## 4 Discussion  
The key findings of this study were a significant loss of ChAT+ neurons in the MP of mice with EAE as opposed to an increase in ChAT+ neurons in the MP of patients with MS. Interestingly, while these changes pertained to the MP, no pathological alterations were evident in the SP. In addition, there was a significant increase in the expression of ANO1+ ICCs in patients with MS and a decrease in the MP of mice with late-stage EAE. A decrease in the number of cholinergic neurons within the MP has previously been reported in the ENS of aged rats [26]. The same observation was made in colonic tissue samples from elderly people, where a decline in ChAT+ neurons was accompanied by an increased number of nNOS+ neurons, which might serve as an explanation for the higher prevalence of constipation with age [25]. Along these lines, ChAT-/- mice exhibited colonic dysmotility [37], and myenteric ChAT+ neurons were diminished in patients with  
Figure 6: Immunohistological staining of the myenteric plexus (MP) of patients with multiple sclerosis (MS) (\(n=5\)) and non-MS controls (CTRLs) (\(n=10\)). Paraffin-embedded human colon tissue was obtained from patients with MS and matched CTRLs. For analysis of anoctamin 1 (ANO1), only tissue containing MP was included. (**a**) Schematic representation of ICCs and their location within the gastrointestinal wall. (**b**,**c**) Representative images stained for ANO1 (_brown arrow_) were selected from (**b**) a CTRL patient and (**c**) a patient with MS. Scale bars denote 50 μm (**d**,**f**) Areas of (**d**) plexus expressed in relation to the area of longitudinal and circular muscle, (**e**) ANO1+ signal in relation to the plexus area, and (**f**) ANO1+ signal in relation to the area of longitudinal and circular muscle. Individual data points are patient mean values. The box and whisker plots show median values, together with the interquartile range. Statistical significance was determined by Mann–Whitney \(U\) test with * \(p<0.05\). slow-transit constipation [38]. Of note, constipation is also a frequent symptom observed in patients with MS [4; 5], which may be linked to ENS alterations. Conflicting with these findings and the observations made in EAE, we found a trend toward an increase in ChAT\({}^{+}\) neurons in patients with MS vs. CTRL patients. There may be several reasons for the discrepancy between EAE and MS. On the one hand, the MS patient sample size was limited in our study. Our cohort consisted of only few individuals from different age groups and with different comorbidities. Important information regarding the clinical subtype of MS and MS treatment prior to abdominal surgery was not available. While relapsing-remitting MS is characterized by predominant inflammation, the progressive stages of MS mainly show degenerative pathology [39]. This switch in pathomechanisms may also affect the ENS differentially so that it will be worthwhile to distinguish between different disease stages of MS in future studies on ENS involvement in MS. The same applies to the impact of MS treatment on the ENS, since some therapies are primarily immune modulatory and suppressive, while others may also have beneficial effects on neurodegeneration [40]. On the other hand, our methods of analysis differed between EAE and MS. While only paraffin-embedded tissue sections were available from MS patients, we were able to study whole mounts from EAE mice. The whole mount technique allows for three-dimensional analysis and hence provides a more complete picture of the ENS. Thus, it is likely to be better suited for studying ENS alterations compared to paraffin-embedded tissue sections. Finally, it needs to be considered that EAE can be induced by immunization with different CNS antigens. While MP4 was used in our study, immunization with, e.g., whole spinal cord homogenate may lead to results that are more similar to what we have observed in MS patients. It will be subject to future studies to dissect the involvement of different target antigens in ENS pathology.