Interleukin-1ß mRNA expression in ischemic rat cortex

Abstract

Background and Pur pose: Interleukin-1ß is a proinftammatory cytokine produced by blood-borne and resident brain inftammatory cells. The present study was conducted to determine if interleukin-1ß mRNA was produced in the brain of rats subjected to permanent focal ischemia. Methods: Rat interleukin-1ß cDNA, synthesized from stimulated rat peritoneal macrophage RNA by reverse transcription and polymerase chain reaction and c10ned in plasmid Bluescript KS+, was used to evaluate the expression of interleukin-1ß mRNA in cerebral cortex from spontaneously hypertensive rats and normotensive rats subjected to permanent middle cerebral artery occlusion. Interleukin-1ß mRNA was quantified by Northern blot analysis and compared with rat macrophage RNA standard. To correct for gel loading, blots were also analyzed with cyclophilin cDNA, which encodes an abundant, conserved protein that was unchanged by the experimental conditions. Results: Interleukin-1ß mRNA produced in the ischemic zone was significantly increased from 6 hours to 120 hours, with a maximum of211±24% ofinterleukin-1ß reference standard, ie, 0.2 ng stimulated rat macrophage RNA, mRNA compared with the level in nonischemic cortices (4±2%) at 12 hours after ischemia (P<.OI; n=6). Interleukin-1ß mRNA at 12 hours after ischemia was markedly elevated in hypertensive rats over levels found in two normotensive rat strains. Neurological deficits were also apparent only in the hypertensive rats. Conclusions: Brain interleukin-1ß mRNA is elevated acutely after permanent focal ischemia and especially in hypertensive rats. These data suggest that this potent proinflammatory and procoagulant cytokine might have a role in brain damage following ischemia.

Full text

Feurestein
T Liu, P C McDonnell, P R Young, R F White, A L Siren, J M Hallenbeck, F C Barone and G Z
Interleukin-1 beta mRNA expression in ischemic rat cortex.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
Copyright © 1993 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Stroke doi: 10.1161/01.STR.24.11.1746
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1746
Interleukin-1/3
mRNA
Expression
in
Ischemic
Rat
Cortex
T.
Liu,
MD;
P.C.
Mc
Donnell,
MS;
P.R.
Young,
PhD;
R.F.
White,
BA;
A.L.
Siren,
MD,
PhD;
J.M.
Hallenbeck,
MD;
F.C.
Barone,
PhD;
G.Z.
Feuerstein,
MD
Background
and
Purpose:
Interleukin-1lj
is
a
proinflammatory
cytokine
produced
by
blood-borne
and
resident
brain
inflammatory
cells.
The
present
study
was
conducted
to
determine
if
interleukin-13
mRNA
was
produced
in
the
brain
of
rats
subjected
to
permanent
focal
ischemia.
Methods:
Rat
interleukin-lj
cDNA,
synthesized
from
stimulated
rat
peritoneal
macrophage
RNA
by
reverse
transcription
and
polymerase
chain
reaction
and
cloned
in
plasmid
Bluescript
KS+,
was
used
to
evaluate
the expression
of
interleukin-1.3
mRNA
in
cerebral
cortex
from
spontaneously
hypertensive
rats
and
normotensive
rats
subjected
to
permanent
middle
cerebral
artery
occlusion.
Interleukin-1f3
mRNA
was
quantified
by
Northern
blot
analysis
and
compared
with
rat
macrophage
RNA
standard.
To
correct
for
gel
loading,
blots
were
also
analyzed
with
cyclophilin
cDNA,
which
encodes
an
abundant,
conserved
protein
that
was
unchanged
by
the
experimental
conditions.
Results:
Interleukin-10
mRNA
produced
in
the
ischemic
zone
was
significantly
increased
from
6 hours
to
120
hours,
with
a
maximum
of
211±24%
of
interleukin-lf
reference
standard,
ie,
0.2
ng
stimulated
rat
macrophage
RNA,
mRNA
compared
with
the
level
in
nonischemic
cortices
(4±2%)
at
12
hours
after
ischemia
(P<.01;
n=6).
