranges from highly ramified cells in normal central nervous system (CNS) tissue, over rod-shaped cells in inflamed cerebral cortex, to full-blown phagocytes in tissue with necrotic or apoptotic cells. This enormous hurdle was finally overcome by the development of ‘specific’stains, including lectins and monoclonal antibodies against epitopes expressed by microglia. This rekindled interest in microglial cells in the 1980s and set the stage for our current understanding of microglial cells in health and disease. However, one major caveat, inherent to all currently available microglial markers, should be pointed out early on. Virtually all available markers to identify microglia also detect other myeloid cells, such as macrophages and monocytes. The so-called microglia-specific labeling, as used here and in virtually all literature, actually only indicates that these labels do not cross-react with other CNS resident cells, such as neurons, astrocytes, or oligodendrocytes.
Lectins Lectins are carbohydrate-binding proteins ubiquitously found in species from plants to humans. They play an important role in cell adhesion and control of protein levels in the blood. Their ability to recognize specific terminal carbohydrates on glycoproteins or glycolipids makes them valuable markers. The a-Dgalactosyl-specific isolectin B4 (IB4) of Griffonia simplicifolia, an African medicinal plant, is a lectin widely used to label microglial cells in the rat, guinea pig, sheep, salamander, and even leech. Ricinus communis (castor bean) agglutinin-1 (RCA-1), a b-D-galactosylpreferring lectin, is frequently used to stain microglial cells in normal and pathological human tissue. The lectin from Lycopersicon esculentum (tomato) has a high affinity to poly-N-acetyllactosamine and is a good marker for mouse microglia. It has been used for fixed tissue but also for labeling of living microglia in brain slices. One common caveat for all lectins is their cross-reactivity with endothelial cells. While blood vessels are rarely mistaken for microglial cells, abundant staining of endothelial cells can hamper the identification of the smaller microglia. However, because of their ease of use, lectin stainings are frequently used in routine histology.
Antibodies The advent of monoclonal antibodies directed against specific epitopes paved the way to improved and more specific ways of microglial cells staining. Due to the myeloid lineage of microglial cells, most of the markers frequently used and presented here are also found in macrophages and monocytes. These markers are linked to common tasks of myeloid cells, such as phagocytosis or other immune functions. Even though most of the antibodies were developed against peripheral myeloid cells, protocols were successfully adapted to stain microglia in the CNS. Antibodies can be used in a variety of methods in immunocytochemistry and immunohistochemistry, flow cytometry, immunopanning, and fluorescent and magnetic cell sorting. Many antibodies can be used on fixed and/or frozen tissue and on paraffin-embedded sections (an antigen retrieval step is sometimes required for the most sensitive antibodies). Furthermore, primary antibodies from different species or different primary antibody isotypes allow for multiple labeling in the same sample. Among the most used antibodies to detect microglia are CD11b, CD68, and F4/80 antibodies, which recognize, respectively, antigens from the integrin family, a lysosomal glycoprotein, and the F4/80 antigens. CD11b is part of the complement receptor 3
850 Microglia Identification Methods
and is also known, under the name of the antibody clones, as Mac-1 (antimouse) and OX-42 (antirat). CD68, also known as macrosialin, is a lysosomal protein also found on the cell surface. Interestingly, the antibody ED1, used for a long time to stain rat microglia, was found to recognize rat CD68. F4/80 antibody is mainly used in mouse tissue and the F4/80 protein was recently found to be involved in immune functions within the eye, which constitutes a CNS-like functional entity. Antibodies against all three epitopes have been extensively used and stain both resting and activated microglia, along with circulating monocytes and macrophages. Interestingly, differentiation between monocyte-derived macrophages invading the CNS and microglial cells was made possible by using antiCD45 antibodies. CD45, also known as the leukocyte common antigen (LCA), is a member of the receptor protein tyrosine phosphatase family. Although expressed widely by microglia, monocytes, macrophages, and other hematopoietic cells, CD45 expression levels differ between microglia and macrophages. Microglia have a low level of expression, whereas circulating monocytes and peripheral tissue macrophages express high levels of CD45. This feature has been successfully exploited for the identification of microglial cells via ex vivo flow cytometry. ED2 and ED3 antibodies, raised against macrophage-specific lysosomal markers with still unknown function, stain activated microglia and macrophages in rats. However, the ED2 antibody does not label resting parenchymal microglia, and ED3 staining of resting microglia is variable. Other antibodies recognize epitopes linked to immune functions of microglial cells and are upregulated when microglia become activated. For example, major histocompatibility complex class (MHC) II antigen is virtually not
