Volume 129, Issue 6, Pages 1817-1824 (December 2005) Neural Stem Cell Transplantation in the Stomach Rescues Gastric Function in Neuronal Nitric Oxide Synthase–Deficient Mice  Maria–Adelaide Micci, Kristen M. Kahrig, Rochelle S. Simmons, Sushil K. Sarna, M. Rosario Espejo–Navarro, Pankaj Jay Pasricha  Gastroenterology  Volume 129, Issue 6, Pages 1817-1824 (December 2005) DOI: 10.1053/j.gastro.2005.08.055 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Transplanted NSCs survive in the pyloric wall of nNOS−/− mice and improve gastric emptying. (A) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week posttransplantation. GFP-positive NSCs are shown in green. PGP9.5 immunoreactivity is indicated in red. (B) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week posttransplantation. GFP-positive NSCs are shown in green. GFAP immunoreactivity is indicated in red. (A and B) lm, longitudinal muscle; cm, circular muscle. Scale bar = 20 μm. (C) Gastric emptying of liquids measured 20 minutes after gavage of dye in nNOS−/− mice 1 week following NSC transplantation or vehicle. *P < .01 by 2-tailed Student t test, 2-sample equal variance. Data are means ± SEM. n = 6 mice for vehicle and n = 8 mice for NSCs. Gastroenterology 2005 129, 1817-1824DOI: (10.1053/j.gastro.2005.08.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 NSC transplantation results in increased relaxation of the pyloric muscle. (A) EFS of pyloric circular muscle under NANC conditions in nNOS−/− mice 1 week after vehicle injection or NSC transplantation. The examples shown are from a representative experiment. (B) Quantification of NANC-induced relaxations in response to EFS in nNOS−/− mice 1 week after vehicle injection or NSC transplantation. Analysis of the results by 2-way analysis of variance showed significant effects of both frequency (P < .001) and NSC transplantation (P = .012). Post hoc analysis with a Tukey multiple comparison test showed significant differences between the vehicle and NSC group at stimulation frequencies of 8 and 16 Hz. *P = .003; **P = .001. Data are means ± SEM. n = 7 mice for vehicle and n = 6 mice for NSCs. (C) Quantification of the effect of 100 μmol/L l-NAME on NANC-induced relaxations in response to EFS in nNOS−/− mice 1 week after vehicle injection or NSC transplantation. Data are means ± SEM. n = 7 mice for vehicle and n = 6 mice for NSCs. Gastroenterology 2005 129, 1817-1824DOI: (10.1053/j.gastro.2005.08.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Improved relaxation of the pyloric muscle is NO dependent and neurally mediated. Quantification of the effect of 1 μmol/L TTX on NANC-induced relaxations in response to EFS in nNOS−/− mice 1 week after NSC transplantation. Analysis of the results by 2-way analysis of variance for repeated measures showed a significant effect of TTX (P = .02). Post hoc analysis with a Tukey multiple comparison test showed significant differences in NANC-induced relaxations measured without (untreated) and in the presence of TTX at stimulation frequencies of 8 and 16 Hz. *P = .02; **P = .001. Data are means ± SEM. n = 7 for untreated and n = 6 for TTX. Gastroenterology 2005 129, 1817-1824DOI: (10.1053/j.gastro.2005.08.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Grafted NSCs express nNOS. (A) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week post-transplantation. GFP-positive NSCs are shown in green. nNOS immunoreactivity is indicated in red. (B) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week post-transplantation. GFP-positive NSCs are shown in green. Control obtained omitting the primary anti-nNOS antibody is shown in the middle panel. (C) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week post-transplantation. GFP-positive NSCs are shown in green. VIP immunoreactivity would be in red, but it was absent in grafted cells. VIP immunoreactivity was found in intrinsic ganglia (data not shown). lm, longitudinal muscle; cm, circular muscle. Scale bar = 20 μm. Gastroenterology 2005 129, 1817-1824DOI: (10.1053/j.gastro.2005.08.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions