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Animal Model |
From the Intestinal Disease Research Program and Department ofMedicine,* McMaster University, Hamilton, Ontario, Canada;and the Institute of Medical Anatomy,University of Copenhagen, Copenhagen, Denmark
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Abstract |
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ICC associated with Auerbach’s plexus (ICC-AP) in the small intestine perform a pacemaker function for intestinal motor activity.1,2 ICC-AP are associated with slow-wave-driven peristalsis, which is prominent during gastric emptying and small bowel transit.3 In fact, normal peristaltic activity in the small bowel after gastric emptying of a liquid was absent in W/Wv mutant mice that do not have ICC-AP.3 ICC-AP are characterized by elongated cell bodies with extremely long, thin, ramified cell processes interconnected by close apposition contacts and gap junctions. ICC-AP have many ultrastructural features in common with smooth muscle cells and fibroblasts, but can be identified because of a unique combination of ultrastructural features together with unique structural associations with nerves and smooth muscle cells.4-6
ICC harbor the c-Kit protein and can therefore be identified at the light microscopy level using immunohistochemistry with c-Kit antibodies.2,7 In fact, almost all current studies on the pathology of ICC are performed by immunostaining.8,9 By using both electron microscopy (EM) and immunohistochemistry we discovered in the present study that marked changes in ICC at the EM level, associated with previously observed functional changes,10 did not lead to any changes at the light microscopy level using Kit immunohistochemistry.
The mouse small intestine harbors resident macrophages that have a close structural relationship with ICC.11 It was therefore of interest to follow the interaction between macrophages and ICC-AP during and after the inflammatory process.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free, male, 5- to 7-week-old, C57BL/6 mice (Taconic Farms, Germantown, NY) maintained in sterilized filtered cages were inoculated with 375 to 500 T. spiralis larvae using the procedures previously described by Castro and Fairbairn12 and modified by Vermillion and Collins.13 All of the experiments were approved by the McMaster University Animal Care Committee and the Canadian Council on Animal Care.
Electron Microscopy
EM was performed on tissue from non-infected control mice (n = 7), and mice infected with T. spiralis 24 hours (n = 12), 48 hours (n = 12), 3 days (n = 8), 6 days (n = 6), 8 days (n = 6), 15 to 17 days (n = 8), 21 to 23 days (n = 4), 40 days (n = 4), and 60 days (n = 3) previously. 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 the 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 (CM) layer in either the cross or longitudinal direction. To locate suitable areas, 0.5-mm thick sections were cut and stained with 2% toluidine blue. After examination of the toluidine blue-stained sections, ultrathin sections were cut, mounted on 200-mesh grids, and stained with lead citrate. The grids were examined in a Jeol-1200 EX Biosystem electron microscope at 80 kV.
This study focuses on changes in ICC-AP that are involved in gut pacemaker activity, it does not discuss the other types of ICC. The nomenclature conforms to that of Faussone-Pellegrini and Thuneberg6 and Komuro.14
Immunohistochemistry for ICC-AP
Whole mounts were made from the musculature of the proximal jejunum (1- to 8-cm distal from the pylorus) from T. spiralis-infected and control mice. Tissues were processed from control mice (n = 6), day 1 (n = 4), day 2 (n = 4), day 15 (n = 6), day 40 (n = 3), and day 60 (n = 3) 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 the primary antibody rat monoclonal anti-c-Kit (ACK2, 1:200; Life Technologies, Inc., Gaithersburg, MD). After the incubation with primary antibody for 2 nights at 4°C, fluorescein isothiocyanate-coupled rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) was added to the tissue with a dilution of 1:100 and left for 1 hour. Tissues were examined with a confocal microscope (LSM 510; Zeiss, Germany) with an excitation wavelength appropriate for fluorescein isothiocyanate (494 nm). Final images were constructed with Carl Zeiss software. Control tissues were prepared by omitting primary antibody from the incubation solution. All of the antibodies were diluted with 0.05 mol/L phosphate-buffered saline with 0.3% Triton X-100.
