Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 2007–2012, March 1997
Inhibition of interleukin 1? converting enzyme family proteases
reduces ischemic and excitotoxic neuronal damage
(infarction?neurological deficit?caspase inhibitors?transient focal ischemia)
HIDEAKI HARA*, ROBERT M. FRIEDLANDER†‡, VALERIA GAGLIARDINI†, CENK AYATA*, KLAUS FINK*,
ZHIHONG HUANG*, MASAO SHIMIZU-SASAMATA*, JUNYING YUAN†§, AND MICHAEL A. MOSKOWITZ*¶
*Stroke and Neurovascular Regulation, Neurosurgical Service, Departments of Surgery and Neurology, and†Cardiovascular Research Center, Department of
Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129; and‡Neurosurgical Service, Department of Surgery,
Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Communicated by H. Robert Horvitz, Massachusetts Institute of Technology, Cambridge, MA, December 23, 1996 (received for review November 11, 1996)
family plays a pivotal role in programmed cell death and has
been implicated in stroke and neurodegenerative diseases. Dur-
ing reperfusion after filamentous middle cerebral artery occlu-
sion, ICE-like cleavage products and tissue immunoreactive
interleukin 1? (IL-1?) levels increased in ischemic mouse brain.
Ischemic injury decreased after intracerebroventricular injec-
tions of ICE-like protease inhibitors, N-benzyloxycarbonyl-Val-
Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-
Asp-chloromethylketone, or a relatively selective inhibitor of
CPP32-like caspases, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-
fluoromethylketone, but not a cathepsin B inhibitor, N-
IL-1? levels in ischemic mouse brain and reduced tissue damage
when administered to rats as well. Only z-VAD.FMK and acetyl-
Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling,
did not attenuate the ischemia-induced increase in tissue IL-1?
levels. The three cysteine protease inhibitors significantly im-
proved behavioral deficits, thereby showing that functional re-
covery of ischemic neuronal tissue can follow blockade of en-
zymes associated with apoptotic cell death. Finally, we examined
the effect of z-VAD.FMK on excitotoxicity and found that it
protected against ?-amino-3-hydroxy-5-methyl-4-isoxazole pro-
pionate-induced or to a lesser extent N-methyl-D-aspartate-
induced excitotoxic brain damage. Thus, ICE-like and CPP32-
like caspases contribute to mechanisms of cell death in ischemic
and excitotoxic brain injury and provide therapeutic targets for
stroke and neurodegenerative brain damage.
The interleukin 1? converting enzyme (ICE)
Apoptosis is a cell suicide mechanism under active cell control.
In the nematode Caenorhabditis elegans, the product of the
ced-3 gene is essential for programmed cell death (1, 2).
Members of the mammalian interleukin 1? (IL-1?) converting
enzyme (ICE) family are homologs of C. elegans Ced-3 (3).
Apoptosis has been observed in striatal and cortical neurons
in animal models of stroke (4, 5) and may play a role in
neuronal injury induced by ischemia. ICE-like proteases pro-
mote neuronal cell death induced by trophic factor deprivation
in vitro (6, 7). Blocking ICE-like protease activity delays
motoneuron death induced in vitro by trophic factor depriva-
tion and in vivo during development (8).
Although resistance to ischemia in transgenic mice overex-
pressing Bcl-2 or deficient in p53 provides evidence for the
importance of gene regulation to brain injury and repair, the
molecular mechanisms of apoptosis after ischemic brain injury
are unknown (9, 10). A role for apoptosis in ischemic injury
was proposed based on morphological (terminal deoxynucle-
otidyltransferase-mediated UTP end labeling staining) and
biochemical (DNA fragmentation) evidence (4, 5, 11). More-
over, IL-1? mRNA and protein expression increases in isch-
emic tissue during permanent focal (12, 13) and global isch-
emia (14, 15). Endogenously produced mature IL-1? plays an
important role in hypoxia-mediated apoptosis in vitro (16).
Furthermore, IL-1 receptor antagonist administration inhibits
ischemic and excitotoxic neuronal damage in the rat, a fact that
implicates IL-1? in ischemic pathophysiology (17, 18).
The ICE family now consists of 11 members (19–33) that can
be divided into three subfamilies, the ICE subfamily, the CPP32
subfamily, and the Ich-1 subfamily, which have recently been
named caspases (34). Each of the 11 family members contains a
QACRG at the active site. Peptide (acyloxy) methylketones,
which are active site inhibitors of ICE-like and CPP32-like
caspases, have recently been developed (35). N-benzyloxycar-
a relatively nonselective inhibitor that blocks both ICE-like and
CPP32-like caspase activity. Acetyl-Tyr-Val-Ala-Asp-chloro-
methylketone (YVAD.CMK) is more selective for ICE, whereas
residue in the P4 position, inhibits CPP32-like caspases more
selectively (36). We examined the effects of these three compet-
itive irreversible synthetic peptide inhibitors on brain injury as
well as behavioral deficits after middle cerebral artery (MCA)
occlusion to determine whether inhibition of proteins associated
with programmed cell death can lead to functional neurological
recovery. We also examined whether histological damage due to
?-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), a
model of excitotoxic brain damage in stroke and neurodegenera-
members. Our findings have important therapeutic implications
for the treatment of stroke and neurodegeneration.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
Copyright ? 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA
PNAS is available online at http:??www.pnas.org.
Abbreviations: AMPA, ?-amino-3-hydroxy-5-methyl-4-isoxazole
propionate; i.c.v., intracerebroventricular(ly); IL-1?, interleukin 1?;
ICE, IL-1? converting enzyme; MCA, middle cerebral artery;
NMDA, N-methyl-D-aspartate; rCBF, regional cerebral blood flow;
YVAD.CMK, acetyl-Tyr-Val-Ala-Asp-chloromethylketone; z-
ylketone; z-DEVD.FMK, N-benzyloxycarbonyl-Asp(OMe)-
Glu(OMe)-Val-Asp(OMe)-fluoromethylketone; z-FA.FMK, N-
§Present address: Department of Cell Biology, Harvard Medical
School, Boston, MA 02115.
