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Activation of the JAK-STAT signaling pathway in the rat basilar artery after subarachnoid hemorrhage
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Activation of the JAK-STAT signaling pathway in the rat basilar artery after subarachnoid hemorrhage Koji Osuka a , Yasuo Watanabe b,⁎, Katsuaki Yamauchi a , Ayami Nakazawa c , Nobuteru Usuda c , Masaaki Tokuda b , Jun Yoshida a a
Department of Neurosurgery, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Cell Physiology, Kagawa University Faculty of Medicine, 1750-1 Ikenobe Miki-cho Kita-gun 761-0793 Kagawa, Japan c Department of Anatomy II, Fujita Health University School of Medicine, Aichi, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
The Janus kinase-signal transducer and activator of transcription (JAK-STAT) is one of the
Accepted 4 December 2005
most important signaling pathways transducing signals from the cell surface in response to
Available online 17 January 2006
cytokines. Subarachnoid hemorrhage (SAH) produces cytokines in the CSF. We investigated whether this signaling pathway is activated in the rat basilar artery after SAH by cytokines.
Keywords:
In a rat single-hemorrhage model of SAH, basilar arteries and CSF were obtained until 7 days
Subarachnoid hemorrhage
after SAH. The concentration of interleukin-6 (IL-6) in CSF was measured by ELISA. Western
Interleukin-6
blot analysis with JAK1, phosphospecific-JAK1, STAT3, phosphospecific STAT3 at Tyr705 and
Signal transduction
Ser727, cyclooxygenase-2 (COX-2), and actin antibodies was performed in basilar artery. The
Phosphorylation
expressions of STAT3, phosphospecific STAT3 at Tyr705 and Ser727, and COX-2 in basilar artery were examined by immunohistochemical studies. The concentration of IL-6 immediately increased after SAH and Western blot analysis revealed that JAK1 was phosphorylated within 2 h, accompanied by phosphorylation of STAT3 at Tyr705, extending to Ser727 at days 1–2. Immunohistochemistry revealed phosphorylation of STAT3 to occur in endothelial and smooth muscle cells of the basilar artery. In addition, intracisternal injection of IL-6 by itself significantly increased phosphorylation of STAT3 at Tyr705 and Ser727. Expression of COX-2 was also upregulated in endothelial cells of the basilar artery. These results indicate that SAH produces the proinflammatory cytokine IL-6 in the CSF, which activates the JAK-STAT signaling pathway in the basilar artery and induces transcription of immediate early genes. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Over the past decade, it has become increasingly apparent that the effects of subarachnoid hemorrhage (SAH) are mediated by a complex signaling network, among which cytokines play a prominent role. Janus kinase-signal transducer and activator of transcription (JAK-STAT) proteins are
transcriptional factors responsible for transmitting messages from the cell surface to the nucleus, thereby modulating gene expression (Horvath 2000), which are activated by cytokines. After focal cerebral ischemia, phosphorylation of STAT3 occurs in neurons and endothelial cells, with a suspected neuroprotective effect against ischemic injury (Suzuki et al., 2001). Upregulation of glycoprotein (gp)-130 and JAK1 has also
⁎ Corresponding author. Fax: +81 87 891 2096. E-mail address: [email protected] (Y. Watanabe). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.003
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been confirmed in activated astrocytes after cerebral ischemia, indicating the response to be controlled by the JAK-STAT pathway (Choi et al., 2003; Justicia et al., 2000). Activation of JAK-STAT has been reported in rat models of myocardial infarction and recent evidence indicates that JAK-STAT activation by myocardial ischemia induces transcriptional activation of the inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) genes and the protective effects of late phase preconditioning (Bolli et al., 2003; Xuan et al., 2001). Increased cytokines in CSF have been reported not only in stroke but also in SAH patients (Mathiesen et al., 1993; Osuka et al., 1998a; Tarkowski et al., 1995; Vila et al., 2000). To date, however, the role of the JAK-STAT pathway after SAH still remains to be detailed. The present study was therefore designed to examine activation of the JAK-STAT signaling pathway in the rat basilar artery after SAH. We also studied the distribution of STAT3 and phosphorylated STAT3 at Tyr705 and Ser727 with an immunohistochemical technique to provide further information on its function in the basilar artery.
2.
Results
2.1.
Physiological parameters
No significant changes in mean arterial blood pressure, temperature, or injected arterial blood gas data were detected in any of the experimental groups (data not shown).
2.2.
Change of interleukin-6 concentrations after SAH
Levels of IL-6 in CSF after SAH were analyzed in 4 rats (Fig. 1). In the control CSF samples, the mean value was 22.8 ± 4.2 pg/ mL and the concentration was hundreds of times higher in the acute stage, and decreased gradually after a peak at 6 h. On day 7, the level was almost the same as in controls.
2.3.
Effects of SAH on phosphorylation of JAK1 and STAT3
We first examined the phosphorylation of JAK1 and STAT3 at Tyr705 and Ser727 in the basilar artery after SAH. Significant enhancement in the JAK1 case was noted at 2 h after injection of blood, relative to control samples (Fig. 2A). As for STAT3, SAH resulted in a 2.5-fold enhancement in the phosphorylation at Tyr705 at 2 h after SAH with gradual decrease thereafter (Fig. 2B), while phosphorylation of STAT3 at Ser727 gradually increased after the onset of SAH during 1–2 days, then decreased by 7 days (Fig. 2C). Almost equal levels of JAK1, STAT3, and actin were detected throughout, indicating that a SAH episode modulates JAK and STAT3 primarily through phosphorylation in the basilar artery.
2.4. Effects of IL-6 on the phosphorylation of STAT3 at Tyr705 and Ser727 To further investigate the involvement of IL-6 in the phosphorylation of STAT3, we injected IL-6, a well-known proinflammatory cytokine, into the cisterna magna and removed basilar arteries 1 h after injection. Immunoblot analyses showed increased phosphorylation of STAT3 compared to the control, with a twofold increase in the densitometric ratio for Tyr705 and a lesser but still significant elevation at Ser727 (Fig. 3).
2.5. Endothelial cells and smooth muscle cells in basilar arteries contain phosphorylated STAT3 after SAH To examine where phosphorylation of STAT3 after SAH occurs in the basilar artery, we performed immunohistochemistry of tissue 2 h after injection of blood and control. Immunoreactivity against STAT3 was observed mainly in the endothelial cells and cytoplasm of smooth muscle cells (Figs. 4A and B). Immunoreactivity of phosphorylated STAT3 at Tyr705 was markedly increased in the endothelial cells and cytoplasm of smooth muscle cells (Figs. 4C and D). Intense staining of STAT3 phosphorylated at Ser727 was also observed in the endothelial cells and nuclei of smooth muscle cells at 2 h after SAH (Fig. 4F, arrowheads).
