(Stroke. 2001;32:212.)
© 2001 American Heart Association, Inc.
Original Contributions |
Quantitative Analysis of Gene Expressions Related to Inflammation in Canine Spastic Artery After Subarachnoid Hemorrhage
From the Department of Neurosurgery (Y.A., H.K., H.O., T.H.), Tokyo Women’s Medical University, Tokyo, Japan, and the Department of Cell Biology (Y.A., H.O., J.T.), Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
Correspondence to Hidetoshi Kasuya, MD, Department of Neurosurgery, Tokyo Women’s Medical University, Kawada-cho 8-1, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail hkasuya{at}nij.twmu.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Background and Purpose—The possible role of inflammatory reaction of the cerebral artery in the pathogenesis of cerebral vasospasm has been noted in recent studies. We quantitatively measured the levels of expression of genes related to inflammation in the spastic artery in a canine double-hemorrhage model.
Methods—Twenty dogs
were assigned to 4 groups: group D0, control; group D2, dogs killed 2
days after cisternal injection of blood; group D7, dogs given double
cisternal injections of blood and killed 7 days after the first
injection; and group D14. Angiography was performed twice: on the first
day and before the animals were killed. Total RNA was extracted from
the basilar artery. The expressions of interleukin (IL)-1
, IL-6,
IL-8, IL-10, tumor necrosis factor-, E-secretin, fibronectin,
intercellular adhesion molecule (ICAM)-1, vascular cell adhesion
molecule-1, transforming growth factor-ß, basic fibroblast growth
factor, and collagen types I, III, and IV were examined with TaqMan
real-time quantitative reverse transcription–polymerase chain
reaction.
Results—Prolonged
arterial narrowing peaking on 7 day was observed. There was
a significant difference in vessel caliber between D0, D2, D7, and D14
groups (P<0.0001). There were
significant differences in mRNA expression in the basilar artery for
IL-1, IL-6, IL-8, ICAM-1, and collagen type I between D0, D2, D7,
and D14 groups (P=0.0079,
0.0196, 0.0040, 0.0017, and <0.0001, respectively). The average level
of mRNA was highest in D7 for IL-1, IL-6, IL-8, and ICAM-1 (17-,
16-, 131-, and 1.7-fold compared with those of D0, respectively) and in
D14 for collagen type I (10.9-fold).
Conclusions—Increased expression of genes related to inflammation in the spastic artery suggests that inflammatory reaction of the cerebral artery is associated with sustained contraction.
Key Words: cerebral vasospasm • cytokines • inflammation • subarachnoid hemorrhage
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Recently, we1 performed mRNA differential display and screening of a cDNA expression array to identify the genes differentially expressed in vasospastic arteries of a canine double-hemorrhage model. The known genes include 5 upregulated inflammation-related genes encoding monocyte chemotactic protein-1, cystain B, inter-
-trypsin inhibitor family
heavy chain–related protein, serum amyloid A protein, and
glycoprotein 130, suggesting that inflammatory reaction in
the artery may be involved in the development of cerebral
vasospasm. Therefore, we chose 15 genes among genes related to inflammation such as cytokines, chemokines, adhesion molecules, growth factors, and extracellular matrixes to clarify the possible role of inflammatory reaction of cerebral artery in the pathogenesis of cerebral vasospasm. We quantitatively measured the levels of expression of these genes in the spastic artery in the canine double-hemorrhage model by using a recent kinetic quantitative polymerase chain reaction (PCR) method based on the fluorescent TaqMan (Perkin Elmer, PE applied Biosystems) methodology and real-time measurement of fluorescence.2 The use of quantitative expression analysis based on real-time analytical reverse transcription (RT)-PCR offers several advantages over other current quantitative methods. It proved to be a rapid, reliable, and highly sensitive method to quantify the simultaneous expression levels of a large number of genes.2
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Cerebral Vasospasm Models
The experimental protocol used for animals was evaluated and approved by the Institutional Animal Care and Use Committee of Tokyo Women’s Medical University. Care of the animals and surgical procedures were performed according to the standards of Tokyo Women’s Medical University Protocol on Laboratory Animals. Twenty mongrel dogs weighing between 17 and 28 kg were anesthetized with intravenous sodium pentobarbital (25 mg/kg). In 15 dogs, the cisterna magna was punctured percutaneously, and 0.3 mL/kg of cerebrospinal fluid (CSF) was removed by spontaneous egress. Subsequently, 0.5 mL/kg of fresh autologous arterial nonheparinized blood was injected into the cisterna magna at a rate of 5 mL/min. The dogs were tilted with the tail up for 15 minutes. The catheter then was removed, and the animals were allowed to recover. The 20 dogs were divided into 4 groups with 5 animals in each group. Group D0 comprised control animals, killed on day 0 without cisternal blood injection. Group D2 animals were killed on day 2 after the first injection on day 0. Group D7 and D14 animals were killed on day 7 and 14 after injections on days 0 and 2. Angiography of the cerebral arteries of each animal was performed on day 0 and before the animal was killed. The diameters of the basilar arteries were measured on angiographic film for evaluation of vasospasm.
