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Table 2 Astrocytic markers and stains
| Procedures | Characteristics | Results | Observation |
|---|---|---|---|
| Hematoxylin and eosin stain (H-E) [23] | Routine staining for basic morphology | Nuclear details | • Astrocytes are difficult to identify (nuclei: small, pale, ovoidal, euchromatic and centrally situated, are mimicking those of small neurons; cytoplasm and cellular processes are undifferentiated from those of neighbouring neurons) |
| Cytoplasm extracellular protein components | • The occasionally pericellular hallo (autolitic modification) impose a differential diagnosis with the oligodendrocytes [23] | ||
| Mallory’s (phosphotungstic acid – hematoxylin) stain [24] | Special stain | Astrocyte processes (deep blue) | |
| Orange-acridine stain [24] | Special stain | Cellular body | • Reveals the astrocytic hyperplasia, without the modification of the cytoplasm aspects [24] |
| Metallic impregnations [23] | Nuclei | • Reveals the cellular characteristic star- shaped aspect | |
| • Del Rio Hortega method | • Special technique with ammonia silver carbonate | Cytoplasm processes | • The abundant cytoplasm surrounding the nuclei differentiates the astrocytes from oligodendrocyte |
| • Ramon y Cajal method (see Figures 1 and 2) | • Special technique with gold chloride | • The fibrillar aspect of the cytoplasm is due to the material formed by the aggregation of GFAP intermediate filaments | |
| • Golgi stain | • Special technique with silver nitrate | • The vascular endfeet are easy to identify. | |
| • Protoplasmic astrocytes, due to their proximity to the blood vessels, are able to contact the vessel directly by their cell body | |||
| • The perivascular hallo is considered to be an artefact [23]. | |||
| Electron microscopy [24] | Cytoplasm intermediate GFAP | • Cytoplasm pale , with lack of organelles | |
| • The clear, perivascular spaces indicate excessive dilatation of astrocytic processes due to water imbibitions | |||
| • The ultrastructural resemblance between normal and well differentiated neoplastic astrocytes is one of the arguments against the use of this method for positive diagnosis of low grade glioma [24] | |||
| Immunohistochemistry | GFAP represents an integrator of the cellular space, but it is also implicated in complex cellular events, such as cytoskeleton reorganisation, myelination, cellular adhesion and several signalling pathways [23, 24]. | ||
| • GFAP (intracytoplasmic protein, with 50 Kda molecular weight, considered the major component of glial fibrils and a marker of astrocytic differentiation) [23, 24] (see Figure 3) | • Golden standard for the definition of astrocytes | Cell body | • Fibrillary astrocytes contain a massive amount of GFAP in their cell bodies and processes unlike protoplasmic astrocyte. |
| • There are different clones of antiGFAP antibodie, characteristic to the different research | Cell processes (positive immunostaining reaction: brown spots) | • Protoplasmic astrocytes are much larger than their GFAP-defined profiles due to the presence of numerous fine processes that are GFAP-negative | |
| • Laboratories (e.g. GF2 DAKO clone; Astro 1) [23, 24] | • In astrocytomas, along with the enhancement of malignity, the intracellular quantity of GFAP is progressively reduced; therefore the evaluation of GFAP immunohistochemical staining will enable the immunophenotypic characterisation of the investigated glial tumors and the confirmation of histopathological diagnosis | ||
| • Not all the cells in the CNS that express GFAP are astrocytes (e.g: astrocyte-like cells from the SVZ-derived from radial glia, ependymal cells) [1, 25, 26] | |||
| • GFAP has also been located in rat kidney glomeruli and peritubular fibroblasts [1, 27], Leydig cells of the testis [1, 28], skin keratinocytes [1, 29], osteocytes of bones, chondrocytes of epiglottis, bronchus [1, 30], and stellate-shaped cells of the pancreas and liver [1] | |||
| S100B (belongs to the S100 family of EF-band calcium binding proteins [1, 31]). | There are different clones of anti S100 antibodies, characteristic to the different research laboratories (e.g. MAB079, CBL410.) | Cell membrane | • Expressed by a subtype of mature astrocytes that ensheath blood vessels and by NG2-expressing astrocytes [1, 31] |
| Other astrocytic markers | |||
| • GLT-1 (the glutamate transporters GLAST) [6] | • GLT-1 is expressed by all astrocytes and provide punctuate staining [6] | ||
| • Human EAAT2 (excitatory amino acids, 1 and 2 for human brain) [6] | |||
| • Glycogen granules [6] | Cytoplasm | ||
| • Gglutamine synthase (GS) [1, 32–35] | GS- enzyme that catalyzes the conversion of ammonia and glutamate to glutamine | Cytoplasm | GS is expressed also by oligodendrocytes [1, 32–35] |
| Kir4.1 (inwardly rectifying K+ channels) [1, 36, 37] | Kir4.1 are only expressed by a subset of astrocytes [37] | ||
| • Aquaporin 4 channels [1, 38] | Cell processes | • Aquaporin 4 channels is localized in some parts of the astrocytic processes rendering identification of the whole cell difficult to interpret [38] | |
| • AldhL1 (aldehyde dehydrogenase 1 family, member L1) [1, 39]. | Genome | • All astrocytes | |
| Battery of tests [40]• GFAP-driven GFP (green fluorescent protein) expressionGFAPprotein expression, S100ß immunostaining | Combinatorial approach | • Nine different classes of astrocytes has been identified, that included Bergmann glia, ependymal glia, fibrous astrocytes, marginal glia, perivascular glia, protoplasmic astrocytes, radial glia, tanycytes and velate glia [3, 40] | |
| • GFAP expression glutamate response [41] | • Define the phenotype of an astrocyte population as (GFAP+/NG2-; T+/R-) which is distinct from NG2-glia (GFAP-/NG2+ T-/R+) [41] | ||
| Dye-filling techniques [6, 42](e.g. sharp electrode, patch clamp recordings, single cell electroporation) | Special techniques that identify cells recorded in situ after filling them with a dye present in a micro-electrode | Cell body | • This technique has the advantage that the cells to be studied can be preselected in living tissue [6, 42] |
| It is suplemented by use of presumed astrocyte- | Cell processes | • However, proteins and promoter activation are subjects to change. Hence one can have a GFAP(-) cell that one should call an astrocyte because it has these other properties [6, 42] | |
| Specific promoters to drive synthesis of fluorescent proteins | • Using these procedures the domain organisation of astrocytes has been demonstrated along with the fusiform morphology of astrocyte nucleus, both playing a possible role in pathology [3, 43, 44] | ||
| Transgenic techniques (use transgenic mice) [1] | Visualize fluorescent astrocytes | Cell body | • Mice specific for astrocytes express [1] |
| Cell processes | - GFP | ||
| - Enhanced GFP under the human GFAP promoter (hGFAP-GFP mice) | |||
| - GLT-1-GFP | |||
| - BLBP-dsRed2 |