present on resting microglia, but is strongly upregulated in activated cells. However, MHC II upregulation varies depending on the type of CNS injury. MHC II is highly expressed during bacterial infection whereas its level is usually low in neurodegenerative diseases. Thus antibodies against the MHC II antigen, such as OX-6 (antirat), reveal activated microglial cells. However, they can also label other MHC IIexpressing cells, such dendritic cells, which have been shown to be involved in the CNS immune response. A recent addition to the microglial marker repertoire is ionized calcium-binding protein adaptor molecule 1(Iba1). Iba1 is a calcium-elongation factor (EF) hand protein, and is specific to the monocytic lineage, including microglia and macrophages. Iba1 protein function is still not completely understood; however, the Iba1 gene is highly conserved throughout evolution, suggesting an important role for this protein. Recent molecular studies pointed out a possible role in membrane ruffling and phagocytosis. The ubiquitous intracellular localization of Iba1 provides good staining, delineating whole microglial cells when used in immunohisto- and immunocytochemistry, as shown in Figure 1. It is worth noting that there is yet no available single marker to reliably distinguish resting from activated microglia. The distinction between the two still mainly relies on morphological criteria. Another way to distinguish resting from activated microglia is to detect the expression of activation-associated cytokines. However, cytokine secretion is not restricted to activated microglia. For example, astrocytes can secrete some of the same cytokines secreted by microglia. In order to identify the source of cytokine secretion, it is necessary to combine the intracellular cytokine
Figure 1 Fluorescent microscopy images of microglia stained with Iba1 antibody. (a) In the microglial N9 cell line, Iba1 antibody stains cytoplasm and processes, and the nucleus is clearly distinguished. (b) Iba1 staining of microglial cells (arrows) in the healthy adult mouse brain. Cells present a ramified morphology, with a small cell body and highly branched processes. (c) In the spinal cord of amyotrophic lateral sclerosis model mice with end-stage disease, Iba1 staining reveals activated microglial cells (arrows), with larger cell bodies and shortened processes. Scale bars ¼ 5 mm (a), 10 mm (b, c).
Microglia Identification Methods 851
staining with a cell-selective (e.g., microglia) marker. Double labeling is a useful approach that can help to distinguish resting and activated microglia. Many antibodies were developed and used before their corresponding antigen was actually known. Consequently, when used as cell markers, they are often referred to by the name of the antibody clone rather than the name of the antigen. This has inevitably led to some confusion. For example, the CD11b antigen, which together with CD18 forms the CR3 receptor, is also known as Mac-1 (mouse) and OX-42 (rat), the names of the respective antibody clones. The Table 1 gives an overview of the different categories of markers currently in use, with a focus on a classification by antigen names instead of clone names.
Genetic Labels Specific labeling of microglia (and all other cells) depends on the restricted expression of certain molecules, which are inherent only to particular cells. For example, the expression of Iba1 in microglial cells, but not in neurons, macroglia, or endothelial cells, is what makes the antibody directed against it such a valuable marker. If the genetics of a cell-type-specific
molecule are known and a promoter has been identified, this information can be used to devise transgenic animals which express a readily identifiable molecule – for example, green fluorescent protein (GFP) – under the control of the promoter. Such approaches have been used to make GFP-labeled neurons, astrocytes and oligodendrocytes. However, transgene expression in microglia and closely related macrophages has proved to be more challenging than expression in neuroectodermal cells. Early experiments using CD11b or CD14 promoters were disappointing. The first report of GFP-expressing microglia was a by-product of engineering a CX3CR1 knockin/knockout mouse. The investigators sought to interrupt the functional expression of CX3CR1 by inserting GFP into this gene. While originally constructed to investigate the role of CX3CR1, the engineered mice have been used by the microglial community to investigate microglial behavior in vivo. However, results from these animals have to be interpreted carefully. Even though these studies used heterozygous animals (CX3CR1þ/), the disruption of one genomic copy of CX3CR1 might reduce CX3CR1 expression, and thereby influence microglial motility. Other animals with GFPexpressing microglia have been reported since, using
