Toll-Like Receptor 4 Antagonist Attenuates Intracerebral Hemorrhage–Induced Brain Injury
Background and Purpose—Accumulating evidence indicates that inflammatory responses cause secondary injury after intracerebral hemorrhage (ICH). We recently demonstrated the involvement of toll-like receptor 4 (TLR4) signaling in these processes. The purpose of the current study was to investigate the protective effect and mechanism of TAK-242 (Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1 -carboxylate, Takeda), a TLR4 antagonist, in an ICH mouse model.
Methods—TAK-242 was intraperitoneally injected 6 hours after ICH once daily for 5 successive days. We assessed neurological deficit scores; changes in brain water content; and levels of inflammatory factors, DNA damage, and neuronal degeneration in perihematomal region 1, 3, and 5 days after ICH. Peripheral inflammatory cell infiltration was determined using flow cytometry; and the expression of TLR4 downstream signaling molecules was assessed by Western blot.
Results—TAK-242 significantly reduced brain water content, neurological deficit scores, and levels of inflammatory factors. The levels of DNA damage and neuronal degeneration were also significantly decreased, as was peripheral inflammatory cell infiltration. The expression of TLR4 downstream signaling molecules, including myeloid differentiation primary response gene 88, toll/IR-1(TIR)-domain-containing adaptor protein inducing interferon-beta IκBα, nuclear factor-κBp65, and phosphorylated nuclear factor-κBp65, was significantly downregulated.
Conclusions—The results suggest that TLR4 antagonist reduced inflammatory injury and neurological deficits in a mouse model of ICH. The mechanism may involve decreased expression of signaling molecules downstream of TLR4.
- ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1-carboxylate
- intracerebral hemorrhage
- toll-like receptor 4
Intracerebral hemorrhage (ICH) accounts for ≈10% to 15% of all strokes worldwide, and this proportion is even higher in Asia.1,2 Because of the high incidence, fatality rate, and disability rate, ICH represents a major threat to human health currently.1,2 Although considerable progress has been made in our understanding of ICH pathogenesis, we still lack effective treatment options for ICH.3,4
ICH is mainly caused by mechanical injury that results in compression because of the mass effect of hematoma.5 Numerous studies have demonstrated that secondary brain injury after ICH is a leading cause of neurological deficits, including hematoma toxicity, high metabolic injury, excitotoxicity, oxidative stress, and inflammatory injury.6 ICH-induced inflammatory processes play an important role in perpetuating secondary injury after ICH.6,7 Glial cells are activated by hematoma,5,8 and blood–brain barrier disruption and the subsequent infiltration of peripheral inflammatory cells into cerebral tissues,9,10 lead to the production of a large number of inflammatory cytokines that induce brain edema, which ultimately results in neuronal death and secondary neurological deficits.
Toll-like receptors (TLRs) are a class of transmembrane signal transduction molecules that belong to the interleukin receptor superfamily. TLRs activate downstream adaptor signaling molecule myeloid differentiation primary response gene 88 (MyD88) and toll/IR-1(TIR)-domain-containing adaptor protein inducing interferon-beta (TRIF) and nuclear factor κB (NF-κB) through recognition of both exogenous (pathogen-associated molecular patterns) and endogenous ligands (damage-associated molecular pattern molecules), which leads to the production of a large number of inflammatory factors11,12 that play key roles in the innate immune response.13,14 To date, 13 TLRs have been identified. TLR4 is one of the most well-studied receptors, and it has been found that a TLR4-mediated inflammatory reaction is involved in cerebral ischemia-reperfusion injury and Alzheimer disease.15,16 In addition, the TLR4/NF-κB signaling pathway is reported to play a crucial role in ICH-induced neurological deficits.17,18 We previously demonstrated that heme, a metabolite of the primary component of hematoma, potentiates microglial activation via TLR4, which subsequently activates NF-κB via the downstream MyD88/TRIF signaling pathway.19 These events ultimately increase cytokine expression and inflammatory injury in ICH, resulting in brain edema and aggravating neurological deficits.19 In addition, it is reported that TLR4 expressed by peripheral inflammatory cells after ICH plays a critical role in ICH-induced inflammatory damage,17 which further validates the important role of TLR4 signaling in secondary neurological deficits after ICH. These observations indicate that TLR4 inhibition may protect against inflammatory damage at the initial stage of ICH and highlight TLR4 as a novel target for ICH treatment.
