Figure 7

Impact of denervation of cell proliferation. (A) Representative pictures H&E stains of healthy colonic tissue (upper panel), and colonic tissue with an anastomosis (lower panel). Magnifications: 20x and 40x. (B,C). Mice were subjected to surgery in which the mesenteric nerve bundle was cut and an anastomosis was created. Sham group underwent anastomotic surgery without cutting the mesenteric nerve bundle. Mice were euthanized after 5 days (Sham n = 9; Sx n = 10). (B) Experimental set-up. (C) Visualization of anastomotic complication score (ACS). Data are shown as individual data points with median. P = 0.004. (D–F). Mice were treated with UK 14,304 (green) or vehicle control (grey) via oral gavage for 3 days after which they were subjected to surgery for creation of an anastomosis. Mice were euthanized after 5 days (n = 10). (D) Experimental set-up. (E) Visualization of anastomotic complication score (ACS). Data are shown as individual data points with median. (F) Quantitative RT-PCR (qPCR) analysis of epithelial cell markers Bmi, Lrig1, Muc2, Tff3, and proliferation marker Mki67. Data are shown as individual data points with median and interquartile range. (G) Single gene expression analysis of Adra2a in UK 14,304 versus vehicle control treated organoids assessed by bulk RNA-sequencing (experiment Fig. 2A) (n = 5). Data are shown in a boxplot. (H) qPCR analysis of expression of Adra2a in mice treated with UK 14,304 or vehicle control via oral gavage for 3 or 21 days (experiment Fig. 4A) (n = 8). Data are shown as individual data points with median and interquartile range. Vehicle control versus UK 14,304 3 days P = 0.020. (I) qPCR analysis of expression of Adra2a in mice treated with UK 14,304 or vehicle control via oral gavage for 3 days after which they underwent anastomotic surgery (experiment Fig. 7D) (n = 10). Data are shown as individual data points with median and interquartile range. (J) In situ hybridization (RNAscope) for Adra2a mRNA in colonic tissue of mice subjected to anastomotic surgery in combination with immunofluorescent stainings with DAPI for nuclei (blue). Single dots (red) represent expression of a single Adra2a mRNA copy. Magnification 10x. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01.