Interleukin-1f3
mRNA
at
12
hours
after
ischemia
was
markedly
elevated
in
hypertensive
rats
over
levels
found
in
two
normotensive
rat
strains.
Neurological
deficits
were
also
apparent
only
in
the
hypertensive
rats.
Conclusions:
Brain
interleukin-1j3
mRNA
is
elevated
acutely
after
permanent
focal
ischemia
and
especially
in
hypertensive
rats.
These
data
suggest
that
this
potent
proinflammatory
and
procoagulant
cytokine
might
have
a
role
in
brain
damage
following
ischemia.
(Stroke.
1993;24:1746-1751.)
KEY
WoRDs
*
cerebral
ischemia
*
cytokines
*
neuronal
damage
*
rats
I
nterleukin-1,3
(IL-1,3)
is
a
cytokine
with
multiple
proinflammatory,
procoagulant,
and
cell
growth
modulatory
actions.'
The
presence
of
IL-l,3
in
the
central
nervous
system
is
believed
to
reflect
synthesis
by
diverse
cells
such
as
endothelium,
microglia,
astrocytes,
and
neurons.2
Interleukin-1,8
acts
via
specific
brain
IL-1
receptors
that
demonstrate
significant
spatial
distribution.3
As
in
peripheral
organs,
the
proinflammatory
and
prothrom-
botic
actions
of
IL-1f31
are
expected to
promote
acute
neuropathological
changes
at
excessive
levels
in
the
brain.
Brain
ischemia
has
been
shown
to
be
associated
with
an
acute
inflammatory
response,4-6
but
the
nature
of
the
inflammatory
mediators
involved
in
brain
isch-
emia
is
still
unknown.
While
numerous
reports
have
dealt
with
the
putative
roles
of
various
inflammatory
mediators
such
as
thromboxane
A2,
leukotrienes,
and
Received
August
14,
1992;
final
revision
received
April
26,
1993;
accepted
May
17,
1993.
From
the
Departments
of
Cardiovascular
Pharmacology
(T.L.,
R.F.W.,
F.C.B.,
G.Z.F.)
and
Molecular
Genetics
(P.C.Mc
D.,
P.R.Y.),
SmithKline
Beecham
Pharmaceuticals,
King
of
Prussia,
Pa;
and
the
Department
of
Neurology,
Uniformed
Services
Uni-
versity
of
the
Health
Sciences
(A.L.S.),
and
the
Stroke
Branch,
National
Institute
of
Neurological
Disorders
and
Stroke
(J.M.H.),
National
Institutes
of
Health,
Bethesda,
Md.
Correspondence
to
Giora
Z.
Feuerstein,
MD,
Department
of
Cardiovascular
Pharmacology,
SmithKline
Beecham
Pharmaceu-
ticals,
709
Swedeland
Rd,
PO
Box
1539,
King
of
Prussia,
PA
19406.
See
Editorial
Comment,
page
1750
platelet-activating
factor78
in
brain
injury,
few
attempts
have
been
made
to
explore
the
role
of
proinflammatory
cytokines
in
brain
injury.
Evidence
supporting
the
involve-
ment
of
cytokines
in
central
nervous
system
injury
in-
cludes
demonstrations
of
IL-1l3
mRNA
expression
in
mouse
brain
3
hours
after
endotoxin
administration,9
release
of
IL-1
in
mechanically
injured
brain
produced
by
intraparenchymal
implantation
of
a
microdialysis
probe,10
expression
of
IL-1,8
mRNA
in
rat
brain
in
response
to
direct
intraparenchymal
administration
of
the
neurotoxin
kainate,"1
or
combination
of
endotoxin
and
y-interferon.12
IL-1i8
mRNA
expression
was
also
shown
in
a
rat
model
of
transient
global
brain
ischemia
induced
by
permanent
bilateral
vertebral
artery
occlusion
followed
by
bilateral
carotid
occlusion
with
reperfusion.13
However,
cytokine
transcription
in
permanent
focal
ischemia
has
not
been
previously
evaluated.
