(Stroke. 1995;26:1093-1100.)
© 1995 American Heart Association, Inc.
Articles |
Cerebral Hypoxia-Ischemia Stimulates Cytokine Gene Expression in Perinatal Rats
From the Departments of Pediatrics and Neurology, University of Michigan, Ann Arbor.
Correspondence to Dr F.S. Silverstein, Room 8301, MSRB III Bldg, University of Michigan, Ann Arbor, MI 48109-0646. E-mail faye.silverstein@umich.edu.
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Abstract |
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Background and Purpose We tested the hypothesis that cerebral hypoxia-ischemia selectively stimulates interleukin-1ß (IL-1ß) and tumor necrosis factor-
(TNF-) gene
expression in brain regions susceptible to irreversible injury in
perinatal rats.
Methods To elicit focal hypoxic-ischemic brain injury,
7-day-old perinatal (P7) rats were subjected to right carotid artery
ligation followed by 3 hours of 8% O2 exposure and were
killed 0 to 48 hours after hypoxia. Regional tissue IL-1ß and
TNF- mRNA content were measured by reverse transcription followed by
polymerase chain reaction amplification (RT-PCR) in samples prepared
from cortex and hippocampus of the lesioned and contralateral
hemispheres. cDNAs were amplified with primers specific for IL-1ß,
TNF-, and the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), which served as an internal control. The RT-PCR
products were subjected to Southern blot analysis and hybridized
with 32P-labeled gene-specific probes. Radioactivity was
measured in excised bands, and results were normalized on the basis of
levels of GAPDH expression.
Results In unlesioned P7 brain, IL-1ß mRNA was barely
detectable. In lesioned forebrain, there was a marked, transient
stimulation of IL-1ß mRNA expression, peaking at 4 hours after
hypoxia. Hybridization signal was increased 16- to 30-fold over
values from contralateral hemisphere samples in three independent
assays (P<.05 comparing values in left and right cortex and
in left and right hippocampus with the Kruskal-Wallis ranking test); by
24 hours after hypoxia, levels returned to normal. Similar
transient increases in TNF- mRNA expression were detected. In a
closely related model of perinatal brain injury elicited by focal
intracerebral
N-methyl-D-aspartate injection, there was a
corresponding acute stimulation of IL-1ß and TNF- mRNA expression
at 4 hours after injection.
Conclusions These results suggest that IL-1ß and TNF- may
play important roles in the response of the developing brain to acute
hypoxic-ischemic injury.
Key Words: interleukins • excitotoxicity • newborn • tumor necrosis factor • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interleukin-1ß (IL-1ß) and tumor necrosis factor-
(TNF-) are multifunctional cytokines that may
play important roles both in normal central nervous system development
and in the response of the brain to diverse forms of
injury.1 2 3 4 5 6 7 8 IL-1ß and TNF- are among the
best-characterized early-response cytokines; they are both small
proteins (each about 17 000 D) and are frequently expressed in
concert. Recent data suggest that they may be synthesized and secreted
by several central nervous system cell types including microglia,
astrocytes, and neurons. IL-1ß and TNF- share potent
proinflammatory actions and the potential to modulate cell growth.
Biological effects of these cytokines that could influence both
progression of injury and regulation of wound healing in the brain
after injury include stimulating synthesis of other cytokines and
soluble injury mediators, inducing leukocyte infiltration, influencing
glial gene expression, and stimulating local synthesis of trophic
factors.
Recent reports based on data obtained in adult animals suggest
that IL-1ß contributes directly to the pathogenesis of
ischemic brain injury, in that pharmacological antagonism of
IL-1ß attenuates cerebral ischemic injury.9 10
Markedly increased levels of expression of IL-1ß mRNA have been
detected acutely in ischemic forebrain in adult
animals.11 12 13 14 Excitotoxic injury elicits a similar
transient increase in levels of expression of mRNA's encoding IL-1ß
and several related cytokines.15 16 Acute stimulation of
expression of TNF- mRNA and protein after focal ischemic
injury in adult rats was also recently reported; of particular interest
were data indicating transient induction of neuronal TNF-
expression.6
Giulian et al7 8 initially demonstrated IL-1ß
bioactivity in immature rodent brain on the basis of functional assays
in forebrain homogenates; they also reported that direct
intracerebral injection of recombinant IL-1ß protein
stimulated gliosis and angiogenesis. However, no previous studies have
evaluated whether ischemic and excitotoxic neuronal injury
stimulate expression of these proinflammatory cytokines in the immature
rodent nervous system. To address this question, we developed
semiquantitative reverse transcriptase–polymerase chain reaction
(RT-PCR) assays16 17 to enable us to estimate changes in
levels of expression of IL-1ß and TNF- mRNA in perinatal rat
brain.
