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From the Intestinal Disease Research Program and Department of Medicine,* McMaster University, Hamilton, Ontario, Canada; and the Department of Anatomy, Histology, and Forensic Medicine, University of Florence, Florence, Italy
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Specific pathogen-free, male, 6 to 8 weeks old, C57BL/6 mice (Taconic, Germantown, NY) maintained in sterilized filtered cages were inoculated with 375 to 500 T. spiralis larvae using the procedures previously described by Castro and Fairbairn19 and modified by Vermillion and Collins.20 Animals were sacrificed at 2, 3, 8, 10, 15, 23, 40, 60, and 90 days after infection. Both control and infected animals were sacrificed by cervical dislocation. All of the experiments were approved by the McMaster University Animal Care Committee and the Canadian Council on Animal Care.
Transmission Electron Microscopy
Electron microscopy was performed on tissue from both noninfected control mice and mice from 2 to 60 days after infection. Under terminal anesthesia with Fluothane (Ayerst, Montreal, Canada), a fixative solution containing 2% glutaraldehyde, 0.075 mol/L sodium cacodylate buffer, pH 7.4, 4.5% sucrose, and 1 mmol/L CaCl2, was gently injected into the peritoneal cavity as well as the lumen of the proximal 10 cm of small intestine already tied at both ends. After 5 minutes of initial fixation, the proximal 8-cm segment of jejunum beginning 1 cm distal to the pylorus, was removed, opened lengthwise, and placed in the same fixative for an additional 2 hours at room temperature. After fixation, the tissues were cut into 2 x 5 mm circular and longitudinal strips, washed overnight in 0.1 mol/L sodium cacodylate buffer, containing 6% sucrose and 1.24 mmol/L CaCl2 (pH 7.4) at 4°C, postfixed with 1% OsO4 in 0.05 mol/L sodium cacodylate buffer (pH 7.4) at room temperature for 90 minutes, stained with saturated uranyl acetate for 60 minutes at room temperature, dehydrated in graded ethanol and propylene oxide, and embedded in Spurr. Tissue strips were oriented in molds to cut the circular muscle layer in either the cross or longitudinal direction. To locate suitable areas, 3-µm-thick sections were cut and stained with 2% toluidine blue. After the examination of the toluidine blue-stained sections, ultrathin sections of the selected areas were obtained with the LKB NOVA ultramicrotome using a diamond knife, mounted on 200-mesh grids, and stained with lead citrate or with an alcoholic solution of uranyl acetate, followed by a solution of concentrated bismuth subnitrate. The grids were examined under either a JEOL-1200 EX Biosystem or JEOL 1010 electron microscopes at 80 kV and photographed.
Double-Immunohistochemical Labeling of c-Kit and Vesicular Acetylcholine Transporter (VAChT)
Whole mounts were made from jejunum musculature with mucosa and submucosa removed of both control and T. spiralis-infected mice (10, 30, and 60 days after infection). All whole mount preparations were fixed in ice-cold acetone at 4°C for 10 minutes. After fixation, preparations were incubated with 1% bovine serum albumin for 1 hour to reduce the nonspecific staining before addition of primary antibodies. For double-labeling immunohistochemistry, tissues were incubated in each of the following primary antibodies for 48 hours at 4°C in a sequential manner. The first incubation was performed with rat monoclonal anti-c-Kit (ACK2, 1:200; Life Technologies, Inc., Gaithersburg, MD; currently ACK4 1:200 from Cederlane, Canada) The second one was with polyclonal rabbit anti-VAChT (1:100; Chemicon Inc., Temecula, CA). For the secondary antibodies, fluorescein isothiocyanate-coupled rabbit anti-rat IgG (1:100) was used to detect c-Kit labeling and Texas Red-conjugated goat anti-rabbit IgG (1:100) to detect VAChT labeling. All of the secondary antibodies were purchased from Vector Laboratories (Burlingame, CA) with the incubation time of 1 hour at room temperature. All of the antisera were diluted with 0.3% Triton X-100 in 0.05 mol/L phosphate-buffered saline (PBS, pH 7.4). Control tissues were prepared by omitting primary antibodies from the incubation solution. Tissues were examined with a confocal microscope (Zeiss LSM 510, Germany) with an excitation wavelength appropriate for fluorescein isothiocyanate (494 nm) and Texas Red (595 nm). Final images were constructed with Carl Zeiss software.
