Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, which are dependent on the platelet glycoprotein Ib-von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion model in Adamts13 ؊/؊ and wild-type mice. After reperfusion for 0.5 hours, the regional cerebral blood flow in the ischemic cortex was decreased markedly in Adamts13 ؊/؊ mice compared with wild-type mice (P < .05), which also resulted in a larger infarct volume after 24 hours for Adamts13 ؊/؊ compared with wild-type mice (P < .01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke. (Blood. 2010; 115:1650-1653) Introductionvon Willebrand factor (VWF) is a large multimeric protein that plays a key role in thrombus formation by tethering platelets at sites of vascular injury. 1 Smaller VWF multimers are less active, and the potent thrombogenic activity of ultra-large VWF (ULVWF) secreted from endothelium is regulated in vivo through cleavage by ADAMTS13. 2,3 The importance of this mechanism for normal hemostasis is supported by evidence that patients with deficiency of ADAMTS13 function, diagnosed with thrombotic thrombocytopenic purpura, have ULVWF in circulating blood and VWFdependent microvascular thrombosis. 2 Recently, we demonstrated that ADAMTS13 cleaves VWF on the surface of platelet thrombi in a shear force-dependent manner, which limits thrombus growth in vitro. 4 These data suggest that ADAMTS13 is a key molecule that maintains a physiologic balance between hemostasis and thrombosis through regulation of VWF function in vivo.ADAMTS13 function is crucial for preventing thrombosis in the cerebral microvasculature, as indicated by the occurrence of neurologic deficits in thrombotic thrombocytopenic purpura, but the role of ADMTS13 in the pathogenesis of reperfusion injury after arterial thrombosis has not been established. To address this issue, we investigated the role of ADAMTS13 in a transient middle cerebral arterial occlusion (MCAO) model of ischemia-reperfusion injury in the mouse brain 5 using Adamts Ϫ/Ϫ mice. 6 Because brain ischemia-reperfusion injury is dependent on the platelet glycoprotein Ib-VWF axis 7 and platelet thrombosis adversely affects the postischemic cerebral microcirculation 8-11 leading to secondary brain damage, 10 ADAMTS13 may reduce platelet thrombus growth and thereby ameliorate ischemic brain injury by improving the postischemic no-reflow phenomenon. 12 Here we demonstrate that Adamts13 gene deletion aggravates postischemic cerebral blood reflow, resulting in larger infarct volume. This result suggests that ADAMTS13 may indeed supp...

Glial scarring is traditionally thought to be detrimental after stroke. But emerging studies now suggest that reactive astrocytes may also contribute to neurovascular remodeling. Here, we assessed the effects and mechanisms of metabolic inhibition of reactive astrocytes in a mouse model of stroke recovery. Five days after stroke onset, astrocytes were metabolically inhibited with fluorocitrate (FC, 1 nmol). Markers of reactive astrocytes (glial fibrillary acidic protein (GFAP), HMGB1), markers of neurovascular remodeling (CD31, synaptophysin, PSD95), and behavioral outcomes (neuroscore, rotarod latency) were quantified from 1 to 14 days. As expected, focal cerebral ischemia induced significant neurological deficits in mice. But over the course of 14 days after stroke onset, a steady improvement in neuroscore and rotarod latencies were observed as the mice spontaneously recovered. Reactive astrocytes coexpressing GFAP and HMGB1 increased in peri-infarct cortex from 1 to 14 days after cerebral ischemia in parallel with an increase in the neurovascular remodeling markers CD31, synaptophysin, and PSD95. Compared with stroke-only controls, FC-treated mice demonstrated a significant decrease in HMGB1-positive reactive astrocytes and neurovascular remodeling, as well as a corresponding worsening of behavioral recovery. Our results suggest that reactive astrocytes in peri-infarct cortex may promote neurovascular remodeling, and these glial responses may aid functional recovery after stroke.