The
purpose
of
the
present
study
was
to
determine
whether
the
initiation
phase
of
IL-1f3
production,
ie,
the
transcription
of
its
mRNA,
takes
place
after
permanent
middle
cerebral
artery
occlusion
(MCAO);
furthermore,
this
study
also
aimed
to
explore
the
differences,
if
any,
between
rats
carrying
risk
factors
for
stroke
(eg,
hypertension)
and
normotensive
rats.
Materials
and
Methods
To
provide
IL-1l8
mRNA
for
use
as
a
standard
and
to
create
the
necessary
IL-1,3
cDNA
probe
for
Northern
by guest on June 6, 2013http://stroke.ahajournals.org/Downloaded from

Liu
et
al
IL-13
mRNA
in
Focal
Ischemia
1747
blot
analysis,
rat
peritoneal
macrophages
were
collected
by
lavage
with
phosphate-buffered
saline
and
incubated
with
5
,g/mL
Escherichia
coli
lipopolysaccharide
(Sig-
ma
Chemical
Co,
St
Louis,
Mo)
at
37°C
for
4
hours.
Cells
were
lysed
in
4
mol/L
guanidinium
thiocyanate,
and
total
RNA
was
isolated
by
centrifugation
over
5.8
mol/L
(2sCl.'4
IL-1p
cDNA
was
synthesized
by
reverse
transcription
and
polymerase
chain
reactions
(RT-PCR;
Boehringer
Mannheim
Biochemica,
Indianapolis,
Ind)
using
the
5'
and
3'
synthetic
oligonucleotide
primers
5
'-CAGCGGCCGCCTTGTGCAAGTGTCTGAAG-
CAG-3'
and
3'-GAACAGCTCTTACCCGTCAGAG-
GTCCAGATCTCGCCGGCGAC-5',
respectively.
The
primers
were
engineered
to
contain
a
Noti
restriction
site
for
cloning
purposes.
The
fragment
obtained
by
RT-PCR
was
digested
with
Noti
and
ligated
into
Blue-
script
KS+
(Stratagene,
La
Jolla,
Calif).
The
identity
of
the
cDNA
insert
was
confirmed
by
sequencing
and
agrees
with
the
known
sequence.12
For
Northern
blot
hybridization,
the 0.9-kb
rat
IL-13
cDNA
insert
was
used
(isolated
by
Noti
digestion
of
Bluescript
K+
that
contained
the
IL-1,8
cDNA
fragment
and
purified
by
agarose
gel
electrophoresis).
Similarly,
a
human
0.9-kb
EcoRI
cyclophilin
cDNA
fragment
was
prepared
and
also
used
for
hybridization
according
to
Bergsma
et
al.15
Both
rat
IL-113
and
cyclophilin
cDNA
fragments
were
labeled
using
an
oligolabeling
kit
(Pharmacia
LKB
Biotechnology,
Piscataway,
NJ)
via
random
primer
ex-
tension
using
Klenow
DNA
Polymerase
I
and
purified
using
a
Nuctrap
push
column
(Stratagene).
Focal
ischemia
or
sham
surgery
was
carried
out
in
spontaneously
hypertensive
rats
(SHR;
Taconic
Farms,
Germantown,
NY)
and
in
two
normotensive
rat
strains
(F-344
or
Wistar-Kyoto;
Taconic
Farms)
(weight,
250
to
300
g).
Animals
were
anesthetized
with
sodium
pento-
barbital
(Steris
Laboratories,
Inc,
Phoenix,
Az;
60
mg/kg
IP)
and
prepared
for
surgery
as
previously
described.6
Briefly,
via
a
2-
to
3-mm
craniotomy
the
right
middle
cerebral
artery
was
isolated
on
the
hooked
tip
of
a
platinum-irridium
wire
(0.0045
-in
diameter;
Medwire,
Mount
Vernon,
NY)
and
then
simultaneously
occluded
and
cut
dorsal
to
the
lateral
olfactory
tract
(n=
18,
SHR;
n=4,
F-344;
n=4,
Wistar-Kyoto).