2.6.
Expression of COX-2 after SAH
COX-2 can be considered to be an immediate early gene. We therefore examined its expression in our one-hemorrhage SAH model. Immunoblot analysis revealed increase, albeit not significant, from 6 h after onset of SAH (Fig. 5A). Immunoreactivity of COX-2 was located in the endothelial cells in controls, where it appeared upregulated 2 days after SAH (Fig. 5C, arrows) compared to control (Fig. 5B).
3. Fig. 1 – Serial changes in concentrations of interleukin-6 (IL-6) in CSF after intracisternal injection of autologous blood (300 μL). Mean ± SE values from four animals are shown. Control; control CSF sample without subarachnoid hemorrhage (SAH). *P b 0.05 denotes a significant difference between the control and SAH groups on analysis of variance followed by a Fisher's post hoc test.
Discussion
In the present study, we showed rapid activation of STAT3 in endothelial and smooth muscle cells of rat basilar arteries after SAH. In particular, phosphorylation of JAK1 and STAT3 at Tyr705 was paralleled by increase in the concentrations of the inflammatory cytokine IL-6 in the CSF, while phosphorylation of STAT3 at Ser727 was significantly increased at days 1–2.
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Furthermore, intracisternal administration of IL-6 significantly induced the phosphorylation of STAT3 compared with controls. These findings suggest that SAH produces proinflammatory cytokines, which activate the JAK-STAT signaling pathway in the basilar artery from acute stages. In recent years, rat SAH models have become popular in the study of cerebral vasospasm. Cerebral vasospasm after SAH in rat shows biphasic patterns with early and late phases (Delgado et al., 1985; Suzuki et al., 1999) and pathological changes in major cerebral arteries (Delgado et al., 1986; Gules
Fig. 3 – Effects of intracisternal injection of interleukin-6 (IL-6) on the phosphorylation of STAT3 at Tyr705 and Ser727. At 1 h after intracisternal injection of IL-6 (2 μg/2 μL, n = 4) or Tris–HCl pH 7.6 (2 μL; control, n = 3), basilar arteries were removed and subjected to Western blotting with anti-actin (α-Actin), anti-STAT3 (α-STAT3), anti-phosphospecific STAT3 at Tyr705 (α-phospho-STAT3 Tyr705), and anti-phosphospecific STAT3 at Ser727 (α-phospho-STAT3 Ser727) antibodies. The histogram shows the amount of α-phoshp-STAT3 relative to that of α-STAT3. Mean ± SE values are shown; the asterisk indicates a significant difference by the Mann–Whitney's U test (P b 0.05).
et al., 2002; Jackowski et al., 1990), as found in humans. Genetic or genomic information is more easily available in rats than in other large animals (Harada et al., 1997; Nikaido et al., 2004; Suzuki et al., 1999). The endovascular puncture model Fig. 2 – Phosphorylation of JAK1 (A), STAT3 at Tyr705 (B), and STAT3 at Ser727 (C) in the basilar artery after subarachnoid hemorrhage (SAH). At 2, 6, 12 h, 1, 2, or 7days after intracisternal injection of autologous blood (300 μL), as indicated below the panel, crude samples (25 μg of protein each) were subjected to Western blotting with anti-JAK1 (α-JAK1), anti-phosphospecific JAK1 (α-phospho-JAK1), anti-STAT3 (α-STAT3) and anti-phosphospecific STAT3 at Tyr705 (α-phospho-STAT3 Tyr705), anti-phosphospecific STAT3 at Ser727 (α-phospho-STAT3 Ser727), and anti-actin (α-Actin) antibodies. The histogram shows the amount of α-phospho-JAK1 relative to that of α-JAK1 (A), the amount of α-phospho-STAT3 Tyr705 relative to that of α-STAT3 (B), and the amount of α-phospho-STAT3 Ser727 relative to that of α-STAT3 (C) in the membrane. Mean ± SE values from four animals are shown. Control; control basilar artery without SAH. *P b 0.05 versus control by Fisher's post hoc test.
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this study, we adopted a single-hemorrhage model to examine the relationship between inflammatory cytokine and JAK-STAT signaling pathway in basilar artery immediately after SAH. SAH initiates an inflammatory response regulated by specific signaling molecules, among which cytokines in CSF may play a prominent role. Proinflammatory cytokines, such as IL-1 and IL-6, demonstrate increased expression in the CSF after SAH (Mathiesen et al., 1993; Osuka et al., 1998a). Our data are consistent with previous clinical reports that production of IL-6 is induced from the immediately early stage of SAH (Osuka et al., 1998a). Increased expression of genes related to inflammation, such as IL-1, IL-6, and IL-8, was confirmed in the spastic basilar artery in a canine SAH model (Aihara et al., 2001). We previously demonstrated that IL-1β vasodilates the basilar artery through the prostaglandin cascade, especially prostacyclin, stimulated by the induced COX-2 (Osuka et al.,
Fig. 4 – Immunohistochemical findings for STAT3 in basilar arteries after subarachnoid hemorrhage (SAH). Rats were perfused with 4% paraformaldehyde after a sham operation (A, C, and E) or 2 h after injection of autologous blood (B, D, and F). Ten-micrometer coronal slices were immunostained with polyclonal antibodies recognizing STAT3 (A and B), phosphospecific-STAT3 at Tyr705 (C and D), or phosphospecific STAT3 at Ser727 (E and F) by the ABC method. Note more intense staining of phosphospecific-STAT3 at Tyr705 in the endothelial cells and cytoplasm of smooth muscle cells at 2 h after SAH (D) as compared to the control case (C), and similarly phosphospecific STAT3 at Ser727 (F, arrowheads) as compared to the control case (E). L, lumen of basilar artery. S, subarachnoid space. Original magnification ×1000.
