Background Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models, leading to a serious concern regarding the safety of pediatric anesthesia. However, if and how ketamine induces human neural cell toxicity is unknown. Recapitulation of neurogenesis from human embryonic stem cells (hESCs) in vitro allows investigation of the toxic effects of ketamine on neural stem cells (NSCs) and developing neurons which is impossible to perform in humans. In the present study we assessed the influence of ketamine on the hESC-derived NSCs and neurons. Methods hESCs were directly differentiated into neurons via NSCs. NSCs and two-week-old neurons were treated with varying doses of ketamine for different durations. NSC proliferation capacity was analyzed by Ki67 immunofluorescence staining and bromodeoxyurindine assay. Neuroapoptosis was analyzed by TUNEL staining and caspase 3 activity measurement. The mitochondria-related neuronal apoptosis pathway including mitochondrial membrane potential, cytochrome c distribution within cells, mitochondrial fission, and reactive oxygen species (ROS) production were also investigated. Results Ketamine (100 μM) increased NSC proliferation after 6 h exposure. However, significant neuronal apoptosis was only observed after 24 h of ketamine treatment. In addition, ketamine decreased mitochondrial membrane potential and increased cytochrome c release from mitochondria into cytosol. Ketamine also enhanced mitochondrial fission as well as ROS production compared with no-treatment control. Importantly, Trolox, a ROS scavenger, significantly attenuated the increase of ketamine-induced ROS production and neuronal apoptosis. Conclusions These data for the first time demonstrate that (1) ketamine increases NSC proliferation and causes neuronal apoptosis; (2) mitochondria are involved in ketamine-induced neuronal toxicity which can be prevented by Trolox; and (3) the stem cell-associated neurogenesis system may provide a simple and promising in vitro model for rapidly screening anesthetic neurotoxicity and studying the underlying mechanisms as well as prevention strategies to avoid this toxic effect.

Myocardial ischemia-reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: 1) ROS are upstream initiators of mitochondrial fission; and 2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨ m ), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose-and timedependently caused cell death. In addition, ketamine decreased ΔΨ m and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨ m changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time-and dosedependently induces human neurotoxicity via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies.

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Plasma procarboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is converted by thrombin into the active enzyme, carboxypeptidase B (CPB)/activated TAFI. Plasma CPB down-regulates fibrinolysis by removing carboxy-terminal lysines, the ligands for plasminogen and tissue-type plasminogen activator (tPA), from partially degraded fibrin. To target thrombosis in a new way, we have identified and optimized a phosphinic acid-containing inhibitor of CPB,heptanoic acid] and determined both the pharmacological profile and pathophysiological role of CPB in rat thrombolysis. EF6265 specifically inhibited plasma CPB activity with an IC 50 (50% inhibitory concentration) of 8.3 nM and enhanced tPA-mediated clot lysis in a concentrationdependent manner. EF6265 decreased detectable thrombi (percentage of glomerular fibrin deposition; control, 98 Ϯ 1.1; EF6265, 0.1 mg/kg, 27 Ϯ 9.1) that had been generated by tissue factor in a rat microthrombosis model with concomitant increases in plasma D-dimer concentration (control, Ͻ0.5 g/ml; EF6265, 0.1 mg/kg, 15 Ϯ 3.5 g/ml). EF6265 reduced plasma ␣2-antiplasmin activity to a lesser extent than tPA. In an arteriovenous shunt model, EF6265 (1 mg/kg) enhanced exogenous tPA-mediated thrombolysis under the same conditions that neither EF6265 nor tPA (600 kIU/kg) alone reduced thrombi. EF6265 (1 and 30 mg/kg) did not affect the bleeding time in rats. Moreover, it did not prolong the bleeding time evoked by tPA (600 kIU/kg). These results confirm that circulating procarboxypeptidase B functions as a fibrinolysis inhibitor's zymogen and validates the use of CPB inhibitors as both an enhancer of physiological fibrinolysis in microcirculation and as a novel adjunctive agent to tPA for thromboembolic diseases while maintaining a small effect on primary hemostasis.Thrombosis-related diseases, including myocardial infarction, cerebral infarction, and disseminated intravascular coagulation are life-threatening and the search for treatments remains challenging. Although thrombolytics including tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator and anticoagulants including heparin have been developed over the last few decades, the risk of hemorrhaging with these antithrombotic agents restricts their clinical use. The dose of tPA must be high enough to overcome the inhibitory effects of plasminogen activator inhibitor-1 in the plasma, and results in the generation of plasmin in circulating blood (Rijken and Sakharov, 2001). Consequently, these large quantities of generated plasmin can induce thrombolysis and result in hemorrhaging as a side effect (Bloom et al., 1988). Many efforts have therefore been made to identify and develop pharmacologically distinct antithrombotic agents while maintaining a low risk of hemorrhaging.Plasma procarboxypeptidase B (proCPB; EC 3.4.17.20), also known as thrombin-activatable fibrinolysis inhibitor (TAFI) or procarboxypeptidase U, is produced in the liver and Article, publication date, and citation informa...