Aganglionosis of the Colon

Path. Res. Pract. 178, 543-547 (1984)

Aganglionosis of the Colon Morphologic investigations in 524 patients H. W. Ziegler, Ph. U. Heitz, M. Kasper, H.-P. Spichtin and J. Ulrich Department of Pathology, University of Basel, Basel, Switzerland

SUMMARY

2014 biopsies and surgical specimens of 524 patients with suspected aganglionosis of the colon were analyzed using enzyme histochemical techniques for acetylcholinesterase and lactate dehydrogenase. The diagnosis could be confirmed in 70 patients. Hypoganglionosis was found in 6, neuronal dysplasia (or hyperganglionosis) in 2, and a completelack of intramural neurons and nerve fibers in 1 patient. The disease was found to occur most often during the first year of life (65.7%) and to be more common in males than females (3.3 : 1).

Enzyme histochemistry is useful for the [mal diagnosis of aganglionosis provided the biopsy contains mucosa and parts of the submucosa of the colon.

Introduction The coordination of bowel activities is effected by a highly complex enteric nervous system". Impaired regulation often results in impedence of the passage of the bowel contents. In 1887 the danish physician Hirschsprung described 2 patients with severe obstipation caused by congenital megacolon. Many years later Hirschsprung's disease was Eostulated to be a developmental defect of the distal colon 8.

Aganglionosis is characterized by a lack of neurons of the plexus submucosus (Meissner's plexus) and myentericus (Auerbach's plexus) and hyperplasia of cholinergic parasympathetic nerve fibers in the circular muscle layer, muscularis mucosae and mucosa 19. These findings could be confirmed by biochemical determination of acetylcholinesterase levels in the rectal mucosa of patients with aganglionosis'. The aganglionotic part of the colon IS a functional stenosis caused by the permanent contraction of the longitudinal muscle layer. This is due to the lack of intramural neurons. The colon proximal to the stenosis is markedly dilated (secondary megacolon."). The agang© 1984 by GustavFischerVerlag, Stuttgart

lionotic segment of the colon is most often localized in the rectum or sigmoid colon. This is probably due to an arrest during the development of the innervation of the colon wall, normally progressing from the proximal to distal parts. In rare cases the entire colon is devoid of intramural neurons". In a small number of patients, an agan~ionosis of the colon and ileum", of the entire intestine':" ,27, and the stomach I? was reported. Hypoganglionosis ("Pseudo-Hirschsprung's disease") is defined by a decreased number of intramural neurons. Groups of neurons are lacking. The disease is uncommon. The symptoms closely resemble those of aganglionosis. The disease may occur isolated or combined with aganglionosis. A third disease which clinically resembles aganglionosis is neuronal dysplasia. It is characterized by a hyperplasia of neurons and cholinergic nerve fibers in all layers of the gut wall. Neuronal bodies associated with muscle fibers in the mucosa have been observed'< 19, 21, 25. Despite the well-known clinical picture, the diagnosis of aganglionosis is not self-evident. Biopsy of the colon is often necessary and the pathologist's diagnosis is therefore conclusive. Histochemical reactions for acetylcholinester0344-0338/84/0178-0543$3.50/0

544

. H. W. Ziegler, Ph. U. Hertz, M. Kasper, H.-P. Spichtin and

ase (E.C. 3.1.1:7) and lactate dehydrogenase (E.C. 1.1.1.27) were introduced during the sixties in order to make more accurate diagnosis of aganglionosis'" 19. The purpose of this paper is to describe our experience over a 10 year period with the diagnosis of aganglionosis of the colon by means of enzyme histochemistry.

Material and Methods During 10 years (1.4.73- 31.3.83) we received 2014 suction biopsy and surgical samples of 524 patients (331 = 63.2 % males, 193 = 36.8% females) for diagnosis of aganglionosis. The age of the patients varied between 1 day and 51 years. The biopsies were taken for clinical evidence of an aganglionotic segment or for control of the remaining colon after an operation for aganglionosis. They were sent by special mail in humid Petri dishes on ice in a Dewar flask. In the laboratory the biopsies were quenched in melting isopentane (2-methyl-butane) at - 160 °C and cut in a cryostate (12 urn) perpendicularly to the surface of the mucosa. The sections were then stained with H + E and 6 to 24 sections were incubated for acetylcholinesterase", and for lactate dehydrogenase!' . All sections were viewed by two pathologists. The diagnosis of aganglionosis was based on the lack of intramural neuron s and on the hyperplasia of cholinergic nerve fibers In the mucosa, muscularis mucosae, submucosa and, if present, in the circular muscle layer.

