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Published in final edited form as: J Pediatr Surg. 2016 Aug 9;51(11):1834–1838. doi: 10.1016/j.jpedsurg.2016.08.001

Bowel dysfunction following pullthrough surgery is associated with an overabundance of nitrergic neurons in Hirschsprung disease

Lily S Cheng a,1, Dana M Schwartz a, Ryo Hotta a, Hannah K Graham a, Allan M Goldstein a,*
PMCID: PMC5065396  NIHMSID: NIHMS817515  PMID: 27570241

Abstract

PURPOSE

Recent evidence suggests that patients with Hirschsprung disease (HD) have abnormal neurotransmitter expression in the ganglionated proximal colon. These alterations may cause persistent bowel dysfunction even after pullthrough surgery. We sought to quantify the proportion of nitrergic neurons in the ganglionic colon of HD patients and relate these findings to functional outcome.

METHODS

The proximal resection margin from 17 patients with colonic HD who underwent a pullthrough procedure and colorectal tissue from 4 age-matched controls were immunohistochemically examined to quantify the proportion of nitrergic neurons. The incidence of constipation, incontinence, and enterocolitis in HD patients was assessed retrospectively and correlated with the proportion of nitric oxide synthase (NOS) expressing neurons. Neuronal subtypes in the ganglionic colon of the Edrnb−/− mouse model of HD were also studied.

RESULTS

Mice with HD had a significantly higher proportion of NOS+ neurons in ganglionic colon than normal littermates (32.0 ± 5.6% vs. 19.8 ± 1.2%, p<0.01). Patients with HD also had significantly more NOS+ neurons than controls (18.4 ± 4.6% vs. 13.1 ± 1.9%, p<0.01). Patients who experienced constipation or enterocolitis postoperatively tended toward a higher proportion of NOS+ neurons (21.4 ± 3.9% vs. 17.1 ± 4.1%, p=0.06). Furthermore, patients with a proportion of NOS+ neurons above the median of all HD patients (18.3%) were significantly more likely to have constipation than those below the median (75% vs. 14%, p<0.05).

CONCLUSION

An overabundance of nitrergic neurons in the proximal resection margin is associated with HD and may predict bowel dysfunction following pullthrough surgery.

Keywords: Hirschsprung disease, Constipation, Enteric nervous system, Myenteric plexus, Nitric oxide synthase, Calretinin

1. INTRODUCTION

Hirschsprung disease (HD) is a common congenital disease characterized by the absence of enteric ganglia in the distal intestine [1]. Even after pullthrough surgery, patients often suffer from persistent problems with constipation, fecal incontinence, and Hirschsprung-associated enterocolitis (HAEC) [2]. Though functional outcome may improve with age, long-term studies have found that many patients with HD continue to have impaired bowel function and, as a result, suffer psychosocial stress into adulthood [3, 4].

Recent studies have suggested that the remaining ganglionic intestine in HD patients may not be normally innervated and that these latent abnormalities could be responsible for persistent bowel dysfunction in some patients [5]. Abnormalities in neurotransmitter expression have been described in the ganglionic bowel of HD patients, with a relative overabundance of nitrergic innervation and a deficit of cholinergic innervation [6]. The overexpression of nitric oxide synthase (NOS) and its related proteins is unique to HD and are not a result of bowel dilatation alone [7]. Similar alterations in NOS expression have been observed in several mouse models of HD and are linked to intestinal dysmotility [8-10]. Intestinal motility relies on a careful balance between inhibitory (e.g. nitrergic) and excitatory (e.g. cholinergic) innervation, and any perturbation of that balance could lead to dysmotility [11-13].

Though it has been suggested that these abnormalities may cause postoperative bowel dysfunction, no studies have yet examined the association between functional outcomes and NOS expression in HD. In addition, the proportion of nitregic neurons in the proximal resection margin of HD patients has not been quantified immunohistochemically, and this is necessary to establish the overabundance of NOS+ neurons as a pathological marker of disease. In this study, we sought to quantify the proportion of nitrergic neurons in ganglionic colon of mice and children with HD and correlate these abnormalities with functional outcome after pullthrough in children.

