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Hirschsprung's disease

Hirschsprung's disease, or aganglionic megacolon, involves an enlargement of the colon, caused by bowel obstruction resulting from an aganglionic section of bowel (the normal enteric nerves are absent) that starts at the anus and progresses upwards. The length of bowel that is affected varies but seldom stretches for more than a foot or so. more...

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This disease is named for Harald Hirschsprung, the Danish physician who first described the disease in 1886, describing two infants who had died with swollen bellies. "The autopsies showed identical pictures with a pronounced dilatation and hypertrophy of the colon as the dominant features" (Madsen 17).

Hirschsprung’s disease is a congenital disorder of the colon in which certain nerve cells, known as ganglion cells, are absent, causing chronic constipation (Worman and Ganiats 487). The lack of ganglion cells, proven by Orvar Swenson to be the cause of the disease, disables the muscular peristalsis needed to move stool through the colon, thus creating a blockage. One in five thousand children suffer from Hirschsprung’s. Four times as many males get this disease than females. Hirschsprung’s develops in the fetus during the early stages of pregnancy. Typical symptoms for infants include not having their first bowel movement (meconium) within 48 hours of birth, and repeated vomiting. Some infants may have a swollen abdomen. Two thirds of the cases of Hirschsprung’s are diagnosed within three months of the birth. Occasionally symptoms do not appear until early adulthood. A barium enema is the mainstay of diagnosis of Hirschsprung’s.

The usual treatment is "pull-through" surgery where the portion of the colon that does have nerve cells is pulled through and sewn over the part that lacks nerve cells (National Digestive Diseases Information Clearinghouse). For a long time, Hirschsprung’s was considered a multi-factorial disorder, where a combination of nature and nurture were considered to be the cause (Madsen 19). However, in August of 1993, two articles by independent groups in Nature Genetics said that Hirschsprung’s disease could be mapped to a stretch of chromosome 10 (Angrist 351). This research also suggested that a single gene was responsible for the disorder. However, the researchers were unable to isolate the single gene that they thought caused Hirschsprung’s.

Genetic basis

In 2002, scientists thought they found the solution. According to this new research, the interaction of two variant genes caused Hirschsprung’s. RET was isolated as the gene on chromosome 10, and it was determined that it could have dominant mutations that cause loss of function (Passarge 11). An important gene that RET has to interact with in order for Hirschsprung’s to develop is EDNRB, which is on chromosome 13. Six other genes were discovered to be associated with Hirschsprung’s. According to the study, these genes are GDNF on chromosome 5, EDN3 on chromosome 20, SOX10 on chromosome 22, ECE1 on chromosome 1, NTN on chromosome 19, and SIP1 on chromosome 2. These scientists concluded that the mode of inheritance for Hirschsprung’s is oligogenic inheritance (Passarge 11). This means that two mutated genes interact to cause a disorder. Variations in RET and EDNRB have to coexist in order for a child to get Hirschsprung’s. However, although six other genes were shown to have an effect on Hirschsprung’s, the researchers were unable to determine how they interacted with RET and EDNRB. Thus, the specifics of the origins of the disease are still not completely known.


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NADPH diaphorase-containing nerve fibers and neurons in the myenteric plexus are resistant to postmortem changes: Studies in Hirschsprung's disease and
From Archives of Pathology & Laboratory Medicine, 5/1/98 by Wester, Tomas

Studies in Hirschsprung's Disease and Normal Autopsy Material

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 wholemount 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 (

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 wholemount 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.

