Yusuke Egashira

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy.

BACKGROUND AIMS Transplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model. METHODS ASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10(5) cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed. RESULTS ASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group. CONCLUSIONS Compared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.

The growth factor progranulin attenuates neuronal injury induced by cerebral ischemia-reperfusion through the suppression of neutrophil recruitment.

BackgroundTo improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice.MethodsIn vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-α, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-κB (NF-κB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells.ResultsWe found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-κB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-α in endothelial cells.ConclusionsPGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.

Pharmacological inhibition of TLR4-NOX4 signal protects against neuronal death in transient focal ischemia

Recent data have shown that TLR4 performs a key role in cerebral ischemia-reperfusion injury which serves as the origin of the immunological inflammatory reactions. However, the therapeutic effects of pharmacological inhibitions of TLR4 and its immediate down-stream pathway remain to be uncovered. In the present study, on mice, intracerebroventricular injection of resatorvid (TLR4 signal inhibitor; 0.01 μg) significantly reduced infarct volume and improved neurological score after middle cerebral artery occlusion and reperfusion. The levels of phospho-p38, nuclear factor-kappa B, and matrix metalloproteinase 9 expressions were significantly suppressed in the resatorvid-treated group. In addition, NOX4 associates with TLR4 after cerebral ischemia-reperfusion seen in mice and human. Genetic and pharmacological inhibitions of TLR4 each reduced NOX4 expression, leading to suppression of oxidative/nitrative stress and of neuronal apoptosis. These data suggest that resatorvid has potential as a therapeutic agent for stroke since it inhibits TLR4-NOX4 signaling which may be the predominant causal pathway.

The conditioned medium of murine and human adipose-derived stem cells exerts neuroprotective effects against experimental stroke model

This study investigated the possible ameliorative effects of adipose-derived stem cells-conditioned medium (ASC-CM) on experimental ischemic stroke. In vivo ischemic stroke was induced in mice after 2h of middle cerebral artery occlusion (MCAO) followed by 22 h reperfusion. Culture of SH-SY5Y human neuroblastoma cells with 100 μM glutamate for 24h was used as an in vitro neuronal apoptosis model. Intracerebroventricular (i.c.v.) administration of 30- and 100-fold concentrated murine ASC-CM 1h prior to MCAO resulted in a dose-dependent reduction in the infarct volume and the brain swelling. The administration of murine ASC-CM immediately after MCAO was also effective, but administration 2h after MCAO was not. Neuroprotective effects of murine ASC-CM were also confirmed in an in vitro model. Pretreatment with 100-fold concentrated murine ASC-CM at 10% of the total culture volume significantly reduced glutamate-induced excitotoxicity in the SH-SY5Y cells. Similar reduction in the MCAO-induced infarction volume was seen following i.c.v. administration of 100-fold concentrated human ASC-CM or murine ASC-CM. In conclusion, ASC-CM appears to exert ameliorative effects on experimental ischemic stroke i\n both in vivo and in vitro models. These findings suggest the feasibility of ASC-CM administration as a therapy for acute stage stroke.

A Rho kinase (ROCK) inhibitor, fasudil, prevents matrix metalloproteinase-9-related hemorrhagic transformation in mice treated with tissue plasminogen activator.

Thrombolysis with tissue plasminogen activator (tPA) is the only FDA-approved therapy for acute ischemic stroke. However, hemorrhagic transformation, neurotoxicity, and a short treatment time window comprise major limitations for thrombolytic therapy. The purpose of the present study was to investigate whether fasudil, a Rho kinase (ROCK) inhibitor, would prevent tPA-associated hemorrhagic transformation and extend the reperfusion window in an experimental stroke model in mice. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone, with combined tPA plus fasudil, or with a vehicle. We used histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To investigate the mechanism of fasudils beneficial effects further, we also performed an in vitro study with tPA and fasudil in human brain microvascular endothelial cells. Combination therapy with tPA plus fasudil prevented the development of hemorrhagic transformation, but did not reduce the infarct volumes. These changes significantly reduced mortality and increased locomotor activity at 7 days after the reperfusion. Furthermore, the administration of both drugs prevented injury to the human brain endothelial cells via the reduction of matrix metalloproteinase-9 (MMP-9) activity. These findings indicate that fasudil prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA, at least in part, by inhibiting the increased activity of MMP-9 in endothelial cells.

