Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 24

detect the presence of laddering through amplifi cation of the frag-
ments via ligation-mediated polymerase chain reaction (LMPCR).
Two excellent, very detailed resources for TUNEL and related
protocols can be found here: ( 57, 58 )
Caveats . There are several issues one should consider when employ-
ing the TUNEL staining. First, though it is often used as a unilat-
eral indicator of apoptosis, TUNEL does not discriminate between
apoptotic and non-apoptotic mechanisms of cell death, as DNA
strand breaks have been demonstrated in necrotic (and other types)
cell death ( 59 ). Second, although it is commonly thought that
TUNEL preferentially labels double-stranded breaks over single-
stranded, it has been shown to label both types. Third, there are
cases in which apoptosis proceeds in the absence of single-stranded
DNA breaks. Lastly, the sensitivity of TUNEL staining is about
10-fold lower than the more technically involved ISEL, leading in
certain cases to signifi cant, even gross underestimations of the timing
and extent of cell death in the mammalian CNS ( 60 ). To be reason-
ably certain of a particular type of cell death, one should, therefore,
employ the TUNEL method in combination with at least two
other mechanistically distinct assessments.

5.2. Infl ammation-
Induced PCD

Methods . Hallmarks: Induction of activated ICE occurs within
hours of permanent MCAO and TBI, while a peak in IL-1 produc-

tion at 24 h post-injury; both can be detected via Western blot.
An increase in activated microglia will occur even many weeks
following the injury which, along with reactive astrocytes, can
release both neuroprotective (IGF-1, BDNF) and neurotoxic (TNF-

β

α ,

IL-1 , IL-6, NO, ROS, etc.) agents. Within 4-6 h following injury,

peripheral blood leukocytes can be detected adhering to the vessel
walls surrounding the site injury with subsequent infi ltration and
can be identifi ed histologically.

β

Method . Two reliable and unequivocal markers for autophagy are
beclin-1, which, together with phosphatidyl inositol kinase III (PIK
III), forms a complex that initiates the formation of the autopha-
gosome, and LC3 (microtubule-associated protein light chain 3),
a membrane-bound marker for phagophores and autophagosomes.
Antibodies to both of these are commercially available and both
can be visualized histologically and/or via Western blot, though
the latter method requires subcellular fractionation for reliable
quantifi cation. An excellent treatment of these methods is provided
in Chu et al. ( 61 ).
Caveats . The autophagic pathway is often triggered in concert with
other PCD pathways, in particular apoptosis. When devising a
strategy for identifying the pathway(s) responsible for cellular/tis-
sue damage resulting from any new experimental protocol, it may
be useful to employ a panel of markers to identify all of the (well
established) pathways that have been involved.

5.3. Autophagy