Immunoelectron Microscopy for VAChT
Tissues from both control and T. spiralis-infected mice (10, 30, and 60 days after infection) were studied. Whole mount tissues were prepared in the same way as for immunohistochemistry and fixed by immersion in 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid in 0.1 mol/L phosphate buffer (PB, pH 7.4) for 1 hour at room temperature. After a brief rinse in 0.1 mol/L PB, tissue was washed vigorously at room temperature in several changes of 50% ethanol until the picric acid staining of the tissue had disappeared (
20 minutes). The tissue was then washed in 0.1 mol/L PB and incubated in 0.1% NaBH3CN (Aldrich Chemical Co., Milwaukee, WI) in 0.1 mol/L PB for 30 minutes at room temperature. After washing in PB several times, nonspecific binding blocking was performed with 1% bovine serum albumin for 1 hour at room temperature. Tissues were then incubated with the same VAChT primary antibody as that for immunohistochemistry with the same time and dilution. On the third day, after washing in PBS several times, biotinylated goat anti-rabbit IgG, ABC reagent (with Vectastain ABC kit, Vector Laboratories) and peroxidase substrate (0.05% 3,3' diaminobenzidine plus 0.01% H2O2 in 0.05 mol/L Tris-buffered saline, pH 7.6) were used for the subsequent immunoreactions. All of the antisera for immunoelectron microscopy were diluted with 0.05 mol/L PBS (without Triton X-100, pH 7.4). Tissues were continuously checked under the light microscope for suitable reaction, before being postfixed, block-stained, dehydrated, embedded, and grid-stained with lead citrate for conventional electron microscope examination.
Quantification of c-Kit and VAChT Immunoreactivities
For quantitative evaluation of c-Kit-positive cells and VAChT-positive nerve fibers at the level of DMP, pictures were taken with the confocal microscope in the whole mount tissues and 50 areas were selected from five cases and scanned at the level of the DMP. The whole mount preparations with known thickness were chosen from control, and 10-, 30-, and 60-day, respectively, animals after infection. Quantification of Kit and VAChT positivity was performed using the KS400 program (Zeiss, Germany). Kit- and VAChT-immunopositive reactions were identified and highlighted using density slicing on color scale images. The area of immunopositive cells on each scanning picture was measured and the volume of positive cells/varicosities at the level of the DMP on whole mount musculature (composed of different scanning pictures with total thickness of 3 to 5 µm) was calculated. Due to the known scanning depth of each whole mount preparation, the data can be expressed as percentage of total volume of the tissues (positive cell volume/total volume). The Triton X-100 allowed full penetration of antibodies into the DMP area. Slight variations in staining intensity were not a problem for quantification. After elimination of background staining both cells that stained intense and somewhat weaker were allowed to be incorporated in the computer analysis.
Quantification of Ultrastructural Features of ICC-DMP and the Inner Circular Muscle Layer
The coated vesicles located at the level of the Golgi apparatus plus those distributed along the ICC-DMP periphery were counted in controls and in mice at 2 to 15 days and 40 to 60 days after infection (two animals each group). Results are expressed as mean number of coated vesicles per ICC profile. To evaluate the caveolae number in the smooth muscle cells of the inner circular muscle layer, the smooth muscle cell perimeters, expressed in µm, were measured in 50 smooth muscle cell profiles of controls, in 57 mice 2 days and after infection, and in 70 animals 40 to 60 days after infection (two animals each group). Each micrograph had the same enlargement and the smooth muscle cells were cross-sectioned. Caveolae were counted in the same micrographs as above. The total areas expressed in µm2 occupied by the inner circular muscle layer, the smooth muscle cells of this layer, and the intercellular stroma, were measured in controls (11 micrographs), and in 2 (10 micrographs), 23 (10 micrographs), 40 (12 micrographs), and 60 (10 micrographs) days in animals after infection (two animals each group). Each micrograph had the same enlargement and the smooth muscle cells were cross-sectioned. The measurements were done by using Scion Image for Windows (Scion Image Corp., Bethesda, MD). Statistical analysis was performed by means of Student’s t-test. A probability value of less than 0.05 was regarded as significant. The results were expressed as the mean values ± SEM.
