## 3 Results
### Increase in the Percentage of ChAT\({}^{+}\) Neurons in the MP of Patients with MS  
To translate the results obtained in the EAE model to MS itself, intestinal resectates and biopsies were studied. As shown in Tables 1 and 2, patients with MS and matched CTRL patients underwent abdominal surgery for various medical reasons, which were divided into two groups: cancer versus inflammatory diseases, such as appendicitisis, [PERSON]'s disease or ulcerative colitis. In contrast to the whole-mount preparations of murine colonic tissue, the 5 \(\upmu\)m-thick sections of human tissue did not allow for the acquisition of three-dimensional z-stacks. In addition, and particularly in CTRL patients, human colon was often cut in a longitudinal direction--i.e., parallel to longitudinal muscle layers and orthogonal to circular muscle layers--showing only a fraction of the broad web that makes up the MP. This explains why the number of neurons per ganglion was lower in the human ENS compared with the murine ENS. Figure 3: Immunohistological staining of murine myenteric plexus (MP). Whole mounts were obtained from the colons of female C57 BL/6J mice with late-stage experimental autoimmune encephalomyelitis (EAE) (\(n=3\)) or from a non-immunized control group (CTRL) (\(n=3\)). For analysis of anoctamin 1 (ANO1), only tissue containing MP was included. The level of the MP was determined by co-staining of glial fibrillary acidic protein (GFAP). (**a**) Schematic representation of ICCs and their location within the gastrointestinal wall. (**b**) The percentage of the ANO1\({}^{+}\) area per image was determined. Individual data points display mean values per mouse. The box and whisker plots show median values, together with the interquartile range. Statistical significance was determined by unpaired Student’s \(t\) test. (**c**,**d**) Representative single-optical sections derived from z-stacks (z-steps: 2.4 \(\upmu\)m) with positive ANO1 staining (_red arrows_) were selected from (**c**) a non-immunized CTRL and (**d**) a mouse with late-stage EAE. For each mouse, six images from different locations were analyzed. Scale bars denote 50 \(\upmu\)m.  
For the IHC analyses, all HU\({}^{+}\) ganglia were imaged, which resulted in 156 ganglia in the SP (Figure 4) and 86 in the MP of patients with MS (Figure 5). In the CTRL group, 289 SP ganglia (Figure 4) and 331 MP ganglia (Figure 5) were analyzed. In the SP, a median of 3.02 (min-max: 2.31-4.67) neurons per ganglion were counted in CTRL patients compared with 3.89 (1-8.5) in patients with MS (Figure 4b). VIP was dismissed from analysis because in the human MP, inhibitory VIP\({}^{+}\) neurons are generally also nNOS\({}^{+}\)[16]. While nNOS was barely detectable in the neurons of the SP in both groups (CTRL: 0.00% (min-max: 0.00-28.89%); MS: 2.28% (0.00-5.28%)) (Figure 4c), the percentage of ChAT\({}^{+}\) cells was higher (CTRL: 23.96% (7.37-60.24%); MS: 18.90% (0.00-75.00%)) (Figure 4d). Overall, the neurochemical signature of the SP showed no significant difference between patients with MS and CTRL patients. Figure 4: Immunohistological staining of the submucosal plexus (SP) of patients with multiple sclerosis (MS) (\(n\) = 4-5) and non-MS controls (CTRLs) (\(n\) = 14). (**a**) Paraffin-embedded human colon tissue was obtained from patients with MS and matched CTRLs. (**b**) Numbers of neurons per ganglion analyzed in each group. (**c**,**d**) Percentage of (**c**) neuronal nitric oxide synthase (nNOS)\({}^{+}\) and (**d**) choline acetyltransferase\({}^{+}\) neurons. Individual data points are patient mean values. The box and whisker plots show median values together with the interquartile range. For normally distributed data, statistical significance was determined by unpaired Student’s \(t\) test; otherwise, the Mann–Whitney \(U\) test was applied.