Institute of Veterinary Pathology at the Center for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität München, Munich, Germany.
Unit 73 Aquatic Ecotoxicology, Microbial Ecology, Bavarian Environment Agency, Wielenbach, Germany.
PLoS One. 2020 Dec 9;15(12):e0243462. doi: 10.1371/journal.pone.0243462. eCollection 2020.
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.
虹鳟（Oncorhynchus mykiss）常用于生态毒理学研究中的实验动物，在实验中，它们会被暴露于定义浓度的测试物质中，例如重金属、农药或药物。在暴露于各种水生污染物后，早期形态学上可检测到的毒性效应通常表现为鳃的改变。因此，准确和无偏的定量描述形态学鳃改变的类型和程度的合适方法是识别、客观评估和比较鳃损伤严重程度的必要前提。本指南的目的是提供实用、标准化和详细的协议，用于对虹鳟鳃的相关形态学参数进行无偏定量体视学分析。这些鳃参数包括初级和次级鳃瓣的总体积、次级鳃瓣上皮的表面积（即呼吸表面）和扩散屏障的厚度等。所介绍的方案适用于常用体型类别（300-2000 克）的鱼类。它们包括经过验证的、常规的采样方法、用于无偏定量体视学分析光镜和电子显微镜 2D 鳃切片的探头和测试系统，以及作为一种创新、快速和有效的定量形态分析方法的光学透明鳃样本的现代 3D 光片荧光显微镜（LSFM）的应用。这里展示的方法为标准化和具有代表性的现代定量形态学分析虹鳟鳃提供了基础，确保了分析结果的无偏性和可重复性，以及研究内和研究间的可比性。它们的广泛实施将极大地促进在生态毒理学研究中可靠地确定无观察效应浓度（NOEC）限值，并通过减少不必要的实验重复来减少实验动物的数量。
