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Keywords:

  • enteric neurons;
  • nitric oxide synthase;
  • substance P;
  • vasoactive intestinal peptide

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Background  Slow-transit constipation (STC) is recognized in children but the etiology is unknown. Abnormalities in substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide (NO) have been implicated. The density of nerve fibers in circular muscle containing these transmitters was examined in colon from children with STC and compared to other pediatric and adult samples.

Methods  Fluorescence immunohistochemistry using antibodies to NO synthase (NOS), VIP and SP was performed on colonic biopsies (transverse and sigmoid colon) from 33 adults with colorectal cancer, 11 children with normal colonic transit and anorectal retention (NAR) and 51 with chronic constipation and slow motility in the proximal colon (STC). The percentage area of nerve fibers in circular muscle containing each transmitter was quantified in confocal images.

Key Results  In colon circular muscle, the percentage area of nerve fibers containing NOS > VIP > SP (6 : 2 : 1). Pediatric groups had a higher density of nerve fibers than adults. In pediatric samples, there were no regional differences in NOS and VIP, while SP nerve fiber density was higher in sigmoid than proximal colon. STC children had lower SP and VIP nerve fiber density in the proximal colon than NAR children. Twenty-three percent of STC children had low SP nerve fiber density.

Conclusions & Inferences  There are age-related reductions in nerve fiber density in human colon circular muscle. NOS and VIP do not show regional variations, while SP nerve fiber density is higher in distal colon. 1/3 of pediatric STC patients have low SP or VIP nerve fiber density in proximal colon.


Abbreviations:
ChAT

choline acetyl transferase

HAPC

high amplitude propagating contractions

IR

immunoreactivity

LTC

left transverse colon

NOS

nitric oxide synthase

RTC

right transverse colon

SIG

sigmoid colon

STC

slow-transit constipation

SP

substance P

VIP

vasoactive intestinal peptide

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Chronic constipation causes major morbidity in children and is responsible for 3% of pediatrician visits and 25% of pediatric gastroenterology consultations.1–5 Despite its high prevalence, the etiology of chronic constipation in children remains poorly understood.6–8 In the absence of obvious organic disease, children with chronic constipation are diagnosed with functional conditions. Gastrointestinal transit studies have become an important part of evaluating patients with chronic constipation.9 Using plastic markers and X-rays or using radioisotope and gamma camera images (scintigraphy), three transit patterns have been described in patients with chronic constipation: normal transit, colonic inertia [slow-transit constipation (STC)] and rectosigmoid obstruction (outlet obstruction, anorectal retention).9–14 In patients with outlet obstruction there is normal or slightly slowed colonic transit with prolonged storage of stool in the rectum.15 It has been demonstrated that children with slow colonic transit, as opposed to normal transit constipation or outlet obstruction generally respond poorly to standard therapies.13

Colonic specimens from adults with STC demonstrate markedly reduced contractile activity in vitro,16,17 and reduced contractions with electrical field stimulation.18–20 Excessive production of nitric oxide (NO) may inhibit colonic motility in STC colon.21 Both increased22 and decreased23 NO-producing neurons and reduced vasoactive intestinal peptide (VIP)24,25 have been reported in adults with idiopathic constipation. Low numbers of substance P-immunoreactive (SP-IR) nerve fibers have been found in colonic circular muscle from adult21 and pediatric26–28 STC patients.

This study aimed to determine if there is a change in nitric oxide synthase (NOS), VIP or SP innervation in colon from STC children. To address this aim, we needed to: (i) establish a quantitative method of assessing the percentage area of nerve fibers, (ii) compare pediatric and adult control groups, (iii) determine regional variation and (iv) compare children with STC to the pediatric and adult control groups. This study was limited to three transmitters shown in previous studies to be altered in adult or pediatric STC (NOS, VIP and SP).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Adult samples

Full thickness biopsies were obtained from transverse colon (= 14) and sigmoid colon (SIG) (= 19) from 33 adult colorectal cancer patients,29 from the normal margin during resection.