RNA Isolation
The animals were killed by injection of 100 mg/kg
pentobarbital, exsanguinated, and perfused with 1500 to 2000 mL normal
saline. Total RNAs were extracted from individual basilar arteries from
each group with the use of TRIzol (GIBCO BRL), according to the
manufacturer’s instructions. Possible traces of genomic DNA
contaminating RNA preparations were removed by DNase I (Promega)
digestion.
Preparation of Dog-Specific Primers and Probes
for Quantitative RT-PCR
The dog sequences of mRNAs for interleukin (IL)-1,
IL-6, IL-8, and IL-10, tumor necrosis factor (TNF)-, E-secretin,
fibronectin, intercellular adhesion molecule (ICAM)-1, vascular cell
adhesion molecule (VCAM)-1, and collagen types I and IV were obtained
by a database search with the Entrez program at NCBI
(http://www.ncbi.nlm.nih.gov/Entrez). The dog sequences of
transforming growth factor (TGF)-ß, basic fibroblast growth factor
(bFGF), collagen type III, and ß-actin were not known, so partial
nucleotide sequences for these genes were determined. The
cDNA was synthesized from 1 µg total RNA extracted from canine middle
cerebral arteries by the murine myeloma leukemia virus RT (Gibco BRL)
and oligo (dT) primer (Gibco BRL). The cDNAs were amplified by PCR with
degenerate oligo primers that were designed on the basis of the human
and mouse nucleotide sequences of the target genes. The PCR
products were directly sequenced with an Applied Biosystem DNA
Sequencer model 377 with a Taq Dye Deoxy Termination Cycle Sequence kit
(Perkin Elmer). The dog sequences obtained, which
represented >90% identity to the human sequences, were
subsequently used to design the dog-specific primers and probes for
quantitative RT-PCR
(Table
).
|
Quantitative RT-PCR With Real-Time TaqMan
Technology
To evaluate the expression level of the target genes,
quantitative RT-PCR was performed with real-time TaqMan
technology2 with a Sequence
Detection System model 7700 (Perkin Elmer). Five serial dilutions of
each total RNA sample (100, 50, 25, 12.5, and 6.25 ng total RNA) were
analyzed for each target gene. The dog-specific detection
probes were labeled with a reporter fluorescent dye, FAM
(6-carboxyfluorescein), on the 5' nucleotide
and a quenching fluorescent dye, TAMRA
(6-carboxy-tetramethyl-rhodamine), on the 3' nucleotide.
Amplification reactions (50 µL) contained total RNA samples,
1xTaqMan EZ buffer, 300 µmol/L dATP, dCTP, and dGTP, 600 µmol/L
dUTP, 3 mmol/L manganese acetate, 5.0 U rTth DNA polymerase, 0.5
AmpErase uracil N-glycosylase
(UNG), 200 nmol/L each primer, and 100 nmol/L of each detection probe.
