From the Departments of Anesthesia (J.E.B.) and Internal Medicine and
Pharmacology (F.M.F.), Cardiovascular Center, University of Iowa College of
Medicine, Iowa City.
Correspondence to J.E. Brian, Jr, MD, Department of Anesthesia 6 JCP, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail eddie-brian{at}uiowa.edu
Methods—Cranial windows were implanted in anesthetized
rats and used to measure the diameter of cerebral arterioles. Windows
were flushed every 30 minutes for 4 hours with artificial cerebrospinal
fluid (aCSF) (n=6); aCSF with TNF
Results—Application of TNF
Conclusions—The present study indicates that TNF
An important component of the systemic response to endotoxin is
production of tumor necrosis factor-
In brain, TNF
A cranial window was prepared in a fashion similar to that described in
rabbits.22 The scalp, muscle, and
periostium overlying the parietal area of the skull were reflected, and
bleeding was controlled with ferric chloride solution. A
craniotomy (approximately 3x4 mm) was made in the
parietal bone with an air-cooled drill, and bone bleeding was
controlled with bone wax. The dura overlying an arteriole was incised.
Two blunt needles were affixed to a dam of bone wax surrounding the
craniotomy, and a circular glass cover slip (12
mm) was fused to the wax. The window was reinforced with dental
acrylic. An outlet tube was affixed to one needle and set to maintain
intracranial pressure at 10 cm H2O
pressure. A stopcock was attached to the other needle, and the window
was filled with aCSF warmed to 37°C and equilibrated with 90%
N2/5% O2/5%
CO2 (pH 7.28±0.01;
PO2, 69±1 mm Hg;
PCO2, 41±0.2 mm Hg). Cerebral
arterioles were observed with a microscope equipped with a video
camera, and images were recorded on videotape. Arteriolar diameter
was measured with a calibrated video micrometer. The
preparation was allowed to equilibrate for 30 minutes, during which
time the window was flushed with 2 mL of aCSF every 15 minutes.
Flushing the window with aCSF did not alter the diameter of cerebral
arterioles.
After the equilibration period, arteriolar diameter was measured under
control conditions and in response to topical ADP
(10-5 and
10-4 mol/L), an activator of
endothelial NO synthase. Responses to ADP were examined
to test responsiveness of the preparation. The window was then flushed
with aCSF several times, and the preparation was allowed to recover for
30 minutes. After a second measurement of baseline vessel diameter,
animals were randomly allocated to receive (1) aCSF alone (n=6); (2)
aCSF containing TNF
The absolute diameter of cerebral arterioles was recorded at
baseline and compared between groups. After baseline measurements, the
cranial windows were flushed every 30 minutes for 4 hours with 2 mL of
vehicle (aCSF) or TNF
To evaluate the effect of prolonged exposure to aminoguanidine on
arteriolar diameter, a separate group of rats had cranial windows
treated with aminoguanidine alone (0.3 mmol/L; n=4). After
creation of the window, dilatation to ADP
(10-5 and
10-4 mol/L) was tested, followed by a
30-minute recovery period. Windows were then flushed with
aminoguanidine every 30 minutes for 4 hours, and diameter was
recorded as above.
To test the specificity of the effect of aminoguanidine and
dexamethasone on TNF
At the conclusion of the 4-hour experimental protocol, some animals in
the aCSF and the TNF
During some studies (n=4), aCSF samples were frozen in liquid nitrogen
and stored at -80°C for later analysis for endotoxin
concentration. aCSF was sampled before being flushed into cranial
windows as well as when it was flushed from beneath windows at 2 and 4
hours. Endotoxin concentrations were measured with a limulus amebocyte
lysate assay (BioWhittaker). Samples of aCSF (50 µL) were incubated
with limulus amebocyte lysate at 37°C for 10 minutes; then a
chromogenic substrate was added, and the solution was
incubated for an additional 6 minutes. The reaction was terminated with
25% acetic acid, and absorption was read at 410 nm on a
spectrophotometer. Unknown concentrations were determined by linear
regression from concurrent standards, and all samples were assayed in
duplicate. aCSF endotoxin concentration was not different at any
measurement point, averaging 52±2 pg/mL.
