Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 21

Calcium-Dependent
Proteases: The Calpains

Of the 15 members of the calpain family of proteases that have
been characterized since their initial discovery in 1964, only two,
the - and m-isoforms, are found in the CNS. There is a wide range

of activated calpain substrates in multiple neuronal subcellular
compartments in the post-ischemic neuron, including the synapse,
plasma membrane, lysosomes, ER, mitochondria, and nucleus ( 48 ).
Method . Calpains themselves are induced within 1 h after the
administration of transient focal ischemic in the adult rat brain
and can be visualized both by western blot and IHC ( 49 ). A more
useful and defi nitive assay for the induction of calpain activity—
though more involved technically—is to analyze subcellular frac-
tions, including synaptosomes, membrane and microsomal, and
cytoplasmic, for calpain enzymatic activity using one of many reliable
substrates, such as the collapsin family ( 50 ).

μ

In the well-documented model of classic apoptosis, both intrinsic
and extrinsic pathways lead to the activation of proteolytic and
nucelolytic enzymes—the effector caspases—which can target and
degrade specifi c cellular components/structures in a manner that
minimizes an infl ammatory response. Virtually all cases involve caspase
3 in this process, and demonstrating that the 32-kD pro-form of
this enzyme has been cleaved to its 19- and 17-kD active forms has
gained wide acceptance in the literature as evidence of apoptosis in
the CNS (but see below for exceptions). Apoptosis in the neonatal
brain is highly correlated with caspase-3 activation, enabling
the use of this marker as a predictor of long-term outcomes after
neonatal hypoxic-ischemic injury. Detecting the immediate upstream
activators of caspase-3, activated caspase 8 and caspase 9 are useful
for identifying whether the extrinsic or intrinsic pathways, respec-
tively (or both) are involved.
Method . Many excellent sources exist for protocols and detection
reagents for visualizing the presence of activated caspase 3; good
places to start are ( 51, 52 ). The use of pan caspase inhibitors, as
well as specifi c caspase inhibitors, is a particularly useful method to
identify the species involved.
Caveats . There are several documented cases where hypoxic-
ischemic-activation of caspase 3 does not result in cell death ( 1 ).
Studies have also shown that nonlethal activation of caspase 3 is
required for ischemic preconditioning via transient occlusion of
the mid-cerebral artery ( 53 ), as well as other forms of cyto-protec-
tion. There is increasing evidence for a role for Caspase-3 in non-
death-related functions in the CNS, such as differentiation (synaptic
pruning) and neuronal plasticity (learning and memory). All of
these points emphasize the need to pay close attention to the spe-
cifi c region being analyzed for an ischemic response and should
reinforce the need for multiple assessments or tests to verify that a
particular cell death pathway has been activated.

Activation of Caspases