Patient data
HD patients who were included in this retrospective, monocentric study underwent transanal pull through (TAPT) surgeries at the Department of Pediatric Surgery, University Hospital Frankfurt/Main, Germany. In all cases, the diagnosis of HD was confirmed by transanal rectal biopsies prior to TAPT surgeries. The specimens (for detailed information please see Table 1). were independently reviewed by at least two experienced neuropathologists (AKB, PNH, MM) from the Institute of Neurology, according to the diagnostic criteria that were proposed by Knowles [19]. Patients with long-segment diseases were excluded from this study. The ages at the time of the surgeries ranged from 28 days to 25 months (mean age: 6.5 months). The study cohort consisted of 11 male (m) and 3 female (f) patients.
Ethical statement
The parents of the patients gave written informed consent for the surgery. The use of patient material was approved by the ethical committee of the Goethe University Frankfurt, Germany (GN 438/19).
Sampling
Partial colectomy specimens were examined and measured (n = 14). Additionally, each resected bowel segment was macroscopically analyzed with regard to the changes in diameter in the zones of bowel obstruction. The length between the proximal resection margin and the beginning of the dilated colon was documented (Fig. 1a). Swiss roll specimens were prepared according to the following guidelines. First, from each of the entire rectosigmoidal specimens, a longitudinal, 0.5–1 cm wide strip was excised in paramedian orientation to the antimesocolic tenia. Afterwards, the strip of the intestinal wall was coiled in caudocranial orientation [20] in cryogel (Tissue Tek O.C.T., Sakura, Alphen aan den Rijn, Netherlands) attached to a cork plate and snap frozen (see below). Swiss rolls were then used to analyze the overall innervation pattern. The “serial biopsies” (referring to the specimens from the transition zone (TZ): TZ 0 cm, 5 cm, 10 cm and 15 cm) were prepared as follows: the beginning of the change in the diameter was defined as the beginning of the transition zone and referred to as TZ 0 cm. A horizontal incisional antimesocolic biopsy of approximately 3 cm × 1 cm was excised at 0 cm. Further rostral biopsies were performed at 5 cm, 10 cm and 15 cm (TZ 5 cm, TZ 10 cm, TZ 15 cm) (Fig. 1a). The biopsies were fixed in cryogel on the cryostat specimen holder. All specimens were frozen at − 80 °C in isopentane, which was precooled in liquid nitrogen. Then, 15-μm-thick sections were cut with a manual cryostat system (Leica CM 1900, Wetzlar, Germany).
Staining and enzyme histochemistry
Cryosections were stained with H&E, NADH reaction, LDH reaction and AChE reaction, according to standard protocols (Fig. 1b). The incubations were performed at 37 °C for 120 min (NADH), 45 min (LDH) and 90 min (AChE). Stained sections were mounted in Entellan (Merck Millipore, Darmstadt, Germany).
Scoring
Before performing semi-quantitative and quantitative analyses, a joint training was done to reduce inter-observer variability (AKB, PNH, MM). In case of doubt during quantification, a joint analysis was performed until agreement was reached. A semi-quantitative scoring system was applied to evaluate the pathological cholinergic innervation in the mucosal layer (Fig. 1c, d). The four grades of the AChE score were defined as follows: score 0, no AChE-positive nerve fibers in the mucosal layer (Fig. 2a, e); score 1, mild positivity (1 fiber per crypt within the lamina propria mucosae (LP)) (Fig. 2b, f); score 2, moderate positivity (up to 2 fibers per crypt) (Fig. 2c, g) and score 3, strong positivity with 3 or more fibers per crypt (Fig. 2d, h) in the LP. GCs were counted within the myenteric plexus using NADH enzyme histochemistry (Fig. 3).
Based on the AChE scoring, two different analyses were performed. First, we determined the number of GCs/mm in the NADH stainings in areas corresponding to each distinct AChE score in the swiss roll (Fig. 1c. For raw values see Supplementary Table 1). In most specimens, areas meeting each grade of the AChE score were present. For the second analysis, transition zone samples were analyzed for AChE scores, as well as for the number of GCs/mm in the corresponding area (Fig. 1d. For raw values see Supplementary Table 2). The length of the myenteric plexus segment was determined by an open polygon line using image analysis software analySIS docu (Olympus, Hamburg, Germany). Depending on the quality of the specimens, the amount GCs in a segment of 20–40 mm were counted. Absolute GC count was then normalized to the length of the respective myenteric plexus segment to compare GC density in GC/mm. The imaging and evaluation were performed using a light microscope (Olympus BX41) at a magnification of 40x (length) or 400x (cell count).
Statistical analysis
For statistical analyses, we used single case overlay diagrams (Fig. 4b). Significance levels were calculated by using an ANOVA and Tukey’s test (Fig. 4a, c, d). A significance level of α = 0.05 was determined for all tests. Statistical analyses were performed by using the JMP 11.0 software (SAS, Cary, NC, USA).