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The Pathophysiology of Aganglionosis of the Entire Colon (Zuelzer-Wilson Syndrome) Morphometric Investigations of the Extent of Sacral Parasympathetic Innervation of the Circular Muscles of the Aganglionic Colon
Beitr. Path. Bd. 147,228-236 (1972)
Exper. Pathology and Histochemistry, Dept. of Medical Basic Research, Sandoz Ltd., Basle, Switzerland
The Pathophysiology of Aganglionosis of the Entire Colon (Zuelzer-Wilson Syndrome) Morphometric Investigations of the Extent of Sacral Parasympathetic Innervation of the Circular Muscles of the Aganglionic Colon W. MEIER-RUGE,
O.
HUNZIKER, H.-J. TOBLER
and CR.
WALLISER
With 5 Figures and I Table' Received April 8, 1972 . Accepted April 24, 1972
Abstract Nerve fibre density in the circular muscle fibres of 7 sections of completely aganglionic colon was determined morphometrically by means of a Leitz-Classimat. The specimens were cut into longitudinal strips and coiled; the following findings were made: I. The circular muscles and mucous membrane of the aganglionic colon are innervated from the anus to the splenic flexure by sacral parasympathetic nerve fibres. 2. The density of these nerves decreases exponentially (y = a . e- bx) from the anus to the splenic flexure. 3. The ratio of nerve fibres in the descending colon to those in the rectum is about I: 10.
4. The sacral parasympathetic nerves in the circular muscle fibres show one of three tendencies: hyperplasia (exponential factor = ~O.02), normoplasia (= ~O.04) or hypoplasia (=~o. I). The aetiology of the disease is discussed in the light of these findings. Hereditary and environmental factors are assessed as possible causes of colonic aganglionosis. The sharp increase in nerve fibre density in the direction of the rectum is related to the analogous increase in the propulsive power of the colon in the same direction. In view of the morphometric findings made in the descending colon the diagnosis of Hirschsprung's disease on the basis of enzyme-histochemical biopsies is only possible in the rectosigmoid.
Our knowledge of the normal and diseased distal colon is not very extensive. Despite the work of GOERTTLER (1932, 195 I) on the mechanics of the colonic muscles, the progressive increase in the propulsive power of the
Aganglionosis of the Colon, 229
intestinal muscles from the splenic flexure to the anus in this condition remained unexplained. According to earlier pathological findings the descending colon and the rectosigmoid are supplied with parasympathetic nerve fibres from the sacral nerves (ADAMSON and AIRD, 1932). In addition, there is enzyme-histotopochemical evidence of a marked rise in the secretion of acetylcholine and of acetylcholinesterase activity in the aganglionic colon segment (KAMIJO, 1953; KOELLE, 1954; NIEMI et aI., 1961; MEIER-RuGE, 1968, etc.). These findings were verified in embryological investigations by OKAMOTO and UEDA (1967), who demonstrated that vagus nerves are formed pararectally in the 5-week-old embryo and that in the 6th week, nerve fibres of the vagus become embedded in the circular muscles of the rectum and descending colon only as far as the splenic flexure (Fig. 1).
Fig. I. Distribution of extrinsic parasympathetic nerves in the gastro-intestinal tract in the '/7-week-old embryo.
Recently we were able to investigate surgical specimens of aganglionic large intestine. In these it was possible to confirm that the rectosigmoid and the descending colon possess extended parasympathetic nerve fibres (ADAMSON and AIRD, 1932). Furthermore we were able to show that the average number of nerves in the circular muscles decreases exponentially 16
Beitr. Path. Bd. 147
230. W. MEIER-RuGE, O. HUNZIKER, H.-J. TOBLER and CH. WALLlSER
between the rectum and the splenic flexure and, by demonstrating acetylcholinesterase, to show why it is possible to diagnose Hirschsprung's disease by enzyme-histotopochemical methods, on evidence from the rectosigmoid only.
Method 7 specimens of totally anganglionic colon were available from children aged
8/12
to
21/12 years. Their average age was 14/12 years but only 2 children were under I year.