¶To whom reprint requests should be addressed. e-mail: Moskowitz@
MATERIALS AND METHODS
Drugs. z-VAD.FMK, z-DEVD.FMK, and N-benzyloxycar-
bonyl-Phe-Ala-fluoromethylketone (z-FA.FMK) were obtained
from Enzyme Systems Products (Dublin, CA), and YVAD.CMK
was obtained from Bachem. Compounds were dissolved in 0.2–
0.6% dimethyl sulfoxide (MCB Chemical, prepared with 0.1 M
PBS, pH 7.4) and made fresh daily. AMPA and NMDA were
obtained from Sigma and dissolved in 0.1 M PBS.
Physiology. Regional cerebral blood flow (rCBF) was deter-
mined by laser-Doppler flowmetry (PF2B; Perimed, Stockholm)
using a flexible 0.5-mm fiber optic extension to the master probe
from the bregma) on the intact skull. rCBF, blood pressure, and
heart rate were monitored as described (37), and heart rate was
monitored from the arterial blood pressure pulse. Arterial blood
samples (30 ?l) were analyzed for oxygen (PaO2) and carbon
dioxide (PaCO2) before and during ischemia and 30 min after
reperfusion using a blood gas?pH analyzer (Corning 178; Ciba
Corning Diagnostics, Medford, MA). Core temperature was
maintained at approximately 37?C with a thermostat (FHC,
Brunswick, ME) and a heating lamp (Skytron, Daiichi Shomei,
blood pressure, and heart rate were determined as described
ischemic cortex at the bregma and 8 mm from midline.
Ischemia Model: Mouse. Spontaneously ventilating adult male
SV-129 mice (n ? 173; 19–23 g; Taconic Farms) were initially
anesthetized with 1.0% and maintained on 0.4–0.8% halothane
in 70% N2O and 30% O2using a Fluotec 3 vaporizer (Colonial
nylon monofilament (Ethicon, Somerville, NJ) coated with a
mixture of silicone resin (Xantopren; Bayer Dental, Osaka) and
38). The procedure lasted 15 min, and the anesthesia was dis-
continued. Two hours later, animals were briefly re-anesthetized
with halothane, and the filament was withdrawn. Eighteen hours
after reperfusion, the forebrains were divided into five coronal
(2-mm) sections using a mouse brain matrix (RBM-2000C; Ac-
tivational Systems, Warren, MI), and the sections were stained
with 2% 2,3,5-triphenyltetrazolium chloride (Sigma). The in-
farcted areas were quantitated by an image-analysis system
(Bioquant IV; R & M Biometrics, Nashville, TN) and calculated
by summing the volumes of each section determined directly (39)
or indirectly by the following formula: contralateral hemisphere
statistical significance was achieved by both methods of analysis,
only data from the direct method are presented. Brain swelling
was calculated according to the following formula: [(infarct
? 100?contralateral volume (%)]. For histological evaluation of
neuronal damage at 18 hr and 3 days after ischemia, sections (12
?m) were stained with hematoxylin?eosin.
z-VAD.FMK (13.5, 40, or 120 ng), z-FA.FMK (120 ng), and
z-DEVD.FMK (40 or 120 ng) were injected intracerebroven-
tricularly (i.c.v.) twice (2 ?l per dose; bregma: 0.9 mm lateral,
0.1 mm posterior, 3.1 mm deep) 15 min before ischemia and
immediately after reperfusion. YVAD.CMK (100 or 200 ng)
was injected i.c.v. 45 min before ischemia and immediately
after reperfusion. In separate experiments, z-VAD.FMK (a
single 80-ng dose) was injected i.c.v. either immediately or 1 hr
after reperfusion, and the animal killed 18 hr later.
Ischemia Model: Rat. Adult male Sprague–Dawley rats (n ?
39; 280–330 g; Charles River Breeding Laboratories) were
initially anesthetized with 2.0% and maintained by 1.0%
The left MCA was occluded with a 3–0 nylon monofilament
(Ethicon) with its tip rounded by heating near a flame. The
filament (23 mm long) was inserted from the left external
carotid artery and advanced into the internal carotid artery.
The distance from the suture tip to the left common carotid
artery bifurcation was approximately 20 mm. Two hours after
ischemia, animals were briefly re-anesthetized with halothane,
and the filament was withdrawn. Twenty-two hours later, the
brains were stained with 2,3,5-triphenyltetrazolium chloride
for morphometric analysis. z-VAD.FMK (8, 27, or 80 ng) was
injected i.c.v. 15 min before ischemia and 10 min after reper-
fusion in 2 equal doses (4 ?l per dose; 1.5 mm lateral, 0.8 mm
posterior from bregma, 4.0 mm below bone surface).
Neurological Deficit. Mice and rats were tested for neuro-
logical deficits and scored as described by Bederson et al. (41)
with the following minor modification: 0, no observable neu-
rological deficits (normal); 1, failure to extend right forepaw
(mild); 2, circling to the contralateral side (moderate); 3, loss
of walking or righting reflex (severe). The rater was naive to
the treatment group, and assessments were made at 10 min and
2 hr during ischemia, and again 18 hr after reperfusion.