Fig. 1 a. Suction biopsy of the colon with normal innervation. Acetylcholinesterase, counterstained with H. x 50.

J.

Ulrich Results

In biopsies taken from 470 patients with clinically suspected aganglionosis the diagnosis could be confirmed by histologic and histochemical examination (Fig. 1 a and b) in 70 patients (Table 1). In 46 patients the disease was diagnosed during the first year of life, in 60 patients before the age of 5 years. In 49 patients the length of the aganglionotic segment was less than 5 em , in additional 15 patients, less than 10 em (T able 2). In 12 patients, the diagnosis of aganglionosis could be confirmed subsequently on surgically resected specimens. In the 2 patients with a questionable diagnosis of aganglionosis (Table 1), the biopsies consisted only of mucosal tissue. The diagnosis was therefore considered to be questionable. In 2 additional patients, a false negative diagnosis was first made. In 1 patient, the biopsies were severely damaged due to a longlasting transport. In the second patient the false negative diagnosis was possibly due to a technical error during processing of the biopsies. In both patients the diagnosis of aganglionosis could be confirmed by subsequent biopsies (bot h patients are included under the diagnosis "aganglionosis" in Table 1).

Fig. 1 b. Aganglionosis of the colon. Large number of cholinergic nerve fibers in submucosa, musucularis mucosae and mucosa. Lack of neuronal bodies in the submucosa. Acetylcholinesterase, counterstained with H. x 70.

Aganglionosis of the Colon

. 545

ill

Table 1. Morphological diagnosis in 470 patients

d'

'i?

d'+'i?

Aganglionosis Aganglionosis questionable Hypoganglionosis Hypoganglionosis questionable Neuronal dysplasia Lack of innervation No morphological diagnosis "Normal" innervation

54 1 5 0 0 0 5 222

16 1 1 1 2 1 2 159

70 2 6 1 2 1 7 382

Total Percentage of aganglionosis

287 18.8

183 8.7

470 14.9

Table 2. Length of the aganglionotic segment 10 70 patients Age at biopsy 0-1 (years) d' 'i?

1-5

d' 'i?

total

5-12

cJ' 'i?

d' 'i?

length of aganglionotic segment (ern)

0-

5

0- 10 0->10

Total Percentage

24 9

7 4

2 0

90 1 1 1 1

33 13 65.7%

112 18.5%

5 1 2 0 1 0

1 0 0 0 1 0

39 10 10 5 5 1

8 1 12.8%

2 0 2.8%

54 16 100%

Fig. 2. Neuronal dysplasia (or hyperganglionosis) of the colon. Very large bundles of nerve fibers and large groups of neuronal bodies in the muscle layers. Lactate dehydrogenase. x 100.

Hypoganglionosis was found in 6 patients. The diagnosis was based on the presence of stenosis of a colonic segment and on the finding of a decreased number of neurons in presence of a large number of cholinergic nerve fibers in the submucosa of the colon. Neuronal dysplasia (Fig. 2) was detected in 2 girls at the age of 3 days and 7 years respectively. The symptoms were identical to those found in aganglionosis. A striking hyperplasia of neurons in the submucosa and of cholinergic nerve fibers in the submucosa and mucosa were found.

Some neurons were present in the mucosa. The muscularis mucosae was very thin. In 7 patients, no decision could be made because the biopsies were taken within the area of anal mucosa, because of formaldehyde fixation or because of a longlasting transport. In 54 patients with known aganglionosis, a control biopsy was taken within 1-20 years after surgery. In 19 patients a normal innervation was found, whereas in 25 patients an aganglionotic segment remained. In 1 patient, an aganglionosis of the entire colon could be detected. In 3 patients, a hypoganglionosis persisted and in 5 patients, no decision could be made. In 1 patient, the persistence of neuronal dysplasia was confirmed. A particularly striking picture was found in a female, i.e. a total lack of nerve fibers and neurons in the wall of the entire gastrointestinal tract (Fig. 3).

Fig. 3. Ileum. Lack of irmervation of the entire intestinal wall. Acetylcholinesterase, counterstained with H. x 42.