(Bederson et al., 1995; Matz et al., 1996), single-hemorrhage model (Delgado et al., 1985, 1986; Gules et al., 2002; Jackowski et al., 1990; Nikaido et al., 2004; Suzuki et al., 1999), and doublehemorrhage model (Gules et al., 2002; Suzuki et al., 1999) have been established in rats. High mortality rates, up to 50%, is one of the existing problems for the endovascular puncture model (Bederson et al., 1995; Gules et al., 2002), while easy controllability of blood injection results in less mortality in injected models (Gules et al., 2002). Discrepancy in the time course of vasospasm between rats and humans still remains an important problem. In humans, angiographic cerebral vasospasm becomes maximum between 6 and 8 days after SAH (Heros et al., 1983; Kassell et al., 1985). The rat singlehemorrhage model and endovascular puncture model show maximum narrowing at day 2, followed by a gradual reduction and then disappearance by day 7 (Delgado et al., 1985; Gules et al., 2002; Nikaido et al., 2004; Suzuki et al., 1999), whereas the rat double-hemorrhage model shows maximum vasospasm at day 7 (Gules et al., 2002; Suzuki et al., 1999). In
Fig. 5 – Immunoblot analysis of inducible cyclooxygenase-2 (COX-2) in basilar arteries after subarachnoid hemorrhage (SAH), induced as for Fig. 2. Crude samples (25 μg of protein each) were subjected to Western blotting with anti-actin (α-Actin) and anti-COX-2 (α-COX-2) antibodies. The histogram shows the amount of α-COX-2 relative to that of α-actin in the membrane. Mean ± SE values from four animals are shown. Control; control basilar artery without SAH. Note the increase (not significant) in immunoreactivity against COX-2 from 6 h after SAH (A). Immunohistochemical analysis of COX-2 in basilar arteries after SAH. Rats subjected to sham operation (B) or 2 day (C) after intracisternal injection of autologous blood were perfused with 4% paraformaldehyde. Ten-micrometer coronal slices were immunostained with antibody against COX-2 by the ABC method. Note remarkable increase in immunoreactivity against COX-2 in the endothelial cells of basilar artery 2 day after SAH (C, arrows). L, lumen of basilar artery. S, subarachnoid space. Original magnification ×1000.
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1997). We also have revealed that inflammatory cytokines, not only IL-1β but also IL-6, immediately induced by SAH may be responsible for expression of COX-2 in basilar arteries after SAH, although the responsible mechanisms still remained to be clarified (Osuka et al., 1998b). IL-6 signaling occurs through binding to an IL-6 receptor and coupling to two gp-130. Then, JAKs are activated and phosphorylate STAT proteins, which dimerize and translocate into the nucleus, binding to promoters of specific target genes (Heinrich et al., 1998; Horvath 2000). Onda et al. (1999) reported the gp130 gene to be upregulated in vasospastic arteries in a canine SAH model. Inflammation in response to SAH may play an important role in vasospasm (Peterson et al., 1990) and our results confirmed for the first time, that the JAK-STAT pathway is activated in basilar arteries after SAH. JAK-STAT signaling appears to play an important cardioprotective role in ischemic preconditioning through upregulation of COX-2 (Bolli et al., 2003; Xuan et al., 2001) and our present data are in agreement with the previous observation of COX-2 expression in rabbit basilar artery endothelial cells after SAH (Tran Dinh et al., 2001). Blanco et al. (1995) confirmed that protein–tyrosine phosphorylation plays a role in the expression of COX-2 in endothelial cells. In addition, we have reported that IL-6 induces expression of the COX-2 in basilar artery (Osuka et al., 1998b) and our results showed intracisternal injection of IL-6 by itself activates JAK-STAT signaling pathway in vivo. From our data, expression of COX-2 in the endothelial cells through JAK-STAT signaling pathway was upregulated from 6 h after onset of SAH. These induced COX-2 plays vasodilator effects in antagonizing delayed cerebral vasospasm after SAH, which occurs maximum at day 2 in this single-hemorrhage model. Other IL-6 family members, such as IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor (CNTF), and cardiotrophin may also be involved with activation of the JAK-STAT signaling pathway (Heinrich et al., 1998). Tyrosine phosphorylation of STAT3 mediated by a JAK and Src family members has been studied (Vignais and Gilman 1999; Zhang et al., 2000) and activation of the tyrosine kinase Src, one of the MAPK cascade, has been shown to play an important role in mediation of cerebral vasospasm (Kusaka et al., 2003). On the other hand, phosphorylation of STAT3 at Ser727 is activated by the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogenactivated protein kinase (MAPK) (Levy and Lee 2002). Activation of ERK and p38 MAPK has been reported in the arterial wall after SAH (Sasaki et al., 2004; Zubkov et al., 1999), and therefore MAPK inhibitors may be useful for treatment of cerebral vasospasm. The single-hemorrhage model shows maximum vasospasm at day 2 (Delgado et al., 1985; Suzuki et al., 1999). A recent study provided evidence that shear flow induces the phosphorylation of STAT3 at Ser727 via the ERK1/2 pathway in endothelial cells (Ni et al., 2003) and in the present study significant increase of phosphorylated STAT3 at Ser727 at this site was evident on days 1–2 when basilar arteries became spastic. Increased shear stress evoked by narrowing of the arterial lumen would be expected to stimulate the ERK pathway and phosphorylation of STAT3 at Ser727 might be increased.
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The present investigation has clarified the time course of activation of the JAK-STAT pathway in rat basilar arteries after SAH and their locations for the first time. Subsequent to the increase in the concentration of IL-6 in the CSF, JAK-STAT signaling is activated with translocation of STAT3 to the nucleus and upregulation of immediate genes, including COX-2. Further studies using IL-6 deficient mice should tell us the roles of this JAK-STAT signaling pathway in vasospasm after SAH and its possible relationship with other immediate early genes, providing clues to potential therapeutic measures.
4.
Experimental procedures
4.1.
Materials
Recombinant rat interleukin-6 (IL-6) was purchased from Pepro Tech (Rochy Hill, Nj). Other chemicals, unless otherwise specified, were from Sigma Chemicals Co Ltd. (St. Louis, MO). 4.2.
Experimental model of SAH and study protocol
All experiments were carried out in accordance with guidelines for the care and use of laboratory animals at the Nagoya University Graduate School of Medicine. Anesthesia was induced in male Sprague–Dawley rats (300 to 350 g, Chubu Kagaku Shizai Ltd., Nagoya, Japan) using chloral hydrate (400 mg/kg, IP). Animals were then intubated and ventilated with 1.0% halothane in an oxygen/nitrous oxide (30%/70%) gas mixture. Temperature was monitored with a rectal probe and maintained between 36.5 and 37.5 °C with a heating pad and lamp. The right femoral artery was exposed and catheterized with polyethylene tubing (PE-50) to allow blood sampling and the monitoring of arterial blood pressure during SAH. Injected arterial blood gases analyses were examined. A midline skin incision was made from the middle of the calvarium to the cervical spine under the stereotactic operation. The atlanto-occipital membrane was exposed under a microscope, and a 26-gauge needle was inserted into the cisterna magna. SAH was induced by injection of 300 μL of autologous arterial blood. Infusions were performed over a 5min period. Rats were kept in a head-down position for 5 min to ensure that the blood had contact with the basilar artery, after which the needle was withdrawn and all wounds were sutured. Animals without injection of blood were used as controls. Tissue samples for Western blot analysis were obtained by decapitation under deep anesthesia at 2, 6, 12, 24, 48, and 168 h after SAH. Basilar arteries were immediately isolated on ice, frozen in liquid nitrogen, and kept at −80 °C until use. Basilar arteries without injection of blood were used as controls. 4.3.
