Problem
Millions of people display colonic dysfunction. For instance, irritable bowel syndrome (IBS) and functional constipation affect 1 out of 6 Americans, resulting in decreased quality of life, and a substantial economic burden. Yet, effective therapies are lacking. The enteric nervous system (ENS) plays a key role in regulating colonic functions. The ENS communicates with the central nervous system, intrinsic neuronal circuits, circulating hormones and a wide range of cells including the interstitial cells of Cajal (ICCs) which modulate muscle activity and neuromuscular transmission. There is also a large population of enteric glia cells (EGCs) and immune cells that modulate colonic barrier function and inflammation. The integration of all of these systems is essential for colonic homeostasis, however, the current understanding of them is far from being complete, particularly in humans. While neuromodulatory therapies have shown promise, further investigation is needed to identify the neuroanatomy of cellular targets, and to optimize parameters for effective neuromodulation. This entails a better understanding of the fundamental organization of the peripheral nervous circuits that innervate the colon in humans and relevant experimental models.
Solution
Immunohistochemical labeling is a well-established approach to localize a wide variety of molecules in cells, with high spatial resolution. As such, it is a powerful means to dissect neural pathways at the cellular level. The SPARC team at the University of California in Los Angeles, United States, Flinders University in Adelaide, Australia, Technical University in Munich, and University Veterinary Medicine in Hannover, Germany adapted and developed cutting-edge approaches to obtain a level of detail on neural circuits in the human, pig and mouse colonic tissues that has never been achieved before. Methods included multi-layer immunohistochemistry, tissue clearing (CLARITY), high resolution confocal microscopy, light sheet microscopy and 3D imaging. The first step in developing these approaches was to characterize the suitability of antibodies for labeling structures adequately in different colonic preparations of multiple species (mouse, pig and human) as detailed in SPARC Antibody Labeling Database: Colonic enteric nervous system.
Impact
The comprehensive list of antibodies tested in various preparations of the human colon will provide valuable information to future researchers in choosing antisera to investigate human tissue in health and diseases. A few examples using these approaches are highlighted.
Figure 1 shows three dimensional (3D) features of intrinsic cholinergic innervation in the human sigmoid submucosal plexus (left panel) and myenteric plexus (right panel) for the first time in videos generated from CLARITY-cleared samples combined with immunofluorescence using a novel mouse anti-human peripheral ChAT (hpChAT).
Figure 2 illustrates double immunostaining with the combination of vesicular acetylcholine transporter (VAChT) antibody to label extrinsic/intrinsic (red arrows) and human peripheral choline acetyltransferase (hpChAT) antibody to label selectively intrinsic innervation (green arrows) allowing to simultaneous visualize the extrinsic and intrinsic cholinergic innervation in the human inner submucosal plexus (A) and outer submucosal plexus (B) of human sigmoid colon.
Figure 3 displays other antibodies tested after CLARITY with multiple labeling of the myenteric neurons in the pig proximal colon (upper figure) triple-labeled by pChAT (green), neuronal nitric oxide synthase (nNOS, red) and pan neuronal marker, Hu C/D, blue) and mouse (lower figure) double-labeled by pChAT (red) and nNOS (green).
Multi-layer immunohistochemistry
The novel multi-layer immunohistochemistry approach produces a comprehensive immunohistochemical coding of human colonic myenteric plexusThis novel approach uses analysis of conventional immunofluorescence staining, followed by an elution step in which the antisera are removed from the preparation. A second layer of primary and secondary antisera can then be applied, followed by analysis and elution. With appropriate controls, up to 8 layers of antisera (localizing 24 markers) can be applied to the same population of cells.
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These new developments open new venues to assess the structure-function relationships, and pathological alterations in colonic diseases.
