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Therapeutic potential of peroxisome proliferator-activated receptor gamma agonist rosiglitazone in cerebral vasospasm after a rat experimental subarachnoid hemorrhage model
Journal of the Neurological Sciences 305 (2011) 85–91
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Therapeutic potential of peroxisome proliferator-activated receptor gamma agonist rosiglitazone in cerebral vasospasm after a rat experimental subarachnoid hemorrhage model Yi Wu, Ke Tang, Ren-Qiang Huang, Zong Zhuang, Hui-Lin Cheng, Hong-Xia Yin, Ji-Xin Shi ⁎ Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, China
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Article history: Received 2 May 2010 Received in revised form 18 February 2011 Accepted 1 March 2011 Available online 26 March 2011 Keywords: Vasospasm PPAR-gamma TLR4 Subarachnoid hemorrhage Rosiglitazone Inflammation
a b s t r a c t The pathogenesis of cerebral vasospasm is closely associated with inflammation and immune response in arterial walls. Recently, the authors proved the key role of Toll-like receptor (TLR)4 in the development of vasospasm in experimental subarachnoid hemorrhage (SAH) model. Because peroxisome proliferatoractivated receptor (PPAR) gamma agonists are identified as effective inhibitors of TLR4 activation, we investigated the anti-inflammation properties of PPAR-gamma agonist rosiglitazone in basilar arteries in a rat experimental SAH model and evaluated the effects of rosiglitazone on vasospasm. Inflammatory responses in basilar arteries were assessed by immunohistochemical staining for intercellular molecule (ICAM)-1 and myeloperoxidase (MPO). Expression of TLR4 was determined by western blot analysis. The degree of cerebral vasospasm was evaluated by measuring the mean diameter and cross-sectional area of basilar arteries. Rosiglitazone suppressed the SAH-induced inflammatory responses in basilar arteries by inhibiting the TLR4 signalling. Furthermore, rosiglitazone could attenuate cerebral vasospasm following SAH. Therefore, we suggested that PPAR-gamma agonists may be potential therapeutic agents for cerebral vasospasm. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Subarachnoid hemorrhage (SAH) following aneurysm rupture leads to the development of cerebral vasospasm, which is a principal cause of mortality and morbidity in relation to SAH. Radiographic evidence vasospasm is found in 50% to 70% of patients surviving SAH, and half of those are associated with delayed neurological deficits [1]. The management of SAH patients hence focuses on the prevention of vasospasm and those secondary complications. Although intensive research has been made to explore the underlying pathophysiology of vasospasm, the precise molecular mechanism is still far from being completely understood. Rapidly increased evidences show that inflammation and immune response play a significant role in the development of cerebral vasospasm. Elevated levels of inflammatory constituents including leukocytes, proinflammatory cytokines, adhesion molecules, chemokines, and matrix metalloproteinases have been demonstrated to be implicated in the pathogenesis of cerebral vasospasm [2–6]. Moreover, proinflammatory transcription factor Nuclear Factor kappa B (NF-κB) and various kinds of signalling pathways, such as MAPK, JNK, JAK-STAT pathway, were observed to be activated in the basilar artery in response to experimental SAH [7–10].
⁎ Corresponding author. Fax: +86 25 84817581. E-mail address: [email protected] (J.-X. Shi). 0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.03.006
Toll-like receptors (TLRs) initiate inflammatory signalling and innate immune responses in response to conserved microbial molecules and endogenous agonists [11,12]. Accumulating literatures on this subject showed that TLRs are potential links between inflammation and vascular dysfunction. Inhibition of TLR4 has been shown to protect against inflammation and atherosclerotic lesion formation [12–14]. Meanwhile, there has been evidence for the involvement of TLR4 in initiating neuroinflammation related to infectious diseases, neurodegenerative diseases and stroke [15–17]. We have reported that increased expression of TLR4 was detected in the arterial wall in experimental SAH model both in vivo and in vitro [18,19]. These results indicated that TLR-induced activation of immune responses and the release of proinflammatory molecules may be responsible for neurotoxic processes in the course of vasospasm following SAH. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily [20]. PPAR-gamma, as a member of this family, was characterized initially as a regulator of lipid and glucose metabolism. Whereas, expanding evidence displayed that PPAR-gamma also plays a critical role in regulating inflammation and immune reactions [21,22]. Therefore, particular attention was paid to study the therapeutic potential of PPAR-gamma agonists in neuroinflammatory diseases like stroke, Alzheimer's disease, infection, spinal cord injury (SCI) and traumatic brain injury (TBI) [23–27]. Several possible mechanisms have been reported to explain such a neuroprotective effect: PPAR-
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gamma activators could suppress the activation of proinflammatory transcription factors, especially NF-κB signalling [28]; PPAR-gamma agonists act inhibitors of proinflammatory mediators, such as cytokines, matrix metalloproteinases, and adhesion molecules [29,30]; activation of PPAR-gamma could decrease microglial and astroglial reactivity to endogenous and exogenous stress and attenuate the injury produced by excessive immunoreaction [31,32]; PPAR-gamma agonists up-regulated superoxide dismutase activity and reduce the generation of reactive oxygen species [23]. Our previous study showed that the up-regulation of TLR4 correlated with the development of cerebral vasospasm [19]. On the other hand, PPAR-gamma activators are known to be effective inhibitors of TLR4 [33,34]. We have documented recently that selective PPAR-gamma agonists rosiglitazone could suppress TLR4 expression and cytokine release in vascular smooth muscle cells (VSMCs) after OxyHb exposure [18]. For these reasons, it becomes possible to predict that PPAR-gamma may exert its anti-inflammation function through TLR4 pathway to prevent cerebral vasospasm following SAH. Moreover, the potential role of PPAR-gamma in SAH and vasospasm has not been studied. In the present work, we shall evaluate the anti-inflammatory effect of rosiglitazone, a potent PPAR-gamma agonist, on inflammatory response following experimental SAH in basilar arteries and exam the vascular changes of basilar arteries after rosiglitazone treatment. 