Sample preparation for Western blot analysis
Samples were prepared from four different animals in each group. Basilar arteries were homogenized using a homogenizer in 15 volumes of homogenization buffer containing 50 mmol/L Tris base/HCl (pH 7.5), 0.1 mmol/L dithiothreitol, 0.2 mmol/L EDTA, 0.2 mmol/L EGTA, 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1.25 μg/mL pepstatin A, 0.2 μg/mL aprotinin, 1 mmol/L sodium orthovanadate, 50 mmol/L sodium fluoride, 2 mmol/L sodium pyrophosphate, and 1% Nonidet P-40 (NP-40). The homogenates were then centrifuged at 15,000 rpm at 4 °C for 10 min. Protein concentrations of the supernatants were determined by the method of Bradford using bovine serum albumin as the standard.
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Western blotting analysis
Crude samples (25 μg of protein each) were subjected to 7.5% SDSPAGE, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes and incubated with primary polyclonal antibodies against phospho-JAK1 and phospho-STAT3 at Ser727 (Cell signaling technology, Beverly, MA) at a dilution of 1:500 overnight at 4 °C and polyclonal phospho-STAT3 at Tyr705 (Cell Signaling Technology, Beverly, Mass) or monoclonal antibodies against inducible cyclooxygenase (COX-2, Transduction Laboratories, Lexington, KY) at a dilution of 1:500 for 45 min at room temperature. The membranes were then incubated for 30 min at room temperature with horseradish peroxidase-conjugated secondary antibodies, and binding was visualized using an enhanced chemiluminescence (ECL) or ECL plus Western blotting detection system (Amersham). Phospho-JAK1 and phospho-STAT3 immunoblots were stripped from PVDF membranes and reblotted with primary monoclonal JAK1 (Transduction Laboratories, Lexington, KY), polyclonal STAT3 (Cell signaling technology, Beverly, MA), and polyclonal actin (Sigma, St. Louis, MO) at a dilution of 1:500 for 45 min at room temperature. Finally, the membranes were developed with the ECL or ECL plus system and band intensities were quantitated by densitometric scanning using the NIH IMAGE program. 4.5.
Measurement of cytokine levels
For the assay of IL-6 levels in CSF, 50 μL CSF samples were obtained through the atlanto-occipital membrane before decapitation at same time course after SAH and immediately centrifuged. Supernatant fluid was collected and stored at −80 °C until assayed. IL-6 was measured using ELISA kit (BIOSOURCE). CSF samples from rats without SAH were used as controls. 4.6.
Interleukin-6 injection model
Under anesthesia, rats were set on a stereotactic operation frame and the atlanto-occipital membrane was exposed under a microscope. IL-6 (2 μg/2 μL) was then slowly injected into the cisterna magna through a 26-gauge needle. The needle was kept in place for 10 min to allow the drug to diffuse into the whole cisterns and Tris–HCl (pH 7.6, 2 μL) was employed as the vehicle control. Basilar arteries were removed 1 h after injection of IL-6 or vehicle for Western blot analyses, as described previously. The following primary antibodies were used: polyclonal antibodies for phosphorylated STAT3 at Tyr705 and at Ser727; a polyclonal antibody for STAT3; and a polyclonal antibody for actin. 4.7.
Immunohistochemistry
The rats were perfused with ice-cold 200 mL 4% paraformaldehyde in 0.1 mol/L sodium phosphate (pH7.4) at 2 h or 2 days after SAH. The basilar arteries with brain stems were removed and preserved in the fixation solution for 3 h. Serial coronal cryostat sections (10 μm) were stained according to the avidin-biotinylated peroxidase complex (ABC) technique at room temperature. The staining sequence was as follows: 2% goat or horse serum for 30 min; primary polyclonal antibodies against phosphorylated STAT3 at Tyr705 and at Ser727 at a dilution of 1:200 each, STAT3 at a dilution of 1:1000, and monoclonal antibody against COX-2 at a dilution of 1:500; biotinylated anti-rabbit or mouse IgG for 1 h; and ABC for 1 h. Sera for the blocking step, biotinylated antibodies, and ABC were purchased from Vector Laboratories (Burlingame, Ca). Reaction products were developed by incubation in 0.05% 3,3′-diaminobendizine tetrachloride and 0.01% H2O2 in 50 mM Tris–HCl (pH 7.5) for 10 min. Rats with all surgical procedures, but without injection of blood, were used as controls.
4.8.
Statistical analysis
Data are expressed as mean ± SE values. Statistical differences between the groups were assessed by one-way ANOVA with Fisher's post hoc test and a Mann–Whitney's test. Statistical significance was concluded at the P b 0.05 level.
Acknowledgments We thank Dr. Malcolm Moore for critical reading of the manuscript and Kenmei Mizutani for technical assistance. This work was supported by grants from the General Insurance Association of Japan to KO.
