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Published in final edited form as: Neurogastroenterol Motil. 2010 Aug 19;23(1):36–44. doi: 10.1111/j.1365-2982.2010.01590.x

Changes in Interstitial Cells of Cajal with Age in the Human Stomach and Colon

Pedro J Gomez-Pinilla 1, Simon J Gibbons 1, Michael G Sarr 3, Michael L Kendrick 3, K Robert Shen 3, Robert R Cima 3, Eric J Dozois 3, David W Larson 3, Tamas Ordog 1, Maria J Pozo 2, Gianrico Farrugia 1
PMCID: PMC2999641  NIHMSID: NIHMS254123  PMID: 20723073

Abstract

Background

Aging produces inevitable changes in function of most organs including the gastrointestinal tract. Together with enteric nerves and smooth muscle cells, interstitial cells of Cajal (ICC) play a key role in control of gastrointestinal motility, yet little is known about the effect of aging on ICC. Our aim was to determine the effect of aging on ICC number and volume in the human stomach and colon.

Methods

Gastric and colonic tissues from patients age 25–70 and 36–92 years old respectively and with no co-existent motility disorders were immunolabeled with an anti-Kit antibody and ICC counted in the circular muscle and myenteric regions. Network volumes were measured using 3D reconstructions of confocal stacks. Effects of age were determined by testing for linear trends using regression analysis.

Key Results

In both stomach and colon, the number of ICC bodies and volume significantly decreased with age at a rate of 13% per decade of life. ICC size was only affected in the myenteric plexus in the colon. The changes associated with age were not differentially affected by sex or colonic region.

Conclusions and Inferences

The number and volume of ICC networks in the normal human stomach and colon decline with age. This decrease in ICC likely reduces the functional capacity of the gastrointestinal motor apparatus, may contribute to changes in gastrointestinal motility with aging and may influence intestinal responses to insults such as disease, operative interventions and medications in older patients. Tissue specimens must be carefully age-matched when studying ICC in disease.

Keywords: healthy aging, motility, myenteric plexus

INTRODUCTION

Aging of our population is one of the most distinctive recent demographic events. The structure and function of organs change with age and these changes are reflected in the increase in the prevalence of certain diseases associated with aging, such as hypertension.1 In the gastrointestinal tract, gastroesophageal reflux, irritable bowel syndrome, constipation, and fecal incontinence are all more prevalent with age.2–4 There are aging associated changes in the gastrointestinal tract, such as the ability to eat large boluses of food that decrease with age 5 and gastrointestinal function may decline in the elderly even in the absence of an overt motility disorder. These changes may negatively affect general well-being and even morbidity in older people.6 Nevertheless, for the most part, the gastrointestinal function remains relatively well preserved with age. There appears to be a decrease in gastric emptying with age, but the magnitude of change is still controversial (review in 7, 8). While it is well known that constipation is a common problem in older people,4, 9, 10 the effect of age on colonic transit is unclear with both a slowing of, and no change in transit reported.11, 12

Gut function is controlled by a dynamic interaction between different cell types, including epithelia that form the mucosal barrier, immune cells, smooth muscle, neurons, glia, and interstitial cells of Cajal (ICC). Gastrointestinal motility requires the normal distribution and function of ICC. Indeed, several human gastrointestinal motility disorders have been associated with depletion of ICC.13 In spite of the role that ICC play in the control of motility, no data address the effect of age on ICC number and network volumes. Careful characterization of age-related changes in humans without overt motility disorders is critical for proper evaluation and understanding of pathologic changes in diseases, especially those that affect preferentially elderly people. Here we show that in both human stomach and colon, there is a marked decrease in the number of ICC with age, and this decrease is accompanied by an overall decrease in ICC network volume. This decrease with age was independent of sex and region and may contribute to any changes in gastrointestinal motor activity associated with aging.

MATERIALS AND METHODS

Human tissues

Collection and use of tissues was approved by the Mayo Clinic Institutional Review Board. Human gastric mid-body tissue was obtained from 20 female patients aged 25–70 years undergoing bariatric surgery for morbid obesity. Colonic tissue was obtained from colectomies carried out for non-obstructing cancer. We prospectively collected 23 colonic tissues from patients aged 36–92 years old (fourteen male, nine female, Table 1).The patients’ ages were normally distributed across the range for all tissues. None of the colon tissues came from patients with familial adenomatous polyposis or inflammatory bowel disease. Studies on these tissues have not been previously reported. In order to blind the investigator performing data acquisition and analysis to the age of the patients, the slides and images were mixed and assigned codes unknown to the investigator prior to counting.

