The Effect of the Ganglionic Segment Inflammatory Response to Postoperative Enterocolitis in Hirschsprung Disease

Abstract Introduction We examined the relationship between proinflammatory cytokines that occur in the inflammatory reaction in the intestine in Hirschsprung disease (HD) and Hirschsprung-associated enterocolitis (HAEC). Methods Thirty cases (M:27, F:3) operated on due to HD. The cases were divided into three groups: group 1 with pre and post operative EC, group 2 with post-operative, and group 3 with pre-operative EC. The intestinal segments were evaluated by immunohistochemistry for interleukin 1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6). Results IL-1β staining was significantly higher in the ganglionic zone of groups with enterocolitis compared to the control group (p = 0.012). TNF-α staining in the transitional zone of Group 3 and IL-1β staining in the ganglionic zone of Group 1 was significantly higher than the control group (p = 0.030, p = 0.020). Conclusion In our study, older age at diagnosis and more than 20% IL-1ß staining in the ganglionic segment were found to be risk factors for HAEC. It is noteworthy that the increase in IL-1ß can be associated with HAEC.


Introduction
Hirschsprung's disease is an intestinal motility disorder resulting from incomplete migration of neural crest cells (which are precursors of intestinal ganglion cells) during intestinal development [1].While the distal rectum is commonly affected, the length of the affected bowel can vary from a short segment to total aganglionosis.The absence of ganglion cells and the nitric oxide synthase enzyme in the myenteric and submucosal plexuses of the affected area leads to impaired intestinal relaxation and functional obstruction [2].Hirschsprung-associated enterocolitis (HAEC) is the primary cause of mortality and morbidity in both pre-and post-operative periods.Even after resection of the intestinal segment lacking ganglion cells, some patients experience postoperative HAEC attacks [3].
Although the pathogenesis of HAEC involves mucosal irregularities, inflammatory cell migration, chronic stress, increased intestinal wall permeability, and bacterial translocation (such as Escherichia coli and Clostridium difficile), this situation remains incompletely understood.Previous studies have shown a relationship between inflammatory cytokines and HAEC, including increased levels of IL-36 and IL-17R and decreased IL-8 [4][5][6].Proinflammatory cytokines IL-1β, TNF-α, IFN-γ, and IL-6 are recognized for initiating inflammation in HAEC and necrotizing enterocolitis, stimulating the release of other proinflammatory mediators, and causing cellular damage [7][8][9].
In this study, we evaluated the clinical and laboratory factors influencing HAEC and investigated the potential use of immunohistochemical stains as a predictive marker of postoperative HAEC.

Materials and methods
This study was conducted jointly by the Pediatric Surgery and Pediatric Pathology departments of a university hospital and received approval from the institutional review board (Istanbul University-Cerrahpasa Ethics Committee, No: 2022-351619).All surgeries for the cases were performed by two senior pediatric surgeons (GTT, RO).
Patients who underwent primary surgery using the transanal endorectal pull-through (TERP) method for Hirschsprung's Disease (HD) between 2012 and 2022, and who had a transition zone at the rectosigmoid and descending colon level were retrospectively analyzed.Cases involving other systemic diseases or chromosomal anomalies, total colonic aganglionosis, long segment aganglionosis, stoma opening, or non-TERP surgical techniques were excluded from the study.
The cases were categorized into three groups based on the timing of their enterocolitis: Group 1: Cases experiencing enterocolitis both before and after surgery.Group 2: Cases with postoperative enterocolitis.Group 3: Cases with preoperative enterocolitis.The control group consisted of HD patients who had never encountered enterocolitis.The aim was to ascertain differences in the inflammatory response between HD patients with and without enterocolitis.
The cases were evaluated in terms of gender, age at diagnosis, age at surgery, severity and numbers of enterocolitis, and inflammatory markers in intestinal segments resected.
Enterocolitis was defined using the HAEC score, clinical assessment, C-reactive protein (CRP) levels, hospitalization duration, requirement for intravenous antibiotic therapy, intravenous hydration, and rectal irrigation needs.The HAEC scoring system developed by Pastor et al. [10] was employed.