Western Blot Analysis. Mouse brain tissue was lysed in a
RIPA buffer (0.15 M NaCl?0.05 M Tris?HCl, pH 7.2?1%
Triton X-100?1% sodium deoxycholate?0.2% SDS) in the
presence of a protease inhibitor cocktail (leupeptin, phenyl-
4?C. After ultracentrifugation at 4?C (TLA 45 type rotor at
35,000 ? g), supernatants were diluted, boiled for 3 min, and
loaded on an SDS?PAGE gel (12%; 10 ?g per lane). An
affinity purified polyclonal antibody against ICE (M8; 2 mg?
temperature. This antibody was raised in rabbits by injection of
a p45 full-length ICE protein fused to a His tag. Resultant
antiserum was evaluated by Western blotting or immunopre-
cipitation and found to recognize endogenous ICE. It did not
cross-react with ICH-1, ICH-3, or members of the CPP32
family (unpublished data), although it recognized an ICE-like
protein in brain and other tissues of an ICE knockout mouse.
IL-1? Immunoassay. Immunoreactive IL-1? was deter-
mined using an ELISA kit (Genzyme; lot B6499F). Male
SV-129 mice (18–23 g) were injected twice i.c.v. with z-
VAD.FMK (120 ng in 1 ?l per dose) 15 min before occlusion
and immediately after reperfusion. Each hemisphere was
homogenized for 15 sec in PBS (0.1 M; pH 7.4; 4?C) containing
2 mM phenylmethylsulfonyl fluoride (stock dissolved in di-
methyl sulfoxide, diluted 1:100 in PBS), 1 ?g?ml leupeptin, 1
?g?ml antipain, 1 ?g?ml aprotinin, 1 ?g?ml pepstatin, 0.05%
(wt?vol) sodium azide, and 4 mM ethylenediaminetetraacetic
acid. The homogenates were centrifuged (30 min at 50,000 ?
g), and 100 ?l of the supernatant was used for each determi-
nation. Immunoreactive IL-1? data are expressed as the
difference between ischemic and contralateral hemispheres.
Neurotoxicity. Adult male SV-129 mice (20–28 g) were anes-
thetized with halothane (2.5% for induction, 1–1.5% for main-
tenance), and the head was fixed in a stereotaxic frame (Kopf
Instruments, Tujunga, CA). AMPA (20 mM, 6 nmol) or NMDA
mm anterior and 2 mm lateral to the bregma, and 2.5 mm below
the dura in 0.3 ?l. Rectal temperature was monitored during this
10-min procedure. To assess the effects of ICE-like inhibition,
AMPA plus z-VAD.FMK were co-injected intrastriatally fol-
lowed 3 hr later by an injection of z-VAD.FMK alone (0.3 ?l
each). z-VAD.FMK was also administered i.c.v. 15 min before
48 hr, and tissue sections (20 ?m) were stained by hematoxylin?
eosin. The lesion was measured by an image analysis system (M4;
integrated to calculate volume.
Statistical Analysis. Data are presented as the mean ? SE.
Statistical comparisons were made by one- or two-way
ANOVA followed by Student’s t test, Dunnett’s test, or
Mann–Whitney U test using the software SUPER ANOVA or
STAT VIEW, version 4.5 (Abacus Concepts, Berkeley, CA). P ?
0.05 was considered statistically significant.
2008Neurobiology: Hara et al.Proc. Natl. Acad. Sci. USA 94 (1997)
Reduction of Ischemic Infarction by the Inhibitors of the ICE
Family. To determine the roles of the ICE-like proteases in
inhibitors in stroke, we determined the ability of three peptide
inhibitors of the ICE-like proteases to reduce brain injury in a
mouse model of stroke. When the peptide inhibitors were in-
jected into ventricle 15 min before and upon reperfusion, a
significant reduction in infarct size was noted. z-VAD.FMK, a
nonselective inhibitor of the ICE family and perhaps some serine
proteases as well, reduced infarction volume by approximately
76 to 128 mm3in vehicle-treated brains, whereas values ranged
from 31 to 80 mm3after treatment with 240 ng of z-VAD.FMK.
Infarct sparing was particularly noted in posterior forebrain
(coronal sections from 6 to 10 mm). Neurological deficits were
significantly reduced after 18 hr but not immediately upon
reperfusion. The neurological score was somewhat better after
were not statistically significant.
Neuroprotection sustained for at least 3 days [152 ? 5 (n ? 4)
vs. 75 ? 30 mm3(n ? 5) for vehicle-treated and z-VAD.FMK-
treated (80 ng) groups, respectively]. Some of the damage at 18
hr and 3 days was attributed to brain swelling which was detected
in the control group. z-VAD.FMK significantly decreased brain
swelling at 18 hr [12.9 ? 2.7% (n ? 12) vs. 2.9 ? 1.4% (n ? 8)
for vehicle and z-VAD.FMK, respectively] and 3 days as well.
To determine if more specific inhibitors of the ICE family
may also reduce ischemic brain injury, we examined whether
YVAD.CMK, which is specific for the ICE-like caspases, can
also reduce ischemic injury in our model. YVAD.CMK was
injected i.c.v. 45 min before MCA occlusion and upon reper-
fusion after 2 hr of occlusion. YVAD.CMK (400 ng but not 200
ng) decreased infarction volume by 64%. Significant decreases
were measurable in each of the five coronal sections except in
the most anterior 2 mm. Brain swelling at 18 hr was statistically
less after injection of YVAD.CMK [14.0 ? 1.4% (n ? 8)
YVAD.CMK (400 ng) also ameliorated the behavioral deficits
assessed during reperfusion (Table 1).
To assess the contribution of the subfamily of CPP32-like
caspases to ischemic brain injury, we evaluated the ability of
z-DEVD.FMK to inhibit ischemic brain injury. z-DEVD.FMK
(240 ng but not 80 ng) reduced infarct volume by 27%, whereas
infarct volume ranged from 72 to 129 mm3in the vehicle groups
(n ? 10), and from 9 to 89 mm3after the higher dose of
z-DEVD.FMK (n ? 9). Injury was reduced only in section 6 mm
(4–6 mm from anterior pole) that contained the largest infarct
area. Lower values were also measured in sections 8 and 10 mm,
not decrease brain swelling but reduced behavioral deficits as
effectively as YVAD.CMK or z-VAD.FMK.