Table 1.

Details of colon samples studied

Age Sex Region
36 F Sigmoid
39 M Transverse
40 F Ascending
40 M Sigmoid
41 M Ascending
49 M Sigmoid
50 M Ascending
53 M Ascending
53 M Sigmoid
57 F Sigmoid
58 M Ascending
60 F Sigmoid
62 F Ascending
68 M Descending
69 M Sigmoid
72 F Ascending
72 F Ascending
73 M Ascending
79 M Ascending
82 F Sigmoid
83 F Ascending
83 M Ascending
92 M Ascending
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Immunocytochemistry

Tissues were obtained in the operating room to minimize ischemic time, transferred in F12 medium over ice to the laboratory and fixed overnight in 4% paraformaldehyde in 0.1 mol L−1 phosphate buffer (pH 7.2). The tissue was washed 4 × 15 min with 0.1 mol L−1 phosphate buffered saline (PBS, pH 7.2) then incubated in 30% sucrose in PBS overnight. Tissue was stored at −80 C until needed. For colonic tissue, the tissue was obtained at least 5 cm from the tumor. There was no tumor present in the sections examined. Multiple, 12 µm sections were cut from each tissue block and the studies were done on non-adjacent sections (each > 60 µm apart).

Cryostat sections from frozen tissue were studied. Sections were warmed to room temperature, rinsed with PBS then blocked for 2 h in 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) in PBS. The sections were incubated overnight at 4°C with mouse monoclonal antibodies against Kit (0.5 µg/ml in 0.3% Triton X-100, 1% BSA, PBS, LabVision, Fremont, CA). After washing, tissues were incubated with Cy3-donkey, anti-mouse secondaries (3.5 µg/ml in Triton/BSA/PBS, Jackson ImmunoResearch, West Grove, PA), washed, and counterstained with 4',6-diamidino-2-phenylindole dilactate (DAPI dilactate, Invitrogen, Carlsbad, CA) to label nuclei.

Quantification of ICC bodies

ICC bodies, defined as Kit-positive structures surrounding a DAPI-positive nucleus, were counted in the circular muscle for the stomach and colon and for colon in the myenteric region. Human stomach does not have a well-defined myenteric ICC network. Mast cells, identified by their round or oval shape and lack of processes, were excluded from the counts. Intramuscular ICC bodies in 13 fields per slide from three non-adjacent sections were counted using a 40× (NA 0.75) air objective (Olympus America, Center Valley, PA) on a BX51WI microscope (Olympus). Fields were 390 × 314 µm (0.12 mm2). Myenteric ICC in the colon were examined in 5 fields per slide from three nonadjacent sections. Because myenteric ICC networks are relatively thin, we used a smaller field (268 × 134 µm; 0.035 mm2) to ensure that counts were not contaminated by intramuscular ICC. These images were obtained by using a 60× (NA 1.2) Olympus objective mounted on a confocal microscope (Olympus FV1000). Fields were randomly selected to sample the full area of the region under study on each slide.

Determination of Kit-positive ICC network volumes

Immunostained tissues were examined with a 60× (NA 1.2) objective (Olympus) on the confocal microscope (Olympus). For all regions, 15 confocal stacks (5 per slide and per region) were taken, with a Z-axis step of 0.49 µm and XY dimensions of 211 × 211 µm and 268 × 134 µm for the circular muscle and myenteric regions respectively. Fluorescence was visualized using a 543 nm laser with an emission bandwidth of 575–630 nm.

The volume occupied by Kit-positive ICC networks was calculated from the confocal stacks using ANALYZE™ (Mayo Foundation, Rochester, MN). The images were volume rendered in 3 dimensions and thresholded to remove background noise. The ICC volume was determined from the number of Kit-positive voxels in bitmaps of the positively labeled structures. . Mast cells were excluded from this quantification.

Data analysis

Data are presented as mean ± SEM. ICC bodies/field are given as bodies/0.122 mm2 or bodies/0.035 mm2 for the circular muscle and myenteric regions respectively. ICC volumes are presented as percentage of Kit-positive voxels per total volume of the stack. The ratio of volume (number of Kit-positive voxels/number of cell bodies) was used to define ICC size. Data were plotted and analyzed using Prism 4 (GraphPad software Inc., San Diego, CA) using iterative linear regression that minimizes the sum-of-squares. To determine effect of sex and colonic region, the slope of regression lines were compared. P values of < 0.05 were considered statistically significant.

