Tissue microarray: an effective high-throughput method to study the placenta for clinical and research purposes.

OBJECTIVE: Tissue microarray (TMA) technology allows simultaneous examination of the expression of many molecular markers (protein, mRNA, DNA, etc.) with high-throughput. The application of this technology, to date, has been largely confined to the study of cancer. Placental pathology poses unique challenges because of the size of the organ, its complex anatomy, as well as its histological heterogeneity. The objective of this study was to assess the feasibility and efficiency of TMAs for immunohistochemistry and in situ hybridization of placental tissues. STUDY DESIGN: TMAs were constructed using an automated tissue arrayer. Standard 0.6-mm or 1-mm microarray needles were used. Villous parenchyma, basal plate, and chorioamniotic membranes were targeted in each block. Five mum-thick TMA sections underwent immunohistochemical analysis of both cytoplasmic and nuclear antigens using a panel of antibodies against a variety of cytoplasmic [cytokeratin-7, vascular endothelial growth factor (VEGF), and protein Z], membranous (endoglin), and nuclear (c-fos and c-jun) antigens. mRNA in situ hybridization for surfactant protein A (SP-A) and chromogenic in situ hybridization for the Y chromosome (DYZ1) were also performed. RESULTS: Validation of TMA immunoreactivity demonstrated comparable results with corresponding whole sections. When a two-tiered scoring system (positive/negative) was employed, there was agreement between two and three cores and whole tissue sections (kappa>0.7). When a three-tiered scoring system (negative, weak-positive, or strong-positive) was used, the data from three cores showed the highest agreement with whole tissue sections (kappa >0.7). In situ hybridization experiments for mRNA and DNA were also successful in that the signals were readily detectable. Successful transfer from the donor block to the recipient block differed according to the anatomical compartment. The transfer efficiency of villous parenchyma, basal plate, and chorioamniotic membranes were 96.9% (875/903), 76.7% (115/150), and 75.4% (224/297), respectively. CONCLUSION: TMA is a practical and effective tool for high-throughput molecular analysis of the human placenta. Duplicate and triplicate cores offer agreement with whole tissue sections for two-category distinction immunostaining. TMA also affords relevant results from in situ hybridization experiments for mRNA and DNA. The major advantages are the conservation of tissues and reagents, simultaneous comparison of molecular markers in different anatomical compartments of the placenta, and reduction of experimental error.     

Synergistic antinociceptive interaction between acetaminophen or metamizol and B vitamins in the formalin test

There is evidence that B vitamins produce antinociception in animals. However, potentiation of NSAID‐induced antinociception by B vitamins is unclear. The current study was designed to investigate the antinociceptive interaction between a mixture of B vitamins and either acetaminophen or metamizol. Acetaminophen (56–316 mg/kg), metamizol (32–178 mg/kg), and the mixture of B vitamins (32–178 mg/kg of thiamine, pyridoxine, and cyanocobalamin in a 100:100:1 proportion, respectively) or a combination of each drug with the B vitamins mixture was administered orally to female Wistar rats, and the antinociceptive effect determined in the formalin test. Isobolographic analyses were used to define the nature of the interaction between NSAIDs and B vitamins. Oral administration of either drug produced a dose‐related antinociceptive effect. Isobolographic analyses revealed that both acetaminophen or metamizol and the B vitamins mixture interacted synergistically in the formalin test, suggesting that these two combinations could be useful in treating inflammatory pain states. Drug Dev. Res. 66:286–294, 2006. © 2006 Wiley‐Liss, Inc.     

Multiexponential T2 analyses in a murine model of hepatic fibrosis at 11.7 T MRI

This study evaluated the effects of hepatic fibrosis on the multiexponential T₂ (MET₂) relaxation of ex vivo murine liver specimens using an 11.7 T MRI. This animal study was approved by the Institutional Animal Care and Use Committee. Eighteen male C57BL/6 mice were divided into control (n = 3) and experimental (n = 15) groups; the latter group was fed a 3,5‐dicarbethoxy‐1,4‐dihydrocollidine‐supplemented diet to induce hepatic fibrosis. Ex vivo liver specimens were imaged using an 11.7 T MRI scanner. A multi‐echo spin‐echo sequence was utilized for subsequent MET₂ analysis. Degrees of fibrosis were determined by a pathologist, as well as by digital image analysis. Scatterplot graphs comparing various features of the MET₂ signal decay with the degrees of fibrosis were generated, and correlation coefficients were calculated. Two distinct peaks of the MET₂ signal decay were identified in all liver specimens: a short T₂ component with a geometric mean T₂ (GMT₂) approximating 30 ms; and a long T₂ component with GMT₂ approximating 400 ms. Strong correlation was found between the degree of hepatic fibrosis and the amplitude of the short T₂ component, with a higher degrees of fibrosis associated with a lower amplitude. Moderate correlation was also found between hepatic fibrosis and the GMT₂ values of the long T₂ component, with higher degrees of fibrosis associated with lower GMT₂ values. The study of hepatic microenvironments using MET₂ analyses offers potential utility in the ongoing development of the noninvasive assessment of hepatic fibrosis using MRI. Copyright © 2012 John Wiley & Sons, Ltd.     

Melatonin enhances neural stem cell differentiation and engraftment by increasing mitochondrial function

Neural stem cells (NSCs) are regarded as a promising therapeutic approach to protecting and restoring damaged neurons in neurodegenerative diseases (NDs) such as Parkinson's disease and Alzheimer's disease (PD and AD, respectively). However, new research suggests that NSC differentiation is required to make this strategy effective. Several studies have demonstrated that melatonin increases mature neuronal markers, which reflects NSC differentiation into neurons. Nevertheless, the possible involvement of mitochondria in the effects of melatonin during NSC differentiation has not yet been fully established. We therefore tested the impact of melatonin on NSC proliferation and differentiation in an attempt to determine whether these actions depend on modulating mitochondrial activity. We measured proliferation and differentiation markers, mitochondrial structural and functional parameters as well as oxidative stress indicators and also evaluated cell transplant engraftment. This enabled us to show that melatonin (25 μM) induces NSC differentiation into oligodendrocytes and neurons. These effects depend on increased mitochondrial mass/DNA/complexes, mitochondrial respiration, and membrane potential as well as ATP synthesis in NSCs. It is also interesting to note that melatonin prevented oxidative stress caused by high levels of mitochondrial activity. Finally, we found that melatonin enriches NSC engraftment in the ND mouse model following transplantation. We concluded that a combined therapy involving transplantation of NSCs pretreated with pharmacological doses of melatonin could efficiently restore neuronal cell populations in PD and AD mouse models depending on mitochondrial activity promotion.     

Outcomes of Donation After Circulatory Death Heart Transplantation in Australia

Background

Transplantation of hearts retrieved from donation after circulatory death (DCD) donors is an evolving clinical practice.