Abstract: TH-PO994
Advanced Optical Clearing Protocols Enable Fine Analysis of Glomerulus Three-Dimensionally with Confocal Microscopy
Session Information
- Pathology and Lab Medicine: Basic
October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Pathology and Lab Medicine
- 1501 Pathology and Lab Medicine: Basic
Authors
- Yamada, Hiroyuki, Chiba University Graduate School of Medicine, Chiba, Japan
- Makino, Shinichi, Chiba University Graduate School of Medicine, Chiba, Japan
- Miyake, Takafumi, Kyoto University Graduate School of Medicine, Kyoto, Japan
- Yanagita, Motoko, Kyoto University Graduate School of Medicine, Kyoto, Japan
- Asanuma, Katsuhiko, Chiba University Graduate School of Medicine, Chiba, Japan
Background
In order to visualize slit diaphragm of podocyte, it is necessary to use an electron microscopy or super-resolution microscopy. However, there are some disadvantages to these approaches in terms of economics and viewable dimensions. We hypothesized that advanced optical clearing methods with confocal microscopy could enable the three-dimensional (3D) analysis of the fine structure of glomerulus and podocyte.
Methods
From among several advanced optical clearing methods, the following protocols were selected based on the simplicity of the technique and the number of citations: CUBIC, PACT, ScaleS, BABB, iDISCO and SeeDB2. Each protocol with immunostaining against Nephrin were applied to a 1-mm-thick slice of adult rat kidney. We investigated which method was most suitable for observing the fine structure of podocyte with confocal microscopy three-dimensionally. Next, the optimal method among them was applied to anti-glomerular basement membrane (anti-GBM) nephritis rats.
Results
All of these protocols could turn the rat kidney sample transparent. Although each protocol made it possible to visualize the 3D image of the whole glomerulus, a few protocols were capable of visualizing their fine structure of podocytes with confocal microscopy. When analyzing anti-GBM nephritis rat with 3D image, the ratio of the glomeruli with crescent formation in 3D analysis was significantly higher than that in the conventional two-dimensional histological analysis (34.1% versus 14.6%, p<0.01). High magnification of the 3D images showed that the meandering pattern of the slit diaphragm was sustained in the vehicle group while it was partially effaced in the anti-GBM nephritis group.
Conclusion
Advanced optical clearing methods enables a pathological 3D diagnosis of glomeruli and has a high sensitivity for pathological lesions. Moreover, using even confocal microscopy, it could visualize the fine structure of podocyte three-dimensionally. Optical clearing methods could make an outstanding contribution to our clinical practices in many aspects.