To determine if the ability to reduce ischemic brain injury was
specific for the protease inhibitors of the ICE family, we admin-
istered z-FA.FMK (240 ng), a cathepsin B inhibitor, as a control
peptide to test for possible effects of the fluoromethylketone
moiety, although the degree of cathepsin B inhibition was not
determined at the dosage tested. This drug did not reduce infarct
volume or neurological deficits (Fig. 1). Thus, we conclude that
the ability to reduce ischemic injury is most likely due to specific
inhibition of the ICE family proteases.
To determine if administering an ICE family inhibitor upon
recirculation has therapeutic value, we injected z-VAD.FMK
upon reperfusion after 2 hr of occlusion. z-VAD.FMK (80 ng),
administered as a single dose, diminished infarct size [114 ?
7 (n ? 11) and 79 ? 10 mm3(n ? 8, P ? 0.05) for vehicle and
z-VAD.FMK, respectively]. Sparing was observed in the three
coronal sections [4–6 mm, 10.7 ? 1.1 and 6.9 ? 1.3 mm2; 6–8
mm, 18.6 ? 0.7 and 16.3 ? 1.0 mm2; and 8–10 mm, 20.5 ? 1.7
8), respectively]. The protection was less than when z-
VAD.FMK (40 ng ? 2) was administered before and upon
reperfusion. Neurological deficits, although improved, did not
reach statistical significance 18 hr later [2.1 ? 0.2 (n ? 11) vs.
1.5 ? 0.3 (n ? 8) for the vehicle- and z-VAD.FMK-treated
groups, respectively]. When z-VAD.FMK (80 ng) was injected
1 hr after reperfusion, the decrease in infarct volume did not
reach significance [114 ? 7 (n ? 11) and 87 ? 10 mm3(n ?
5) for vehicle and z-VAD.FMK, respectively], although a
significant reduction was measured in the most anterior coro-
nal section [4 mm; 10.7 ? 1.1 (n ? 11) and 6.2 ? 1.8 mm2(n ?
5, P ? 0.05) for vehicle and z-VAD.FMK, respectively].
injected into the lateral ventricle of Sprague–Dawley rats 15 min
before ischemia (2 hr of MCA occlusion) and upon reperfusion.
in rat cortex and striatum (Fig. 2) and decreased neurological
FA.FMK) on infarct volume (A) and area (B), and neurological deficits
Vehicle (E), z-VAD.FMK (27 ng; F), z-VAD.FMK (80 ng; Ç), z-
VAD.FMK (240 ng; å), and z-FA.FMK (240 ng; ?) were injected i.c.v.
15 min before ischemia and immediately after reperfusion. Infarct area
was determined in each of five coronal sections (2 mm) from anterior (2
mm from anterior pole) to posterior (10 mm; B). z-VAD.FMK (80 and
240 ng) decreased infarct volume and neurological deficits, whereas
z-FA.FMK (240 ng) did not. After treatment with z-FA.FMK and
z-VAD.FMK (27 ng), infarct areas did not differ from vehicle in the 5
coronal sections (data not shown). Data are presented as means ? SE
(n ? 5–12). ?, P ? 0.05; ??, P ? 0.01 vs. vehicle.
Effects of z-VAD.FMK and cathepsin B inhibitor (z-
Neurobiology: Hara et al.Proc. Natl. Acad. Sci. USA 94 (1997) 2009
deficits [2.4 ? 0.2 (n ? 9) vs. 1.4 ? 0.4 (n ? 8) for vehicle- and
z-VAD.FMK (160 ng)-treated groups, respectively; P ? 0.05],
which indicates that inhibition of ICE family members amelio-
rates injury in more than a single species.
We monitored physiology in some of the mice before, during,
and after operation. rCBF decreased to approximately 20% of
baseline immediately after MCA occlusion and sustained during
2 hr of ischemia in both mice and rats. After reperfusion, rCBF
increased immediately to 90–100% of baseline, and hyperemia
(approximately 30% above the baseline) was observed ipsilater-
ally over the subsequent 10 min in both vehicle- and z-
VAD.FMK-treated groups. There were no significant blood flow
differences between vehicle- and drug-treated groups at any time
point, nor were there differences in mean arterial blood pressure,
heart rate, rectal (core) temperature, and blood gases detected
between groups after drug administration to mice (Table 2) or
rats (data not shown). Thus, reduction of ischemic infarct by the
inhibitors of the ICE family does not result from hemodynamic
effects on cerebral blood flow, blood pressure, or heart rate. In
our experiments, less than 5% of mice died after drug treatment
or MCA occlusion. In preliminary experiments, 3 of 6 mice died
after large doses (800 ng) of YVAD.CMK, presumably due to a
low toxic?therapeutic ratio (2:1).
ICE-Like Proteases in Ischemic Brain. To examine if ischemic
injury is accompanied by activation of ICE-like proteases, we
determined whether immunoreactive ICE cleavage products
appear on a Western blot in ischemic brain using polyclonal
antisera. ICE cleavage is associated with the appearance of p20
and p10 bands (42). Ischemia increased the ICE-like cleavage
product (p20) on immunoblots at 2 and 18 hr after reperfusion
after ischemia and reperfusion. This additional band may repre-
sent the activation of ICE-like proteases in ischemic brain.
recirculation decreased p20 and p35 bands 3 hr and 18 hr after
reperfusion (Fig. 3).