2. MATERIALS AND METHODS

2.1 Animal Subjects

With approval from the Institutional Animal Care and Use Committee, tissue was obtained from 3-week old homozygous Ednrbtm1Ywa (Ednrb−/−) mice on a hybrid C57BL6/J-129Sv background (B6;129-Ednrbtm1Ywa/J; Jackson Labs) or their wild-type (Ednrb+/+) littermates. Ednrb−/− mice, characterized by distal colonic aganglionosis and a piebald color, are a commonly used model for human HD [14]. After euthanasia, colons were removed, opened along the mesenteric border, and flushed of enteric contents. The whole colon was fixed overnight in 4% paraformaldehyde and tissue embedded in 15% sucrose containing 7.5% gelatin for cryosection.

2.2 Human Subjects

With approval from the Institutional Review Board, paraffin-embedded biopsies of the proximal resection margin were obtained from 17 patients with partial colonic HD who had undergone a pullthrough procedure (Soave) between September 2003 and April 2016 at a single institution. All resection margins had been examined by pathologists and considered to be normoganglionic. For comparison, colorectal biopsies were obtained from 4 age-matched control patients, including 2 patients with chronic constipation who had rectal biopsies to rule out HD, 1 patient with anorectal malformation (ARM), and 1 patient with a sigmoid stricture after necrotizing enterocolitis.

For patients with HD, charts were retrospectively reviewed to determine length of bowel resection and clinical outcome including need for reoperation, constipation, fecal incontinence, and HAEC. A senior surgical resident (D.M.S.) reviewed patient outcomes blinded to the immunohistochemical findings for each patient.

2.3 Immunohistochemistry

Immunohistochemical studies were performed on either 12 μm cryosections (mice) or 5 μm paraffin-embedded sections (humans). For cryosections, slides rehydrated in warmed PBS to remove surrounding gelatin. For paraffin-embedded tissue sections, slides were heated at 65 °C for 1 hour, then rehydrated by sequentially soaking in xylene (5 minutes × 3), ethanol (100% for 5 minutes × 3, followed by 90%, 75%, 50%, 25%, 10% for 1 minute each), water (5 minutes × 3), and phosphate-buffered saline (PBS; 5 minutes × 3). Antigen retrieval was performed by heating slides in a 1 M sodium citrate buffer solution (15 minutes).

For both cryosections and paraffin-embedded sections, tissue was permeabilized and blocked with 0.1% Triton X-100, 10% donkey serum, and 10% bovine serum albumin in PBS for 1 hour. Primary antibodies were applied overnight for 12-16 hours at 4 °C and secondary antibodies were applied for 1 hour at room temperature. Primary antibodies included: mouse anti-neuronal class III β-tubulin (Tuj1, 1:500; Covance, Dedham, MA), rabbit anti-neuronal nitric oxide synthase 1 (NOS, 1:500; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-calretinin (CR, 1:200; Invitrogen, Carlsbad, CA). Neuronal class III β-tubulin (Tuj1) marks all enteric neurons; calretinin (CR) immunoreactivity labels enteric, and not extrinsic, nerves [15, 16]; and NOS immunoreactivity indicates nitrergic neurons in the gut. Secondary antibodies used were: donkey anti-mouse Alexa Fluor 488 (1:1000; Invitrogen) and donkey anti-rabbit Alexa Fluor 546 (1:1000; Life Technologies, Carlsbad, CA). Cell nuclei were stained with DAPI. Images were taken using a Nikon Eclipse 80i microscope. Three or more randomly-selected myenteric ganglia were imaged for each subject. Cell numbers were quantified using ImageJ software (NIH, Bethesda, Maryland).

2.4 Statistics

Data are presented as mean ± standard deviation. Results were compared using Student’s unpaired t-test for continuous variables and Fisher’s exact test for dichotomous variables. Statistical significance was considered at p<0.05. Statistical analysis was performed using using Graphpad Prism version 7.0 for Windows (Graphpad Software, La Jolla, CA).

3. RESULTS

3.1 Ednrb−/− mice have a greater proportion of nitrergic neurons in the ganglionic colon than Ednrb+/+ littermates

In the aganglionic distal colon of Edrnb−/− mice, hypertrophic extrinsic nerves were immunoreactive for Tuj1, but negative for CR (Fig. 1A) and NOS (Fig. 1D), distinguishing them from intrinsic ganglia. In the transition zone, small ganglia consisting of CR+Tuj1+ (Fig. 1B) and NOS+Tuj1+ (Fig. 1E) cells were present along with scant NOS+Tuj1+ and CR+Tuj1+ double-positive fibers in the circular muscle. Proximal to the transition zone, we observed normal-appearing ganglia immunoreactive for CR (Fig. 1C) and NOS (Fig. 1F), along with abundant NOS+Tuj1+ and CR+Tuj1+ fibers throughout the muscularis propria.