2. Materials and methods 2.1. Animal preparation The experimental protocol using animals was approved by the Animal Care and Use Committee of Nanjing University and conformed to the Guide for the Care and Use of Laboratory Animals from the National Institute of Health. The method of producing SAH was performed as a double hemorrhage injection method [35]. Male Sprague–Dawley rats (300 to 350 g) were anesthetized with an intraperitoneal injection of pentobarbital (40 mg/kg), and allowed to breathe spontaneously. Aided by a surgical microscope, a small suboccipital incision was made, exposing the arch of the atlas, the occipital bone, and the atlantooccipital membrane. With a 27-gauge needle, the atlantooccipital membrane was tapped carefully into the cisterna magna. Freshly autologous nonheparinized blood (0.2 ml) withdrawn from the femoral artery was injected over a period into the cisterna magna. Immediately after the injection of blood, the hole was sealed with glue to prevent firtula and the incision was sutured. The rats were then placed in a 30° head down prone position for 30 min to permit a good distribution of blood around the basal intracranial arteries. After 24 h (day 1), the same procedure were repeated and 0.2 ml of autologous blood injected. The animals were allowed to recover from the effects of anesthesia and returned to their cages. 2.2. Drug administration Rosiglitazone (Cayman Chemical Co., Ann Arbor, MI, USA) was first dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Inc.) (vehicle for rosiglitazone) and then diluted with saline (1:3 ratio), and administrated intraperitoneally (3 mg/kg) at 30 min before surgery plus 30 min after SAH induction. After 24 h, the same administration of rosiglitazone was repeated when the animals received the second intracisternal injection of autologous blood. Seventy-eight male Sprague–Dawley rats received administration of equal volumes of rosiglitazone or vehicle (DMSO) on day 0 and day 1 were assigned randomly to three groups: (1) the control group (n= 26) with intracisternal saline injection, intraperitoneal administration of vehicle; (2) the SAH group (n = 26) with SAH, intraperitoneal administration of vehicle; and (3) the rosiglitazone group (n= 26) with SAH, intraperitoneal administration of rosiglitazone (3 mg/kg).
2.3. Perfusion–fixation On day 5, the rats (n=20, each group) were anesthetized, followed by the perfusion–fixation procedure. After a thoracotomy was performed, a cannula was placed in the left ventricle. Subsequently, the abdominal aorta clamped, and the right atrium was opened widely. Perfusion was performed with 500 ml of physiological phosphate buffer solution (pH 7.4) at 37 °C, followed by 500 ml of 10% buffered formaldehyde under a perfusion pressure of 120 cm H2O. After perfusion–fixation, the whole brain with the basilar artery was removed and immersed in the same fixative solution at 4 °C overnight. 2.4. Measurement of basilar artery The degree of cerebral vasospasm was evaluated by measuring the mean diameter and cross-sectional area of each basilar artery. The formalin-fixed and paraffin-embedded basilar artery sections (4 μmin thickness) were deparaffinized, hydrated, washed and stained with H&E. Then the stained basilar arteries were measured using Image Pro-Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). We calculated the mean diameter and the internal area of each slice. For each vessel, three sequential sections (midpoint of the proximal, the middle and the distal) were taken, measured and averaged. 2.5. Immunohistochemical study To evaluate the involvement of PPARγ in the regulation of intercellular adhesion molecule (ICAM-1), and myeloperoxidase (MPO), immunocytochemistry on formalin-fixed paraffin-embedded sections was employed to determine those immunoreactivities. Coronal sections through the basilar artery were deparaffinized and rehydrated in graded concentrations of ethanol to distilled water. Endogenous peroxidase activity was blocked with 3% H2O2 for 5 min, followed by a brief rinse in distilled water and a 15 min wash in PBS. Sections were placed in 10 mmol/l citrate buffer (pH 6.0), and heated in microwave oven at 95 °C for 30 min, then cooled at room temperature for 20 min and rinsed in PBS. Non-specific protein binding was blocked by 40 min incubation in 5% horse serum. The basilar artery sections were incubated with primary antibodies (anti-MPO and anti-ICAM-1, prediluted and diluted in 1:100, respectively, both from Abcam, Cambridge, UK) for 1 h at room temperature, followed by a 15 min wash in PBS. Sections were incubated with horseradish peroxidase (HRP)-conjugated IgG (1:500 dilution, Santa Cruz Biotechnology, Inc., California, USA) for 60 min at room temperature. DAB was used as chromogen and counterstaining was done with hematoxylin. Sections incubated in the absence of primary antibody were used as negative controls. For qualitative analysis, immunohistochemical stained sections were analyzed with standard light microscope, The number of ICAM-1 and MPO positive cells in each section was counted in four microscopic fields (at ×400 magnification) and averaged to determine the number of positively immunostained cells per field. 2.6. Harvest of cerebral arteries The rats for protein determination were anesthetized and then killed by decapitation. Afterward, their brains were removed. With the aid of a microscope, the basilar arteries and circle of Willis arteries were carefully dissected free from each brain, cleared of connective tissue, and snap frozen in liquid nitrogen until use. 2.7. Western blot analysis The frozen basilar arteries from each group (n = 6) were mechanically lysed in 20 mM Tris, pH 7.6, which contains 0.2% SDS, 1% Triton X-100, 1% deoxycholate, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.11 IU/ml aprotinin (all purchased from Sigma-Aldrich,
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Inc., St. Luis, MO, USA). Lysates were centrifuged at 12,000 ×g for 20 min at 4 °C. The protein concentration was determined by the Bradford assay (Bio-Rad Lab, Hercules, CA, USA). Equal amounts of protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membrane. The membranes were blocked with 3% (wt/vol) low-fat milk and then incubated overnight at 4 °C with primary antibodies for TLR4 (Bioworld Technology, Inc., Minneapolis, MN, USA), and at the dilution of 1:500. The β-actin (diluted in 1:6000, Sigma-Aldrich, Inc.) was used as a loading control. After the membranes were washed and further incubated with HRP-conjugated secondary antibody at 1:400 for 2 h, the blotted protein bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham, Arlington Heights, IL, USA). Optical densities of the resulting bands were quantified using Glyko Bandscan software (Glyko, Novato, CA, USA).
