Age-related changes in innervation in hypertrophic pyloric stenosis

Age-Related

Changes

in Innervation

By Hiroyuki

Kobayashi,

in Hypertrophic Tomas

Dublin,

Background/Purpose: Recent reports have identified abnormal innervation of the circular muscle layer involving the fine intramuscular nerve fibers in hypertrophic pyloric stenous (HPS). HPS presenting after 3 months of age is rare. The aim of this study was to determine the distribution pattern of nerve fibers in the smooth muscle layers in HPS and correlate this with age at presentation. Methods: Full-thickness pyloric muscle biopsy specimens were obtained from eight patients with HPS (five age 3 to 5 weeks, two age 3 months, and one age 7 months) and five controls with normal pylorus (age 5 days to 3 years). All specimens were stained with monoclonal antibody to the neural cell adhesion molecuie (NCAM) using immunohistochemistry. Resu/ts:There were many NCAM-positive nerve fibers in the circular and longitudinal muscle layers in the controls. No NCAM positive nerve fibers were seen in the circular or

H

YPERTROPHIC PYLORIC STENOSIS (HPS) is the most common condition requiring surgery in the first few months of life. ’ Usually, the onset of clinical symptoms occurs at 3 to 6 weeks of age.’ Pyloric stenosis is rare after 3 months of age, although older cases involving older patients have been reported in the literature.2-4 In 1,481 consecutive cases of pyloric stenosis treated at three children’s hospitals in Dublin between 1969 and 1990, 3.6% of patients presented with pyloric stenosis between 12 and 30 weeks of age.’ Neural cell adhesion molecule (NCAM) is a cell surface glycoprotein involved in cell-cell adhesion during development.5 NCAM appears on early embryonic cells and is important in the formation of cell collectives and their boundaries at the sites of morphogenesis.6 It is involved in adhesion between several types of neural cells and their processes, and formation of initial contacts between nerve and muscle cells play an essential role in the development and function of the peripheral nervous system.’ NCAM immunoreactivity appears in human large bowel development according to a predetermined

From the Children’s Research Centre, Our Lady’s Hospital for Sick Children, Dubliir, Ireland. Address reprint requests to Prem Puri, Director of Research, Childl-en’s Research Centre, Our Lady’s Hospital for Sick Children, Crumlin, Dublin 12, Ireland. Copyright o 1997 by WB. Saunders Company 0022-3468/97/3212-0008$03.00/O

1704

Wester,

and

Prem

Pyloric

Stenosis

Puri

Ireland

longitudinal muscle layers in the five cases of HPS in which the patients were less than 5 weeks old. In the two cases in which the patients were 3 months old, occasional NCAMpositive nerve fibers were seen in the circular layer, and moderate numbers of NCAM positive fibers were seen in the longitudinal muscle layers. Moderate numbers of NCAMpositive nerve fibers in the circular muscle layer and many NCAM positive nerve fibers in the longitudinal muscle layers were identified in the 7-month-old HPS patient. Conclusions: The data suggest that the pyloric muscle lacks innervation in the young HPS infant. Whereas, at 3 months of age the hypertrophied pyloric muscle is partially innervated, and at 7 months of age the pyloric muscle has practically normal innervation. J Pediatr Surg 32:1704-1707. Copyright o 1997 by W.B. Saunders Company. INDEX

WORDS:

Pyloric

stenosis,

innervation,

NCAM.

pattern.7 Kobayashi et al8 recently described a marked decrease of NCAM immunoreactivity in the circular and longitudinal muscle layers in HPS infants, whereas the NCAM immunoreactivity of the myenteric plexus was normal. It is unknown whether older HPS infants present the same innervation abnormalities. The aim of this study was to examine the NCAM immunoreactivity in the smooth muscle layers in HPS and to correlate this with age at presentation. MATERIALS Tissue

AND

METHODS

Specimens

Full-thickness pyloric muscle biopsy specimens were obtained from eight patients (seven boys and one girl) who had HPS. Five patients were 3 to 5 weeks old, two patients were 3 months old, and one patient was 7 months old. Control tissue was obtained from five patients (age 5 days to 3 years) with a normal pylorus, in two cases during pyloroplasty at the time of surgery for gastroesophageal reflux and in three during postmortem examination, which was performed less than 4 hours after death. For NCAM staining, fresh specimens were fixed in periodatelysine paraformaldehyde (PLP), embedded in OCT-compound and snap frozen in liquid nitrogen. The specimens were stored at -80°C until they were sectioned. Fresh sections of 10pm thickness were cut serially and used for immunohistochemistry.