The untreated specimens were cut into narrow longitudinal strips and coiled with the anal end at the centre. The strips were then placed unfixed on the freezing platforms of a Dittes-Duspiva Cryostat 880 T and frozen. When the temperature of the specimens had reached that of the cryostat (-20°C), 14 iJ. sections were prepared. The following enzymes were studied histotopochemically: acetylcholinesterase (ACE) by the method of KARNOVSKY and ROOTS, and lactic dehydrogenase by the method of HESS and his colleagues. All enzyme reactions were performed by a technique modified for routine laboratory use (MEIER-RuGE et aI., 1971). Morphometric examinations were carried out on sections in which (without counterstaining) acetylcholinesterase-positive parasympathetic structures had been demonstrated. All sections were incubated for 90 min. For morphometric analyses a Leitz-Classimat was used (HUNZIKER et aI., 1972). By the light optical electronic analysis system of this apparatus it was possible to pick out and count automatically the percentage of acetylcholinesterase (ACE)-positive parasympathetic nerve fibres in the circular muscles of the rectum, sigmoid colon and descending colon. The test area was set at a constant scanning width of 100 iJ. and the height was adjusted in accordance with variations in the thickness of the circular muscles. The most marked ACE activity was found in the distal part of the colon at the centre of the coil. The constant setting allowed scanning of the strips of muscle from the centre outwards IOO!l at a time; the findings were transferred directly to a desk calculator attached to the Leitz-Classimat. Since these findings comprised statistically dependent values, the mean of 10 of them (representing I mm of intestine) was calculated at a later stage and used as an independent measure in further mathematical-statistical processing. A regression was calculated for the relationship between intestinal length and the density of nerve fibres, according to the function y = a . e- bx • The closeness of the regression curve to the individual values can be seen from the distribution of the latter round the curve. The constancy of uniformity of the regression was tested by means of a series of trigonometric functions.
Results The percentage of nerve fibres in the circular muscles of the colon as measured by a Leitz-Classimat led us to conclude that: I. The muscles and mucosa of the aganglionic colon are innervated by sacral (extrinsic) parasympethetic nerve fibres between the anus and the splenic flexure.
Aganglionosis of the Colon . 231 density of nerve fibres Vol. %
9
7
5
3
Noln Patrick 1!'112 l.
Masino Lucia 1'Y12
y=B,99 ' e-O,036x
y=4.71· e-O,099x
Oem 2 4 Specimens from distal end at colon
6
B
l.
10
Fig. 2. Exponential decrease in the density of (extrinsic) sacral parasympathetic nerve fibres in the circular muscle between the rectum and the splenic flexure. The straight regression curves show the juxtaposition of a case of normal distribution of nerve fibre density with an expontential factor of - o.o36x and a case of hypoplasia of the extrinsic parasympathetic fibres (-o.099x).
sacral p ra ympathetic n rves .......... sympathetic ner¥8
Fig. 3. Percentage density of sacral parasympathetic nerve fibres in rectal muscles.
232' W. MEIER-RuGE,
O.
HUNZIKER, H.-J. TOBLER
and CH.
WALLISER
Table I. Exponential functions for nerve fibre density in aganglionosis of the entire colon. Density varies in accordance with rectal length ]. Fistric U. Walz L. Masina E. Zoch A. Hochuli A. Grass P. Nein
8/12 16/ 12 18/ 12 21/12 16/ 12 2°/ 12 10/ 12
years years years years years years years
y= y= y= y= y= y= Y=
2.85 2.3 6 8.99 4. 61 3. 02 3. II 4.71
. e-O,019x . e-O,022x . e-O,036x . e-O,038x . e- O,039x . e- O,039x . e- O,099x
The density of these fibres decreases exponentially from the anus to the splenic flexure, in accordance with the function y = a . e- bx (Fig. 2). Detailed analysis of fibre density in serial sections showed that the density of the nerve fibres corresponded highly among the individual sections. The variations in nerve fibre density are approximately equivalent to a sinus curve. The density of nerve fibres was greatest in the middle of each muscle segment falling off towards the inner and outer margins. 3. The ratio of nerve fibre density in the descending colon to that in the rectum is about I: 10 (Fig. 3). 4. There are 3 different forms of extrinsic or sacral parasympathetic nerve fibres in complete aganglionosis (Table I): a) hyperplasia of the extrinsic parasympathetic nerves with an exponential factor b of -0.02 (Fig. 4). b) normoplasia with an exponential factor b ~ --0.04, c) hypoplasia with an extremely sharp decline in mean fibre density (in this case proportional to e- O. 1x - Fig. 5). The factor 'a' in the exponential function varies, the reason being that the lower extremity of the section was arbitrarily taken as the starting point in measuring the length of the section, because in all cases the lower resection line was difficult to locate. But since the angle of the regression curve as an expression of the variations in nerve fibre density is determined by the negative exponential factor, 'a' can be ignored for this purpose. 2.
Discussion Our results show that there is an exponential decrease in the density of sacral parasympathetic nerve fibres in the circular muscles of the colon from the anal sphincter to the splenic flexure. This phenomenon can be plausibly explained with reference to the growth of the sacral parasympathetic nerves in the rectum during the
"...It, 01 Kl
Aganglionosis of the Colon . 23 3 ••••• I,D ...
