The Onset of Apoptosis of Neurons Induced by Ischemia-Reperfusion
Injury Is Delayed by Transient Period of Hypertension in Rats
M. SMRČKA1, M. HORKÝ, F. OTEVŘEL, Š. KUCHTIČKOVÁ,
V. KOTALA, J. MUŽÍK
Institute of Pathophysiology, Medical School, Masaryk
University, and 1Department of Neurosurgery,
University Hospital, Brno, Czech Republic
Received January 16, 2002
Accepted May 29, 2002
Summary
We investigated the potential neuroprotective effect of
transient hypertension on neuronal cell death induced by
ischemia-reperfusion. Recovery of neurons, terminally
differentiated cells, is almost entirely dependent upon active
transcription and repair of DNA damage. We focused on the
histochemical detection of distribution of NOR (argyrophylic
nucleolar proteins) reflecting nucleolar integrity,
immunohistochemical detection of PARP-1 (poly(ADP-ribose)
polymerase-1), MADD (mitogen-activated death domain), a protein
accumulated in nucleoli upon stimulation by ischemia, the active
form of caspase-3, a universal proteolytic enzyme of apoptosis.
The terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin
nick-end-labeling method (TUNEL) proved the presence of in situ
DNA fragmentation. We used the model of transient focal cerebral
ischemia in rats with occlusion of middle cerebral artery. In
experimental group of rats, the transient hypertension was
induced by constriction of the abdominal aorta. The period of
ischemia lasted 15, 30, 60 and 120 min followed by 48 h of
reperfusion. We examined the frontal lobe of the ipsilateral
hemisphere for apoptosis of neurons and compared it with the
intact brain tissue. In normotensive rats with transient focal
cerebral ischemia, we found disintegrated nucleoli of cortical
as well as subcortical neurons at all investigated periods of
ischemia, whereas the neurons of intact animals showed compact
nucleoli with a few satellites. Nuclear positivity for MADD and
PARP-1 was apparent in the neocortex after 15 min and peaked
after 30 min of ischemia. On the other hand, the subcortical
neurons showed nuclear positivity after 60 and 120 min. The
immunohistochemical reaction for active caspase 3 was apparent
after 30 min onwards predominantly in the cortex. The TUNEL
staining was distinct after 60 and 120 min. In hypertensive
rats, we found nucleolar disintegration, positivity for MADD,
PARP-1 and caspase 3 after 30 min cortically and subcortically,
followed by TUNEL positive staining of cortical neurons after 60
and 120 min. In summary, we detected delayed activation of
neuronal apoptosis in transiently hypertensive rats with focal
cerebral ischemia compared to normotensive animals. The
apoptotic phenotype was confirmed by a panel of complementary
methods showing rapid proteolysis-nucleolar segregation, MADD,
PARP-1 and caspase-3 positivity as well as ultimate DNA
fragmentation proved by the TUNEL assay.
Key
words
Neuroprotection • Hypertension • MADD • PARP-1 •
Caspase-3 • Apoptosis
Reprint
requests
Marcel Horký,
MD, Department of Pathological Physiology, Faculty of Medicine,
Masaryk University, Komenského náměstí 2, 662 43 Brno, Czech
Republic, Fax +420-5-42126550, E-mail:
mhorky@med.muni.cz
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