In
sham-operated
animals
(n=3;
SHR)
the
same
surgical
procedures
were
exercised,
but
the
artery
was
not
occluded,
and
animals
were
killed
at
12
hours.
In
addition,
nonoperated
animals
(n=3;
SHR)
were
also
studied.
Two
separate
neurological
examinations
were
per-
formed
on
the
rats
before
euthanasia
to
determine
the
severity
of
motor
deficits.
Contralateral
forelimb
def-
icits
were
measured
using
a
neurological
grade
as
previously
described,16
while
contralateral
hind-limb
deficits
were
measured
using
a
standard
hind-limb
placement
test.6
At
various
times
post-MCAO
rats
were
overdosed
with
pentobarbital,
and
forebrains
were
removed
and
dissected
as
described
previously.5'6
A
segment
of
the
ipsilateral
frontoparietal
cortex
was
sliced
from
the
hemisphere;
an
identical
segment
was
sliced
from
the
contralateral
cortical
hemisphere
(nonischemic
control).
The
segments
were
immedi-
ately
frozen
in
liquid
nitrogen
and
stored
at
-80°C.
For
Northern
blot
analysis
of
IL-1,3
mRNA
in
the
focal
ischemic
and
nonischemic
tissue,
total
RNA
from
peritoneal
macrophages
(ie,
0.2
,ug
isolated
as
described
above
and
used
in
the
Northern
blots
as
an
IL-1X3
mRNA
reference
standard
for
quantitation
purposes)
was
fractioned
through
a
1%
agarose
gel,
containing
6%
formaldehyde
in
MOPS
(3-[N-morpholino-]propane-
sulfonic
acid;
Sigma).
The
RNA
was
then
transferred
onto
a
nitrocellulose
membrane
(Keene,
NH;
BA83,
0.2
,um)
by
capillary
blotting
in
20x
saline-sodium
phos-
phate-ethylenediaminetetraacetic
acid
(EDTA)
(SSPE)
(lx
SSPE
is
150
mmol/L
NaCl,
2
mmol/L
NaH2P04,
and
1
mmol/L
EDTA).
RNA
was
immobi-
lized
on
the
filter,
prehybridized,
and
then
hybridized
to
the
32P-deoxycytidine
triphosphate-labeled
rat
IL-1p
cDNA
probes
at
42°C
for
14
to
18
hours
in
the
buffer
containing
50%
formamide,
6x
SSPE,
5x
Denhardt's
solution,
0.5%
sodium
dodecyl
sulfate
(SDS),
100
,g/mL
denatured
salmon
sperm
DNA,
and
10%
dex-
trane
sulfate
and
then
washed
to
a
final
stringency
of
0.5x
SSPE,
0.1%
SDS,
at
50°C
for
15
minutes.
The
membrane
was
exposed
to
Amersham
Hyperfilm-MP
for
2
days
with
intensifying
screen
and
developed
by
a
Kodak
M35A-OMAT
processor.
All
the
membranes
were
stripped
in
boiling
0.05
x
SSPE
and
rehybridized
to
32P-deoxycytidine
triphosphate-labeled
human
cyclo-
philin
cDNA
to
control
for
differences
in
RNA
loading.
Radioactivity
of
the
hybridized
blots
was
counted
by
a
Betascope
603
blot
analyzer
(Betagen
Corp,
Waltham,
Mass).
Cortical
tissue
IL-lp
mRNA
was
quantitated
for
between-blot
comparisons
as
the
percent
relative
radio-
activity
of
the
reference
standard
(ie,
macrophage
RNA
loaded
on
the
same
gel
normalized
to
the
actual
amount
of
RNA
loaded
that
was
determined
from
the
cyclo-
philin
mRNA
counts
from
the
same
cortical
samples).
For
within-blot
comparisons
of
ischemic
cortex
IL-1f3,
percent
relative
radioactivity
was
normalized
directly
in
relation
to
the
loaded
RNA
determined
from
the
cyclo-
philin
mRNA
in
each
sample.
All
data
are
expressed
as
mean±SEM.
