We found loss of calretinin immunostaining as a result of the absence of ganglion cells in the aganglionic colon of HD.
The pathological diagnosis of HD is established by demonstrating the lack of ganglion cells in the colonic neural plexus. The traditional approach is a full thickness segment,14
but alternatives such as “suction” or mucosal rectal biopsies are also frequently used.4,15
Suction biopsies are more difficult to interpret than full thickness biopsies because they only show the superficial portion of the submucosal neural plexus, and do not contain the more abundantly ganglionated and more easily evaluated myenteric plexus.8
The loss of ganglion cells is usually associated with the proliferation of nerve fibres. Acetylcholinesterase stains, mainly in frozen tissues, demonstrate the increased network of coarse, thickened, and irregular cholinergic nerve fibres within the muscularis mucosa in affected segments.3
The nerve fibres may also be highlighted using immunohistochemical stains such as PGP 9.5 or antibodies to S100, NSE, neurofilament protein, or the microtubule associated protein MAP5. NSE immunostains produce intense staining of ganglia, facilitating the recognition of small immature ganglion cells. S100 immunostaining highlights ganglion cells as prominent negatively stained cells surrounded by positive Schwann cells. However, despite the fact that these immunomarkers can facilitate the diagnosis in some cases, many specimens are still difficult to evaluate and may require careful and repeat deeper sections. In addition, the fact that the loss of calretinin expression in aganglionic segments was evident both in the submucosal and myenteric plexus, might help in the interpretation of smaller or submucosal biopsies.
The enteric nervous system, which controls the gastrointestinal tract, is composed of different types of neurones, which subserve various functions.7
In the normal colon, these neurones include motor neurones to the circular and longitudinal muscle layers, sensory neurones, ascending and descending interneurones, and secretomotor neurones.9
Immunohistochemical markers of many types have been used in an attempt to differentiate the functional subclasses of neurones in the enteric nervous system. These include antibodies directed against the following: (1) various peptides, such as the C-terminal region of substance P, tachykinins, vasoactive intestinal peptide, enkephalin, and neuropeptide Y; (2) enzymes, such as tyrosine hydroxylase, choline acetyltransferase, and nitric oxide synthase; and (3) the calcium binding proteins calretinin and calbindin.6,7
The calcium binding proteins are involved in the physiological buffering of excess cytosolic calcium ions, calcium transport, and protection against calcium ion overload.16–18
In their absence, there is an accumulation of excess calcium ions inside the cytoplasm, causing hyperexcitability, which often leads to neurodegeneration.16,18
Nerve cell bodies in both submucosa and myenteric ganglia of guinea pig19,20
and in the human gastrointestinal tract were found to show immunopositivity for calretinin and calbindin.21
In a previous study, immunoreactivity to calretinin was present in 23% of neurones projecting anally and 3% projecting orally within the myenteric plexus.9
Myenteric neurones with calretinin immunoreactivity were demonstrated with projections to the circular muscle in the myenteric ganglia in both the mucosa and submucosa. In addition, the interneurones with calretinin immunoreactivity were found to have anal polarity.9
“The fact that the loss of calretinin expression in aganglionic segments was evident both in the submucosal and myenteric plexus, might help in the interpretation of smaller or submucosal biopsies”
In our study, there was an absence of immunostaining for calretinin in the aganglionic colon of HD. No ganglion cells were noted. In addition, immunoexpression of calretinin by nerve fibres, which was present in the ganglionic colon of HD and in normal colon, was absent. This contrasts with the prominent nerve fibre proliferation demonstrated by S100 immunostaining. Therefore, the lack of calretinin immunostaining in the nerve fibres also represents the absence of related ganglion cells.
Take home messages
- Ganglion cells and nerve fibres express calretinin in ganglionic areas of Hirschsprung’s disease (HD) and in normal colon, whereas in patients with HD aganglionic segments and their associated nerve fibres show a lack of expression
- The absence of calretinin expression may serve as a diagnostic aid in identifying aganglionic segments in HD
ICCs are distributed throughout the gastrointestinal tract and are thought to be pacemaker cells, which control neural and muscular intestinal activity.22
A recent study has demonstrated an altered distribution of ICCs in HD. Moreover, abnormalities in ICC organisation were found not only in the aganglionic segments, but in the entire resected bowel, suggesting that the persistent dysmobility problems after surgery in HD may be the result of the altered distribution and impaired function of ICCs throughout the colon.8
Special attention was given to the transitional zone, which was characterised by small and sparse ganglia accompanied by hypertrophic nerve bundles. The ICCs were evident as single cells or cell clusters closely related to the small myenteric ganglia, without the formation of the typical network of the normal bowel. In our study, in addition to the abnormalities of the organisation of the ICCs already described, some of the ganglion cells were immunonegative for calretinin in the transitional zone, whereas the nerve fibres were mostly positive. Calretinin immunopositive nerve fibres may represent connections to proximal calretinin positive ganglion cells. However, the abnormalities in the organisation of the ICCs in combination with the alterations in calretinin immunoreactivity in these regions may be some of the developmental abnormalities that occur between the ganglionic colon and the obvious aganglionic colon.
In summary, there was a lack of immunostaining for calretinin in aganglionic segments in patients with HD and in the nerve fibres in these areas, whereas both ganglion cells and nerve fibres showed calretinin expression in ganglionic areas of HD and in normal colon.