## 4 Discussion
Alternatively, our results obtained in MS patients could be observed from a different, more theoretical perspective, and one could speculate about the occurrence of neurogenesis in the ENS, which is currently heavily debated. Postnatal neurogenesis originating from enteric glial cells has been reported in mice after chemical injury [41] as well as in zebrafish under physiological circumstances [42]. In addition, in the context of colitis, [PERSON] et al. showed the expression of markers indicative of neurogenesis in the human ENS, with subsequent studies of a corresponding mouse model suggesting that the newborn neurons were predominantly excitatory [43]. Compared to mice with early-stage EAE, which were analyzed 12 days after immunization and before the appearance of any symptoms, patients with MS in our study had been affected by the disease for a prolonged time. Hence, it is conceivable that an initial loss of neurons in patients with MS might be compensated for by the preferential genesis of new excitatory neurons. In addition to ChAT, we detected nNOS\({}^{+}\) neurons in the ENS. In CTRL patients, a median of 20.4% of myenteric neurons were nNOS\({}^{+}\), and a median of 22.5% neurons were ChAT\({}^{+}\). The MP of patients with MS contained slightly more nNOS\({}^{+}\) neurons (median 30.8%) and markedly more ChAT\({}^{+}\) neurons (median 45.2%). Earlier studies of the human MP have described equal proportions of nitrorgic and cholinergic neurons, with each of the two populations accounting for approximately 50% of the total ENS neuronal population [13; 14; 15; 38]. However, in prior studies, sample sizes were larger, and whole mounts were used instead of sections of paraffin-embedded tissue [13; 14; 15; 38], which may explain why our results differ from the literature. Similar to our findings, [PERSON] et al. reported divergent numbers, detecting only about 35% ChAT\({}^{+}\) neurons in their subjects, for which they suspected different strategies of tissue processing before staining as an explanation [23]. In fact, in two of the studies cited above, tissue was cultured before fixation and staining in order to enhance ChAT immunoreactivity [14; 38]. In our study, the MP seemed to be affected more than the SP in both EAE and MS. Indeed, there are other diseases in which alterations are predominantly shown in the MP. Among such diseases is enteric ganglionitis, which is characterized by the inflammation and infiltration of lymphocytes into the ENS and the development of circulating anti-neuronal antibodies targeting the ENS [44]. Anti-MP autoantibodies were recently also proposedas the origin of idiopathic achalasia, which is characterized by disturbed esophageal peristalsis [22]. A further main result of our study was that the area of ICCs, which are located at the level of the MP and in the muscle layers surrounding it [21], was increased in patients with MS, while it was decreased in EAE mice. Increased networks of ICCs were found in early stages of type 1 diabetes, where they were associated with accelerated emptying of the stomach [45]. On the contrary, in conditions such as constipation, in which a slower passage of the gastrointestinal system prevails, ICCs are typically less abundant [21]. Consistent with our findings in the murine colon, a decrease in the number of ICCs was previously observed in the bladder of EAE mice [46]. Interestingly, ICCs are not independent in their role as intestinal pacemakers, but they interact, inter alia, with inhibitory and excitatory neurons [21]. A study in nNOS'/ mice demonstrated a significant reduction in the volume of ICCs in the MP, while incubation in vitro with an NO donor increased the number of ICCs [47]. Another study showed that the terminal endings of excitatory neurons were close to networks of ICCs, which have receptors for ACh [48]. Thus, interrelations between ICCs and this type of neuron are conceivable.