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Background: Apelin-12 has been proposed as a novel adipocytokine with vital roles in metabolic disorders. In this study, we aimed to explore the relationship between serum apelin-12 and metabolic inflammatory syndrome (MIS) in the elderly. Methods: The cross-sectional study involved 224 subjects aged over 60. According to their diagnoses of the four components of MIS (type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis, and obesity/overweight), they were divided into two groups: the control group (0 or 1 component n=63) and the MIS group (≥2 components, n=161). Serum levels of apelin-12, adiponectin and C-reactive protein were measured by ELISA. Results: Serum apelin-12 was significantly higher in the MIS group than in the control group (1.83 ng/mL (1.59-2.07) vs 1.66 ng/mL (1.45-1.92), P=0.001). Partial correlation analysis showed that apelin-12 levels were positively correlated with fasting insulin (ra=0.169), HOMA-IR (ra=0.183), ALT (ra=0.180), AST (ra=0.145) and CRP (ra=0.140, all Para=-0.211) and HDL-C (ra=-0.156) after controlling age, sex and body mass index. Besides, after adjustment for age, sex, fasting blood glucose, diastolic blood pressure and total triglyceride, apelin-12 was still determined as an independent predictor for MIS. Conclusion: Serum apelin-12 can be an independent risk factor predicting metabolic inflammatory syndrome and may serve as a useful potential marker for the diagnosis of MIS in elderly Chinese.

Although taurine is known to exert an antihypertensive effect, it is unclear whether it is involved in the mechanism for hypertension-related target organ injury. To reveal the role of endogenous taurine in renal injury formation during salt-sensitive hypertension and clarify its mechanisms, both salt-sensitive Dahl rats and salt-resistant SS-13BN rats were fed a high-salt diet (8% NaCl) and given 2% taurine for 6 weeks. Rat systolic blood pressure (SBP) was measured by the tail-cuff method and artery catheterization. Kidney ultrastructure was observed under an electron microscope. Taurine content and mRNA and protein levels of taurine synthases, cysteine dioxygenase type 1 (CDO1) and cysteine sulfinic acid decarboxylase (CSAD), were decreased in Dahl rats fed a high-salt diet. However, taurine supplementation and the resulting increase in renal taurine content reduced the increased SBP and improved renal function and structural damage in high-salt diet-fed Dahl rats. In contrast, taurine did not affect SS-13BN SBP and renal function and structure. Taurine intervention increased the renal H2S content and enhanced cystathionine-β-synthase (CBS) expression and activity in Dahl rats fed a high-salt diet. Taurine reduced the renin, angiotensin II, and aldosterone contents and the levels of oxidative stress indices in Dahl rat renal tissues but increased antioxidant capacity, antioxidant enzyme activity, and protein expression. However, taurine failed to achieve this effect in the renal tissue of SS-13BN rats fed a high-salt diet. Pretreatment with the CBS inhibitor HA or renal CBS knockdown inhibited H2S generation and subsequently blocked the effect of taurine on renin, superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2) levels in high-salt-stimulated Dahl renal slices. In conclusion, the downregulation of endogenous taurine production resulted in a decrease in the renal CBS/H2S pathway. This decrease subsequently promoted renin-angiotensin-aldosterone system (RAAS) activation and oxidative stress in the kidney, ultimately contributing to renal injury in salt-sensitive Dahl rats.

Purpose: Apelin-12 has been proposed as a novel adipocytokine with vital roles in metabolic disorders. In this study, we aimed to explore the relationship between serum apelin-12 and metabolic inflammatory syndrome (MIS) in the elderly.Methods: The cross-sectional study involved 224 subjects aged over 60. According to their diagnoses of the four components of MIS (type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis, and obesity/overweight), they were divided into two groups: the control group (0 or 1 component n=63) and the MIS group (≥2 components, n=161). Serum levels of apelin-12, adiponectin and C-reactive protein were measured by ELISA.Results: Serum apelin-12 was significantly higher in the MIS group than in the control group (1.83 ng/mL (1.59-2.07) vs 1.66 ng/mL (1.45-1.92), P=0.001). Partial correlation analysis showed that apelin-12 levels were positively correlated with fasting insulin (ra=0.169), HOMA-IR (ra=0.183), ALT (ra=0.180), AST (ra=0.145) and CRP (ra=0.140, all Para=-0.211) and HDL-C (ra=-0.156) after controlling age, sex and body mass index. Besides, after adjustment for age, sex, fasting blood glucose, diastolic blood pressure and total triglyceride, apelin-12 was still determined as an independent predictor for MIS.Conclusion: Serum apelin-12 can be an independent risk factor predicting metabolic inflammatory syndrome and may serve as a useful potential marker for the diagnosis of MIS in elderly Chinese.

Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

Qingsong Wang,1 Xiu-ling Zhou,2 Guang-hai Mei,3 Jiantao Wang,1 Zhongyu Yu,1 Yue Hu,1 Xinyu Guo,1 Hanyu Zhang,1 Xiaodie Xu,4 Chengfeng He,4 Jingchun Guo,4 Yinghong Tao,5 Houguang Zhou1 1Department of Geriatrics, Huashan Hospital, National Clinical Research Center for Aging and Medicine, Fudan University, Shanghai, 200040, People’s Republic of China; 2Department of Ultrasonics, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China; 3Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China; 4National Key Laboratory of Medical Neurobiology, Institute of Brain Science, Fudan University, Shanghai, 200030, People’s Republic of China; 5Department of Medical Examination Center, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of ChinaCorrespondence: Houguang ZhouDepartment of Geriatrics of Huashan Hospital, National Clinical Research Center for Aging and Medicine, Fudan University, No. 24 Ward, Building 6, No.12 Middle WuLuMuQi Road, JingAn District, Shanghai, 200040, People’s Republic of ChinaTel +86-21-52887246Email zhouhouguang@huashan.org.cnYinghong TaoDepartment of Medical Examination Center of Huashan Hospital, Fudan University, Building 4, No. 12 Middle WuLuMuQi Road, JingAn District, Shanghai, 200040, People’s Republic of ChinaTel +86-21-52889087Email taoyinghong@huashan.org.cnAim: Metabolic inflammation syndrome (MIS) can lead to a series of complications, but its exact inflammatory mechanism is still unclear. The aim of this study was to explore the correlation between heparanase (HPA) and MIS, and the close relationship between HPA and other chronic low-grade inflammation index, such as C-reactive protein (CRP) and interleukin-6 (IL-6).Methods: A total of 105 patients with MIS in the physical examination population of Huashan Hospital affiliated to Fudan University from May to June 2018 were selected as the MIS group, and 52 patients who were relatively healthy during the same period were used as the control group. The basic clinical data of the selected candidates were collected, the levels of serum HPA, CRP and IL-6 were measured by ELISA, and the levels of blood glucose and blood lipids were also detected.Results: Compared with the control group, the levels of HPA, CRP, IL-6, FBG, HbA1C, and TG of MIS group were all significantly elevated (all P< 0.05), and HDL-C levels were considerably reduced (P< 0.05). Correlation analysis showed that there was a noticeably positive correlation between serum HPA level and CRP, IL-6 levels (P< 0.05).Conclusion: Higher HPA levels might play a certain role in the occurrence and development of MIS. There was a certain close correlation between serum HPA level and CRP and IL-6 levels, and which indicated that HPA was involved in the chronic low-grade inflammatory reaction process of MIS.Keywords: metabolic inflammatory syndrome, MIS, heparanase, HPA, C-reactive protein, CRP, interleukin-6, IL-6, chronic low-grade inflammation, CLGI

