Objective: Nitric oxide is considered to be the most important messenger of inhibitory nonadrenergic, noncholinergic nerves in the enteric nervous system. Histochemical studies have shown that nitric oxide synthase is identical to reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. Histochemical staining with NADPH diaphorase has been widely used to study nitrergic innervation of the gastrointestinal tract, but fresh tissue is considered a prerequisite for satisfactory results. The purposes of this study were to evaluate whether whole-mount specimens of human bowel obtained after death are suitable for histochemical staining with NADPH diaphorase and to compare the staining properties with those of specimens of resected bowel from patients with Hirschsprung's disease
Methods: Whole-mount preparations of the myenteric plexus were examined using NADPH diaphorase histochemical staining of bowel specimens obtained at autopsy from 18 pediatric subjects (31 specimens). Fresh tissue was also obtained from the colon of four patients with Hirschsprung's disease. The staining properties of postmortem specimens were assessed in relation to the postmortem time before fixation (<12 hours, 13-24 hours, or 25-48 hours) and were compared with those of specimens of ganglionic bowel from patients with Hirschsprung's disease. Specimens of aganglionic bowel were also stained and examined.
Results: Strong NADPH diaphorase staining was achieved in 26 of the 31 postmortem bowel specimens, including all specimens from patients who underwent autopsy 25 to 48 hours after death. Staining properties were similar to those obtained in ganglionic bowel specimens from patients with Hirschsprung's disease. In aganglionic bowel the normal myenteric plexus meshwork was absent and was replaced by weakly staining nerve fibers.
Conclusion: Histochemical staining with NADPH diaphorase is a robust technique suitable for use with whole-mount preparations to demonstrate nitrergic innervation in motility disorders such as Hirshsprung's disease. The technique may be used with both fresh tissue and specimens obtained up to 48 hours after death.