Visualization of internal carotid artery atherosclerotic plaques in symptomatic and asymptomatic patients: a comparison of optical coherence tomography and intravascular ultrasound.

BACKGROUND AND PURPOSE: OCT has been reported as a high-resolution imaging tool for characterizing plaque in the coronary arteries. The present study aimed to evaluate the ability of OCT to visualize carotid artery plaques compared with that of IVUS in asymptomatic and symptomatic patients. MATERIALS AND METHODS: OCT was performed for 34 plaques (17 symptomatic, 17 asymptomatic) in 30 patients during CAS under a proximal cerebral protection method. OCT was performed before balloon angioplasty and after stent placement. IVUS was also performed just after OCT. RESULTS: No technical or neurologic complications were encountered by using OCT. An inner catheter was used in 12 of 34 procedures (35.3%) for advancing the OCT image wire beyond the site of stenosis. OCT clearly visualized intraluminal thrombus in 15 of 34 plaques (44.1%), whereas IVUS detected a thrombus in 1 plaque (2.9%, P < .001). Neovascularization was demonstrated in 13 of 34 plaques (38.2%) by OCT, but not by IVUS (0%, P < .001). Intraluminal thrombus was more frequently observed in symptomatic plaques (13 of 17, 76.5%) than in asymptomatic plaques (2 of 17, 11.8%; P < .001). Interobserver and intraobserver variability with OCT diagnosis was excellent for thrombus, ulceration, neovascularization, and lipid pool. CONCLUSIONS: The present findings suggest that OCT can safely and precisely visualize human carotid plaques during CAS and that intraluminal thrombus and neovascularization are more frequently detected in symptomatic plaques.

Experience of Staged Angioplasty to Avoid Hyperperfusion Syndrome for Carotid Artery Stenosis

Hyperperfusion syndrome (HPS) after carotid artery stenting (CAS) may cause hemorrhagic or ischemic events leading to serious sequelae. We previously reported the staged angioplasty (SAP) to prevent HPS. In the present study, we analyzed our treatment results of SAP to know its effectiveness and problems. The study included 43 patients scheduled for SAP in whom preoperative single photon emission computed tomography (SPECT) showed severely impaired cerebral blood flow (CBF). The analyzed subjects were 38 males and 4 females, mean age was 73 ± 8.5 years old. SAP was indicated for the patients whose CBF ratio in the affected/unaffected hemisphere (asymmetry index) was below 0.8, and cerebrovascular reactivity measured by acetazolamide challenge was below 10%. First, percutaneous transluminal angioplasty (PTA) was performed. If PTA was successful, CAS was performed 2 weeks later. If PTA was not successful due to inadequate dilatation or extensive dissection, a stent was placed. SPECT was performed immediately after PTA and CAS to confirm the presence or absence of hyperperfusion phenomenon (HPP) indicating radiologic hyperperfusion. In 39 of 43 patients (91%), SAP was successfully performed and HPP was not observed. On the other hand, in the other four patients (9%), immediate stent placement was added due to inadequate dilatations in three patients and vascular dissection in one. Among 43 candidates for SAP, 41 patients (95.4%) had favorable course, but one hemorrhagic and one ischemic complications were observed after PTA. SAP was a relatively simple procedure, and its clinical results seemed acceptable.

Current perioperative management of anticoagulant and antiplatelet use in neuroendovascular therapy: analysis of JR-NET1 and 2.

To evaluate current perioperative antithrombotic management in neuroendovascular therapy in Japan, we analyzed perioperative anticoagulant and antiplatelet use in various procedures and examined their relationships with periprocedural adverse events. Patients data from nationwide surveys administered by the Japanese Registry of Neuroendovascular Therapy (JR-NET) between January 2005 and December 2007 (JR-NET1) and January 2008 and December 2009 (JR-NET2) were retrospectively analyzed. Compared to JR-NET1, the frequency of perioperative antiplatelet therapy and dual or triple therapy were increased for either aneurysm coiling and percutaneous transluminal angioplasty or stenting in JR-NET2. Although ischemic complications were significantly decreased (4.2% vs. 2.1%, p < 0.001), hemorrhagic complications (2.1% vs. 5.3%, p < 0.001), severe adverse events (1.5% vs. 2.1%, p < 0.001), and total perioperative complications (8.3% vs. 10.3%, p < 0.001) were significantly increased in JR-NET2. The rate of hemorrhagic complications was significantly higher in patients with triple or more perioperative antiplatelet therapy (preoperative: 5.3% vs. 9.2%, p < 0.0001, postoperative: 5.7% vs. 12.7%, p < 0.0001). Perioperative antithrombotic therapy was performed more frequently and intensively in neuroendovascular therapy in Japan. While ischemic complications were decreased, hemorrhagic complications and severe adverse events were increased. These results suggest that intensive antithrombotic therapy has a potential risk of hemorrhagic complications for Japanese patients.

