Biology Reference
In-Depth Information
Caveats . This pathway might be expected to be activated in any
ischemic tissue in the CNS in which excitotoxicty is involved.
PARP-1 activation can be confused with necrosis or apoptosis
morphologically, depending on the particular PARP-1 pathway
and the timing of tissue fi xation/examination. Biochemical techniques,
such as analysis of cleavage products, co-immunoprecipitations, or
pull-downs, while informative, will not distinguish the particular
cell type in which the event is occurring, and one should, in parallel,
carry out immunostaining to make this assessment.
Method . Widely available markers that have been used to characterize
the intrinsic apoptotic pathway that is activated in delayed PCD
include active Caspase 2, the p53-inducible proteins PUMA and
PIDD, and the caspase-2 target protein bid. These can be identifi ed
using standard Western blotting, immunoprecipitations or pull-
downs, but require isolation of the appropriate brain regions (e.g.,
CA1 and CA3), and, in some cases, subcellular fractionation of the
subsequent cellular extracts. Mitochondrial fractions derived from
such extracts have been used to analyze the MPT and localization
of BH-3-only pro-apoptotic bcl-2 family members ( 56 ).
5.1.3. Late Hallmarks
Caspase-2 and p53-
Targeting Genes
Whether caused enzymatically or via ROS, detecting or visualizing
disruptions in nuclear DNA is a well-established and widely used
technique to assess the state of cells after ischemic insults. Positive
results for, in particular, TUNEL staining are, however, often used
as defi nitive proof of apoptotic (or other types of) cell death in
circumstances where such an assertion is clearly not warranted (see
below).
Method . Among the well-established methods for detecting the
presence of single- or double-stranded breaks in chromosomal DNA,
including in situ end labeling (ISEL), terminal deoxy-transferase
dUTP nick end labeling (TUNEL), and ligation-mediated PCR
(LMPCR), the TUNEL method, perhaps because it is technically
the most straightforward and readily available in reliable and afford-
able kit-form, is, by far, the most widely used and reported. Both
TUNEL staining and in-situ nick translation (ISNT) assay can be
carried out on either frozen sections or sections from paraformal-
dehyde perfusion-fi xed brains. Fragmentation of chromosomal
DNA into discrete fragments of increasing length—laddering—is,
likewise, an accepted hallmark of apoptotic cell death. While tech-
nically simple, in cases where levels of apoptotic cell death are low
or occur over an extended period of time, a signifi cant amount of
fresh wet tissue is required for DNA isolation and subsequent anal-
ysis via agarose gel electrophoresis, and so cannot always be
employed where the extent of apoptotic cell death is minimal or
confi ned to a small region of the CNS (i.e., the CA1 region of the
hippocampus following a global ischemic insult). In cases where
sample size, availability or signal-to-noise issues arise, one can still
Detection of Single-
and Double-Stranded
Breaks in DNA
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