Standardized tissue sampling guidelines for histopathological and molecular analyses of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

In ecotoxicology, evaluation of toxicities and no observed effect concentrations (NOEC) of test compounds in experimental fish is commonly based on molecular-, biochemical- and analytical chemistry analyses of organ/tissue samples and the assessment of (histo-) pathological lesions. Standardization of organ/tissue sampling locations, sample numbers, and sample processing contributes to warrant the reproducibility and inter- and intra-study comparability of analysis results. The present article provides the first comprehensive tissue sampling guidelines specifically adapted to rainbow trout (Oncorhynchus mykiss) as a frequently used fish species in ecotoxicological studies. A broad spectrum of ~40 different organs and tissues is covered. Appropriate sampling locations, sample sizes and sample numbers for subsequent routine histopathological evaluation (all organs/tissue) and for molecular analyses (~30 organs/tissues) are described in detail and illustrated with schematic drawings and representative macroscopic and histological images. These field-proven sampling guidelines were developed based on the pertinent literature and practical experience in ecotoxicological fish studies. They are intended to serve as a standard reference for any routine ecotoxicological study using rainbow trout as a test system. A broad application of the featured tissue sampling procedures will help to improve the reproducibility of analyses and to reduce inter- and intra-study variability induced by sampling bias and (normal) inter-sample morphological variation, and will therefore provide a robust basis for reliable characterization of toxicity and NOEC identification of diverse test substances and aquatic pollutants.

A new method for physical disector analyses of numbers and mean volumes of immunohistochemically labeled cells in paraffin sections.

BACKGROUND: In the neurosciences, the physical disector method represents an established quantitative stereological method for unbiased sampling and counting of cells in histological tissue sections of known thickness. Physical disector analyses are conventionally performed using plastic-embedded tissue samples, because plastic-embedding causes a comparably low and definable shrinkage of the embedded tissue, and the thickness of thin plastic sections can be determined adequately. However, immunohistochemistry protocols often don't work satisfactorily in sections of plastic-embedded tissue. NEW METHOD: Here, a new methodological approach is presented, allowing for physical disector analyses of immunohistochemically labeled cells in paraffin sections. The embedding-related tissue shrinkage is standardized by using defined tissue sample volumes and paraffin volumes, and the extent of tissue shrinkage can be determined accurately from the sample volumes prior to and after embedding. Co-embedding of polyethylene section thickness standards together with the tissue samples allows the precise determination of individual paraffin section thicknesses by spectral reflectance measurements. RESULTS AND COMPARISON WITH EXISTING METHOD(S): The applicability of the new method is demonstrated by physical disector analysis of immunohistochemically identified somatotroph cells in paraffin sections of porcine pituitary gland tissue. With consideration of individual shrinkage factors and section thicknesses, the cell numbers and mean volumes estimated in paraffin disector sections do not significantly differ from the results obtained by analyses of plastic-embedded pituitary tissue samples of the identical animals (2.4% average difference). CONCLUSIONS: The featured method enables combination of paraffin section immunohistochemistry and physical disector analyses for unbiased quantitative stereological analyses of different cell types.

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy.

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species.

A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300-2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.