Among currently identified TLR4 antagonists,20,21 TAK-242 (Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1 -carboxylate, Takeda) is a specific antagonist of TLR4 that binds to Cys747 in the intracellular domain, thereby inhibiting TLR4 signaling. Because TAK-242 has a low molecular weight and is liposoluble, it exhibits remarkable protection against lipopolysaccharide-induced Gram-negative sepsis and peritonitis.21–23 However, whether TLR4 inhibition alleviates secondary injury after ICH remains unknown.
In the present study, we generated a mouse model of ICH by injection of autologous blood. We performed intraperitoneally injections of TAK-242 and observed whether TAK-242 could cross the blood–brain barrier and assessed brain water content, neurological deficit score (NDS), and inflammatory factor levels. The terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) technique was performed to detect DNA damage, and Fluoro-Jade B staining was used to detect neuronal degeneration in perihematoma tissues. We also measured the infiltration of peripheral blood inflammatory cells into the cerebral tissues and changes in the expression of signaling molecules downstream of TLR4. The results indicated that TLR4 inhibition was protective and significantly reduced neurological deficits. The mechanism may involve decreased expression of signaling molecules downstream of TLR4 and suggest that TAK-242 may be a potential drug for treating ICH.
A total of 186 male C57BL/6 mice (18–22 g) were purchased from the Laboratory Animal Center of the Third Military Medical University (Chongqing, China). All animals were housed in a specific pathogen-free facility with free access to water and food. Experiments were conducted in accordance with the animal care guidelines approved by the Animal Ethics Committee of the Third Military Medical University. Some animals were used in several experiments.
The ICH model was established as described previously.19,24,25 Briefly, mice were anesthetized with an intraperitoneal injection of 400 mg/kg chloral hydrate (σ) and fixed on a mouse stereotaxic frame (Stoelting). A 20-μL volume of autologous nonanticoagulated blood was collected from the tail vein of the mouse and then injected into the caudate nucleus at 2 μL/min under stereotactic guidance at the following coordinates relative to bregma: 0.8 mm anterior, 2 mm left lateral, and 3.5 mm deep during a period of 10 minutes. The needle was held in place for 10 minutes after injection, and the microsyringe was pulled out after the blood had coagulated. The craniotomy was then sealed with bone wax, and the scalp was closed with sutures. Body temperature was maintained at 37°C throughout the procedure, and the mice were given free access to food and water after they woke up. The mice that died because of anesthesia were excluded. The physiological parameters of mice 1 hour before and 6 hours after ICH were measured (Table I in the online-only Data Supplement).
Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1 -carboxylate, Takeda
TAK-24226 was formulated with 1% dimethyl sulfoxide (σ) and double-distilled water to a final concentration of 0.4 mg/mL. TAK-242 was intraperitoneally injected after ICH. For liquid chromatography–mass spectrometry (LC–MS), 3 mg/kg TAK-242 was administered by intraperitoneal injection 6 hours after ICH, and TAK-242 concentrations were determined 0.5, 1, 3, and 5 hours later. When other indexes were detected, TAK-242 was intraperitoneally injected at a dose of 3 mg/kg once daily for 5 successive days beginning 6 hours after ICH. The protocol was based on previous report26; moreover, we did experiments to test the effect of different dose of TAK-242 (1, 3, or 10 mg/kg) at different administration protocols (twice a day, once daily, or administration once every 2 days for 5 successive days) on ICH, we found that 3 mg/kg once daily achieved the maximum inhibitory effect on the mice’s NDS after ICH (data were not shown). Mice were randomly assigned into 1 of 3 groups: sham operation group (sham group), ICH+vehicle group (administration of the same volume of solvent used to formulate the TAK-242 solution), and ICH+TAK-242.
Liquid Chromatography–Mass Spectrometry
Mice were intraperitoneally injected with TAK-242 at a dose of 3 mg/kg 6 hours after ICH and perfused with 0.01 mol/L phosphate-buffered saline 0.5, 1, 3, or 5 hours later (n=3). The cerebral tissues were sampled, and the perihematoma tissue was isolated. TAK-242 levels were measured with LC–MS (Agilent Techonologies 6410B Triple Quad LC/MS). The first time point was selected on the basis of time needed for TAK-242 to distribute throughout the entire body (15 minutes).26
Neurological deficits were assessed 1, 3, and 5 days (n=9) after TAK-242 administration using a 28-point neurological deficit scale, including circling behavior, climbing, front limb symmetry, and body symmetry. Detailed procedures were performed as previously described.27,28 Scoring was performed by 2 trained investigators who were blind to animal grouping, and the mean score of the subscales was the final score of each mouse.