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Peterson, J.W., Kwun, B.D., Hackett, J.D., Zervas, N.T., 1990. The role of inflammation in experimental cerebral vasospasm. J. Neurosurg. 72, 767–774. Sasaki, T., Kasuya, H., Onda, H., Sasahara, A., Goto, S., Hori, T., Inoue, I., 2004. Role of p38 mitogen-activated protein kinase on cerebral vasospasm after subarachnoid hemorrhage. Stroke 35, 1466–1470. Suzuki, H., Kanamaru, K., Tsunoda, H., Inada, H., Kuroki, M., Sun, H., Waga, S., Tanaka, T., 1999. Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats. J. Clin. Invest. 104, 59–66. Suzuki, S., Tanaka, K., Nogawa, S., Dembo, T., Kosakai, A., Fukuuchi, Y., 2001. Phosphorylation of signal transducer and activator of transcription-3 (Stat3) after focal cerebral ischemia in rats. Exp. Neurol. 170, 63–71. Tarkowski, E., Rosengren, L., Blomstrand, C., Wikkelso, C., Jensen, C., Ekholm, S., Tarkowski, A., 1995. Early intrathecal production of interleukin-6 predicts the size of brain lesion in stroke. Stroke 26, 1393–1398. Tran Dinh, Y.R., Jomaa, A., Callebert, J., Reynier-Rebuffel, A.M., Tedgui, A., Savarit, A., Sercombe, R., 2001. Overexpression of cyclooxygenase-2 in rabbit basilar artery endothelial cells after subarachnoid hemorrhage. Neurosurgery 48, 625–633. Vignais, M.L., Gilman, M., 1999. Distinct mechanisms of activation of Stat1 and Stat3 by platelet-derived growth factor receptor in a cell-free system. Mol. Cell. Biol. 19, 3727–3735. Vila, N., Castillo, J., Davalos, A., Chamorro, A., 2000. Proinflammatory cytokines and early neurological worsening in ischemic stroke. Stroke 31, 2325–2329. Xuan, Y.T., Guo, Y., Han, H., Zhu, Y., Bolli, R., 2001. An essential role of the JAK-STAT pathway in ischemic preconditioning. Proc. Natl. Acad. Sci. U. S. A. 98, 9050–9055. Zhang, Y., Turkson, J., Carter-Su, C., Smithgall, T., Levitzki, A., Kraker, A., Krolewski, J.J., Medveczky, P., Jove, R., 2000. Activation of Stat3 in v-Src-transformed fibroblasts requires cooperation of Jak1 kinase activity. J. Biol. Chem. 275, 24935–24944. Zubkov, A.Y., Ogihara, K., Tumu, P., Patlolla, A., Lewis, A.I., Parent, A.D., Zhang, J., 1999. Mitogen-activated protein kinase mediation of hemolysate-induced contraction in rabbit basilar artery. J. Neurosurg. 90, 1091–1097.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Activation of the JAK-STAT signaling pathway in the rat basilar artery after subarachnoid hemorrhage Koji Osuka a , Yasuo Watanabe b,⁎, Katsuaki Yamauchi a , Ayami Nakazawa c , Nobuteru Usuda c , Masaaki Tokuda b , Jun Yoshida a a
Department of Neurosurgery, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Cell Physiology, Kagawa University Faculty of Medicine, 1750-1 Ikenobe Miki-cho Kita-gun 761-0793 Kagawa, Japan c Department of Anatomy II, Fujita Health University School of Medicine, Aichi, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
The Janus kinase-signal transducer and activator of transcription (JAK-STAT) is one of the
Accepted 4 December 2005
most important signaling pathways transducing signals from the cell surface in response to
Available online 17 January 2006
cytokines. Subarachnoid hemorrhage (SAH) produces cytokines in the CSF. We investigated whether this signaling pathway is activated in the rat basilar artery after SAH by cytokines.
Keywords:
In a rat single-hemorrhage model of SAH, basilar arteries and CSF were obtained until 7 days
Subarachnoid hemorrhage
after SAH. The concentration of interleukin-6 (IL-6) in CSF was measured by ELISA. Western
Interleukin-6
blot analysis with JAK1, phosphospecific-JAK1, STAT3, phosphospecific STAT3 at Tyr705 and
Signal transduction
Ser727, cyclooxygenase-2 (COX-2), and actin antibodies was performed in basilar artery. The
Phosphorylation
expressions of STAT3, phosphospecific STAT3 at Tyr705 and Ser727, and COX-2 in basilar artery were examined by immunohistochemical studies. The concentration of IL-6 immediately increased after SAH and Western blot analysis revealed that JAK1 was phosphorylated within 2 h, accompanied by phosphorylation of STAT3 at Tyr705, extending to Ser727 at days 1–2. Immunohistochemistry revealed phosphorylation of STAT3 to occur in endothelial and smooth muscle cells of the basilar artery. In addition, intracisternal injection of IL-6 by itself significantly increased phosphorylation of STAT3 at Tyr705 and Ser727. Expression of COX-2 was also upregulated in endothelial cells of the basilar artery. These results indicate that SAH produces the proinflammatory cytokine IL-6 in the CSF, which activates the JAK-STAT signaling pathway in the basilar artery and induces transcription of immediate early genes. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Over the past decade, it has become increasingly apparent that the effects of subarachnoid hemorrhage (SAH) are mediated by a complex signaling network, among which cytokines play a prominent role. Janus kinase-signal transducer and activator of transcription (JAK-STAT) proteins are
transcriptional factors responsible for transmitting messages from the cell surface to the nucleus, thereby modulating gene expression (Horvath 2000), which are activated by cytokines. After focal cerebral ischemia, phosphorylation of STAT3 occurs in neurons and endothelial cells, with a suspected neuroprotective effect against ischemic injury (Suzuki et al., 2001). Upregulation of glycoprotein (gp)-130 and JAK1 has also
⁎ Corresponding author. Fax: +81 87 891 2096. E-mail address: [email protected] (Y. Watanabe). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.003
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been confirmed in activated astrocytes after cerebral ischemia, indicating the response to be controlled by the JAK-STAT pathway (Choi et al., 2003; Justicia et al., 2000). Activation of JAK-STAT has been reported in rat models of myocardial infarction and recent evidence indicates that JAK-STAT activation by myocardial ischemia induces transcriptional activation of the inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) genes and the protective effects of late phase preconditioning (Bolli et al., 2003; Xuan et al., 2001). Increased cytokines in CSF have been reported not only in stroke but also in SAH patients (Mathiesen et al., 1993; Osuka et al., 1998a; Tarkowski et al., 1995; Vila et al., 2000). To date, however, the role of the JAK-STAT pathway after SAH still remains to be detailed. The present study was therefore designed to examine activation of the JAK-STAT signaling pathway in the rat basilar artery after SAH. We also studied the distribution of STAT3 and phosphorylated STAT3 at Tyr705 and Ser727 with an immunohistochemical technique to provide further information on its function in the basilar artery.
2.
Results
2.1.
Physiological parameters
No significant changes in mean arterial blood pressure, temperature, or injected arterial blood gas data were detected in any of the experimental groups (data not shown).
2.2.
Change of interleukin-6 concentrations after SAH
Levels of IL-6 in CSF after SAH were analyzed in 4 rats (Fig. 1). In the control CSF samples, the mean value was 22.8 ± 4.2 pg/ mL and the concentration was hundreds of times higher in the acute stage, and decreased gradually after a peak at 6 h. On day 7, the level was almost the same as in controls.
2.3.
Effects of SAH on phosphorylation of JAK1 and STAT3
We first examined the phosphorylation of JAK1 and STAT3 at Tyr705 and Ser727 in the basilar artery after SAH. Significant enhancement in the JAK1 case was noted at 2 h after injection of blood, relative to control samples (Fig. 2A). As for STAT3, SAH resulted in a 2.5-fold enhancement in the phosphorylation at Tyr705 at 2 h after SAH with gradual decrease thereafter (Fig. 2B), while phosphorylation of STAT3 at Ser727 gradually increased after the onset of SAH during 1–2 days, then decreased by 7 days (Fig. 2C). Almost equal levels of JAK1, STAT3, and actin were detected throughout, indicating that a SAH episode modulates JAK and STAT3 primarily through phosphorylation in the basilar artery.
