Increasing Trend of Calretinin-Positive Mucosal Innervation from Aganglionic Zone toward Transition Zone in Hirschsprung's Disease

Abstract

Introduction Diagnosis of very short-segment Hirschsprung's disease (vsHD) by rectal suction biopsy is challenging as its aganglionic zone (AZ) overlaps with physiologic hypoganglionic zone and calretinin-positive mucosal nerves may extend from the transition zone (TZ) into AZ. We studied whether an increasing trend/gradient of calretinin-positive mucosal nerves along the distance from AZ toward TZ aids in diagnosis of HD. Materials and Methods In this study, 46 rectal suction biopsies from non-HD and HD, and 15 pull-through specimens from short-segment HD were evaluated by mucosal calretinin immunostain (CI) and image processing and analysis (IPA) to measure pixel count (PC, the percentage of calretinin stained pixels in the mucosa). Consecutive longitudinal sections of proximal AZ toward distal TZ in HD pull-through specimens were utilized as a vsHD surrogate model. First, we studied variability of mucosal CI in non-HD biopsies along the distance from dentate line. Second, we determined a cutoff point of mucosal CI by IPA that separated non-HD versus HD and applied this cutoff to longitudinal sections from proximal AZ to distal TZ segments in HD pull-through specimens. Third, we studied whether an increasing trend of mucosal CI was universally observed in HD pull-through. Results Our findings included a significant variability in PC along the biopsy distance in non-HD cases. Positive mucosal CI was found in proximal AZ in 6 (43%) of 14 HD pull-through, among which 1 case lacked submucosal nerve hypertrophy in the proximal AZ. All 14 HD pull-through cases showed an increasing trend/gradient of PC from AZ toward TZ. Conclusion Based on our findings, the presence or absence of mucosal CI positivity and submucosal nerve hypertrophy may not reliably diagnose vsHD in rectal suction biopsy. While we acknowledge that the density of mucosal innervation in variable contexts and anatomical locations is unknown and yet to be explored, our study suggests that an increasing trend of positive mucosal CI from AZ toward TZ by IPA might prove to be a useful tool for the diagnosis of vsHD in the future.

Inflammation and tissue remodeling contribute to fibrogenesis in stricturing Crohn’s disease: image processing and analysis study

“…We postulate that the submucosal neural hypertrophy seen in CD is a reactive process to compensate for impeded bowel movement, similar to Hirschsprung disease. In conventional Hirschsprung disease, submucosal nerve hypertrophy is predominantly seen in the transition zone and aganglionic bowel segment, but usually not in the proximal ganglionated bowel [ 21 ].…”

“…Pixel count (PC) was defined as the percentage of immunostained pixels out of the total number of pixels in an image. PC for calretinin (PC-calr), CD34 (PC-CD34), and calprotectin (PC-calp) were calculated using Image Processing and Analysis (MATLAB) as previously described [19][20][21].…”

“…Calretinin immunohistochemistry highlights mucosal innervation in ganglionated bowel. Image processing and analysis has been utilized in recent studies of Hirschsprung disease wherein the amount of mucosal calretinin immunostaining was quantified by counting the pixels with immunostaining [19][20][21].…”

Do Histologic Features of the Proximal Margin of Resected Specimens Predict Clinical Outcomes in Hirschsprung Disease?

“…Hirschsprung disease (HSCR) is a congenital aganglionosis of the rectum and colon that extends for a variable distance proximally from the internal anal sphincter (IAS). It is associated with the absence of ganglion cells in the submucosal and myenteric plexus, the absence of mucosal calretinin staining, and an increase in acetylcholinesterase nerve fiber activity [1][2][3][4]. The high levels of excitatory neurotransmitters, such as acetylcholine, lead to a spastic contraction of the diseased bowel, causing a functional obstruction [5].…”