The thermal cycling conditions were as follows: 2 minutes at 50°C for
the initial step, 30 minutes at 60°C for reverse transcription, 5
minutes at 95°C for deactivation of UNG, 40 cycles of 20 seconds at
94°C for denaturation, and 1 minute at 60°C for annealing and
extension. CT values corresponded to the cycle
number at which the fluorescent emission monitored in real time
reached the threshold, which was set at 10 SD above the mean of
baseline emission calculated from cycles 5 to 15. The
CT values decreased linearly with increasing
target quantity and could be used as a quantitative measurement of the
input target number.
Total RNA concentrations from each sample were normalized by the quantity of ß-actin mRNA, and the expression levels of target genes were evaluated by the ratio of the number of target mRNA to ß-actin mRNA.
Statistical Analysis
Statistical comparisons of arterial
diameters and levels of mRNA in target genes between groups were
evaluated by ANOVA and the Kruskal-Wallis test. Probability values of
<0.05 were considered statistically
significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Vasospasm Model
Figure 1
shows the change in vessel caliber of basilar
arteries after cisternal injection of blood. A significant reduction in
vessel caliber was recognized on angiograms in the D2, D7, and D14
groups, compared with the D0 baseline. There was no difference in
vessel caliber of baseline between groups. There was a significant
difference in vessel caliber between D0, D2, D7, and D14 groups
(P<0.0001).
|
Expression Levels of Cytokine/Chemokine
(IL-1, IL-6, IL-8, IL-10, and TNF-)
The ratios of these mRNA to ß-actin in the
normal basilar artery were <0.1. There were significant differences of
IL-1, IL-6, IL-8, and TNF- mRNA between D0, D2, D7, and D14
groups (P=0.0075, 0.1039,
0.0310, and 0.0354, ANOVA;
P=0.0079, 0.0196, 0.0040, and
0.0949, Kruskal-Wallis). The patterns of difference in each mRNA were
similar; the level of these gene expressions in the basilar artery was
highest in D7. The average level of IL-8 mRNA in D7 was extremely high
(131-fold) compared with that of D0: 17-fold for IL-1, 16-fold for
IL-6, and 2.2-fold for TNF-
(Figure 2).
|
Expression Levels of Adhesion Molecule
(E-secretin, fibronectin, ICAM-1, and VCAM-1)
The ratios of fibronectin and ICAM-1 mRNA to ß-actin
in the normal basilar artery were >1, whereas those of E-secretin and
VCAM-1 were <0.1. There was a significant difference of ICAM-1 mRNA
between D0, D2, D7, and D14 groups
(P=0.0017, ANOVA;
P=0.0095, Kruskal-Wallis). The
average level of ICAM-1 mRNA was highest in D7 (1.67-fold) compared
with that of D0
(Figure 3). The patterns of difference in E-Secretin and
VCAM-1 mRNA were similar to that of ICAM-1, but there were no
significant differences between groups. There was also no significant
difference of fibronectin mRNA between groups.
|
Expression Levels of Growth
Factor/Extracellular Matrix (TGF-ß, bFGF, and Collagen Types I, III,
and IV)
The ratios of TGF-ß, bFGF, and collagen types I, III,
and IV mRNA to ß-actin were 0.145, 0.516, 1.343, 0.030, and 0.095,
respectively. There was a significant difference in collagen type I
mRNA between D0, D2, D7, and D14 groups
(P<0.0001, ANOVA;
P=0.0016, Kruskal-Wallis). The
average level of collagen type I mRNA was 4.1- and 4.5-fold for D2 and
D7, respectively, and highest in D14 (10.9-fold) compared with that of
D0
(Figure 4). There were no significant differences of TGF-ß,
bFGF, and collagen types III and IV mRNA between
groups.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Inflammatory cytokines, such as IL-1, IL-6, and IL-8, have been reported to be induced in the CSF beginning in the acute stage after subarachnoid hemorrhage (SAH).3 Increased inflammatory cytokine mRNA expression has been shown to occur after several types of injury to the brain such as ischemia and trauma. Astrocytes, endothelial cells, and neurons can synthesize cytokines, but microglia and macrophages appear to be the dominating sources of these cytokines.4 Because elevated levels of these cytokines in CSF are correlated with neurological damage, it is likely that they contribute to brain damage after SAH5 6 and may not be related to elevated levels of cytokines mRNA in the spastic artery.