Mechanical and Electrophysiological Studies on Stretch-Induced Peristalsis
Both control and infected male mice were sacrificed by cervical dislocation. Infected mice were inoculated with T. spiralis at day 1. The distention-induced motor patterns were determined on days 10, 30, 60, and 80 after infection. The small intestine was exposed by a mid-line abdominal incision. The intestine was placed in a continuously oxygenated (95% O2 and 5% CO2) Krebs solution (pH 7.3 to 7.4). A 6-cm segment was taken 2 cm distal of the pyloric sphincter. The segment was flushed gently with Krebs solution to remove luminal contents. The segment was placed in a 60-ml organ bath filled with continuously oxygenated (95% O2 and 5% CO2) Krebs solution at 37°C, pH 7.3 to 7.4. The composition of the Krebs solution was (in mmol/L): 120.3 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 20.0 NaHCO3, 1.2 NaH2PO4, and 11.5 glucose.
The oral part was attached to a pressure column, a large Krebs-filled beaker that allowed for the maintenance of constant pressure. The distal part was attached to an open vertical tube that did not allow outflow of contents. The lumen of the segment was filled with Krebs solution. The pressure within the segment was kept constant. The electrical pattern and luminal pressure were measured. The pressure within the segment was increased by increasing the level in the Krebs-filled beaker gradually to 5 cm. The intraluminal pressure was measured at two points with two Krebs solution-filled plastic open end tubes whose openings were located 3 and 5 cm from the oral end of the segment. Three suction electrodes attached to the serosal side of the segment measured the electrical activity in the segment. Electrical activity is measured from both circular and longitudinal muscle because of the small diameter of the mouse small intestine musculature. Electrical activities of the circular and longitudinal muscle are similar21 in contrast to those of the colonic muscle layers.22 The ground and recording electrodes were silver chloride-coated silver wires 0.06 mm in diameter. The recording electrodes were insulated by placing them in an open-ended flexible plastic tubing of 0.2-mm inner diameter and an outer diameter of 1.0 mm. The suction on the recording electrodes was 380 to 390 mmHg. All signals were amplified and recorded by a Grass ink writing amplifier-recorder (7 PCM 12 C). All recordings were made within 2 hours after removal of the tissue from the animal. The duration of active and quiet periods and the amplitude of the slow waves in the active and quiet periods were measured. The frequency in the active and quiet periods and the overall frequency were determined. The number of action potentials superimposed on the omnipresent slow waves per minute and the number of bursting periods within a 5-minute period were determined. These parameters were determined before and after increasing the distention of the intestinal segment.
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Confocal and EM Immunohistochemistry
In control mice, c-Kit-positive ICC-DMP were bipolar and were connected to each other by long processes to form a mesh-like structure (Figure 1a)
. ICC-DMP were significantly reduced in the tissues 10 days after infection (Figure 1d)
, primarily recovered at 30 days after infection (Figure 1g)
, and returned to normal at 60 days after infection (Figure 1j)
. Numerous VAChT-immunoreactive nerve fibers, rich in varicosities were seen at the DMP level (Figure 1b)
. VAChT-immunoreactive nerve fibers were significantly reduced in the tissues 10 days after infection (Figure 1e)
, primarily recovered 30 days after infection (Figure 1h)
, and returned to normal 60 days after infection (Figure 1k)
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; c, f, i, and l) and transmission electron microscope confirmed this (Figure 2
; a to c). Moreover, ultrastructural examination confirmed that, compared with control tissue (Figure 2a)
, VAChT-immunoreactive varicosities were reduced 10 days after infection (Figure 2b)
, and recovery was seen at 30 days after infection (Figure 2c)
although significant structural damage was still present. Complete recovery occurred 60 days after infection (not shown).
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shows the result of quantification of c-Kit- and VAChT-immunoreactive structures at the level of the DMP of control and infected tissues. Both c-Kit (P < 0.05)- and VAChT (P < 0.01)-immunoreactive elements were significantly decreased in the tissues 10 days after infection. No significant difference with control was observed at 30 days after infection (P = 0.38 for c-Kit and P = 0.71 for VAChT) and 60 days after infection (P = 0.97 for c-Kit and P = 0.91 for VAChT).