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3.2. Measurement of basilar artery To exam the vascular changes of basilar arteries, mean diameter and cross-sectional area were measured. Representative images of each group were showed in Fig. 1. Compared with the control group, notable vasospasm was found in the SAH group. Whereas, the severity of the vasospasm was markedly attenuated in the rosiglitazone group (Fig. 1A–C). Besides, the spastic and thicken appearance of vessel wall following SAH was attenuated in the rosiglitazone group (Fig. 1D–F). Quantitative analysis showed that, there was a significant difference in the basilar artery cross-sectional area among groups (P b 0.001, ANOVA). The cross-sectional area of basilar artery were found to be 55,508 ± 4630 μm2 in the control group, 32,139 ± 3239 μm2 in the SAH group, 45,969 ± 4464 μm2 in the rosiglitazone group (Fig. 2A) Similar results were also detected while measuring of mean diameter (Fig. 2B). A significant difference was observed in the mean diameter among groups (Pb 0.001, ANOVA).
2.8. Statistical analysis All results were presented as the mean ± SD. All data were subjected to one-way ANOVA. Fischer's LSD post hoc test was used for multiple comparisons. A probability value of less than 0.05 was considered statistically significant.
3. Results 3.1. General observation and mortality No significant changes in body weight and blood glucose were detected in any of the experimental groups (P N 0.05, ANOVA, data not shown). No animals died in the control group. Within the SAH groups, mortality rate was 20% (6 of 30) in the vehicle treated group, compared to 13.3% (4 of 30) in the rosiglitazone treated group. The animals died in the course of anesthesia or blood injection were excluded from the analysis of mortality.
3.3. Immunocytochemical study for intercellular adhesion molecule and myeloperoxidase Immunochemistry was performed to evaluate the immunoreactivity of ICAM-1 and MPO. The result displayed that both ICAM-1 and MPO were expressed at low levels in the control group (Fig. 3A and D). Whereas, increased ICAM-1-positive and MPO-positive cells in the SAH group could be observed in the basilar artery (Fig. 3B and E). Compared with that, both the degree of vasospasm and the range of positive staining for ICAM-1 and MPO were attenuated in the basilar artery after rosiglitazone treatment (Fig. 3C and F). Moreover, ICAM-1 immunoreactivity was mainly expressed in the intimal and adventitial cells, while MPO immunoreactivity was located in all the intimal, medial, and adventitial cells. The positive staining for ICAM-1 was almost disappeared in intimal cells after rosiglitazone treatment (Fig. 3C), which may indicated the capacity of rosiglitazone to inhibit endothelial inflammation.
Fig. 1. Histological changes of basilar arteries on day 5 after SAH induction. (A–C) Representative images of basilar arteries of the control rats or rats subjected to SAH alone or SAH plus intraperitoneal injection with rosiglitazone (3 mg/kg). Vasospasm could be detected in the SAH group, which was attenuated in the rosiglitazone group. (D–F) Magnified images of the square area from A through C, respectively. The spastic and thicken appearance of vessel wall after SAH, which was attenuated in the rosiglitazone group. Scale bar = 200 and 20 μm, respectively.
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Fig. 2. Histogram of the mean diameter and cross-sectional area of the basilar arteries. (A) Cross-sectional area of basilar arteries from the control, SAH, and rosiglitazone groups. (B) Mean diameter of basilar arteries from three groups. The results indicated that rosiglitazone could attenuate cerebral vasospasm following SAH. Bars represent the mean ± SD (n = 20, each group). **P b 0.01 compared with rats treated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.
Semi-quantitative analysis for the immunohistochemistry showed that administration of rosiglitazone remarkably inhibited SAHinduced upregulation of ICAM-1 and MPO expression in the arterial wall. Significant differences were both found between the control or SAH groups, and the rosiglitazone group (both P b 0.01) (Fig. 3G). 3.4. Western blot analysis for TLR4 Western blot was performed to assess the effect of rosiglitazone on the expression of TLR4 in the basilar artery after SAH. A significant increase in TLR4 expression was observed in the SAH group as compared with that in the control group (P b 0.01). Rosiglitazone treatment was found to reduce TLR4 expression dramatically in the basilar artery following SAH (Pb 0.01), although there was still a difference in the production of TLR4 between rosiglitazone group and control group (Pb 0.05) (Fig. 4). 4. Discussion The present work coincides with our previous findings in VSMCs after OxyHb exposure in vitro. The VSMCs pretreated with PPARgamma agonist rosiglitazone exhibited significant reduction of TLR4 expression and cytokine release in response to OxyHb [18]. In present study, we demonstrated that the activation TLR4 and downstream adhesion molecules could be induced in basilar arteries in the rat SAH model. The PPAR-gamma agonist, rosiglitazone, could significantly suppressed the SAH-induced inflammatory responses by blocking the expression of TLR4 and adhesion molecules. Most importantly,
rosiglitazone could attenuate the SAH-induced cerebral vasospasm. These current data support the involvement of TLR4 in SAH-mediated inflammatory activation in basilar arteries, which may facilitate the progression of cerebral vasospasm after SAH. The study also reveals the potential therapeutic role of PPAR-gamma agonists in SAHinduced cerebral vasospasm by negative regulation of inflammatory responses through TLR4 signalling. Vasospasm remains a leading cause of morbidity and mortality following SAH. It refers to a circumstance that is more complex than simple constriction of vessels. Pathological changes occur in cerebral arteries following SAH that lead to additional lumen narrowing, vessel wall thickening and impaired vascular reactivity [36]. Despite investigating a myriad of compounds, very few have made it to clinical application. So far, Nimodipine, a calcium channel antagonist, is the only drug approved by the US Food and Drug Administration for use in treatment of vasospasm. However, it has a limited improvement on the overall morbidity and mortality [37]. Thus, there is a great need to explore new therapeutic