Statistical Analysis
Effect of TNF
Effect of Aminoguanidine on Cerebral Arterioles
Effect of Dexamethasone on Cerebral Arterioles
Immunocytochemistry for Inducible NO Synthase
TNF
TNF
In the present study, examination of TNF
Dexamethasone has been reported to suppress expression of
inducible NO synthase in several
systems.36 37 38 39 Dexamethasone
appears to prevent expression of inducible NO synthase by inducing
overexpression of the cytoplasmic inhibitory unit I
Aminoguanidine has been reported to be a relatively selective
inhibitor of inducible (but not
endothelial) NO synthase.44
Relative to the constitutive isoforms of NO synthase
(endothelial and neuronal NO synthases), aminoguanidine
has a 50- to 500-fold higher affinity for inducible NO
synthase.45 Others have reported that
aminoguanidine does not constrict piglet cerebral arterioles and does
not reduce cerebral blood flow in rats, which suggests that
aminoguanidine has minimal effect on basal activity of constitutive NO
synthase.46 47 In addition, aminoguanidine
does not inhibit hypercapnia-induced cerebrovasodilatation, which has
been previously shown to be dependent on neuronal NO
synthase.47 We previously reported in
rabbits that aminoguanidine inhibits endotoxin-mediated dilatation of
cerebral arterioles but not dilatation due to activation of
endothelial NO synthase by acetylcholine, even after 2
hours of exposure to aminoguanidine.3 We
tested vasodilatation to ADP after 1 and 2 hours of aminoguanidine
exposure because the effect of aminoguanidine has been reported to have
a slow onset.44 Prior investigations have
shown that in rats, ADP-induced dilatation of cerebral arterioles is
mediated by activation of endothelial NO
synthase.48 49 In the present study,
exposure of cerebral arterioles to aminoguanidine for 4 hours did not
alter the diameter of cerebral arterioles, which suggests minimal
effect on the basal activity of constitutive NO synthase. However,
dilatation of cerebral arterioles in response to ADP was reduced after
2 hours but not 1 hour of aminoguanidine treatment. This suggests that
the effect of aminoguanidine on endothelial NO synthase
may have a relatively slow onset and that the inhibition of
endothelial NO synthase is modest compared with
inducible NO synthase. However, we observed significant reduction of
TNF
Recent data suggest that in the neonatal cerebral circulation, exposure
to a high concentration of TNF
In the present study we did not prepare cranial windows under
aseptic conditions, and low levels of endotoxin were present in
aCSF. We cannot rule out a potential role for endotoxin contamination
in the observed response. It is possible that endotoxin contamination
could enhance TNF
Before this study, little information was available regarding the
vascular effects of TNF
Received September 9, 1997;
revision received October 30, 1997;
accepted October 30, 1997.
Cardiovascular
Pharmacology WW,
SmithKline Beecham Pharmaceuticals,
King of Prussia, Pennsylvania
Several issues, however, must be pointed out concerning this
pharmacological demonstration so that further investigations provide
evidence that the data presented in the accompanying article
are not merely a pharmacological demonstration but may have biological
significance. First, what are the cellular sources of TNF
In summary, the present investigation is important because it draws
attention to possible biological function of a cytokine that
has been given little attention due to dominance of the
cytokine "dogma"—ie, the immune/inflammatory function.
Another example of the "changing flavor" of TNF
Received September 9, 1997;
revision received October 30, 1997;
accepted October 30, 1997.
© 1998 American Heart Association, Inc.