Immunohistochemical staining
We conducted immunohistochemistry to assess the expression of inflammatory markers (IL-1β, IL-6, and TNF-α) in resection specimens separately, specifically in the ganglion-negative, transitional, and ganglion-positive zones.One block per segment was chosen through slide reassessment.
The staining ratio and intensity of epithelial cells were evaluated for IL-1β, IL-6, and TNF-α.Cytoplasmic staining was regarded as positive.The staining ratio was assessed semiquantitatively as 10% increments.The staining intensity also scored as weak, moderate, and strong.

Statistical analysis
Statistical analyses were performed using SPSS 15.0 for Windows.Descriptive statistics were given as numbers and percent for categorical variables for categorical variables and mean, standard deviation, minimum, maximum, and median for numerical variables.The distribution of numerical variables was not normal, and independent samples were compared with the Whitney U test.Subgroup analyses were interpreted by using Bonferroni correction.Spearman correlation analysis analyzed the relationships between numerical variables since the parametric test condition was unmet.Cutoff value analyzed with ROC curve analysis.The statistical alpha significance level was regarded as p < 0.05.The correlation coefficient (cc) was evaluated as >0.3 positive and <0.3 negative correlation.
There was no statistically significant difference between the diagnosis ages among all groups (p = 0.10).When comparing the groups separately, only Group 3 exhibited a statistically significantly higher age at diagnosis (p = 0.016) compared to the control group.The age of surgery did not significantly differ between the groups (p = 0.29).A pairwise comparison revealed that the patients in Group 3 underwent surgery at an older age compared to the control group (p = 0.024).Age at diagnosis, age at surgery, and time from diagnosis to surgery showed no correlation with HAEC count and score (each with cc < 0.3).No correlation was found between CRP value and HAEC score (cc = 0.06).There was a correlation observed between the interval from diagnosis to surgery and CRP value (cc = 0.51).

Immunohistochemical staining
The staining frequency of each group for IL-1β, IL-6, and TNF-α were summarized in Table 2.
Since staining intensity showed no variance among cases, statistical analysis relied on the staining frequency scored in 10% increments.
When comparing the control group and groups with enterocolitis separately, IL-1β staining in the transitional zone of patients with only preoperative enterocolitis (Group 3) was significantly higher than patients with both pre and postoperative enterocolitis (Group 1) (p = 0.008).IL-1β staining in the ganglionic zone in Group 1 was higher than in the control group (p = 0.020).Analysis of IL-1β staining in the ganglionic zone of Group 1 and the control group did not reveal a statistically significant cutoff value.(AUC: 0.646 p = 0.157).
In the analyses of IL-6, no statistically significant differences were observed in all intestinal zones.This held for comparisons between the control group and Groups 1, 2, and 3, as well as for comparisons among Groups 1, 2, and 3 themselves (p > 0.05) (Figure 7).
No relation was found between the number of pre and postoperative enterocolitis episodes and the level of staining (Table 5).