RESULTS

Kit-immunoreactivity

The distribution of Kit-positive cells had the characteristic pattern of ICC and mast cells in human stomach and colon.14 Mast cells were identifiable by their size, round shape and lack of processes. Human gastric intramuscular ICC are found scattered in both muscle layers and were more prominent in the circular muscle, while an ICC-myenteric network was patchy and not well defined. In human colon, isolated intramuscular ICC were present in the longitudinal and circular muscle layers including in septa. In the muscle layers, Kit-positive cells were oriented parallel to the long axis of the myocytes and had branching processes running out from the cell body. ICC also formed distinct networks in the myenteric and submucosal regions.

Effect of age on ICC number

Representative images from the circular muscle of the stomach of two female patients are shown in Figure 1A. A total of 3771 intramuscular ICC from 780 fields from 20 patients were counted in the stomach. The mean ICC bodies per field was 4.9±0.04. The youngest patient (25 yr old) had 6.8±0.7 ICC bodies per field while the oldest (70 yr old) had 2.97±0.4 ICC bodies per field. Figure 1C shows all the data, showing that aging resulted in a decrease in number of intramuscular ICC with a slope of −0.88 ICC bodies/field/10 years (r2 = 0.55, P = 0.0005 and n = 20 female patients). This represents a 12.9 % decrease in ICC per 10 years of life after age 25.

Figure 1.

Figure 1

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ICC numbers and volumes decrease with age in the circular muscle layer of the human stomach. Panel A shows Kit-immunoreactivity (red pseudocolor) in representative fields (0.122 mm2) from a 25 yr old female (left) and a 70 yr old female (right). DAPI-stained nuclei are shown in blue pseudocolor. Round Kit-positive cells with mast cell morphology (arrowheads) were not counted or included in the volume reconstructions. Panel B represents 3-D object maps (0.044 mm2) of Kit-immunoreactivity in the gastric circular muscle from a 30 yr old female (left) and a 58 yr old female (right). Note decreased numbers of ICC in the older subjects. Panel C shows age-related changes in the number of ICC bodies per field. Each point is the mean ± SEM of 39 fields observed. Some error bars are smaller than the symbol size. Regression line (solid) and limits of the 95% confidence interval (dotted) are shown. Panel D represents age-related changes in the volume of intramuscular ICC. Note significant decline in ICC numbers and volumes. Scale bar, 50 µm.

A similar finding was present in human colon. Figure 2A shows representative images of Kit-positive circular muscle ICC of human colon from a 36 yr old female and a 79 yr old male. A total of 2894 ICC bodies were counted in 897 fields from 23 tissue specimens. In circular muscle the mean of ICC bodies per field was 3.2±0.1. Figure 2C shows that the youngest patient (36 yr old female) had 5.5±0.4 ICC bodies per field while the oldest one (92 yr old male) had 1.3±0.2 ICC bodies per field. The number of ICC in the circular muscle layer declined with age, with a slope of −0.69 ICC bodies/field/10 years, that is a 12.6% decrease per 10 yrs of age after the age of 36 (r2 = 0.55, P < 0.0001, n= 23 patients, solid black line, Figure 2C). Plotting the data separately for males (n=14) and females (n=9) showed no significant differences in the regression line by sex (−0.87 female vs −0.57 males ICC bodies/field/10 years, P=0.31, Figure 2C).

Figure 2.

Figure 2

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Aging is associated with decrease independent of sex in the number of ICC in the circular muscle layer and myenteric plexus region of the human colon. Panel A shows Kit immunoreactivity (red pseudocolor) in representative fields (0.122 mm2) in the circular muscle layer from a 36 yr old female (left) and a 79 yr old male subject (right). Panel B shows Kit immunoreactivity in representative fields (0.035 mm2) in the myenteric plexus region from a 49 yr old male (left) and a 83 yr old female patient (right). DAPI-stained nuclei are shown in blue pseudocolor. CM, circular muscle; LM, longitudinal muscle. Note decreased ICC numbers in both regions in the older patients. Panel C and D summarize the age-related changes in the number of ICC bodies per field in the circular muscle and the myenteric region, respectively. Each point represents the mean ± SEM of 39 (circular muscle) or 15 (myenteric region) fields observed. Some error bars are smaller than the symbol size. Open and closed symbols depict data from female and male subjects, respectively. Black solid lines are regression lines fitted to all data; dotted lines delimit the 95% confidence interval. Red and blue solid lines are regression lines fitted to data from female and male patients, respectively. The number of Kit-positive structures per field decreased significantly with age in both the circular muscle layer and the myenteric region. Scale bar, 50 µm for all images.

