Su2029 Medium Conditioned With Conventionally-Activated M1 Macrophages Inhibits Survival of Mouse Interstitial Cells of Cajal in Primary Culture

Background: Application of the surfactant benzylkonium chloride (BAC) to the serosal surface of the intestine causes ablative neurodegeneration within two days. Modest neurogenesis within 4-8 weeks of ablation has been observed in this model with neurons derived from glial precursors. We hypothesized that non-ablative concentrations of BAC would spare potential neural precursors and allow a more robust neuroregeneration. Aims: To determine whether low concentration BAC causes neurodegeneration and subsequent neurogenesis and whether markers of neural progenitors are expressed following BAC treatment. Methods: In C57Bl/6 mice, following laparotomy, a 1x0.2cm piece of absorbent paper soaked in 0.0001% BAC was applied to the serosal surface of the jejunum for 5 min. Following various periods of recovery, fixed segments of the external muscle layers were immunostained for HuC/D (mature enteric neurons), S100β (enteric glia), doublecortin (DCX; immature neurons), and/or SOX2 (neural pluripotency transcription factor). Immunoreactive (IR) cells were enumerated as the number of cells per ganglion. In some mice, EdU (100mg/kg IP) was administered daily between 12-16d post-BAC before animals were euthanized 21d postBAC. Results: In mice two days after BAC treatment there was a significant reduction in the number of HuC/D-IR cells per ganglion in the region of treatment (27 ± 8) compared to an orally-adjacent region (61 ± 7; P<0.05). In mice 21 days after BAC treatment, the number of neurons per ganglion in the treated region (45 ± 5) were no longer significantly less than the orally-adjacent region (61 ± 6; P<0.05). There was no evidence for EdU incorporation in HuC/D-IR cells. Although DCX-IR was detected in the rostral migratory stream of the CNS, DCX-IR was rarely detected in the myenteric plexus. SOX2-IR was increased in HuC/ D-IR neurons at 21d post-BAC. SOX2-IR was detected in S100β-IR cells as early as 2d postBAC and S100β-IR was detected in a proportion of HuC/D-IR cells in the BAC-treated region between 2-14d post-BAC. Conclusion: Low concentration BAC caused moderate neurodegeneration in the myenteric plexus of the mouse jejunum. The number of myenteric neurons increased by 21d post-BAC. The pluripotency transcription factor SOX2, which in the CNS is capable of reprogramming astrocytes to mature neurons, is expressed in enteric glial cells early following BAC treatment and in a proportion of mature neurons by 21d post-BAC. These data support the concept of neuroregeneration of the enteric nervous system, and suggest that SOX2 expression in enteric glia may drive adult neurogenesis without cell proliferation.