Volume 119, Issue 6, Pages 1590-1599 (December 2000) Interstitial cells of Cajal and inflammation-induced motor dysfunction in the mouse small intestine  Tara Der, Premysl Bercik, Graeme Donnelly, Tim Jackson, Irene Berezin, Stephen M. Collins, Jan D. Huizinga  Gastroenterology  Volume 119, Issue 6, Pages 1590-1599 (December 2000) DOI: 10.1053/gast.2000.20221 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Changes in slow wave activity, 2–15 days PI. Intracellular recordings of the electrical activity of the circular muscle layer. All recordings were made 6 cm distal to the pylorus, except where otherwise noted. (A) Control recording. (B) Normal slow wave activity with extensive generation of action potentials superimposed on the slow wave. (C) Slow wave activity at 120 cycles/min. (D) Slow wave activity 3 days PI, at reduced frequency and irregular amplitude. (E) Slow wave activity at normal frequency (dotted line) alternating with slow waves at high (double) frequency, indicating that pacemaker activity from 2 locations summates at the recording electrode. This occurs when there are irregularities in intercellular coupling. (F) Waxing and waning of slow wave activity. This pattern never occurs in control tissue but is expected when more or less independent pacemaker sites interact. (G) An example of reduced frequency slow wave activity at 15 days PI. (H) Normal slow wave activity at 60 days PI recorded 0.5 cm from the pylorus. Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Slow wave–like activity occurring 2 days PI. The activity was abolished by nifedipine and recovered by the addition of Bay K8644. Note the depolarizing prepotentials preceding the slow wave–like oscillations in the presence of Bay K8644. Note changes in recording speed. Activity was recorded 6 cm distal to the pylorus. Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Slow wave frequency before, during, and after infection measured 0.5 and 5 cm distal to the pylorus and the terminal ileum. The average slow wave frequency at 5 cm markedly increased 2 days PI as a consequence of areas of very high frequency, which also occur at 0.5 cm. By day 15, the average slow wave activity decreased at 0.5 cm, as also reflected in the in vivo recordings (Figure 5). Note the marked increase in variability in the slow wave frequency during the period of inflammation. Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Peristaltic contractions in vivo and percent retrograde contractions. (A) The frequency within bursts of regular peristaltic contractions was assessed, not taking into account periods of mechanical quiescence or tonic contraction. This frequency decreased at day 15 PI. (B) The propagation velocity of anterograde peristaltic contractions also decreased. (C) In control animals, occasionally an orally propagating wave of contraction was seen after the stomach had emptied. In contrast, on days 2 and 15 PI, a significant percentage of contractions were aboral throughout the period of gastric emptying. Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Patterns of peristaltic contractions in control and infected mice in vivo. Light areas represent contraction. (A) The x-axis represents distance, with the arrowhead showing the position of the pylorus. The y-axis shows time from bottom to top with 10 seconds between the peristaltic contraction waves observed in the stomach. (A) Regular aborally propagating contraction in antrum (thick arrow) and in duodenum and jejunum (thin arrow) in a control animal. (B) At 2 days PI, contractions originate in the duodenum and propagate in both directions (arrows). (C) At 2 days PI, contractions originate close to the pylorus and propagate aborally (thin arrow). Retrograde contractions originate distally (thick arrow) and encounter oral contractions in a shifting collision zone (*). (D) At 15 days PI, aborally propagating contractions can be seen in duodenum (thin line) with irregular frequency. Orally propagating contractions (thick line) originate in jejunum. (E) At 15 days PI, intestinal contractions propagate orally. The periods of retrograde peristalsis alternate with periods of mechanical quiescence. Note the stomach skips a peristaltic movement. (F) At 60 days PI, regular, aborally propagating contractions can be seen in the antrum and small intestine (arrow). Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Ultrastructural changes in ICC processes after infection. (A) A group of ICC processes located between the longitudinal muscle layer (LM) and circular muscle layer (CM) 2 days PI show structural evidence of cellular injury. The injured ICC processes (☆) are characterized by loss of all intermediate filaments and most of the thin filaments. Few free ribosomes (r) are present in their cytoplasm. The double arrows indicate multiple membranes resulting from plasma membrane blebbing. Note 2 close apposition contacts (large arrows) between a longitudinal muscle cell and an intact ICC process. Undamaged ICC process cytoplasm includes intermediate filaments (small arrows), thin filaments (circle), and mitochondria (m) and is easily distinguished from other cell types by the presence of caveolae (arrowheads; magnification 37,900×). (B) By day 15 PI, many ICC processes, as well as some ICC cellular bodies, show structural evidence of cellular injury suggesting a prominent disruption of the ICC network. An injured ICC cell body (ICC, ☆) and a group of severely injured processes (☆) are shown at the level of Auerbach's plexus. The injured ICC perinuclear region and ICC injured processes are characterized by partial disintegration of the plasma membrane (arrow), presence of multimembrane blebs (double arrows), and lysosomes (arrowheads), as well as a partial loss of their cytoplasmic contents. LM, longitudinal muscle cell; CM, circular muscle cell; Nu, ICC nucleus (magnification 36,700×). Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Infiltration of immune cells into Auerbach's plexus region 6 days PI. Lymphocytes (Ly) and macrophages (Ma) penetrate the Auerbach's plexus region at close proximity to ICC processes (ICC), ganglions of the Auerbach's plexus (Ap), and blood vessels (Bv). LM, longitudinal muscle layer; CM, circular muscle layer (magnification 5500×). Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Recovery of ICC associated with Auerbach's plexus 60 days PI. No structural evidence of cellular injury is seen in ICC at this time. The ICC cytoplasm contains abundant intermediate filaments (large arrows), numerous mitochondria (m), and a few ribosomes (r). Small arrows, caveolae; Nu, nucleus; LM, longitudinal muscle; CM, circular muscle (magnification 42,660×). Gastroenterology 2000 119, 1590-1599DOI: (10.1053/gast.2000.20221) Copyright © 2000 American Gastroenterological Association Terms and Conditions