10 1
fllir ic 110' ""
1
y_ 2.85 . -o.OI!.hc 0.1
nDl+---_,----_r----r_--~----~--_,----_r----r_--_r--__,
Oem
2
,
SpecIlTll!nS from d t 1 lid d colon
8
6
Fig. 4. Hyperplasia of extrinsic parasympathetic nerves in the circular muscles, showing an extremely flat line of regression and an exponential factor of about 0.02.
d''''II, of I)
••
n.
tab •••
100
• lad! Ie hald " ,
y. '.61
0.1
J
I
-O.OJh
.N ,,'ohdlO/u'
yo l..?I.-o. 099 I(
Ml+----r--~----r_--,_---r--~----._--,_--_r--_;
Oem 2 , Specimens from d stll end of colon
6
8
Fig. 5. Juxtaposition of hypoplasia (Pat. Nein, expo ~ -0.1) and normoplasia (exp. ~ -0'04) of the sacral parasympathetic nerves in the circular muscles of rectum and descending colon.
234·
W. MEIER-RuGE,
O. HUNZIKER, H .-J .
TOBLER
and CH. WALLISER
development of the embryo, if we assume that the growth of all fibres is equivalent to a dichotomic ramification. This means that the highest fibre density can be expected at the point where the extrinsic parasympathetic nerves enter the intestinal wall (distal rectum) and that the fibres gradually became less dense in a caudocranial direction. Our observations in serial sections, that the density of the fibres oscillated in strict correlation around the regression line almost in a sinuscurve might conform with GOERTTLER'S findings that the circular muscles of the distal colon are arranged in annular segments. In view of the fact that there are 3 different types of aganglionosis in which the formation of the extrinsic parasympathetic nerves varies considerably, the question inevitably arises as to aetiology. Which of the 3 are due to genetic, and which to environmental factors? There is no doubt that aganglionosis of the colon can be congenital (ALTHOFF, 1962; BomAN and CARTER, 1963; EMANUEL et aI., 1965), but the incidence of the disorder suggests that environmental factors must also be taken into account (P ASSARGE, 1967; EHRENPRElS, 1966, 1970), and experiments with folic acid antagonists (MEIER-RuGE, I 968 b) have shown that environmental factors can also affect the growth of sacral parasympathetic neurones in the embryo. Surgical and toxicological experiments, such as those done by McELHANNON (1960), HUKUHARA et aI. (1961) and OKAMOTO et ai. (1967), however, have failed to provide convincing corroboratory evidence and it seems that such methods are less well suited to the provocation of complete or partial aganglionosis of the colon than are the teratogenic studies of NELSON. The exponential distribution of mean nerve fibre density in the circular muscle fibres with maximum density in the rectal ampulla is largely in agreement with observations made by manometer of a craniocaudal increase in the contractile and propulsive power of the rectum (RITCHIE, 1968; SCHAERLI, 1971) and with the findings of ADAMSON and AIRD (1932) that megacolon results from transection of the sacral parasympathetic nerves. This explains why the contractile force of the lower part of the rectum (HOWARD and GARRETT, 1970) is such a crucial parameter in diagnosing aganglionic disorders of the colon. Our observations that hypoplasia of the parasympathetic nerves of the rectosigmoid occurs in some cases of aganglionosis of the entire colon (Fig. 5), might explain the surprisingly long survival of isolated patients. RIKER (1957) reported cases of patients who received no treatment for 3 ~ years. In Hirschsprung's disease we have seen three cases in which surgery was necessary only after 15, 30 and 64 years (s. STONE et aI., 1965; EHRENPREIS, 1970). These cases lend powerful support to the theory that the effect of the sacral parasympathetic nerves on the circular muscle fibres of the rectum
Aganglionosis of the Colon .
23 5
can vary enormously (ADAMSON and AIRD, 1932). We were unable to determine any relationship between the development of these nerves and the commencement of treatment, since all our patients in this study had been under treatment since birth. Finally, our observation that nerve fibre density decreased exponentially between the rectosigmoid and the splenic flexure (Figs. 2 and 3) both in the circular muscle and in the lamina propria mucosae explains why only the rectosigmoid can be taken into account when diagnosing Hirschsprung's disease on the basis of increased acetylcholinesterase activity associated with the extrinsic parasympathetic nerve fibres (MEIER-RuGE, 1968a; GARRETT et aI., 1969a, b; MORGER and MEIER-RuGE, 1968, 1971; MOSER et aI., 1971; MEIER-RuGE et aI., 1972). The observation that the density of sacral parasympathetic nerve fibres is practically nil at the level of the splenic flexure underlines why a biopsy of mucosa from the transverse colon (e.g., in colostomy) cannot be judged by the same diagnostic criteria as a rectal biopsy.