Table 1 Commonly used microglial markers Marker
Name
Other names
Antigen (function)
Staining pattern
Lectins
Griffonia simplicifolia isolectin B4 Ricinus communis agglutinin-1 lectin Lycopersicon esculentum lectin CD11b
IGSA-IB4 IB4
a-D-Galactosyl group (cell adhesion and control of immune function)
RCA-1
b-D-Galactosyl group (cell adhesion and control of immune function)
Resting and activated microglia (rat) Vascular endothelium Patchy staining Resting and activated microglia (human) Vascular endothelium Patchy staining
Tomato lectin TL
Poly-N-acetyllactosamine group (cell adhesion and control of immune function) Complement receptor CR3; integrin family (adhesion) Coreceptor for lipopolysaccharide (endotoxin immune response) Receptor protein tyrosine phosphatase family (T cell receptor/ B cell receptor-mediated activation) Lysosomal glycoprotein (phagocytosis) Macrophage-specific lysosomal marker (unknown function)
Antibodies
CD14 CD45
Mac-1 OX-42 (rat) rmC5-3; Sa2-8 (mouse) LCA OX-1 (rat)
CD68
ED1 (rat)
ED2 ED3
ED2, ED3 (rat)
F4/80
F4/80; BM8 (mouse) I-A; I-E (mouse) RT1B; OX-6 (rat) Ionized calciumbinding adaptor molecule 1
MHC II Iba1
Resting and activated microglia (mouse) Vascular endothelium Patchy staining Resting and activated microglia Macrophages Activated microglia Monocytes Resting and activated microglia (low) Monocytes/macrophages (high)
Glycoprotein (role in immune regulation) MHC class II (antigen presentation)
Resting and activated microglia Macrophages Resting and activated microglia; not parenchymal microglia Macrophages Resting and activated microglia Macrophages Activated microglia
Calcium-EF hand protein (membrane ruffling; phagocytosis)
Resting and activated microglia Monocytes/macrophages
852 Microglia Identification Methods
Iba1, CD68, and F4/80 as microglia-specific promoters. Again, one has to keep in mind that the term ‘microglia-specific’ only refers to ‘not expressed in other CNS resident cells.’ All these animals express GFP in other myeloid cells (e.g., macrophages and monocytes). A variation in these transgenic approaches to drive expression of a marker gene is the use of microgliaspecific promoters such as F4/80 or CD11b in a cyclization recombinase/locus of ‘X-over’ P (Cre/Lox) system. Expression of Cre recombinase in microglial cells allows the microglia-specific knockdown of a gene flanked by loxP sites.
PK11195 The isoquinoline PK11195 is a ligand for the peripheral benzodiazepine receptor expressed in myeloid cells. In the CNS it selectively labels microglial cells, and PK11195 binding strongly increases in activated microglia. Based on these observations, radiolabeled PK11195 was successfully used in positron emission tomography (PET) studies to investigate microglial cells in human disease. For example, in Alzheimer’s disease (AD), hot spots with PK11195-labeled activated microglia can be readily identified, providing a prognostic tool for disease progression. In patients with multiple sclerosis (MS), activated microglial cells are found in active lesions identified in gadolinium-enhanced magnetic resonance images (MRIs). Similar results have been obtained for many other neurological diseases and have kindled the interest of neuroradiologists.
Other Labels Before specific antibodies against microglial epitopes became widely available, enzymes such as nucleoside diphosphatase or thiamine pyrophosphatase were used to identify microglial cells in tissue sections. Ecto-50 nucleotidase (CD73), another enzymatic marker used, is found on microglia cell membranes, and its expression is strongly upregulated in activated cells; however, it is not cell-type specific. The same is true for NADPH diaphorase, which has frequently been used to detect oxidative burst in activated microglial
cells. Furthermore, due to the necessity of maintaining enzyme activity, such stainings cannot be used on paraffin-embedded tissue. A less frequently used method to identify microglial cells exploits the increased pinocytosis of myeloid cells. Microglial cells incubated with a dye solution such as lucifer yellow will take up this dye into pinocytic vesicles, and therefore the cells become labeled over time. Another indirect labeling technique takes advantage of the phagocytic capability of fully activated microglia. Fluorescent carbocyanine dye (Di-I) injected into axons retrogradely labels neuronal cell bodies. When neuronal cell death is induced, Di-I is found in cells with microglial morphology. Although interesting for studies focusing on phagocytic functions of microglial cells, this type of indirect labeling will only mark cells which have differentiated into phagocytes. See also: Glial Cells: Microglia During Normal Brain Aging; Inflammation in Neurodegenerative Disease and Injury; Microglia Properties; Microglial Response to Injury; Neural Repair and Regeneration: Inflammatory Mechanisms and Cytokines.