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Electron Microscopy
In control animals, ICC-DMP (Figure 4a)
are identified as spindle-shaped cells located in between the inner and outer subdivisions of the circular muscle layer and closely apposed to the nerve bundles of the DMP.1,6
The cytoplasm is electron-dense and rich in mitochondria, and numerous caveolae are aligned along the plasma membrane (Figure 4b)
. The Golgi apparatus is small and cis-ternae of the rough endoplasmic reticulum (RER) scarce. All ICC-DMP have a continuous basal lamina (Figure 4, a and b)
. Moreover, in the mouse the ICC-DMP are frequently in contact with each other and the smooth muscle cells of the outer circular muscle layer by means of gap junctions (Figure 4a)
.
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; a, c, and d) characterized the ICC and coated vesicles, located as in controls at the budding face of the Golgi apparatus and close to the plasma membrane, were significantly more numerous (Figure 5, b and d
; Figure 6
). Moreover, in the cytoplasm of some ICC-DMP there were lamellar bodies (Figure 5, c and d)
and autophagosomes (Figure 7c)
. Loss of cell-to-cell contacts was widespread. Gap junctions were virtually absent and lamellar bodies were seen between ICC as well as between ICC and nerve varicosities (Figure 7a)
. These injuries were the most extensive at 10 days after infection, when wide areas of cytoplasmic depletion devoid of organelles were observed (Figure 8
; a to d). By day 2 up to 15 days after infection the basal lamina was lacking or discontinuous.
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. At 60 days after infection, all ICC-DMP had normal features and the number of coated vesicles was similar to controls (Figure 6)
. Statistical analysis revealed that the mean number of coated vesicles in ICC-DMP per ICC profile was 0.42 ± 0.11 in controls, 3.06 ± 0.32 at 2 to 15 days after infection (P < 0.01), and 0.47 ± 0.11 at 40 to 60 days after infection (Figure 6)
. Nerve Varicosities of the DMP
In control animals, the nerve varicosities of the DMP contained either small agranular vesicles or large granular vesicles and were frequently in close contact with the ICC-DMP (Figure 4, a and b)
. Two to fifteen days after infection, many nerve varicosities remained normal (Figure 5, a and b)
but others, especially those close to the damaged ICC-DMP, were reduced (Figure 7a)
or had completely lost their content (Figure 8, b and c)
or contained lamellar bodies (Figure 7a)
. Moreover, scattered areas were devoid of nerve structures (Figure 8d)
. By 30 to 60 days after infection, most nerve varicosities were similar to control and only a few of them still showed lamellar bodies (Figure 7b)
.
Macrophages
In the control pathogen-free animals, macrophages or other inflammatory cells were not present in the DMP area. Three to fifteen days after infection, inflammatory cells, mainly macrophages, were present, at variance with the submucosa where there were also many granulocytes and lymphocytes. The ICC-DMP and the DMP nerves were frequently contacted by macrophages (Figure 8
; a to d). Lamellar bodies were particularly numerous when ICC-DMP and nerves were contacted by macrophages (Figure 8b)
. Occasionally, there was also a loss of the cell content (Figure 8, c and d)
. Macrophages were less frequently observed at 23 to 40 days after infection and continued to be present day 60 after infection.
Inner Circular Muscle Layer
The inner circular muscle layer in control mice is only one cell thick, no close contacts with nerve varicosities nor specialized cell-to-cell junctions with the ICC-DMP were present (Figure 4, a and b
; Figure 9a
). Two to fifteen days after infection, the smooth muscle cells contained markedly extended RER and Golgi apparatus (Figure 9b)
and were increased in size (Figures 9b, 10b, and 11b)
. Days 23 to 60 after infection constituted a recovery phase of synthetic activity as judged by a gradual return to a normal RER and Golgi apparatus extension. However, this period also witnessed structural changes that remained at day 60 after infection. Smooth muscle cells formed thin, short protrusions; that gradually increased in number, extended into the intercellular space (Figure 9, c and d)
and finally formed branches obliquely oriented (Figure 9e)
. Because of this increase in branching, the smooth muscle cells acquired a ramified shape and their perimeter increased significantly and this maintained up to day 60 after infection (Figure 11b)
. Moreover, an overlapping of these ramifications also occurred and, consequently, the inner circular muscle layer gradually stratified to double its thickness (Figure 9, d and e
; Figure 10
; Table 1
).