Cilostazol Ameliorates Warfarin-Induced Hemorrhagic Transformation After Cerebral Ischemia in Mice

Background and Purpose— Although long-term treatment with the oral anticoagulant warfarin is widely used to prevent cardioembolic ischemic stroke, it has been reported that warfarin can exacerbate hemorrhagic transformation (HT) after cerebral ischemia. We investigated whether cilostazol, a phosphodiesterase-III inhibitor, suppressed the warfarin-induced HT after cerebral ischemia in mice. Methods— Male ddY mice were treated with oral warfarin before 3-hour middle cerebral artery occlusion followed by 21-hour reperfusion to induce HT. The duration of warfarin pretreatment was determined by measurement of prothrombin time-international normalized ratio value. Cilostazol or vehicle was administered by intraperitoneal injection immediately after reperfusion. The infarct volume, brain swelling, and brain hemoglobin content were evaluated at 24 hours after middle cerebral artery occlusion. We also evaluated the survival rate of each treated group for 7 days after surgery. To investigate the mechanism underlying cilostazol’s effects, the proteins involved in vascular endothelial integrity were investigated using Western blotting. Results— HT volume was exacerbated by warfarin treatment, and cilostazol (3 mg/kg, IP) suppressed this exacerbation (sham, mean±SD, 29.2±13.4 mg/dL; vehicle, 33.3±11.9 mg/dL; warfarin, 379.4±428.9 mg/dL; warfarin+cilostazol 1 mg/kg, 167.5±114.2 mg/dL; warfarin+cilostazol 3 mg/kg, 116.9±152.3 mg/dL). Furthermore, cilostazol improved survival rate and upregulated the expression of tight junction proteins and vascular endothelial cadherin. Conclusions— Cilostazol reduced the warfarin-related risk of HT after ischemia by protecting the vascular endothelial cells. This result suggested that cilostazol administration in patients with acute ischemic stroke might reduce HT.

Retrospective Nationwide Survey of Acute Stroke due to Large Vessel Occlusion in Japan: A Review of 1,963 Patients and the Impact of Endovascular Treatment

Background: The purpose of this study was to clarify the clinical impact of endovascular treatment (EVT) on acute cerebral large vessel occlusion using a nationwide survey of Japan conducted in 2009. Methods: Patients admitted within 24 h after stroke onset were registered retrospectively. Treatment selection, methods, and clinical results were analyzed. Results: A total of 1,963 patients (855 women, 1,108 men) treated in 2008 were registered from 68 medical centers in Japan. Mean age on admission was 74.1 ± 12.2 years (range 7–100 years). The first treatment was conservative therapy in 68%, intravenous tissue plasminogen activator (IV-tPA) in 21%, EVT in 9%, and combined IV-tPA + EVT in 2%. EVT mainly comprised angioplasty, intra-arterial thrombolysis and/or the combination of both. Patients treated ≤3 h after onset (1,286 cases) showed better clinical outcomes with combined IV-tPA + EVT than with conservative therapy, and significant differences in outcomes were seen for patients with occlusion of the basilar artery (p < 0.01) or middle cerebral artery (p < 0.01), but not the internal carotid artery. At >3 h after onset (677 patients), no IV-tPA was performed, and EVT was performed in 11%. Among the patients treated by EVT, there were significant differences in clinical outcome between complete recanalization (TIMI grade 3) and partial/no recanalization (TIMI grade 0–2) (p < 0.001; OR 5.98; 95% CI 3.27–10.96) and between any recanalization (TIMI grade 1–3) and no recanalization (TIMI grade 0) (p < 0.001; OR 36.15; 95% CI 4.88–267.53). Conclusions: This nationwide survey showed the efficacy of EVT with IV-tPA in patients with occlusion of the basilar or middle cerebral artery, but not of the internal carotid artery. The effects of new endovascular devices should be clarified in the near future.