C. J. Greig1, N. Gandotra1, J. J. Tackett1, M. C. Bamdad1, R. A. Cowles1 1Yale University School Of Medicine,Department Of Surgery, Section Of Pediatric Surgery,New Haven, CT, USA
Introduction:
The intestinal mucosa recovers from injury by accelerating enterocyte proliferation resulting in villus growth. A similar phenomenon is seen in the adapting small intestine after massive bowel resection. Serotonin (5-HT), a ubiquitous neurotransmitter in the bowel, has been implicated as an important regulator of intestinal mucosal homeostasis by promoting growth in the epithelium. The impact of 5-HT on other components of growing villi is not known. We hypothesized that 5-HT-stimulated growth in the intestinal epithelium would be associated with growth in other components of the villus such as enteric neural axonal processes.
Methods:
Enteric serotonergic signaling is inactivated by the SERT molecule located on enterocytes and enteric nerves. Knock out of SERT (SERTKO) or treatment with serotonin reuptake inhibitors (SSRIs) effectively enhances serotonergic signaling by blocking its inactivation. Therefore, 8 week-old wild type (WT), SERTKO, and SSRI-treated wild type mice were used for experiments. For SSRI treatment, a miniosmotic pump containing citalopram was inserted subcutaneously and left in place for 7 days before sacrifice. Paraffin-embedded sections were created from predefined segments of ileum harvested from each experimental group. Villus height and crypt depth were assessed in H&E-stained sections. Crypt proliferation index was calculated using Ki67 staining. Immunofluorescence microscopy with antibodies targeting Gap43, a marker of axonal growth, was performed and tiled images were obtained to visualize multiple villi in cross section. Images were analyzed with ImageJ software using integrated optical density and region of interest (ROI) tool. Adequate villi with a visualized central lacteal were identified and a ratio of Gap43 to DAPI signal was obtained to normalize differences in villus size. Data for Gap43/DAPI ratio were analyzed using one way ANOVA and two-sample Student’s t-test to ascertain statistical differences. The study protocols were approved by the Institutional Animal Care and Use Committee.
Results:
SERTKO and SSRI-treated mice displayed increased epithelial growth with taller villi, deeper crypts and increased enterocyte proliferation rates when compared to WT mice. Eight adequate villi per animal group were analyzed for Gap43 expression. Gap43/DAPI ratios within villi were calculated and average Gap43/DAPI signal ratios (%) were 4.0(SEM 0.39, 95% CI 3.24-4.76) for WT, 5.3(SEM 0.47, 95% CI 4.39-6.21) for SERTKO and 11.0(SEM 1.53, 95% CI 8.0-14.02) for SSRI-treated animals. The differences in Gap43 expression between all animal groups were statistically significant with p<0.05 by ANOVA and t-test.
Conclusion:
Enhanced 5-HT signaling results in intestinal mucosal growth in both the epithelial cell compartment as well as the enteric nervous system. 5-HT appears be an important regulator of intestinal mucosal growth and enteric neuronal plasticity.