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

Abstract

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.

Oxidative stress is involved in the activation of NF-κB signal pathway and immune inflammatory response in grass carp gill induced by cypermethrin and/or sulfamethoxazole

“…The morphological changes of gills are an indicator of early toxicity (Fiedler, et al, 2020). However, it is still unclear whether SMZ and CMN at environmental concentrations can damage sh gills.…”

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

“…To evaluate the comparability of the results of LSFM-based quantitative morphological analyses of cleared adipose tissue samples to the results obtained by unbiased stereological methods, both analysis approaches were performed on adipose tissue specimens SUR sampled from the same adipose tissue depots of the identical pigs. Previously, few other studies have already successfully applied LSFM analyses for examination of distinct quantitative morphological parameters in other types of tissues, e.g., to count the number of glomeruli in murine kidneys [62], to analyse the volume of bronchus associated lymphoid tissue in mice [63], or to determine the volume and surface of fish gill lamellae [64]. A large variety of different techniques for tissue clearing has been developed [65], such as CLARITY, iDISCO, 3DISCO, FRUIT, CLEAR T [20], vDISCO [66], Adipo Clear [27], and ECi [62], optimized for different sample types and various downstream applications.…”

Detection of Diclofenac‐Induced Alterations in Rainbow Trout (Oncorhynchus mykiss) Using Quantitative Stereological Methods

“…A statistically valid comparison of histomorphological alterations requires an objective, unbiased assessment of quantitative (numerical) data of relevant, defined (three‐dimensional) morphological parameters (e.g., numbers, surfaces, lengths, and volumes) of the tissue elements of interest (D. L. Brown, 2017a, 2017b; Gundersen et al, 1988; West, 2012). The gold standards for accurate (i.e., unbiased and precise) quantitative determination of such histomorphological parameters are design‐based quantitative stereological methods, which are widely applied in diverse biomedical research disciplines (Albl et al, 2016; D. L. Brown, 2017b; Fiedler et al, 2020; Gundersen et al, 1988; Hume et al, 2020; Resende et al, 2010). In the present study, gills and trunk kidney, the main target organs for DCF‐induced alterations in fish, were investigated with unbiased stereological methods (1) to gain objective, quantitative results, and (2) to prove reproducibility of data.…”