Further Reading Banati R (2002) Visualising microglial activation in vitro. Glia 40: 206–217. del Rio-Hortega P (1932) Microglia. In: Penfield W (ed.) Cytology and Cellular Pathology of the Nervous System, pp. 482–534. New York: Hoeber. del Rio-Hortega P (1939) The microglia. Lancet 1: 103–106. Hirasawa T, Ohsawa K, Imai Y, et al. (2005) Visualization of microglia in living tissues using Iba1-EGFP transgenic mice. Journal of Neuroscience Research 81(3): 357–362. Imai Y, Ibata I, Ito D, et al. (1996) A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochemistry and Biophysics Research Communications 224(3): 855–862. Kettenmann H and Ransom BR (eds.) (2004) Neuroglia, 2nd edn. Oxford: Oxford University Press. Ramo´n y Cajal S (1911, 1995) Histology of the Nervous System of Man and Vertebraters (History of Neuroscience, No. 7), Swanson N and Swanson L (trans.). New York: Oxford University Press. Streit W (1990) An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4). Journal of Histochemistry and Cytochemistry 38(11): 1683–1696.
Microglia Identification Methods E Raibon and T Mo¨ller, University of Washington, Seattle, WA, USA ã 2009 Elsevier Ltd. All rights reserved.
Brief History of Microglial Identification Utilizing the silver staining method developed by Golgi, Ramo´n y Cajal first described an uncharacterized ‘third element’ within the central nervous system in 1913. In his definition, this term applied to all cells morphologically different from either neurons (first element) or astrocytes (second element). As we know today, this term actually applies to two distinct cell groups, oligodendrocytes and microglia. Pio del Rio Hortega formally described ‘microglia’ as their own unique entity in 1921 and provided the first systematic investigation of microglial cells in 1932. He described microglia as cells that ‘‘possess liberal ramified expansions and display migratory and phagocytic activity.’’ Both Ramo´n y Cajal and del Rio Hortega used silver staining and relied on cell morphology for their assessments. Their method reveals resting microglia as cells with characteristically elongated, almost bipolar, cell bodies with spine-like processes having many short branches. The nucleus of microglial cells is large in comparison with the small perikaryon. While this early morphological description of microglial cells still holds true today, the staining methods of Ramo´n y Cajal and del Rio Hortega were neither specific nor reproducible. Consequently, for an extended period of time there was little progress in our understanding of microglial cells. This quest was further hampered by the fact that microglia show a remarkable morphological plasticity, which ranges from highly ramified cells in normal central nervous system (CNS) tissue, over rod-shaped cells in inflamed cerebral cortex, to full-blown phagocytes in tissue with necrotic or apoptotic cells. This enormous hurdle was finally overcome by the development of ‘specific’stains, including lectins and monoclonal antibodies against epitopes expressed by microglia. This rekindled interest in microglial cells in the 1980s and set the stage for our current understanding of microglial cells in health and disease. However, one major caveat, inherent to all currently available microglial markers, should be pointed out early on. Virtually all available markers to identify microglia also detect other myeloid cells, such as macrophages and monocytes. The so-called microglia-specific labeling, as used here and in virtually all literature, actually only indicates that these labels do not cross-react with other CNS resident cells, such as neurons, astrocytes, or oligodendrocytes.
Lectins Lectins are carbohydrate-binding proteins ubiquitously found in species from plants to humans. They play an important role in cell adhesion and control of protein levels in the blood. Their ability to recognize specific terminal carbohydrates on glycoproteins or glycolipids makes them valuable markers. The a-Dgalactosyl-specific isolectin B4 (IB4) of Griffonia simplicifolia, an African medicinal plant, is a lectin widely used to label microglial cells in the rat, guinea pig, sheep, salamander, and even leech. Ricinus communis (castor bean) agglutinin-1 (RCA-1), a b-D-galactosylpreferring lectin, is frequently used to stain microglial cells in normal and pathological human tissue. The lectin from Lycopersicon esculentum (tomato) has a high affinity to poly-N-acetyllactosamine and is a good marker for mouse microglia. It has been used for fixed tissue but also for labeling of living microglia in brain slices. One common caveat for all lectins is their cross-reactivity with endothelial cells. While blood vessels are rarely mistaken for microglial cells, abundant staining of endothelial cells can hamper the identification of the smaller microglia. However, because of their ease of use, lectin stainings are frequently used in routine histology.