Brain Water Content Measurement
Brain water content was measured in mouse cerebral tissues after ICH.29 Briefly, mice were randomly sampled from each group and anesthetized by intraperitoneal injection with chloral hydrate (n=5). Next, the cerebral tissues were removed, and the surface water on the cerebral tissues was blotted with filter paper. The brains were divided into 5 parts (ipsilateral and contralateral cortex, ipsilateral and contralateral basal ganglia, and cerebellum). Brain samples were immediately weighed on an electric analytic balance to obtain the wet weight and then dried at 100°C for 24 hours to obtain the dry weight. Brain water content was calculated using the following formula: brain water content (%)=(wet weight−dry weight)/wet weight×100%.
Enzyme-Linked Immunosorbent Assay
Perihematoma tissues collected from each group were homogenized and centrifuged, and the supernatant was collected for analysis (n=6). The concentrations of inflammatory factors, including tumor necrosis factor-α, interleukin-1β, and interleukin-6, were measured using an ELISA reagent kit (Dakewe Biotech Company) following the manufacturer’s instructions.
TUNEL and Fluoro-Jade B Staining
To further assess the protective effect of TAK-242 on ICH-induced injury in mice, we assessed DNA damage and neuronal degeneration in cerebral tissue collected from the perihematomal region (n=9). After the mice were perfused and fixed, the cerebral tissues were collected, fixed in 4% paraformaldehyde (σ,) for 24 hours, dehydrated in 30% sucrose solution for 48 hours, embedded, frozen, and cut into 25-μm sections using a Leica CM1900 cryostat (Mannheim). TUNEL-positive cells were detected using the In situ Cell Death Detection Kit (Roche Molecular Biochemicals), and neuronal degeneration was detected using a Fluoro-Jade B staining reagent kit (Millipore) following the manufacturers’ instructions. Sections were observed and photographed under a fluorescence microscope (Olympus BX-60). Four quadrants were selected from each section, the number of positive cells was observed in each quadrant, and the mean value was calculated.
Peripheral blood inflammatory cell infiltration of the perihematomal region were detected using a flow cytometer as previously described.17 Briefly, cerebral tissues were collected, lightly homogenized, digested with pancreatin, and prepared into a single-cell suspension using GentleMACS dissociator and Neural tissue Dissociation Kit(P) (Miltenyi; n=5). The suspension was blocked in blocking buffer, followed by successive addition of CD45.2-APC (1:40), CD11b-PerCp Cy5.5 (1:80), CD11c-PECy7 (1:80), CD19-FITC (1:50), CD3-FITC (1:100), NK1.1-FITC (1:100), Gr1-PE (1:600; all from eBioscience), and Ly6G-v450 (1:500; BD Biosciences) and then incubated at 4°C in the dark for 30 minutes. BD FACSVerse flow cytometer (BD Biosciences) was used for detection, and the counts of each cell type were calculated using BD FACSuite software (BD Biosciences). The tissues sampled at different time points were measured at least in triplicate at various time points. The parameters for various inflammatory cells were as follows: leukocytes (CD45hi), neutrophils (CD45hiCD3−CD19−NK1.1−CD11b+Ly6G+), monocytes (CD45hiCD3−CD19−NK1.1−CD11b+Ly6G+CD11c−GR1−), inflammatory monocytes (CD45hiCD3−CD19−NK1.1−CD11b+Ly6G+CD11c−GR1+), and dendritic cells (CD45hiCD3−CD19−NK1.1−CD11b+Ly6G−CD11c+).
