## 3 Results
### Increase in the Percentage of ChAT\({}^{+}\) Neurons in the MP of Patients with MS
(**e**,**f**) Representative images of the SP showing staining for (**e**) anti-HuC/D (HU) (_red arrow_, first picture from the left) and nNOS (_green arrow_, second picture from the left), combined with Hoechst 33258 (third picture from the left), and (**f**) HU (_red arrow_, first picture from the left) and ChAT (_green arrow_, second picture from the left), combined with Hoechst 33258 (third picture from the left). Scale bars denote 50 μm. In the MP, a median of 4.25 (min-max: 2-8.09) neurons per ganglion were detected in CTRL patients compared with 6.25 (3.29-11.41) in patients with MS (Figure 5b). In CTRL patients, 20.40% (min-max: 0.00-63.89%) of neurons were nNOS\({}^{+}\) compared with 30.81% (8.33-55.00%) in patients with MS (Figure 5c); and 22.51% (0.00-66.67%) of neuronal cells were ChAT\({}^{+}\) in CTRL patients compared with 45.16% in patients with MS (Figure 5d). While no statistically significant observations were made comparing CTRL and MS patients, a trend toward an increase in ChAT\({}^{+}\) neurons in patients with MS was noted. Interestingly, the percentage of ChAT\({}^{+}\) neurons in patients with MS showed the smallest variation of all human data sets, ranging only from 44.44% to 48.57%. Figure 5: Immunohistological staining of the myenteric plexus (MP) of patients with multiple sclerosis (MS) (\(n=4\)) 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 acetyltransferaseane (ChAT)\({}^{+}\) 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. (**e,f**) Representative images of the MP showing staining for (**e**) anti-HuC/D (HU) (_red arrow_, first picture from the left) and nNOS (_green arrow_, second picture from the left), combined with Hoechst 33258 (third picture from the left), and (**f**) HU (_red arrow_, first picture from the left) and ChAT (_green arrow_, second picture from the left), combined with Hoechst 33258 (third picture from the left). Scale bars denote 50 μm. Significant Increase in the Area of ANO1+ ICCs in Relation to the Area of Muscle Tissue in Patients with MS  
The relative areas of longitudinal and circular muscle tissue, MP, and ANO1+ ICCs were investigated (Figure 6). Overall, when the area of the MP was compared with the area of muscle tissue, the MP covered a median area of 0.65% (min-max: 0.28-2.3%) in CTRL patients compared with 0.98% (0.51-1.196%) in patients with MS (Figure 6d). In relation to the MP area, the ICC area was larger in patients with MS than in CTRL patients: a median of 16.19% (min-max: 5.61-57.89%) of the size of MP area was ANO1+ in CTRL patients compared with 61.7% (6.67-108.82%) in patients with MS (Figure 6e), yet, no statistical significance was reached. However, relative to the muscle tissue area, the ICC area was significantly higher in patients with MS than in CTRL patients: median 0.56% (min-max: 0.05-0.92%) versus 0.09% (0.03-0.41%) (\(p<0.05\); Figure 6f).