Statistical
analysis
of
the
data
was
performed
using
analysis
of
variance
and
Tukey's
multiple
comparison
tests.
Signif-
icant
differences
were
accepted
at
P<.05.
Results
Neurological
deficits
after
focal
ischemia
occurred
in
SHR
(Fig
1)
but
not
in
normotensive
animals.
Both
forelimb
and
hind-limb
dysfunction
in
SHR
were
ob-
served.
Partial
spontaneous
recovery
was
evidenced
at
5
days
for
forelimb
scores
and
at
1
and
5
days
for
hind-limb
scores.
Fig
2
illustrates
the
results
of
a
representative
North-
ern
blot
of
rat
cortical
RNA
12
hours
after
MCAO.
IL-1,8
mRNA
is
significantly
more
abundant
in
ipsilat-
eral
(top
IL-1,3
lanes
1
through
6)
ischemic
cortices
than
in
the
contralateral
cortices
(bottom
IL-1p
lanes
1
through
6)
of
the
same
animals.
RNA
from
macro-
phages
was
used
as
the
reference
standard
for
quanti-
tation
purposes
(Fig
2);
correcting
for
total
loaded
RNA
was
done
by
measurement
of
the
cyclophilin
mRNA
(cyclophilin
lanes
1
through
6)
from
the
ischemic
(top)
and
nonischemic
(bottom)
regions.
Fig
3
depicts
the
quantitated
IL-lp
mRNA
levels
in
the
cortices
taken
at
various
time
points
after
MCAO.
IL-1i8
mRNA
was
not
detectable
in
cortical
samples
from
nonoperated
rats
(0±0%,
n=3).
Sham
surgery
produced
no
significant
effect.
In
the
contralateral
cortical
tissues
(50
lxg)
and
total
RNA
obtained
from
by guest on June 6, 2013http://stroke.ahajournals.org/Downloaded from

1748
Stroke
Vol
24,
No
11
November
1993
2.5
-
0
C1
n
jo
0
IL
2.0
-
1.5
-
1.0
-
0.5
1.2
-
0
'm
C
I
E
V5
.r
1.0
0.8
-
0.6
-
0.4
-
0.2
-
*
*
*
Sham
6
hr
12
hr
24
hr
5
day
T
T
0.0-
Sham
6
hr
12
hr
24
hr
5
day
FIG
1.
Bar
graphs
illustrate
graded
forelimb
(top)
and
hind-limb
(bottom)
deficits
in
sham
(n-3)
and
permanent
middle
cerebral
artery
occlusion
(n=3
to
6)
in
spontane-
ously
hypertensive
rats.
*P<.05
compared
with
sham.
Neurological
deficits
at
1
hour
after
middle
cerebral
artery
occlusion
could
not
be
determined
because
of
anesthe-
sia.
Vertical
bar
lines
represent
SEM.
nonischemic
cortex
after
MCAO,
a
weak
trend
for
expression
of
IL-i83
was
detected
that
did
not
reach
statistical
significance.
Permanent
MCAO
dramatically
increased
the
IL-1f8
mRNA
level
in
the
ipsilateral
ischemic
cortex
compared
with
the
contralateral
control
cortex
from
6
hours
after
MCAO
and
up
to
5
days
later.
Peak
IL-1if
mRNA
expression
was
noticed
12
hours
after
MCAO
(P<.01),
but
significantly
elevated
levels
were
still
monitored
5
days
later
(P<.05).
To
examine
whether
the
elevated
levels
of
IL-1i8
found
in
the
ischemic
cortices
are
specific
to
SHR
only,
we
have
monitored
IL-1i6
mRNA
in
three
groups
(n=4
per
group)
of
rats
studied
simultaneously
(ie,
killed
at
12
hours
after
MCAO
and
quantitated
together).
The
two
normotensive
strains,
WKY
and
F-344
rats,
responded
with
much
less
IL-Ip
mRNA
expression
compared
with
the
SHR
group
(Fig
4).
