Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction

Abstract

1. Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction. 2. Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques. 3. Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. 4. Ultrastructural analysis revealed no evidence of cell death in regions where the lesion in ICC networks was developing. Cells with a morphology intermediate between smooth muscle cells and fibroblasts were found in locations that are typically populated by ICC. These cells may have been the redifferentiated remnants of ICC networks. 5. Removal of the obstruction led to the redevelopment of ICC networks and recovery of slow wave activity within 30 days. Neural responses were partially restored in 30 days. 6. These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype.

Figure 1

A, gross distension of the murine small intestine 14 days after insertion of a clip to create a partial occlusion. Distension of the intestine occurred proximal to the site of the clip. B, the gastrointestinal tract after its removal from the animal. Scale bars are as indicated in each panel.

Figure 2. Kit-like immunoreactivity (Kit-LI) in ICC networks (arrows) shown in confocal images of whole-mounts at various sites oral and aboral to the site of partial occlusion lasting 14 days

A, ICC networks of normal appearance at a distance 5 mm aboral to the occlusion clip. ICC were also observed at the site of the occlusion clip (B). Immediately proximal, and for distances up to 50 mm oral to the occlusion clip, ICC networks were disrupted (C and D). ICC were absent at 1 mm oral to the clip, and only weak Kit-LI was observed at 25 mm. At 75–100 mm oral to the clip (E and F) ICC networks were essentially normal. G–I, cryostat sections through regions aboral and oral to the occlusion clip. These sections demonstrate that ICC networks between the circular and longitudinal muscle layers at the level of the myenteric plexus (ic-my) and those at the level of the deep muscular plexus in the circular muscle layer (ic-dmp) were present aboral to (G) and at 100 mm oral to (I) the site of partial obstruction. However, at 25 mm oral to the obstruction (H), IC-MY and IC-DMP were greatly reduced in number. The arrow and arrowhead in H represent the levels of the myenteric plexus and the deep muscular plexus across the section, respectively. Scale bars are as indicated in each panel. A, B, E and F–I are reconstructions from 10 × 1 μm optical sections. C and D are reconstructions from 30 × 1 μm optical sections.

Figure 3. Electron microscopy confirmed the loss of ICC oral, but not aboral to the site of the occlusion clip applied for 14 days

A, a typical IC-MY interposed between the circular muscle layer (cm) and a myenteric ganglion (mg). This section was from a tissue sample taken 5 mm aboral to the site of the occlusion clip. As typically observed, the ICC has an abundance of mitochondria (m). Intermediate cells (arrowhead) with less-dense cytoplasm and an abundance of rough ER were also occasionally observed in this region. At the same distance aboral to the clips, ICC were found in the deep muscular plexus (IC-DMP) within the circular muscle layer. These cells were in close association with nerve bundles (nb; B). Immediately oral to the site of the occlusion clips, cells with ultrastructural features of IC-MY and IC-DMP were absent. Intermediate cells (see text for description of features) were found in this region (arrowhead; C) in both the myenteric plexus and deep muscular plexus (C and D, respectively). E and F show IC-MY and IC-DMP, respectively, that were occasionally observed 50 mm oral to the site of partial occlusion. IC-MY and IC-DMP at this level were intermixed with intermediate cells (arrowheads; E). At 100 mm oral to the occlusion clip, IC-MY and IC-DMP with ultrastructural features typical of ICC were observed in all animals. Scale bar in H represents 1 μm and is the same for all panels.

Figure 4. Electrical slow waves recorded oral and aboral to the site of a 14 day partial occlusion

Slow waves were present 5 mm aboral (A) and at the site of the occlusion (B). Oral to the occlusion (1–5 mm), slow waves were either absent or greatly reduced in amplitude (C and D). From 25–50 mm slow wave activity was attenuated (E and F). Between 75 and 100 mm oral to the occlusion slow waves were normal (G and H).