We incorporated studies using two closely related, well-characterized perinatal rat brain injury models. To elicit hypoxic-ischemic injury at this developmental stage, a perinatal rat stroke model was used: 7-day-old perinatal (P7) rats underwent right carotid artery ligation followed by timed exposure to moderate hypoxia (8% O2 for 3 hours).18 19 This lesioning method elicits extensive ipsilateral forebrain injury; in animals killed 5 days later, there is widespread neuronal necrosis and substance loss in cortex, hippocampus, and striatum. To elicit focal excitotoxic lesions, stereotaxic intracerebral injections of N-methyl-D-aspartate (NMDA) were performed in P7 rats.20 In P7 rats that receive intracerebral injections of NMDA (5 to 25 nmol) and are killed 5 days later, there are consistent dose-dependent ipsilateral lesions, characterized by neuronal necrosis and tissue loss.20 Intrahippocampal injection of 7.5 nmol NMDA elicits a well-circumscribed focal necrotic lesion in the dorsal hippocampus with loss of pyramidal cells extending from the injection site and a reduction of approximately 25% in hippocampal cross-sectional area; the overlying cortex is also invariably injured.
Levels of expression of IL-1ß and TNF- mRNA were compared in
tissue samples from the lesioned and contralateral cerebral hemispheres
of animals killed 0 to 48 hours after the initial insult. Our data
demonstrated that in P7 rats both focal hypoxic-ischemic and
excitotoxic injury resulted in marked, transient stimulation of IL-1ß
and TNF- mRNA expression in lesioned forebrain structures.
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Materials and Methods |
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Animal Methods
All lesioning was performed in P7 Sprague-Dawley rats. To elicit unilateral hypoxic-ischemic forebrain injury, the right carotid artery was ligated in ether-anesthetized animals; 1 to 2 hours later animals were exposed to 8% O2/balance N2 for 3 hours in plastic chambers set in water baths (temperature, 36.5°C).18 19 21 Ligation alone does not reduce regional cerebral blood flow, elicit disruption of any metabolic markers, or result in tissue injury. However, ligation in combination with sustained exposure to 8% O2 for
2.5 hours results
in prominent ipsilateral forebrain injury. Animals were killed by
decapitation immediately after hypoxia or after recovery
periods of 1, 2, 4, 8, 12, 24, or 48 hours (n=3 to 4 per group). Three
independent sample groups were evaluated at 4 and 24 hours after
hypoxia.
To elicit excitotoxic injury, NMDA (7.5 nmol NMDA dissolved in
phosphate-buffered saline [PBS], pH 7.4) was stereotaxically injected
into the right cerebral hemisphere, and rats were killed 4 or 24 hours
after injection, along with PBS-injected littermate controls (n4 per
group). Injections were performed in ether-anesthetized P7 rats using
coordinates relative to bregma: anteroposterior, -2.0 mm; lateral, 2.5
mm; depth, 4 mm; 1 µL was injected over 5 minutes with a 26-gauge
Hamilton syringe.
Surgical protocols were approved by the University of Michigan Committee on Care and Use of Laboratory Animals.