In human colon, ICC form a distinct network in the myenteric region.14 In the myenteric region, a total of 1846 ICC bodies in 345 fields from 23 specimens of different ages were counted. The mean number of ICC bodies per field was 5.3±0.1. Note that a smaller field size was used when counting ICC in the myenteric region compared to the circular muscle. Taking field size into account, the density of ICC in the myenteric region was 5-fold higher than the density of ICC in the circular muscle. As Figure 2D shows, the youngest patient (36 yr old female) had 7.9 ± 0.9 ICC bodies per field and the oldest one (92 yr old male) 3.1 ± 0.2 ICC bodies per field. Plotting the numbers of ICC bodies per field against age showed a decrease (slope −0.9 ICC bodies/field/10 years, r2 = 0.77, P < 0.0001, solid black line, n= 23 patients, Figure 2D) signifying an 11.5% loss per 10 yrs of age. Similarly to the circular muscle there was no difference in the slope of the regression lines when analyzed by sex of the patient (−0.87 female vs −0.93 male ICC bodies/field/10 years, n= 9 and 14 respectively, P= 0.77, Figure 2D).

Effect of age on ICC network volume

Figure 1B shows representative object maps of intramuscular ICC in the circular muscle of human stomach. Kit-positive ICC volume for the youngest patient was 1.03 % of the total stack volume while for the oldest it was 0.15%. The impact of age on the ICC network volume was a decrease of 85% in 45 years due to a decrease of 15.5% per 10 years. (r2 = 0.70, P < 0.0001; n = 20 female patients, Figure 1D). A representative set of object maps corresponding to the circular muscle and myenteric regions in the human colon are shown in Figures 3A and B respectively. ICC network volumes for the youngest patient were 1.0±0.3% (circular) and 1.3±0.05% (myenteric region) of the total stack volume while for the tissue from the oldest individual the network volumes were 0.19 ± 0.01% (circular) and 0.2 ± 0.01% (myenteric region) of total stack volume. Figures 3C and D show a significant decrease with age in ICC network volume for both regions of the colon tissues. The slope for circular muscle was −0.17% of the total stack volume/10 years, resulting in an 81% decrease over 56 years or 17%/10 years for circular and 18%/10 years for the myenteric region. (P= 0.0008 and P= 0.0006, r2 = 0.42 and 0.43, respectively n= 23 patients, Figure 3C and D). Similarly to the ICC count, there was no sex difference (circular muscle; female: −0.19% of the total stack volume/field/10 years; male: −0.15% of the total stack volume/field/10 years, n= 9 and 14, respectively, P= 0.70; myenteric plexus region; female: −0.30% of the total stack volume/field/10 years; male: −0.21% of the total stack volume/field/10 years, n= 9 and 14 respectively, P= 0.48; Figure 3C and D).

Figure 3.

Figure 3

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ICC volumes decrease with age in the circular muscle layer and myenteric plexus region of the human colon. Panels A and B show representative 3-D object maps of Kit-immunoreactivity in the circular muscle layer and myenteric plexus region, respectively, from a 36 yr old (left) and a 82 yr old (right) female subject. CM, circular muscle; LM, longitudinal muscle. Panels C and D summarize age-dependent changes in ICC volumes expressed as percentage of total stack volume in the circular muscle layer and myenteric plexus region, respectively (n=23 patients). Each point is the mean ± SEM of 15 stacks acquired. Open and closed symbols depict data from female and male subjects, respectively. Solid lines are regression lines fitted to all data; dotted lines delimit the 95% confidence interval. ICC volumes were not significantly different by sex. Scale bar, 50 µm.

Ascending versus sigmoid colon

In this study, we used 12 tissues from ascending and 10 from sigmoid colon (Table 1). Changes in ICC number with age were not different when the data from the two colonic regions were compared. Changes in circular muscle ICC network volumes with age were also not different between the ascending and sigmoid colon. In contrast, the volume of the Kit-positive ICC networks declined more quickly with age in the myenteric region of the ascending colon compared to the same region of the sigmoid colon (−0.39 ascending vs −0.14 sigmoid in % of the total stack volume/10 years, equivalent to a reduction of 16.4% and 10.7% of the volume present in the youngest patient per 10 years, n= 12 and 10 respectively, P= 0.026).