targets for vasospasm. Growing documents indicate that inflammation and immunity after SAH play a crucial role in the development of vasospasm. A complex series of inflammatory constituents including cytokines, adhesion molecules, chemokines and transcription factors have been reported to be implicated in the pathogenesis of cerebral vasospasm [2,5,10]. Moreover, notable therapeutic effects of anti-inflammatory agents for vasospasm have been testified in many animal SAH model, whereas the similar efficacy have not been supported in the clinical trial [38,39]. It is probably because the precise role of the inflammatory responses in the artery accompanying SAH and cerebral vasospasm had not yet been well clarified. TLR4 is increasingly recognized as a critical factor in initiating inflammatory signalling and innate immune responses. It is considered to activate signalling pathways leading to the expression of pro-inflammatory cytokines implicated in the etiology of vascular lesions [40,41]. Recently, upregulated expression of TLR4 was detected in the endothelial cell layer of human cerebral aneurysm walls, and the TLR4 expression coincided well with NF-κB activation. These findings indicated TLR4 may play an important role in cerebral aneurysm formation [42]. Further, in our previous study, the activation of TLR4 signalling was observed both in VSMCs after OxyHb stimuli and in basilar arteries in the rabbit SAH model [18,19]. Likewise, in the present work, elevated expression of TLR4 was detected in the basilar arteries in the rat SAH model. Moreover, we evaluated the degree of leukocyte infiltration in the vessel wall through the assessment of MPO and adhesion molecules immunoreactivity. The results proved that inflammation was markedly triggered in the basilar arteries following SAH. Therefore, it is reasonable for us to believe that TLR4 signalling may tightly correlate with the development of aneurysmal SAH and aggravation of vasospasm. PPAR-gamma, an essential transcriptional mediator of adipogenesis, lipid metabolism, and glucose homeostasis, is increasingly recognized as a key player in the regulation of various type of inflammatory response. These functions are mediated through its ability to regulate energy homeostasis, cell proliferation, membrane lipid composition and transrepress the activities of many transcription factors, including NF-κB, STATs and AP1 [43], which are all known to be involved in inflammation and immune processes. The therapeutic effects of PPAR-gamma agonists have been testified in many different CNS diseases, including cerebral ischemia, intracerebral hemorrhage, Alzheimer's disease, Multiple sclerosis and trauma [23,32,44–46]. Additionally, PPAR-gamma was reported to plays critical roles in vascular system. PPAR-gamma activation inhibits endothelial inflammation and therefore improves endothelial dysfunction. Meanwhile, it suppresses vascular smooth muscle cells proliferation, migration, and vascular remodeling, which is of benefit to atherosclerosis and hypertension [47,48]. PPAR-gamma agonists have been identified as effective inhibitor of TLR4. LPS-induced TLR4 signalling and NF-κB activation in monocytes could be inhibited by PPAR-gamma ligands [14]. Rosiglitazone could
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Fig. 3. Immunohistochemical study for ICAM-1 (A–C) and MPO (D–F) on cross section of basilar arteries. ICAM-1 and MPO were expressed at low levels in the vessel walls in the control group (A and D). Increased positive cells were observed in the arterial wall in the SAH group (B and E). The degree of vasospasm and the range of positive expression were declined in rosiglitazone group (C and F). The arrows indicate positively staining cells in the intima. Scale bar = 20 μm. Semi-quantitative analysis shows that ICAM-1 and MPO positive cells in rosiglitazone groups are significantly fewer than in SAH (G). Bars represent the mean ± SD (n = 5, each group). **P b 0.01 compared with rats treated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.
regulate angiotensin II-induced or OxyHb-induced vascular inflammation through the TLR4-dependent signalling pathway [18,49]. Present data showed that pretreatment with rosiglitazone could significantly reduce SAH-mediate upregulation of TLR4 and increased production of MPO and ICAM-1. We also analyzed the vascular changes of basilar arteries by measuring mean diameter and crosssectional area of vessels. The results revealed that rosiglitazone could effectively attenuate cerebral vasospasm. These findings suggested that rosiglitazone may exert its neuroprotective effect through blocking TLR4 pathway and diminishing downstream inflammatory mediators release in vessels. It therefore may prevent vascular inflammation and attenuate cerebral vasospasm associated with SAH. It has been recognized that TLR4 induced activation of inflammatory cells and the release of proinflammatory molecules are responsible for tissue damage during various CNS diseases. Paradoxically, TLR ligands administered systemically induce a state of tolerance to later ischemic injury [50]. Thus, modulating the activation of TLR4 and holding a precise balance between protective and harmful effects for the host may be a feasible strategy for prevention of neurotoxic damage relate to
inflammation. PPAR-gamma agonist rosiglitazone could modulate TLR4 activation, decrease inflammatory response in the cerebral vessels and attenuate vasoconstriction in experimental SAH model. Furthermore, Our previous study demonstrated that the anti-inflammation effects of rosiglitazone on OxyHb-treated VSMCs were effectively counteracted by the PPARγ antagonist GW9662. Thus the neuroprotective effects of rosiglitazone may at least partly dependent on the PPAR-gamma activation. Overall, we have shown that the TLR4 mediated inflammatory response has occurred in basilar arteries following SAH. Pretreatment with rosiglitazone protects the cerebral vessels against inflammation and attenuates vasospasm by inhibiting TLR4 signalling, which suggests that rosiglitazone may have therapeutic potential for the treatment of cerebral vasospasm. Finally, administration of rosiglitazone did not attenuate vasospasm completely, which coincided with the degree of TLR4 inhibition. It suggested that restraint at multiple levels for TLR4 and bringing the level of TLR4 to get close to those in normal condition may provide more protection from vasospasm.
