C. J. Greig1,2, C. J. Park1, S. J. Armenia1, L. Zhang1, R. A. Cowles1 1Yale University School Of Medicine,New Haven, CT, USA 2University Of Massachusetts Medical School – Baystate Medical Center,Springfield, MA, USA
Introduction:
The enteric nervous system (ENS) has long been considered a static environment, lacking significant neurogenesis under steady-state conditions. Recent evidence suggests, however, that certain stimuli can induce neurogenesis originating from enteric neuronal precursors. While much about this process remains unknown, emerging data suggest the generation of new neurons may occur via two different mechanisms: differentiation from existing neuronal precursors or generation of new mature neurons via cell division. Previous experiments suggested that bowel resection could induce the generation of new mature neurons, but whether this occurred due to cell division was unclear. In the current study, we hypothesized that ischemia-reperfusion injury would induce neuronal precursor cells to generate new neurons via cell division.
Methods:
Wild type (WT) C57Bl/6 controls and Nestin_CreER/eGFP bitransgenic mice expressing eGFP in a tamoxifen-inducible manner and under the control of the actin promoter were used for experiments. Following tamoxifen induction, bitransgenic mice underwent ischemia-reperfusion (IR) injury via superior mesenteric artery occlusion (T=30 and 45 min). WT and bitransgenic controls received tamoxifen injections alone without IR, and bitransgenic sham laparotomy animals received the same surgical procedure as IR animals without SMA clamping. IR animals received injections of EdU at the start of the reperfusion period and again at day 5. After the reperfusion period (3 or 10 days), distal ileal segments were obtained from all animals. Whole mounts of the myenteric and submucosal plexuses were created by mechanically peeling these layers after fixation. Peeled specimens were then stained with fluorescent antibodies targeting a marker of mature neurons, HuC/D, and GFP. A click chemistry assay was used to detect Edu in the whole mount specimens.
Results:
WT and bitransgenic controls demonstrated no evidence of GFP-expressing mature neurons, suggesting no demonstrable neurogenesis occurred at baseline. Bitransgenic sham laparotomy mice demonstrated a low incidence of neurogenesis, as evidenced by mature neurons expressing GFP in one of four high-powered fields. In IR animals, we found evidence of extensive neurogenesis with GFP-expressing mature neurons in nearly all high-powered fields. Moreover, EdU was detected in most mature, GFP-expressing neurons, suggesting that these neurons had been generated from precursors via cell division.
Conclusion:
Ischemia-reperfusion injury appears to induce a substantial mitotic response in the ENS of the murine ileum, and this process appears to occur via cell division of neuronal precursors. Interestingly, anesthesia and laparotomy alone appear to represent a sufficient stimulus to induce neurogenesis, but to a lesser extent than ischemia-reperfusion. While preliminary in nature, these data provide insight for future study into the mechanisms of enteric neurogenesis.