References ADAMSON, W. A. D. and AIRD, ].: Brit. J. Surg. 20, 220 (1932) ALTHOFF, W.: Z. mensch!. Vererb.- u. Konstit.-Lehre 36, 314 (1962) BODIAN, M. and CARTER, C. 0.: A family study of Hirschsprung's disease. Ann. hum. Genet. 26, 261 (1963) EHRENPREIS, TH.: J. pediat. Surg. 1, 329 (1966) EHRENPREIS, TH.: Hirschsprung's disease. Year Book Medical Pub!. Inc., Chicago (1970) EMANUEL, B., PADORR, M. P. and SWENSON, 0.:]. Pediat. 67, 381 (1965) GARRETT,]. R., HOWARD, E. R. and NIXON, H. H.: Arch. Die. Childh. 44,406 (1969a) GARRETT, ]. R. and HOWARD, E. R.: Proc. Roy. microsc. Soc. 4, 76 (I 969 b) GOERTTLER, K.: Gegenbaurs morpho ]b. 69, 327 (1932) GOERTTLER, K.: Gegenbaurs morpho lb. 90,33 (1951) HESS, R., SCARPELLI, D. G. and PEARSE, A. G. A.: J. biophys. biochem. Cyto!. 4, 753 (195 8a) HOWARD, E. R. and GARRETT, J. R.: Gut II, 1007 (1970) HUKUHARA, T., SUMI, T. and KOTANl, S.: Jap.]. Physio!. II, 281 (1961) HUNZIKER, 0., DEL POZO, E., WIESINGER, D. und MEIER-RuGE, W.: Die Anwendung des Leitz-Classimaten in der quantitativen Histomorphologie und in der experimentellen Pathologie. Leitz-Mitt. 5,5 I (1972), Sonderheft "Classimat" (197 2) KAMIlO, K., KlATT, R. B. and KOELLE, F. B.: Gastroenterology 24,173 (1952) KARNOVSKY, M. J. and ROOTS, L.: J. Histochem. Cytochem. 12, 219 (1964) KOELLE, G. B.: Ann. N.Y. Acad. Sci. 58, 307 (1954) McELHANNON, F. M.: Surg. Forum 10, 218 (1960) MEIER-RuGE, W.: Virchows Arch. path. Anat. 344, 67 (1968a) MEIER-RuGE, W.: Schweiz. med. Wschr. 98, II22 (1968b) MEIER-RuGE, W.: The pathology of megacolon in "Progress in proctology". Proc. 3rd Internat. Congress Hedrologicum Conlegium. J. HOFERICHTER, pp. 72-77. Springer, Berlin (1969)
236. W. MEIER-RuGE, O. HUNZIKER, H.-J. TOBLER and CH. WALLlSER MEIER-RUGE, W., BIELSER jun., W., WIEDERHOLD, K. H. und MEYENHOFER, M.: Beitr. Path. 144,409 (197 1) MEIER-RUGE, W., LUTTERBECK, P. M., HERZOG, B., MORGER, R., MOSER, R. and SCHARLI, A.: J. pediat. Surg. 1, I I (1972) MORGER, R. und MEIER-RuGE, W.: Sandorama (Basel) (1968) MORGER, R. und MEIER-RuGE, W.: Schweiz. Rundschau Med. (Praxis) 60, 799 (1971) MOSER, R., WIDMER, A. und MEIER-RuGE, W.: Schweiz. med. Wschr. 101, 109 (1971) NELSON, M. M.: Teratogenic effects of pteroylglutamic acid deficiency in the rat. Ed. G. E. W. WOLSTENHOLME and C. M. O'CONNOR, pp. 134-157. (Ciba Foundation Symposium "Congenital malformations".) J. & A . Churchill, London (1960) NIEMI, M., KOUVALAINEN, K. and HJELT, L. : J. Path. Bact. 82, 363 (1961) OKAMOTO, E., UEDA, T.: J. pediat. Surg. 2,437 (1967) OKAMOTO, E., IWASAKI, T., KUK UTANI, T. and UEDA, T .: J. pediat. Surg. 2,444 (1967) PASSARGE, E.: New Engl. J. Med. 276, 138 (1967) RIKER, W. L.: Arch. Surg. 7J, 362 (1957) RITCHIE, J. A.: Gut 9, 442 (1968) SCHARLl, A. F.: Angeborene MiBbildungen des Rectums und Anus. Diagnose, Physiologie und Therapie. ("Aktuelle Probleme in det Chirurgie", Bd. 15.) Hans Huber, Bern-Stuttgart-Wien (1971) STONE, W. D., HENDRIX, T . R. and SCHUSTER, M. M . : Gastroenterology 48,636 (1965) Privatdozent Dr. W. MEIER-RuGE, Medizin. Grundlagenforschung, Abt. fUr expo Pathologie, Sandoz AG, CH-4000 Basel, LichtstraBe
Exper. Pathology and Histochemistry, Dept. of Medical Basic Research, Sandoz Ltd., Basle, Switzerland
The Pathophysiology of Aganglionosis of the Entire Colon (Zuelzer-Wilson Syndrome) Morphometric Investigations of the Extent of Sacral Parasympathetic Innervation of the Circular Muscles of the Aganglionic Colon W. MEIER-RUGE,
O.