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Fig. 4. Western blot analysis for TLR4 protein in the basilar arteries. (A) Representative autoradiogram of TLR4 expression in basilar arteries. (B) Quantitative analysis of the Western blot results for TLR4. Results were expressed as the mean ± SD of six experiments done in triplicate (n = 6, each group). *P b 0.05 or **P b 0.01 compared with rats treated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Therapeutic potential of peroxisome proliferator-activated receptor gamma agonist rosiglitazone in cerebral vasospasm after a rat experimental subarachnoid hemorrhage model Yi Wu, Ke Tang, Ren-Qiang Huang, Zong Zhuang, Hui-Lin Cheng, Hong-Xia Yin, Ji-Xin Shi ⁎ Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, China
a r t i c l e
i n f o
Article history: Received 2 May 2010 Received in revised form 18 February 2011 Accepted 1 March 2011 Available online 26 March 2011 Keywords: Vasospasm PPAR-gamma TLR4 Subarachnoid hemorrhage Rosiglitazone Inflammation
a b s t r a c t The pathogenesis of cerebral vasospasm is closely associated with inflammation and immune response in arterial walls. Recently, the authors proved the key role of Toll-like receptor (TLR)4 in the development of vasospasm in experimental subarachnoid hemorrhage (SAH) model. Because peroxisome proliferatoractivated receptor (PPAR) gamma agonists are identified as effective inhibitors of TLR4 activation, we investigated the anti-inflammation properties of PPAR-gamma agonist rosiglitazone in basilar arteries in a rat experimental SAH model and evaluated the effects of rosiglitazone on vasospasm. Inflammatory responses in basilar arteries were assessed by immunohistochemical staining for intercellular molecule (ICAM)-1 and myeloperoxidase (MPO). Expression of TLR4 was determined by western blot analysis. The degree of cerebral vasospasm was evaluated by measuring the mean diameter and cross-sectional area of basilar arteries. Rosiglitazone suppressed the SAH-induced inflammatory responses in basilar arteries by inhibiting the TLR4 signalling. Furthermore, rosiglitazone could attenuate cerebral vasospasm following SAH. Therefore, we suggested that PPAR-gamma agonists may be potential therapeutic agents for cerebral vasospasm. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Subarachnoid hemorrhage (SAH) following aneurysm rupture leads to the development of cerebral vasospasm, which is a principal cause of mortality and morbidity in relation to SAH. Radiographic evidence vasospasm is found in 50% to 70% of patients surviving SAH, and half of those are associated with delayed neurological deficits [1]. The management of SAH patients hence focuses on the prevention of vasospasm and those secondary complications. Although intensive research has been made to explore the underlying pathophysiology of vasospasm, the precise molecular mechanism is still far from being completely understood. Rapidly increased evidences show that inflammation and immune response play a significant role in the development of cerebral vasospasm. Elevated levels of inflammatory constituents including leukocytes, proinflammatory cytokines, adhesion molecules, chemokines, and matrix metalloproteinases have been demonstrated to be implicated in the pathogenesis of cerebral vasospasm [2–6]. Moreover, proinflammatory transcription factor Nuclear Factor kappa B (NF-κB) and various kinds of signalling pathways, such as MAPK, JNK, JAK-STAT pathway, were observed to be activated in the basilar artery in response to experimental SAH [7–10].
⁎ Corresponding author. Fax: +86 25 84817581. E-mail address: [email protected] (J.-X. Shi). 0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.03.006
Toll-like receptors (TLRs) initiate inflammatory signalling and innate immune responses in response to conserved microbial molecules and endogenous agonists [11,12]. Accumulating literatures on this subject showed that TLRs are potential links between inflammation and vascular dysfunction. Inhibition of TLR4 has been shown to protect against inflammation and atherosclerotic lesion formation [12–14]. Meanwhile, there has been evidence for the involvement of TLR4 in initiating neuroinflammation related to infectious diseases, neurodegenerative diseases and stroke [15–17]. We have reported that increased expression of TLR4 was detected in the arterial wall in experimental SAH model both in vivo and in vitro [18,19]. These results indicated that TLR-induced activation of immune responses and the release of proinflammatory molecules may be responsible for neurotoxic processes in the course of vasospasm following SAH. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily [20]. PPAR-gamma, as a member of this family, was characterized initially as a regulator of lipid and glucose metabolism. Whereas, expanding evidence displayed that PPAR-gamma also plays a critical role in regulating inflammation and immune reactions [21,22]. Therefore, particular attention was paid to study the therapeutic potential of PPAR-gamma agonists in neuroinflammatory diseases like stroke, Alzheimer's disease, infection, spinal cord injury (SCI) and traumatic brain injury (TBI) [23–27]. Several possible mechanisms have been reported to explain such a neuroprotective effect: PPAR-
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gamma activators could suppress the activation of proinflammatory transcription factors, especially NF-κB signalling [28]; PPAR-gamma agonists act inhibitors of proinflammatory mediators, such as cytokines, matrix metalloproteinases, and adhesion molecules [29,30]; activation of PPAR-gamma could decrease microglial and astroglial reactivity to endogenous and exogenous stress and attenuate the injury produced by excessive immunoreaction [31,32]; PPAR-gamma agonists up-regulated superoxide dismutase activity and reduce the generation of reactive oxygen species [23]. Our previous study showed that the up-regulation of TLR4 correlated with the development of cerebral vasospasm [19]. On the other hand, PPAR-gamma activators are known to be effective inhibitors of TLR4 [33,34]. We have documented recently that selective PPAR-gamma agonists rosiglitazone could suppress TLR4 expression and cytokine release in vascular smooth muscle cells (VSMCs) after OxyHb exposure [18]. For these reasons, it becomes possible to predict that PPAR-gamma may exert its anti-inflammation function through TLR4 pathway to prevent cerebral vasospasm following SAH. Moreover, the potential role of PPAR-gamma in SAH and vasospasm has not been studied. In the present work, we shall evaluate the anti-inflammatory effect of rosiglitazone, a potent PPAR-gamma agonist, on inflammatory response following experimental SAH in basilar arteries and exam the vascular changes of basilar arteries after rosiglitazone treatment. 