Figure 5. Summary of electrical parameters from cells oral and aboral to the 14 day partial occlusion

A, resting membrane potential (RMP) decreased immediately oral to the site of the occlusion, and returned to normal approximately 75–100 mm oral to the occlusion clip. B, slow wave amplitudes were greatly depressed immediately oral to the occlusion site. Slow waves were essentially normal 75–100 mm oral to the occlusion site. C, the effect of obstruction on slow wave frequency. D, the effect of obstruction on slow wave duration. Both frequency and duration were decreased immediately oral to the site of obstruction, but were normal 25–50 mm oral to the site of occlusion. – indicates sites aboral and + indicates sites oral to the obstruction clip. Levels of significance compared to controls are indicated by asterisks (*P < 0.05; **P < 0.001).

Figure 6. Post-junctional neural responses were attenuated in tissues oral to the site of partial occlusion

A, neural responses to electrical field stimulation (EFS: 1–20 Hz for 1 s, 0.5 ms pulse duration, indicated by the arrow and bars) recorded from the circular muscle layer of a control ileum. Responses typically consisted of a frequency-dependent hyperpolarization in membrane potential and inhibition of slow wave electrical activity during or immediately following stimulation, which was followed by a slower developing membrane depolarization. B, response to EFS in a muscle taken 5 mm oral to a partial occlusion clip (14 days after insertion of the clip). Neural responses were greatly attenuated at all frequencies tested (1–20 Hz, for 1 s, 0.5 ms pulse duration; indicated by the arrow and bars). C, recovery of neural responses 30 days after removal of the partial obstruction clip. Inhibitory and excitatory neural responses were recorded 5 mm oral to the site where the partial obstruction clip had been placed.

Figure 7. Restoration of normal anatomy after removal of the partial occlusion

A partial occlusion clip was placed on the intestine for 14 days and then removed. A period of 30 days was allowed before the animal was killed. The distension of the small intestine caused by 14 days of partial occlusion was greatly reduced during the recovery period. Scale bar, 5 mm.

Figure 8. ICC networks recovered after removal of the partial obstruction

A, Kit-LI 5 mm aboral to the site of the clip after a 30 day recovery period. B, ICC networks (arrows) at the site of the partial obstruction. C, Kit-LI 5 mm oral to the partial obstruction site. D, Kit-LI 25 mm oral to the partial obstruction. Scale bar, 50 μm in all panels. A–D are 1 μm z-steps through a tissue thickness of 10 μm.

Figure 9. Electron microscopy confirmed recovery of ICC 30 days after removal of the partial obstruction clips

A and B, typical IC-MY adjacent to myenteric ganglia (mg) in tissues within 5 mm of the site of the obstruction clip 30 days after removal of the clip. IC-MY had a characteristic electron-dense cytoplasm, abundance of mitochondria, caveolae and gap junctions with adjacent IC-MY (A; inset). After this period of recovery, occasional intermediate cells (arrowhead) were still present (B). This cell was found in the myenteric plexus region and lies adjacent to a circular muscle cell (cm). The cytoplasm of the intermediate cell contained mitochondria and an abundance of rough ER. C and D, restoration of IC-DMP oral to the site of partial obstruction 30 days after clip removal. The cytoplasm of IC-DMP was electron dense and contained numerous mitochondria. Caveolae were observed along the plasma membrane. IC-DMP also formed gap junctions (arrow) with circular smooth muscle cells (C and inset) and were in close association with nerve bundles (nb: D). Circular smooth muscle cells also returned to more normal morphology after removal of the partial obstruction clip (A–D). Scale bars are as indicated in each panel.

Figure 10. Recovery of slow waves after removal of partial obstruction clips

A, electrical slow waves 5 mm aboral to the site of obstruction 30 days after removal of the clip. B, slow waves at the site of obstruction. C–E, partial recovery of slow waves 1–25 mm oral to the site of occlusion. F–H, normal slow wave activity 50–100 mm oral to the occlusion.

Figure 11. Summary of electrical parameters 30 days after removal of the partial occlusion clips

A, the RMP was partially restored oral to the occlusion site during the recovery period. Slow wave activity was also partially restored (B–D) during the recovery period. – indicates sites aboral and + indicates sites oral to the obstruction site. Levels of significance compared to controls are indicated by asterisks (*P < 0.05; **P < 0.001).