Amniotic fluid stem (AFS) cells modulate stromal cells expressing COX-2 in the lamina propria. (A) Representative cryosections of the terminal ileum from breastfed (BF) and necrotising enterocolitis (NEC) rats receiving phosphate buffered saline (PBS) and AFS cells stained with anti-COX2 Ig (red) and DAPI (blue). Scale bars: 20 μm. (B) In NEC rats treated with AFS cells, the number of COX-2+ cells per villus unit (4.97±0.46; n=7, p=n.s.) was similar to that of BF rats (4.25±0.52; n=8), but higher compared with NEC rats treated with PBS (2.37±0.29; n=8, p<0.05). (C, D) This difference was not determined by the quantity of COX-2+ cells in the villi, which was similar between the BF and AFS (1.95±0.28 vs 1.32±0.16, p=ns) rats, but by their number underlying the cryptae which was higher in NEC rats treated with AFS cells (3.01±0.41) compared with BF rats (1.41±0.23, p<0.01) and NEC rats injected with PBS (1.05±0.18, p<0.001). (E, F) The number of COX-2+ cells per villus unit and in the cryptae inversely correlated with the histological grade of NEC by linear regression. (G) A survival study employing selective and non-selective COX inhibitors showed that COX inhibitors did not modify the high survival rate of BF rats (n=32; BF+DMSO vs BF+sc-560, BF+ibuprofen, BF+celecoxib: p=n.s.) and the low survival rate of PBS-treated NEC rats (n=77; PBS+DMSO vs PBS+sc-560, PBS+ibuprofen, PBS+celecoxib: p=n.s.). However, the improved survival of NEC rats receiving AFS cells (n=78) was annulled by COX-2 (AFS cells+celecoxib vs AFS cells+DMSO: p<0.0001; AFS cells+celecoxib vs PBS+DMSO: p=n.s.) and COX-1+2 inhibitors (AFS cells+ibuprofen vs AFS cells+DMSO: p<0.01; AFS cells+ibuprofen vs PBS+DMSO: p=n.s.), but conserved in rats receiving COX-1 inhibitor (AFS cells+sc-560 vs AFS cells+DMSO: p=n.s.; AFS cells+sc-560 vs PBS+DMSO: p=0.001). (H) The clinical sickness score improvement observed in NEC rats treated with AFS cells (0.77±0.36) was abolished by COX-2 inhibitor (7.15±0.89; AFS cells+celecoxib vs AFS cells+DMSO: p<0.001), diminished by COX-1+2 inhibitor (2.86±0.91; AFS cells+ibuprofen vs AFS cells+vehicle: p=n.s.) and unaltered by COX-1 inhibitor (0.92±0.39; AFS cells+sc-560 vs AFS cells+vehicle: p=n.s.).