Enhanced IL-1? Level in Ischemic Brain. Processing of
pro-IL-1? to generate mature IL-1? is a specific function of
ICE in vitro and in vivo, and thus, secretion of mature IL-1? is
a specific indication of ICE activation (16). To examine
whether ischemic injury induces IL-1? secretion, we measured
the brain IL-1? levels using an ELISA kit that detects mature
IL-1?. We found that brain IL-1? levels reached a peak 30 min
to 1 hr after reperfusion, and then decreased, suggesting that
ICE is transiently activated upon reperfusion. We assessed the
relative specificity of z-VAD.FMK and z-DEVD.FMK on
IL-1? production. Since z-VAD.FMK is an inhibitor of the
ICE family, whereas z-DEVD.FMK prefers the CPP32 sub-
family, we anticipated that z-VAD.FMK but not z-
DEVD.FMK would reduce the augmentation of IL-1?. We
found that z-VAD.FMK (240 ng) reduced IL-1? by 76% vs.
control, whereas z-DEVD.FMK treatment (240 ng) did not
prevent the rise at 30 min after reperfusion (Fig. 4). No
differences were measured between ipsilateral and contralat-
eral hemispheres in sham animals, or between control groups.
The Effects of ICE Inhibitor on Glutamate Receptor-
Mediated Neurotoxicity. The importance of glutamate neu-
rotoxicity to cerebral ischemia has been well documented (43).
NMDA and AMPA cause tissue injury when added to neu-
ronal cultures or when injected into the striatum, and this can
be blocked by NMDA and AMPA receptor antagonists.
NMDA or AMPA was microinjected under stereotaxic control
into the striatum in anesthetized mice. Two days later, we
determined that intrastriatal co-injection of z-VAD.FMK (24
ng) attenuated AMPA-induced neuronal damage [32.2 ? 2.5
mm3(n ? 8) vehicle vs. 24.5 ? 1.8 mm3(n ? 8) z-VAD.FMK;
P ? 0.05]. However, z-VAD.FMK attenuated the NMDA-
induced neuronal damage only when administered at a higher
dose (80 ng) and directly into the ventricle [32.1 ? 1.6 mm3
(n ? 5) vehicle vs. 24.1 ? 2.1 mm3(n ? 6) z-VAD.FMK; P ?
0.05]. When z-VAD.FMK was co-injected intrastriatally with
NMDA, there was no difference from control [22.7 ? 1.0 mm3
(n ? 5) vehicle vs. 24.3 ? 1.5 mm3(n ? 5) z-VAD.FMK]. We
ischemia in rats. Animals were subjected to filament MCA occlusion
for 2 hr and reperfused for 22 hr. z-VAD.FMK was injected i.c.v. 15
min before ischemia and 10 min after reperfusion. Open bar, vehicle;
hatched bar, z-VAD.FMK (16 ng); shaded bar, z-VAD.FMK (54 ng);
solid bar, z-VAD.FMK (160 ng). Data are represented as means ? SE
(n ? 7–9). ?, P ? 0.05; ??, P ? 0.01 vs. vehicle.
Effects of z-VAD.FMK on infarct size after transient focal
brain after left MCA filament occlusion and reperfusion. Immunoreac-
tive products p35 and p20 increased in the ipsilateral hemisphere at 3 hr
after reperfusion. z-VAD.FMK (80 ng) administered 15 min before
ischemia and immediately upon reperfusion decreased immunoreactive
products p35 and p20 in the ipsilateral hemisphere at 3 and 18 hr after
reperfusion. There was no significant difference between normal brain
(sham) and the nonischemic contralateral hemisphere (data not shown).
L, Left (ischemic); R, right (nonischemic) hemisphere.
Effects of z-VAD.FMK on ICE-like protease expression in
a relatively selective CPP32-like caspase inhibitor (z-DEVD.FMK)
on infarct volume and neurological deficits after temporary MCA
occlusion (2 hr) and reperfusion (18 hr)
Effects of ICE-like caspase inhibitor (YVAD.CMK) and
100 ? 9
96 ? 13
36 ? 18**
90 ? 6
83 ? 7
66 ? 8*
1.8 ? 0.2
1.5 ? 0.3
0.8 ? 0.4*
2.1 ? 0.1
1.6 ? 0.2
1.1 ? 0.3**
YVAD.CMK (200 ng)
YVAD.CMK (400 ng)
z-DEVD.FMK (80 ng)
z-DEVD.FMK (240 ng)
Neurological deficits were decreased by YVAD.CMK and z-
DEVD.FMK after 18 hr, although no significant group differences
were detected at 10 min or 2 hr of ischemia (data not shown). Data are
presented as means ? SE. ?, P ? 0.05; ??, P ? 0.01 vs. vehicle.
2010 Neurobiology: Hara et al.Proc. Natl. Acad. Sci. USA 94 (1997)
conclude that the nonselective inhibitor of ICE-like caspases
z-VAD.FMK can reduce toxicity by AMPA and to a lesser
extent NMDA, suggesting the importance of ICE family
members to glutamate neurotoxicity.