Objective: The objective of this study was to evaluate the effects of earlier intervention by an antimicrobial stewardship team (AST) on antimicrobial use, antimicrobial resistance rates, and the clinical outcomes, without changing the weekly intervention schedule. Methods: A retrospective study was conducted at Fukuoka University Hospital between April 2013 and March 2016. The effects were compared among three study periods (SP): SP1 (patients receiving antimethicillin-resistant Staphylococcus aureus agents and carbapenems for 14 days), SP2 (patients receiving specific antimicrobials for 14 days), and SP3 (patients receiving specific antimicrobials regardless of the duration of treatment). Results: The timing of AST intervention was shortened from an average of 15.5 days after administration in SP1 to 4.2 days in SP3. The antimicrobial use density (AUD) of carbapenems and piperacillin-tazobactam decreased significantly (SP2 vs. SP3, p < 0.05), and the costs of specific antimicrobials decreased (SP1, US$ 1 080 000; SP2, US$ 944 000; SP3, US$ 763 000). The rates of carbapenem resistance among Pseudomonas aeruginosa isolates showed a significant reduction from 16.2% in SP2 to 8.7% in SP3 (p < 0.05). The mortality rate and length of stay did not change during the study period. Conclusions: Earlier intervention by an AST could contribute to the proper use of antimicrobials without adversely affecting patient outcomes.

Summary. Background: Microthrombosis and reactive inflammation contribute to neuronal injury after subarachnoid hemorrhage (SAH). ADAMTS-13 cleaves von Willebrand factor multimers, and inhibits thrombus formation and, seemingly, inflammatory reactions. Objective: To investigate the effect of ADAMTS-13 in experimental SAH. Methods: A total of 100 male C57/BL6 mice were randomly assigned to four groups: sham (n = 15), SAH (n = 27), vehicle (n = 25), and ADAMTS-13 (n = 23; 100 lL per 10 g of body weight of 100 lg of ADAMTS-13 per 1 mL of 0.9% NaCl; 20 min after SAH). Neurologic performance was assessed on days 1 and 2 after SAH. Animals were killed on day 2. The amounts of subarachnoid blood, microthrombi, apoptosis and degenerative neurons were compared. The degree of neuronal inflammation and vasospasm was also compared. In five mice each (SAH and ADAMTS-13 groups), bleeding time was assessed 2 h after SAH. Results: Systemic administration of ADAMTS-13 achieved significant amelioration of microthrombosis and improvement in neurologic performance. ADAMTS-13 reduced the amount of apoptotic and degenerative neurons. A tendency for decreased neuronal inflammation was observed. ADAMTS-13 did not show any significant effect on vasospasm. The degree of systemic inflammation was not changed by ADAMTS-13 administration. ADAMTS-13 neither increased the amount of subarachnoid blood nor prolonged the bleeding time. Conclusions: ADAMTS-13 may reduce neuronal injury after SAH by reducing microthrombosis formation and neuronal inflammation, thereby providing a new option for mitigating the severity of neuronal injury after SAH.

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Highly adhesive glycoprotein von Willebrand factor (VWF) multimer induces platelet aggregation and leukocyte tethering or extravasation on the injured vascular wall, contributing to microvascular plugging and inflammation in brain ischemia-reperfusion. A disintegrin and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) cleaves the VWF multimer strand and reduces its prothrombotic and proinflammatory functions. Although ADAMTS13 deficiency is known to amplify post-ischemic cerebral hypoperfusion, there is no report available on the effect of ADAMTS13 on inflammation after brain ischemia. We investigated if ADAMTS13 deficiency intensifies the increase of extracellular HMGB1, a hallmark of post-stroke inflammation, and exacerbates brain injury after ischemia-reperfusion. ADAMTS13 gene knockout (KO) and wild-type (WT) mice were subjected to 30-min middle cerebral artery occlusion (MCAO) and 23.5-h reperfusion under continuous monitoring of regional cerebral blood flow (rCBF). The infarct volume, plasma high-mobility group box1 (HMGB1) level, and immunoreactivity of the ischemic cerebral cortical tissue (double immunofluorescent labeling) against HMGB1/NeuN (neuron-specific nuclear protein) or HMGB1/MPO (myeloperoxidase) were estimated 24 h after MCAO. ADAMTS13KO mice had larger brain infarcts compared with WT 24 h after MCAO (p < 0.05). The rCBF during reperfusion decreased more in ADAMTS13KO mice. The plasma HMGB1 increased more in ADAMTS13KO mice than in WT after ischemia-reperfusion (p < 0.05). Brain ischemia induced more prominent activation of inflammatory cells co-expressing HMGB1 and MPO and more marked neuronal death in the cortical ischemic penumbra of ADAMTS13KO mice. ADAMTS13 deficiency may enhance systemic and brain inflammation associated with HMGB1 neurotoxicity, and aggravate brain damage in mice after brief focal ischemia. We hypothesize that ADAMTS13 protects brain from ischemia-reperfusion injury by regulating VWF-dependent inflammation as well as microvascular plugging.

