ABSTRACT
Liver sinusoidal endothelial cells (LSECs) represent a highly specialized and unique type of endothelial cell in terms of their morphology and function. The biochemical and functional characterization of LSECs in vitro is restrained by the rapid change of LSECs' phenotype upon culturing under classical experimental conditions. In this work, we present a novel approach to characterize the biochemical content of murine LSECs, freshly isolated from the liver, with the use of microspectroscopic analysis. For comparison, hepatocytes and Hepatic Stellate Cells (HSCs) were analyzed. Our approach, based on label-free confocal Raman imaging of live cells combined with chemometric analysis, provided insight into the biochemical content of freshly isolated LSECs on a subcellular level. LSECs were featured by a distinct biochemical signature in comparison with other major cell types of the liver. Based on our work we claim that the non-invasive and non-destructive confocal Raman imaging may assist in obtaining chemical information spatially distributed within the cells that characterize the phenotype of primary LSECs as well as other types of liver cells. Furthermore, our approach provides a unique insight into LSECs' morphology and chemical composition that may help to understand their functions.
Subject(s)
Endothelial Cells/cytology , Hepatic Stellate Cells/cytology , Hepatocytes/cytology , Spectrum Analysis, Raman , Animals , Liver/cytology , Male , Mice , Mice, Inbred C57BLABSTRACT
In this work we apply FT-IR imaging of large areas of liver tissue cross-section samples (â¼5 cm × 5 cm) for quantitative assessment of steatosis in murine model of Non-Alcoholic Fatty Liver (NAFLD). We quantified the area of liver tissue occupied by lipid droplets (LDs) by FT-IR imaging and Oil Red O (ORO) staining for comparison. Two alternative FT-IR based approaches are presented. The first, straightforward method, was based on average spectra from tissues and provided values of the fat content by using a PLS regression model and the reference method. The second one the chemometric-based method enabled us to determine the values of the fat content, independently of the reference method by means of k-means cluster (KMC) analysis. In summary, FT-IR images of large size liver sections may prove to be useful for quantifying liver steatosis without the need of tissue staining.
Subject(s)
Fats/analysis , Liver/chemistry , Liver/pathology , Non-alcoholic Fatty Liver Disease/pathology , Spectroscopy, Fourier Transform Infrared , Animals , Male , Mice , Mice, Inbred C57BLABSTRACT
In this study, we report an approach to characterize individual BoLA haplotypes using cells from parthenogenetic bovine embryos derived from slaughterhouse ovaries. Eight of the 15 parthenogenetic embryos so obtained had not undergone meiotic recombination on the BoLA region and were suitable to describe BoLA haplotypes. Detailed analysis of the BoLA class IIa region identified seven different class IIa haplotypes, including six not previously described and two new alleles of BoLA-DQA and one BoLA-DQB. Our method provided reliable sources of homozygous DNA to describe BoLA haplotypes.
Subject(s)
Cattle/genetics , Genes, MHC Class II , Haplotypes , Alleles , Animals , Embryo, Mammalian , ParthenogenesisABSTRACT
Confocal Raman microspectroscopy was used in this study to identify hepatic stellate cells (HSCs) from healthy mice and mice with untreated and treated liver steatosis. We have identified the main form of occurrence of vitamin A in healthy liver and confirmed its absence in the pathological state. Additionally, we have reported the reappearance of vitamin A in the tissue after treatment of liver steatosis.
Subject(s)
Liver/metabolism , Spectrum Analysis, Raman , Vitamin A/analysis , Vitamin A/metabolism , Animals , Biomarkers/metabolism , Fatty Liver/metabolism , Fatty Liver/therapy , Hepatic Stellate Cells/metabolism , Liver/chemistry , Liver/cytology , Male , Mice , Mice, Inbred C57BLABSTRACT
The composition of the lung tissue of mice was investigated using Raman confocal microscopy at 532 nm excitation wavelength and was supported with various staining techniques as well as DFT calculations. This combination of experimental and theoretical techniques allows for the study of the distribution of lung lipofibroblasts (LIFs), rich in vitamin A, as well as the chemical structure of vitamin A. The comparison of the Raman spectra derived from LIFs with the experimental and theoretical spectra of standard retinoids showed the ability of LIFs to store all-trans retinol, which is partially oxidized to all-trans retinal and retinoic acid. Moreover, we were able to visualize the distribution of other lung tissue components including the surfactant and selected enzymes (lipoxygenase/glucose oxidase).
Subject(s)
Lung/chemistry , Optical Imaging , Spectrum Analysis, Raman , Vitamin A/analysis , Vitamin A/chemistry , Animals , Fibroblasts/chemistry , Isomerism , Lung/cytology , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Conformation , Quantum TheoryABSTRACT
We have developed a microagglutination test for typing rhesus monkey erythrocytes that is sensitive, accurate and easy to perform. The technique requires only microliter quantities of antiserum and cells, and agglutination is easily detected using an inverted microscope. An advantage of this technique is that the typing plates can be stored at -70 degrees C without loss of activity. The results of typing over 400 rhesus blood samples with this technique were 95% concordant with results using the standard microtitre agglutination technique. Preliminary results indicate that this test is also adaptable to typing human blood.