Immunohistochemistry for Macrophages
Proximal jejunum was removed from both infected and control mice as described above. Tissues from each animal were divided into two groups, one for whole-mount preparations and another for frozen sections. Muscle whole mounts were made by peeling away the mucosa and submucosa. To prepare frozen sections, after freezing the mouse intestine in isopentane and liquid nitrogen, sections of 5 µm were cut with the cryostat, both whole mounts and frozen sections were fixed in ice-cold acetone for 5 minutes, and then fixed with ice-cold 3.75% periodate-lysine-paraformaldehyde for 10 minutes at 4°C immediately before the immunostaining. Endogenous peroxidase was quenched by submerging the tissues in 1% hydrogen peroxide for 15 minutes and nonspecific antibody binding was blocked by the incubation with 8% normal goat serum plus 1% bovine serum albumin for 1 hour. The tissues were then incubated with monoclonal rat anti-mouse F4/80 (diluted to 1:15 with 8% normal goat serum and 1% bovine serum albumin; Serotec, Oxford, UK) overnight at 4°C plus 1 hour at room temperature. Biotinylated rabbit anti-rat IgG (mouse adsorbed; DAKO, Glostrup, Denmark) was added to the tissues at a 1:400 dilution (diluted with 1% bovine serum albumin) and left for 1 hour at room temperature. After incubating the tissues with ABC-complex (Vector Laboratories) for 1 hour, 3,3' diaminobenzidine (0.05% DAB plus 0.01% H2O2 in 0.05 mol/L Tris buffer saline, pH 7.6) was used as peroxidase substrate. Frozen sections were then counter-stained with Mayer’s hematoxylin and pictures were taken with the digital camera (Sony 3CCD, model no. DXC-930; Sony, Japan). For whole-mount preparations, pictures were taken focused on the level of AP. Because of the very thin longitudinal muscle (LM) layer, F4/80 immunoreactivity in the serosa was inevitably present in the picture. Control experiments involved incubation of the sections with a rat IgG2b isotypic control (Serotec) instead of anti-F4/80 serum.
Quantification of F4/80 positivity was performed on a G3 Macintosh computer using the public domain NIH Image 1.62 program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). F4/80-immunopositive cells were identified and highlighted using density slicing on gray scale images. The area of immunopositive cells was calculated and then expressed as percentage of total area of the image. Regions with increased background staining, occasionally found at the borders of the image, were excluded from the analysis.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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ICC-AP were seen as c-Kit-positive multipolar cells connected to
each other by long processes to form a mesh-like structure associated
with AP (Figure 2a)
. This structural
arrangement remained essentially unaltered during and after the mucosal
infection (Figure 2b
, day 15 after infection).
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Resident macrophages were present primarily in the subserosal
space as well as in the AP region but not within the muscle layers as
observed in whole-mount muscle preparations from pathogen-free control
mice (Figure 3a
and Figure 4a
). There was no variability in the
distribution of macrophages in control mice. After 24 hours of
infection, macrophages infiltrated the AP region (see EM results) and
after 48 hours of infection macrophages were present in both muscle
layers (not shown). Figures 3b and 4b
show the dramatic increase in F
4/80-positive cells at day 15 after infection. A high concentration was
found in the AP region but they were also scattered throughout the
muscle layers and at the level of the deep muscular plexus. After 23
days, the number of macrophages gradually decreased although even at
day 40, there still was a marked increase greater than control values
(Figures 3c and 4c)
. At day 60 after infection, the number of
macrophages in the AP region was still markedly increased and
macrophages within the muscle layers could still be found (Figures 3d and 4d)
. Quantitatively, F4/80 immunoreactivity occupied 1.1 ±
0.4% of total area (n = 4) in control tissue,
on day 15 after infection this was 7.7 ± 1.0%
(n = 4) (P < 0.0001), on
day 40 after infection it was 5.8 ± 0.7%
(n = 3) (P < 0.0001),
and on day 60 after infection it was 5.1 ± 0.8%
(n = 3) (P < 0.003).
Immunoreactivity on day 40 after infection was significantly reduced
compared to day 15 after infection, there was no significant difference
between days 40 and 60 after infection.
