Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 603

of positive expression of CD3 (CD3+) and CD4 (CD4+) or CD8
(CD8+) cells indicates helper or cytotoxic T cells, respectively
( 15, 21 ) (Table 2 ). Some other leukocytes also express these CD
molecules in different combinations: macrophages express low
levels of CD4; dendritic cells express high levels of CD8 and
monocytes express both CD4 and CD8 proteins ( 15, 22 )
(Table 2 ). In addition, CD3 markers can label Purkinje cells and
cytoplasm of plasma cells and macrophages, while CD4 proteins
are expressed on granulocyte surfaces ( 17 ).
To avoid confusion between T-lymphocyte identity as cyto-
toxic and helper T-lymphocyte subsets, double immunostaining of
CD3/CD8 or CD3/CD4 is more appropriate while single CD4
or CD8 labeling is not unique for only these cell types (Table 2 ).
The advantage of multiple immunostaining is its ability to char-
acterize the location of specifi c cellular components by virtue of the
degree of overlap between multiple fl uorescent labels where each has
a separate emission wavelength (i.e. colocalization analysis). However,
the immunohistochemical results can be affected by autofl uores-
cence when immunolabeled tissue is excited with light of short
wavelengths ( 23 ). Autofl uorescence is the natural property of living
cells (see Fig. 2 ). Endogenous fl uorophores (fi bronectin, lipofuscin,
and elastin) naturally emit light which increase with aging and/or
disease ( 24, 25 ). When older animals are used for ICH research,
autofl uorescence can pose a serious problem. Several solutions can
help to solve this problem: (a) using thinner sections if possible;
(b) chemically suppressing the autofl uorescence signal prior to
blocking for protein (c) utilizing technical advantages of imaging
software: fi lters, background correction, modulation of exposure
time, etc. (for details see ref. 29 ) (d) using an infrared scanner which
emits light with a high wavelength (700-800 nm) thus eliminating
autofl uorescence. Immunohistochemistry with infrared immunola-
beling allows analysis of the spatial T-cytotoxic and T-helper cell in
ICH-injured brain tissue and is suitable for quantitative studies.
While protocols for the above discussed techniques are well estab-
lished and published elsewhere ( 26-28 ), also see Subheading 2.11
below, the analysis of the brain tissue immunoreactivity by infrared
systems is a relatively new approach. Its primary advantages are
accuracy, simplicity, and low cost. In the detailed protocol described
below, we demonstrate assessment of T-lymphocyte immunoreac-
tivity following ICH. These data were extracted from 6-month-old
Sprague-Dawley rats that underwent intracerebral collagenase
injection to induce ICH. One brain section per animal at each time
point was used to demonstrate the immunohistochemistry tech-
niques. The two techniques presented here are immunofl uores-
cence and infrared immunolabeling quantifi cation. As the study of
brain immunoreactivity following cerebral hemorrhage is the sub-
ject of intensive research, no clear timeline is suggested to identify
T-cell activities. Existing information about the time course of
immune system activation is summarized in Table 1 .