Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 19

5. Analyzing Cell
Death

Because of the widespread commercial availability of many standard
tests and assessments for each stage, rather than list an exhaustive
catalog of protocols, we highlight those that have either worked
well in our hands or have gained widespread use in the stroke lit-
erature. To reliably determine which type of cell death has occurred
as a result of an ischemic insult, one should employ a minimum of
three biochemically/morphologically distinct assessments.

Among the well-documented biochemical events occurring during
the initiation of the apoptotic pathway is the non-physiological
translocation of phosphatidyl serine (PS) from the inner to the
outer leafl et of the plasma membrane. Compelling evidence also
shows that excessive potassium effl ux (with or without alteration in
intracellular calcium) is another common early event largely respon-
sible for apoptotic cell volume shrinkage and caspase activation
( 33-35 ). The cellular protein Annexin V specifi cally binds to PS in
the presence of calcium and its fi rst use as an indicator of apoptosis
was in a FACS-based analysis of B-cells ( 36 ). Based on its homology
to the calcium-binding Annexin family of proteins, this phospholipid-
binding protein was called Annexin V.

5.1. Apoptosis

5.1.1. Early Hallmarks

Method . There are many reliable and affordable Annexin V-based
kits available to test for the presence of PS on the outer leaf of the
plasma membrane. For an excellent treatment of this topic, including
step-by-step protocols, the reader should consult ( 37 ).
Caveats . Inversion of PS has also been observed during autophagic
cell death, and one should bear this in mind when designing,
carrying out and evaluating any particular assessment strategy.
Cells undergoing autophagy will not have demonstrable caspase
activation, chromatin condensation, or DNA laddering. It is there-
fore critical (to reiterate an earlier point), that at least two, and in
most cases, three independent analytical/ histological tests should
be conducted in order to be reasonably certain that one has identi-
fi ed a particular type of cell death.

Detection of Annexin V

Pro-apoptotic intracellular K + depletion has been identifi ed in neu-
ronal and non-neuronal cells ( 34, 38 ). Based on the K + mechanism
of apoptosis, it has become important to measure intracellular K +
depletion in dying cells. Intracellular and extracellular K + concen-
tration/content in brain tissues and cultured cells can be assessed
using the K + -selective electrodes ( 39-41 ) or cell-permeable ace-
toxymethyl ester derivative of the fl uorescent potassium-sensitive
dye 1,3-benzenedicarboxylic acid, 4,4¢-[1,4,10,13-tetraoxa-7,16-
diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]
bis-, tetrakis [(acetyloxy) methyl] ester (PBFI-AM). PBFI-AM stock

Cytofl uorometric
Determination
of Intracellular K +
by PBFI-AM Imaging