Discussion
Hirschsprung disease (HD) is a congenital enteric neuropathy resulting in functional bowel obstruction due to distal intestinal aganglionosis [11].It commonly presents as short-segment aganglionosis, affecting only the rectosigmoid colon in approximately 80% of cases.Less frequently, it may extend proximally to the sigmoid colon (15%), involve the entire colon (total colonic aganglionosis, 5%), or exceptionally, encompass the entire intestine (total intestinal aganglionosis) [12].HAEC poses a significant challenge, being the primary concern in both preoperative and postoperative periods and a leading cause of mortality and morbidity.The precise mechanism underlying HAEC and its predisposing factors remain elusive [13].Understanding the pathogenesis and influencing factors of HAEC will also enable new treatment options.Roorda et al.'s study did not establish any correlation between postoperative HAEC and the demographic data or clinical findings [14].Conversely, Sakurai's study indicated an association between HAEC and long-segment HD as well as older age at surgery [15].In our series, a higher frequency of enterocolitis was observed in patients diagnosed at an older age during  the preoperative period.No discernible difference was found between the patients experiencing enterocolitis before and after surgery and those with solely postoperative enterocolitis concerning the age at While an older age at diagnosis was identified as a risk factor for preoperative enterocolitis, it did not influence postoperative HAEC.
The HAEC scoring systems serve as a guide for clinicians in diagnosing the condition and establishing a common terminology but may lack information regarding the disease's severity [10].As CRP levels are often linked to the severity of infectious or inflammatory diseases, they offer a potential means to assess the severity of enterocolitis [16,17].Our study revealed a positive correlation between the duration from age at diagnosis to surgery and CRP values across all groups.These findings suggest that the period between surgery and diagnosis age does not pose a risk factor for HAEC but correlates with increased severity and clinical symptoms.
While the pathogenesis of HAEC remains controversial, current research is shedding light on factors such as deficiency in intestinal mucosal immune response, compromised defense barriers, and inadequate cholinergic activity.Keck et al. [18] reported that in the aganglionic intestinal segment or transition zone lacking mucosal cholinergic innervation, there is a reduction in the release of Th17, a factor known for its anti-inflammatory properties, leading to the dominance of pro-inflammatory cytokines (such as IL-1ß, IL-6).Inflammation associated with IL-1β is identified as a primary contributor to irreversible tissue damage and non-healing cellular reactions.
In the study of Tomuschat et al. [19], they reported that the deficiency of NLRP6, the primary inflammasome complex that aids protective immunity and inhibits IL-1ß synthesis, plays a role in HAEC.Similarly, our study found significantly higher IL-1β staining in the ganglionic zone of groups with enterocolitis (both pre and postoperative) compared to those without enterocolitis.IL-1β staining in the ganglionic segment of the pre + postoperative enterocolitis group was higher than the control group, suggesting IL-1β's involvement in HAEC pathogenesis.Our study proposes that IL-1β staining above 20% can serve as a predictive marker for HAEC.If further studies validate these results, initiating prophylactic anti-inflammatory treatment in patients exhibiting high IL-1β levels in rectal biopsy and resection material could potentially reduce the frequency and severity of HAEC attacks.
TNF-α is recognized for its role in maintaining the mucosal barrier and balancing between protective immunity and inflammatory responses [20].Elevated TNF-α levels contribute to the release of other pro-inflammatory cytokines (IL-1β, IL-6), exhibiting a synergistic effect.In our study, the TNF-α levels in the transition zone of patients with preoperative enterocolitis only were notably higher compared to both the control group and those with preoperative + postoperative enterocolitis.Simultaneously, we observed heightened IL-1β levels in the transition zone of the preoperative enterocolitis group.Due to the limited case number, we did not observe a significant difference in TNF-α levels between the ganglionic and aganglionic zones of the enterocolitis groups and the control group.Based on the current dataset, it's challenging to conclude whether TNF-α predisposes to HAEC.Future studies with larger sample sizes may provide better insight into this aspect.
There are some limiting factors in our study.The retrospective nature restricted access to all tissue blocks in the pathology archive, resulting in a lower case count and varying numbers among groups.
As a result, in our study, we identified older age at diagnosis and more than 20% IL-1ß staining in the ganglionic segment as risk factors for HAEC.IL-1ß demonstrated potential as a predictive marker for postoperative HAEC.These findings offer guidance for future HAEC prevention and treatment studies, particularly post-surgery.A comprehensive delineation of pro-inflammatory cytokines' role in HAEC pathogenesis could pave the way for prophylactic anti-inflammatory agent use in high-risk cases.

Figure 3 .
Figure 3. negative staining for il-1β in inflammatory cells in the epithelial area and lamina propria (100X).

Figure 4 .
Figure 4. roc analysis for il-1β in the ganglionic zone in enterocolitis groups (aUc: area under the roc curve).

Table 1 .
the characteristics of the groups.

Table 2 .
the amount of staining of each group for il-1β, il-6, and tnF-α.

Table 3 .
comparison of control group and enterocolitis groups.

Table 4 .
comparison between groups.

Table 5 .
comparison of the number of enterocolitis and the ratio of cytokines.