However,
the
levels
of
IL-1,i
mRNA
in
both
normotensive
rat
strains
were
signifi-
cantly
elevated
over
background
levels.
Discussion
This
article
demonstrates
that
IL-1if
mRNA
in
rat
brain
is
significantly
increased
in
ischemic
cerebral
lschemic
Cortex
1
2
3
4
5 6
7
IL-1,
Ps
Cyclophilin
w
*
Nonischemic
Cortex
1
2 3
4
5 6
7
IL-1l
S
Cyclophilin
50i
@
0
M
*
FIG
2.
Northern
blot
of
interieukin-1,/
(IL-1i8)
from
cere-
bral
cortices
after
12-hour
permanent
middle
cerebral
artery
occlusion
(n=6).
Lanes
1
through
6,
50
gg
of
total
RNA
from
ipsilateral
(ischemic)
or
contralateral
(nonisch-
emic)
cortex
was
separated
on
1
%
agarose
gel
contain-
ing
6%
formaldehyde,
blotted
into
nitrocellulose
mem-
brane,
and
then
hybridized
with
rat
cDNA
IL-1p8
probe.
Lane
7,
0.2
gg
of
total
RNA
was
isolated
from
rat
macrophages
stimulated
with
5
gg/mL
lipopolysaccha-
ride
loaded
on
the
same
gel.
After
stripping,
the
blot
was
hybridized
with
a
human
cyclophilin
cONA
probe
and
used
in
quantitation
of
the
data
to
control
for
amount
of
loaded
RNA.
tissue.
The
time
course
of
IL-1:
mRNA
expression
demonstrated
a
trend
toward
increased
IL-1/3
mRNA
expression
as
early
as
1
hour
after
permanent
MCAO,
a
definite
increase
at
6
hours,
and
persistently
elevated
IL-1,3
mRNA
levels
up
to
5
days
after
ischemia.
The
early
IL-1p
mRNA
expression,
ie,
1
to
6
hours,
pre-
cedes
the
time
of
leukocyte
infiltration
(measured
by
mycloperoxidase
accumulation)
from
blood
vessels
out-
side
the
ischemic
zone,
which
only
begins
to
occur
after
12
hours
and
then
dramatically
increases
for
a
period
of
5
days
after
infarction
in
this
model.'7
These
data
are
further
supported
by
a
detailed
histological
study
show-
ing
significant
elevation
of
neutrophils
in
ischemic
cor-
tex
at
48
hours
after
ischemia.'8
Our
data
also
suggest
that
IL-1,B
mRNA
expression
in
focal
ischemic
brain
tissue
is
not
specific
to
the
SHR
strain
since
significant
IL-1:3
mRNA
expression
has
been
clearly
demonstrated
in
the
two
normotensive
rat
strains.
The
lesser
IL-1J3
mRNA
expression
in
the
two
normotensive
rat
strains
may
represent
the
lesser
vulnerability
of
brain
cortex
of
normotensive
rats
to
permanent
MCAO,6
as
also
re-
flected
in
the
present
study
by
lack
of
neurological
deficits
in
the
normotensive
rats
and
previous
reports
on
infarct
volume.6
The
data
presented
in
this
study
are
in
accord
with
recent
reports
demonstrating
IL-1,3
mRNA
expression
in
several
different
brain
injury
models.9-'3
Thus,
Mi-
nami
et
al13
have
shown
acute
biphasic
IL-18
mRNA
expression
in
various
brain
regions
after
transient
global
ischemia.
However,
several
important
differences
be-
tween
the
two
studies
must
be
pointed
out.
First,
the
present
data
represent
transcription
of
IL-1f3
in
perma-
nent
focal
ischemia.
Second,
the
previous
study'3
has
0.0
by guest on June 6, 2013http://stroke.ahajournals.org/Downloaded from

Liu
et
al
IL-1f
mRNA
in
Focal
Ischemia
1749
300 -
>.h
0
0
cc
._
0
._
cc
z
Cc
250
-
200
-
150
-
100
-
50
-
*
ischemic
cortex
E
nonischemic
cortex
**
I
**
.