Original Contributions
Tumor Necrosis Factor-
–Induced Dilatation of Cerebral Arterioles
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Background and Purpose—In brain,
several cell types produce tumor necrosis factor- (TNF) after
injury or exposure to endotoxin. TNF alone or in combination with
endotoxin or other cytokines can cause expression of inducible
nitric oxide (NO) synthase. We have previously demonstrated that
endotoxin caused NO-dependent dilatation of cerebral arterioles in
vivo. In the present study we examined the hypothesis that TNF
causes NO-mediated dilatation of cerebral arterioles in vivo. (100 ng/mL; n=10); aCSF with
TNF and aminoguanidine (0.3 mmol/L; n=5), an
inhibitor of inducible NO synthase; or aCSF with TNF and
dexamethasone (1 µmol/L; n=6), which attenuates
expression of inducible NO synthase. In some animals, brain from
beneath the cranial window was examined by immunocytochemistry for
inducible NO synthase expression. caused marked, progressive
dilatation of cerebral arterioles, with a maximum increase in diameter
of 46±9% (mean±SEM) at 4 hours. Coapplication of either
aminoguanidine or dexamethasone with TNF prevented
dilatation of cerebral arterioles compared with TNF alone (4±2%
and 1±1% dilatation at 4 hours, respectively; P<.05).
Dexamethasone did not inhibit dilatation of cerebral
arterioles in response to adenosine diphosphate. However, 2
hours of aminoguanidine treatment produced moderate inhibition of
adenosine diphosphate–induced dilatation of cerebral
arterioles. After treatment with TNF, immunocytochemistry for
inducible NO synthase demonstrated expression in perivascular and
arachnoid cells but not brain cells. There was no detectable expression
of inducible NO synthase after treatment with aCSF. causes
cerebral vasodilatation and expression of inducible NO synthase in
perivascular and arachnoid cells. Inhibition of TNF-induced
dilatation by aminoguanidine and dexamethasone suggests
that the vasodilatation was due predominantly to expression of
inducible NO synthase. These findings support the concept that cerebral
vasodilatation that occurs during
pathophysiological conditions associated with
increased TNF production in brain is mediated by expression
of inducible NO synthase.
Key Words: cerebral arteries • dexamethasone • nitric oxide synthase • tumor necrosis factor • vasodilation
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Early bacterial
meningitis is accompanied by dilatation of cerebral arterioles and
marked increases in cerebral blood flow.1 2
Recently, we demonstrated that topical application of endotoxin
(lipopolysaccharide) to cerebral arterioles in vivo caused
marked, time-dependent dilatation that was mediated by nitric oxide
(NO).3 4 Dexamethasone and
aminoguanidine inhibited endotoxin-induced cerebrovasodilatation in
these studies, which suggested that the NO was derived from inducible
NO synthase (type II NO synthase).3 4 Most brain
cell types in culture can express inducible NO synthase after exposure
to endotoxin and/or cytokines.5 6 7 8 9 Thus,
it appears that expression of inducible NO synthase and NO
production in brain is an important element in the response of
brain arterioles to endotoxin. (TNF). During
systemic sepsis, TNF is released from macrophages and
systemic TNF levels increase.10 Systemic
administration of TNF mimics hypotension seen during
sepsis,11 and TNF-induced hypotension can be
attenuated by inhibitors of NO
synthase.12 Administration of anti-TNF
antibodies attenuates both endotoxin-induced hypotension and expression
of inducible NO synthase.13 Thus, during systemic
sepsis, it appears that TNF is an important mediator of