2. Materials and methods 2.1. Animal preparation The experimental protocol using animals was approved by the Animal Care and Use Committee of Nanjing University and conformed to the Guide for the Care and Use of Laboratory Animals from the National Institute of Health. The method of producing SAH was performed as a double hemorrhage injection method [35]. Male Sprague–Dawley rats (300 to 350 g) were anesthetized with an intraperitoneal injection of pentobarbital (40 mg/kg), and allowed to breathe spontaneously. Aided by a surgical microscope, a small suboccipital incision was made, exposing the arch of the atlas, the occipital bone, and the atlantooccipital membrane. With a 27-gauge needle, the atlantooccipital membrane was tapped carefully into the cisterna magna. Freshly autologous nonheparinized blood (0.2 ml) withdrawn from the femoral artery was injected over a period into the cisterna magna. Immediately after the injection of blood, the hole was sealed with glue to prevent firtula and the incision was sutured. The rats were then placed in a 30° head down prone position for 30 min to permit a good distribution of blood around the basal intracranial arteries. After 24 h (day 1), the same procedure were repeated and 0.2 ml of autologous blood injected. The animals were allowed to recover from the effects of anesthesia and returned to their cages. 2.2. Drug administration Rosiglitazone (Cayman Chemical Co., Ann Arbor, MI, USA) was first dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Inc.) (vehicle for rosiglitazone) and then diluted with saline (1:3 ratio), and administrated intraperitoneally (3 mg/kg) at 30 min before surgery plus 30 min after SAH induction. After 24 h, the same administration of rosiglitazone was repeated when the animals received the second intracisternal injection of autologous blood. Seventy-eight male Sprague–Dawley rats received administration of equal volumes of rosiglitazone or vehicle (DMSO) on day 0 and day 1 were assigned randomly to three groups: (1) the control group (n= 26) with intracisternal saline injection, intraperitoneal administration of vehicle; (2) the SAH group (n = 26) with SAH, intraperitoneal administration of vehicle; and (3) the rosiglitazone group (n= 26) with SAH, intraperitoneal administration of rosiglitazone (3 mg/kg).
2.3. Perfusion–fixation On day 5, the rats (n=20, each group) were anesthetized, followed by the perfusion–fixation procedure. After a thoracotomy was performed, a cannula was placed in the left ventricle. Subsequently, the abdominal aorta clamped, and the right atrium was opened widely. Perfusion was performed with 500 ml of physiological phosphate buffer solution (pH 7.4) at 37 °C, followed by 500 ml of 10% buffered formaldehyde under a perfusion pressure of 120 cm H2O. After perfusion–fixation, the whole brain with the basilar artery was removed and immersed in the same fixative solution at 4 °C overnight. 2.4. Measurement of basilar artery The degree of cerebral vasospasm was evaluated by measuring the mean diameter and cross-sectional area of each basilar artery. The formalin-fixed and paraffin-embedded basilar artery sections (4 μmin thickness) were deparaffinized, hydrated, washed and stained with H&E. Then the stained basilar arteries were measured using Image Pro-Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). We calculated the mean diameter and the internal area of each slice. For each vessel, three sequential sections (midpoint of the proximal, the middle and the distal) were taken, measured and averaged. 2.5. Immunohistochemical study To evaluate the involvement of PPARγ in the regulation of intercellular adhesion molecule (ICAM-1), and myeloperoxidase (MPO), immunocytochemistry on formalin-fixed paraffin-embedded sections was employed to determine those immunoreactivities. Coronal sections through the basilar artery were deparaffinized and rehydrated in graded concentrations of ethanol to distilled water. Endogenous peroxidase activity was blocked with 3% H2O2 for 5 min, followed by a brief rinse in distilled water and a 15 min wash in PBS. Sections were placed in 10 mmol/l citrate buffer (pH 6.0), and heated in microwave oven at 95 °C for 30 min, then cooled at room temperature for 20 min and rinsed in PBS. Non-specific protein binding was blocked by 40 min incubation in 5% horse serum. The basilar artery sections were incubated with primary antibodies (anti-MPO and anti-ICAM-1, prediluted and diluted in 1:100, respectively, both from Abcam, Cambridge, UK) for 1 h at room temperature, followed by a 15 min wash in PBS. Sections were incubated with horseradish peroxidase (HRP)-conjugated IgG (1:500 dilution, Santa Cruz Biotechnology, Inc., California, USA) for 60 min at room temperature. DAB was used as chromogen and counterstaining was done with hematoxylin. Sections incubated in the absence of primary antibody were used as negative controls. For qualitative analysis, immunohistochemical stained sections were analyzed with standard light microscope, The number of ICAM-1 and MPO positive cells in each section was counted in four microscopic fields (at ×400 magnification) and averaged to determine the number of positively immunostained cells per field. 2.6. Harvest of cerebral arteries The rats for protein determination were anesthetized and then killed by decapitation. Afterward, their brains were removed. With the aid of a microscope, the basilar arteries and circle of Willis arteries were carefully dissected free from each brain, cleared of connective tissue, and snap frozen in liquid nitrogen until use. 2.7. Western blot analysis The frozen basilar arteries from each group (n = 6) were mechanically lysed in 20 mM Tris, pH 7.6, which contains 0.2% SDS, 1% Triton X-100, 1% deoxycholate, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.11 IU/ml aprotinin (all purchased from Sigma-Aldrich,
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Inc., St. Luis, MO, USA). Lysates were centrifuged at 12,000 ×g for 20 min at 4 °C. The protein concentration was determined by the Bradford assay (Bio-Rad Lab, Hercules, CA, USA). Equal amounts of protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membrane. The membranes were blocked with 3% (wt/vol) low-fat milk and then incubated overnight at 4 °C with primary antibodies for TLR4 (Bioworld Technology, Inc., Minneapolis, MN, USA), and at the dilution of 1:500. The β-actin (diluted in 1:6000, Sigma-Aldrich, Inc.) was used as a loading control. After the membranes were washed and further incubated with HRP-conjugated secondary antibody at 1:400 for 2 h, the blotted protein bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham, Arlington Heights, IL, USA). Optical densities of the resulting bands were quantified using Glyko Bandscan software (Glyko, Novato, CA, USA).