Effect of age on ICC size

The mean individual ICC size for all patients and for both stomach and colon are shown in Figure 4. For gastric circular muscle the slope of the regression line was not different from zero (P = 0.18, Figure 4A), and a similar result was obtained for colonic intramuscular ICC (P = 0.27, Figure 4B) suggesting that aging was not associated with a change in ICC size. For the colonic myenteric region, however, there was a borderline decrease in ICC size with age with a slope of −801 voxels/ICC/10 years representing a decrease of 53% over 56 years and a mean decrease of ICC size of 11.4 % per 10 years (r2 = 0.20, P= 0.048, n=23 patients, Figure 4C). Colonic ICC size was independent of sex at any age (P= 0.15 and 0.81 for circular muscle and myenteric region respectively).

Figure 4.

Figure 4

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Effects of aging on ICC size. ICC size was calculated by dividing the Kit-positive volume in each stack by the number of ICC bodies within the same stack. Age-related changes in ICC size in the circular muscle of the stomach (A), the circular muscle of the colon (B) and the myenteric plexus region of the colon (C) are shown. A declining trend was only found in the myenteric region of the colon. In panels B and C, data from both males and females were included in the regression analysis.

DISCUSSION

The main finding of this report is the marked loss of ICC number and network volume in normal human stomach and colon with age. These decreases are substantial and occur in both men and women. The rate of decline is similar in colon and stomach, with a decrease of 55% over 45 years for ICC numbers in stomach and 60% over 56 years for colon. The decline in ICC occurred at similar rates in the myenteric region and the circular muscle of colon and in both ascending and sigmoid colon.

These observations are important in several respects. Particularly, decreases in ICC were not associated with clear indications of abnormal gastrointestinal motility. While the elderly may attribute changes in gastrointestinal function to ‘normal aging’ and underreport symptoms none of the tissues used here came from patients with gastrointestinal symptoms clinically important enough to be notified to the health care provider. The stomach tissues came from obese patients. However it is unlikely that changes in ICC with age were a function of patient weight. Similar changes were observed for ICC in colon and previous examinations of ICC numbers in obese and normal weight patients identified no such differences (unpublished observations). Although the patient cohort covered ages 25 to 70 for stomach tissues and 36 to 92 for colon tissues, there were no increases in the size of the processes of the remaining cells to compensate for the decrease in cell numbers. In fact, there was a small but significant decrease with age in average Kit positive volume per ICC cell body (ICC size) in ICC from the myenteric region of the colon. Thus, there may be a functional reserve of ICC in young healthy gastrointestinal tracts, and with aging, this reserve is depleted steadily. The size of the reserve is not known, but it is notable that in a preliminary study,15 two groups of individuals with gastroparesis were detected. Some patients with gastroparesis had normal ICC numbers in the gastric body, but other patients with gastroparesis had distinctly fewer ICC, and the numbers of ICC (≤ 3 cells per field) were all lower than those in the stomach of the oldest healthy specimens studied for this current work. This observation suggests that 3 ICC per field (0.12 mm2 in a 12 µm section) represents the lower limit of ICC numbers for normal function in the gastric body.

With the exception of enteric nerves, there are few systematic reports that have assessed changes with age in the cell types that contribute to normal motility. There have been no published studies on changes in ICC in aging experimental animals. Enteric nerves decrease in number with age.16–24 We reported studies on a separate cohort of tissues showing a 38% decrease in myenteric neurons between ages 30 and 60 with selective sparing of neurons positive for nitric oxide synthase.17 There was no significant change in the density of PGP 9.5 immunoreactive volumes with age, suggesting that the remaining neurons in the tissue had increased in size. This finding contrasts with the observed decrease in volume of Kit positive ICC structures in the present study and the reduced average size of myenteric ICC from the colon of older patients.

The presence of an apparent surplus of ICC in younger tissues is consistent with a greater ability to sustain function after injury in those young patients. Younger people may consequently be less likely to experience motility problems after insults that are known to damage ICC, including obstruction or pseudo-obstruction,25, 26 diabetes 27–31 and after infections from, for example intestinal parasites.32 This resilience of young networks makes identifying the initial cause of ICC related motility disorders difficult because an early insult can deplete networks but not decrease them to a level of causing dysfunction. Only after a period of aging will ICC numbers decline to the point that motility is impaired, and the disorder becomes apparent. In older patients, gastrointestinal motility problems may be more common because of the impact of earlier injuries on ICC networks that become manifest with aging but also because there is little reserve to buffer against further injury. Dysfunctions develop typically when an organ’s capacity to function normally no longer matches demand. For example, reduced ICC in the stomach may allow processing of smaller meals but not processing of normal portions and such a mismatch may lead to early satiety and consequently the moderately decreased food intake that occurs almost uniformly in the elderly.6, 33 Under-nutrition may contribute to the development and progression of chronic diseases and increase morbidity and mortality.6