endotoxin-induced expression of inducible NO synthase. levels in CSF are elevated in both clinical and
experimental meningitis.14 15 16 After endotoxin
stimulation, astrocytes, microglia, and neurons produce
TNF.17 18 19 In addition, TNF is produced in
brain during other conditions including head
injury20 and
ischemia.21 Because TNF is produced
during inflammatory conditions in brain and because TNF is capable
of causing expression of inducible NO synthase, we hypothesized that
local exposure of cerebral arterioles in vivo to TNF would cause
dilatation mediated by inducible NO synthase.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Male Sprague-Dawley rats (n=47; weight, 363±6 g) were
anesthetized with pentobarbital (50 mg/kg IP), a tracheostomy
was performed, and ventilation was maintained with a small animal
ventilator. PaCO2 was adjusted to
approximately 40 mm Hg by altering minute ventilation, and
PaO2 was maintained at >100
mm Hg by supplementing room air with oxygen. Anesthesia
was supplemented by administration of additional pentobarbital (5 to 15
mg/kg per hour) through the femoral vein. Rectal temperature was
measured and maintained at 37±0.5°C with a heating pad. (Calbiochem; 100 ng/mL, n=10 or 10 ng/mL, n=3);
(3) aCSF containing TNF (100 ng/mL) and an inhibitor of
NO synthase, aminoguanidine (Sigma; 0.3 mmol/L; n=5); or (4) aCSF
containing TNF (100 ng/mL) and dexamethasone (Sigma;
1 µmol/L; n=6). with or without inhibitors. The
diameter of arterioles was measured at 30 minutes and 1, 2, 3, and 4
hours before flushing of the window. Changes in arteriolar diameter are
expressed as percent change in diameter compared with baseline.
Arterial blood pressure was continuously monitored, and
arterial blood gases were measured at regular
intervals. -induced vasodilatation, two other
groups of animals had ADP vasodilatation tested before and after
topical treatment with aminoguanidine or dexamethasone. In
one group (n=9), vasodilatation to ADP
(10-5 and
10-4 mol/L) was tested, followed by a
30-minute recovery period. Windows were then flushed with
aminoguanidine (0.3 mmol/L) initially and every 30 minutes.
Vasodilatation to ADP was retested in the presence of aminoguanidine
after 1 and 2 hours of exposure to aminoguanidine. In a second group
(n=4), vasodilatation to ADP (10-5 and
10-4 mol/L) was tested, followed by a
30-minute recovery period. Windows were then flushed with
dexamethasone (1 µmol/L) every 30 minutes for 4
hours. Dilatation to ADP was retested in the presence of
dexamethasone after 4 hours of exposure to
dexamethasone. 100-ng/mL groups (n=2 per group) underwent
transcardial perfusion fixation (Histochoice, Amersco). To delineate
the area of cortical exposure in the window, windows were filled with
Evans blue solution (3%) for 10 minutes before perfusion fixation.
Brain tissue from the window was removed, postfixed, and embedded in
paraffin. With a microtome, sections were cut (7 µm) through the
Evans blue–stained cortical area, deparaffinized, and rehydrated.
Sections were blocked for 1 hour (Secondary Detection Kit, Kirkegaard)
and rinsed, incubated overnight at 4°C with antibody to inducible NO
synthase (1:200 dilution, Upstate Biotechnology), then washed and
incubated with a secondary antibody (Secondary Detection Kit,
Kirkegaard) for 30 minutes. Endogenous peroxidase activity
was quenched with 3% hydrogen peroxide for 15 minutes, followed by
washing. Antibody binding was visualized with diaminobenzidine
chromagen peroxidase reaction (Secondary Detection Kit, Kirkegaard).
Positive staining is denoted by a brown color. Sections were
counterstained with eosin. In some sections, the primary antibody was
omitted.