We demonstrated here that lipid soluble cysteine protease inhib-
MCA occlusion in mice. Decreased infarct volume was observed
18 hr after reperfusion and sustained for at least 72 hr after
z-VAD.FMK treatment, and was consistently present in the
coronal section containing frontal cortex, striatum, and anterior
hippocampus. rCBF, arterial blood pressure, heart rate, core
temperature, blood gases, or arterial pH did not change with
treatment, suggesting that the observed protection reflects in-
creased resistance of ischemic tissue. Moreover, neurological
deficits also improved. In nearly every experiment showing a
significant reduction in infarct volume, motor deficits were no-
ticeably better. Hence, deterioration of neurological function
after acute injury may be reduced by administering cysteine
protease inhibitors. Inhibition of ICE-like caspases attenuated
ischemic cell death in rat cortex and striatum, as described in a
preliminary report after permanent ischemia (44). Our findings
were associated with behavioral improvement as well. Taken
together, these data are consistent with our recent preliminary
MCA occlusion (45).
z-VAD.FMK decreased brain swelling, at least in part due to
reduced formation of the proinflammatory cytokine IL-1?. Ac-
tive ICE is a tetramer composed of two subunits, p20 and p10,
processed from p45 precursor peptide (42). The subunits are
cleaved from a single proenzyme, p45 (42). Ischemia promoted
activation of ICE or ICE-like proteins, as evidenced by increased
of cleavage products was decreased by z-VAD.FMK as was brain
IL-1? levels 30 min after reperfusion. Treatments that reduce or
antagonize the action of IL-1? (e.g., recombinant IL-1? antibod-
ies or zinc protoporphyrin administration) decrease brain water
content in ischemic brain (46).
We tested the effects of two other irreversible inhibitors.
YVAD.CMK is more selective for ICE-like than for CPP32-
like caspases (47), and this drug decreased infarct size, edema
formation, and behavioral deficits. z-DEVD.FMK shows
greater selectivity for CPP32-like than for ICE-like caspases
(48). A related compound, Ac-DEVD.CHO, inhibits cleavage
of a CPP32 substrate, poly (ADP ribose) polymerase 10,000
times more potently than Ac-YVAD.CHO (47). Moreover,
z-DEVD.FMK did not reduce IL-1? levels in ischemic brain
(Fig. 4), thereby suggesting that the cleavage of ICE or
pro-IL-1? may not be an important action of CPP32-like
caspases in vivo under the stated condition. The most potent
(and least selective) among the three inhibitors was z-
VAD.FMK. z-VAD.FMK inhibits anti-Fas antibody-induced
apoptosis in Jurkat cells by blocking activation of CPP32-like
caspases indirectly (49). Both z-VAD.FMK and YVAD.CMK
afforded similar protection (57% vs. 64% decrease in volume,
respectively), although the dose of z-VAD.FMK required was
approximately 4-fold lower (0.17 nmol vs. 0.74 nmol, respec-
nonselective cysteine protease inhibitors may offer advantages
over those showing greater selectivity.
We are not aware of published studies addressing whether
CPP32 is activated during ischemia, although this would not be
unexpected since ischemia activates ICE, and ICE cleaves CPP32
induced immunoreactive IL-1? levels in brain. Treatment with z-
VAD.FMK (120 ng ? 2) but not z-DEVD.FMK (120 ng ? 2) 15 min
before ischemia and at reperfusion reduced the expected augmentation of
Control animals were treated with the same volume of vehicle. Data are
presented as the mean ? SEM (n ? 6 or 7). ??, P ? 0.01 vs. vehicle.
Effect of z-VAD.FMK and z-DEVD.FMK on ischemia-
Physiological variables before and during ischemia, and during reperfusion in SV-129 mice under halothane
19 ? 5
18 ? 5
93 ? 6
95 ? 4
482 ? 26
484 ? 27
36.7 ? 0.3
36.8 ? 0.1
125 ? 20
113 ? 11
80 ? 7
87 ? 4
509 ? 48
477 ? 19
36.3 ? 0.3
36.8 ? 0.2
MABP 86 ? 5
92 ? 3
502 ? 13
505 ? 18
36.4 ? 0.2
36.9 ? 0.2
7.31 ? 0.02
7.31 ? 0.003
47 ? 4
47 ? 2
130 ? 16
123 ? 7
7.32 ? 0.02
7.29 ? 0.01
48 ? 4
52 ? 1
133 ? 10
125 ? 7
7.33 ? 0.02
7.30 ? 0.01
44 ? 3
49 ? 2
155 ? 9
151 ? 9
millimeters of mercury; 1 mmHg ? 133 Pa), heart rate (beats per min), core temperature (?C), and arterial blood gases [pH,
PaCO2(torr), PaO2(torr)] were measured before occlusion, during ischemia (2 hr), and during up to 30 min reperfusion. Data
are presented as the mean ? SE (n ? 5 per group). There are no significant differences between groups. Values were averaged
over a 15-min time period immediately before ischemia, during ischemia (45–60 min), and after reperfusion (15–30 min).
Neurobiology: Hara et al.Proc. Natl. Acad. Sci. USA 94 (1997) 2011
(47).Weobservedthatz-DEVD.FMKreproduciblyreducedboth Download full-text
infarct size and behavioral deficits, although z-DEVD.FMK ap-
peared weaker (27% protection at 0.36 nmol) than either z-
VAD.FMK or YVAD.CMK. z-DEVD.FMK treatment did not
block edema formation, suggesting mechanistic differences be-
tween inhibitors of CPP32-like and ICE-like caspases and IL-1?
synthesis as noted above. Moreover, improved neurological func-
tion after z-DEVD.FMK treatment suggested that an effect on
ment after z-VAD.FMK and YVAD.CMK administration. Inhib-
itors of both ICE-like and CPP32-like caspases confer ischemic
brain protection, although their mechanism of action seems to
differ. Inhibition of ICE-like caspases and IL-1? production can
reduce the appearance of inflammation and apoptosis as a con-
sequence of ischemia, whereas inhibition of CPP32-like caspases
may be more specifically related to blockade of apoptosis.
An inhibitor of ICE and ICE-like activity (z-VAD.FMK, 80
ng) successfully reduced tissue injury when given immediately
as a single dose upon reperfusion. These findings suggest the
therapeutic potential for combining ICE family protease in-
hibition with recombinant tissue plasminogen activator during
clot lysis and subsequent reperfusion. However, the therapeu-
tic window for this peptide inhibitor appears relatively short as
the data after z-VAD.FMK was administered 1 hr after
recirculation did not reach statistical significance.