Figure 1. The ganglionic colon of the Ednrb−/− mouse contains an overabundance of nitrergic neurons.

Figure 1

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Hypertrophic extrinsic nerve fibers in the aganglionic colon of Ednrb−/− mice are immunoreactive for pan-neuronal maker Tuj1, but negative for CR (A) and NOS (D). Small ganglia in the transition zone (B, E) and normal ganglia in the proximal colon (C, F) are immunoreactive for Tuj1, CR, and NOS. The proportion of CR+Tuj1+ cholinergic neurons in the proximal ganglionic colon of Ednrb−/− and Ednrb+/+ littermates is similar (G). However, ganglionic proximal colon in Ednrb−/− mice contains a significantly higher percentage of NOS+Tuj1+ nitrergic neurons (H) and a greater ratio of NOS+Tuj1+ to CR+Tuj1+ neurons than the proximal colon of Ednrb+/+ littermates (I).

Scale bar in A is 50 μm and applies to A-F.

**p<0.01; *p<0.05; CR, calretinin; NOS, nitric oxide synthase

The presence of NOS+Tuj1+ and CR+Tuj1+ neurons in the myenteric ganglia of ganglionated colon, approximately 1-2 cm proximal to the transition zone, was analyzed quantitatively. The proportion of CR+Tuj1+ neurons in the ganglionic segment did not differ significantly in Ednrb−/− mice and Ednrb+/+ littermates (19.2 ± 5.8% vs. 25.1 ± 4.3%, p=0.15; Fig. 1G). However, the ganglionic colon of Ednrb−/− mice had a greater proportion of NOS+Tuj1+ neurons compared to the equivalent colonic segment of Ednrb+/+ littermates (32.0 ± 5.6% vs. 19.8 ± 1.2%, p<0.01; Fig. 1H). The ratio of NOS+Tuj1+ to CR+Tuj1+ neurons in Ednrb−/− mice was also significantly greater than in Ednrb+/+ littermates (1.8 ± 0.6% vs. 0.8 ± 0.2; p<0.05; Fig. 1I).

3.2 Children with HD have a greater proportion of nitrergic neurons in the ganglionic colorectum than control patients

Patients with HD ranged in age from 9 days old to 7 months old (median = 2 months old) at the time of pullthrough, 88% were male, and 12% had Down syndrome. In comparison, control patients ranged in age from 1-9 months old (median = 3 months old), 100% were male, and none had Down syndrome. In colorectal samples from the ganglionic segment of HD patients and controls, all ganglia exhibited immunoreactivity for CR (Fig. 2D-E). In contrast, hypertrophied extrinsic nerves from the distal aganglionic segments of HD patients were negative for CR (Fig. 2B-C). All ganglia were also immunoreactive for NOS (Fig. 2H-I), whereas extrinsic nerves were not (Fig. 2F-G). No hypertrophied nerves were seen in specimens obtained from the proximal resection margin.

Figure 2. Ganglionic colon of patients with Hirschsprung disease contains NOS+ and CR+ neurons, whereas aganglionic colon does not.

Figure 2

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A biopsy from the aganglionic segment of a 1 month-old male with Hirschsprung disease (A, black box) shows hypertrophic nerves that are immunoreactive for pan-neuronal marker Tuj1, but negative for neuronal subtype markers CR (B, C) and NOS (F, G). A biopsy from the proximal ganglionic segment (A, white box) of the same patient shows normal myenteric and submucosal ganglia and nerve fibers throughout the muscularis propria with immunoreactivity for Tuj1, CR (D, E), and NOS (H, I).

Scale bar in B is 100 μm and applies to B, D, F, H.

Scale bar in C is 50 μm and applies to C, E, G, I.

CR, calretinin; NOS, nitric oxide synthase

We found no statistical difference in the proportion of CR+Tuj1+ neurons in the ganglionic segment (Fig. 3A-B) compared to controls (8.7 ± 3.4% vs. 8.5 ± 1.7%, p=0.82; Fig. 3E). In contrast, the proportion of NOS+Tuj1+ enteric ganglion cells in HD patients (Fig. 3B-C) was significantly greater than controls (18.4 ± 4.6% vs. 13.1 ± 1.9%, p<0.01; Fig. 3F). In addition, the ratio of NOS+Tuj1+ to CR+Tuj1+ neurons was also greater in HD patients, although this did not reach statistical significance (2.4 ± 1.0 vs. 1.6 ± 0.7, p=0.11; Fig. 3G).