*
0
1
=
Z
WR
E
:
Sham
lhr
MCAO
6hr
MCAO
12hr
MCAO
24hr
MCAO
5day
MCAO
FIG
3.
Bar
graph
illustrates
quantitative
analysis
of
the
time
course
for
interleukin-1,p
(IL-1
p)
mRNA
production
in
cerebral
cortices
ipsilateral
or
contralateral
to
permanent
middle
cerebral
artery
occlusion
(MCAO)
(n=3
to
6
per
time
point)
or
sham
surgery
(n=3
killed
at
12
hours).
The
}-emissions
from
bands
on
hybridized
Northern
blots
were
counted
using
a
Betascope
counter.
Data
(mean+±SE)
from
different
blots
are
presented
as
percentage
of
loaded
rat
macrophage
IL-1
p
mRNA
(reference
standard)
normalized
to
the
actual
amount
of
loaded
RNA
(see
"Materials
and
Methods").
*P<.05
compared
with
contralateral
cortex;
**P<.01
compared
with
contralateral
cortex
and
compared
with
ipsilateral
cortex
of
5-day
MCAO.
No
detectable
IL-1,8
mRNA
was
observed
in
either
cortex
of
nonoperated
rats
(n=3;
data
not
shown).
Vertical
bars
represent
SEM.
not
used
a
reference
message
(eg,
cyclophilin),
nor
did
it
standardize
its
hybridization
probe
against
a
single
IL-lp
mRNA-containing
sample
such
as
rat
macrophage
IL-113
mRNA.
Third,
and
most
importantly,
the
present
study
provides
quantitated
data,
whereas
only
qualitative
(visu-
al)
representative
gels
were
provided
in
the
global
isch-
emia
study.13
These
differences
may
underlie
the
lack
of
significant
IL-1,8
mRNA
in
the
brain
between
1
and
7
days
after
global
brain
ischemia,
whereas
significant
IL-1i8
mRNA
was
clearly
demonstrated
in
our
study
at
5
days
1400
-
C)
0
a)
1200-
1i000
800
600
I
400
_
_
~~~**
200-
SHR
WKY
Fisher
FIG
4.
Bar
graph
illustrates
quantitative
analysis
of
isch-
emic
cortex
interleukin-1,p
mRNA
production
in
groups
(n=4)
of
hypertensive
(spontaneously
hypertensive
rats
[SHR])
and
normotensive
(Wistar-Kyoto
[WKY]
and
Fisher)
rats
12
hours
after
middle
cerebral
artery
occlu-
sion.
Data
from
one
blot
are
presented
as
relative
change
normalized
to
the
actual
amount
of
loaded
RNA
(see
"Materials
and
Methods").
*P<.05
compared
with
SHR;
**P<.05
compared
with
WKY
rats.
after
MCAO.
The
lack
of
standardization
and
quantifica-
tion
of
IL-1X
mRNA
in
the
global
ischemia
report
also
makes
it
difficult
to
correlate
the
magnitude
of
the
IL-1p
mRNA
expression
in
the
focal
ischemic
brain
cortex
described
here
to
the
changes
described
previously
in
the
global
ischemia
model.13
The
significance
of
the
IL-18
mRNA
expression
de-
scribed
in
the
present
study
must
be
interpreted
with
caution
because
no
evidence
has
been
provided
to
indicate
that
translation
of
the
message
into
the
functional
cyto-
kine
has
occurred
either
at
the
time
of
the
IL-1,8
mRNA
expression
or
thereafter.
However,
the
de
novo
expression
of
IL-1p
mRNA
in
the
nonperfused
ischemic
cortex
supports
the
possibility
that
endogenous
IL-lp
might
be
produced
in
ischemic
brain.
This
possibility
draws
further
credence
from
reports
demonstrating
the
capacity
of
the
brain
to
synthesize
cytokines,
including
IL-1,
in
several
other
brain
injury
models.
For
example,
mechanical
inju-
ry'0
resulted
in
significant
IL-1
production
24
hours
after
injury,
which
compares
favorably
with
the
earlier
IL-1,3
mRNA
shown
in
our
study.