Among genes related to inflammation, we showed that
the levels of cytokines such as IL-1, IL-6, and IL-8 that
were not expressed much in the normal cerebral arteries were extremely
highly elevated in the spastic artery. The pattern of maximum
cytokine levels fits remarkably well with the time course of
vasospasm. The relation between cytokine synthesis in the
spastic artery and cerebral vasospasm could be explained by a causative
role of cytokines in the cascade
phosphorylation of intracellular kinases. IL-1,
IL-2, and IL-4 were detected in chronic periaortitis by PCR-assisted
mRNA analysis but were not detected in normal
aorta.7 Chronic adventitial
treatment with IL-1, IL-1ß, and TNF- was reported to induce
selective hyperconstrictive responses to autacoids and coronary
arteriosclerosis–like
changes.8 These responses
were significantly suppressed in a dose-dependent manner by cotreatment
with a selective tyrosine kinase inhibitor, suggesting that
tyrosine kinase activation may play an important role in mediating
these effects.9 As general
features, the cellular inflammatory cytokine production
in response to various cellular stressors provided second-messenger
signaling through a cascade of protein phosphorylations
involving the mitogen-activated protein kinase
pathway.4 Accumulating
evidence shows that the activation of tyrosin kinase and/or the
mitogen-activated protein kinase pathway leads to sustained
contraction after SAH through actin-regulatory
proteins.10 11
Thus, inflammatory cytokines in the cerebral artery exposed to
periarterial clots may be involved in cerebral vasospasm
through the process of signal transduction to contraction.
ICAM-1 is a member of the immunoglobulin superfamily
that is expressed on the endothelial surface in the
early phase after tissue
injury.12 ICAM-1 expression
can be stimulated by various cytokines including
lipopolysaccharides, TNF-, interferon-
, and IL-1. ICAM-1
mediates adherence and transendothelial migration of
neutrophils in the area of tissue injury. The increased expression of
endothelial ICAM-1 was reported in response to the
deposition of blood around arteries in the rat femoral artery model of
vasospasm.13
Endothelial ICAM-1 expression increased 3 hours after
blood deposition, remained elevated for 24 hours, and returned to
baseline levels by 48 hours. Antibodies to ICAM-1 administered
intracisternally and systemically inhibited vasospasm in a rabbit
single-hemorrhage and rat femoral artery
model.12 14 It is
conceivable that cellular adhesion is an important step in the
initiation of vasospasm, but it is not necessarily important throughout
the entire duration of the
phenomenon.12 However, Handa
et al15 reported that there
was greater expression of ICAM-1 on the endothelial
layer of the basilar artery in SAH rats and that the expression was
observed also in the medial layer of the artery from 2 to 5 days after
SAH. The current results are in accordance with this observation and
did not indicate upregulation of ICAM-1 in the
endothelium in the early stage of SAH, probably because
the mRNA extracted from endothelial cells was
negligible.
We previously observed that the expression of
procollagen types I and III mRNA was increased in rat femoral arteries
exposed to periarterial blood on day 7 and day
14.16 We now observed in the
canine double-hemorrhage model that collagen type I mRNA
expression started increasing at day 2 (4.1-fold), and reached its peak
(10.9-fold) at day 14. Onoda et
al17 reported that the
application of antisense oligonucleotides for collagen
type I gene inhibited arterial contraction in the rat
femoral artery model and resulted in a marked decrease in 1(I)
procollagen mRNA expression. These findings are consistent with
the concept of increased collagen deposition in the vessel wall after
SAH, on the basis of light and electron microscopic
observations.18 19 20 21 22
Although the peak of increased expression of collagen type I mRNA was
later than that of vasospasm, the expression had already elevated
4-fold at days 2 and 7. Increased synthesis of collagen type I may be
related to increased artery wall stiffness, which may contribute to the
sustained arterial narrowing of cerebral
vasospasm.20
The expression of the collagen type I gene was
upregulated by cytokines such as IL-1, interferon-, TNF-,
and TNF-ß and growth factors such as platelet-derived growth
factor, TGF-ß, in human vascular smooth muscle
cells.17 We previously
reported that TGF-ß gene expression in rat femoral artery is
stimulated after exposure to
blood.16 The expression
increased >3-fold 3 days after the application of blood, at which time
the procollagen gene expression remained unchanged. The gene expression
of TGF-ß in the current model may have been elevated more before 2
days after SAH. Because of the stimulatory effects of IL-1 and
TNF- on collagen formation, the increase in IL-1 and TNF- at 7
days is probably related to the maximum increase in collagen type I
gene expression 14 days after SAH.
