Linkage between growth retardation and pituitary cell morphology in a dystrophin-deficient pig model of Duchenne muscular dystrophy.

OBJECTIVE: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model. METHODS: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods. RESULTS: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls. CONCLUSION: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.

In Situ Metabolomics in Cancer by Mass Spectrometry Imaging.

Metabolomics is a rapidly evolving and a promising research field with the expectation to improve diagnosis, therapeutic treatment prediction, and prognosis of particular diseases. Among all techniques used to assess the metabolome in biological systems, mass spectrometry imaging is the method of choice to qualitatively and quantitatively analyze metabolite distribution in tissues with a high spatial resolution, thus providing molecular data in relation to cancer histopathology. The technique is ideally suited to study tissues molecular content and is able to provide molecular biomarkers or specific mass signatures which can be used in classification or the prognostic evaluation of tumors. Recently, it was shown that FFPE tissue samples are also suitable for metabolic analyses. This progress in methodology allows access to a highly valuable resource of tissues believed to widen and strengthen metabolic discovery-driven studies.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

Anatomical and histomorphometric observations on the transfer of the anterior interosseous nerve to the deep branch of the ulnar nerve.

This study focuses on the anatomical and histomorphometric features of the transfer of the anterior interosseous nerve to the deep motor branch of the ulnar nerve. The transfer was carried out in 15 cadaver specimens and is described using relevant anatomical landmarks. Nerve samples of donor and target nerves were histomorphometrically analysed and compared. The superficial and the deep ulnar branches had to be separated from each other for a length of 67 mm (SD 12; range 50-85) to reach the site of coaptation. We identified a suitable site for coaptation lying proximal to the pronator quadratus muscle, 202 mm (SD 15; range 185-230) distal to the medial epicondyle of the humerus. The features of the anterior interosseous nerve included a smaller nerve diameter, smaller cross-sectional area of fascicles, fewer fascicles and axons, but a similar axon density. The histomorphometric inferiority of the anterior interosseous nerve raises a question about whether it should be transferred only to selected parts of the deep motor branch of the ulnar nerve.Level III.

A rapid ex vivo tissue model for optimising drug detection and ionisation in MALDI imaging studies.

The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.

Optoacoustic imaging and staging of inflammation in a murine model of arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is one of the most frequent inflammatory diseases, causing pain and disability in the affected joints. Early diagnosis is essential for the efficiency of symptom-targeting treatments, but its diagnosis requires careful clinical, serologic, and imaging examinations, such as magnetic resonance imaging (MRI), which is both expensive and time consuming. In an effort to provide the biomedical community with a more accessible way to assess the advancement of arthritis, this study sought to investigate the use of multispectral optoacoustic tomography (MSOT) in a murine arthritis model, to visualize the extent of inflammation in vivo through an L-selectin/P-selectin-targeting contrast agent. METHODS: Mice with collagen-induced arthritis were studied as a model of RA. MSOT was performed using an L-selectin/P-selectin-targeting contrast agent, polyanionic dendritic polyglycerol sulfate (dPGS) labeled with a near-infrared (NIR) fluorophore, to increase the contrast of the arthritic joint. The signal intensity ratios between healthy legs and arthritic legs were calculated. Findings on contrast-enhanced MRI, clinical observations, the lymphocyte:granulocyte ratio, and histologic findings served as referents for comparison. RESULTS: MSOT using an inflammation-targeting contrast agent, dPGS-NIR, allowed for accurate diagnosis of inflammation in the mouse joints. In addition, use of this technique resulted in significant differentiation of the inflamed joints from the healthy joints (P = 0.023). The observed advancement of arthritis on the MSOT images was confirmed by clinical observation, blood analysis, contrast-enhanced MRI, and ex vivo histologic examinations. CONCLUSION: This study demonstrates that the combination of an inflammation-targeting contrast agent and optoacoustic tomographic imaging presents a promising means for the diagnosis of RA and the staging of arthritis-related inflammation.

High STEAP1 expression is associated with improved outcome of Ewing's sarcoma patients.

BACKGROUND: Ewing's sarcoma (ES) is the second most common bone or soft-tissue sarcoma in childhood and adolescence and features a high propensity to metastasize. The six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is a membrane-bound mesenchymal stem cell marker highly expressed in ES. Here, we investigated the role of STEAP1 as an immunohistological marker for outcome prediction in patients with ES. PATIENTS AND METHODS: Membranous STEAP1 immunoreactivity was analyzed using immunohistochemistry in 114 primary pre-chemotherapy ES of patients diagnosed from 1983 to 2010 and compared with clinical parameters and patient outcome. Median follow-up was 3.85 years (range 0.43-17.51). RESULTS: A total of 62.3% of the ES samples displayed detectable STEAP1 expression with predominant localization of the protein at the plasma membrane. High membranous STEAP1 immunoreactivity was found in 53.5%, which correlated with better overall survival (P=0.021). Accordingly, no or low membranous STEAP1 expression was identified as an independent risk factor in multivariate analysis (hazard ratio 2.65, P=0.036). CONCLUSION: High membranous STEAP1 expression predicts improved outcome and may help to define a specific subgroup of ES patients, who might benefit from adapted therapy regimens.