Pediatric groups

Scintigraphic analysis and biopsy collection.  Sixty-two children with chronic constipation (fulfilling Rome II Criteria30) underwent both scintigraphy12 and laparoscopic seromuscular colonic biopsy31 between January 2000 and December 2005. Three investigators, including an experienced pediatric nuclear medicine physician (DJC), blindly reviewed the scintigraphic data at 6, 24, 30 and 48 h12 and subdivided the patients into those with STC, outlet obstruction (anorectal retention) and normal colonic motility.9,32 STC was defined as retention of radioisotope proximal to the splenic flexure at 24 and 48 h, while anorectal retention was defined as normal transit through the colon at 24 h and with retention in the anorectum at 48 h.32 Anorectal manometry and defecography were not performed.

Fifty-one children had slow motility in the proximal and mid colon at 24 and 48 h and were defined as STC32 (M : F = 35 : 16, mean age 8.2 years, range 2.0–14.8 years). Eleven children had no slowing of the radioisotope in the proximal or mid colon. Seven of these had visible accumulation of radioisotope in the rectum at 24 or 48 h (M : F = 8 : 3, mean age 7.8 years, range 3.0–16.8 years). As motility was normal in the proximal and mid colon, with retention in the anorectum in some, this combined group was named ‘normal transit with anorectal retention (NAR)’.

Seromuscular biopsies were collected laparoscopically from hepatic flexure (right transverse colon, RTC), splenic flexure (left transverse colon, LTC) and SIG.31 These biopsies contain longitudinal muscle and circular muscle but not mucosa or submucosa. The biopsies are collected from the serosal side without forming a hole in the mucosa. The biopsy site closes over in a few days. Biopsies were collected from three defined positions in all pediatric patients to allow an assessment of regional variation.

In addition, SIG samples were collected from children with familial adenomatous polyposis (FAP, = 2), and the normal margin of Hirschsprung’s Disease (HD, = 9). The density of nerve fibers in these samples has previously been reported.33 The data are included in this study to allow the comparison of colon from children with constipation and ‘normal’ pediatric colon.

The study was performed with ethical approval from the Institutional Ethics Committee (EHRC 96037, 98072, 23081, 24105, STV 074/03). All samples were collected with informed consent.

Immunohistochemical technique

Immunofluorescence staining methods for SP and VIP have been reported previously.26 Since 2000, techniques have been standardized in our department.34 All biopsies were placed into ice-cold Zamboni’s fixative (2% formaldehyde and 0.2% picric acid in 0.1 mol L−1 sodium phosphate buffer, pH 7.1) within 30 min of removal (O/N, 4 °C). Samples were then washed in dimethyl sulfoxide (DMSO) (3 × 10 min), followed by phosphate-buffered saline (PBS) (3 × 10 min) and incubated in PBS/sucrose (30% w/v, O/N, 4 °C). Samples were then placed in 50% Optimal Cutting Temperature compound (OCT, Tissue-Tek; Electron Microscopy Sciences, Hatfield, PA, USA): 50% PBS/sucrose (3 h, 4 °C), before being embedded into cryomoulds in 100% OCT frozen over isopentane in liquid nitrogen. Frozen blocks were sectioned within 1 month of collection. Cryostat sections (10 μm) were air-dried (O/N, 4 °C) and incubated in either rabbit antineuronal NOS (nNOS, AB 5380; Chemicon International, Temecula, CA, USA, 1 : 1000), rabbit anti-VIP (NCL VIPp; Novacastra Laboratories, Newcastle upon Tyne, UK, 1 : 200) or rabbit anti-SP (18-0091; Zymed Laboratories, South San Francisco, CA, USA, 1 : 50) (O/N, RT), followed by sheep anti-rabbit-FITC (AP332F; Silenus, Melbourne, Australia, 1 : 200) (2 h, RT) and mounted in Mowiol. Sections from one patient with FAP (12 years old), were stained for NOS, VIP and SP in every staining round to establish positive results and insure consistent immunoreactivity. The colon was removed from this patient prior to developing bowel cancer.35 This provided a positive control of known intensity of labeling and density of nerve fibers. With this sample included we could be confident that low staining in an individual sample was not due to poor staining in that staining run.