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3.2. Measurement of basilar artery To exam the vascular changes of basilar arteries, mean diameter and cross-sectional area were measured. Representative images of each group were showed in Fig. 1. Compared with the control group, notable vasospasm was found in the SAH group. Whereas, the severity of the vasospasm was markedly attenuated in the rosiglitazone group (Fig. 1A–C). Besides, the spastic and thicken appearance of vessel wall following SAH was attenuated in the rosiglitazone group (Fig. 1D–F). Quantitative analysis showed that, there was a significant difference in the basilar artery cross-sectional area among groups (P b 0.001, ANOVA). The cross-sectional area of basilar artery were found to be 55,508 ± 4630 μm2 in the control group, 32,139 ± 3239 μm2 in the SAH group, 45,969 ± 4464 μm2 in the rosiglitazone group (Fig. 2A) Similar results were also detected while measuring of mean diameter (Fig. 2B). A significant difference was observed in the mean diameter among groups (Pb 0.001, ANOVA).
2.8. Statistical analysis All results were presented as the mean ± SD. All data were subjected to one-way ANOVA. Fischer's LSD post hoc test was used for multiple comparisons. A probability value of less than 0.05 was considered statistically significant.
3. Results 3.1. General observation and mortality No significant changes in body weight and blood glucose were detected in any of the experimental groups (P N 0.05, ANOVA, data not shown). No animals died in the control group. Within the SAH groups, mortality rate was 20% (6 of 30) in the vehicle treated group, compared to 13.3% (4 of 30) in the rosiglitazone treated group. The animals died in the course of anesthesia or blood injection were excluded from the analysis of mortality.
3.3. Immunocytochemical study for intercellular adhesion molecule and myeloperoxidase Immunochemistry was performed to evaluate the immunoreactivity of ICAM-1 and MPO. The result displayed that both ICAM-1 and MPO were expressed at low levels in the control group (Fig. 3A and D). Whereas, increased ICAM-1-positive and MPO-positive cells in the SAH group could be observed in the basilar artery (Fig. 3B and E). Compared with that, both the degree of vasospasm and the range of positive staining for ICAM-1 and MPO were attenuated in the basilar artery after rosiglitazone treatment (Fig. 3C and F). Moreover, ICAM-1 immunoreactivity was mainly expressed in the intimal and adventitial cells, while MPO immunoreactivity was located in all the intimal, medial, and adventitial cells. The positive staining for ICAM-1 was almost disappeared in intimal cells after rosiglitazone treatment (Fig. 3C), which may indicated the capacity of rosiglitazone to inhibit endothelial inflammation.
Fig. 1. Histological changes of basilar arteries on day 5 after SAH induction. (A–C) Representative images of basilar arteries of the control rats or rats subjected to SAH alone or SAH plus intraperitoneal injection with rosiglitazone (3 mg/kg). Vasospasm could be detected in the SAH group, which was attenuated in the rosiglitazone group. (D–F) Magnified images of the square area from A through C, respectively. The spastic and thicken appearance of vessel wall after SAH, which was attenuated in the rosiglitazone group. Scale bar = 200 and 20 μm, respectively.
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Fig. 2. Histogram of the mean diameter and cross-sectional area of the basilar arteries. (A) Cross-sectional area of basilar arteries from the control, SAH, and rosiglitazone groups. (B) Mean diameter of basilar arteries from three groups. The results indicated that rosiglitazone could attenuate cerebral vasospasm following SAH. Bars represent the mean ± SD (n = 20, each group). **P b 0.01 compared with rats treated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.
Semi-quantitative analysis for the immunohistochemistry showed that administration of rosiglitazone remarkably inhibited SAHinduced upregulation of ICAM-1 and MPO expression in the arterial wall. Significant differences were both found between the control or SAH groups, and the rosiglitazone group (both P b 0.01) (Fig. 3G). 3.4. Western blot analysis for TLR4 Western blot was performed to assess the effect of rosiglitazone on the expression of TLR4 in the basilar artery after SAH. A significant increase in TLR4 expression was observed in the SAH group as compared with that in the control group (P b 0.01). Rosiglitazone treatment was found to reduce TLR4 expression dramatically in the basilar artery following SAH (Pb 0.01), although there was still a difference in the production of TLR4 between rosiglitazone group and control group (Pb 0.05) (Fig. 4). 4. Discussion The present work coincides with our previous findings in VSMCs after OxyHb exposure in vitro. The VSMCs pretreated with PPARgamma agonist rosiglitazone exhibited significant reduction of TLR4 expression and cytokine release in response to OxyHb [18]. In present study, we demonstrated that the activation TLR4 and downstream adhesion molecules could be induced in basilar arteries in the rat SAH model. The PPAR-gamma agonist, rosiglitazone, could significantly suppressed the SAH-induced inflammatory responses by blocking the expression of TLR4 and adhesion molecules. Most importantly,
rosiglitazone could attenuate the SAH-induced cerebral vasospasm. These current data support the involvement of TLR4 in SAH-mediated inflammatory activation in basilar arteries, which may facilitate the progression of cerebral vasospasm after SAH. The study also reveals the potential therapeutic role of PPAR-gamma agonists in SAHinduced cerebral vasospasm by negative regulation of inflammatory responses through TLR4 signalling. Vasospasm remains a leading cause of morbidity and mortality following SAH. It refers to a circumstance that is more complex than simple constriction of vessels. Pathological changes occur in cerebral arteries following SAH that lead to additional lumen narrowing, vessel wall thickening and impaired vascular reactivity [36]. Despite investigating a myriad of compounds, very few have made it to clinical application. So far, Nimodipine, a calcium channel antagonist, is the only drug approved by the US Food and Drug Administration for use in treatment of vasospasm. However, it has a limited improvement on the overall morbidity and mortality [37]. Thus, there is a great need to explore new therapeutic targets for vasospasm. Growing documents indicate that inflammation and immunity after SAH play a crucial role in the development of vasospasm. A complex series of inflammatory constituents including cytokines, adhesion molecules, chemokines and transcription factors have