In P7 rats, cytochrome oxidase histochemistry provides a sensitive
indicator of acute ischemic and excitotoxic
injury21 ; in brain regions in which irreversible neuronal
injury evolves, focal suppression of cytochrome oxidase activity is
detectable within the first 12 hours after the insult and precedes loss
of Nissl's staining. To illustrate the anatomic distribution of acute
injury elicited by the two lesioning methods used, lesions were induced
in two animals that were killed 4 hours after
hypoxia-ischemia or NMDA injection. Cytochrome oxidase
histochemistry assays were performed by incubation of 20-µm frozen
brain sections in 0.1 mol/L phosphate buffer, pH 7.4, containing 50 mg
3,3'-diaminobenzidine, 25 mg cytochrome C, 20 mg catalase, and 4 g
sucrose/100 mL for 3 hours. Representative photomicrographs that
demonstrate the widespread distribution of ipsilateral forebrain injury
are presented in Fig 1
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RNA Isolation
Total RNA was prepared from pooled tissue samples of left or
right cortex, hippocampus, and in some cases, also from striatum,
immediately after death by homogenizing tissue in guanidinium
thiocyanate and extracting RNA using acidified phenol.22
The entire structure was included, and typically samples were pooled
from three to four animals. RNA concentrations were estimated from
optical density measurements at 260 nm, and typical yields were 0.6 to
0.8 µg/mg tissue.
Reverse Transcriptase–Polymerase Chain Reaction
Total RNA (1 µg) was used for cDNA synthesis. First-strand
cDNA synthesis was primed with random hexamers and carried out
according to manufacturer specifications (RT-PCR kit,
Perkin-Elmer).
IL-1ß PCR was performed using 200 µmol/L of each deoxynucleotide, 2
U Taq DNA polymerase (Perkin-Elmer), and 1 µmol/L of
IL-1ß–specific primers that amplified a 520-bp fragment (Table 1).15 16 With the following amplification
conditions, IL-1ß mRNA was detected in most samples: 94°C for 90
seconds, 60°C for 45 seconds, and 72°C for 45 seconds for 35
cycles. Primers for a 528-bp fragment of the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (0.4 µmol/L) (Table 1) were included in each amplification reaction to allow
standardization.23 GAPDH expression did not change with
any of the experimental manipulations performed.
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To assay TNF- mRNA expression, the above reaction mix was
modified to include 0.5 µmol/L of primers specific for a 295-bp
TNF- fragment (Clontech Laboratories) and 0.2 µmol/L of the GAPDH
primers. Amplification conditions were 94°C for 100 seconds, 60°C
for 40 seconds, and 72°C for 40 seconds for 32 cycles. TNF- mRNA
was detected in all samples.
In preliminary experiments, the influence of the number of
amplification cycles on the yields of PCR products was assessed. RT
samples were used in which high levels of expression of the cytokine
mRNAs had been detected (P7 right hippocampus, 4 hours after NMDA
injection20 ). Aliquots of the RT product were amplified
with primers for IL-1ß and GAPDH or TNF- and GAPDH concurrently
for 20 to 40 cycles. Final amplification conditions were within the
linear range for amplification of IL-1ß and TNF- cDNAs.
Southern Blot Analysis
A semiquantitative method was developed to compare levels of
mRNA expression in different samples on the basis of Southern blot
analysis of RT-PCR products.17 The probes used were a
648-bp BamHI fragment of rat IL-1ß cDNA (the generous gift
of Dr Masui, Otsuka Pharmaceuticals, Japan), labeled with
32P-dCTP by the random prime method (Promega), and
oligonucleotide probes for TNF- and GAPDH (Table 1
), labeled with
32P-dATP, using terminal deoxynucleotide transferase
(Boehringer Mannheim).
Duplicate 25-µL aliquots of each PCR product were electrophoresed through 1% agarose gels in parallel, transferred to Nytran membranes (Schleicher and Schuell, according to manufacturer's protocol), and baked at 80°C for 2 hours. Standard conditions for Southern blot hybridization were used.24 The procedure for the cDNA probe included prehybridization for 3 hours in buffer containing 50% deionized formamide, 6x SSC, 1x Denhardt's reagent, and 1% SDS and 1 mg/mL salmon sperm DNA and overnight hybridization in 10 mL of the same buffer with 107 cpm probe at 42°C. The hybridization procedure for oligonucleotide probes included prehybridization in buffer containing 6x SSC, 1% SDS, and 1x Denhardt's reagent and 0.5 mg/mL salmon sperm DNA for 3 hours at 55°C and hybridization in 10 mL of 6x SSC/1% SDS buffer with 3x106 cpm probe overnight at 55°C. Posthybridization washes were performed with 2x SSC/0.1% SDS at RT.