Western Blotting Analysis
Western blotting analysis was performed as previously described.19 Briefly, the mice were perfused with 0.01 mol/L phosphate-buffered saline 1, 3, or 5 days after ICH, and the cerebral tissues from the perihematomal region were isolated (n=5). Changes in the expression of the total and cleaved caspase 3 and the signaling molecules downstream of TLR4, including MyD88, TRIF, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), IκBα, NF-κBp65, and phosphorylated NF-κB p65 (phospho-NF-κB p65), were determined. The perihematoma tissues were lysed in 1-mL radioimmunoprecipitation assay lysis buffer, and then the protein was extracted, electrophoresed, and transferred onto polyvinylidene fluoride membranes (Amersham Pharmacia). The polyvinylidene fluoride membranes were incubated with primary antibodies (Table II in the online-only Data Supplement) overnight, followed by incubation with peroxidase-conjugated secondary antibodies (Table II in the online-only Data Supplement) for 6 hours. The same membranes were probed with an antibody for GAPDH. Protein signals were detected with an enhanced chemiluminescence system. The signals were quantified by scanning densitometry and computer-assisted image analysis. Protein levels were expressed as the ratio of the values of the detected protein band to the GAPDH band.
All data are expressed as mean± SD. Statistical analyses were performed using the statistical software SPSS version 13.0 (SPSS Inc). Differences among multiple groups were compared using Kruskal–Wallis H tests or 1-way ANOVA with post hoc Bonferroni test for normally distributed data. A P value <0.05 was considered statistically significant.
TAK-242 Crosses the Mice Blood–Brain Barrier
We first determined whether intraperitoneally administered TAK-242 could cross the blood–brain barrier. The TAK-242 level in cerebral tissues from the perihematomal region was determined using LC–MS, and the TAK-242 level was estimated by calculating the area under the peak. The concentrations of TAK-242 in cerebral tissues were 2.5280, 1.979, 1.2837, and 0.9363 ng/mL at 0.5, 1, 3, and 5 hours, respectively (Figure 1A–1D). These results demonstrate that systematically administered TAK-242 can cross the blood–brain barrier of the mouse after ICH.
TAK-242 Significantly Reduces NDS and Brain Edema
After ICH, brain edema and NDS are important indexes for assessing ICH severity. Here, we observed NDS improvements in mice after TAK-242 administration. The results showed that the NDS of mice in the ICH+vehicle group were significantly increased in comparison with the sham group, and the highest NDS was observed 3 days after TAK-242 administration. The NDS of mice in the ICH+TAK-242 group was significantly reduced compared with the ICH+vehicle group (12.22±1.39 versus 15.22±0.97; P<0.001) 3 days after the administration, whereas the NDS of mice in the ICH+TAK-242 group were also significantly lower than that in the ICH+vehicle group both 1 day (9.44±1.13 versus 13.22±1.09; P<0.001) and 5 days (6.22±0.97 versus 7.89±1.05; P<0.001) after treatment. These results demonstrate that TAK-242 significantly reduce the NDS of mice with ICH (Figure 2A).
The most severe neurological deficits were observed in the ICH+vehicle group at 3 days. Therefore, we used the dry-wet weight method to determine the effect of TAK-242 on brain edema 3 days after ICH. We found that brain water content in the ipsilateral cortex and basal ganglia of mice on day 3 after intraperitoneal injection of TAK-242 was significantly reduced compared with the ICH+vehicle group (80.62±0.71 versus 81.64±0.62; P=0.006, cortex and 79.78±0.69 versus 81.22±0.48; P=0.019, basal ganglia; Figure 2B). These findings suggest that TAK-242 significantly reduced the formation of brain edema in mice with ICH.
TAK-242 Significantly Reduced Inflammatory Factor Levels
We determined changes in expression levels of inflammatory factors, including tumor necrosis factor-α, interleukin-1β, and interleukin-6, in the perihematoma tissue of mice after ICH using the ELISAs. The results showed that the levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6 were significantly increased in the ICH+vehicle group compared with the sham group 1, 3, and 5 days (Figure 2C) after ICH. Again, the increase was greatest 3 days after ICH. Compared with mice in the ICH+vehicle group, the levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6 were significantly reduced in the ICH+TAK-242 group 1, 3, and 5 days (Figure 2C). These results indicate that TAK-242 significantly reduces the production of inflammatory factors in perihematoma tissue in a mouse model of ICH.
