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- 1. ©2013 MFMER |slide-1 Alterations in Networks of Interstitial Cells of Cajal Following Partial Obstruction in the Mouse Kayleigh Kresse Grinnell College, Grinnell, IA July 30, 2013
- 2. ©2013 MFMER |slide-2 Introduction • Interstitial Cells of Cajal (ICC) • Pacemaker cells of the gastrointestinal tract Longitudinal Muscle Circular Muscle ICC-MY ICC-DMP Mouse small intestine muscularis propria Electrical slow wave recordings from ICC and SMC in mouse small intestine Lees-Green et.al. (2011)
- 3. ©2013 MFMER |slide-3 Depleted ICC-MY networks in human jejunum Diabetic Gastroparesis Depleted ICC networks in mouse small intestine Partial Obstruction Depleted ICC networks in human colon Slow Transit Constipation Depleted ICC-MY networks in human colon Pseudo-obstruction Introduction • Interstitial Cells of Cajal (ICC) • Associated with gastrointestinal motility disorders Feldstein et.al. (2003), He et.al. (2000 & 2001), Chang et.al (2001),
- 4. ©2013 MFMER |slide-4 Aims • Improve methods in quantifying ICC networks • Determine how networks are altered before, during, and after partial obstruction • Assess changes to ICC proliferation in response to obstruction and after recovery • Density • Thickness • Size of ICC-negative spaces • Contact between ICC and non-ICC • Connectivity • Anisotropy
- 5. ©2013 MFMER |slide-5 Methods • Female BALB/c mice (n=4), 4-6 wks old • Laparotomy • Polyethylene clip (3.4 mm OD, 2.4 mm ID, 5 mm length) placed over intestine 15-25 mm oral to the ileocaecal valve Figure 7. Mouse gut 7 days post-surgery
- 6. ©2013 MFMER |slide-6 Methods • Killed 7 days post-surgery, ileum and distal jejunum dissected out • Fixed and incubated in antibodies against c-Kit (ICC), Ki67 (proliferation), and DAPI (nuclei) • Confocal microscopy • 60X 1.2 NA water immersion objective • 10mm aboral; 2, 25, and 76mm oral • 3 regions each: right of mesentery, left of mesentery, midline Figure 8. Dissected ileum/distal jejunum 7 days post-surgery
- 7. ©2013 MFMER |slide-7 Results Ki67-positive ICC Count and Network Quality Score 0.25 4.85 3.512 5 4.3 0 2 4 6 0 1 2 10mm Aboral 0.5 0.5 2.9 2.225 2.787 5 0 2 4 6 0 1 2 2mm Oral 0.5 0.5 0.75 2.937 5 4.325 4.375 0 2 4 6 0 1 2 25mm Oral 1.0 0.75 1.25 6.163 3.8 2.48 0 2 4 6 0 1 2 76mm Oral Oral Aboral
- 8. ©2013 MFMER |slide-8 0.25 4.85 3.5125 4.3 0 1 2 3 4 5 6 7 0 0.5 1 1.5 2 Left of mesentery Midline Right of mesentery Network Quality (0-poor, 10- excellent) Ki67-positive ICC/ 60X Field 10mm Aboral n=4 0.5 0.5 2.9 2.225 2.7875 0 1 2 3 4 5 6 7 0 0.5 1 1.5 2 Left of mesentery Midline Right of mesentery Network Quality (0-poor, 10- excellent) Ki67-positive ICC/ 60X Field 2mm Oral n=4 0.5 0.5 0.75 2.9375 4.325 4.375 0 1 2 3 4 5 6 7 0 0.5 1 1.5 2 Left of mesentery Midline Right of mesentery Network Quality (0-poor, 10- excellent) Ki67-positive ICC/ 60X Field 25mm Oral n=4 1.0 0.75 1.25 6.1625 3.8 2.475 0 1 2 3 4 5 6 7 0 0.5 1 1.5 2 Left of mesentery Midline Right of mesentery Network Quality (0-poor, 10- excellent) Ki67-positive ICC/ 60X Field 76mm Oral n=4
- 9. ©2013 MFMER |slide-9 Conclusion • Chang 2001 paper supported • H&E staining • Networks closer to mesentery might appear to be healthier overall • More data needed • Loss of proliferation may be contributing to the ICC loss • More data needed
- 10. ©2013 MFMER |slide-10 Acknowledgements • Mayo Clinic SURF program • My mentor, Simon • My PI, Gianrico • Seth • Gary, Sha Lei, Kmu, Cheryl
- 11. ©2013 MFMER |slide-11 Questions?
- 12. ©2013 MFMER |slide-12 References • 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 • 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 • 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 • Chang IY, Glasgow NJ, Takayama I, Horiguchi K, Sanders KM, et al. (2001) Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction. J Physiol 536: 555-68 • Lees-Green R, Du P, O'Grady G, Beyder A, Farrugia G, Pullan AJ. (2011) Biophysically based modeling of the interstitial cells of cajal: current status and future perspectives. Front Physiol 2:29
Editor's Notes
- #2 Hey everyone, I’m Kayleigh and I’m a rising junior at Grinnell College in Iowa. My project this summer focused on alterations in the Interstitial cells of Cajal networks in the small intestine in mice after partial obstruction.
