Myenteric Neurons Do Not Replicate in Small Intestine Under Normal Physiological Conditions in Adult Mouse

Abstract

Background & aims: The enteric nervous system (ENS) is the largest part of the peripheral nervous system; moreover, abnormal ENS development and function are associated with multiple human pathologies. Data from several groups suggest that under normal physiological conditions in adult animals, enteric nerve cells do not replicate. A study by Kulkarni et al in 2017 challenged this view and proposed that nearly 70% of enteric neurons in the myenteric ganglia are born in 1 week. The authors of this study suggested that differences in DNA labelling times and DNA denaturation conditions might explain discrepancies with previous reports. Previous studies were carried out using different conditions and labelling techniques in various regions of the gastrointestinal tract; thus, conclusions have remained elusive.

Methods: Here, we have eliminated those variables by analyzing the whole small intestine using the reagents and conditions that Kulkarni et al used. To exclude variables related to immunohistochemistry, we carried out parallel experiments with "click chemistry"-based detection of DNA replication.

Results: Although proliferation was readily detected in the epithelium, we found no evidence of neuronal replication in the myenteric ganglia.

Conclusions: We conclude that within 1 week under normal physiological conditions, myenteric neurons in the small intestine do not replicate.

Keywords: DNA Labelling; ENS; EdU; IdU; Proliferation.

Graphical abstract

Figure 1

Study design to analyze ENS proliferation in the small intestine. (A) IdU or EdU was given in drinking water for 1 week, after which the mice were euthanized. (B) The small intestine (IdU, N = 3 animals/EdU, N = 3–4 animals) was divided into 3 anatomic segments of about equal length to represent the duodenum, jejunum, and ileum and processed for longitudinal immunohistochemistry using both cryosections and paraffin embedding systems and for LM-MPs for whole mount analysis. (C, E, and G) Epithelial cells show a strong positive signal, demonstrating success of labelling and label detection for both nucleotide analogues. (D and F) Note background fluorescence in both cryosections and paraffin sections in IdU samples indicating background staining. (H) No signal is observed when EdU is omitted, validating specificity. Scale bar, 50 μm.

Figure 2

Analysis of ENS proliferation in the small intestine. (A–C) Longitudinal cryosections and (D–F) paraffin sections of the small intestine were immunostained for IdU (green) and for HuD (blue) to reveal enteric neurons that had undergone replication. Cells in the epithelium show signal for IdU as expected. (A–F) Myenteric ganglia are indicated with white arrowheads and submucosal ganglia with yellow arrowheads. No double-positive HuD+/IdU+ enteric neurons were detected (n = 300 ganglia analyzed in n = 3 animals, age = 24 weeks). Dashed rectangle indicates magnified area next to the panel. Scale bar, 20 μm, 10 μm on insets. (G–I) Analysis of LM-MPs from IdU-labelled animals revealed no double-positive HuD+/IdU+ enteric neurons (n = 3790 neurons analyzed in n = 3 animals, age = 21 weeks). Scale bar, 20 μm. (J–L) Analysis of longitudinal paraffin sections from the small intestine of EdU-labelled animals, HuD (green) and EdU (red). Cells in the epithelium show signal for EdU as expected. No double-positive HuD+/EdU+ enteric neurons were detected (n = 451 ganglia counted in n = 3 animals, age = 17 weeks). Myenteric ganglia are indicated with white arrowheads and submucosal ganglia with yellow arrowheads. Dashed rectangle indicates magnified area next to the panel. Scale bar, 20 μm, 10 μm on insets. (M–O) Analysis of LM-MPs from EdU-labelled animals (n = 1474 neurons analyzed in n = 4 animals, age = 8–10 weeks) revealed no double-positive HuD+/EdU+ enteric neurons. (P and Q) Dashed rectangle indicates magnified images below. (R) In 11 neurons in EdU-labelled animals we observed a putative overlap of HuD+/EdU+ labelling. 3D ApoTome analysis revealed that in all cases (n = 11) cells were layered on top of each other along the Z axis, appearing as false positives in 2D microscopy. Scale bar, 20 μm, 10 μm on insets.