Data are expressed as mean±SEM. Data between groups were
compared by ANOVA and Duncan's post hoc test. Data within groups were
analyzed by repeated-measures ANOVA and post hoc comparison by
means contrast. P<.05 was considered significant.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Baseline arteriolar diameter was not different between groups and
averaged 61±2 µm. Dilatation of cerebral arterioles in response
to ADP (10-5, 10-4 mol/L)
performed at the beginning of each study averaged 8±1% and 17±1%,
respectively. on Cerebral Arteriolar Diameter
In control animals treated with aCSF (n=6), there was no change in
arteriolar diameter over the 4 hours of study (Fig 1A
). However, TNF (n=10; 100 ng/mL)
caused time-dependent dilatation of cerebral arterioles, reaching
46±9% at 4 hours (Fig 1A). TNF-induced changes in arteriolar
diameter were significantly different than those in the aCSF group at
hours 1 to 4 (P<.05). Treatment of windows with a lower
concentration of TNF (10 ng/mL; n=3) produced 36±15% dilatation at
4 hours. When aminoguanidine (n=5; 0.3 mmol/L) was coapplied with
TNF (100 ng/mL), there was no significant change in arteriolar
diameter compared with aCSF over the 4 hours of the study (Fig 1B;
P>.05). There was also no significant change in arteriolar
diameter compared with aCSF when dexamethasone (n=6; 1
µmol/L) was coapplied with TNF (100 ng/mL) over the 4 hours of
study (Fig 1C; P>.05). There were no differences either
across time or between groups in mean arterial pressure or
arterial blood gas values, which averaged 121±1
mm Hg, pH 7.39±0.02, PaCO2
40±0.2 mm Hg, and PaO2
209±4 mm Hg (P>.05).
View larger version (13K):
[in a new window]
Figure 1. A, Effects of aCSF ( 
; n=6) or TNF (100
ng/mL; 
; n=10) on diameter of cerebral arterioles compared with
baseline (0 hours). B, Effect of aminoguanidine (AG) (0.3 mmol/L;
; n=5) and TNF (100 ng/mL) on diameter of cerebral arterioles. C,
Effect of dexamethasone (Dex) (1 µmol/L; 
; n=6)
and TNF (100 ng/mL) on diameter of cerebral arterioles. Data are
mean±SEM. *P<.05 compared with aCSF.
In a separate group of animals, application of aminoguanidine
alone for 4 hours (n=4; 0.3 mmol/L) did not alter the diameter of
cerebral arterioles (5±4% change at 4 hours). In other animals (n=9),
topical application of aminoguanidine (0.3 mmol/L) did not reduce
ADP-induced dilatation after 1 hour but modestly reduced ADP-induced
dilatation after 2 hours of treatment (Fig 2; P<.05). Mean
arterial pressure and arterial blood gases did
not vary during the study and averaged 116±2 mm Hg, pH
7.39±0.01, PaCO2 38±1 mm Hg,
and PaO2 197±13 mm Hg.
View larger version (14K):
[in a new window]
Figure 2. Effect of ADP (10-5 mol/L,
closed bars; 10-4 mol/L, open bars) on diameter
of cerebral arterioles (n=6) before (control) and after 1 and 2 hours
of treatment with aminoguanidine (0.3 mmol/L). Treatment of
cranial windows with aminoguanidine for 2 hours but not 1 hour
significantly reduced dilatation of cerebral arterioles to both
concentrations of ADP. Data are mean±SEM. *P<.05
compared with control.
In a separate group of animals (n=4), application of
dexamethasone for 4 hours (1 µmol/L) did not affect
baseline arteriolar diameter (56±5 µm before and 61±5
µm after 4 hours of dexamethasone exposure) or
ADP-induced dilatation (Fig 3). Mean
arterial pressure and arterial blood gases did
not vary during the study and averaged 123±1 mm Hg, pH
7.38±0.01, PaCO2 41±1 mm Hg,
and PaO2 204±4 mm Hg.
View larger version (10K):
[in a new window]
Figure 3. Effect of ADP (10-5 mol/L,
closed bars; 10-4 mol/L, open bars) on diameter
of cerebral arterioles (n=4) before (control) and after 4 hours
treatment with dexamethasone (Dex) (1 µmol/L).
Data are mean±SEM. There were no differences in dilatation to either
dose of ADP.
In tissue sections from windows treated with TNF, perivascular
cells as well as the arachnoid membrane exhibited positive staining for
inducible NO synthase (Fig 4). In tissue
sections from windows treated with aCSF alone, no staining was noted.
