Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 14

can easily mislead one into believing that cells are dead or dying
when, in fact, they recover and survive (demonstrated unequivo-
cally in vitro). Before undertaking the sometimes arduous task of
identifying the mechanistic basis for cell death, one should, there-
fore, fi rst verify that the administered insult does indeed cause a
signifi cant level of cell death in the target region. This can be
accomplished via standard histological staining (e.g., cresyl violet,
Nissal, etc.) to quantify, if necessary, the loss of cells (compared to
controls) in a given region over time. It should also be recognized
that at the early stages of PCD, there may be very few morphological
changes despite signifi cant genetic and protein modulations
(such as early caspase activation) having already occurred. In any
event, one is strongly encouraged to employ at least two, and if
possible three, biochemically/histologically distinct methods to
determine with reasonable certainty that a particular cell death
pathway has been activated.

An increasing understanding of the molecular details of the various
cell death pathways and their use as markers or indicators of those
pathways is steering the stroke, TBI and spinal cord injury fi elds
away from reliance on morphological criteria. This is a welcomed
development as distinct pathways can often manifest in identical or
similar neural/neuronal morphology, leading to confl icting and
confusing results in the literature. Overlaps in the effects on cellular
appearance between pathways and/or the triggering of multiple
pathways by a given insult can confound any assessment based
solely on morphology. Several of the most recently discovered
pathways—necroptosis for one—manifest phenotypic characteris-
tics of the “classic,” established cell death morphologies: apoptosis
and necrosis ( 14 ). The benefi ts of a morphological assessment for any
particular experimental protocol are (usually) low cost, speed, and
reproducibility. One should, however, regard most cellular appearances
as “skin deep”; they should (almost always) never be the sole
criterion for deciding on any particular pathway. The appear-
ance of a fl ashy car in your neighbor's driveway may be the result
of years of hard work or yesterday's lottery win.

3.1. Morphological
Assessments

In general, fresh frozen tissue is a faster, more convenient method
when comparing large groups of animals, and, as perfusion-fi xation-
based section-to-section variation is not an issue, frozen sections
are the method of choice for fl uorescence-based, quantitative, and
autoradiographic-based analysis, as well as for comparing adjacent
sections via in situ hybridization histochemistry. Brain perfusion
and paraffi n embedding is preferred if one needs to analyze many
samples from the same animal, allowing greater versatility in their
handling. One can also take advantage of the storage capabilities
of paraffi n-embedded tissue for comparative analysis, even years
later. As staining artifacts can arise due to variations in perfusion,

3.2. Tissue Fixation