In conclusion, expression of genes related to inflammation, especially cytokines, was increased in the spastic artery, suggesting that inflammatory reaction of the cerebral artery may cause sustained contraction of the cerebral artery, probably through both the process of signal transduction to contraction and the change of components in the cerebral artery.
|
Acknowledgments |
|---|
This study was supported in part by a Grant-in-Aid for Scientific Research (H. Kasuya) from the Ministry of Education, Science, Sports, and Culture of Japan. We thank Takashi Sakayori, Dr Atsushi Sasahara, and Dr Akitsugu Kawashima, Department of Neurosurgery, Tokyo Women’s Medical University, and Hiroshi Miyazaki, Department of Cardiovascular Science, the Heart Institute of Japan, for technical assistance. We also thank Dr Ituro Inoue, Department of Cell Biology, Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation, Gunma University, for his helpful comments.
Received May 18, 2000; revision received August 18, 2000; accepted September 26, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Onda H, Kasuya H, Takakura K, Hori T, Imaizumi T, Takeuchi T, Inoue I, Takeda J. Identification of genes differentially expressed in canine vasospastic cerebral arteries after subarachnoid hemorrhage. J Cereb Blood Flow Metab. 1999;19:1279–1288.[Medline] [Order article via Infotrieve]
2.
Gibson UE,
Heid CA, Williams PM. A novel method for real time quantitative RT-PCR.
Genome Res. 1996;6:995–1001.
3.
Osuka K,
Suzuki Y, Watanabe Y, Takayasu M, Yoshida J. Inducible
cyclooxygenase expression in canine basilar artery
after experimental subarachnoid hemorrhage.
Stroke. 1998;29:1219–1222.
4. Barone FC, Feuerstein GZ. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab. 1999;19:819–834.[Medline] [Order article via Infotrieve]
5. Mathiesen T, Andersson B, Loftenius A, von Holst H. Increased interleukin-6 levels in cerebrospinal fluid following subarachnoid hemorrhage. J Neurosurg. 1993;78:562–567.[Medline] [Order article via Infotrieve]
6. Mathiesen T, Edner G, Ulfarsson E, Andersson B. Cerebrospinal fluid interleukin-1 receptor antagonist and tumor necrosis factor-alpha following subarachnoid hemorrhage. J Neurosurg. 1997;87:215–220.[Medline] [Order article via Infotrieve]
7.
Ramshaw AL,
Roskell DE, Parums DV. Cytokine gene expression in aortic
adventitial inflammation associated with advanced
atherosclerosis (chronic periaortitis).
J Clin Pathol. 1994;47:721–727.
8. Fukumoto Y, Shimokawa H, Ito A, Kadokami T, Yonemitsu Y, Aikawa M, Owada MK, Egashira K, Sueishi K, Nagai R, Yazaki Y, Takeshita A. Inflammatory cytokines cause coronary arteriosclerosis-like changes and alterations in the smooth-muscle phenotypes in pigs. J Cardiovasc Pharmacol. 1997;29:222–231.[Medline] [Order article via Infotrieve]
9. Ito AS, H. Kadokami T, Fukumoto Y, Owada MK, Shiraishi T, Nakaike R, Takayanagi T, Egashida K, Takeshita A. Tyrosine kinase inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses induced by chronic treatment with interleukin-1b in pigs in vivo. J Clin Invest. 1995;96:1288–1294.