(Arch Pathol Lab Med. 1998;122:461-466)

Motility disorders of the gastrointestinal tract occur frequently, but in many cases the pathogenesis remains incompletely understood. These disorders range from Hirschsprung's disease (HD) and diverticular disease to such rare conditions as hollow visceral myopathy. In irritable bowel syndrome, perhaps the most frequent motility disorder of all, no pathogenesis has been identified with current techniques. Factors involved in bowel motility are difficult to study by morphologic methods because of the complexity of the innervation and the musculature, particularly because most current investigational techniques use two-dimensional preparations. Three-dimensional techniques, in which the relationships of branching and interconnecting nerve fibers to each other and to muscle can be clearly seen, are generally believed to be technically complex and suitable for use only with fresh surgically resected tissue, which is rarely available. Tissue obtained at autopsy is often regarded as unsuitable for study, particularly because some antigens are difficult to identify by immunohistochemical techniques in such tissue although many antigens are surprisingly resistant to postmortem autolysisl, and fresh tissue is usually considered a prerequisite for satisfactory enzyme histochemical studies.

Nitric oxide (NO) has recently been recognized as a major neurotransmitter, being the primary mediator of nonadrenergic, noncholinergic neurotransmitter of the gastrointestinal tract.2 The role of NO as a neurotransmitter was supported by results of immunochemical studies showing that NO synthase was expressed in neurons of the myenteric plexus.3 In both brain and peripheral neuronal tissue, NO synthase has been shown to colocalize with reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase.4,5 Histochemical staining with NADPH diaphorase, described in brain tissue by Scherer-Singler et al6 in 1983, has facilitated the identification of neuronal NO synthase. Neuronal NO synthase catalyzes the oxidation of L-arginine to form L-citrulline and NO, a reaction that depends on Ca^sup 2+^ / calmodulin and NADPH. Nitric oxide synthase reduces nitroblue tetrazolium to a water-insoluble, intensely blue formazan, using NADPH as substrate.7

Hirschsprung's disease, a relatively common cause of neonatal bowel obstruction, is characterized by the absence of enteric ganglia in the distal hindgut.8,9 It was recently demonstrated that nitrergic fibers are severely reduced in the aganglionic segments in patients with HD, with nerve fibers of extrinsic origin in the intermuscular plexus staining weakly with NADPH and the absence of fibers in the circular muscle layer.10,11 In a recent study of autonomic nerve maturation, we used a previously described technique to prepare thick, whole-mount sections of gut muscle layers to study the distribution of nerve fibers containing NADPH.l2 This technique gives a threedimensional view of the fibers coursing between and within muscle layers. In this study we applied the technique both to further delineate the pattern of loss of NADPH staining in freshly resected tissue from patients with HD and to compare the staining properties with those found in less optimally preserved tissue. We found that the technique is readily applicable to pathologic tissue such as that from patients with HD and, in particular, is applicable to postmortem material, even after a surprisingly long autolysis period.


Specimens of small bowel and/or proximal colon were obtained at postmortem examination of 18 infants and children (age, 7 days to 9 years) who died of nongastrointestinal causes. Seven patients (13 specimens) underwent postmortem examination within 12 hours after death; eight patients (14 specimens), between 13 and 24 hours after death; and three patients (4 specimens), 25 to 48 hours after death. Tissue was also obtained from the ganglionic and aganglionic segments of the sigmoid colon of four patients with HD; these tissues were fixed within 1 hour of resection. The specimens were opened along the mesenteric border, rinsed, and cut into 2 x 2-cm pieces. Subsequently, they were prefixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.3), stored overnight at +4 deg C, rinsed in phosphate-buffered saline, and stored at -70 deg C until use. Whole-mount preparations of the longitudinal muscle layer and myenteric plexus were made by separating the muscular layers from the submucosal layer, then removing the circular muscle layer from the longitudinal muscle layer with the adherent myenteric plexus. The longitudinal muscle-myenteric plexus laminae were pinned flat and slightly distended on silicone elastomer (Sylgard, Dow Corning, Wiesbaden, Germany) in plastic dishes. For histochemical staining with NADPH diaphorase, the laminae were incubated in 1 mg/mL beta-NADPH (Sigma Chemical Company, Dorset, UK), 0.25 mg/mL nitroblue tetrazolium (Sigma), and 0.3% Triton X-100 in 0.05 mol/L Tris-HCl buffer (pH 7.6) at 37 deg C. The specimens obtained at autopsy and those from the ganglionic segment of patients with HD were examined with a dissecting microscope after 10, 30, 60, 90, and 120 minutes of incubation in the staining solution. Staining intensity of the myenteric plexus was semiquantitatively assessed as strong, moderate, weak, or absent. Background staining intensity, or the diffuse staining of the muscular layer, was also assessed as strong, moderate, weak, or absent. Tissue from the aganglionic segment of the bowel was incubated until the staining intensity of the neuronal structures was assessed as optimal. Staining did not occur in negative control specimens, in which NADPH was omitted from the staining solution.