Antibodies The advent of monoclonal antibodies directed against specific epitopes paved the way to improved and more specific ways of microglial cells staining. Due to the myeloid lineage of microglial cells, most of the markers frequently used and presented here are also found in macrophages and monocytes. These markers are linked to common tasks of myeloid cells, such as phagocytosis or other immune functions. Even though most of the antibodies were developed against peripheral myeloid cells, protocols were successfully adapted to stain microglia in the CNS. Antibodies can be used in a variety of methods in immunocytochemistry and immunohistochemistry, flow cytometry, immunopanning, and fluorescent and magnetic cell sorting. Many antibodies can be used on fixed and/or frozen tissue and on paraffin-embedded sections (an antigen retrieval step is sometimes required for the most sensitive antibodies). Furthermore, primary antibodies from different species or different primary antibody isotypes allow for multiple labeling in the same sample. Among the most used antibodies to detect microglia are CD11b, CD68, and F4/80 antibodies, which recognize, respectively, antigens from the integrin family, a lysosomal glycoprotein, and the F4/80 antigens. CD11b is part of the complement receptor 3
850 Microglia Identification Methods
and is also known, under the name of the antibody clones, as Mac-1 (antimouse) and OX-42 (antirat). CD68, also known as macrosialin, is a lysosomal protein also found on the cell surface. Interestingly, the antibody ED1, used for a long time to stain rat microglia, was found to recognize rat CD68. F4/80 antibody is mainly used in mouse tissue and the F4/80 protein was recently found to be involved in immune functions within the eye, which constitutes a CNS-like functional entity. Antibodies against all three epitopes have been extensively used and stain both resting and activated microglia, along with circulating monocytes and macrophages. Interestingly, differentiation between monocyte-derived macrophages invading the CNS and microglial cells was made possible by using antiCD45 antibodies. CD45, also known as the leukocyte common antigen (LCA), is a member of the receptor protein tyrosine phosphatase family. Although expressed widely by microglia, monocytes, macrophages, and other hematopoietic cells, CD45 expression levels differ between microglia and macrophages. Microglia have a low level of expression, whereas circulating monocytes and peripheral tissue macrophages express high levels of CD45. This feature has been successfully exploited for the identification of microglial cells via ex vivo flow cytometry. ED2 and ED3 antibodies, raised against macrophage-specific lysosomal markers with still unknown function, stain activated microglia and macrophages in rats. However, the ED2 antibody does not label resting parenchymal microglia, and ED3 staining of resting microglia is variable. Other antibodies recognize epitopes linked to immune functions of microglial cells and are upregulated when microglia become activated. For example, major histocompatibility complex class (MHC) II antigen is virtually not
present on resting microglia, but is strongly upregulated in activated cells. However, MHC II upregulation varies depending on the type of CNS injury. MHC II is highly expressed during bacterial infection whereas its level is usually low in neurodegenerative diseases. Thus antibodies against the MHC II antigen, such as OX-6 (antirat), reveal activated microglial cells. However, they can also label other MHC IIexpressing cells, such dendritic cells, which have been shown to be involved in the CNS immune response. A recent addition to the microglial marker repertoire is ionized calcium-binding protein adaptor molecule 1(Iba1). Iba1 is a calcium-elongation factor (EF) hand protein, and is specific to the monocytic lineage, including microglia and macrophages. Iba1 protein function is still not completely understood; however, the Iba1 gene is highly conserved throughout evolution, suggesting an important role for this protein. Recent molecular studies pointed out a possible role in membrane ruffling and phagocytosis. The ubiquitous intracellular localization of Iba1 provides good staining, delineating whole microglial cells when used in immunohisto- and immunocytochemistry, as shown in Figure 1. It is worth noting that there is yet no available single marker to reliably distinguish resting from activated microglia. The distinction between the two still mainly relies on morphological criteria. Another way to distinguish resting from activated microglia is to detect the expression of activation-associated cytokines. However, cytokine secretion is not restricted to activated microglia. For example, astrocytes can secrete some of the same cytokines secreted by microglia. In order to identify the source of cytokine secretion, it is necessary to combine the intracellular cytokine
Figure 1 Fluorescent microscopy images of microglia stained with Iba1 antibody. (a) In the microglial N9 cell line, Iba1 antibody stains cytoplasm and processes, and the nucleus is clearly distinguished. (b) Iba1 staining of microglial cells (arrows) in the healthy adult mouse brain. Cells present a ramified morphology, with a small cell body and highly branched processes. (c) In the spinal cord of amyotrophic lateral sclerosis model mice with end-stage disease, Iba1 staining reveals activated microglial cells (arrows), with larger cell bodies and shortened processes. Scale bars ¼ 5 mm (a), 10 mm (b, c).
