Claudin domain containing 1 contributing to endothelial cell adhesion decreases in presence of cerebellar hemorrhage.

The claudin family comprises four-pass transmembrane proteins involved in the formation of tight junctions (TJs). Relatively recently, claudin domain containing (CLDND) 1, also known as claudin-25, was identified as a novel member of the claudin family. In the present study, we revealed that in the adult murine brain, CLDND1 is abundant in the cerebellum among common sites of intracerebral hemorrhage. Thus, the dynamics of CLDND1 after cerebellar hemorrhage were examined. Both CLDND1 mRNA and protein levels transiently decreased at 24 hr after hemorrhagic insult. For immunostaining, an anti-CLDND1 antibody that recognizes the specific epitope in the extracellular first loop was prepared. Dual immunohistochemical staining with CD31 using coronal cryosections of intact murine cerebellum tissue revealed that CLDND1 is expressed on endothelial cells. We therefore performed an in vitro permeability test using a human brain endothelial cell (HBEC) line to reveal whether CLDND1 contributes to cell adhesion like other claudins. CLDND1 was expressed on HBECs as well as in murine cerebellum tissue, and a strong signal was observed at TJs. RNA interference against CLDND1 decreased both the mRNA and protein levels without cytotoxicity. The permeability to small molecules, but not to large ones, across confluent HBECs increased on CLDND1 knockdown compared with mock-treated cells. These results suggest that the transient decrease of CLDND1 after cerebellar hemorrhage is responsible for low-molecular-weight selective vascular hyperpermeability. © 2017 Wiley Periodicals, Inc.

Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia.

The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3', 5'-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction.

Development of analytical method for simultaneous determination of five rodent unique bile acids in rat plasma using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry.

Bile acids (BAs) are crucial for the diagnosis, follow-up, and prognostics of liver injuries and other BA metabolism related diseases. In particular, rodent unique BAs, α-muricholic acid (α-MCA), ß-MCA, ω-MCA, tauro-α-MCA (α-TMCA), and ß-TMCA, are valuable biomarkers for preclinical drug development. To the best of our knowledge, however, a simple, selective, sensitive, and robust analytical method for ω-MCA and taurine-conjugated MCAs has never been reported. We have developed a simple, selective, and sensitive analytical method for measurement of 16 BAs including the five rodent unique BAs in rat plasma using an ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method. Activated charcoal was utilized to prepare BA-free plasma, which served as the surrogate matrix for the preparation of calibration standards and quality control (QC) samples. Results of matrix effects evaluation suggested that the BA-free plasma could be adequate as a surrogate matrix for BAs determination. Three stable isotope labelled internal standards were separated by reverse phase UPLC using gradient elution and were detected by TOF-MS in negative ion mode. The calibration curve was linear for all BAs over a range of 10-25ng/mL to 1000-10,000ng/mL, with overall imprecision below 15% and 20% at lower limit of quantification (LLOQ), respectively. This analytical method was used to determine BA concentrations in more than 300 plasma samples from rats with liver injuries induced using α-naphthylisocyanate, carbon tetrachloride, or flutamide. The alteration of BA concentrations was most evident for necrosis, and cholestasis hepatotoxins, with more subtle effects by steatosis and idiosyncratic hepatotoxins. In conclusion, we have developed a simple, selective, and sensitive analytical method to measure plasma 16 BAs including 5 rodent unique BAs, α-MCA, ß-MCA, ω-MCA, α-TMCA, and ß-TMCA. Our data suggested that α-TMCA and ß-TMCA could be useful for identification or prediction of liver injuries, a currently unmet need in preclinical toxicity. Our method using TOF-MS is useful to determine BAs in rat plasma and of use in structural analyses of metabolites in early stage of drug development.

A serious clinical course of a very elderly patient with takotsubo cardiomyopathy.

An 86-year-old woman was admitted to our hospital because of vomiting and anorexia. Although serum cardiac markers, an electrocardiogram, and echocardiography suggested acute myocardial infarction, emergency cardiac catheterization revealed akinesis of the left ventricular apex without significant coronary artery stenosis. She was diagnosed as having takotsubo cardiomyopathy. The left ventricular dysfunction was considered transient and reversible but did not improve at all, contrary to our expectations. She died of worsening heart failure on day 14. We discuss this serious clinical course of a very elderly patient with takotsubo cardiomyopathy.

Ventricular septal perforation in a patient with takotsubo cardiomyopathy.

An 84-year-old woman was admitted with anorexia and because the serum cardiac markers, electrocardiogram and echocardiography suggested acute myocardial infarction she underwent emergency cardiac catheterization. Coronary angiography revealed no significant coronary artery stenosis, but left ventriculography revealed akinesis of the left ventricular apex with shunt flow to the right ventricle. The diagnosis was a rare case of takotsubo cardiomyopathy complicated by ventricular septal perforation. The patient died of cardiogenic shock on the day of admission day.

Catheter interventional therapy in an elderly patient with deep vein thrombosis and a brain tumor.

A 92-year-old woman with a brain tumor developed swelling of the left lower extremity. Venography showed considerable thrombi from the left common iliac vein to the femoral vein. Following implantation of a temporary inferior vena cava filter, catheter aspiration therapy and catheter-directed thrombolysis were performed. Venography after 3 days showed disappearance of the thrombi and an improvement in vein flow. A permanent inferior vena cava filter was implanted. Local intensive thrombectomy and thrombolysis by catheter together with a temporary inferior vena cava filter were effective treatments in this elderly patient with deep vein thrombosis.