Whole-mount NADPH-diaphorase histochemistry is a reliable technique for the intraoperative evaluation of extent of aganglionosis

Abstract

Multiple seromuscular biopsies at three levels (narrow segment, transitional zone, and dilated segment) were taken and investigated intraoperatively to determine the extent of aganglionosis. Using the whole-mount preparation technique, circular muscle fibers were separated from the specimens. After a short prefixation, the muscle fibers were stained by the NADPH-diaphorase technique and were examined within 20-25 min. A fine and dense neuronal meshwork was observed between circular muscle fibers in the normal and ganglionic part of the bowel. In contrast, there was a complete lack of NADPH-diaphorase-positive fibers in the circular muscle of aganglionic colon. In the transitional zone, NADPH-diaphorase-positive fibers were markedly reduced compared to the ganglionic region. The density of these fibers increased and attained normal levels in the proximal bowel above the transition zone. These results suggest that whole-mount NADPH-diaphorase histochemistry is a three-dimensional technique suitable for the intraoperative evaluation of extend of aganglionosis. The technique is sufficiently rapid to be used in conjunction with routine frozen sections to assist in the diagnosis and in selecting the optimal level of resection at the time of pull-through operation.

Calretinin, β‐Tubulin Immunohistochemistry, and Submucosal Nerve Trunks Morphology in Hirschsprung Disease

“…The transition zone interposed between aganglionic and ganglionic segments is a critical area from a surgical point of view because it is not yet clearly established whether the presence of ganglion cells itself is a sufficient evidence of good intestinal innervation. For this reason, some authors suggest the use of additional investigations, as intraoperative rapid histoenzymatic/immunohistochemical stainings or determination of nerve hypertrophy, to obtain further anatomic information about the surgical margin (27–31). In our study, the presumptive transition zone was accurately studied with HE, calretinin, and morphometric analysis of nerve trunk diameter.…”

“…For this reason, some authors suggest the use of additional investigations, as intraoperative rapid histoenzymatic/ immunohistochemical stainings or determination of nerve hypertrophy, to obtain further anatomic information about the surgical margin (27)(28)(29)(30)(31). For this reason, some authors suggest the use of additional investigations, as intraoperative rapid histoenzymatic/ immunohistochemical stainings or determination of nerve hypertrophy, to obtain further anatomic information about the surgical margin (27)(28)(29)(30)(31).…”

Acetylcholinesterase in Hirschsprung?s disease

“…Further modifications involved the use of the specific BChE inhibitor ethopropazine to eliminate reaction of the acetylthiocholine substrate with this enzyme as well as AChE [13] and the use of magnesium sulphate suppression to counteract the staining of contaminating red blood cell AChE in haemorrhagic specimens [67]. Rapid staining AChE techniques have been devised for use during surgery [21,50,73] to eliminate difficulties that may be encountered with frozen sections.…”

The Expression of Enteric Nerve Markers and Nerve Innervation in Total Colonic Aganglionosis

“…Sporadic reports have stated that peripherin was seen, via a positive immune reaction, in hypertrophic nerve trunks in the aganglionic segment of the HD group, whereas no or only weak expression was found in the TCA group. 9 Our study suggests that PGP9.5, S-100, NSE, and peripherin have similar expression levels in the ganglionic (near-end) enteric segment of TCA, HD, and controls, with the strongest expression being of PGP9.5, possibly because of its ability to be expressed in submucous nerve plexi and ganglion cells. However, PGP9.5, S-100, NSE, and peripherin all had significantly lower levels of expression in the aganglionic enteric segments and transitional segments of the TCA group compared with the HD group.…”

“…For both clinicians and pathologists, presurgical determination of the length of the affected segment in patients with TCA, especially for the transitional segment (in which there are diminishing numbers of ganglion cells), remains a challenge. 5,9 Other than a lack of ganglion cells in the whole colonic wall, there does not exist a universal standard for the morphological diagnosis of TCA. Techniques commonly used in diagnosing common-segment-type HD include HE staining and AchE staining; however, AchE staining is atypical in TCA, and research shows that the intestinal muscularis of TCA has only low-density cholinergic fibers.…”