Gluhovic Vladimir, Timm Sara, Kuebler Wolfgang M, Lopez-Rodriguez Elena, Ochs Matthias
Institute of Functional Anatomy, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Core Facility Electron Microscopy, Charité-Universitätsmedizin Berlin, Berlin, Germany.
J Anat. 2025 May;246(5):770-781. doi: 10.1111/joa.14129. Epub 2024 Sep 8.
The alveolar surface of the lung is lined by an epithelium consisting of type I (AECI) and type II alveolar epithelial cells (AECII). This epithelium is covered by a liquid alveolar lining layer (ALL). Besides intra-alveolar surfactant, ALL also contains the alveolar epithelial glycocalyx on the apical side of AECI and AECII. To better understand the alveolar epithelial glycocalyx, its ultrastructural visualization by transmission electron microscopy is required. The aim of this study was to systematically re-evaluate routine cytochemical methods for visualization of the alveolar epithelial glycocalyx and specifically its glycan components. For this purpose, we used chemical fixation by vascular perfusion with aldehydes as a common routine approach in mice. After fixation, staining is needed for glycocalyx visualization. Cytochemical staining agents such as alcian blue, ruthenium red, and lanthanum nitrate were compared. In addition, SNL (Sambucus nigra lectin) and UEA1 (Ulex europaeus agglutinin I) were used for sialic acid and fucose-specific labeling. Alcian blue showed the strongest staining, with cloud-like structures, whereas ruthenium red appeared as thread-like structures. On the other hand, lanthanum nitrate did not stain the alveolar epithelial glycocalyx. For specific sialic acid and fucose labeling, both lectins presented a specific signal. In conclusion, these methods can be used routinely for assessing ultrastructural changes of the alveolar epithelial glycocalyx in experimental in vivo models under different physiological and pathological conditions. In addition, cytochemical staining by tissue massage and post-embedding lectin labeling after vascular perfusion support 3R (reduction, refinement, replacement) principles of animal welfare.
肺的肺泡表面由包含I型（AECI）和II型肺泡上皮细胞（AECII）的上皮细胞排列而成。该上皮细胞被液体肺泡内衬层（ALL）覆盖。除了肺泡内表面活性物质外，ALL在AECI和AECII的顶端还含有肺泡上皮糖萼。为了更好地理解肺泡上皮糖萼，需要通过透射电子显微镜对其进行超微结构观察。本研究的目的是系统地重新评估用于观察肺泡上皮糖萼及其聚糖成分的常规细胞化学方法。为此，我们采用醛类血管灌注化学固定法，这是小鼠常用的常规方法。固定后，需要进行染色以观察糖萼。比较了阿尔辛蓝、钌红和硝酸镧等细胞化学染色剂。此外，还使用了黑接骨木凝集素（SNL）和荆豆凝集素I（UEA1）进行唾液酸和岩藻糖特异性标记。阿尔辛蓝染色最强，呈云状结构，而钌红呈线状结构。另一方面，硝酸镧未对肺泡上皮糖萼染色。对于特异性唾液酸和岩藻糖标记，两种凝集素均呈现特异性信号。总之，这些方法可常规用于评估不同生理和病理条件下实验体内模型中肺泡上皮糖萼的超微结构变化。此外，血管灌注后通过组织按摩进行细胞化学染色和包埋后凝集素标记符合动物福利的3R（减少、优化、替代）原则。
