Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 597

and heme catabolism products, all aggravate an initial impact
contributing to secondary ICH-injury development ( 7, 8 ).
Current concepts outline a two-step immune system impact:
initially, it exacerbates ICH-injury progression, while at later time
points it appears to be involved in brain tissue repair and is benefi -
cial to post-ICH brain remodeling ( 2, 3, 8, 9 ). The infl ammation
and complement cascade improves post-stroke neuroregeneration
via release of the growth factors and clearance of cellular debris ( 2 ).
Adaptive immunity via bystander mechanisms are involved in main-
tenance of administrated stem cell activity in experimental ICH
( 10, 11 ).
Since the immune system is extensively involved in ICH injury
progression and recovery, it is a potential therapeutic target. It is
worthy to note that under experimental settings, the method of
hemorrhage induction and species choice alters the ability to detect
the level and time course of immune system markers ( 12 ).
Comparison of collagenase and blood injection ICH models dem-
onstrates accelerated cytokine release (tumor necrosis factor-alpha,
TNF-alpha), delayed expression of matrix metalloproteinase
(MMP)-related genes, and cytotoxic T-cell markers (CD8-alpha)
in collagenase-induced ICH, while expression of microglia/
macrophage markers does not differ between these two models
( 4, 8, 13, 14 ) (Table 1 ).
Dependent on the research focus, different tissues can be
obtained and studied. Numerous methods such as immunohis-
tochemistry (IHC), histology, Western blot (WB), and polymerase
chain reaction (PCR) can be performed on the fi xed and stored
tissues, while others (fl ow cytometry (FC), blood cell counting)
require an immediate processing of the freshly harvested tissue
samples. To avoid technical artifacts, special care should be taken
during sample collection as immune cells are highly sensitive to
tissue degradation and can be easyily activated.
The assessment techniques differ in their sensitivity and are
refl ective of the time course of immune response to ICH brain
injury (Table 1 ). For instance, cytokine-related gene expression
precedes expression of cytokine proteins as detected by immuno-
histochemistry in paraffi n-embedded brain sections (IP, Table 1 ).
While IP identifi ed the peak of interleukin 6 (IL-6) and TNF-alpha
activity at 2 days following intracerebral blood injection, it was
detected at 6 h post-ICH using messenger ribonucleic acid assay
(mRNA, Table 1 ) ( 8, 13 ). In addition, the peak cytokine protein
levels, revealed by enzyme-linked immunosorbent assay (ELISA),
appeared to precede mRNA detection (interleukin 1 beta (IL-1
beta), Table 1 ). There are no clear visible differences between
Western Blot and immunohistochemistry (using Avidin Biotin
Complex (ABC) method) sensitivity in detection of activated com-
plement cascade markers ( 3, 6 ). In contrast, the sensitivity of FC
for detecting ICH brain injury is not clear. At days 1 and 4, post-
ICH no CD8-alpha positive cells in animal brains were identifi ed