Microglia Identification Methods 851
staining with a cell-selective (e.g., microglia) marker. Double labeling is a useful approach that can help to distinguish resting and activated microglia. Many antibodies were developed and used before their corresponding antigen was actually known. Consequently, when used as cell markers, they are often referred to by the name of the antibody clone rather than the name of the antigen. This has inevitably led to some confusion. For example, the CD11b antigen, which together with CD18 forms the CR3 receptor, is also known as Mac-1 (mouse) and OX-42 (rat), the names of the respective antibody clones. The Table 1 gives an overview of the different categories of markers currently in use, with a focus on a classification by antigen names instead of clone names.
Genetic Labels Specific labeling of microglia (and all other cells) depends on the restricted expression of certain molecules, which are inherent only to particular cells. For example, the expression of Iba1 in microglial cells, but not in neurons, macroglia, or endothelial cells, is what makes the antibody directed against it such a valuable marker. If the genetics of a cell-type-specific
molecule are known and a promoter has been identified, this information can be used to devise transgenic animals which express a readily identifiable molecule – for example, green fluorescent protein (GFP) – under the control of the promoter. Such approaches have been used to make GFP-labeled neurons, astrocytes and oligodendrocytes. However, transgene expression in microglia and closely related macrophages has proved to be more challenging than expression in neuroectodermal cells. Early experiments using CD11b or CD14 promoters were disappointing. The first report of GFP-expressing microglia was a by-product of engineering a CX3CR1 knockin/knockout mouse. The investigators sought to interrupt the functional expression of CX3CR1 by inserting GFP into this gene. While originally constructed to investigate the role of CX3CR1, the engineered mice have been used by the microglial community to investigate microglial behavior in vivo. However, results from these animals have to be interpreted carefully. Even though these studies used heterozygous animals (CX3CR1þ/), the disruption of one genomic copy of CX3CR1 might reduce CX3CR1 expression, and thereby influence microglial motility. Other animals with GFPexpressing microglia have been reported since, using
Table 1 Commonly used microglial markers Marker
Name
Other names
Antigen (function)
Staining pattern
Lectins
Griffonia simplicifolia isolectin B4 Ricinus communis agglutinin-1 lectin Lycopersicon esculentum lectin CD11b
IGSA-IB4 IB4
a-D-Galactosyl group (cell adhesion and control of immune function)
RCA-1
b-D-Galactosyl group (cell adhesion and control of immune function)
Resting and activated microglia (rat) Vascular endothelium Patchy staining Resting and activated microglia (human) Vascular endothelium Patchy staining
Tomato lectin TL
Poly-N-acetyllactosamine group (cell adhesion and control of immune function) Complement receptor CR3; integrin family (adhesion) Coreceptor for lipopolysaccharide (endotoxin immune response) Receptor protein tyrosine phosphatase family (T cell receptor/ B cell receptor-mediated activation) Lysosomal glycoprotein (phagocytosis) Macrophage-specific lysosomal marker (unknown function)
Antibodies
CD14 CD45
Mac-1 OX-42 (rat) rmC5-3; Sa2-8 (mouse) LCA OX-1 (rat)
CD68
ED1 (rat)
ED2 ED3
ED2, ED3 (rat)
F4/80
F4/80; BM8 (mouse) I-A; I-E (mouse) RT1B; OX-6 (rat) Ionized calciumbinding adaptor molecule 1
MHC II Iba1
Resting and activated microglia (mouse) Vascular endothelium Patchy staining Resting and activated microglia Macrophages Activated microglia Monocytes Resting and activated microglia (low) Monocytes/macrophages (high)
Glycoprotein (role in immune regulation) MHC class II (antigen presentation)
Resting and activated microglia Macrophages Resting and activated microglia; not parenchymal microglia Macrophages Resting and activated microglia Macrophages Activated microglia
Calcium-EF hand protein (membrane ruffling; phagocytosis)
Resting and activated microglia Monocytes/macrophages
852 Microglia Identification Methods
Iba1, CD68, and F4/80 as microglia-specific promoters. Again, one has to keep in mind that the term ‘microglia-specific’ only refers to ‘not expressed in other CNS resident cells.’ All these animals express GFP in other myeloid cells (e.g., macrophages and monocytes). A variation in these transgenic approaches to drive expression of a marker gene is the use of microgliaspecific promoters such as F4/80 or CD11b in a cyclization recombinase/locus of ‘X-over’ P (Cre/Lox) system. Expression of Cre recombinase in microglial cells allows the microglia-specific knockdown of a gene flanked by loxP sites.