Strong NADPH staining of nerve fibers and a subpopulation of ganglion cells in the myenteric plexus of the ganglionic area occurred in all four specimens from patients with HD. Surprisingly, staining of similar intensity was observed in 26 of the 31 bowel specimens obtained at autopsy (Fig 1, a and b, and 2, a). In contrast, in specimens of the aganglionic gut, the myenteric plexus meshwork was absent and was replaced by nerve fibers that stained weakly with NADPH diaphorase (Fig 2, b). Remaining circular muscle fibers were present in almost all the whole-mount preparations; circular muscle in ganglionic bowel contained numerous NADPH diaphorase-positive nerve fibers, whereas the circular muscle layer in preparations of aganglionic gut lacked these nerve fibers.

The intensity of staining after incubation periods ranging from 10 to 120 minutes is shown in the Table, which reveals that the staining intensity at the various times for the majority of the tissues obtained at autopsy was identical to that for specimens from patients with HD.

Staining was strong in all four specimens from patients who underwent autopsy 25 to 48 hours after death. After 120 minutes of incubation, the staining intensity was moderate in two specimens from two patients and weak in two specimens from one patient who underwent autopsy 13 to 24 hours after death. One specimen from one patient who underwent autopsy less than 12 hours after death stained weakly. Staining was never strong after 10 minutes of incubation but was strong after 30 minutes in 1 of the 31 specimens obtained at autopsy. Diffuse staining of muscular fibers never made visualization of the myenteric plexus difficult if the circular muscle layer was adequately removed. In 3 of the 31 autopsy specimens, the diffuse background staining was strong after 90 to 120 minutes, but in these cases the neuronal structures stained strongly with weak or moderate background staining at 60 minutes. In 6 of the 31 autopsy specimens, the staining of nerve fibers was very strong after 120 minutes, which made it difficult to distinguish the nerve cell bodies.


Whole-mount preparations elegantly demonstrated the meshlike structure of the myenteric plexus in both normal bowel specimens obtained at autopsy and specimens of the ganglionic portion from the four patients with HD. In contrast, in the aganglionic segments, not only were ganglion cells absent, but the meshlike structure of the nerve plexuses was readily seen to be deficient. These findings confirm those presented in a previous report on the neuronal structure.13 The weak NADPH diaphorase staining of the nerve fibers in the intramuscular space is in line with reports of reduced staining in conventional two-dimensional preparations.10,11 Intramuscular nerve fibers stained with NADPH diaphorase were clearly seen to be abundant in ganglionic bowel but depleted in aganglionic areas, confirming that nitrergic innervation is deficient in HD.10,11 This deficiency of NO, a transmitter that has a major role in muscle relaxation, at the level of individual muscle fibers is likely to be the principal pathophysiological basis for the characteristic motility disorder of HD.l4