Immunocytoclzemisty Immunocytochemical studies were performed using the avidin-biotinperoxidase complex (ABC) method, with NCAM monoclonal antibody (1:100 DAKO, Denmark). All sections were incubated in the antiserum overnight at 4°C. The second and third layers were incubated for 1 hour JournalofPediatn’cSurgery,

Vol32,

No 12 (December),

1997:

pp 1704.1707

PYLORIC

Table

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STENOSIS

1. Distribution Circular and

of NCAM Longitudinal

lmmunoreactive Fibers Muscles of the Pylorus

Normal Controls

wk

-

++ ++

Circular muscle Longitudinal muscle

Pyloric

<5

*Abbreviations: -, no fibers visible; ate number of fibers; ++, many fibers.

?, occasional

each at room temperature. Visualization for achieved by the addition of 3,3’-diaminobenzidine

Stenosis

in the

Patients

3mo

7mo

f +

++

fibers;

t

+, moder-

the peroxidase as substrate.

was

Quantitation of Results The number of NCAM-positive no visible fibers (0), occasional (+),andmanyfibers(++).

nerve fibers were graded as follows: fibers (t-), moderate number of fibers

RESULTS

Normal Pylorus Numerous NCAM-positive nerve fibers were seenin circular and longitudinal musclelayers. In the myenteric plexus, ganglion cells also showedstrong NCAM immunoreactivity (Fig 1, Table 1).

Fig 2. Pyloric muscle specimen at the age of 1 month. There is strong staining of the myenteric plexus, there are no NCAM-positive fibers in circular and longitudinal muscle layers. (Peroxidase technique, original magnification x160.1

Hypertrophic Pyloric Stenosis No NCAM-positive nerve fibers were seenin either the circular or the longitudinal musclelayers in the five HPS caseswith patients age3 to 5 weeks (Fig 2, Table 1). OccasionalNCAM-positive fibers in the circular muscle layer and moderate numbersof NCAM-positive fibers in the longitudinal muscle layer were seenin two patients who were 3 months old (Fig 3). In the 7-month-old patient, there were moderate numbers of NCAM-positive nerve fibers in the circular muscle layer and many NCAM-positive nerve fibers in the longitudinal musclelayer (Fig 4). In all casesof HPS, there was strong NCAM immunoreactivity in the myenteric plexus identical to that in the control cases(Table 1). DISCUSSION

Fig 1. Normal pylorus. There is strong staining of the myenteric plexus. Finer fibers within the circular muscle and longitudinal muscle are abundant. (Peroxidase technique, original magnification x

160.1

The etiology of HPS remainsunknown. Several recent studiesindicate that there is a deficient innervation of the muscular layers in HPS, whereas the myenteric plexus appearsto be normal. Malmfors and Sandler9 Tam,r” and Shen et al” have describedabnormalitiesof the peptidergic innervation in HPS. Reduced immunoreactivity has

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which may be an important observation because the glial cells are essential for maintenance of the basic physiological function of the neurons. Vanderwinden et alI9 studied the interstitial cells of Cajal in HPS and found decreased immunoreactivity of the interstitial cell-specific c-kit immunoreactivity. Interstitial cells of Cajal are generally recognized as the pacemaker cells of the enteric nervous system. In the avian bowel, this cell type has recently been described to have mesodermal origin and develop independently of the enteric nervous system.20 The lack of interstitial cells may influence the motility of the hypertrophied pylorus. The majority of the quoted reports have used biopsy specimens from HPS patients less than 2 months old. Several investigators do not describe the age distribution of their patients. This study confirms the previously reported finding9 that NCAM immunoreactivity is markedly decreased or absent in the muscular layers in HPS patients age 3 to 5 weeks, whereas the myenteric plexus expresses normal NCAM immunreactivity. In the 3-month-old patients, the

Fig 3. Pyloric strong staining positive nerve positive nerve nique, original

muscle specimen at the age of 3 months. of the myenteric plexus. There are occasional fibers in circular muscle layers and moderate fibers in longitudinal muscle layers. (Peroxidase magnification x160.)

There is NCAMNCAMtech-

been observed using immunohistochemical methods for substance P, vasoactive intestinal peptide (VIP), and enkephalin. Nitric oxide has recently been recognized as the most important messenger of nonadrenergic noncholinergic inhibitory nerves in the gastrointestinal tract.12 Vanderwinden et alI3 found that nitric oxide synthase activity was markedly decreased in the circular muscle layer in HPS, using NADPH diaphorase histochemistry, which has been recognized as an accurate marker for nitric oxide synthase immunoreactivity.‘4.15 The deficient nitrergic innervation may explain the pylorospasm in HPS. Furthermore. abnormal nerve terminals have been found in HPS using antibodies against the presynaptic vesicles.16 Several investigators have also described abnormalities that do not directly involve the ganglion cells or the nerve fibers. Cass and Zhang17 found changes in the extracellular matrix involving mainly a marked increase of the chondroitin sulphate content in the hypertrophied pylorus. However, the pathophysiological relevance of this finding is unclear. Kobayashi et ails reported a reduction of nerve-supporting cells in HPS,

Fig 4. Pyloric muscle specimen at the age of 7 months. There is strong staining of the myenteric plexus. There are moderate NCAMpositive nerve fibers in circular muscle and many NCAM-positive nerve fibers in longitudinal muscle layers. (Peroxidase technique, original magnification x 160.)

