- Partial obstruction of the small intestine in mice resulted in distance-dependent disruption of interstitial cells of Cajal (ICC) networks, with number of proliferating ICC appearing to be lower closest to the occlusion. - Proliferating ICC were unexpectedly low 10mm from the occlusion. - This study aims to improve quantification methods of ICC networks and examine proliferation as a potential factor in ICC network depletion using a mouse model of partial small intestine obstruction.

1. Use of a small intestinal Partial Obstruction Mouse model to
study Interstitial cells of Cajal networks
© 2013 Mayo Foundation for Medical Education and Research
Kayleigh Kresse1, Jerry Gao2, Seth Eisenman, Simon J Gibbons, Gianrico Farrugia,
Grinnell College1, Auckland Bioengineering Institute2, Division of Gastroenterology and Hepatology, Department of Physiology and
Biophysics and Center for Individualized Medicine
Mayo Clinic, Rochester, MN
• Partial obstruction of the small intestine resulted in a distance
dependent disruption of the ICC networks
• Number of proliferating ICC’s appear to be less in the area
closest to the occlusion
• Proliferating ICC’s were unexpectedly low 10mm aboral of the
occlusion.
Summary
• This study provides more evidence that the partial obstruction
model is a good model to study for disruption of ICC networks
• Loss of proliferation may be contributing to the ICC loss
• In this study, the clip used for obstruction did not result in visible
distension of the intestine. H&E staining is still in progress to
confirm that the muscle layer was morphologically altered.
Statistical analyses to confirm results also in progress.
• These data need to be expanded and the networks used to
create automated ways to analyze the data.
Conclusions
1. Thomsen L, Robinson TL, Lee JC, Farraway LA, Hughes MJ, Andrews
DW, Huizinga JD (1998) Interstitial cells of Cajal generate a rhythmic
pacemaker current. Nat Med 4:848-851.
2. He CL, Soffer EE, Ferris CD, Walsh RM, Szurszewski JH, Farrugia G
(2001) Loss of interstitial cells of Cajal and inhibitory innervation in
insulin-dependent diabetes. Gastroenterology 121:427-434
3. He CL, Burgart L, Wang L, Pemberton J, Young-Fadok T, Szurszewski
J, Farrugia G (2000) Decreased interstitial cell of Cajal volume in
patients with slow-transit constipation. Gastroenterology 118:14-21
4. Feldstein AE, Miller SM, El-Youssef M, Rodeberg D, Lindor NM,
Burgart LJ, Szurszewski JH, Farrugia G (2003) Chronic intestinal
pseudoobstruction associated with altered interstitial cells of Cajal
networks. J Pediatr Gastroenterol Nutr 36: 492-497
5. Gao J, O’Grady G, Archer R, Farrugia G, Gibbons SJ, Cheng LK
(2013) Numerical metrics for automated quantification of interstitial
cells of Cajal network structural properties.
References
Interstitial Cells of Cajal (ICC) are cells located
within the smooth muscle tissue of the
gastrointestinal tract. ICC interact with enteric nerve
cells and smooth muscle cells to control
gastrointestinal motility. They function as a network
of electrical pacemaker cells [1] that transmit a
series of slow wave signals to smooth muscle,
regulating smooth muscle function and initiating
contractile movement. Loss of ICC is an area of
great interest as loss of ICC is associated with
several gastrointestinal motility disorders such as
diabetic gastroparesis [2], slow transit constipation
[3], and intestinal pseudo-obstruction [4]. There are
two types of ICC in the small intestine: those found
between the circular and longitudinal muscle layers
associated with the myenteric plexus (ICC-MY) and
those found in the inner circular muscle layer
associated with the deep muscular plexus (ICC-
DMP).
How ICC are lost in motility disorders and the
consequences of alterations in ICC networks on
contractile activity are not well understood.
Numerical metrics have recently been formulated
and used to quantitatively determine jejunal ICC
network changes in mice with decreased (5-HT2B
receptor knockout (KO)) and normal (Ano1 KO) ICC
numbers [5]. However, quantification of ICC
networks remains difficult and mostly subjective.