In histochemical studies using NADPH diaphorase, fresh tissue from animals or fresh surgical specimens have usually been used.15-17 Some authors have reported using postmortem tissue but did not specify the time from death to autopsy.18 To our knowledge, this is the first study showing that histochemical staining with NADPH diaphorase is a robust technique for demonstrating nitrergic innervation in the human enteric nervous system subject to postmortem changes. Our results indicate that the level of NADPH diaphorase activity is acceptable for histochemical studies using material when the autopsy is performed up to 48 hours after death. Strong NADPH diaphorase staining was observed in 26 of the 31 bowel specimens obtained at autopsy, including all specimens from patients who underwent autopsy 25 to 48 hours after death. It was not obvious why the staining results were unsatisfactory in specimens from four subjects in the study. In one subject bile in the bowel also stained the bowel wall. One died of sudden infant death syndrome, and the other two died of epileptic disease (one of these had cerebral edema at autopsy as a consequence of hypoxia). Smith19 found that the conditions of death were important for the quality of silver impregnations of the myenteric plexus in postmortem material and that anoxia before death resulted in altered staining properties. Events occurring before death may also be a plausible explanation for the decreased NADPH diaphorase activity in some postmortem specimens.

In this study, optimal staining was obtained after 60 to 120 minutes of incubation, which is in accordance with our previous experience with the technique using fresh surgical specimens and postmortem tissue. However, other investigators have reported considerably shorter incubation times (10-30 minutes) for whole-mount preparations of human myenteric plexus.17,18

Several reports describe the influence of postmortem time on the activity of various enzymes in humans and animals. Yamazaki and Wakasugi2 analyzed the activity of several drug-metabolizing enzymes of rat liver microsomes and found that some enzymes had no activity 48 hours after death but that others had almost unchanged activity. Mello de Oliveira and Santos-Martin21 also reported varied sensitivity of liver enzymes to autolysis in human autopsy material and suggested that the time-related decrease of catalytic activity of various enzymes may be a possible method for estimation of time elapsing between death and autopsy. Lindena et al22 examined the catalytic activity of up to 15 enzymes in tissue from different organs in humans and found that, except for pancreas, the influence of postmortem changes on catalytic activity was of minor importance. Goffin23 found that hydrolytic enzymes in human skin are very stable and resistant to postmortem autolysis until putrefaction of the body occurs. Similar results were found when Goffin and Beekmans24 studied the influence of postmortem time on oxidative enzymes, including NADPH tetrazolium reductase, in human skin.

The whole-mount technique requires care in dissecting the circular muscle to avoid damaging the nerve plexus but produces three-dimensional preparations that are more informative than conventional sections in assessing neuronal distribution. This technique facilitates study of whole ganglia in both the normal myenteric plexus and pathologic conditions such as HD. It is particularly suitable for morphometric studies, such as estimation of neuron density, and for the study of nerve fiber distribution and connections. Nitric oxide, a newly recognized neurotransmitter, remains incompletely studied in the gastrointestinal tract. As a marker of NO, NADPH diaphorase has the advantages of being applicable to conventional and whole-mount preparations and suitable for use in studies of motility disorders using material obtained up to 48 hours after death.

This study was supported in part by HKH Kronprinsessan Lovisas forening for bamsjukvird, Ake Wibergs Stiftelse, and the Swedish Medical Research Council (Dr Wester).


1. Knudsen LM, Pallesen G. The preservation and loss of various non-haematopoietic antigens in human post-mortem tissues as demonstrated by monoclonal antibody immunohistological staining. Histopathology. 1986;10:10071014.

2. Sanders KM, Ward SM. Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission. Am J Physiol. 1992;262:G379-G392.

3. Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature.1990;347:768-770.

4. Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissue. Proc Natl Acad Sci USA. 1991;88:7797-7801.

5. Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA. 1991;88:28112814.

6. Scherer-Singler U, Vincent SR, Kimura H, McGeer EG. Demonstration of a unique population of neurons with NADPH diaphorase histochemistry. J Neurosci Methods. 1983;9:229-234.

7. Blottner D, Grozdanovic Z, Gossrau R. Histochemistry of nitric oxide synthase in the nervous system. Histochem J. 1995;27:785-811.

