Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 25

5.4. Necrosis

Evidence of necrotic cell death can be found in all experimental
models of ischemia, and virtually all assessments are based on
morphological criteria. The hallmarks of necrotic cell death are
well established: loss of membrane integrity, nuclear, cytoplasmic
and organelle swelling (leading eventually to lysis), disintegration
of chromatin structure into a particulate, amorphic structures
(fl occulation), and breakdown of cytoplasmic and organelle struc-
ture and function. In addition, the release of cellular contents into
the extracellular space will invariably result in an infl ammatory
response around the site of injury.
Method . Compared to regulated forms of cell death, disruption of
the plasma membrane happens relatively early in necrosis. One
can take advantage of this through intra-cerebroventricular injec-
tion (following a time-course after the adminstration of the insult)
of membrane-impermeable dye markers such propidium iodide
(PI). Upon sacrifi ce, cells in the region of interest that are
PI-positive and negative for apoptotic (or other PCD) markers
are considered to have undergone necrosis. Another hallmark of
necrosis, lysosomal disruption, releasing organelle-specifi c contents
into the cytoplasm, is accomplished through immunological detec-
tion (via histology or Western blot) of an appropriate antigen,
such as cathepsin b. Release of this protease will produce a unique
50-kD cleavage product of PARP-1.
Caveats . There have been a few reports of lysosomal disruption as
a consequence of PCD, including apoptosis.

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