Excitotoxins cause apoptosis in cell culture (50) or after
intrastriatal injection (51), particularly when the injury is mild
(50). Neuronal damage due to AMPA was attenuated when
z-VAD.FMK and AMPA were co-injected intrastriatally. The
same protocol, however, was ineffective after NMDA injection
despite the presence of a smaller (submaximal) lesion. MK-
801, a selective NMDA receptor antagonist, however, blocked
lesion development completely (data not shown). When ad-
ministered at a higher dose and directly into the ventricle, the
lipid protease inhibitor attenuated NMDA-induced neuronal
damage. Whether this means that NMDA is more closely
associated with necrotic mechanisms of cell death compared
with AMPA neurotoxicity is unknown at this time, but it
suggests important mechanistic differences between the two
excitotoxins (43). Nevertheless, the protection we observed in
the presence of excitotoxin injection is consistent with the
results after administering an endogenous IL-1 receptor an-
tagonist (17, 18), and this further implicates the ICE family
proteases in events related to ischemic pathophysiology.
ICE family proteases prevent ischemic and excitotoxic neuro-
function. Inhibition of ICE-like and?or other caspase family
members, particularly by non-peptide inhibitors that cross the
blood–brain barrier and easily penetrate neurons and glia,
could provide novel treatments for stroke and other forms of
brain and spinal cord injury in humans.
We thank Drs. Seth P. Finkelstein and Bradley T. Hyman (Massa-
chusetts General Hospital) and Dr. Pierre A. Henkart (National
Cancer Institute, National Institutes of Health, Bethesda) for their
advice and Drs. Gamze Ayata and Albert Kim for helpful technical
assistance. Our studies were supported by Massachusetts General
Hospital Interdepartmental Stroke Project Grant NS10828 and by an
unrestricted award in Neuroscience from Bristol-Myers Squibb (to
M.A.M.). K.F. was supported by the Deutsche Forschungsgemein-
schaft (Fi600?2-1). J.Y. was supported by the National Institute of
Neurological Disorders and Stroke, American Heart Association
Established Investigatorship Award and by Bristol–Myers Squibb.
R.M.F. was supported by a postdoctoral training fellowship from the
National Institutes of Health and by an Upjohn award from the joint
section on Cerebrovascular Surgery, the Congress of Neurological
Surgeons, and the American Association of Neurological Surgeons.
Ellis, R. E., Yuan, J. & Horvitz H. R. (1991) Annu. Rev. Cell Biol. 7, 663–698.
Yuan, J. & Horvitz H. R. (1990) Dev. Biol. 138, 33–41.
Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. (1993) Cell 75, 641–652.
Li, Y., Chopp, M., Jiang, N., Yao, F. & Zaloga, C. (1995) J. Cereb. Blood Flow Metab.
Charriaut-Marlangue, C., Margaill, I., Represa, A., Popovici, T., Plotkine, M. & Ben-Ari,
Y. (1996) J. Cereb. Blood Flow Metab. 16, 186–194.
Gagliardini,V.,Fernandez,P.-A.,Lee,R. K.K.,Drexler,H. C.A.,Rotello,R.,Hartwieg,
E. A. & Yuan, J. (1994) Science 263, 826–828.
Li, W., Fishman, M. C. & Yuan, J. (1995) Cell Death Differ. 3, 105–112.
Milligan, C. E., Prevette, D., Yaginuma, H., Homma, S., Cardwell, C., Fritz, L. C.,
Tomaselli, K. J., Oppenheim, R. W. & Schwartz, L. M. (1995) Neuron 15, 385–393.
Martinon, J.-C., Dubois-Dauphin, M., Staple, J. K., Rodriguez, I., Frankowski, H.,
Missotten, M., Albertini, P., Talabot, D., Catsicas, S., Pietra, C. & Huarte, J. (1994)
Neuron 13, 1017–1030.
Crumrine, R. C., Thomas, A. L. & Morgan, P. F. (1994) J. Cereb. Blood Flow Metab. 14,
Johnson, E. J., Greenlund, L. J., Akins, P. T. & Hsu, C. Y. (1995) J. Neurotrauma 12,
Liu, T., McDonnell, P. C., Young, P. R., White, R. F., Siren, A. L., Hallenbeck, J. M.,
Barone, F. C. & Feuerstein, G. Z. (1993) Stroke 24, 1746–1751.
Buttini, M., Sauter, A. & Boddeke, H. W. G. M. (1994) Mol. Brain Res. 23, 126–134.
Minami, M., Kuraishi, Y., Yabuuchi, K., Yamazaki, A. & Satoh, M. (1992) J. Neurochem.
Bhat, R. V., DiRocco, R., Marcy, V. R., Flood, D. G., Zhu, Y., Dobrzanski, P., Siman,
R., Scott, R., Contreras, P. C. & Miller, M. (1996) J. Neurosci. 16, 4146–4154.
Friedlander, R. M., Gagliardini, V., Rotello, R. J. & Yuan, J. (1996) J. Exp. Med. 184,
Rothwell, N. J. & Relton, J. K. (1993) Neurosci. Biobehav. Rev. 17, 217–227.
Relton, J. K. & Rothwell, N. J. (1992) Brain Res. Bull. 29, 243–246.
Kumar, S., Kinoshita, M., Noda, M., Copeland, N. G. & Jenkins, N. A. (1994) Genes Dev.
Fernandez-Alnemri, T., Litwack, G. & Alnemri, E. S. (1995) Cancer Res. 55, 2737–2742.