2.4. Effects of IL-6 on the phosphorylation of STAT3 at Tyr705 and Ser727 To further investigate the involvement of IL-6 in the phosphorylation of STAT3, we injected IL-6, a well-known proinflammatory cytokine, into the cisterna magna and removed basilar arteries 1 h after injection. Immunoblot analyses showed increased phosphorylation of STAT3 compared to the control, with a twofold increase in the densitometric ratio for Tyr705 and a lesser but still significant elevation at Ser727 (Fig. 3).
2.5. Endothelial cells and smooth muscle cells in basilar arteries contain phosphorylated STAT3 after SAH To examine where phosphorylation of STAT3 after SAH occurs in the basilar artery, we performed immunohistochemistry of tissue 2 h after injection of blood and control. Immunoreactivity against STAT3 was observed mainly in the endothelial cells and cytoplasm of smooth muscle cells (Figs. 4A and B). Immunoreactivity of phosphorylated STAT3 at Tyr705 was markedly increased in the endothelial cells and cytoplasm of smooth muscle cells (Figs. 4C and D). Intense staining of STAT3 phosphorylated at Ser727 was also observed in the endothelial cells and nuclei of smooth muscle cells at 2 h after SAH (Fig. 4F, arrowheads).
2.6.
Expression of COX-2 after SAH
COX-2 can be considered to be an immediate early gene. We therefore examined its expression in our one-hemorrhage SAH model. Immunoblot analysis revealed increase, albeit not significant, from 6 h after onset of SAH (Fig. 5A). Immunoreactivity of COX-2 was located in the endothelial cells in controls, where it appeared upregulated 2 days after SAH (Fig. 5C, arrows) compared to control (Fig. 5B).
3. Fig. 1 – Serial changes in concentrations of interleukin-6 (IL-6) in CSF after intracisternal injection of autologous blood (300 μL). Mean ± SE values from four animals are shown. Control; control CSF sample without subarachnoid hemorrhage (SAH). *P b 0.05 denotes a significant difference between the control and SAH groups on analysis of variance followed by a Fisher's post hoc test.
Discussion
In the present study, we showed rapid activation of STAT3 in endothelial and smooth muscle cells of rat basilar arteries after SAH. In particular, phosphorylation of JAK1 and STAT3 at Tyr705 was paralleled by increase in the concentrations of the inflammatory cytokine IL-6 in the CSF, while phosphorylation of STAT3 at Ser727 was significantly increased at days 1–2.
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Furthermore, intracisternal administration of IL-6 significantly induced the phosphorylation of STAT3 compared with controls. These findings suggest that SAH produces proinflammatory cytokines, which activate the JAK-STAT signaling pathway in the basilar artery from acute stages. In recent years, rat SAH models have become popular in the study of cerebral vasospasm. Cerebral vasospasm after SAH in rat shows biphasic patterns with early and late phases (Delgado et al., 1985; Suzuki et al., 1999) and pathological changes in major cerebral arteries (Delgado et al., 1986; Gules
Fig. 3 – Effects of intracisternal injection of interleukin-6 (IL-6) on the phosphorylation of STAT3 at Tyr705 and Ser727. At 1 h after intracisternal injection of IL-6 (2 μg/2 μL, n = 4) or Tris–HCl pH 7.6 (2 μL; control, n = 3), basilar arteries were removed and subjected to Western blotting with anti-actin (α-Actin), anti-STAT3 (α-STAT3), anti-phosphospecific STAT3 at Tyr705 (α-phospho-STAT3 Tyr705), and anti-phosphospecific STAT3 at Ser727 (α-phospho-STAT3 Ser727) antibodies. The histogram shows the amount of α-phoshp-STAT3 relative to that of α-STAT3. Mean ± SE values are shown; the asterisk indicates a significant difference by the Mann–Whitney's U test (P b 0.05).
et al., 2002; Jackowski et al., 1990), as found in humans. Genetic or genomic information is more easily available in rats than in other large animals (Harada et al., 1997; Nikaido et al., 2004; Suzuki et al., 1999). The endovascular puncture model Fig. 2 – Phosphorylation of JAK1 (A), STAT3 at Tyr705 (B), and STAT3 at Ser727 (C) in the basilar artery after subarachnoid hemorrhage (SAH). At 2, 6, 12 h, 1, 2, or 7days after intracisternal injection of autologous blood (300 μL), as indicated below the panel, crude samples (25 μg of protein each) were subjected to Western blotting with anti-JAK1 (α-JAK1), anti-phosphospecific JAK1 (α-phospho-JAK1), anti-STAT3 (α-STAT3) and anti-phosphospecific STAT3 at Tyr705 (α-phospho-STAT3 Tyr705), anti-phosphospecific STAT3 at Ser727 (α-phospho-STAT3 Ser727), and anti-actin (α-Actin) antibodies. The histogram shows the amount of α-phospho-JAK1 relative to that of α-JAK1 (A), the amount of α-phospho-STAT3 Tyr705 relative to that of α-STAT3 (B), and the amount of α-phospho-STAT3 Ser727 relative to that of α-STAT3 (C) in the membrane. Mean ± SE values from four animals are shown. Control; control basilar artery without SAH. *P b 0.05 versus control by Fisher's post hoc test.
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this study, we adopted a single-hemorrhage model to examine the relationship between inflammatory cytokine and JAK-STAT signaling pathway in basilar artery immediately after SAH. SAH initiates an inflammatory response regulated by specific signaling molecules, among which cytokines in CSF may play a prominent role. Proinflammatory cytokines, such as IL-1 and IL-6, demonstrate increased expression in the CSF after SAH (Mathiesen et al., 1993; Osuka et al., 1998a). Our data are consistent with previous clinical reports that production of IL-6 is induced from the immediately early stage of SAH (Osuka et al., 1998a). Increased expression of genes related to inflammation, such as IL-1, IL-6, and IL-8, was confirmed in the spastic basilar artery in a canine SAH model (Aihara et al., 2001). We previously demonstrated that IL-1β vasodilates the basilar artery through the prostaglandin cascade, especially prostacyclin, stimulated by the induced COX-2 (Osuka et al.,
Fig. 4 – Immunohistochemical findings for STAT3 in basilar arteries after subarachnoid hemorrhage (SAH). Rats were perfused with 4% paraformaldehyde after a sham operation (A, C, and E) or 2 h after injection of autologous blood (B, D, and F). Ten-micrometer coronal slices were immunostained with polyclonal antibodies recognizing STAT3 (A and B), phosphospecific-STAT3 at Tyr705 (C and D), or phosphospecific STAT3 at Ser727 (E and F) by the ABC method. Note more intense staining of phosphospecific-STAT3 at Tyr705 in the endothelial cells and cytoplasm of smooth muscle cells at 2 h after SAH (D) as compared to the control case (C), and similarly phosphospecific STAT3 at Ser727 (F, arrowheads) as compared to the control case (E). L, lumen of basilar artery. S, subarachnoid space. Original magnification ×1000.