Membranes were apposed to x-ray film (XAR, Kodak, or REFLECTION NEF, EI
Dupont) (1 to 3 hours of exposure for TNF- and GAPDH and 24 to 48
hours for IL-1ß); the autoradiograms were used to identify bands of
interest, corresponding regions of the membranes were excised, and
accumulated radioactivity was measured by liquid scintillation
spectroscopy. In each assay, values for IL-1ß and TNF- were
normalized on the basis of GAPDH counts in the same sample.
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Results |
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Using these RT-PCR assay conditions, IL-1ß mRNA was detected at very low levels in most samples from unlesioned P7 to P10 rat cortex, hippocampus, and striatum; TNF-
and GAPDH mRNA were readily detected
in all samples. Of note, before initiating these studies we had
attempted Northern blot analysis of total RNA (up to 30 µg per
sample) and did not detect any IL-1ß mRNA in our samples with this
much less sensitive method (not shown).
IL-1ß mRNA expression was compared in samples from the left and right
cortex (Fig 2, top) and corresponding left and right
hippocampus (Fig 2, bottom) of animals that underwent
hypoxic-ischemic lesioning and were killed 0 to 48 hours after
hypoxia. Fig 3 summarizes the quantitative data,
normalized for GAPDH expression in each sample; in both regions, values
peaked at 4 hours in samples from the lesioned hemisphere. Samples from
lesioned animals killed 1 or 2 hours after hypoxia were assayed
in independent experiments; there were variable increases of lesser
magnitude in samples from the lesioned hemisphere at these times (not
shown).
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To confirm these trends, data were incorporated from three
independent assays of IL-1ß mRNA in samples from left and right
cortex, hippocampus, and striatum of P7 animals that underwent right
carotid artery ligation plus 3 hours of 8% O2 exposure and
were killed 4 hours after hypoxia (Fig 4). In
all three regions of the lesioned hemisphere, there were increased
levels of expression of similar magnitudes (mean 16- to 30-fold
increases). Statistical analysis demonstrated significant
interhemispheric differences in the cortex and hippocampus
(P<.05 comparing values in left and right cortex and in
left and right hippocampus with the Kruskal-Wallis ranking test). The
striatal trends were similar, but there was somewhat greater variation
among the results of the three assays. Quantitative data from two
independent assays of samples from lesioned animals killed 24 hours
after hypoxia demonstrated that levels of expression returned
to the normal range (mean values [cpm]: left cortex, 332; right
cortex, 519; left hippocampus, 379; right hippocampus, 396; left
striatum, 350; right striatum, 432).
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In a single experiment, levels of TNF- mRNA were compared in samples
from left and right cortex (Fig 5, top) and
corresponding left and right hippocampal samples (Fig 5, bottom) of
animals that underwent hypoxic-ischemic lesioning and were
killed 0 to 48 hours after hypoxia. These assays were performed
using aliquots from the same RT products as were used in the assays of
IL-1ß mRNA presented in Fig 2. Fig 6 summarizes
the quantitative data, normalized for GAPDH expression in each sample.
TNF- mRNA was detectable in all samples from P7 to P10 cortex and
hippocampus; at 4 hours after hypoxia, in both cortex and
hippocampus values were twofold to threefold higher in samples from the
lesioned hemisphere than in the contralateral hemisphere.
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Fig 7 summarizes results of assays of TNF- mRNA
in three independent samples from left and right cortex and hippocampus
of P7 rats that underwent right carotid artery ligation followed by 3
hours of 8% O2 exposure and were killed 4 or 24 hours
after hypoxia. Increased levels of expression of similar
magnitudes (mean threefold increases) were found in both regions of the
lesioned hemisphere at 4 hours but not at 24 hours after
hypoxia. Differences in the left and right 4-hour
posthypoxia hippocampal samples were significant
(*P<.05, Kruskal-Wallis ranking test); the trends for the
4-hour cortical values were similar but not statistically
significant.