Fernandes-Alnemri, T., Takahashi, A., Armstrong, R., Krebs, J., Fritz, L., Tomaselli, K.,
Wang, L., Yu, Z., Croce, C. M., Earnshaw, W. C., Litwack, G. & Alnemri, E. S. (1995)
Cancer Res. 55, 6045–6052.
Fernandes-Alnemri, T., Armstrong, R. C., Krebs, J., Srinivasula, S. M., Wang, L.,
Bullrich, F., Fritz, L. C., Trapani, J. A., Tomaselli, K. J., Litwack, G. & Alnemri, E. S.
(1996) Proc. Natl. Acad. Sci. USA 93, 7464–7469.
Wang, L., Miura, M., Bergeron, L., Zhu, H. & Yuan, J. (1994) Cell 78, 739–750.
Faucheu, C., Diu, A., Chan, A. W., Blanchet, A.-M., Miossec, C., Herve, F., Collard-
Dutilleul, V., Gu, Y., Aldape, R. A., Lippke, J. A., Rocher, C., Su, M. S.-S., Livingston,
D. J., Hercend, T. & Lalanne, J.-L. (1995) EMBO J. 14, 1914–1922.
Tewari, M. & Dixit, V. M. (1995) J. Biol. Chem. 270, 3255–3260.
Kamens, J., Paskind, M., Hugunin, M., Talanian, R. V., Allen, H., Banach, D., Bump,
N., Hackett, M., Johnston, C. G., Li, P., Mankovich, J. A., Terranova, M. & Ghayur, T.
(1995) J. Biol. Chem. 270, 15250–15256.
Munday, N. A., Vaillancourt, J. P., Ali, A., Casano, F. J., Miller, D. K., Molineaux, S. M.,
Yamin, T.-T., Yu, V. L. & Nicholson, D. W. (1995) J. Biol. Chem. 270, 15870–15876.
Wang, S., Miura, M., Jung, Y., Zhu, H., Gagliardini, V., Shi, L., Greenberg, A. H. &
Yuan, J. (1996) J. Biol. Chem. 271, 20580–20587.
Lippke, J. A., Gu, Y., Sarnecki, C., Caron, P. R. & Su, M. S.-S. (1996) J. Biol. Chem. 271,
Duan,H. J.,Chinnaiy,A. M.,Hudson,P. L.,Wing,J. P.,He,W. W.&Dixit,V. M.(1996)
J. Biol. Chem. 271, 1621–1625.
Duan, H., Orth, K., Chinnaiyan, A. M., Poirier, G. G., Froelich, C. J., He, W. & Dixit,
V. (1996) J. Biol. Chem. 271, 16720–16724.
Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J.,
Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Kreammer, P. H., Peter, M. E.
& Dixit, V. M. (1996) Cell 85, 817–827.
Boldin, M. P., Goncharov, T. M., Goltsev, Y. V. & Wallach, D. (1996) Cell 85, 803–815.
Alnemri, E. S., Livingston, D. J., Nicholson, D. W., Salvesen, G., Thornberry, N. A.,
Wong, W. W. & Yuan, J. (1996) Cell 87, 171.
Thornberry, N. A., Peterson, E. P., Zhao, J. J., Howard, A. D., Griffin, P. R. & Chap-
man, K. T. (1994) Biochemistry 33, 3934–3940.
Cain, K., Inayat-Hussain, S. H., Couet, C. & Cohen, G. M. (1996) Biochem. J. 314, 27–32.
Hara, H., Huang, P. L., Panahian, N., Fishman, M. C. & Moskowitz, M. A. (1996) J.
Cereb. Blood Flow Metab. 16, 605–611.
Yang, G. Y., Chen, S. F., Kinouchi, H., Chan, P. H. & Weistein, P. R. (1992) Stroke 23,
Huang, Z., Huang, P. L., Panahian, N., Dalkara, T., Fishman, M. C. & Moskowitz, M. A.
(1994) Science 265, 1883–1885.
Swanson, R. A., Morton, M. T., Tsao-Wu, G., Savalos, R. A., Davidson, C. & Sharp,
F. R. (1990) J. Cereb. Blood Flow Metab. 10, 290–293.
Bederson, J. B., Pitts, L. H., Tsuji, M., Nishimura, M. C., Davis, R. L. & Bartkowski,
H. M. (1986) Stroke 17, 472–476.
Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D. et al.
(1992) Nature (London) 356, 768–774.
Choi, D. W. (1992) J. Neurobiol. 23, 1261–1276.
Loddick, S. A., MacKenzie, A. & Rothwell, N. J. (1996) NeuroReport 7, 1465–1468.
Hara, H., Friedlander, R. M., Gagliardini, V., Ayata, C., Ayata, G., Yuan, J. &
Moskowitz, M. A. (1996) Circulation Suppl. 94, 2283 (abstr.).
Yamasaki, Y., Matsuura, N., Shozuhara, H., Onodera, H., Itoyama, Y. & Kogure, K.
(1995) Stroke 26, 676–681.
Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant,
M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M.,
Smulson, M. E., Yamin, T. T., Yu, V. L. & Miller, D. K. (1995) Nature (London) 376,
Sarin, A., Wu, M.-L. & Henkart, P. A. (1996) J. Exp. Med. 185, 2445–2450.
Slee, E. A., Zhu, H., Chow, S. C., MacFarlane, M., Nicholson, D. W. & Cohen, G. M.
(1996) Biochem. J. 315, 21–24.
Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. & Lipton, S. A. (1995) Proc. Natl.
Acad. Sci. USA 92, 7162–7166.
Ferrer, I., Martin, F., Serrano, T., Reiriz, J., Perez-Navarro, E., Alberch, J., Macaya, A.
& Planas, A. M. (1995) Acta Neuropathol. 90, 504–510.
2012 Neurobiology: Hara et al. Proc. Natl. Acad. Sci. USA 94 (1997)