Another conclusion of this study is that samples from diseased tissues need to be closely age-matched with appropriate controls. The observed decrease in ICC of greater than 15% for every decade in healthy individuals could easily obscure a real change or result in a false correlation depending on how the diseased and control groups are chosen. It is not likely that the decline in ICC numbers in the colon is due to stretching of tissue from older patients as these tissues were handled in the same way as tissues used for a previous study.17 In that study a variety of measurements changed in different ways that were not correlated with any possible changes in the tissue dimensions.

The underlying mechanism for the decrease in ICC networks with age is not known. This decrease was not limited to intramuscular or myenteric ICC. We did not quantify ICC from the sub-muscular region of the colon because the low density of those cells meant we did not have a sufficient sample size to detect changes. The decreases were also not restricted a colonic region, with both ascending and sigmoid colon affected equally, suggesting that dietary causes may not be a dominant factor given the widespread and equal changes. It is appreciated increasingly that ICC networks turn over throughout life and that a balance exists between processes for repairing and maintaining ICC networks and the processes that lead to de-differentiation of or clearance of dead and damaged ICC.13 In adult mice, proliferation of ICC has been detected,34, 35 and this proliferation may decline with age. Replacement of ICC from CD34+/CD44+, weakly Kit+ progenitor cells 36 may also decrease as these stem cells become less effective at self-renewal and differentiation with age.37 Similarly it is not known whether rates of cell death increase with age due to processes, such as oxidative injury and apoptosis, both of which are known to contribute to loss of ICC.27, 38

While all the stomach tissues were from female patients, we had the opportunity to study the effect of sex on colonic ICC. Two findings bear highlighting. Firstly, there were no significant differences in ICC numbers and network volumes between females and males at any age. While it is possible that there may be different susceptibilities to loss of ICC in males or females, a lower start-off point for females does not appear to underlie the increased predominance of constipation in females.39, 40 Secondly, the rate of loss of ICC with age in colon is equal for both females and males. While we did not have sufficient power to analyze separately different age groups, the distribution of the data suggests a leveling off of ICC loss at about age 65. This leveling off may represent a floor effect, but given that it occurs for both sexes and there is no acceleration of loss of ICC at this point, it argues against a sexual hormonal component contributing to the ICC loss.

This study used Kit to label ICC. Kit is a well accepted marker for ICC with the caveat that mast cells are also Kit positive and must be excluded from the analysis based on morphology. Ano1 has recently been identified as specifically expressed on ICC 41 and immunolabeling for Ano1 can be used to quantify ICC. We examined Ano1 immunoreactivity in colonic and gastric samples from the 4 oldest and 4 youngest patients in the two cohorts. Total numbers of Ano1 positive ICC were reduced in the tissues from older patients but differences in the small numbers of a group of Ano1 positive, Kit-dim cells were not seen (data not shown). In summary, ICC networks decline significantly with age. The changes occur in both stomach and colon and in both myenteric region and intramuscular ICC. This decrease in ICC numbers may contribute to changes in gastrointestinal motility associated with aging and may make the elderly more susceptible to acute insults that result in changes in ICC number.

Acknowledgements

We thank Kristy Zodrow for secretarial assistance and Peter Strege for technical assistance. Grant Support: Supported by a research grant from Novartis and by DK57061, DK68055, and P30DK84567. Maria J. Pozo is supported by grant BFU2007-60563 from the Spanish MEC and RETICEF (RD060013/1012). Pedro J. Gomez-Pinilla is funded by the Spanish MEC and FECYT (2007-0637). Tamas Ordog is supported by DK58185. Pedro J. Gomez-Pinilla performed the research. Pedro J. Gomez-Pinilla, Maria J. Pozo, Tamas Ordog, Gianrico Farrugia and Simon J. Gibbons designed the research study and wrote the paper. Pedro J. Gomez-Pinilla and Simon J. Gibbons analyzed the data. Michael Sarr, Michael Kendrick, K. Robert Shen, Robert Cima, Eric Dozois and David Larson contributed essential reagents and tools and critically reviewed the paper.

Footnotes

Disclosures

Competing interests: The authors have no competing interests.

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