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The influence of excitotoxic injury on IL-1ß and TNF- mRNA
expression in P7 rats was also assessed. In Fig 8, two
autoradiograms compare IL-1ß and TNF- expression in samples
prepared from left and right hippocampus of rats killed 4 or 24 hours
after they had received right intrahippocampal injections of NMDA (7.5
nmol) or equal volumes of saline. The dose of NMDA used elicits a
well-circumscribed focal necrotic lesion in the dorsal hippocampus,
whereas injection of PBS results in a focal mechanical injury along the
needle track.25 In the NMDA-lesioned hippocampus, IL-1ß
was markedly increased at 4 hours (1956 versus 243 cpm in the
contralateral hippocampus [values normalized for GAPDH expression]);
in the saline-injected hippocampus, values were slightly but
consistently about twofold higher than in the contralateral hippocampus
(341 versus 128 cpm); values were equal bilaterally in both groups at
24 hours after lesioning. With respect to TNF- expression, trends
were similar, but the magnitude of stimulation at 4 hours was
considerably less (151 030 versus 67 075 cpm in the contralateral
hippocampus); findings were similar in cortical samples (J.S. and
F.S.S., unpublished data, 1994).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These data provide the first experimental evidence that ischemic and excitotoxic lesioning stimulate cytokine gene expression in the developing mammalian brain.
In adult rats, acute ischemia-induced stimulation of IL-1ß
mRNA has been detected by Northern blot analysis after permanent
middle cerebral artery occlusion11 and after transient
global ischemia with recirculation at 15
minutes.12 In situ hybridization assays have documented
acute increases in regional expression of IL-1ß mRNA after permanent
middle cerebral artery occlusion in spontaneously hypertensive
rats13 and a delayed stimulation, peaking at 3 to 7 days
after transient global ischemia with recirculation at 30
minutes in adult Wistar rats.14 Differences in the
temporal sequence, anatomic distribution, and magnitude of stimulation
of IL-1ß mRNA expression among cerebral ischemia models
studied (summarized in Table 2) likely are attributable
predominantly to intrinsic differences in the pathophysiology of injury
among the experimental paradigms and to a lesser degree to differences
in the mRNA assay methods used.
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The highly sensitive RT-PCR assay developed for this study enabled measurement of much lower concentrations of mRNA than could be assayed by Northern analysis. Yet, the pattern of acute hypoxia-ischemia–induced IL-1ß mRNA expression in perinatal rats was quite similar to findings reported in adult brain after transient global ischemia with recirculation at 15 minutes.12 In both settings, increased IL-1ß mRNA was detected in all forebrain structures in which injury evolved, and peak expression occurred within the first 4 hours after the insult. In situ hybridization assays of IL-1ß mRNA in adult rodent brain after kainic acid treatment suggest that its major cellular source is microglial26 ; in lesioned P7 rat brain the predominant cellular source(s) remains to be determined.
Increased tissue content of specific mRNAs could reflect increased
synthesis and/or decreased degradation, and we did not attempt to
distinguish these mechanisms. The similar temporal features of peak
expression of IL-1ß and TNF- suggest that stimulation of their
transcription may be regulated by a common mechanism. Of note, in a
group of complementary experiments (J.S., unpublished data, 1994) in
which TGF-ß mRNA content was assayed using a similar RT-PCR assay
(Clontech Laboratories), we found that levels of expression of TGF-ß
mRNA did not change in the first 48 hours after injury. These results
provide evidence of the specificity of acute injury-induced increases
in IL-1ß and TNF- expression.
In this perinatal ischemic injury model, considerable data
indicate that endogenous excitatory amino acids play a
pivotal role in the progression of neuronal injury.19 That
direct intracerebral injection of NMDA elicited a
remarkably similar pattern of transient stimulation of cytokine gene
expression raises the possibility that NMDA-receptor overactivation may
be the initiating mechanism for ischemia-induced stimulation of
IL-1ß and TNF- mRNA transcription. Alternatively, in both
perinatal models, stimulation of cytokine gene expression also could be
a consequence of one or more of the events in the cascade of
irreversible neuronal injury that evolve after NMDA-receptor
overactivation.