Sections were viewed on a Zeiss Axioplan II microscope (Oberkochen, Germany) with a BioRad 1024 laser scanning confocal microscope (488 nm excitation and emission settings of 522–532 nm, Zeiss), an Olympus FV1000 laser scanning confocal microscope (Mount Waverely, Vic., Australia) (543 nm excitation and emission filters between 555 and 625 nm), or on a Leica TCS SP2 SE laser scanning confocal microscope (Wetzlar, Germany) (488 nm excitation and emission settings of 500–550 nm). Single confocal images were collected using 20× objective and 512 × 512 pixel images (dimensions 548.9 μm × 548.9 μm).

Visual analysis of nerve fiber density

Our pathologist (CWC) performed visual analysis as in previous studies26,29,36 using a standard fluorescence microscope on samples from 44 STC children. Samples were graded as normal or low density of SP nerve fibers in the circular muscle. Density was classed as low, when nerve fibers were absent or difficult to find. We have previously shown that the samples graded as very low density have significantly less SP-IR profiles than colon from adults or the normal margin of HD children.29

Quantitative analysis of nerve fiber density

Investigators performed quantitative analyzes blinded to patient and biopsy details. Only sections cut with circular muscle nerve fibers in true cross-section were quantified. We have previously described this method of quantitation in detail for guinea-pig colon37 and human SIG.33 Neurotransmitter quantification was performed using ‘Image J’ (NIH, http://rsb.info.nih.gov/ij/). A region of interest was drawn around the muscle excluding ganglia and damaged tissue. Total circular muscle area was measured. Thresholds were set so only fluorescence in nerve fibers was included. The thresholded image was converted to a binary image, the number of pixels above threshold was counted and the percentage area containing IR nerve fibers calculated.33,37

Statistical analysis

Regional variations within the pediatric STC cohort, pediatric NAR cohort and adult controls were examined, and comparisons between the three cohorts were made using anova (followed by Tukey’s multiple comparison test) and between the two pediatric groups using 2-tail unpaired t-test. All measurements are expressed as mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Distribution of nerve fibers

Nitric oxide synthase-immunoreactivity (IR), VIP-IR and SP-IR was visible in neurons in the myenteric ganglia and in nerve fibers in circular and longitudinal muscle (Fig. 1). Abundant nerve fibers with VIP-IR and SP-IR were also present in the submucosal ganglia and mucosa. The distribution of NOS, VIP and SP was consistent with previous reports.33,38–40

image

Figure 1.  Distribution of nerve fibers containing (A) nitric oxide synthase (NOS), (B) vasoactive intestinal peptide (VIP) and (C) substance P (SP) immunoreactivity in human colon. Colonic biopsy from splenic flexure (left transverse colon) from a child with familial adenomatous polyposis. MG, myenteric ganglia; CM, circular muscle; SMG, submucosal ganglia; M, mucosa. Scale bar = 100 μm.

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Nerve fiber density in circular muscle in adults

We first quantified density of nerve fibers in a cohort (= 33) of adult samples for the percentage area of NOS-IR, VIP-IR and SP-IR in circular muscle (Fig. 2A). The ratio of NOS : VIP : SP was 6 : 2 : 1. There was no difference in the density of each group of nerve fibers in the transverse and SIG.

image

Figure 2.  Regional variation in percentage area of nitric oxide synthase-immunoreactivity (NOS-IR), vasoactive intestinal peptide (VIP)-IR and substance P (SP)-IR in adult and pediatric sigmoid colon. (A) Adult transverse (trans) and sigmoid (SIG) colon. (B1–3) Pediatric sigmoid colon from children with familial adenomatous polyposis (FAP), Hirschsprung’s Disease (HD), slow-transit constipation (STC) and normal colonic transit and anorectal retention (NAR). (B1) NOS, (B2) VIP, (B3) SP. *P < 0.05, **P < 0.01, ***P < 0.001. Mean (SEM). Adult sig, = 14, NAR, = 11, STC, = 51, FAP, = 2, HD, = 9. (Data from FAP, HD and adult samples is previously published33 and repeated here for comparison with data from the children with constipation.)