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Injury to ICC was extremely rare in tissue from properly fixed control mice that were processed in the same manner and at the same time as tissue from infected mice. Furthermore, in inflamed tissue, many ICC-AP not associated with blood vessels did not show any structural abnormalities, ruling out fixation artifacts. ICC were identified as described by Thuneberg.4 A key feature was the presence of caveoli. Throughout the cytoplasm of the cell body and processes, intermediate and thin filaments were present but thick filaments were absent. Lysosomes were rarely observed. Rough endoplasmic reticulum (rER) was found to be sparse in all regions of the ICC, and Golgi complexes were observed only in the perinuclear region, hence not in the processes.
From days 1 to 3 after infection, ICC-AP displayed ultrastructural
abnormalities primarily restricted to their processes without
involvement of the nucleus and perinuclear space (Figure 5, a and b)
. Loss of the intermediate
filament network was observed, as well as a partial depletion of thin
filaments (Figure 5a)
. Small lysosomes were present (Figure 5a)
and
multiple membrane structures formed as a result of membrane ruptures
(Figure 5b)
. Many terminal processes were ruptured with complete loss
of their cytoplasmic contents (Figure 5b)
. These changes were seen in a
patchy manner throughout the tissue but
were primarily associated with those
ICC-AP closest to dilated blood vessels,
which were mainly confined to the AP region (Figures 6, 7, and 8)
.
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; and
Figure 6, a to f
), but injury was restricted to the membrane and
filament structures. Injury to nuclei was minimal. Multiple membrane
structures and plasma membrane blebs were displayed in the cytoplasm
and associated with the plasma membrane (Figure 5c
; Figure 6, b to f
;
Figure 7b
; and Figure 8, b and c
). In some perinuclear regions,
cytoplasmic content was partially lost (Figure 5d)
. Nonetheless, injury
to ICC-AP never involved the whole ICC-AP network (see Figure 2b
), and
in many sections, ICC-AP and their processes were not damaged (Figure 5c)
. Injury was again most prevalent close to dilated blood vessels in
the AP (Figures 6a, 7a, and 8a)
.
ICC with damaged processes did not make close contact with smooth
muscle cells or nerve varicosities, features always present in
undamaged ICC. Damage was primarily restricted to ICC, most smooth
muscle cells were not injured (Figures 6 to 8)
.
From day 3 onward, injury to ICC-AP was concurrent with extensive
proliferation of ribosomes, rER, and Golgi complexes in many ICC-AP
(Figure 9
; a to c). In non-infected mice,
rER was sparse in all regions of ICC-AP, and Golgi complexes were
observed only in the perinuclear regions, and were not apparent in the
processes. In infected mice, outgrowth and expansion of rER and Golgi
complexes was present not only in perinuclear regions (Figure 9a)
, but
also in the cell processes (Figure 9, b and c)
resulting in a partial
shifting of filaments from the central cytoplasm to the periphery. At
40 days after infection, the majority of ICC-AP processes were
structurally similar to control. Injured ICC-AP processes were
extremely rare on day 60 after infection but some ICC-AP continued to
display proliferation of rER and ribosomes in their cytoplasm.
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Within the muscle layers and around the AP area, injury to smooth
muscle cells was rare and severe injury did not occur. Except for
resident macrophages,15
immune cells were not present in
non-infected specific pathogen-free control mice. As early as 24 hours
after infection, conspicuously activated macrophages were seen in the
AP region in extensive close contact with ICC-AP processes (Figure 8, a to c
; Figure 10, a to c
). Activated
macrophages were enlarged, with numerous cytoplasmic foldings, and
numerous transport vesicles and lysosomes.16
The first
days after infection, close contact between ICC-AP and macrophages was
not associated with phagocytosis consistent with the fact that these
ICC-AP were not injured. Furthermore, in most macrophages, phagosomes
did not occur indicating that the macrophages were not involved in
phagocytosis during this period. In addition to ICC-AP, macrophages
formed frequent close contacts with adjacent smooth muscle cells of
both muscle layers. Figure 10a
shows a peg-and-socket-like contact
between a LM cell and a macrophage process observed 2 days after
infection. By day 15 after infection, some macrophages containing
numerous large phagosomes and lysosomes were now involved in
phagocytosis of damaged cellular processes. Figure 10c
shows a
macrophage containing secondary lysosomes attached to a severely
injured ICC-AP process. During this period, many macrophages were found
in close association with lymphocytes and occasionally with
eosinophils. Between 40 and 60 days after infection, macrophages
situated between the two muscle layers and in the subserosal space were
distinguished by the presence of large phagosomes, containing residual
bodies, indicating their continuing involvement in phagocytosis of
debris.