Ischemic injury stimulates expression of a range of genes that may have
beneficial and/or detrimental effects on the evolution of neuronal
injury.27 28 The functional significance of injury-induced
expression of factors such as heat-shock proteins and immediate
early-response genes is currently uncertain. In neonatal rat brain,
focal hypoxic-ischemic injury stimulates transient expression
of multiple immediate early gene mRNAs in the lesioned hemisphere and,
to a lesser degree, in the contralateral hemisphere, peaking 1 to 3
hours after hypoxia.29 Whether the immediate
early-response genes, whose peak expression precedes that of IL-1ß
and TNF-, influence transcription of the latter mRNAs is unknown.
Critical experimental questions include identification of the
pathophysiological factors that account for the very brief duration of
stimulation of transcription in the early postinjury period and of any
processes that contribute actively to terminate their effects.
To assess the functional significance of our data, it will be essential
to determine whether increased levels of the encoded proteins are, in
fact, synthesized acutely. The studies of IL-1ß in adult stroke
models11 12 13 14 have not reported results of protein assays.
Similarly, our preliminary immunocytochemistry assays using a
commercial antibody (not shown) did not enable us to consistently
detect IL-1ß in lesioned brain; this may reflect intrinsic
limitations of current antibodies and assay methods. In contrast, Liu
et al6 recently reported important immunocytochemical data
demonstrating that focal ischemic injury in adult rats
stimulated synthesis of TNF- protein as well as mRNA. These results
provide support for the hypothesis that cytokines play a functional
role in the acute postischemic period.
Currently, the most convincing evidence that IL-1ß is
functionally important in the pathogenesis of ischemic injury
is provided by pharmacological studies demonstrating attenuation of
injury by treatment with antagonists. Treatment with an IL-1ß
receptor antagonist attenuated acute ischemic injury in adult
rats9 ; similarly, treatment with the functional IL-1
antagonist zinc protoporphyrin resulted in acute reduction of
ischemia-induced cerebral edema.10 No studies have
been published in which the neuroprotective efficacy of TNF-
antagonists was tested in ischemia models (through December
1994).
Potential cellular targets for cytokines in the early postinjury period
include astrocytes, microglia, endothelial cells, and neurons. In vivo
direct intracerebral injection of IL-1ß stimulates
gliosis (and neovascularization)4 8 ; additional effects of
IL-1ß on astrocytes, documented in vitro, include induction of nitric
oxide synthase.30 In both perinatal brain injury models
studied, there is marked stimulation of glial fibrillary acidic protein
mRNA expression in the acute phase in the lesioned
hemisphere,31 32 and cytokines may be among the molecular
signals that initiate this response. IL-1ß and TNF- may stimulate
microglia to synthesize other cytokines that amplify the injury
response. IL-1ß also may directly influence endothelial gene
expression; in vitro, IL-1ß stimulates endothelial cell expression of
the adhesion molecule intercellular adhesion
molecule–1.33 In addition, IL-1ß may exert direct
effects on neurotransmission.34 Thus, the cumulative
impact of these pleiotropic cytokine effects could depend on a wide
range of interacting factors in the acute postinjury recovery
period.
Recent clinical studies provide preliminary support for the hypothesis that cytokines play a pathogenetic role in neurological injury in the developing human nervous system. A prospective study of cord blood cytokine levels demonstrated that concentrations of IL-1ß were elevated in infants with severe perinatal complications35 ; moreover, in children with bacterial meningitis, acutely elevated cerebrospinal fluid concentrations of IL-1ß were strongly correlated with adverse neurological outcomes.36
Our results demonstrate that in the immature rodent brain,
hypoxic-ischemic and excitotoxic injury markedly and
transiently stimulate acute IL-1ß mRNA expression and that there are
coincident increases in TNF- mRNA levels. These data provide the
impetus to evaluate the potential functional role of cytokines in
perinatal brain injury and to determine whether pharmacological
strategies based on cytokine antagonism are beneficial or deleterious
to the developing nervous system.
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Acknowledgments |
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This research was supported by grants from the United Cerebral Palsy Education and Research Foundation and US Public Health Service NS-26142 (Dr Silverstein).
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Footnotes |
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Preliminary reports of these data were presented at the annual meetings of the Child Neurology Society, Orlando, Fla, October 14-16, 1993, and the Society for Pediatric Research, Seattle, Wash, May 2-5, 1994.
Received October 14, 1994; revision received January 3, 1995; accepted March 9, 1995.
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