Volume 136, Issue 2, Pages 630-639 (February 2009) Changes in the Structure and Function of ICC Networks in ICC Hyperplasia and Gastrointestinal Stromal Tumors  Joong Goo Kwon, Sung Jin Hwang, Grant W. Hennig, Yulia Bayguinov, Conor McCann, Hui Chen, Ferdinand Rossi, Peter Besmer, Kenton M. Sanders, Sean M. Ward  Gastroenterology  Volume 136, Issue 2, Pages 630-639 (February 2009) DOI: 10.1053/j.gastro.2008.10.031 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Hyperplasia of ICC in the stomachs of KitV558Δ/+ mice. In each panel (A–D), cryostat cross sections (Aa–Da), whole mounts (Ab–Db), and volume-rendered micrographs (Ac–Dc; see Materials and Methods section) are shown. In each cryostat cross section (Aa–Da), c-kit-LI is labeled in red to distinguish intramuscular ICC (ICC-IM) and ICC of the myenteric plexus region (ICC-MY) from smooth muscle cells (SMC) that are labeled with smooth muscle myosin II heavy chain (SMMHC) in green. Panels Aa–c show the gastric fundus of +/+ mice. ICC-IM are present in both circular (ICC-IMC; CM) and longitudinal (ICC-IML; LM) muscle layers. Panels Ba–c show the gastric fundus from KitV558Δ/+ mice. Panels Ca–c show ICC-IM within the circular layer and ICC-MY in the gastric antrum of +/+ mice, and panels Da–c show ICC-IM and ICC-MY in the gastric antrum of KitV558Δ/+ mice. Scale bars at bottom apply to panels above. Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 ICC hyperplasia in small intestine of KitV558Δ/+ mice. Cryostat cross sections (Aa–Ba), whole mounts (Ab–Bb), and volume-rendered micrographs (Ac–Bc) are shown. In cryostat cross sections (Aa–Ba), c-kit-LI is labeled in red to distinguish ICC of the deep muscular plexus (ICC-DMP) and ICC-MY from SMC labeled with SMMHC in green. Panel A shows images from +/+ mice, and panel B shows images from KitV558Δ/+ mice. Scale bars at bottom apply to panels above. Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Hyperplasia of ICC in the cecum and colon of KitV558Δ/+ mice. Panels A–D show cryostat cross sections (Aa–Da), whole mounts (Ab–Db), and volume-rendered micrographs (Ac–Dc). In cryostat cross sections (Aa–Da), c-kit-LI is labeled in red (ICC-IM and ICC-MY) and ICC along the submucosal surface of the circular muscle (ICC-SM) and SMMHC is labeled in green to identify smooth muscle cells. Panels Aa–c show the cecum of +/+ mice. Panels Ba–c show images from the cecum of KitV558Δ/+ mice. Panels Ca–c show images from the proximal colon of +/+ mice, and Panels Da–c show images from the proximal colon of KitV558Δ/+ mice. Scale bars at bottom apply to panels above. Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 c-kit Immunohistochemistry of wholemount preparations from KitV558Δ/+ and +/+ E19 fetuses. Panels A and B show confocal images of ICC-IM from the fundus of KitV558Δ/+ mice, and panel C shows ICC-IM from the fundus of an E19 +/+ fetus. Panels D and E show images of ICC-IM and ICC-MY from the antrums of KitV558Δ/+ E19 embryos and panel F from a +/+ E19 fetus. Marked hyperplasia of ICC-IM and ICC-MY occurs prior to birth. Scale bar (100 μm) applies to all panels. Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Intracellular electrical activity of gastric smooth muscle of +/+ and KitV558Δ/+ mice. Panel A shows basal activity and postjunctional neural responses to electrical field stimulation (EFS) in +/+ and KitV558Δ/+ mice. Basal electrical activity was characterized by ongoing unitary potential discharge. Responses to EFS (arrowheads; 10 Hz, 0.3 ms; 1 s) were biphasic IJPs that were largely blocked by L-NNA (100 μmol/L; dashed line). EJPs (arrows) were blocked by atropine (1 μmol/L) and were unmasked by L-NNA. After L-NNA and atropine, only purinergic IJPs were observed (blocked by apamin; not shown). Panel B shows spontaneous electrical activity and responses to EFS in antrums of +/+ and KitV558Δ/+ mice. Slow waves were similar in +/+ and KitV558Δ/+ mice. Neural responses (EFS at arrowheads) in +/+ and KitV558Δ/+ mice were not significantly different (eg, transient IJP that phase advanced but attenuated the amplitude of the next slow wave). L-NNA (100 μmol/L) largely blocked IJPs (not shown). Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Intracellular electrical recordings from small intestines of +/+ and KitV558Δ/+ mice. Panels A and B show slow waves from jejunums and ileums of +/+ and KitV558Δ/+ mice. Panels C and D show neural responses (EFS at arrowheads; 10 Hz, 0.3 ms; 1 s) from ileums of +/+ and KitV558Δ/+ mice. Responses consisted of a transient inhibitory junction potential (IJP) and inhibition of slow wave amplitude for several cycles following EFS. Inhibitory effects of EFS were largely blocked by L-NNA (100 μmol/L). Responses to EFS were not different in +/+ and KitV558Δ/+ mice. Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Electrical activities of cecum of +/+ and KitV558Δ/+ mice. Panel A shows the electrical activity of the circular muscle layer of a control animal. Panel B shows normal and spontaneous action potentials or slow waves in the cecums of KitV558Δ/+ mice in which spontaneous electrical activity consisted of small “unitary potentials.” Spontaneous action potentials or slow wave activity developed in the cecums of KitV558Δ/+ mice in which solid tumors had not yet formed. Action potentials and slow wave-like depolarizations were inhibited by nifedipine (1 μmol/L; panel C). Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Electrical activity and neural responses in proximal colons of +/+ and KitV558Δ/+ mice. Panels A and B show that spontaneous electrical activity in the proximal colon was similar in +/+ and mutant animals. Panels C and D show neural responses in +/+ and KitV558Δ/+ mice. EFS (single pulse, 0.3-ms duration; arrowheads) caused biphasic IJPs, characterized by a rapid transient component followed by a sustained component (dashed lines). The sustained component was blocked by L-NNA (100 μmol/L). Atropine (1 μmol/L) in the presence of L-NNA had little effect on responses. Gastroenterology 2009 136, 630-639DOI: (10.1053/j.gastro.2008.10.031) Copyright © 2009 AGA Institute Terms and Conditions