546 . H. W. Ziegler, Ph. U. Heitz, M. Kasper, H.-P. Spichtin and J. Ulrich

Discussion The diagnosis of aganglionosis is based on the following findings: 1. anamnestic data and the clinical picture of ileus or chronic constipation, 2. an X-ray examination and/or enema disclosing a contracted segment of the colon and a proximal dilation, 3. manometric examination of the rectum'< 22, 24, 26, and 4. histologic and histochemical examination of colon biopsies. In general, biopsies including the mucosa, muscularis mucosae and superficial parts of the submucosa are suitable for a safe diagnosis provided they are sent unfixed on ice and the transport lasts less than 10 hours. In this series a morphologic diagnosis could not be reached in 65 of 2014 biopsies, i.e, in 7 of 470 patients (less than 1.5%). In 2 patients (0.43%), a false-negative diagnosis was corrected by subsequent biopsies. In addition, the diagnosis could be confirmed in 12 surgical specimens.Morphologic diagnosis of aganglionosis is therefore very reliable'", In this series, aganglionosis of the colon was found to be much more common in males than in females. The clinical suspicion of aganglionosis could be confirmed morphologicallyin almost one fifth of the male and in little less than 9% of the female patients. The disease was found to occur most often before the age of 5 years and often during the first months of life. These findings confirm and extend results reported previously'", The diagnosis of hypoganglionosis is difficult without systematic application of morphometric analysis. The diagnosis was made in 6 patients based on the clinical picture, and on the basis of the small number of intramural neurons present in the submucosa of the colon. In our opinion this diagnosis should be avoided unless extensive quantitative analysis can be done. Neuronal dysplasia of the colon is apparently very rare. Its symptoms closely resemble those of aganglionosis, but the morphologic picture is entirely different. The contraction of the colon is possibly caused by the absence of contraction of the longitudinal muscle layer because of hyperplasia of the cholinergic nerve fibers. In analogy to the term "aganglionosis", the term "neuronal dysplasia", a misnomer, could be substituted by the term "hyperganglionosis", as proposed by Garret and Howard (1981). Total lack of innervation of the gastrointestinal tract appears to be extremely uncommon. In our patient, neither cholinergic nor adrenergic fibers could be visualized in the stomach, small or large intestine. The girl died at the age of 2 months. At autopsy no further malformations could be detected. It is of interest to the surgeon that an aganglionotic segment was found to persist after surgery in 26 out of 54 patients. Care must be taken to resect a segment longer than the macroscopically contracted part of the colon. In addition, it is important to realize that the extent of aganglionosis in the submucosa is apparently greater than the hyperplasia of cholinergic nerve fibers detected in a biopsy containing only mucosa, a finding previously reported by Hamoudi et al. (1982).

The histochemical diagnosis of aganglionosis in biopsy material is safe provided an experienced technician processes and an experienced pathologist examines the specimens. On the other hand, the procedure is cumbersome considering the necessarily fast transport on ice of the specimens, the cryostate sectioning, and the histochemical reactions involved. Time and costs can be saved by optimizing aliquoting and storage of the histochemical reagents. Despite these difficulties, the final diagnosis of aganglionosis must be based on a careful morphologic evaluation of colon biopsies since it implies a surgical resection of the aganglionotic segment in often very young infants. Although abnormalities in aganglionosis are very complex', the diagnosis can be made by using two relatively simple enzyme histochemical reactions. In the near future it will probably be possible to supersede enzyme histochemistry by immunocytochemical techniques which can be carried out on routinely fixed tissue. This procedure would cancel the aforementioned disadvantages of enzyme histochemistry. References 1 Ahmed 5, Cohen 5J,Jacobs 51 (1971) Totalintestinal aganglionosis presenting as duodenal obstruction. Arch Dis Childh 46:

868-869

2 Berdon WE, Koontz P, Baker DH (1964) Diagnosis of colonic and terminal ileal aganglionosis. Am J Dis Roentgenol 91:

680-689 3

Bishop AE, Polak JM, Lake BD, Bryant MG, Bloom SR

(1981) Abnormalities of the colonic regulatory peptides in Hirschsprung's disease. Histopathology 5: 679-688 4 Costa M, Furness JB (1982) Neuronal peptides in the intestine. Br MedBull 38: 247-252 5 Dale G, Lowdon P, Rangecroft L, Bonham JR, Wagget J, Scott DJ (1979) Diagnostic value of rectal mucosal acetylcholinesterase levels in Hirschsprung's disease. Lancet 1:

347-349

6 Dalla-Valle A (1920) Ricerche istologiche su di un caso di m1acolon congenito. Pediatria, Napoli 28: 740-752 Dalla-Valle A (1924) Contributo allaconoscenza della forma famigliare del megacolon congenito. Pediatria, Napoli 32:

569-599

8 Ehrenpreis T (1946) Megacolon in the newborn. A clinical and roentgenological study with special regard to the pathogenesis. Acta Chir Scand 94 Suppl112: 1-114 9 Garret JR, Howard ER (1969) Histochemistry and the pathology of Hirschsprung's disease. Proc Roy Micr Soc 4:

76-78

10 Garret JR, Howard ER (1981) Myenteric plexus of the hind-gut: developmental abnormalities in humans and experimental studies. In: Development of the autonomic nervous system, K. Elliott, G. Lawrenson, ed. Ciba Foundation Symposium 83: 326-354. Pitman Medical London 11 Hamoudi AB, Reiner CB, Boles ET, Juhling McClung H, Kerzner B (1982) Acetylthiocholinesterase staining activity of rectal mucosa. Arch Path Lab Med 106: 670-672 12 Herzog B (1970) Die Hirschsprung'sche Krankheit, diagnostische und therapeutische Probleme im Neugeborenen- und friihen Sauglingsalter, Hausmann AG, Med Neuheiten 3: 35-42 13 Hess R, Scarpelli DG, Pearse AGE (1958) Thecytochemical localization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases. J. Biophys Biochem Cytol 4: 753-760

Aganglionosis of the Colon 14 Hirschsprung H (1887) Sruhltraghert Neugeborener III Folge von Dilatation und Hypertrophie des Colon. Jb Kinderheilk 27: 1-7 15 Karnovsky MJ, Roots L (1964) A "direct-coloring" thiocholine method for cholinesterase. J Histochern Cytochem 12: 219-221 16 Lake BD, Pun P, Nixon HH, Claireaux AE (1978) Hirschsprung's disease. Arch Pathol Lab Med 102: 244-247 17 Lee CM (1955) Megacolon with particular reference to Hirschsprung's disease. Surgery 37: 762-777 18 Meier-Ruge W, Hunziker 0, Tobler H-J, Walhser Ch (1972) The pathophysiology of aganghonosis of the entire colon (Zuelzer-Wilson Syndrome). Morphometric investigations of the extent of sacral parasympathetic innervation of the circular muscles of the aganglion colon. Beitr Path 147: 228-236 19 Meier-Ruge W (1972) Fortschritte III der Diagnostik des aganglionaren Segments. Padiatrie und Pathologie. Suppl 2: 55-64 20 Meier-Ruge W (1974) Hirschsprung's disease. Its aetiology, pathogenesis and differential diagnosis. Current topics in pathology 59: 131-179

. 547

21 Puri P, Lake B, Nixon HH, Mishalany H, Claireaux AE (1977) Neuronal colonic dysplasia: an unusual association of Hirschsprung's disease. J Ped Surg 12: 681-685 22 Rehbein F, Halsband H, Hofmann S (1969) Hirschsprungsche Krankheit mit langem engem Segment. Dtsch Med Wschr 14: 1-25 23 Riker WL (1957) Diagnosis and treatment of aganglionosis of the myenteric plexus. Arch Surg 75: 362-375 24 Scharli AF, Kiesewetter WE (1969) Imperforate anus: Anorectosigmoid pressure studies as a quantitative evaluation of postoperative continence. J Pediat Surg 4: 694 25 Scharli AF, Meier-Ruge W (1981) Localized and disseminated forms of neuronal intestinal dysplasia mimicking Hirschsprung's disease. J Ped Surg 16: 164 26 Swenson 0, Rheinlander HF, Diamond I (1949) Hirschsprung's disease: a new concept of the etiology. N Engl J Med 241: 551-556 27 Walker AE, Kempson RL, Ternberg JL (1966) Aganglionosis of the small intestine. Surgery 60: 449-457 28 Zuelzer WW, Wilson JL (1948) Functional intestinal obstruction on a congenital neurogenic basis in infancy. Am] Dis Childh 75: 40-64

Received May 31, 1983 . Accepted December 21, 1983

Key words: Aganglionosis - hypoganglionosis - neuronal dysplasia - enzyme histochemistry Prof. Dr. Ph. U. Heitz, Department of Pathology, Schonbeinstr, 40, CH-4003 Basel, Switzerland