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Nerve fiber density in circular muscle in sigmoid colon in children with chronic constipation

Some children with STC had visibly low numbers of SP-IR nerve fibers in the circular muscle (Fig. 3). Visual analysis of SP nerve fiber density was performed on samples from 44 STC children. SP was low in 9/42 RTC, 10/41 LTC and 10/44 Sigmoid biopsies. Twenty-three percent of STC children had low SP in at least one region.

image

Figure 3.  High and low density of nerve fibers in the circular muscle. Circular muscle from colons from slow-transit constipation (STC) children showing examples of different densities of nerve fibers containing (A, B) nitric oxide synthase (NOS), (C, D) vasoactive intestinal peptide (VIP) and (E, F) substance P (SP). Top panels show high densities and bottom panels show low densities. Scale bar = 50 μm.

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We then quantitatively measured the density of nerve fibers in colon from children with chronic constipation. Children with chronic constipation were divided into those with slow transit in the proximal colon (STC) and those with normal transit in the proximal colon and anorectal retention and (NAR). In the SIG, the density of nerve fibers in the circular muscle of NAR children was similar to the density in colon from FAP and the normal margin of colon from HD children (Fig. 2B1–B3). Furthermore, the density of each nerve fiber subgroup in SIG from STC children was similar to the other pediatric groups (NAR, FAP and HD).

Nerve fiber density in SIG from children was then compared to SIG from adults. The ratios of NOS-IR: VIP-IR: SP-IR were similar in adults and children, but the percentage area of each nerve subgroup was significantly higher (P < 0.05) in children than in adults.

Nerve fiber density in transverse colon

In adults there were no regional differences between transverse and SIG (Fig. 2A). In the biopsies from three regions (RTC, LTC and SIG) from 11 NAR children (Fig. 4A1), there were no regional differences for NOS-IR or VIP-IR. However, SP-IR was significantly higher in nerve fibers in circular muscle from the SIG than from the LTC (Fig. 4A1). Likewise, in the STC group (Fig. 4A2), there were no regional differences in the density of NOS-IR or VIP-IR nerve fibers in the circular muscle, but SP-IR was higher in the SIG (Fig. 4B).

image

Figure 4.  Regional variation in percentage area of nitric oxide synthase-immunoreactivity (NOS-IR), vasoactive intestinal peptide-immunoreactivity (VIP-IR) and substance P-immunoreactivity (SP-IR) nerve fibers in pediatric colon from (A1) normal colonic transit and anorectal retention (NAR) group and (A2) slow-transit constipation (STC) group. (B) Comparison between NAR and STC groups: (B1) NOS, (B2) VIP and (B3) SP. Seromuscular biopsies obtained from right transverse colon (RTC), left transverse colon (LTC) and sigmoid colon (SIG) from children with STC or NAR constipation. Adult samples from transverse and sigmoid colon. *< 0.05, **P < 0.01, ***P < 0.001. Mean (SEM).

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There was no difference in the density of NOS-IR nerve fibers in STC and NAR (Fig. 4B1) or VIP-IR or SP-IR nerve fibers in the LTC or SIG (Fig. 4B2). In the RTC, SP-IR and VIP-IR was significantly lower (P < 0.05) in circular muscle from the STC group compared to NAR children (Fig. 4B3).