Figure 3. Myenteric ganglia in patients with HD contain greater proportions of NOS+ neurons than controls.

Figure 3

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The proportion of CR+Tuj1+ in the myenteric ganglia of patients with HD (A-B) is similar to normal controls (E). In contrast, the proportion of NOS+Tuj1+ neurons is significantly greater in patients with HD (C-D) than controls (F). The ratio of NOS+Tuj1+ to CR+Tuj1+ neurons is also greater in patients with HD (G), but does not reach statistical significance.

**p<0.01; CR, calretinin; HD, Hirschsprung disease; NOS, nitric oxide synthase

3.3 Clinical outcomes in patients with HD

Of 17 patients, 13 had short-segment disease confined to the rectosigmoid and 4 patients had aganglionosis extending to the splenic flexure. All patients underwent bowel resection with transanal Soave endorectal pullthrough. The average length of colon resected was 16.3 ± 6.5 cm. The median length of follow-up was 6 years (range: 1 month to 12 years). Three patients (18%) required reoperation after pullthrough for incisional hernia, anastomotic stricture, and lysis of adhesions. No patient required reoperation for residual aganglionosis or transition zone pullthrough. Eight patients (47%) experienced constipation after pullthrough. Two patients with constipation were also diagnosed with anastomotic stricture; one was successfully treated with dilatation and the other required reoperation. Of the two patients with Down syndrome, one had constipation. Five patients (29%) experienced one or more episodes of HAEC postoperatively and all but one of these patients also had constipation. Eleven patients were toilet-trained at the time of this study and one (9%) had fecal incontinence.

Neither age at time of pullthrough nor length of bowel resected had a significant effect on the incidence of postoperative constipation. Among patients whose constipation could not be attributed to anastomotic stricture, the rate of constipation was similar between those of above and below the median age (2 months old) at time of pullthrough surgery (38% vs. 43%, respectively, p=1.0). Patients with bowel resection length greater than the median (17 cm) tended to have a higher incidence of constipation than those below the median, but this did not reach statistical significance (63% vs. 14%; p=0.12). There was no strong correlation between the proportion of NOS+Tuj1+ neurons and patient age at surgery (R2 = 0.016) or resection length (R2 = 0.098).

Patients with postoperative bowel dysfunction, specifically defined as constipation and/or HAEC, tended to have a greater proportion of NOS+Tuj1+ neurons in the proximal resection margin. Excluding patients with anastomotic stricture, the proportion of NOS+Tuj1+ neurons was greater in patients with bowel dysfunction than patients without (21.4 ± 3.9% vs. 17.1 ± 4.1%; p=0.06). Furthermore, patients in whom the proportion of nitrergic neurons was greater than the median (18.3%) experienced significantly more bowel dysfunction than those below the median (75% vs. 14%; p<0.05). In addition, though the proportion of CR+Tuj1+ neurons did not differ significantly between patients with and without bowel dysfunction (8.9 ± 2.7% vs. 9.8 ± 3.5%, respectively; p=0.55), the ratio of NOS+Tuj1+ to CR+Tuj1+ neurons was nearly 50% greater in patients with bowel dysfunction (2.8 ± 0.9 vs. 1.9 ± 0.8; p<0.05).

4. DISCUSSION

Bowel dysfunction, including symptoms of obstruction, incontinence, and enterocolitis, is common in HD patients even after pullthrough surgery [3, 17]. One possible explanation for these persistent perturbations is an imbalance of neuronal subtypes in the ganglionated proximal colon. Since nitrergic neurons are inhibitory in the gut [18], disproportionately increased expression of this neurotransmitter can lead to colonic dysmotility [13]. We found that the proportion of nitrergic neurons was significantly greater in ganglionic colon of both mice and children with HD and, importantly, a greater proportion of nitrergic neurons in the proximal resection margin was associated with a higher incidence of postoperative constipation and/or HAEC after pullthrough surgery. These findings suggest that previously unrecognized abnormalities in neuronal subtype expression in the ganglionic colon may account for postoperative bowel dysfunction in HD patients and represent a target for future therapy. If validated in a larger cohort of patients, these findings may redefine the transition zone, which is currently based largely on ganglion cell density and the presence of hypertrophic nerve fibers [19].