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; Figure 11a
; Table 1
). This increase could not be accounted for by the increase in cell perimeter. Together with the increased size (cell area) and ramification (cell surface) of the smooth muscle cells, the intercellular space also enlarged (Figure 9, c, d, and e
; Figure 10c
; Table 1
) and by day 23 after infection it became rich in collagen fibrils (Figure 9c)
. The ratio between the space occupied by intercellular connective tissue and smooth muscle cells increased 23 to 40 days after infection but returned to normal at 60 days (Figure 10, a to c
; Table 1
). Outer Circular Muscle Layer
In control tissue, smooth muscle cells of the outer circular muscle layer were frequently connected by gap junctions (Figure 12a)
. Smooth muscle cells formed gap junctions with ICC-DMP (Figure 4a)
and made contact with intramuscular nerve varicosities. Two to ten days after infection, the intercellular space became widened and contained an edematous stroma (Figures 5 and 12b)
; concomitantly, loss of gap junctions occurred. A prominent increase in RER and Golgi apparatus extension was observed in the smooth muscle cells 8 to 15 days after infection (Figure 12c)
, but by day 23 after infection, smooth muscle cells were normal in ultrastructure. Between days 40 and 60 after infection, gap junctions reappeared (Figure 12d)
, but the intercellular space remained larger than in control tissue and the stroma became moderately fibrotic.
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In control mice with distention, a segment of intestine displayed regular slow wave activity with or without action potential generation (Figure 13)
. When action potentials occurred they were seen in bursts. A burst of action potentials was observed when 5 to 10 consecutive slow waves would carry action potentials. The number of bursts per minute was highly variable ranging from 0.3 to 2.2 bursts per minute (1.1 ± 0.5 bursts per minute). Spiking increased by distention. During the burst type activity the spiking amounted to 24.9 ± 1.7 action potentials/minute. The slow waves with superimposed action potentials propagated anally causing peristaltic contractile activity. The slow wave frequency after distention during nonburst or quiet periods and during a burst of action potentials was, respectively, 37.7 ± 1.0 cpm and 39.6 ± 1.0 cpm. Ten days after infection, segments of the intestine did not show rhythmic bursts of action potentials with or without stretch applied to the tissue. The strips of isolated muscle were thickened yet were much more fragile compared to control. In addition, the ability to develop stretch-induced bursts of action potentials and stretch-induced peristalsis was completely abolished. Consistently, no visible rhythmic contractile activity occurred in the intestinal segment. Thirty days after infection, segments of the intestine were much less fragile and in preparations from four infected mice, spontaneous activity was present with bursting activity. However, segments from only one of four animals showed increased bursting on distention. In the segments that showed increased bursting, the slow wave activity was still very irregular so that quantification was not possible. Sixty days after infection, the slow wave pattern was restored. Even without distention (<1 cm) a stable slow wave pattern was observed. Short periodic bursts of action potentials occurred both before and after distention, but distention increased the number of bursting periods and the spiking/minute to a normal level as observed in healthy control tissue. A stable pattern of periodic bursts of action potentials occurred at a burst frequency of 6.0 ± 1.5 per minute and a burst duration ranging from 5.8 to 17.3 seconds. The slow wave activity was so stable that a frequency increase could be observed in response to distention. The slow wave frequency increased from 35.2 ± 0.1 cycles/minute to 45.6 ± 0.5 cycles/minute. These quantitative parameters were all within normal limits.