8. Puri P. Hirschsprung's disease. In: Oldham KT, Colombani PM, Foglia RP,

eds. Surgery of Infants and Children. Philadelphia, Pa: Lippincott-Raven; 1997: 1277-1299.

9. Puri P Hirschsprung's disease. In: Puri P, ed. Newborn Surgery. Oxford, UK: Butterworth-Heinemann; 1996:363-378.

10. Kobayashi H, O'Briain DS, Puri P. Lack of expression of NADPH diaphorase and neural cell adhesion molecule (NCAM) in colonic muscle of patients with Hirschsprung's disease. J Pediatr Surg. 1994;29:301-304.

11. Vanderwinden J-M, De Laet M-H, Schiffmann SN, et al. Nitric oxide synthase distribution in the enteric nervous system of Hirschsprung's disease. Gastroenterology 1993;105:969-973.

12. Wester T, O'Briain S, Puri P. The size of ganglia and ganglion cell density in submucosal wholemount preparations of normal human distal colon. I Pediatr Surg. In press.

13. O'Kelly TJ, Davies JR, Tam PKH, Brading AF, Mortensen NJ. Abnormalities of nitric-oxide-producing neurons in Hirschsprung's disease: morphology and implications. J Pediatr Surg. 1994;29:294-300.

14. Bealer JF, Natuzzi ES, Flake AW, Adzick NS, Harrison MR. Effect of nitric oxide on the colonic smooth muscle of patients with Hirschsprung's disease. I Pediatr Surg. 1994;29:1025-1029.

15. Cracco C, Filogamo G. Quantitative study of the NADPH-diaphorasepositive myenteric neurons of the rat ileum. Neuroscience.1994;61:351-359.

16. O'Kelly TJ, Davies JR, Brading AF, Mortensen NJ. Distribution of nitric oxide synthase containing neurons in the rectal myenteric plexus and anal

canal: morphologic evidence that nitric oxide mediates the rectoanal inhibitory reflex. Dis Colon Rectum. 194;37:350-357.

17. Krammer H-J, Zhang M, Kunel W. Distribution of NADPH-diaphorasepositive neurons in the enteric nervous system of the human colon. Ann Anat. 1994;176:137-141.

18. Timmermans J-P, Barbiers M, Scheuermann DW, et al. Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract. Cell Tissue Res. 1994;275:235-245.

19. Smith B. The Neuropathology of the Alimentary Tract London, UK: Edward Arnold Publishers; 1972.

20. Yamazaki M, Wakasugi C. Postmortem changes in drug-metabolizingenzymes of rat liver microsome. Forensic Sci Int. 1994;67:155-168.

21. Mello de Oliveira JA, Santos-Martin CC. Enzyme histochemistry of the liver in autopsy material at different postmortem time. Med Sci Law.1995;35: 201-206.

22. Lindena J, Sommerfeld U, Hopfel C, Trautschold I. Catalytic enzyme activity concentration in tissues of man, dog, rabbit, guinea pig, rat and mouse: approach to a quantitative diagnostic enzymology, Ill. Communication. I Clin Chem Clin Biochem. 1986;24:35-47.

23. Goffin Y. Postmortem variations and effect of autolysis on some hydrolytic enzymes of the skin and skin appendages. Acta Pathol Microbiol Scand. 1968;73:351-358.

24. Goffin Y, Beekmans Y. Histochemistry of succinate dehydrogenase, glucose-6-phosphate dehydrogenase and NADPH tetrazolium reductase in human skin. Forensic Sci. 1973;2:221-231.

Accepted for publication January 8, 1998. From Children's Research Centre, Our Lady's Hospital for Sick Children, Dublin, Ireland.

Reprint requests to Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin 12, Ireland (Mr Puri).

Copyright College of American Pathologists May 1998
Provided by ProQuest Information and Learning Company. All rights Reserved

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