(Bederson et al., 1995; Matz et al., 1996), single-hemorrhage model (Delgado et al., 1985, 1986; Gules et al., 2002; Jackowski et al., 1990; Nikaido et al., 2004; Suzuki et al., 1999), and doublehemorrhage model (Gules et al., 2002; Suzuki et al., 1999) have been established in rats. High mortality rates, up to 50%, is one of the existing problems for the endovascular puncture model (Bederson et al., 1995; Gules et al., 2002), while easy controllability of blood injection results in less mortality in injected models (Gules et al., 2002). Discrepancy in the time course of vasospasm between rats and humans still remains an important problem. In humans, angiographic cerebral vasospasm becomes maximum between 6 and 8 days after SAH (Heros et al., 1983; Kassell et al., 1985). The rat singlehemorrhage model and endovascular puncture model show maximum narrowing at day 2, followed by a gradual reduction and then disappearance by day 7 (Delgado et al., 1985; Gules et al., 2002; Nikaido et al., 2004; Suzuki et al., 1999), whereas the rat double-hemorrhage model shows maximum vasospasm at day 7 (Gules et al., 2002; Suzuki et al., 1999). In
Fig. 5 – Immunoblot analysis of inducible cyclooxygenase-2 (COX-2) in basilar arteries after subarachnoid hemorrhage (SAH), induced as for Fig. 2. Crude samples (25 μg of protein each) were subjected to Western blotting with anti-actin (α-Actin) and anti-COX-2 (α-COX-2) antibodies. The histogram shows the amount of α-COX-2 relative to that of α-actin in the membrane. Mean ± SE values from four animals are shown. Control; control basilar artery without SAH. Note the increase (not significant) in immunoreactivity against COX-2 from 6 h after SAH (A). Immunohistochemical analysis of COX-2 in basilar arteries after SAH. Rats subjected to sham operation (B) or 2 day (C) after intracisternal injection of autologous blood were perfused with 4% paraformaldehyde. Ten-micrometer coronal slices were immunostained with antibody against COX-2 by the ABC method. Note remarkable increase in immunoreactivity against COX-2 in the endothelial cells of basilar artery 2 day after SAH (C, arrows). L, lumen of basilar artery. S, subarachnoid space. Original magnification ×1000.
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1997). We also have revealed that inflammatory cytokines, not only IL-1β but also IL-6, immediately induced by SAH may be responsible for expression of COX-2 in basilar arteries after SAH, although the responsible mechanisms still remained to be clarified (Osuka et al., 1998b). IL-6 signaling occurs through binding to an IL-6 receptor and coupling to two gp-130. Then, JAKs are activated and phosphorylate STAT proteins, which dimerize and translocate into the nucleus, binding to promoters of specific target genes (Heinrich et al., 1998; Horvath 2000). Onda et al. (1999) reported the gp130 gene to be upregulated in vasospastic arteries in a canine SAH model. Inflammation in response to SAH may play an important role in vasospasm (Peterson et al., 1990) and our results confirmed for the first time, that the JAK-STAT pathway is activated in basilar arteries after SAH. JAK-STAT signaling appears to play an important cardioprotective role in ischemic preconditioning through upregulation of COX-2 (Bolli et al., 2003; Xuan et al., 2001) and our present data are in agreement with the previous observation of COX-2 expression in rabbit basilar artery endothelial cells after SAH (Tran Dinh et al., 2001). Blanco et al. (1995) confirmed that protein–tyrosine phosphorylation plays a role in the expression of COX-2 in endothelial cells. In addition, we have reported that IL-6 induces expression of the COX-2 in basilar artery (Osuka et al., 1998b) and our results showed intracisternal injection of IL-6 by itself activates JAK-STAT signaling pathway in vivo. From our data, expression of COX-2 in the endothelial cells through JAK-STAT signaling pathway was upregulated from 6 h after onset of SAH. These induced COX-2 plays vasodilator effects in antagonizing delayed cerebral vasospasm after SAH, which occurs maximum at day 2 in this single-hemorrhage model. Other IL-6 family members, such as IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor (CNTF), and cardiotrophin may also be involved with activation of the JAK-STAT signaling pathway (Heinrich et al., 1998). Tyrosine phosphorylation of STAT3 mediated by a JAK and Src family members has been studied (Vignais and Gilman 1999; Zhang et al., 2000) and activation of the tyrosine kinase Src, one of the MAPK cascade, has been shown to play an important role in mediation of cerebral vasospasm (Kusaka et al., 2003). On the other hand, phosphorylation of STAT3 at Ser727 is activated by the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogenactivated protein kinase (MAPK) (Levy and Lee 2002). Activation of ERK and p38 MAPK has been reported in the arterial wall after SAH (Sasaki et al., 2004; Zubkov et al., 1999), and therefore MAPK inhibitors may be useful for treatment of cerebral vasospasm. The single-hemorrhage model shows maximum vasospasm at day 2 (Delgado et al., 1985; Suzuki et al., 1999). A recent study provided evidence that shear flow induces the phosphorylation of STAT3 at Ser727 via the ERK1/2 pathway in endothelial cells (Ni et al., 2003) and in the present study significant increase of phosphorylated STAT3 at Ser727 at this site was evident on days 1–2 when basilar arteries became spastic. Increased shear stress evoked by narrowing of the arterial lumen would be expected to stimulate the ERK pathway and phosphorylation of STAT3 at Ser727 might be increased.
5
The present investigation has clarified the time course of activation of the JAK-STAT pathway in rat basilar arteries after SAH and their locations for the first time. Subsequent to the increase in the concentration of IL-6 in the CSF, JAK-STAT signaling is activated with translocation of STAT3 to the nucleus and upregulation of immediate genes, including COX-2. Further studies using IL-6 deficient mice should tell us the roles of this JAK-STAT signaling pathway in vasospasm after SAH and its possible relationship with other immediate early genes, providing clues to potential therapeutic measures.
4.
Experimental procedures
4.1.