ABSTRACT:The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 ؎ 2.1 nmol/cm to 18.1 ؎ 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.The small intestine is a highly differentiated organ with a barrier function against xenobiotics and a gateway function for nutrients. The ATP-binding cassette (ABC) transporter family, including P-glycoprotein/multidrug resistance protein 1 (P-gp/Mdr1/Abcb1), breast cancer resistant protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2), are well known efflux transporters that are located on the brush-border membrane (BBM) of small intestinal epithelia to limit the absorption of a broad range of compounds (Takano et al., 2006). The distinct but sometimes overlapping substrate specificities of these efflux transporters have been shown previously (Chan et al., 2004). Indeed, a significant increase in the oral absorption of their substrates, including drugs and carcinogens, was confirmed in animals genetically deficient in these transporters (Dietrich et al., 2001;Yamaguchi et al., 2002).Several reports indicated the disparity between mRNA and protein expression of Abcc2 in the intestine. Naud et al. (2007) reported that Abcc2 protein expression was reduced to approximately 40% of that of the control during chronic renal failure in rats, but its mRNA expression was unaffected. Dietrich et al. (2004) also reported ABCC2 protein expression was reduced to approximately 27% of that of the control patients in the human intestine du...

Cannabis contains about 60 different cannabinoids, including the psychoactive component, D 9 -tetrahydrocannabinol as well as non-psychoactive components, which include cannabidiol, cannabinol and cannabigerol. Among these components, cannabidiol, a non-psychoactive constituent of cannabis, is known to exert potent anti-inflammatory, immunomodulatory and analgesic effects. 1,2) In addition, cannabidiol has been shown to be protective against global and focal ischemic injury. 3,4) Although cannabidiol is generally known to have a very low affinity (in the micromolar range) for the cannabinoid CB 1 and CB 2 receptors, it has many pharmacological actions, 5) including anxiolytic actions and anti-inflammatory and a neuroprotective effect against ischemic injury. These actions are thought to be dependent on a new abnormal cannabidiol receptor, but not the non-CB 1 or non-CB 2 receptor.6,7) We have also reported that cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent mechanism.8) In addition, cannabidiol has been shown to significantly prevent infarction and myeloperoxidase (MPO) activity after reperfusion via a CB 1 and CB 2 receptor-independent mechanism.9) Recently, we have reported that cannabidiol decreased the cerebral infarction via MPO expressing highmobility group box1 (HMGB1)-inhibiting mechanism. In addition, cannabidiol decreased the level of HMGB1 in plasma in ischemic early phase. 10)HMGB1, a non-histone DNA-binding protein, is widely expressed in various tissues, including the brain. HMGB1 has been implicated in diverse intracellular functions, including the stabilization of nucleosomal structure and the facilitation of gene transcription.11) HMGB1 is known to be massively released into the extracellular space by monocytes and macrophages or necrotic cells immediately after an ischemic insult, and induces expression of several genes related to progressive inflammation, leading to apoptosis in the postischemic brain. [12][13][14][15][16] In addition, we have reported that repeated treatment with minocycline, microglia inhibitor, for 14 d improved functional deficits, and decreased plasma levels of HMGB1 and the expression of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive cells at 14 d after cerebral ischemia, 17) suggesting that the extracellular HMGB1 level is of considerable importance for the treatment of post-ischemic injury.Cannabidiol decreased cerebral infarction and HMGB1 in plasma in ischemic early phase. However, plasma HMGB1 levels in ischemic delayed phase reached higher concentration with the progressing brain injury.10) Therefore, we examined whether cannabidiol can inhibit the progressive inflammation reaction related with HMGB1 and estimated the therapeutic time window of cannabidiol in ischemic delayed phase. MATERIALS AND METHODS Animals Male ddY mice (25-35 g, Kiwa ExperimentalAnimal Laboratory, Wakayama, Japan) were kept under a 12-h light/dark cycle (lights on from 07:00 to 19:00 h) in an airconditioned room (23Ϯ2...