HUNZIKER, H.-J. TOBLER
and CR.
WALLISER
With 5 Figures and I Table' Received April 8, 1972 . Accepted April 24, 1972
Abstract Nerve fibre density in the circular muscle fibres of 7 sections of completely aganglionic colon was determined morphometrically by means of a Leitz-Classimat. The specimens were cut into longitudinal strips and coiled; the following findings were made: I. The circular muscles and mucous membrane of the aganglionic colon are innervated from the anus to the splenic flexure by sacral parasympathetic nerve fibres. 2. The density of these nerves decreases exponentially (y = a . e- bx) from the anus to the splenic flexure. 3. The ratio of nerve fibres in the descending colon to those in the rectum is about I: 10.
4. The sacral parasympathetic nerves in the circular muscle fibres show one of three tendencies: hyperplasia (exponential factor = ~O.02), normoplasia (= ~O.04) or hypoplasia (=~o. I). The aetiology of the disease is discussed in the light of these findings. Hereditary and environmental factors are assessed as possible causes of colonic aganglionosis. The sharp increase in nerve fibre density in the direction of the rectum is related to the analogous increase in the propulsive power of the colon in the same direction. In view of the morphometric findings made in the descending colon the diagnosis of Hirschsprung's disease on the basis of enzyme-histochemical biopsies is only possible in the rectosigmoid.
Our knowledge of the normal and diseased distal colon is not very extensive. Despite the work of GOERTTLER (1932, 195 I) on the mechanics of the colonic muscles, the progressive increase in the propulsive power of the
Aganglionosis of the Colon, 229
intestinal muscles from the splenic flexure to the anus in this condition remained unexplained. According to earlier pathological findings the descending colon and the rectosigmoid are supplied with parasympathetic nerve fibres from the sacral nerves (ADAMSON and AIRD, 1932). In addition, there is enzyme-histotopochemical evidence of a marked rise in the secretion of acetylcholine and of acetylcholinesterase activity in the aganglionic colon segment (KAMIJO, 1953; KOELLE, 1954; NIEMI et aI., 1961; MEIER-RuGE, 1968, etc.). These findings were verified in embryological investigations by OKAMOTO and UEDA (1967), who demonstrated that vagus nerves are formed pararectally in the 5-week-old embryo and that in the 6th week, nerve fibres of the vagus become embedded in the circular muscles of the rectum and descending colon only as far as the splenic flexure (Fig. 1).
Fig. I. Distribution of extrinsic parasympathetic nerves in the gastro-intestinal tract in the '/7-week-old embryo.
Recently we were able to investigate surgical specimens of aganglionic large intestine. In these it was possible to confirm that the rectosigmoid and the descending colon possess extended parasympathetic nerve fibres (ADAMSON and AIRD, 1932). Furthermore we were able to show that the average number of nerves in the circular muscles decreases exponentially 16
Beitr. Path. Bd. 147
230. W. MEIER-RuGE, O. HUNZIKER, H.-J. TOBLER and CH. WALLlSER
between the rectum and the splenic flexure and, by demonstrating acetylcholinesterase, to show why it is possible to diagnose Hirschsprung's disease by enzyme-histotopochemical methods, on evidence from the rectosigmoid only.
Method 7 specimens of totally anganglionic colon were available from children aged
8/12
to
21/12 years. Their average age was 14/12 years but only 2 children were under I year.