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases among the elderly people. The T2DM increases the risk of cardio-cerebrovascular disease (CCD), and the main pathological change of the CCD is atherosclerosis (AS). Meanwhile, the carbonic anhydrases (CAs) are involved in the formation and progression of plaques in AS. However, the exact physiological mechanism of carbonic anhydrase III (CAIII) has not been clear yet, and there are also no correlation study between CAIII protein and T2DM with CCD. The 8-week old diabetic mice (db/db−/− mice) and wild-type mice (wt mice) were feed by a normal diet till 32 weeks, and detected the carotid artery vascular opening angle using the method of biomechanics; The changes of cerebral cortex and myocardium were watched by the ultrastructure, and the autophagy were observed by electron microscope; The tissue structure, inflammation and cell injury were observed by Hematoxylin and eosin (HE) staining; The apoptosis of cells were observed by TUNEL staining; The protein levels of CAIII, IL-17, p53 were detected by immunohistochemical and Western Blot, and the Beclin-1, LC3, NF-κB were detected by Western Blot. All statistical analysis is performed using PRISM software. Compared with wt mice, db/db−/− mice’ carotid artery open angle increased significantly. Electron microscope results indicated that autophagy in db/db−/− mice cerebral cortex and heart tissue decreased and intracellular organelle ultrastructure were damaged. HE staining indicated that, db/db−/− mice’ cerebral cortex and heart tissue stained lighter, inflammatory cells infiltration, cell edema were obvious, myocardial fibers were disorder, and myocardial cells showed different degrees of degeneration. Compared with wt mice, TUNEL staining showed that there was obviously increase in db/db−/− mice cortex and heart tissue cell apoptosis. The results of immunohistochemistry and Western Blot indicated that CAIII, Beclin-1 and LC3II/I expression levels conspicuously decreased in cortex and heart tissue of db/db−/− mice, and the expression level of IL-17, NF-κB and p53 obviously increased. The carotid artery’ vascular stiffness was increased and which was probably related with formation of AS in diabetic mice. And the autophagy participated in the occurrence and development of diabetic CCD. CAIII protein might somehow be involved in the regulation of autophagy probably through affecting cell apoptosis and inflammation, but the underlying mechanism remains to be further studied.

Hypoxic pulmonary vascular remodelling (PVR) is the major pathological basis of aging-related chronic obstructive pulmonary disease and obstructive sleep apnea syndrome. The pulmonary artery endothelial cell (PAEC) inflammation, and pulmonary artery smooth muscle cell (PASMC) proliferation, hypertrophy and collagen remodelling are the important pathophysiological components of PVR. Endogenous sulfur dioxide (SO2) was found to be a novel gasotransmitter in the cardiovascular system with its unique biological properties. The study was aimed to investigate the role of endothelial cell- (EC-) derived SO2 in the progression of PAEC inflammation, PASMC proliferation, hypertrophy and collagen remodelling in PVR and the possible mechanisms. EC-specific aspartic aminotransferase 1 transgenic (EC-AAT1-Tg) mice were constructed in vivo. Pulmonary hypertension was induced by hypoxia. Right heart catheterization and echocardiography were used to detect mouse hemodynamic changes. Pathologic analysis was performed in the pulmonary arteries. High-performance liquid chromatography was employed to detect the SO2 content. Human PAECs (HPAECs) with lentiviruses containing AAT1 cDNA or shRNA and cocultured human PASMCs (HPASMCs) were applied in vitro. SO2 probe and enzyme-linked immunosorbent assay were used to detect the SO2 content and determine p50 activity, respectively. Hypoxia caused a significant reduction in SO2 content in the mouse lung and HPAECs and increases in right ventricular systolic pressure, pulmonary artery wall thickness, muscularization, and the expression of PAEC ICAM-1 and MCP-1 and of PASMC Ki-67, collagen I, and α-SMA ( p < 0.05 ). However, EC-AAT1-Tg with sufficient SO2 content prevented the above increases induced by hypoxia ( p < 0.05 ). Mechanistically, EC-derived SO2 deficiency promoted HPAEC ICAM-1 and MCP-1 and the cocultured HPASMC Ki-67 and collagen I expression, which was abolished by andrographolide, an inhibitor of p50 ( p < 0.05 ). Meanwhile, EC-derived SO2 deficiency increased the expression of cocultured HPASMC α-SMA ( p < 0.05 ). Taken together, these findings revealed that EC-derived SO2 inhibited p50 activation to control PAEC inflammation in an autocrine manner and PASMC proliferation, hypertrophy, and collagen synthesis in a paracrine manner, thereby inhibiting hypoxic PVR.

