Fig. 5

Intestinal epithelial barrier leakage does not occur with acute glafenine treatment. (A,B) Zebrafish larvae at 6 dpf were pretreated with glafenine or vehicle control for 1 or 3 hours, followed by gavage with 1% TxR-dextran or 1% TxR-dextran plus 20 mM EDTA (positive control). ImageJ was used to quantitate the relative mean fluorescence in an ROI in the zebrafish larvae trunk above segments 2 and 3 of the intestine (white dashed rectangles). Values were normalized to an ROI outside of the fish as an internal control to account for background glare. (A) Dextran leaks into circulation and intersomitic spaces when co-gavaged with EDTA. Representative images of larvae from the 1-hour pretreatment group are shown. In larvae gavaged with dextran only, the dextran remains in the intestinal lumen. When co-gavaged with dextran plus EDTA, fluorescent signal is observed in circulation (dorsal aorta, white arrow) and intersomitic vessels and spaces (white arrowheads). BF, brightfield. Scale bars: 200 μm. (B) Treatment with glafenine does not lead to acute intestinal barrier disruption. Mean normalized fluorescence in the trunk was determined following 1 or 3 hours of pretreatment with glafenine or control. Graph represents data averaged from two experimental replicates taking into account the mean, s.d. and n (16–19 larvae/condition/experiment). Two-way ANOVA with Bonferroni post-test was performed to compare each column to all other columns. *P<0.05, **P<0.01.