Background
• To improve methods in quantifying ICC networks.
• To study proliferation as potential factor in the
depletion of ICC networks.
• To examine ICC networks with regard to the
following metrics: density, thickness, size of ICC-
negative spaces within the network, amount of
contact between ICC and non-ICC, connectivity,
and anisotropy, all of which are variables involved
in determining the phenotypes of depleted ICC
networks in order to determine better ways to
quantify ICC network changes.
Objectives
Results
Animal Care & Surgery
Experiments were performed with
approval from the Institutional Animal
Care and Use Committee (IACUC) of the
Mayo Clinic.
• We used fully weaned female BALB/c
mice (n=4) aged between 4-6 wks
• To create a model of ICC loss, we
placed a polyethylene clip 3mm in
internal diameter over the small intestine
15-25mm oral to the ileocecal valve
• Mice were fed a soft diet to prevent total obstruction
• Mice were killed by CO2 inhalation 7 days after surgery
Whole Mount Preparation and Immunohistochemistry
• Tissue was obtained from:
10mm distal to the obstruction
and 2, 25, and 76mm proximal
to the obstruction
Whole mount labeling
procedures were done at 4 °C
and tissues were washed with
cold phosphate buffered saline
(PBS) between each step.
• Fixation in 4% paraformaldehyde in phosphate buffer
• Incubation in 1% Bovine Serum Albumin (BSA) and 0.3%
Triton-X-100 in PBS to minimize non-specific binding
• Overnight incubation in goat anti-c-Kit antibody and
rabbit anti-Ki67, a proliferation marker, in blocking solution
• Overnight incubation with secondary antibodies, donkey
anti-goat IgG conjugated to Cy3 and donkey anti-rabbit
IgG conjugated to FITC in the dark
• 30 min incubation in 4’, 6-diamidino-2-phenylindole
(DAPI) as a nuclear counter stain.
• Tissues mounted with SlowFade® Gold Antifade
Reagent with DAPI
Image acquisition
Images were collected by laser scanning confocal
microscopy (Olympus FV 1000) with a 60X 1.2 NA water
immersion objective.
ICC networks were assessed by 2 independent blinded
reviewers.
Methods
Figure 2. Mouse gut
7 days post-surgery
Figure 3. Dissected ileum &
distal jejunum 7 days post-
surgery
• Many thanks to the Mayo Clinic SURF program and the Mayo
Clinic Laboratories, Gary Stoltz, Lei Sha, Kmu Choi, Cheryl
Bernard, and Kristy Zodrow.
Acknowledgements
Longitudinal Muscle
Circular Muscle
ICC-MY
ICC-DMP
Figure 1. Mouse small intestine muscularis propria
Figures
Figure 4. (a) Healthy ICC
network (red) and
proliferating cells (green)
10mm aboral of occlusion.
(b) Very faint ICC network
(red) and proliferating cells
(green) 2mm oral of
occlusion.
a ba
4.22083
0
1
2
3
4
5
6
7
8
9
10
10mm Aboral
Network
Quality
(0-poor,
10-excellent)
10mm Aboral
0.08333
0
0.5
1
1.5
2
10mm Aboral
Ki-67-
positive ICC/
60X Field
0.33333
0
0.5
1
1.5
2
2mm Oral
Ki-67-
positive
ICC/ 60X
Field
2mm Oral
2.6375
0
1
2
3
4
5
6
7
8
9
10
2mm Oral
Network
Quality
(0-poor,
10-excellent)
0.58333
0
0.5
1
1.5
2
25mm Oral
Ki-67-
positive
ICC/ 60X
Field
25mm Oral
3.87917
0
1
2
3
4
5
6
7
8
9
10
25mm Oral
Network
Quality
(0-poor,
10-excellent)
1
0
0.5
1
1.5
2
76mm Oral
Ki-67-
positive
ICC/ 60X
Field
76mm Oral
4.14583
0
1
2
3
4
5
6
7
8
9
10
76mm Oral
Network
Quality (0-
poor, 10-
excellent)