## 2 Materials and Methods
### Immunohistochemistry  
Sections of human colon were cut at 5 \(\upmu\)m using either a SM2000 R microtome or a Histocore MULTICUT microtome (both Leica, Wetzlar, Germany) and transferred onto HistoBond slides (Roth). Subsequently, two sections per individual were dehydrated in an ascending series of ethanol (Brenntag, Essen, Germany) and xylene (Roth). For the investigation of enteric neurons, Trilogy solution (Sigma-Aldrich) was used for heat-induced epitope retrieval. Sections were blocked using TBS containing 1% BSA (Roth), 0.5% Triton X (Merck Millipore), and 5% normal donkey serum (Dianova). All sections were double-stained with an antibody against HU (Thermo Fisher; dilution 1:40) and an antibody directed either against ChAT (Sigma-Aldrich; 1:20) or nNOS (University of Graz; 1:1500); all primary antibodies were diluted in a solution of TBS, 1% BSA, and 0.5% Triton X. Sections were incubated at room temperature for 24 h. After washing with TBS, secondary antibodies were applied and incubated for 1 h. Secondary antibodies comprised donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher; 1:1000), donkey anti-goat Alexa Fluor 647 (Thermo Fisher; 1:1000), biotinylated donkey anti-mouse immunoglobulin (Ig)G (Dianova; 1:200), and streptavidin-Cy3 (Dianova, 1:1000). For each staining and combination of secondary antibodies, two slides were incubated in the absence of primary antibodies, thus serving as negative controls. Hoechst 33258 (Sigma-Aldrich, dilution of 1:1000 in TBS) was used as a nuclear counterstain. All sections were coverslipped with TBS and glycerol (Roth) (dilution 1:2, pH 8.6). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Patient ID** & **Sex** & **Age (Years)** &
\begin{tabular}{c} **Reason** \\ **for Surgery** \\ \end{tabular} & **Tissue Source** \\ \hline
1 & F & 25 & I & Appendix \\
2 & F & 46 & I & Neoileocolic anastomosis \\
3 & M & 67 & C & Colon \\
4 & M & 68 & C & Ileum and rectum \\
5 & F & 37 & O & Ileum and colon \\
6 & F & 63 & O & Colon \\
7 & F & 65 & O & Colon \\ \hline \hline \end{tabular} \(C\), cancer; F, female; I, inflammatory disease; ID, identification number; M, male; O, other.  
\end{table} 
Table 1: Composition of the Cohort of Patients with Multiple Sclerosis. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Analysis** & **MS** & **CTRL** & _p Value_ \\ \hline
**ChAT and HU (in MP), nNOS and HU** & & & \\
**(in MP), nNOS and HU (in SP)** & & & \\ Number of patients (patient IDs) & 4 (1, 2, 3, 4) & 14 (all) & \\ Age, years—median (min-max) & 56.5 (25–68) & 55 (25–74) & 0.9399 \({}^{*}\) \\ Sex—\% female & 50 & 50 & 1 \({}^{\dagger}\) \\ Reason for surgery—\% C/\% I & 50/50 & 57/43 & 1 \({}^{\dagger}\) \\ \hline
**ChAT and HU (in SP)** & & & \\ Number of patients (patient IDs) & 5 (1, 2, 3, 4, 5) & 14 (all) & \\ Age, years—median (min-max) & 46 (25–68) & 55 (25–74) & 0.8056 \({}^{*}\) \\ Sex—\% female & 60 & 50 & 1 \({}^{\dagger}\) \\ Reason for surgery—\% C/\% I & 40/40 (20\% other) & 57/43 & 1 \({}^{\dagger}\) \\ \hline
**ANO1-positive ICCs** & & & \\ Number of patients (patient IDs) & 5 (1, 2, 4, 6, 7) & 10 (3–7, 9, 10, 12–14) & \\ Age, years—median (min-max) & 63 (25–68) & 48 (25–69) & 0.3866 \({}^{*}\) \\ Sex—\% female & 80 & 50 & 0.5804 \({}^{\dagger}\) \\ Reason for surgery—\% C/\% I & 20/40 (40\% other) & 60/40 & 0.5954 \({}^{\dagger}\) \\ \hline \hline \end{tabular} \(\begin{array}{c}\)ANO1, anoctamin 1; C, cancer; ChaT, choline acetyltransferasefrac; CTRL, control; HU, anti-HuC/D antibody; I, inflammatory disease; ICC, interstitial cell of Cajaj; ID, identification number; MP, myenteric plexus; MS, multiple sclerosis; nNOS, neuronal nitric oxide synthase; SP, submucosal plexus. \({}^{*}\) Mann–Whitney \(U\) Test. \({}^{\dagger}\) [PERSON]’s exact test.  
\end{table} 
Table 2: Composition of the Non-Multiple Sclerosis Control Patient Cohort.