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where a
lymphocyte is shown to be enclosed by a LM cell forming extensive close
apposition contact with it. Within the AP region, lymphocytes
frequently formed close apposition contacts with ICC-AP processes
(Figure 12)
. Lymphocytes were also
observed at the deep muscular plexus and in the subserosa. In addition
to scattered single immune cells, accumulation of macrophages and
lymphocytes was frequently observed between the main muscle
layers usually at close proximity to blood vessels where macrophages
and lymphocytes formed extensive close contacts with each other.
Eosinophils were seen mostly in the CM layer and between the two main
muscle layers (not shown). They did not make special contacts with
smooth muscle cells or ICC-AP. Within the AP region eosinophils were
mostly observed in close proximity to blood vessels. Mast cells were
rarely encountered in the muscle layers and no increase in mast cells
was observed during the period of inflammation and recovery.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The present study shows that the ICC-AP (especially the ICC-AP close to the blood vessels) were one of the first cells in the musculature to show structural damage after a mucosal infection. It is important to note that the observed injury to most ICC-AP did not involve all cellular organelles but primarily the cell membrane and filaments. Other structures such as the mitochondria remained the least affected. Any type of injury because of fixation will involve injury to mitochondria, plus the fact that damage was not observed in adjacent cells and control tissue, as well as the fact that most of the injured ICC-AP were close to the dilated blood vessels, ruling out that damage observed was related to fixation. Patchy damage primarily to ICC-AP processes will leave parts of the ICC network undisturbed, which makes slow-wave-driven peristalsis still possible.10 Indeed, the present study shows that the general network structure of ICC is still intact. The existence of a network structure has clear advantages. When parts of it are damaged, electrical activity may find its way around it. What EM reveals and what immunohistochemistry does not show is that ICC processes are damaged and that contacts between these ICC and smooth muscle cells and nerves are lost.
From day 3 onward, a dramatic change occurred in the ICC-AP as they displayed extensive proliferation of ribosomes and rER, and expansion of Golgi complexes in their cytoplasm. This synthesizing capacity is not only found in the perinuclear space, where normally only a few rER and Golgi complexes are found, but also in the larger processes as well as in the terminal processes where normally no rER nor Golgi complexes are found. An abundance of such terminal processes were found in the AP region 6 to 23 days after infection, where in the early days after infection many damaged processes were found. This indicates a regrowth of the terminal processes. In addition, lymphocytes made specialized contacts with smooth muscle cells and ICC-AP with marked synthetic activity and sometimes were seen to be enclosed by these cells. Importantly, no such intimate contacts were observed between eosinophils and smooth muscle cells or ICC-AP, hence the observed specialized contacts with lymphocytes can not be described as accidental. Such intimate contacts and increased synthetic activity support the concept of immunomodulation of intestinal smooth muscle function20-22 and indicate that ICC-AP might be an active participant in the immune process. Cells with a synthetic phenotype diminished in number between days 23 and 40 but even at day 60 after infection they were still present.
The most dominant immune cell involved was the macrophage that invaded both the AP region and the musculature. Macrophages entered the musculature after 24 hours and made close apposition contacts including peg-and-socket-like contacts with ICC-AP and smooth muscle cells. Macrophages were the most prominent immune cell not confined to the areas around blood vessels but distributed throughout the musculature, although a relatively high concentration was observed in the AP area with intimate contacts with the ICC-AP. Macrophages in the muscle layers of uninfected animals are classified as resident macrophages.11,15,23-26 Although all other immune cells disappeared from the musculature, many macrophages remained 60 days after infection, now concentrated in the AP region and even within the musculature where there were none before infection. Even after 60 days, the macrophages identified by EM were still activated. Whether or not after a longer time the number of resident, non-activated macrophages increases, remains to be seen.