Omission of the primary antibody prevented staining in TNF-treated
sections. We did not detect staining of brain parenchyma in any section
with or without TNF treatment. In addition, staining of vascular
endothelium or neurons was not evident in any
section.
View larger version (91K):
[in a new window]
Figure 4. Immunocytochemistry staining for inducible NO
synthase protein in brain from beneath cranial windows treated with
either TNF (100 ng/mL) or aCSF for 4 hours. In all panels in this
figure, the brain parenchyma is at the bottom of the photograph, and
the arachnoid is the transverse membrane. The blood vessels noted are
located in the pia-arachnoid space. A, Brain from a cranial window
treated with TNF, which demonstrates positive (brown) staining of
the arachnoid membrane (long arrow) and perivascular cells (short
arrow; magnification x400). A large (short arrow) and small arteriole
are present at the right and center of the photograph, and a venule
is present on the left side. B, A higher magnification view of a
cerebral arteriole and arachnoid membrane from a different cranial
window treated with TNF, also demonstrating positive staining of the
arachnoid membrane (long arrow) and perivascular cells (short arrow;
magnification x1250). C, Section from the same cranial window as in B
demonstrating more intense staining of the arachnoid membrane (arrow;
magnification x1250). D, Section from a window treated with aCSF,
demonstrating no detectable staining of the arachnoid or blood vessel
(magnification x1250). The arachnoid membrane is denoted by the
arrow.
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
There are several new findings in this study. First, TNF
produced time-dependent dilatation of cerebral arterioles in vivo.
Second, TNF-mediated vasodilatation was dependent on
production of NO, which appeared to be derived from inducible
NO synthase. Because both aminoguanidine and dexamethasone
completely inhibited TNF-induced dilatation, the entire vasodilator
response may be dependent on NO from inducible NO synthase. Third,
inducible NO synthase appears to be expressed in perivascular and
meningeal cells but not in parenchymal cells. These data suggest that
when TNF levels are increased in the central nervous system, TNF
may lead to increased NO production from inducible NO synthase
and cerebrovasodilatation. Expression in Brain
A number of cells in brain can produce TNF, including
microglia,18
astrocytes,17 19 23 and
neurons.18 During exposure of animal and
human brain to endotoxin, TNF concentration increases in
CSF,24 25 and peak concentrations reach 50
to 100 ng/mL.26 27 When endotoxin is
introduced into CSF, TNF concentration in CSF markedly exceeds
systemic concentration, which suggests local production of
TNF in brain.28 After introduction of
endotoxin into CSF, TNF can be detected in CSF within 15 minutes,
which suggests that TNF is rapidly produced in the central nervous
system.25 In experimental meningitis,
administration of antibodies against TNF into the CSF reduces
leukocytosis and edema formation in brain, which suggests that TNF
plays an important role in the inflammatory
response.29 In addition, TNF
concentration in brain is increased in other conditions with an
inflammatory component such as head
injury20 and
ischemia.21 Thus, there are data to
support the concept that TNF is produced locally in brain during
injury and/or inflammation and that TNF contributes to the
inflammatory response. and Inducible NO Synthase
TNF appears to be an important mediator in expression of
inducible NO synthase during systemic sepsis, as antibodies directed
against TNF attenuate endotoxin-mediated hypotension as well as
reduce inducible NO synthase activity.13 In
cultured noncerebral cells, TNF alone30
or in combination with endotoxin or other
cytokines31 32 33 causes expression
of inducible NO synthase and increases NO production. Cultured
glioma cells (derived from astrocytes)34 as
well as cultured cerebral endothelium and smooth
muscle8 express inducible NO synthase after
exposure to TNF. Others have reported that exposure of cultured
astrocytes or glioma cells to TNF and endotoxin causes expression of
inducible NO synthase and increased NO
production.9 Microglia, when
stimulated with TNF and endotoxin, demonstrate increased NO
production and are cytotoxic to
oligodendrocytes.35 In addition,
TNF-treated meningeal fibroblasts are toxic to cortical neurons, and
the toxicity can be blocked with NO synthase
inhibitors.23 Some studies in
isolated cells in vitro suggest that TNF alone does not to cause
expression of inducible NO synthase. However, in vivo multiple cell
types are present, and each may contribute to the inflammatory
response. -treated tissue sections
stained with a polyclonal antibody against inducible NO synthase
suggests expression of inducible NO synthase. The arachnoid membrane
stained positive, which suggests expression of inducible NO synthase in
meningeal fibroblasts. Positive staining was also noted in perivascular
cells, which could be fibroblasts or pericytes. Cells located in brain
parenchyma did not stain positive for inducible NO synthase, which
suggests that there was no significant expression of inducible NO
synthase in glia. The antibody utilized appears specific, since there
was no staining of the vascular endothelium
(endothelial NO synthase) or neurons (neuronal NO
synthase). 