Materials
Recombinant rat interleukin-6 (IL-6) was purchased from Pepro Tech (Rochy Hill, Nj). Other chemicals, unless otherwise specified, were from Sigma Chemicals Co Ltd. (St. Louis, MO). 4.2.
Experimental model of SAH and study protocol
All experiments were carried out in accordance with guidelines for the care and use of laboratory animals at the Nagoya University Graduate School of Medicine. Anesthesia was induced in male Sprague–Dawley rats (300 to 350 g, Chubu Kagaku Shizai Ltd., Nagoya, Japan) using chloral hydrate (400 mg/kg, IP). Animals were then intubated and ventilated with 1.0% halothane in an oxygen/nitrous oxide (30%/70%) gas mixture. Temperature was monitored with a rectal probe and maintained between 36.5 and 37.5 °C with a heating pad and lamp. The right femoral artery was exposed and catheterized with polyethylene tubing (PE-50) to allow blood sampling and the monitoring of arterial blood pressure during SAH. Injected arterial blood gases analyses were examined. A midline skin incision was made from the middle of the calvarium to the cervical spine under the stereotactic operation. The atlanto-occipital membrane was exposed under a microscope, and a 26-gauge needle was inserted into the cisterna magna. SAH was induced by injection of 300 μL of autologous arterial blood. Infusions were performed over a 5min period. Rats were kept in a head-down position for 5 min to ensure that the blood had contact with the basilar artery, after which the needle was withdrawn and all wounds were sutured. Animals without injection of blood were used as controls. Tissue samples for Western blot analysis were obtained by decapitation under deep anesthesia at 2, 6, 12, 24, 48, and 168 h after SAH. Basilar arteries were immediately isolated on ice, frozen in liquid nitrogen, and kept at −80 °C until use. Basilar arteries without injection of blood were used as controls. 4.3.
Sample preparation for Western blot analysis
Samples were prepared from four different animals in each group. Basilar arteries were homogenized using a homogenizer in 15 volumes of homogenization buffer containing 50 mmol/L Tris base/HCl (pH 7.5), 0.1 mmol/L dithiothreitol, 0.2 mmol/L EDTA, 0.2 mmol/L EGTA, 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1.25 μg/mL pepstatin A, 0.2 μg/mL aprotinin, 1 mmol/L sodium orthovanadate, 50 mmol/L sodium fluoride, 2 mmol/L sodium pyrophosphate, and 1% Nonidet P-40 (NP-40). The homogenates were then centrifuged at 15,000 rpm at 4 °C for 10 min. Protein concentrations of the supernatants were determined by the method of Bradford using bovine serum albumin as the standard.
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Western blotting analysis
Crude samples (25 μg of protein each) were subjected to 7.5% SDSPAGE, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes and incubated with primary polyclonal antibodies against phospho-JAK1 and phospho-STAT3 at Ser727 (Cell signaling technology, Beverly, MA) at a dilution of 1:500 overnight at 4 °C and polyclonal phospho-STAT3 at Tyr705 (Cell Signaling Technology, Beverly, Mass) or monoclonal antibodies against inducible cyclooxygenase (COX-2, Transduction Laboratories, Lexington, KY) at a dilution of 1:500 for 45 min at room temperature. The membranes were then incubated for 30 min at room temperature with horseradish peroxidase-conjugated secondary antibodies, and binding was visualized using an enhanced chemiluminescence (ECL) or ECL plus Western blotting detection system (Amersham). Phospho-JAK1 and phospho-STAT3 immunoblots were stripped from PVDF membranes and reblotted with primary monoclonal JAK1 (Transduction Laboratories, Lexington, KY), polyclonal STAT3 (Cell signaling technology, Beverly, MA), and polyclonal actin (Sigma, St. Louis, MO) at a dilution of 1:500 for 45 min at room temperature. Finally, the membranes were developed with the ECL or ECL plus system and band intensities were quantitated by densitometric scanning using the NIH IMAGE program. 4.5.
Measurement of cytokine levels
For the assay of IL-6 levels in CSF, 50 μL CSF samples were obtained through the atlanto-occipital membrane before decapitation at same time course after SAH and immediately centrifuged. Supernatant fluid was collected and stored at −80 °C until assayed. IL-6 was measured using ELISA kit (BIOSOURCE). CSF samples from rats without SAH were used as controls. 4.6.
Interleukin-6 injection model
Under anesthesia, rats were set on a stereotactic operation frame and the atlanto-occipital membrane was exposed under a microscope. IL-6 (2 μg/2 μL) was then slowly injected into the cisterna magna through a 26-gauge needle. The needle was kept in place for 10 min to allow the drug to diffuse into the whole cisterns and Tris–HCl (pH 7.6, 2 μL) was employed as the vehicle control. Basilar arteries were removed 1 h after injection of IL-6 or vehicle for Western blot analyses, as described previously. The following primary antibodies were used: polyclonal antibodies for phosphorylated STAT3 at Tyr705 and at Ser727; a polyclonal antibody for STAT3; and a polyclonal antibody for actin. 4.7.
Immunohistochemistry
The rats were perfused with ice-cold 200 mL 4% paraformaldehyde in 0.1 mol/L sodium phosphate (pH7.4) at 2 h or 2 days after SAH. The basilar arteries with brain stems were removed and preserved in the fixation solution for 3 h. Serial coronal cryostat sections (10 μm) were stained according to the avidin-biotinylated peroxidase complex (ABC) technique at room temperature. The staining sequence was as follows: 2% goat or horse serum for 30 min; primary polyclonal antibodies against phosphorylated STAT3 at Tyr705 and at Ser727 at a dilution of 1:200 each, STAT3 at a dilution of 1:1000, and monoclonal antibody against COX-2 at a dilution of 1:500; biotinylated anti-rabbit or mouse IgG for 1 h; and ABC for 1 h. Sera for the blocking step, biotinylated antibodies, and ABC were purchased from Vector Laboratories (Burlingame, Ca). Reaction products were developed by incubation in 0.05% 3,3′-diaminobendizine tetrachloride and 0.01% H2O2 in 50 mM Tris–HCl (pH 7.5) for 10 min. Rats with all surgical procedures, but without injection of blood, were used as controls.
4.8.
Statistical analysis
Data are expressed as mean ± SE values. Statistical differences between the groups were assessed by one-way ANOVA with Fisher's post hoc test and a Mann–Whitney's test. Statistical significance was concluded at the P b 0.05 level.
Acknowledgments We thank Dr. Malcolm Moore for critical reading of the manuscript and Kenmei Mizutani for technical assistance. This work was supported by grants from the General Insurance Association of Japan to KO.
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