In the early days after infection, macrophages showed little sign of
phagocytic activity, but made specialized contact with uninjured
ICC-AP, including peg-and-socket-like junctions. Such ICC-AP showed
markedly increased rER, hence it seems that macrophages may be
associated with the induction of rER possibly through secretion of
cytokines or growth factors. After day 6, macrophages showing signs of
phagocytic activity were seen associated with injured cells.
Recent studies on op/op mice may provide a hypothesis as to
the mechanism by which macrophages are involved in tissue injury.
Op/op mice have a mutation in the colony-stimulating
factor-1 (CSF-1) gene rendering this cytokine
nonfunctional.27
Op/op mice do not have
macrophages in the musculature28
except occasionally in
the subserosa, and on a T. spiralis infection, no
macrophages invade the musculature.29
This indicates that
the macrophages that are associated with ICC before and during
infection are CSF-1-dependent macrophages. CSF-1-dependent macrophages
are principally involved in cytokine secretion,30
and in
op/op mice, no production of interleukin-1ß within
the muscle layer (in contrast to control31
) was observed
in response to a Trichinella infection.29
Hence
it is likely that CSF-1-dependent resident macrophages and
CSF-1-dependent-invading macrophages become activated during a T.
spiralis infection, on which interleukin-1ß is released that in
turn initiated a cascade of events involving prostaglandins and tumor
necrosis factor-
, which causes injury to ICC, similar to the
development of cholinergic nerve dysfunction that also occurs in this
model.29
The present study shows that mast cells within
the musculature are not players in the immune response to T.
spiralis.
In several human motility disorders ICC are implicated based on abnormal numbers as observed at the light microscopy level using c-Kit immunohistochemistry but few data are available on the ultrastructural pathology of ICC.9,32 Rumessen33 examined tissues from patients with advanced ulcerative colitis. He showed that ICC at the colonic submuscular border displayed specific abnormalities, whereas the nearby smooth muscle cells appeared normal. The typical changes found in ICC were: occurrence of multiple secondary lysosomes, the presence of large lipid vacuoles, as well as margination and clumping of intermediate filaments—structural signs of severe cellular injury. No structural signs of recovery of ICC were found. In children with infantile hypertrophic pyloric stenosis, ICC were not present in sections of CM, although there were cells with ultrastructural characteristics of underdeveloped ICC.34 Acid-induced inflammation in the dog colon also affected ICC, suggesting that also in the colon, ICC damage may underlie motor abnormalities.35 In a study on Hirschsprung’s disease, ICC were found to be markedly reduced in number by immunohistochemistry and EM showed that the remaining ICC had normal ultrastructural features.36 The present study describes the ultrastructural pathology of ICC associated with AP that serve as pacemaker cells of the small intestine. It is shown that a mucosal infection induces immediate inflammation in the musculature. Within 24 hours, macrophages enter the musculature and ICC-AP processes are structurally damaged. This can be demonstrated ultrastructurally but not by using c-Kit immunohistochemistry. Damage to ICC is correlated with disturbed slow-wave activity and disrupted peristaltic motor patterns.10
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Acknowledgements |
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Footnotes |
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Supported by grants from the Canadian Institutes of Health Research and a Canadian Association of Gastroenterologists fellowship from Janssen-Ortho (to X.-Y. Wang).
Accepted for publication January 9, 2002.
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References |
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  R. Metzger, U. Rolle, H. C Fiegel, F. E Franke, K. Muenstedt, and H. Till C-kit receptor in the human vas deferens: distinction of mast cells, interstitial cells and interepithelial cells Reproduction, March 1, 2008; 135(3): 377 - 384. [Abstract] [Full Text] [PDF]
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