TAK-242 Can Significantly Reduce DNA Damage and Neuronal Degeneration in Perihematoma Tissues
Western blotting was used to assess changes in total and cleaved caspase 3 expression in perihematoma tissue (collection of cerebral tissues from the perihematomal region is shown in Figure 3A). The results show that TAK-242 significantly reduced cleaved caspase 3 expression compared with the ICH+vehicle group (Figure 3B), suggesting that TAK-242 successfully inhibited cell apoptosis after ICH. To further validate this observation, a TUNEL assay was performed to detect DNA damage in the perihematoma tissues of mice. The results showed that the level of DNA damage was significantly decreased in the ICH+TAK-242 group compared with the ICH+vehicle group 1, 3, and 5 days after ICH. The most notable reduction was observed 1 day after ICH (14.33±4.06 versus 42.78±4.14; P<0.001; Figure 3C). Fluoro-Jade B staining was also used to assess neuronal degeneration in perihematoma tissue after ICH, and the results were in agreement with those of the TUNEL assay (Figure 3D). Collectively, these experiments indicate that TAK-242 significantly inhibits neuronal injury after ICH.
TAK-242 Significantly Reduces Inflammatory Cell Infiltration in Perihematoma Tissues
After ICH, the inflammatory response caused by the infiltration of peripheral inflammatory cells is an important factor underlying secondary neurological deficits. We determined changes in the counts of peripheral inflammatory cells, including neutrophils, inflammatory monocytes, monocytes, and dendritic cells, in perihematoma tissue using flow cytometry. The counts of all 4 cell types were significantly increased in the ICH+vehicle group compared with those in the sham group 1, 3, and 5 days after ICH. Compared with the ICH+vehicle group, the counts of the infiltrated inflammatory cells in the perihematoma tissue were significantly reduced after TAK-242 administration (Figure 4A–4D; **P<0.01), and the most notable reduction was observed 3 days after ICH (Figure 4C; **P<0.01).
TAK-242 Significantly Downregulates the Expression of Signaling Molecules Downstream of TLR4
To investigate TAK-242’s mechanism of action further, Western blotting analysis was performed to examine the expression of signaling molecules downstream of TLR4, including MyD88, TRIF, IKKβ, IκBα, NF-κBp65, and phospho-NF-κB p65. The results showed that the expression levels of MyD88 and TRIF were significantly upregulated in the perihematoma tissues of mice in the ICH+vehicle group compared with the sham group 1, 3, and 5 days after ICH (Figure 5A and 5B); the expression levels of IKKβ, IκBα, NF-κBp65, and phospho-NF-κB p65 also increased (Figure 6A–6D). Compared with the ICH+vehicle group, the expression levels of MyD88 and TRIF were significantly lower in the perihematoma tissues of mice in the ICH+TAK-242 group 1, 3, and 5 days after ICH (Figure 5A and 5B), whereas IKKβ, IκBα, NF-κBp65, and phospho-NF-κB p65 were also downregulated (Figure 6A–6D; *P<0.05, **P<0.01).
Inflammatory processes after ICH play a critical role in the secondary damage associated with ICH, which is currently considered one of the major mechanisms of injury.6,7 After ICH, glial cells are activated,5,8 and blood–brain barrier disruption and peripheral blood inflammatory cell infiltration into the cerebral tissues,9,10 lead to the production of a large number of inflammatory cytokines and induce brain edema and neuronal injury. Therefore, inhibiting the initial upstream event that triggers subsequent inflammatory response can significantly alleviate ICH-induced injury and reduce associated neurological deficits. Our results demonstrate that TAK-242, a TLR4 antagonist, significantly reduces brain water content, the production of inflammatory factors, and peripheral blood inflammatory cell infiltration; inhibits DNA damage and neuronal degeneration after ICH; and reduces NDS in mice with ICH. With the reduction of inflammatory events, the neurological recovery in mice is modest at best. This indicated that inflammation injury plays an important role in neurological deficit after ICH, meanwhile, TAK-242 improved the neurological function through reducing the inflammatory injury. In addition, we observed a significant downregulation in the expression levels of MyD88 and TRIF, the downstream signaling molecules of TLR4, thereby decreasing the activation of signaling molecules, such as NF-κB, and reducing the production of inflammatory factors. These results indicate that TLR4 inhibition was cerebral protective in mice with ICH through its effects on 2 TLR4 downstream signaling pathways, MyD88 and TRIF. To our knowledge, this study is the first to demonstrate the neuroprotective effects of TLR4 inhibition after ICH and suggests that preventing the inflammatory response elicited by ICH may be a novel approach for treating ICH-associated inflammatory injury.