B,
which prevents activation and nuclear translocation of nuclear factor
B.40 41 We previously reported that
dexamethasone attenuates endotoxin-induced dilatation of
cerebral arterioles, which suggested that a portion of
endotoxin-induced dilatation of cerebral arterioles was mediated by
expression of inducible NO synthase.4 In
the present study, dexamethasone completely inhibited
TNF-induced dilatation of cerebral arterioles. However,
dexamethasone did not affect the baseline diameter of
cerebral arterioles or vasodilatation due to activation of
endothelial NO synthase by ADP. In addition, others
have reported that dexamethasone does not alter constrictor
or dilator responses in vessels.42 43 These
data strengthen the concept that TNF-induced vasodilatation of
cerebral arterioles is due entirely to NO production from
inducible NO synthase. -induced dilatation after 1 hour of aminoguanidine exposure,
which suggests that the effect of aminoguanidine on inducible NO
synthase may have a more rapid onset. Because aminoguanidine as well as
dexamethasone fully inhibited TNF-induced dilatation, we
believe it is unlikely that the primary mechanism of TNF-induced
dilatation is due to activation of endothelial NO
synthase. causes acute dilatation of cerebral
arterioles.46 Both NG-nitro-L-arginine and
aminoguanidine inhibited the TNF-induced dilatation, which suggests
that NO, possibly derived from inducible NO synthase, was important in
the dilatation.46 However, the concentration of
TNF used in that study (10-7 mol/L; 3.6
µg/mL) is higher than concentrations reported to occur in vivo during
brain inflammation.26 27 In addition, the time
course of the observed vasodilatation (30 minutes) may not be
consistent with expression of inducible NO
synthase.46 The concentration of TNF we used
(100 ng/mL; 2x10-9 mol/L) is within the range
reported in brain in vivo.26 27 -induced dilatation of cerebral arterioles.
However, in windows treated only with aCSF, there was no change in
diameter of cerebral arterioles over the 4 hours of study. Furthermore,
in rat cranial windows the minimum concentration of endotoxin necessary
to produce measurable dilatation of cerebral arterioles over 4 hours
treatment is 1 ng/mL (J.E.B., unpublished data, 1994), approximately
20-fold higher than the contaminating endotoxin concentration measured
in the present study. This suggests that endotoxin contamination
alone has minimal, if any, impact on vascular diameter in our model. In
addition, a recent study reports that preparation of cranial windows
under sterile or nonsterile conditions did not alter the response of
neonatal cerebral arterioles to TNF, which suggests that endotoxin
contamination did not affect TNF-induced
dilatation.46 in intact brain. By use of pharmacological
tools, we have demonstrated that the cerebrovascular effects of TNF
appear to be mediated by NO production from inducible NO
synthase. Furthermore, immunocytochemistry for inducible NO synthase in
brain tissue treated with TNF indicates that inducible NO synthase
is expressed in perivascular and meningeal cells. This suggests that
increases in cerebral blood flow during meningitis or other
inflammatory conditions in brain may be mediated by production
of TNF in the central nervous system and subsequent expression of
inducible NO synthase.