The untreated specimens were cut into narrow longitudinal strips and coiled with the anal end at the centre. The strips were then placed unfixed on the freezing platforms of a Dittes-Duspiva Cryostat 880 T and frozen. When the temperature of the specimens had reached that of the cryostat (-20°C), 14 iJ. sections were prepared. The following enzymes were studied histotopochemically: acetylcholinesterase (ACE) by the method of KARNOVSKY and ROOTS, and lactic dehydrogenase by the method of HESS and his colleagues. All enzyme reactions were performed by a technique modified for routine laboratory use (MEIER-RuGE et aI., 1971). Morphometric examinations were carried out on sections in which (without counterstaining) acetylcholinesterase-positive parasympathetic structures had been demonstrated. All sections were incubated for 90 min. For morphometric analyses a Leitz-Classimat was used (HUNZIKER et aI., 1972). By the light optical electronic analysis system of this apparatus it was possible to pick out and count automatically the percentage of acetylcholinesterase (ACE)-positive parasympathetic nerve fibres in the circular muscles of the rectum, sigmoid colon and descending colon. The test area was set at a constant scanning width of 100 iJ. and the height was adjusted in accordance with variations in the thickness of the circular muscles. The most marked ACE activity was found in the distal part of the colon at the centre of the coil. The constant setting allowed scanning of the strips of muscle from the centre outwards IOO!l at a time; the findings were transferred directly to a desk calculator attached to the Leitz-Classimat. Since these findings comprised statistically dependent values, the mean of 10 of them (representing I mm of intestine) was calculated at a later stage and used as an independent measure in further mathematical-statistical processing. A regression was calculated for the relationship between intestinal length and the density of nerve fibres, according to the function y = a . e- bx • The closeness of the regression curve to the individual values can be seen from the distribution of the latter round the curve. The constancy of uniformity of the regression was tested by means of a series of trigonometric functions.
Results The percentage of nerve fibres in the circular muscles of the colon as measured by a Leitz-Classimat led us to conclude that: I. The muscles and mucosa of the aganglionic colon are innervated by sacral (extrinsic) parasympethetic nerve fibres between the anus and the splenic flexure.
Aganglionosis of the Colon . 231 density of nerve fibres Vol. %
9
7
5
3
Noln Patrick 1!'112 l.
Masino Lucia 1'Y12
y=B,99 ' e-O,036x
y=4.71· e-O,099x
Oem 2 4 Specimens from distal end at colon
6
B
l.
10
Fig. 2. Exponential decrease in the density of (extrinsic) sacral parasympathetic nerve fibres in the circular muscle between the rectum and the splenic flexure. The straight regression curves show the juxtaposition of a case of normal distribution of nerve fibre density with an expontential factor of - o.o36x and a case of hypoplasia of the extrinsic parasympathetic fibres (-o.099x).
sacral p ra ympathetic n rves .......... sympathetic ner¥8
Fig. 3. Percentage density of sacral parasympathetic nerve fibres in rectal muscles.
232' W. MEIER-RuGE,
O.
HUNZIKER, H.-J. TOBLER
and CH.
WALLISER
Table I. Exponential functions for nerve fibre density in aganglionosis of the entire colon. Density varies in accordance with rectal length ]. Fistric U. Walz L. Masina E. Zoch A. Hochuli A. Grass P. Nein
8/12 16/ 12 18/ 12 21/12 16/ 12 2°/ 12 10/ 12
years years years years years years years
y= y= y= y= y= y= Y=
2.85 2.3 6 8.99 4. 61 3. 02 3. II 4.71
. e-O,019x . e-O,022x . e-O,036x . e-O,038x . e- O,039x . e- O,039x . e- O,099x
The density of these fibres decreases exponentially from the anus to the splenic flexure, in accordance with the function y = a . e- bx (Fig. 2). Detailed analysis of fibre density in serial sections showed that the density of the nerve fibres corresponded highly among the individual sections. The variations in nerve fibre density are approximately equivalent to a sinus curve. The density of nerve fibres was greatest in the middle of each muscle segment falling off towards the inner and outer margins. 3. The ratio of nerve fibre density in the descending colon to that in the rectum is about I: 10 (Fig. 3). 4. There are 3 different forms of extrinsic or sacral parasympathetic nerve fibres in complete aganglionosis (Table I): a) hyperplasia of the extrinsic parasympathetic nerves with an exponential factor b of -0.02 (Fig. 4). b) normoplasia with an exponential factor b ~ --0.04, c) hypoplasia with an extremely sharp decline in mean fibre density (in this case proportional to e- O. 1x - Fig. 5). The factor 'a' in the exponential function varies, the reason being that the lower extremity of the section was arbitrarily taken as the starting point in measuring the length of the section, because in all cases the lower resection line was difficult to locate. But since the angle of the regression curve as an expression of the variations in nerve fibre density is determined by the negative exponential factor, 'a' can be ignored for this purpose. 2.
Discussion Our results show that there is an exponential decrease in the density of sacral parasympathetic nerve fibres in the circular muscles of the colon from the anal sphincter to the splenic flexure. This phenomenon can be plausibly explained with reference to the growth of the sacral parasympathetic nerves in the rectum during the
"...It, 01 Kl
Aganglionosis of the Colon . 23 3 ••••• I,D ...