One widely used mouse model of ICH is generated by injecting autologous blood or collagenase, but this has advantages and disadvantages. The present study did not use collagenase because that the collagenase itself can enable the cerebral tissues to produce inflammatory responses, thereby interfering with the mechanism of inflammation associated with ICH and results from intervention studies.30 Therefore, we injected autologous blood to mimic ICH.
Many TLR4 antagonists have been identified, and both TAK-242 and eritoran tetrasodium (E5564) have been extensively studied.20,22 A phase III clinical trial has been conducted to investigate the efficacy of E5564 in the prevention and treatment of sepsis. The present study used TAK-242 for the following reasons. (1) E5564 binds to the TLR4/MD-2 complex, thereby inhibiting TLR4 activation, whereas TAK-242 binds to Cys747 in the intracellular domain of TLR4, thereby inhibiting the protein’s functionality.20,31 (2) E5564 has a large molecular weight and poor liposolubility, which make it difficult for the compound to cross the blood–brain barrier, whereas TAK-242 has a low molecular weight (360.1) and high liposolubility. In the current study, LC–MS revealed that TAK-242 could be detected in the cerebral tissues 0.5 hour after intraperitoneal injection in ICH mice, indicating that TAK-242 is able to access the brain. However, this may be associated with ICH-associated opening of the blood–brain barrier. In this study, LC–MS revealed low levels of TAK-242, which may be because of the fact that we only measured TAK-242 in perihematoma tissues.
Our recent study showed that heme, the primary component of perihematoma tissue after ICH, plays a critical role in the secondary neurological deficits to ICH by inducing inflammatory injury via TLR4 on microglial cells.19 In addition, TLR4 expressed on peripheral inflammatory cells is also thought to be involved in inflammatory injury after ICH.31 The present study demonstrates that TLR4 inhibition by TAK-242 significantly reduced the inflammatory response in perihematoma tissues and markedly reduced the infiltration of the primary peripheral inflammatory cells, suggesting that TAK-242 may reduce neurological deficits by inhibiting the intracellular and peripheral inflammatory responses after ICH.
We previously showed that TLR4 induces NF-κB activation via MyD88 and TRIF signaling pathways, and these events mediated inflammatory injury in ICH.19 Here, we demonstrated that TAK-242 significantly reduces MyD88 and TRIF expression in perihematoma tissue and inhibits NF-κB activation, thereby decreasing the expression of inflammatory factors and reducing neuronal apoptosis and necrosis, which ultimately improved NDS in mice with ICH. Therefore, TAK-242 protects ICH through the mechanism that TAK-242 downregulates the expression of TLR4 downstream signaling molecules and reduces the NF-κB activation, thereby alleviating the neurological deficits after ICH.
In conclusion, our findings reveal that treatment with TAK-242 6 hours after ICH successfully inhibits TLR4 signal transduction and can effectively alleviate neurological deficits in a mouse model of ICH. They provide evidence that TLR4 inhibition exhibits a remarkable cerebral protective effect after ICH and suggest that TAK-242 may be a promising drug candidate for ICH.
Sources of Funding
This work was supported by National Natural Science Foundation of China (No. 81070932, 81271283).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.001038/-/DC1.
- Received February 2, 2013.
- Revision received April 29, 2013.
- Accepted May 20, 2013.
- © 2013 American Heart Association, Inc.
- Sudlow CL,
- Warlow CP
- Mendelow AD,
- Gregson BA,
- Fernandes HM,
- Murray GD,
- Teasdale GM,
- Hope DT,
- et al
- Qureshi AI,
- Suri MF,
- Nasar A,
- Kirmani JF,
- Ezzeddine MA,
- Divani AA,
- et al
- Aronowski J,
- Zhao X
- Del Bigio MR,
- Yan HJ,
- Buist R,
- Peeling J
- Tahara K,
- Kim HD,
- Jin JJ,
- Maxwell JA,
- Li L,
- Fukuchi K
- Ii M,
- Matsunaga N,
- Hazeki K,
- Nakamura K,
- Takashima K,
- Seya T,
- et al
- Zhou Y,
- Xiong KL,
- Lin S,
- Zhong Q,
- Lu FL,
- Liang H,
- et al
- Xue M,
- Hollenberg MD,
- Demchuk A,
- Yong VW
- Clark W,
- Gunion-Rinker L,
- Lessov N,
- Hazel K