- #3 Interstitial Cells of Cajal (ICC) are cells located within the smooth muscle tissue of the gastrointestinal tract, which interact with enteric nerve cells and smooth muscle cells to control gastrointestinal motility. They function as a network of electrical pacemaker cells that transmit a series of slow wave signals to smooth muscle, regulating smooth muscle function and initiating contractile movement.
- #4 The depletion of ICC have become an area of great interest as it is associated with several gastrointestinal motility disorders in humans such as diabetic gastroparesis, slow transit constipation, and intestinal pseudo-obstruction. Past studies of ICC have focused on how electrical slow waves are altered in motility disorders, how those slow waves correlate with contractile patterns, and how they relate to changes in ICC networks. My project specifically relates to a paper by Chang and his group published in 2001, in which partial obstruction showed loss of ICC in mouse small intestine.
- #5 One of the ultimate goals of my project is to improve the methods of quantifying ICC networks. Particularly because it is not well understood how ICC are lost in motility disorders and what the consequences are of the alterations in ICC networks on contractile activity. Jerry Gao and his group at the Auckland Bioengineering Institute in New Zealand have formulated metrics used to quantitatively determine ICC network changes, which are listed here: the density, thickness, size of holes in 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. However, quantification of ICC networks still remains difficult and mostly subjective. We also wanted to look at the role of proliferation in the development of ICC networks- does a decrease in proliferating ICC’s correlate with more depleted ICC networks? Is proliferation a potential factor in ICC depletion?
- #6 For the experiments, we used four female BALB/c mice aged between 4-6 wks. They were fully weaned and on liquid diets. I assisted Gary in performing a laparotomy on them in which a polyethylene clip was placed over piece of intestine 15-25 mm oral to the ileocaecal valve so that we could get enough data oral and aboral of the clip.
- #7 The mice were killed by CO2 inhalation 7 days following the surgery, at which time I dissected out the ileum and distal jejunum. I obtained a 1mm cross section of the ileum just oral of the clip for H&E staining and then removed the mucosa and mesentery from the rest of the intestine, leaving the smooth muscle tissue for staining. I then did the immunohistochemistry in order to view the cells. We used a goat anti-c-Kit antibody as a marker for ICC and a rabbit anti-Ki67 antibody as a marker for proliferation. DAPI was also used as a nuclear counter stain. And then we collected the images by confocal microscopy and a 60X water immersion objective. Tissue was collected from 10mm aboral to the clip and 2, 25, and 76mm oral to the clip. And at each of these distances, we collected images of both sides of the mesentery and the midline, which is just opposite of the mesentery. The ICC networks were then assessed by 2 independent blinded reviewers.
- #8 So these are the four distances we took images from. And you can qualitatively see here that the most faint tissue is the one just oral of the occlusion, which was expected and hoped for. And the others look relatively normal and healthier.
- #9 And if you take a closer look at the graph, the overall scores of the networks at 2mm oral of the occlusion were scored the lowest in quality, while other scores for 10mm aboral, 25mm oral, and 76mm oral report better-looking networks. In terms of network quality being dependent on the specific area (left, right, midline), we hypothesized that the networks closer to the mesentery would be healthier since it has easier access to nutrients. More data would be needed to prove this hypothesis since we only see the midline having the lowest network quality in 10mm aboral and 2mm oral. The average number of proliferating ICC’s seem to increase as we move oral from the occlusion, which would be consistent with our hypothesis that ICC proliferation is positively correlated with network quality. However, there is an unexpected low amount of proliferating ICC 10mm aboral of the clip, which would need more investigation if we really wanted to prove our hypothesis.
- #10 This study was further evidence of what was reported in the Chang 2001 paper: that partial obstruction of the small intestine resulted in a distance dependent disruption of the ICC networks. However, we still need to confirm this because when we opened the mouse for dissection after a week, there was no visible distension of the intestine. So H&E staining is still in progress to confirm that the muscle layer was morphologically altered. With some data supporting the hypothesis that the regions closer to the mesentery might have healthier networks and the midline more depleted networks, I think that if we gathered more data from more mice, and increase n, we might see this overall trend in all areas of the intestine we obtained tissue. We also have some evidence that loss of proliferation may be contributing to the ICC loss, but I think gathering data on this would really confirm this and make this finding more pronounced. One other future direction that we just didn’t have time for is to insert the clip, wait a week, remove the clip, wait another week, and then harvest tissues to study how proliferating ICC’s are altered after the mouse has had a chance to recover from partial obstruction.
- #11 I would like to thank the Mayo Clinic SURF program for having me this summer. I would like to thank my mentor, Simon, and my PI, Gianrico, for giving me such an interesting project and for their guidance. I would like to thank Seth for helping me with nearly every aspect of the project, Gary for performing the surgeries, Sha Lei for taking pictures for me, Kmu for showing me how to make the scoring sheets, and Cheryl for answering my questions and helping me with H&E staining.