10 1
fllir ic 110' ""
1
y_ 2.85 . -o.OI!.hc 0.1
nDl+---_,----_r----r_--~----~--_,----_r----r_--_r--__,
Oem
2
,
SpecIlTll!nS from d t 1 lid d colon
8
6
Fig. 4. Hyperplasia of extrinsic parasympathetic nerves in the circular muscles, showing an extremely flat line of regression and an exponential factor of about 0.02.
d''''II, of I)
••
n.
tab •••
100
• lad! Ie hald " ,
y. '.61
0.1
J
I
-O.OJh
.N ,,'ohdlO/u'
yo l..?I.-o. 099 I(
Ml+----r--~----r_--,_---r--~----._--,_--_r--_;
Oem 2 , Specimens from d stll end of colon
6
8
Fig. 5. Juxtaposition of hypoplasia (Pat. Nein, expo ~ -0.1) and normoplasia (exp. ~ -0'04) of the sacral parasympathetic nerves in the circular muscles of rectum and descending colon.
234·
W. MEIER-RuGE,
O. HUNZIKER, H .-J .
TOBLER
and CH. WALLISER
development of the embryo, if we assume that the growth of all fibres is equivalent to a dichotomic ramification. This means that the highest fibre density can be expected at the point where the extrinsic parasympathetic nerves enter the intestinal wall (distal rectum) and that the fibres gradually became less dense in a caudocranial direction. Our observations in serial sections, that the density of the fibres oscillated in strict correlation around the regression line almost in a sinuscurve might conform with GOERTTLER'S findings that the circular muscles of the distal colon are arranged in annular segments. In view of the fact that there are 3 different types of aganglionosis in which the formation of the extrinsic parasympathetic nerves varies considerably, the question inevitably arises as to aetiology. Which of the 3 are due to genetic, and which to environmental factors? There is no doubt that aganglionosis of the colon can be congenital (ALTHOFF, 1962; BomAN and CARTER, 1963; EMANUEL et aI., 1965), but the incidence of the disorder suggests that environmental factors must also be taken into account (P ASSARGE, 1967; EHRENPRElS, 1966, 1970), and experiments with folic acid antagonists (MEIER-RuGE, I 968 b) have shown that environmental factors can also affect the growth of sacral parasympathetic neurones in the embryo. Surgical and toxicological experiments, such as those done by McELHANNON (1960), HUKUHARA et aI. (1961) and OKAMOTO et ai. (1967), however, have failed to provide convincing corroboratory evidence and it seems that such methods are less well suited to the provocation of complete or partial aganglionosis of the colon than are the teratogenic studies of NELSON. The exponential distribution of mean nerve fibre density in the circular muscle fibres with maximum density in the rectal ampulla is largely in agreement with observations made by manometer of a craniocaudal increase in the contractile and propulsive power of the rectum (RITCHIE, 1968; SCHAERLI, 1971) and with the findings of ADAMSON and AIRD (1932) that megacolon results from transection of the sacral parasympathetic nerves. This explains why the contractile force of the lower part of the rectum (HOWARD and GARRETT, 1970) is such a crucial parameter in diagnosing aganglionic disorders of the colon. Our observations that hypoplasia of the parasympathetic nerves of the rectosigmoid occurs in some cases of aganglionosis of the entire colon (Fig. 5), might explain the surprisingly long survival of isolated patients. RIKER (1957) reported cases of patients who received no treatment for 3 ~ years. In Hirschsprung's disease we have seen three cases in which surgery was necessary only after 15, 30 and 64 years (s. STONE et aI., 1965; EHRENPREIS, 1970). These cases lend powerful support to the theory that the effect of the sacral parasympathetic nerves on the circular muscle fibres of the rectum
Aganglionosis of the Colon .
23 5
can vary enormously (ADAMSON and AIRD, 1932). We were unable to determine any relationship between the development of these nerves and the commencement of treatment, since all our patients in this study had been under treatment since birth. Finally, our observation that nerve fibre density decreased exponentially between the rectosigmoid and the splenic flexure (Figs. 2 and 3) both in the circular muscle and in the lamina propria mucosae explains why only the rectosigmoid can be taken into account when diagnosing Hirschsprung's disease on the basis of increased acetylcholinesterase activity associated with the extrinsic parasympathetic nerve fibres (MEIER-RuGE, 1968a; GARRETT et aI., 1969a, b; MORGER and MEIER-RuGE, 1968, 1971; MOSER et aI., 1971; MEIER-RuGE et aI., 1972). The observation that the density of sacral parasympathetic nerve fibres is practically nil at the level of the splenic flexure underlines why a biopsy of mucosa from the transverse colon (e.g., in colostomy) cannot be judged by the same diagnostic criteria as a rectal biopsy.