Age-related changes in STC

To investigate age-related changes, the STC cohort was stratified into two age-groups: (i) young: 2–8.0 years (= 27) and (ii) old: 8.1–15 years (= 24). There were no age-related differences in the percentage area of NOS-IR or VIP-IR (Fig. 5A1, A2). The percentage area of SP-IR was significantly lower (P < 0.05) in the RTC in the older cohort (Fig. 5A3). When all the biopsies were considered together there was a significant decrease in the density of SP-IR nerve fibers from young to old children and to adults (Fig. 5D).

image

Figure 5.  Age-related changes in nerve fiber area. Density of (A1) nitric oxide synthase-immunoreactivity (NOS-IR), (A2) vasoactive intestinal peptide-immunoreactivity (VIP-IR) and (A3) substance P-immunoreactivity (SP-IR) nerve fibers in slow-transit constipation (STC) children from young (2–8 years) and old (9–15 years) age groups. (B) Density of SP-IR in NAR, STC (young and old) and adults. *P < 0.05, **P < 0.01, ***P < 0.001. Mean (SEM).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Some subgroups of nerve fibers in colon circular muscle have been reported to alter in density in chronic constipation. This study quantified nerve fiber density in colonic circular muscle from adults and from children with chronic constipation. There were no regional differences in adults. The percentage area of NOS-IR, VIP-IR and SP-IR nerve fibers decreased with age. There were no regional differences in NOS-IR or VIP-IR nerve fiber density in colonic circular muscle in children with chronic constipation, but the density of SP-IR nerve fibers was higher in the distal colon. There was a lower density of nerve fibers containing VIP or SP in the proximal colon in children with slow transit than in the proximal colon of NAR children. These results suggest that a subgroup of STC children have disorders in nerve fibers (VIP or SP) in the circular muscle in the proximal colon and that pediatric STC may be composed of subgroups with different neuronal disorders.

Age-related changes in nerve fibers

The area of nerve fibers containing NOS, VIP or SP was consistently higher in children compared to adults. This suggests there is decreasing nerve fiber density with age, as reported in human SIG33 and in animals.37,41–43 Gomes et al. demonstrated that the density of neurons in human colon decreases with age.44 We recently studied changes in nerve fibers in the human sigmoid colon with aging.33 There was a 50% decrease in the density of NOS-IR, VIP-IR and SP-IR nerve fibers from birth to late adolescence with little further change with aging. Importantly, the circular muscle increased in thickness during childhood and the reduction in nerve density was associated with the increase in muscle thickness rather than a loss of nerve fibers.33 As the muscle gets thicker, the number of nerve fibers stayed the same, so the density of nerve fibers decreased.

This means that, children who start with a low density of nerve fibers would be expected to have an even lower density as they grow. This may explain the onset of chronic constipation in some patients after childhood. Women are particularly prone to develop STC as teenagers or after childbirth.45,46 Effects of sex hormones may explain the occurrence of the gender bias in adults but not children.47 Effects of childbirth on the pelvic floor may add additional stress in females.

It has been recognized for some time that appropriate, age-matched controls are important,21 however these studies are handicapped by the lack of access to bowel from normal children.21,33 Due to ethical and access issues it is difficult to obtain colonic muscle from healthy children. In adults, the common ‘control’ tissue is from the margin of bowel removed for cancer. Few children have bowel cancer or have normal bowel removed and we obtained samples from only two FAP patients over 10 years (from a feeder population of 3 million people). Children with HD have the aganglionic bowel removed and it is most commonly SIG. It would be valuable to obtain normal pediatric bowel. In the absence of any truly normal tissue, we collected bowel from children with chronic constipation, where the holdup occurred in the anorectum (NAR) and others where the delay was in the proximal colon (STC). We compared the density of nerve fibers in the bowel in the two groups of children.