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Dramatic transient changes were observed in ICC-DMP after infection, in particular associated with the cell membrane. Although c-Kit immunohistochemistry suggested loss of cells, the ultrastructural findings primarily showed membrane damage; marked cell degeneration was not observed nor differentiating ICC. This suggests that the massive membrane injury is accompanied by loss of c-Kit-positive membrane. It is therefore possible that loss of cell structures is limited and that ICC function is lost because of structural injury and loss of c-Kit positivity. The recovery was not focused on generating new cells but on cell repair clearly reflected in the ultrastructural findings, in particular the enlargement of the organelles responsible for protein synthesis (RER and Golgi apparatus) and a significantly increased number of coated vesicles representing newly formed plasma membrane either at the budding face of the Golgi apparatus, where they form, or close to the plasma membrane that is their final destination. The presence of lamellar bodies at contact areas signified degeneration of gap junctional contact with other ICC and synapse-like contact with nerves. ICC-DMP are innervated by cholinergic (VAChT-positive) nerves as shown previously for humans7 and the guinea pig.26 Close association with nerves has always been a hallmark of ICC-DMP.2 From 8 to 23 days after infection contacts between ICC-DMP and DMP nerves reduced markedly and c-Kit-VAChT double labeling showed a significant reduction of VAChT-positive nerves 10 days after infection indicating markedly reduced communication between excitatory nerves and ICC. Recovery of cholinergic innervation paralleled recovery of ICC. Hence, loss of cell to cell communication is likely a major mechanism underlying loss of function. There are interesting similarities with a previous study on ICC bordering the submucosa in the human colon.27 The structural changes were similar, close association with macrophages was also observed and rather selective injury to ICC and associated nerve structures, with smooth muscle cells and fibroblasts relatively undisturbed. An animal model of colitis showed similar findings that were associated with functional abnormalities.28 Hence, the modalities of recovery are the dramatic increase in rough endoplasmic reticulum in ICC, followed by restoration of cell structure and junctions with neighboring cells, and the withdrawal of macrophages. ICC injury and recovery are an important part of the pathology of intestinal inflammation related to associated motor dysfunction.
The present study shows that injury to ICC-DMP and associated nerve structures was temporally connected to loss of distention-induced patterns of electrical activity that are associated with distention-induced peristalsis.14 This suggests that the ICC-DMP and associated nerves are a critical part of the pathway that links physical distention with stimulation of enteric nerves that provide the periodic motor activity characteristic of distention-induced peristalsis. In the dog29 and in the mouse,2 the ICC-DMP have been suggested to detect muscular tone and intraluminal pressure changes and the present results provide functional support for this hypothesis. Consistently, in Nippostrongylus brasiliensis-infected rats the absence of c-Kit-positive cells was accompanied by a loss of ICC-DMP even after inflammation had spontaneously resolved 30 days after infection.12 The authors speculated that this loss, together with impairment in tachykinergic control of jejunal functions, might be responsible for alterations of motility and, in particular, of sensitivity to distension. It is possible that the inner circular muscle layer that borders the submucosa is also part of the structure that senses stretch induced by increase in intraluminal pressure.30,31 The ICC-DMP and the inner circular muscle layer are specific structures for the small intestine, and their role is reasonably related to a motor function specific of this area. The present results have important similarities with a study on human orthotopic ileal bladders32 where evidence was presented that the unit of DMP nerves, ICC-DMP, and the inner circular smooth muscle cells were involved in detection of distention. Orthotopic ileal bladders are continent urine reservoirs constructed after radical cystectomy by replacing the excised urinary bladder with an ileal loop still attached to its mesentery. Motor patterns, intraluminal pressures, and volume capacity were recorded in situ before corrective surgery and anatomical characteristics were studied in full-thickness specimens from the ileal reservoirs removed during corrective surgery. Morphological examination was performed with both light and electron microscopy. The ileal reservoirs generated phasic activity when filled with liquid, and all retained the ICC-AP; however, after extended periods (eg, 3 to 8 years), the ICC-DMP, together with the inner circular muscle layer and the DMP completely disappeared (ICC-AP remained normal) and the muscle wall lost its normal responsiveness to distention.32 Based on morphological data, a similar role is being proposed for ICC-IM in the stomach.33,34
We have previously reported that ICC-AP are injured by the T. spiralis infection.17
The structural and functional injury was due to cytokine secretion from macrophages and these macrophages were colony-stimulating factor-1 (CSF-1)-dependent.35,36
The ICC-DMP injury presently found was qualitatively similar to injury to the ICC-AP but the injury was much more severe.17,37,38
It is unlikely that the injury to ICC-AP is associated with the functional changes described here. First, the primary role of ICC-AP is the generation of pacemaker activity39,40
and there is no evidence for a role of ICC-AP in detection of tension although this cannot be ruled out. Second, the timing of the recovery of ICC-AP mimics that of the recovery of electrical pacemaker activity, 15 days after infection,17
which is much faster than the recovery of distention-induced peristaltic activity and ICC-DMP.