PYLORIC

STENOSIS

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NCAM immunoreactivity was slightly decreased with occasional fibers in the circular muscle layer and moderate numbers of NCAM-positive fibers in the longitudinal muscle layer. Interestingly, the NCAM immunoreactivity was near normal in the 7-month-old patient with moderate numbers of NCAM-positive fibers in the circular muscle layer and many NCAM-positive fibers in the

longitudinal muscle layers. To our knowledge, the innervation has not been reported in an older HPS infant. The significance of the findings is unclear, but the results suggest that the innervation abnormality in HPS is age dependent. It may be possible that the innervation abnormality normalizes with age and that this normalization precedes the normalization of the muscular hypertrophy.

REFERENCES 1. Puri P, Lakshmanadass G: Hypertrophic pyloric stenosis, in Puri P (ed): Newborn Surgery. Oxford, England, Butterworth-Heinemann, 1996, pp 266-271 2. Rendle-Short J, Zachary RB: Congenital pyloric stenosis in older babies. Arch Dis Child 30:70-71, 1955 3. Konvolinka CW, Wermuth CR: Hypertrophic pyloric stenosis in older infants. Am J Dis Child 122:76-79, 1971 4. Evans AL: Hypertrophic pyloric stenosis presenting in childhood (letter). Postgrad Med J 63:919, 1987 5. Edelman GM: Cell adhesion and the molecular processes of morphogenesis. Am Rev Biochem 43:135-169, 1985 6. Tosney KW, Watanabe M, Landmesser L: The distribution of NCAM in the chick hind limb during axon outgrowth and synaptogenesis. Dev Biol 14:437-452, 1986 7. Romanska HM, Bishop AE, Moscoso G, et al: Neural cell adhesion molecule (NCAM) expression in nerves and muscle of developing human large bowel. .J Pediatr Gastroenterol Nutr 22:351358,

1996

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H, O’Briain DS, Puri P: Immunochemical characterization of neural cell adhesion molecule (NCAM), nitric oxide synthase, and neurofilament protein expression in pyloric muscle of patients with pyloric stenosis. J Pediatr Gastroenterol Nutr 20~3 19-325, 1995 9. Malmfors G, Sundler F: Peptidergic innervation in infantile hypertrophic pyloric stenosis. J Pediatr Surg 21:303-306, 1986 10. Tam PKH: An immunochemical study with neuron specific enolase and substance P of human enteric innervation-The normal developmental pattern and abnormal deviations in Hirschsprung’s disease and pyloric stenosis. J Pediatr Surg 21:227-232, 1986

11. Shen Z, She Y, Wang W, et al: Immunohistochemical study of peptidergic nerves in infantile hypertrophic pyloric stenosis. Pediatr SurgInt5:110-113,199O 12. Sanders KM, Ward SM: Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission. Am J Physiol262:G379-G392, 1992 13. Vanderwinden J-M, Mailleux P, Schiffmann S, et al: Nitric oxide synthase activity in infantile hypertrophic pyloric stenosis. N Engl J Med 327:511-515, 1992 14. Dawson TM, Bredt DS, Fotuhi M, et al: Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissue. Proc Nat1 Acad Sci US A 88:7797-7801, 1991 15. Hope BT, Michael GJ, Knigge KM, et al: Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Nat1 Acad Sci U S A 88:2811-2814, 1991 16. Okazaki T, Yamataka A, Fujiwara T. et al: Abnormal distribution of nerve terminals in infantile hypertrophic pyloric stenosis. J Pediatr Surg 29:655-658, 1994 17. Cass DT, Zhang AL: Extracellular matrix changes in congenital hyperytrophic pyloric stenosis. Pediatr Surg Int 6: 190- 194, 1991 18. Kobayashi H, O’Briain DS, Puri P: Selective reduction in intramuscular nerve supporting cells in infantile hypertrophic pyloric stenosis. J Pediatr Surg 29:651-654, 1994 19. Vanderwinden J-M, Liu H, DeLaet M-H, et al: Study of the interstitial cells of Cajal in infantile hypertrophic pyloric stenosis. Gastroenterology 111:279-28X, 1996 20. Lecoin L, Gabella G, Ledourain N: Origin of the c-kit positive interstitial cells in the avian bowel. Development 122:725-731, 1996