Regional variation in neurotransmitters in pediatric colon

Only a few studies have looked at variation in density of nerves in different regions of colon because of the difficulty of obtaining samples. In a recent detailed study, Wattchow et al. showed that NOS neurons make up 51% of ascending colon and 44% of descending colon myenteric neurons,48 however they did not report total neuron numbers. In an earlier study, they reported that choline acetyl transferase (ChAT), NOS and VIP neurons project into the circular muscle.40 Anlauf et al. 49 reported a constant density of cholinergic and VIP nerves throughout the large bowel circular muscle. In this study, there was also a constant density of NOS, VIP and SP nerve fibers in circular muscle from transverse and SIG in adults. In contrast, Matini et al. 50 reported a lower density of VIP and NOS neurons in right (proximal) than the left colon. In a previous study of ‘normal’ pediatric samples, our group reported 39 a constant density of VIP and SP nerve fibers in the circular muscle in proximal and distal colon. In this study, the 11 NAR children had a similar density of NOS and VIP nerve fibers in all regions. However, they had more SP-IR nerve fibers in the sigmoid than in RTC or LTC (Fig. 4A1).

NOS

Physiological studies suggest there is altered NO transmission in adult STC.18,20 Immunohistochemical studies reported both increases22 and decreases23 in NOS-IR neurons in adult chronic idiopathic constipation. We found no quantitative changes in NOS-IR nerve fibers in circular muscle in children with STC (Fig. 4A3).

VIP

Some NOS-containing nerves also contain VIP.40,51–53 VIP causes direct relaxation of colonic muscle cells by activating receptors to increase cAMP, but also stimulates NO production.54 It has also been suggested that NO mediates VIP release from inhibitory nerves.55 Studies in adult constipation have shown increased56 or decreased VIP-IR.22,24,25

In the biopsies from the hepatic flexure (RTC) of STC children, we found VIP-IR was low compared to NAR children. The hepatic flexure is considered the primary site of colonic motility.57 Decreased VIP might decrease relaxation, producing an increased muscle tone and poor stool propagation and could possibly lead to pseudo-obstruction.

Substance P

SP neurons comprise a small subgroup of myenteric neurons (<6%).58,59 This study showed low SP-IR in the RTC in 23% of STC children using visual qualitative analysis and in 30–40% using quantitative analysis, supporting previous qualitative studies reporting low SP-IR in the proximal colon in STC children.26 Low SP-IR may represent fewer nerve fibers or reduced SP content per axon. Previously in STC, there are reports of hypoganglionosis,60,61 reductions in cholinergic myenteric neurons48 and low density of SP-IR nerve fibers in circular muscle21 In contrast, Sjolund et al. 56 found increased SP-IR in the myenteric plexus in the proximal colon. There were no changes in SP (using RIA) in adults with chronic constipation.24,25

The current study showed lower SP-IR in adults than children and in older STC children compared with the younger group (Fig. 5). We have reported measurements on SP-IR nerves in STC children for over 12 years and during this period, the proportion with low SP has decreased (Table 1). Children in the earliest studies were older and this age bias also may have contributed to the higher incidence of low SP-IR. The reduced SP in the RTC could be due to increases in muscle thickness, however the same reductions did not occur in the LTC and SIG.

Table 1.   Numbers of patients with low levels of SP nerves in studies from our group
Samples fixednPatients with low SP% of patients with low SPAge range (mean)Age range of controlsRef #
1993–1995107/10702–15 years (mean 7.5)6 months–6 years26
1993–19989563/95667 months–19 years (mean 7.9)6 months–6 years36
1998–20004832/48352–18 years (mean 8.7)37 adults – 23–87 years29
2000–20084410/44233–15 years (mean 8.2)3–15 years 33 adults – 23–87 yearsThis study

The tachykinins (TK), SP and neurokinin A (NKA), are present in excitatory motor neurons, interneurons and sensory neurons.62 Although originally identified as excitatory transmitters, recent studies show that TK in enteric neurons also have inhibitory effects,63–66 binding to NK1 receptors on inhibitory neurons,67,68 exerting inhibitory restraint on circular muscle and descending excitatory and inhibitory pathways during propulsion, inhibiting colonic propulsion,69 stimulating VIP release and altering NO production.70,71 Thus reductions in SP could have multiple effects.

