|
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
|