References ADAMSON, W. A. D. and AIRD, ].: Brit. J. Surg. 20, 220 (1932) ALTHOFF, W.: Z. mensch!. Vererb.- u. Konstit.-Lehre 36, 314 (1962) BODIAN, M. and CARTER, C. 0.: A family study of Hirschsprung's disease. Ann. hum. Genet. 26, 261 (1963) EHRENPREIS, TH.: J. pediat. Surg. 1, 329 (1966) EHRENPREIS, TH.: Hirschsprung's disease. Year Book Medical Pub!. Inc., Chicago (1970) EMANUEL, B., PADORR, M. P. and SWENSON, 0.:]. Pediat. 67, 381 (1965) GARRETT,]. R., HOWARD, E. R. and NIXON, H. H.: Arch. Die. Childh. 44,406 (1969a) GARRETT, ]. R. and HOWARD, E. R.: Proc. Roy. microsc. Soc. 4, 76 (I 969 b) GOERTTLER, K.: Gegenbaurs morpho ]b. 69, 327 (1932) GOERTTLER, K.: Gegenbaurs morpho lb. 90,33 (1951) HESS, R., SCARPELLI, D. G. and PEARSE, A. G. A.: J. biophys. biochem. Cyto!. 4, 753 (195 8a) HOWARD, E. R. and GARRETT, J. R.: Gut II, 1007 (1970) HUKUHARA, T., SUMI, T. and KOTANl, S.: Jap.]. Physio!. II, 281 (1961) HUNZIKER, 0., DEL POZO, E., WIESINGER, D. und MEIER-RuGE, W.: Die Anwendung des Leitz-Classimaten in der quantitativen Histomorphologie und in der experimentellen Pathologie. Leitz-Mitt. 5,5 I (1972), Sonderheft "Classimat" (197 2) KAMIlO, K., KlATT, R. B. and KOELLE, F. B.: Gastroenterology 24,173 (1952) KARNOVSKY, M. J. and ROOTS, L.: J. Histochem. Cytochem. 12, 219 (1964) KOELLE, G. B.: Ann. N.Y. Acad. Sci. 58, 307 (1954) McELHANNON, F. M.: Surg. Forum 10, 218 (1960) MEIER-RuGE, W.: Virchows Arch. path. Anat. 344, 67 (1968a) MEIER-RuGE, W.: Schweiz. med. Wschr. 98, II22 (1968b) MEIER-RuGE, W.: The pathology of megacolon in "Progress in proctology". Proc. 3rd Internat. Congress Hedrologicum Conlegium. J. HOFERICHTER, pp. 72-77. Springer, Berlin (1969)
236. W. MEIER-RuGE, O. HUNZIKER, H.-J. TOBLER and CH. WALLlSER MEIER-RUGE, W., BIELSER jun., W., WIEDERHOLD, K. H. und MEYENHOFER, M.: Beitr. Path. 144,409 (197 1) MEIER-RUGE, W., LUTTERBECK, P. M., HERZOG, B., MORGER, R., MOSER, R. and SCHARLI, A.: J. pediat. Surg. 1, I I (1972) MORGER, R. und MEIER-RuGE, W.: Sandorama (Basel) (1968) MORGER, R. und MEIER-RuGE, W.: Schweiz. Rundschau Med. (Praxis) 60, 799 (1971) MOSER, R., WIDMER, A. und MEIER-RuGE, W.: Schweiz. med. Wschr. 101, 109 (1971) NELSON, M. M.: Teratogenic effects of pteroylglutamic acid deficiency in the rat. Ed. G. E. W. WOLSTENHOLME and C. M. O'CONNOR, pp. 134-157. (Ciba Foundation Symposium "Congenital malformations".) J. & A . Churchill, London (1960) NIEMI, M., KOUVALAINEN, K. and HJELT, L. : J. Path. Bact. 82, 363 (1961) OKAMOTO, E., UEDA, T.: J. pediat. Surg. 2,437 (1967) OKAMOTO, E., IWASAKI, T., KUK UTANI, T. and UEDA, T .: J. pediat. Surg. 2,444 (1967) PASSARGE, E.: New Engl. J. Med. 276, 138 (1967) RIKER, W. L.: Arch. Surg. 7J, 362 (1957) RITCHIE, J. A.: Gut 9, 442 (1968) SCHARLl, A. F.: Angeborene MiBbildungen des Rectums und Anus. Diagnose, Physiologie und Therapie. ("Aktuelle Probleme in det Chirurgie", Bd. 15.) Hans Huber, Bern-Stuttgart-Wien (1971) STONE, W. D., HENDRIX, T . R. and SCHUSTER, M. M . : Gastroenterology 48,636 (1965) Privatdozent Dr. W. MEIER-RuGE, Medizin. Grundlagenforschung, Abt. fUr expo Pathologie, Sandoz AG, CH-4000 Basel, LichtstraBe