10. Fujiwara H, Tani E, Ozaki I, Miyaji K, Sato M, Takahashi K, Imajoh-Ohmi S. Activation of protein kinases in canine basilar artery in vasospasm. J Cereb Blood Flow Metab. 1999;19:44–52.[Medline] [Order article via Infotrieve]
11. Epstein AM, Throckmorton D, Brophy CM. Mitogen-activated protein kinase activation: an alternate signaling pathway for sustained vascular smooth muscle contraction. J Vasc Surg. 1997;26:327–332.[Medline] [Order article via Infotrieve]
12.
Bavbek M,
Polin R, Kwan AL, Arthur AS, Kassell NF, Lee KS. Monoclonal antibodies
against ICAM-1 and CD18 attenuate cerebral vasospasm after experimental
subarachnoid hemorrhage in rabbits.
Stroke. 1998;29:1930–1936.
13. Sills AK Jr, Clatterbuck RE, Thompson RC, Cohen PL, Tamargo RJ. Endothelial cell expression of intercellular adhesion molecule 1 in experimental posthemorrhagic vasospasm. Neurosurgery. 1997;41:453–460.[Medline] [Order article via Infotrieve]
14.
Oshiro EM,
Hoffman PA, Dietsch GN, Watts MC, Pardoll DM, Tamargo RJ.
Inhibition of experimental vasospasm with anti-intercellular adhesion
molecule-1 monoclonal antibody in rats.
Stroke. 1997;28:2031–2038.
15. Handa Y, Kaneko M, Tsuchida A, Kobayashi H, Kawano H, Kubota T. Expression of intercellular adhesion molecule 1 (ICAM-1) on the cerebral artery following subarachnoid haemorrhage in rats. Acta Neurochir. 1995;132:92–97.[Medline] [Order article via Infotrieve]
16. Kasuya H, Weir B, Shen Y, Hariton G, Vollrath B, Ghahary A. Procollagen types I and III and transforming growth factor-beta gene expression in the arterial wall after exposure to periarterial blood. Neurosurgery. 1993;33:716–722.[Medline] [Order article via Infotrieve]
17.
Onoda K,
Ono S, Ogihara K, Shiota T, Asari S, Ohmoto T, Ninomiya Y. Role of
extracellular matrix in experimental vasospasm: inhibitory
effect of antisense oligonucleotide on collagen
induction. Stroke. 1996;27:2102–2109.
18. Mayberg MR, Okada T, Bark DH. The significance of morphological changes in cerebral arteries after subarachnoid hemorrhage. J Neurosurg. 1990;72:626–633.[Medline] [Order article via Infotrieve]
19.
Clower BR,
Smith RR, Haining JL, Lockard J. Constrictive endarteropathy following
experimental subarachnoid hemorrhage.
Stroke. 1981;12:501–508.
20.
Vorkapic P,
Bevan RD, Bevan JA. Pharmacologic irreversible narrowing in chronic
cerebrovasospasm in rabbits is associated with functional damage.
Stroke. 1990;21:1478–1484.
21. Vorkapic P, Bevan RD, Bevan JA. Longitudinal time course of reversible and irreversible components of chronic cerebrovasospasm of the rabbit basilar artery. J Neurosurg. 1991;74:951–955.[Medline] [Order article via Infotrieve]
22.
Bevan JA,
Bevan RD, Frazee JG. Functional arterial changes in chronic
cerebrovasospasm in monkeys: an in vitro assessment of the contribution
to arterial narrowing.
Stroke. 1987;18:472–481.
Editorial Comment
Department of Internal Medicine, Cardiovascular Division, University of Iowa College of Medicine, Iowa City, Iowa
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Mechanisms that mediate vasospasm after subarachnoid hemorrhage (SAH) have been studied fairly widely.R1 Despite this effort, changes that occur within the blood vessel wall at the molecular level are poorly defined. For example, relatively few studiesR2 R3 have examined changes in expression of multiple genes in vessels from experimental models of SAH.