Background The study was designed to explore the significance of endogenous H 2 S in the development of high-salt-induced hypertension in rats. Methods High-salt-induced hypertension rat model was made by feeding Dahl rat high-salt diet containing 8% NaCl for 8 weeks with SD rats as control. SBP and aorta structure in rats were observed. Endogenous H 2 S content and expression of cystathionine β-lyase (CBS), cystathionine γ-lyase and mercaptopyruvate sulfurtransferase in renal tissues were detected. Mechanisms for the impact of high-salt on CBS/H 2 S in renal tissues were studied, targeting HIF-1α pathway. The effect of H 2 S on RAS in serum and renal tissue of rats were tested. Results High-salt reduced endogenous H 2 S content and inhibited the expression of CBS in renal tissue in salt-sensitive Dahl rats. H 2 S donor, however, inhibited salt-sensitive hypertension, reversed aortic structural remodeling and inhibited activation of the RAS system in renal tissues in Dahl rats. Expression of HIF-1α was decreased but expression of PHD2 was increased in renal tissue of Dahl rats with high-salt diet, whereas they did not alter in renal tissue of SD rats with high-salt diet. Ex vivo experiment showed that inhibitor of HIF-1α degradation could rescue down-regulated CBS/H 2 S pathway in renal tissue of Dahl rats with high-salt. In contrast, inhibitor of HIF-1α activity decreased the CBS/H 2 S pathway in the renal tissue of SD rats treated with high-salt. Conclusions Down-regulated CBS/H 2 S pathway in renal tissues under high-salt insult might be an important pathogenesis of salt-sensitive hypertension.

Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury.

Aims Although cognitive dysfunction is a common neurological complication in elderly patients with diabetes, the mechanisms underlying this relationship remain unclear, and effective preventive interventions have yet to be developed. Thus, this study investigated the preventive effects and mechanisms of action associated with granulocyte colony-stimulating factor (G-CSF) on cognitive dysfunction in elderly diabetic mice with cerebral small vessel disease. Methods This study included 40 male db/db diabetic and wild-type (WT) mice that were categorized into the following four groups at the age of 3 weeks: db/db group (DG), db/db+G-CSF group (DGG), WT group (WG), and WT+G-CSF group (WGG). The mice were fed normal diets for 4 months and then given G-CSF (75 μg/kg) via intraperitoneal injections for 1 month. At 7.5 months of age, the cognitive abilities of the mice were assessed with the Y-maze test and the Social Choice Test; body weight, blood pressure (BP), and blood glucose measurements were obtained throughout the study. Brain imaging and blood oxygen level-dependent (BOLD) contrast imaging analyses were performed with a small animal magnetic resonance imaging (MRI) system, autophagosome levels were detected with a transmission electron microscope (TEM), hippocampal neurons were assessed with hematoxylin and eosin (HE) staining, and protein expressions and distributions were evaluated using immunohistochemistry and Western blot analyses. Results (i) The body weight and blood glucose levels of the DG and DGG mice were significantly higher than those of the WG and WGG mice; (ii) social choice and spatial memory capabilities were significantly reduced in DG mice but were recovered by G-CSF in DGG mice; (iii) the MRI scans revealed multiple lacunar lesions and apparent hippocampal atrophy in the brains of DG mice, but G-CSF reduced the number of lacunar lesions and ameliorated hippocampal atrophy; (iv) the MRI-BOLD scans showed a downward trend in whole-brain activity and reductions in the connectivities of the hippocampus and amygdala with subcortical structures in DG mice, but G-CSF clearly improved the altered brain activity as well as the connectivity of the hippocampus in DGG mice; (v) HE staining revealed fewer neurons in the hippocampus in DG mice; (vi) TEM analyses revealed significantly fewer autophagosomes in the hippocampi of DG mice, but G-CSF did not increase these numbers; (vii) there were significant reductions in mechanistic target of rapamycin (mTOR) and LC3-phosphatidylethanolamine conjugate (LC3)-II/I levels in the hippocampi of DG mice, whereas p62 was upregulated, and G-CSF significantly enhanced the levels of Beclin1, mTOR, and LC-II/I in DGG mice; and (viii) G-CSF significantly reversed increases in nuclear factor κB (NF-κB) protein levels in DG but not in WG mice. Conclusions In this study, aged diabetic mice were prone to cognitive dysfunction and cerebral small vessel disease. However, administration of G-CSF significantly improved cognitive function in elderly db/db diabetic mice, and this change was likely related to the regulation of autophagy and NF-κB signaling pathways.

