ABSTRACT
PURPOSE: The soft tissue imaging in micro-CT remains challenging due to its low intrinsic contrast. The aim of this study was to create a simple staining method omitting the usage of contrast agents for ex vivo soft tissue imaging in micro-CT. MATERIALS AND METHODS: Hearts and lungs from 30 mice were used. Twenty-seven organs were either fixed in 97% or 50% ethanol solution or in a series of ascending ethanol concentrations. Images were acquired after 72, 168 and 336 h on a custom-built micro-CT machine and compared to scans of three native samples. RESULTS: Ethanol provided contrast enhancement in all evaluated fixations. Fixation in 97% ethanol resulted in contrast enhancement after 72 h; however, it caused hardening of the samples. Fixation in 50% ethanol provided contrast enhancement after 336 h, with milder hardening, compared to the 97% ethanol fixation, but the visualization of details was worse. The fixation in a series of ascending ethanol concentrations provided the most satisfactory results; all organs were visualized in great detail without tissue damage. CONCLUSIONS: Simple ethanol fixation improves the tissue contrast enhancement in micro-CT. The best results can be obtained with fixation of the soft tissue samples in a series of ascending ethanol concentrations.
Subject(s)
Contrast Media , Ethanol , Heart/anatomy & histology , Image Enhancement/methods , Lung/anatomy & histology , X-Ray Microtomography/methods , Animals , Heart/diagnostic imaging , Lung/diagnostic imaging , Male , Mice , Mice, Inbred C57BL , Models, AnimalABSTRACT
BACKGROUND/AIMS: Male C57BL/6 and DBA/2 mice differ in their liver iron content. The aim of this study was to examine possible differences in the expression of hepcidin genes (Hamp and Hamp2) between the two strains. METHODS: Hepatic mRNAs were quantified by real-time PCR. RESULTS: Ferroportin1, transferrin receptor 2 and HAMP mRNA levels displayed no significant strain differences. However, HAMP2 mRNA levels were higher in DBA/2N mice. In both strains, HAMP2 mRNA content was sex-dependent, with higher values in female animals. Both hepatic HAMP and HAMP2 mRNA levels were elevated by iron overload, but treatment with lipopolysaccharide increased only HAMP mRNA. Lipopolysaccharide also elevated the amount of HAMP mRNA in the pancreas, while pancreatic HAMP2 mRNA levels were decreased. Sequence analysis of hepcidin amplicons from DBA/2N mice predicted an Asn-->Lys substitution at position 73 of the HAMP peptide and a Ser-->Phe substitution at position 76 of the HAMP2 peptide. CONCLUSIONS: Hepatic Hamp2 expression displays considerable strain- and sex-dependent variation. Lipopolysaccharide increases expression of Hamp both in the liver and pancreas, but Hamp2 does not respond to lipopolysaccharide treatment. The significance of the amino acid substitutions in hepcidin peptides in DBA/2N mice is at present unknown.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Gene Expression Regulation/physiology , Liver/physiology , Amino Acid Substitution , Animals , DNA Primers , Female , Gene Expression Regulation/drug effects , Hepcidins , Iron/blood , Lipopolysaccharides/pharmacology , Liver/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Pancreas/physiology , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Sex Characteristics , Species SpecificityABSTRACT
Sublethal cyclophosphamide treatment induces unique regeneration patterns in bone marrow and the spleen of a mouse. Colony-forming units spleen (CFU-S)(day 8), CFU-granulocyte-macrophage (GM), nucleated cell counts, and their differentials in bone marrow, spleen, and peripheral blood were determined in mice treated with a single dose of cyclophosphamide. To study further the mechanisms underlying the unique patterns of hematopoietic regeneration after cyclophosphamide, mRNA levels for stem cell factor (SCF), Flt-3 ligand, and macrophage inflammatory factor (MIP)-1 alpha cytokines were determined in bone marrow and spleen. Granulocyte precursor cells were less depleted by cyclophosphamide compared to erythroid nucleated cells and lymphocytes both in bone marrow and spleen. Rapid expansion of granulopoietic cells increased the granulocytic/erythroid ratio significantly during regeneration. CFU-S in the bone marrow and the spleen showed different sensitivity in vivo but not in vitro to cyclophosphamide; CFU-GM were equisensitive in both sites. In bone marrow, an initial fast recovery of CFU-S and CFU-GM on days 2 to 3 was followed by a secondary deep decline in their numbers occurring between days 5 and 7. This decline was accompanied with a depression of CFU-S proliferation and with significantly increased CFU-S numbers in the peripheral blood. In the spleen, absolute CFU-S and CFU-GM numbers were increased several-fold at this time. Seven days after cyclophosphamide, the spleen contained 69% of the total body CFU-S compared to 4% in controls. Splenectomy did not abolish the secondary disease of CFU-S in the bone marrow, but it led to a marked elevation of circulating leukocytes and CFU-S. There was an eight-fold increase in the SCF mRNA level in the bone marrow 2 days after cyclophosphamide, corresponding with a high proliferation rate of CFU-S. No significant changes in mRNAs for Flt-3 ligand and MIP-1 alpha have been found. This in-depth analysis of murine hematopoietic responses to cyclophosphamide provides evidence for the complexity of the involved local and systemic regulations. This represents a significant challenge to experimental hematology, which could now be tackled with methods allowing the study of changes in the gene expression during cyclophosphamide-induced hematopoietic damage.
