Histological Analysis  

Whole Mount v. Cross-Sectional Analysis

Whole mount staining of the ENS allows enteric neurons and neuronal fibers to be more easily seen than with traditional sectioning methods. Image (A) shows a hematoxylin and eosin stained section of the small bowel in a newborn mouse. Although enteric neurons can be seen (blue arrows), they are difficult to see clearly. In addition, with cross-sectional analysis, it is difficult to determine whether the number of neurons in any region of the bowel is normal, and neuronal fibers cannot be appreciated. Image (B) shows a whole mount preparation of the muscle layers in the adult mouse small bowel stained with Cuprolinic Blue. In this image, clustered myenteric ganglion cells are easily appreciated.

(C and D) Within the myenteric plexus, clusters of ganglion cells are connected together by thick nerve fiber bundles. Fine neuronal filaments spread out in the regions between these ganglion cell clusters. There neuronal fibers can be easily seen with Acetylcholinesterase (Achase) histochemistry (C) or by NADPH diaphorase staining (D). Both Achase and NADPH diaphorase staining methods show only a subset of neurons within the wall of the bowel. Nonetheless, these methods allow large regions of the ENS to be visualized at once. In fact, the entire small bowel of the mouse can be stained as a single piece. These methods are also much more sensitive than cross sectional analysis for detecting defects in ENS structure.

Benefits of Whole Mount Staining

The potential for whole mount staining to show defects in the enteric nervous system is demonstrated by examination of the myenteric plexus in Neurturin deficient mice (B). Wild type myenteric plexus is shown in Image (A). Acetylcholinesterase stained Neurturin deficient mouse myenteric plexus is shown in (B). The loss of neuronal fibers in the ENS of the Neurturin deficient mouse is obvious with this staining method, but cannot be appreciated in routine histological sections.

Whole mount staining of the newborn gut is also useful. (C) Shows a low power view of the wild type newborn stomach after acetylcholinesterase staining. (D) The loss of neuronal fibers and neurons is easily seen in the GFRalpha 1 KO mouse gut with this whole mount staining approach, but more difficult to appreciate in cross sections of the bowel even using immunohistochemistry to highlight enteric neurons. In Figure (E and F), immunostaining with PGP9.5 antibody shows a yellow ring of neurons and neuronal fibers around the stomach (S) and esophagus (Eso) in both the wild type and knockout mouse.