PK11195 The isoquinoline PK11195 is a ligand for the peripheral benzodiazepine receptor expressed in myeloid cells. In the CNS it selectively labels microglial cells, and PK11195 binding strongly increases in activated microglia. Based on these observations, radiolabeled PK11195 was successfully used in positron emission tomography (PET) studies to investigate microglial cells in human disease. For example, in Alzheimer’s disease (AD), hot spots with PK11195-labeled activated microglia can be readily identified, providing a prognostic tool for disease progression. In patients with multiple sclerosis (MS), activated microglial cells are found in active lesions identified in gadolinium-enhanced magnetic resonance images (MRIs). Similar results have been obtained for many other neurological diseases and have kindled the interest of neuroradiologists.
Other Labels Before specific antibodies against microglial epitopes became widely available, enzymes such as nucleoside diphosphatase or thiamine pyrophosphatase were used to identify microglial cells in tissue sections. Ecto-50 nucleotidase (CD73), another enzymatic marker used, is found on microglia cell membranes, and its expression is strongly upregulated in activated cells; however, it is not cell-type specific. The same is true for NADPH diaphorase, which has frequently been used to detect oxidative burst in activated microglial
cells. Furthermore, due to the necessity of maintaining enzyme activity, such stainings cannot be used on paraffin-embedded tissue. A less frequently used method to identify microglial cells exploits the increased pinocytosis of myeloid cells. Microglial cells incubated with a dye solution such as lucifer yellow will take up this dye into pinocytic vesicles, and therefore the cells become labeled over time. Another indirect labeling technique takes advantage of the phagocytic capability of fully activated microglia. Fluorescent carbocyanine dye (Di-I) injected into axons retrogradely labels neuronal cell bodies. When neuronal cell death is induced, Di-I is found in cells with microglial morphology. Although interesting for studies focusing on phagocytic functions of microglial cells, this type of indirect labeling will only mark cells which have differentiated into phagocytes. See also: Glial Cells: Microglia During Normal Brain Aging; Inflammation in Neurodegenerative Disease and Injury; Microglia Properties; Microglial Response to Injury; Neural Repair and Regeneration: Inflammatory Mechanisms and Cytokines.
Further Reading Banati R (2002) Visualising microglial activation in vitro. Glia 40: 206–217. del Rio-Hortega P (1932) Microglia. In: Penfield W (ed.) Cytology and Cellular Pathology of the Nervous System, pp. 482–534. New York: Hoeber. del Rio-Hortega P (1939) The microglia. Lancet 1: 103–106. Hirasawa T, Ohsawa K, Imai Y, et al. (2005) Visualization of microglia in living tissues using Iba1-EGFP transgenic mice. Journal of Neuroscience Research 81(3): 357–362. Imai Y, Ibata I, Ito D, et al. (1996) A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochemistry and Biophysics Research Communications 224(3): 855–862. Kettenmann H and Ransom BR (eds.) (2004) Neuroglia, 2nd edn. Oxford: Oxford University Press. Ramo´n y Cajal S (1911, 1995) Histology of the Nervous System of Man and Vertebraters (History of Neuroscience, No. 7), Swanson N and Swanson L (trans.). New York: Oxford University Press. Streit W (1990) An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4). Journal of Histochemistry and Cytochemistry 38(11): 1683–1696.