Granulocyte-colony stimulating factor (G-CSF) has been shown to play a neuroprotective role in ischemic stroke by mobilizing bone marrow (BM)-derived endothelial progenitor cells (EPCs), promoting angiogenesis, and inhibiting apoptosis. Impairments in mobilization and function of the BM-derived EPCs have previously been reported in animal and human studies of diabetes where there is both reduction in the levels of the BM-derived EPCs and its ability to promote angiogenesis. This is hypothesized to account for the pathogenesis of diabetic vascular complications such as stroke. Here, we sought to investigate the effects of G-CSF on diabetes-associated cerebral vascular defect. We observed that pretreatment of the cultured human brain vascular endothelial cells (HBVECs) with G-CSF largely prevented cell death induced by the combination stimulus with high glucose, free fatty acids (FFA) and hypoxia by increasing cell viability, decreasing apoptosis and caspase-3 activity. Cell ultrastructure measured by transmission electron microscope (TEM) revealed that G-CSF treatment nicely reduced combination stimulus-induced cell apoptosis. The results from fluorescent probe Fluo-3/AM showed that G-CSF greatly suppressed the levels of intracellular calcium ions under combination stimulus. We also found that G-CSF enhanced the expression of cell cycle proteins such as human cell division cycle protein 14A (hCdc14A), cyclinB and cyclinE, inhibited p53 activity, and facilitated cell cycle progression following combination stimulus. In addition, activation of extracellular signal-regulated kinase1/2 (ERK1/2) and Akt, and deactivation of c-Jun N terminal kinase (JNK) and p38 were proved to be required for the pro-survival effects of G-CSF on HBVECs exposed to combination stimulus. Overall, G-CSF is capable of alleviating HBVECs injury triggered by the combination administration with high glucose, FFA and hypoxia involving the mitogen-activated protein kinases (MAPK) and Akt signaling cascades. G-CSF may represent a promising therapeutic agent for diabetic stroke.

Summary Aims The damage of human brain vascular endothelial cells (HBVECs) is the key pathogenesis of diabetes-associated cerebral vascular complications. The aim of this study was to elucidate the effects of glutathione (GSH) on free fatty acids (FFAs)-induced HBVECs apoptosis, oxidative stress, and the involved possible signaling pathway. Methods After culturing HBVECs for 72 h with GSH and FFAs, we determined cell proliferation by CCK8, detected apoptosis by caspase-3 and Annexin V-FITC/PI staining, and judged oxygen stress by determining the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP). We investigated whether the Akt pathway was involved in FFAs-induced signaling pathway alteration and whether GSH influenced the above effects. Results After being cultured in 200 μM FFAs for 72 h, the HBVECs proliferation significantly decreased; HBVECs apoptosis increased; the ROS levels increased; and the HBVECs MMP subsequently decreased. FFAs induced a significant decrease in phosphorylated active Akt. These alterations were obviously prevented when 1 mM GSH was added to culture medium containing FFAs, and the above effects of GSH were blocked by Akt inhibitor. Conclusion GSH may prevent FFAs-induced HBVECs damage, oxidative stress, and apoptosis through activating the Akt pathway.

Background Spontaneous intracerebral hemorrhage (ICH) is a devastating form of stroke with the high mortality twofold to sixfold higher than that for ischemic stroke. But the treatment of haematomas within the basal ganglia continues to be a matter of debate among neurologists and neurosurgeons. The purpose of this study is to judge the short-term and long-term clinical value of minimally invasive stereotactic puncture therapy (MISPT) on acute ICH. Methods A prospective controlled study was undertaken. The clinical trial was in compliance with the WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects. According to the enrollment criterion, there were 168 acute ICH cases analyzed, of which 90 cases were performed by MISPT (MISPT group, MG) and 78 cases by Conventional craniotomy (CC group, CG), by means of compare of short-term outcome such as Glasgow Coma Scale (GCS) score, postoperative complications (PC) and rebleeding incidence (RI), moreover, long-term outcome of 1 year postoperation judged by Glasgow Outcome Scale (GOS), Barthel Index (BI), modified Rankin Scale (mRS) and case fatality (CF). Results GCS score of MG patients showed obvious amelioration when compared with that of CG (P = 0.039). The PC incidence of in MG decreased obviously compared with that of CG (P = 0.000). The incidences of rebleeding in MG and CG were 10.0% and 15.4% respectively (P = 0.293). There was no obvious difference between CFs of MG and CG. For three parameters representing long-term outcome,the GOS, BI and mRS in MG were ameliorated significantly than that of CG (P = 0.043, P = 0.011 and P = 0.042 respectively). Conclusion These data indicated that compared with CC, the advantage of MISPT was not only displayed in short-term outcome such as minute trauma and safety, but also seemed to be feasible and had a trend towards improved long-term outcome such as the GOS,BI and mRS. Trial Registration The Australian New Zealand Clinical Trials Registry (ANZCTR), the registration number:ACTRN12610000945022.