Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 602

affect primary antibody immunoreactivity and strengthens the
fl uorescent signal. A disadvantage of the indirect method is that
the secondary antibodies may produce nonspecifi c staining and
result in cross-reactivity which is avoided by labeling with primary
antibodies. However, labeled primary antibodies are more expen-
sive and are not as readily available compared to the variety of
labeled secondary antibodies. Typically, two methods, both quan-
titative and qualitative, should to be used to ensure assessment of
the fi nal results.
Fluorescent phenotyping allows monitoring of the local cellu-
lar immune response by visualizing lymphocytes: their abundance,
morphology and types, as well as the dynamics of spatial and tem-
poral distribution. The number of T-lymphocytes and their subsets
in the brain can be used to evaluate the progress of immune
response to ICH. T-cell subsets can be defi ned by recognition of
cluster of differentiation (CD) molecule proteins on the lympho-
cyte surface ( 15, 17 ) (Table 2 ). There are three CD molecules
which are commonly used as markers to identify T-lympocyte sub-
populations. CD3 is specifi c for all T-lymphocytes and is the basis
of its segregation from B-lymphocytes. CD3 is a multisubunit
complex of proteins that associates directly with the T-cell antigen
receptor (TCR, heterodimer composed of either alpha, beta or
gamma and delta chains) ( 18, 19 ). CD3 is composed of fi ve invari-
ant polypeptide chains that associate to form three dimers: a het-
erodimer of gamma and epsilon chains, a heterodimer of delta and
epsilon chains, and a heterodimer of two zeta chains or zeta and eta
chains. CD8, a cell-surface glycoprotein, is a two-chain complex
(alpha-alpha or alpha-beta) receptor that binds class I major histo-
compatibility complex (MHC) molecules presented by the anti-
gen-presenting cells ( 15, 18 ). A primary function of CD8 is to
facilitate antigen recognition by TCR and to strengthen the affi nity
of TCR-antigen interactions. CD4 is a membrane glycoprotein
that contains four extracellular immunoglobulin-like domains.
CD4 increases the avidity of interaction between the TCR and
antigen II MHC molecules presented by APC ( 20 ). A combination

Table 2
Characterization of lymphocyte cell types by CD surface
molecules

Leukocyte cell types CD3 CD 4 CD8 References

Monocytes − + + ( 15 )

Natural Killer cells − − + ( 21 )

T-cytotoxic cells + − + ( 22 )

T-helper cells + + − ( 20 )