Tachykinin receptors (NK1, NK2 and NK3) are present on muscle cells, nerves and interstitial cells of Cajal (ICC).67,72–76 In adult chronic constipation, the colon is hypo-responsive to TKs.77 The contractile reflex to distension is greatly reduced but can be restored by incubation with NK2r and NK3r agonists, suggesting it normally relies on release of TK that is not occurring in chronic constipation. The maximal contractile response to a selective NK2r agonist [beta-ala8] NKA(4–10) was significantly higher in adult chronic idiopathic constipation.78 In colon from STC children, we found that in vitro muscle contractions induced by TK were diminished, with the greatest reduction via NK2r.29

Immunoreactivity variations with processing

This study demonstrates the difficulty in quantifying neurotransmitters in nerve fibers in pediatric intestine. We compared adults with cancer (= 33) and a small group of children with chronic constipation with normal transit in the proximal colon and anorectal retention (= 11) with a large cohort of children affected by slow transit in the proximal colon (= 51). Immunohistochemistry is highly skilled and variability in fixation and labeling can affect fluorescence. The microscope and the strength and type of light source also affect the signal. To insure consistency, we cut sections of one sample and stained them in every staining run. This systematic approach to processing and quantification resulted in variance due to sampling of 5–10% of the mean value (data not shown).

Individual samples may have antigen loss due to the variations in fixation and storage. For example, SP is unstable at room temperature and may degrade when frozen. To minimize differences, all samples were fixed within 30 min of removal, sectioned and stained the next day, and viewed within 1 month of collection. These logistics are unlikely to be possible in routine hospital pathology laboratories.

Primary vs secondary defect

The role of neurotransmitters in pediatric STC remains controversial, as changes in neurotransmitters may be primary or secondary. A reduction in SP-IR with normal NOS-IR and VIP-IR has been demonstrated in a constipated child with Multiple Endocrine Neoplasia 2B, despite ganglioneuromatosis.79 In this child, the selective loss of SP in only the nerve fibers was thought to be primary, with constipation a secondary phenomenon.

Other possible aetiologies of pediatric STC

While defects in NOS, VIP and SP have been reported in adult STC, low SP-IR and VIP-IR in only 30–40% of STC children suggests that STC is a heterogeneous condition. Other neuronal subgroups may be affected and require further study. Muscle defects,80,81 and loss of ICC have been reported in adult STC,82–84 while autonomic nervous system defects have been reported in upper gastrointestinal tract disorders85 and colonic disorders86–88 and the central release of corticotrophin releasing hormone has been shown to influence colonic motility.89 Children with STC lack a postprandial increase in colonic motor activity,90,91 suggesting normal changes in postprandial hormones might be lacking.

Colonic manometry in children with chronic constipation

Colonic activity has been measured using multilumen catheters to measure pressure changes in the colon. Twenty-four hour studies in STC children show they have less high amplitude propagating contractions (HAPC) and have a reduced frequency of antegrade propagating contractions and show a poor waking and postmeal responses.90,91 Bisacodyl challenge also produces less HAPC in adults with STC.92

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

There are reductions in NOS-IR, VIP-IR and SP-IR nerve fibers in the circular muscle with aging and there are regional differences in SP but not NOS or VIP in pediatric colon. Pediatric STC is associated with low SP-IR or low VIP-IR in the RTC in some patients and reductions in SP in older STC children. 1/3 of STC children have a visible defect in nerve fibers in the circular muscle. These patients have been previously classified as functional and may now be considered organic.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Supported by grants (114215, 216704) and Senior Research Fellowship (436916) from NHMRC Australia, PhD scholarships (SKK) from Royal Australasian College of Surgeons and NHMRC, Murdoch Childrens Research Institute and King’s Fund London (MPS). Thanks to Dr. Patricia Hengel and Mark Antonello for early studies and quantification. There are no other financial interests. Presented at the American Gastroenterology Association Annual meeting, 2008 [Gastroenterology 134 (4) S1: 420. M1793] and the American Motility Society Annual meeting, 2006 [Neurogastroenterology and Motility 18 (8) 669. A13, 2006].

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References