Further study is needed to determine the cause and extent of these neuronal subtype imbalances. One possible explanation is that HD-causing mutations also cause delayed neuronal maturation. NOS+ neurons are one of the first subtypes to appear in embryonic mouse colon [20], and failure of neuronal maturation to progress normally may result in an overabundance of NOS+ neurons. In previous studies in mouse models of HD, neuronal subtype imbalance was found to be limited to the mid and distal colon and was normal in proximal colon and small bowel [8, 9]. The extent of neuronal abnormality in HD patients is not yet known and warrants further study, as this knowledge is essential to determine the appropriate length of resection and to predict functional outcome after pullthrough. If neuronal imbalances are limited to the distal ganglionic intestine, these areas may represent a hitherto unrecognized transition zone with a normal density of ganglia but an abnormal neuronal subtype distribution, and patients may benefit from additional resection.

To account for potential confounding factors, we examined bowel function with respect to length of bowel resected and patient age at pullthrough surgery. Prior studies of functional outcome after pullthrough have suggested that a longer length of aganglionosis is associated with worse outcome, particularly in patients with total colonic aganglionosis [21, 22]. Our study excluded patients with total colonic aganglionosis, but a similar trend was seen toward a higher incidence of constipation in patients with a longer length of aganglionosis. Studies in mice have suggested that the extent of neuronal subtype imbalance also correlates with the length of aganglionosis [8]; however, we found no correlation between the proportion of NOS neurons and the length of bowel resected. In addition, studies have shown that disturbances in NOS expression are greater in older HD mice [9], but we found no similar correlation between NOS and age in our patients. As in prior studies [22], we also found no correlation between constipation and age at surgery. Given these findings, the proportion of nitrergic neurons appears to be an independent predictor of postoperative bowel function.

Though this is the largest study to date examining nitrergic neurons in the ganglionic intestine of HD patients and the first to associate it with clinical outcome, it is still limited by sample size and its retrospective nature. Likely due to low power, the incidence of constipation or HAEC alone was not statistically associated with the proportion of nitrergic neurons. Since HAEC is known to be strongly associated with obstruction [23], and because we found that most of our patients with HAEC also had constipation, we considered HAEC and constipation together as a composite measure of postoperative bowel function. Due to differences in methodology, namely the use of Tuj1 to label neuronal bodies and axons instead of the neuronal cell body marker, Hu, the percentage of neuronal subtypes we observed differs from prior studies [9, 24], but the observed trends are similar. An important caveat is that normal rectal tissue is difficult to obtain at this age, since any resection or biopsy is performed for some clinical indication. While the rectum of children with ARM may not be entirely normal, previous studies have found that the expression of NOS-related proteins is equivalent in the normal and dilated segments of bowel in patients with ARM [7]. Larger studies with more Hirschsprung patients, more “normal” controls, and analysis of additional neuronal subtypes are needed.

In addition to identifying a neuronal subtype imbalance in the setting of HD, this study also lends support to the clinical relevance of the Ednrb mouse model of this disease. We first noted the overexpression of nitrergic neurons in the Edrnb−/− mice, and this is what prompted us to corroborate that observation in human HD. Edrnb−/− mice have been used extensively for many years to deepen our understanding of the developmental etiology and clinical pathophysiology of HD [14]. It is therefore important to know that the HD seen in these mice shares many of the features observed in humans. Confirming the observation in both mice and humans both reinforces our findings and also further validates the utility of this mouse model for HD studies.

In conclusion, our data suggest that the balance of neuronal subtypes in enteric ganglia may not be normal in patients with HD. An overabundance of nitrergic neurons at the resection margin may contribute to postoperative bowel dysfunction and further validation of this observation is needed.

Acknowledgments

FUNDING

This work was supported by the National Institutes of Health (R01DK103785 to A.M.G.) and the Society of University Surgeons Resident Research Award (to L.S.C.).

ABBREVIATIONS

HD

Hirschsprung disease

HAEC

Hirschsprung-associated enterocolitis

NOS

Nitric oxide synthase

CR

Calretinin

PBS

Phosphate-buffered saline

ARM

Anorectal malformation

Footnotes

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LEVEL OF EVIDENCE

Diagnostic, Level III

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