Also
in
this
latter
study,
IL-1
production
was
significantly
lower
at
7
days
after
injury,
again
in
accord
with
the
significant
diminution
of
IL-1ij
mRNA
observed
in
our
study.
In
any
case,
it
will
be
necessary
to
directly
monitor
IL-1p
production
in
focal
brain
ischemia
to
precisely
relate
the
transcriptional
event
to
the
cytokine
production.
The
cellular
elements
expressing
IL-1,i
mRNA
in
the
ischemic
brain
have
not
been
elucidated
in
the
present
study.
However,
IL-1
has
been
shown
to
be
produced
by
microglial
cells
in
vitro19
and
in
vivo2021
and
by
astro-
cytes
in
vitro.22,23
Furthermore,
IL-1
has
been
detected
in
the
cerebrospinal
fluid
of
rats.24
It
is
also
noteworthy
that
a
peripheral
source
for
IL-1
might
gain
access
into
the
brain
since
this
cytokine
was
shown
to
be
trans-
ported
from
the
blood
into
the
brain25;
therefore,
even
i..-
-
by guest on June 6, 2013http://stroke.ahajournals.org/Downloaded from

1750
Stroke
Vol
24,
No
11
November
1993
if
IL-118
is
produced
in
the
brain,
this
may
not
preclude
a
role
for
peripheral
IL-1
in
brain
injury.
If
one
accepts
the
possibility
that
the
increase
in
IL-1p
mRNA
shortly
after
ischemic
stroke
indeed
translates
into
a
robust
production
of
this
potent
cyto-
kine,
one
must
also
anticipate
its
consequences;
these
include
activation
of
microglia
and
astrocytes26
and
transformation
of
the
endothelium
into
a
proinflamma-
tory
and
prothrombotic27
state
that
may
propagate
thrombosis
and
the
extension
of
the
ischemic
zone.
Finally,
it
is
important
to
point
out
that
IL-1p
mRNA
is
only
one
of
many
genes
that
are
overexpressed
in
the
brain
after
injury;
most
notably,
acute immediate
genes
such
as
C-foS,28
heat-shock
protein,2829
and
the
tumor
suppressor
gene
product
p5330
have
been
shown
to
be
overexpressed
in
the
brain
in
response
to
ischemia.
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after
middle
cerebral
artery
occlusion
in
the
rat.
Biochem
Biophys
Res
Commun.
1992;182:1201-1207.
Editorial
Comment
Recent
advances
and
research
interest
in
molecular
biology
have
prompted
stroke
researchers
and
neurosci-
entists
to
study
gene
induction
and
expression
in
ischemic
brain.
A
good
example
of
this
interest
is
the
induction
and
expression
of
the
70-kD
stress
(heat-shock)
gene
at
both
transcriptional
and
translational
levels
in
neurons,
glia,
and
endothelial
cells
that
have
been
intensively
investi-
gated
in
various
models
of
permanent
and
temporary
cerebral
ischemia.1-6
Along
a
similar
line,
using
a
rat
interleukin
(IL)-1l3
cDNA
synthesized
from
stimulated
rat
peritoneal
macrophage
RNA,
Liu
et
al
have
now
reported
the
induction
and
expression
of
IL-1X3
mRNA
in
the
ischemic
zone
in
both
hypertensive
and
normotensive
rats
after
permanent
focal
ischemia.
Furthermore,
the
brain
level
of
IL-1X3
mRNA
is
increased
markedly
in
hyperten-
sive
rats
over
the
levels
found
in
two
different
strains
of
normotensive
rats.
A
couple
of
important
implications
resulting
from
this
interesting
study
might
need
elabora-
tion.
First,
the
elevated
levels
of
IL-1,3
mRNA
in
the
brain
cortex
in
hypertensive
rats
correlate
with
the
severity
of
the
neurological
deficits
after
permanent
focal
ischemia,
which
suggests
that
the
elevated
levels
of
cytokine
mRNA
by guest on June 6, 2013http://stroke.ahajournals.org/Downloaded from