Figure 6

Enteric neural stem cell transplantation protects the intestines in experimental necrotizing enterocolitis. (A) Outline of the experimental protocol used. Rat pups were delivered by Cesarean section and then exposed to repeated episodes of hypoxia, hyperthermia, and hypertonic formula feeding for 3 days to induce experimental necrotizing enterocolitis (NEC). Pups then received intraperitoneal (IP) administration of green fluorescence protein (GFP)-labeled neural stem cells (NSCs) or carrier medium only, after which they were maintained on normal feeds with discontinuation of stress for an additional 4 days. (B) Representative images of intestinal sections from breast-fed rat pups, from pups exposed to NEC, and from pups exposed to NEC but treated with NSC transplantation. GFP-labeled transplanted cells were visualized in intestinal tissue sections by anti-GFP immunohistochemistry, and were further characterized by protein gene product 9.5 (PGP9.5) staining to identify mature neurons. (C) High magnification of the merged image inset (white box) demonstrating that transplanted NSCs have engrafted into the submucosal plexus (S. Plexus; arrows) and myenteric plexus (M. Plexus; arrowheads) of the intestine and have differentiated into PGP9.5-positive mature neurons (merged yellow staining). (D) Western blot analysis of intestinal longitudinal muscle–myenteric plexus strips showing protein expression of peripherin and neuronal nitric oxide synthase (nNOS). (E) Quantification of the intensity of immunoreactive bands: the band intensity ratios of peripherin to β-actin, nNOS to β-actin, and glial fibrillary acid protein (GFAP) to β-actin were calculated and are expressed as mean ± standard error of the mean, n = 4. *P <0.05; ⁺P <0.01. Scale bars: (B) 100 μm; (C) 25 μm.