Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 16

has no auto-fl uorescence artifacts, and can allow for very high
resolution of cyto-architecture. Immuno-fl uorescent signals, on
the other hand, can be quantifi ed, can better enable one to detect
relatively rare cell death events using lower power objectives, and
must be used if double or triple staining of subcellular architecture
(or confocal analysis) is required.
Due to their lack of myelination and high water content, brains
from juvenile animals are prone to trauma-induced artifacts during
dissection and removal, and should be handled accordingly. It is,
likewise, hopefully obvious that looking for more subtle hallmarks
of tissue damage in experimental paradigms involving traumatic
injury can be hindered from artifacts introduced during tissue
dissections in the CNS.

4. Categories/
Types of Cell Death

It is important to emphasize that in addition to the most well-
characterized form of PCD—apoptosis—there are others, including
autophagic and mixed (or hybrid) cell death (such as necroptosis,
apopnecrosis). Some have proposed that some necrotic death may,
as well, be regulated by a cell death program ( 22-24 ). This type of
cell death, however, often appears as the hybrid (or mixed) form.
For example, “programmed necrosis” may be mediated by the caspase-
independent apoptotic gene apoptosis-inducing factor (AIF) ( 23 ).
In general, mixed cell death can be thought of as a regulated form
of cell death having morphological and biochemical characteristics
of both apoptosis and necrosis.
In parallel with the discovery of novel cell death pathways, the
tools and methodologies available for their classifi cation have
evolved considerably. Despite identifi cation of numerous cell death
regulatory genes, morphological alterations remain as the “Gold
Standard” for identifying typical apoptosis and mixed cell death
with both apoptotic and necrotic features. It is widely recognized
that apoptotic cells undergo marked cell body and nuclear shrinkage,
chromatin condensation, formation of membrane blebbing, and
apoptotic bodies. While an apoptotic cell may show swollen mito-
chondria, its membrane and plasma membrane remain intact. Mixed
cell death commonly results in atypical morphological features
of cell body, nuclear, mitochondrial, and cell membranes ( 3, 4, 25 ).
The widespread use of a growing number of reliable, pathway-specifi c
molecular markers is allowing for a more accurate, reproducible
picture of cell fate in the complex, highly dynamic post-ischemic
milieu. To maintain consistency between the many experimental
approaches devoted to studying salvageable tissue in the ischemic
CNS, it will be useful to establish a consensus on the most useful and
accurate types of analyses and assessments for any given lesion; such

4.1. Apoptosis,
Necrosis, and Mixed
Cell Death