been reported to be implicated in the pathogenesis of cerebral vasospasm [2,5,10]. Moreover, notable therapeutic effects of anti-inflammatory agents for vasospasm have been testified in many animal SAH model, whereas the similar efficacy have not been supported in the clinical trial [38,39]. It is probably because the precise role of the inflammatory responses in the artery accompanying SAH and cerebral vasospasm had not yet been well clarified. TLR4 is increasingly recognized as a critical factor in initiating inflammatory signalling and innate immune responses. It is considered to activate signalling pathways leading to the expression of pro-inflammatory cytokines implicated in the etiology of vascular lesions [40,41]. Recently, upregulated expression of TLR4 was detected in the endothelial cell layer of human cerebral aneurysm walls, and the TLR4 expression coincided well with NF-κB activation. These findings indicated TLR4 may play an important role in cerebral aneurysm formation [42]. Further, in our previous study, the activation of TLR4 signalling was observed both in VSMCs after OxyHb stimuli and in basilar arteries in the rabbit SAH model [18,19]. Likewise, in the present work, elevated expression of TLR4 was detected in the basilar arteries in the rat SAH model. Moreover, we evaluated the degree of leukocyte infiltration in the vessel wall through the assessment of MPO and adhesion molecules immunoreactivity. The results proved that inflammation was markedly triggered in the basilar arteries following SAH. Therefore, it is reasonable for us to believe that TLR4 signalling may tightly correlate with the development of aneurysmal SAH and aggravation of vasospasm. PPAR-gamma, an essential transcriptional mediator of adipogenesis, lipid metabolism, and glucose homeostasis, is increasingly recognized as a key player in the regulation of various type of inflammatory response. These functions are mediated through its ability to regulate energy homeostasis, cell proliferation, membrane lipid composition and transrepress the activities of many transcription factors, including NF-κB, STATs and AP1 [43], which are all known to be involved in inflammation and immune processes. The therapeutic effects of PPAR-gamma agonists have been testified in many different CNS diseases, including cerebral ischemia, intracerebral hemorrhage, Alzheimer's disease, Multiple sclerosis and trauma [23,32,44–46]. Additionally, PPAR-gamma was reported to plays critical roles in vascular system. PPAR-gamma activation inhibits endothelial inflammation and therefore improves endothelial dysfunction. Meanwhile, it suppresses vascular smooth muscle cells proliferation, migration, and vascular remodeling, which is of benefit to atherosclerosis and hypertension [47,48]. PPAR-gamma agonists have been identified as effective inhibitor of TLR4. LPS-induced TLR4 signalling and NF-κB activation in monocytes could be inhibited by PPAR-gamma ligands [14]. Rosiglitazone could
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Fig. 3. Immunohistochemical study for ICAM-1 (A–C) and MPO (D–F) on cross section of basilar arteries. ICAM-1 and MPO were expressed at low levels in the vessel walls in the control group (A and D). Increased positive cells were observed in the arterial wall in the SAH group (B and E). The degree of vasospasm and the range of positive expression were declined in rosiglitazone group (C and F). The arrows indicate positively staining cells in the intima. Scale bar = 20 μm. Semi-quantitative analysis shows that ICAM-1 and MPO positive cells in rosiglitazone groups are significantly fewer than in SAH (G). Bars represent the mean ± SD (n = 5, each group). **P b 0.01 compared with rats treated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.
regulate angiotensin II-induced or OxyHb-induced vascular inflammation through the TLR4-dependent signalling pathway [18,49]. Present data showed that pretreatment with rosiglitazone could significantly reduce SAH-mediate upregulation of TLR4 and increased production of MPO and ICAM-1. We also analyzed the vascular changes of basilar arteries by measuring mean diameter and crosssectional area of vessels. The results revealed that rosiglitazone could effectively attenuate cerebral vasospasm. These findings suggested that rosiglitazone may exert its neuroprotective effect through blocking TLR4 pathway and diminishing downstream inflammatory mediators release in vessels. It therefore may prevent vascular inflammation and attenuate cerebral vasospasm associated with SAH. It has been recognized that TLR4 induced activation of inflammatory cells and the release of proinflammatory molecules are responsible for tissue damage during various CNS diseases. Paradoxically, TLR ligands administered systemically induce a state of tolerance to later ischemic injury [50]. Thus, modulating the activation of TLR4 and holding a precise balance between protective and harmful effects for the host may be a feasible strategy for prevention of neurotoxic damage relate to
inflammation. PPAR-gamma agonist rosiglitazone could modulate TLR4 activation, decrease inflammatory response in the cerebral vessels and attenuate vasoconstriction in experimental SAH model. Furthermore, Our previous study demonstrated that the anti-inflammation effects of rosiglitazone on OxyHb-treated VSMCs were effectively counteracted by the PPARγ antagonist GW9662. Thus the neuroprotective effects of rosiglitazone may at least partly dependent on the PPAR-gamma activation. Overall, we have shown that the TLR4 mediated inflammatory response has occurred in basilar arteries following SAH. Pretreatment with rosiglitazone protects the cerebral vessels against inflammation and attenuates vasospasm by inhibiting TLR4 signalling, which suggests that rosiglitazone may have therapeutic potential for the treatment of cerebral vasospasm. Finally, administration of rosiglitazone did not attenuate vasospasm completely, which coincided with the degree of TLR4 inhibition. It suggested that restraint at multiple levels for TLR4 and bringing the level of TLR4 to get close to those in normal condition may provide more protection from vasospasm.
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Fig. 4. Western blot analysis for TLR4 protein in the basilar arteries. (A) Representative autoradiogram of TLR4 expression in basilar arteries. (B) Quantitative analysis of the Western blot results for TLR4. Results were expressed as the mean ± SD of six experiments done in triplicate (n = 6, each group). *P b 0.05 or **P b 0.01 compared with rats treated under the indicated conditions, by one-way ANOVA with Fischer's LSD post hoc comparison.
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