Previously, it has been difficult to accurately and reproducibly quantify mRNA levels using RT-PCR. The recent development of real time PCR methodology has eliminated much of this variability, and should allow for more routine and reliable quantification of PCR products. This method has recently been applied for studies of changes in gene expression in extracranial vessels and in brain tissue after experimental stroke.R4 R5
In the present study, real time RT-PCR was used to
quantify changes in expression of several inflammatory related genes in
the basilar artery after SAH. The results suggest that expression of
mRNA for several genes, including the cytokines IL-1, IL-6,
and IL-8, is increased markedly after SAH. Expression of these genes
was relatively low in vessels under control conditions.
This work appears to be the first using real-time RT-PCR in cerebral vessels. The functional importance of the observed changes in gene expression was not evaluated in this study. However, the results support the concept that expression of components of the inflammatory cascade may contribute to vasospasm after SAH.
Received May 18, 2000; revision received August 18, 2000; accepted September 26, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Dietrich HH, Dacey RG. Molecular keys to the problems of cerebral vasospasm. Neurosurgery. 2000;46:517–530.[Medline] [Order article via Infotrieve]
2. Suzuki H, Kanamaru K, Tsunoda H, Inada H, Kuroki M, Sun H, Waga S, Tanaka T. Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats. J Clin Invest. 1999;104:59–66.[Medline] [Order article via Infotrieve]
3. Onda H, Kasuya H, Takakura K, Hori T, Imaizumi T, Takeuchi T, Inoue I, Takeda J. Identification of genes differentially expressed in canine vasospastic cerebral arteries after subarachnoid hemorrhage. J Cerebral Blood Flow Metab. 1999;19:1279–1288.
4.
Tai JTN,
Brooks EE, Liang S, Somogyi R, Rosete JD, Lawn RM, Shiffman D.
Determination of temporal expression patterns for multiple genes in the
rat carotid artery injury model.
Arterioscler Thromb Vasc Biol. 2000;20:2184–2192.
5. Wang X, Li X, Currie RW, Willette RN, Barone FC, Feuerstein GZ. Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1beta mRNA in ischemic brain tolerance. J Neurosci Res. 2000;59:238–246.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  H.-C. Shih, C.-L. Lin, T.-Y. Lee, W.-S. Lee, and C. Hsu 17{beta}-Estradiol Inhibits Subarachnoid Hemorrhage-Induced Inducible Nitric Oxide Synthase Gene Expression by Interfering With the Nuclear Factor {kappa}B Transactivation Stroke, December 1, 2006; 37(12): 3025 - 3031. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C J M Frijns, R Fijnheer, A Algra, J A van Mourik, J van Gijn, and G J E Rinkel Early circulating levels of endothelial cell activation markers in aneurysmal subarachnoid haemorrhage: associations with cerebral ischaemic events and outcome J. Neurol. Neurosurg. Psychiatry, January 1, 2006; 77(1): 77 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sasaki, H. Kasuya, H. Onda, A. Sasahara, S. Goto, T. Hori, and I. Inoue Role of p38 Mitogen-Activated Protein Kinase on Cerebral Vasospasm After Subarachnoid Hemorrhage Stroke, June 1, 2004; 35(6): 1466 - 1470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yamada, K. Sato, M. Komachi, E. Malchinkhuu, M. Tobo, T. Kimura, A. Kuwabara, Y. Yanagita, T. Ikeya, Y. Tanahashi, et al. Lysophosphatidic Acid (LPA) in Malignant Ascites Stimulates Motility of Human Pancreatic Cancer Cells through LPA1 J. Biol. Chem., February 20, 2004; 279(8): 6595 - 6605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.J.M. Frijns and L.J. Kappelle Inflammatory Cell Adhesion Molecules in Ischemic Cerebrovascular Disease Stroke, August 1, 2002; 33(8): 2115 - 2122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Noel, J. E. Conway, and R. J. Tamargo Vascular Complications of Cocaine Use * Response Stroke, July 1, 2002; 33(7): 1747 - 1748. [Full Text] [PDF]
|
|
|
|
|
|
M. Satoh, A. D. Parent, and J. H. Zhang Inhibitory Effect With Antisense Mitogen-Activated Protein Kinase Oligodeoxynucleotide Against Cerebral Vasospasm in Rats Stroke, March 1, 2002; 33(3): 775 - 781. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||