Selected Abbreviations and Acronyms
aCSF
=
artificial cerebrospinal fluid
CSF
=
cerebrospinal fluid
NO
=
nitric oxide
TNF
=
tumor necrosis factor-
Acknowledgments
This study was supported by National Institutes of Health grants
HL-38901, NS-24621, and GM-08442; by research funds from the Iowa
Affiliate of the American Heart Association (IA-94-GS-29); and by
research funds from the Department of Anesthesia. Dr Faraci
is an Established Investigator of the American Heart Association. The
authors wish to express thanks to Dr Costantino Iadecola for aid with
immunocytochemistry, Dr Bradley J. Hindman for aid with endotoxin
assays, and Paula Ludwig and Alice McAllister for technical
assistance.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
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Editorial Comment
Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
TNF is a pleiotrophic cytokine originally implicated in
immune and inflammatory reactions. TNF has the prime reputation of
enhancing inflammation by activation of the production and
release of other cytokines (eg, interleukin-1, interleukin-6)
and chemokines (eg, interferon-inducible protein 10) from diverse cells
(eg, leukocytes, endothelial, smooth muscle) and
activation of the expression of adhesion molecules on
endothelial cells (eg, intercellular adhesion
molecule-1, endothelial leukocyte adhesion molecule-1,
vascular cell adhesion molecule-1 and leukocytes
(CD11/CD18).1 However, TNF possesses additional, less
studied actions that may bear significantly on the microcirculation.
For example, TNF induces upregulation of potent prothrombotic
molecules such as tissue factor (a key coagulation initiation factor)
and plasminogen activator
inhibitor-1 and suppression of thrombomodulin expression on
the endothelium. Thus, TNF transforms the
blood-endothelium interfaces so that it "flips"
from an antithrombotic surface to a prothrombotic surface, thereby
enhancing clot formation and inflammation. In the accompanying article
by Brian and Faraci, attention is drawn to yet another possible
biological function of this cytokine—regulation of
vasoactivity of the brain microcirculation. In this well-done study,
brain microvessels were superfused with TNF by a "window" method
that allows direct monitoring of vessel diameter. TNF caused
dose-dependent dilation of cerebral microvessels apparently mediated by
NO production after the induction of the inducible NO synthase
isoform. that
provide for the abluminal TNF access? Since neurons and glia
(astrocytes and microglia) have been shown to be capable of
transcribing and translating TNF2 in specialized
conditions (eg, ischemia, endotoxin exposure), it is important
to elucidate these cellular elements and conditions. Second, could
physiological or
pathophysiological conditions result in comparable
levels of TNF that were used to elicit the responses monitored in
the window setup? Third, is the mechanism of TNF-induced
vasodilatation only NO dependent? For example, the time course for NO
synthase induction lags behind the early dilation induced by TNF;
are there downstream or upstream mediators that might be involved?
Fourth, more definite proof for a role that TNF might play in
physiological and/or pathological regulation of
brain microcirculation tone must employ highly specific TNF
antagonists which, if applied to
physiological or pathological conditions that are
associated with modified microvascular tone, could reverse/manipulate
the vascular condition. These studies are critical in view of the
potential large redundancy in vasoactive mediators that seem to have
access to the microcirculation during
pathophysiological conditions. biology has
recently been provided by Nawashiro et al,3 who
diversified the annotation of cytokines and TNF in
particular by showing remarkable neuroprotection against
ischemia using direct application of TNF–binding protein in an
ischemic neuroinjury study. Are there more surprises to be
watched for? Possibly.
Selected Abbreviations and Acronyms
aCSF
=
artificial cerebrospinal fluid
CSF
=
cerebrospinal fluid
NO
=
nitric oxide
TNF
=
tumor necrosis factor-
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
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