ABSTRACT
The enteric nervous system (ENS), sometimes considered as a ‘second brain’ due to its large autonomy from central circuits is made of interconnected plexuses organized in a mesh-like network lining the gastrointestinal tract. Originally described as a leading actor of the regulation of digestion, bowel advance and intestinal secretion, its implication in various neuropathologies has recently been demonstrated. However, with few exceptions, its morphology and functions have been studied on thin sections of the intestinal wall or in dissected explants. Due to its intricate morphology, precious information on its three-dimensional (3-D) architecture and connectivity is often lost. In this context, we have developed a fast, label-free 3-D imaging method of the ENS, based on intrinsic signals of the tissue. We adapted a fast tissue-clearing protocol based on a high refractive-index aqueous solution, and then characterized the autofluorescence signals arising from the various cellular and sub-cellular components of the ENS. Immunofluorescence and spectral recordings complete this characterization. Finally, we demonstrate the fast acquisition of detailed 3-D image stacks of unlabeled mouse intestine, across the whole intestinal wall and including both the myenteric and submucosal enteric nervous plexuses using a new spinning-disk two-photon microscope. The combination of fast clearing (less than 15min for 73 % transparency), autofluorescence imaging and rapid volumetric imaging (less than a minute for the acquisition of s z-stack of 100 planes (150*150 µm) at 300-nm spatial resolution) paves the way for new applications in fundamental and clinical research.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵† these authors share last authorship.
↵# elements of this work were part of the Master 1 internship of Doriane Hazart (2021) « Imagerie d’autofluorescence rapide et tridimensionnelle du système nerveux entérique sur un prototype de microscope biphotonique à disque rotatif unique ».
↵$ this work was initiated when Doriane Hazart was a student at the Faculté des Sciences Fondamentales et Biomédicales, Université Paris Descartes, Paris, F-75006, France (Licence 3: Sciences pour la Santé).