Macrophages were present in control tissue in the external muscle layers, in particular the myenteric plexus area, and are considered to be resident macrophages.41,42 In the DMP area macrophages were absent in our pathogen-free mice although they were encountered rarely in nonpathogen-free mice in which they were associated with ICC-DMP processes.3 After the T. spiralis infection, in areas of severe damage to ICC-DMP and associated nerve structures, macrophages were seen to be abundant. Lymphocytes were also observed in the DMP area after infection but their numbers were far less than macrophages and lymphocytes did not make close contact with ICC-DMP or nerves. In contrast, lymphocytes and granulocytes were prominent in the submucosa. Therefore, it is likely that a major part of the injury to ICC-DMP and related nerves is mediated by macrophages because they were the only immune cell prominent in the DMP area and close contact between them and injured ICC-DMP/nerves was common. The time course of ICC-DMP and nerve injury and recovery and the presence and decline of macrophages coincided as well. This suggests that the structural and functional injury was, at least in part, due to cytokine secretion from macrophages. No enlarged and dilated blood vessels were found in the DMP area as reported in the Auerbach’s plexus after T. spiralis infection.38 Hence the macrophages in this area likely migrated from the submucosa or Auerbach’s plexus area or developed from resident macrophages. An association between macrophages and ICC injury was also observed in ulcerative colitis27 and in an animal model for Hirschsprung’s disease where in the distended part of the colon altered motor activity was associated with an influx of macrophages and injury to ICC.43
T. spiralis infection markedly and permanently affected the inner circular muscle layer that is only one cell layer thick in the mouse and separates the DMP area from the submucosa. Transient changes that occurred were an increase in RER and Golgi apparatus, indicating intense protein synthesis possibly related to the increase in cell size and the synthesis of collagen. Changes that did not subside were enlargement of the muscle cells, development of branches, resulting in a ramified cell shape, a marked increase in the number of caveolae in the smooth muscle cells, and an enlargement of the intercellular space with collagen deposits. The thickening of the inner circular muscle layer was due to enlargement of the muscle cells and the appearance of multiple layers to extensive overlapping of the cell branches. An increase in the number of muscle cells is unlikely because differentiating smooth muscle cells were never observed. Branching of smooth muscle cells might represent an adaptive attempt to maintain mechanical properties of the inner circular muscle layer in the presence of a fibrotic stroma. The increased number of caveolae might represent an increased ability to respond to signaling molecules as part of the attempt to withstand the adverse environment.44 In response to a N. brasiliensis infection, injury to ICC was more severe and longer lasting, although no changes in the inner circular muscle occurred.12 A different immune response may explain the differences between N. brasiliensis and T. spiralis. In contrast to a T. spiralis infection in which macrophages are the dominant immune cell in the musculature, mast cells penetrated the musculature after a N. brasiliensis infection45 and changes in motor function in response to a N. brasiliensis infection depended on the presence of mast cells.46 Interestingly, the presence of mast cells in the musculature was long lasting with a half life of 40 days, which may explain the severity of the damage to ICC.45
The present study provides further evidence for the T. Spiralis-infected mouse being a model for postinfectious enteropathies, a significant problem in clinical gastroenterology. The present study shows persistent changes in the inner circular muscle layer. Long-lasting decreases in excitatory47 and inhibitory48 neurotransmission were also observed. One consequence is the persistence of retro-peristalsis,17,49 which also occurs in IBS.50 Macrophages persisted in the DMP area at least until day 60 after infection similar to the Auerbach’s plexus area noted previously.38 There is also persistence of CD3-positive cells.49 In addition, corticosteroids suppressed some of the postinfectious symptoms giving arguments for the hypothesis that continued immune activation is involved in postinfectious motor dysfunction.49
The present results support the hypothesis that the ICC-DMP, possibly together with the inner circular muscle layer and DMP varicosities, represent the intestinal stretch receptor sensing intraluminal pressure changes. A mucosal T. spiralis infection is particularly affecting the DMP area at the border of the submucosa thereby affecting a major function of the musculature, distention-induced motor activity. The changes in the inner circular muscle layer represent an attempt to restore the mechanical properties of this layer, but may also result in a permanent change in sensitivity to distention after infection.
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Supported by an operating grant from the Canadian Institutes of Health Research and a fellowship from the Canadian Association of Gastroenterology sponsored by the Canadian Institutes of Health Research and Janssen-Ortho (to X.Y.W.).
Accepted for publication March 14, 2005.
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0.01. No significant difference between control and 40 to 60 days after infection.
