AN IMMUNOHISTOCHEMICAL STUDY OF THE INTERSTITIAL CELLS OF CAJAL (ICCS) IN THE DOG INTESTINAL TRACT

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1 Journal of Cell and Tissue Research Vol. 11(2) (2011) ISSN: (Available online at Original Article AN IMMUNOHISTOCHEMICAL STUDY OF THE INTERSTITIAL CELLS OF CAJAL (ICCS) IN THE DOG INTESTINAL TRACT CHOUDHARY, R. K., DAS, P. AND GHOSH. R. K. Department of Veterinary Anatomy and Histology, Faculty of Veterinary and Animal Sciences. West Bengal University of Animal and Fishery Sciences, 37, K. B. Sarani, Kolkata E. Mail: Received: July 1, 2011; Revised: July 20, 2011; Accepted: July 27, 2011 Abstract: In the present investigation the distribution pattern of interstitial cells of Cajal (ICCS) population throughout the canine intestinal tract was explored. ICCS were identified by Kit receptor immunohistochemistry. Kit positive ICCS were present all along the segment of intestine but there was a marked difference in their presence, distribution and morphology. The number of ICCS were counted in different parts of intestine and recorded as ± 7.74, ± 4.17, ± 6.60, ± 10.02, ± 12.42, ± and ± 10.43/mm² in case of duodenum, jejunum, ileum, ileocolic junction, cecocolic junction, caecum and colon respectively. The average number of Kit positive ICCS were significantly (P 0.05) higher in cecocolic junction and less in the ileocolic junction. According to their occurrence within the intestine ICCS were sub typed into three categories, like Submucosal plexus or Meissner s plexus ICCS (SMP-ICCS), Intramuscular ICCS (IM-ICCS), Auerbach s plexus or Myenteric plexus ICCS (AP-ICCS). All the subtypes were not present all along the intestinal tract rather they were region specific. SMP-ICCS were not detected in small intestine. However they were present in the large intestine, ileocolic and cecocolic junctions. At the level of AP, C-Kit positive cells were stellate shaped with long branching process, which formed a network and sometimes extended into circular longitudinal muscle layers. Intramuscular C-Kit positive cells were spindle shaped and ran parallel to the muscle fibre. At the level of SMP C-Kit positive cells were round in shape and formed a network of cells. The IM-ICCS was found in cecum and colon along the outer aspects of the circular muscle bundles. Key words: Intestinal tract, Interstitial cells of Cajal? INTRODUCTION The gastrointestinal (GI) motility is the main characteristic feature of the smooth muscle. A special cell types, known as interstitial cells of Cajal (ICCS) have an immense role in the GI motility. ICCS are mesodermal in origin and not derived from neural crest cells, showing that these cells had the same origin as smooth muscle cells [1]. Three types of C- Kit immunoreactive cells were identified viz., spindleshaped cells in the region of the myenteric plexus, stellate or bipolar cells in the circular muscle layer, and round cells in the submucosa in case of equine [2]. At the level of the Auerbach s plexus (AP), C- Kit positive cells were multipolar with long branching processes, which formed a network and sometimes extended into the circular and longitudinal muscles layers in mice. The processes at the circular muscles side seemed to be in contact with cells in the outer circular muscles layer. Intramuscular C-Kit positive cells were spindle shaped cells and ran parallel to the muscle fibers [3]. The sphincter had a dense innervations and a sparse population of ICCS in case of canine [4]. Kit immunohistochemistry revealed a dense network of ICCS along the submucosal and myenteric borders in the rectum, whereas in the internal anal sphincter, ICCS were located along the periphery of muscle bundles within the circular layer 2865

2 J. Cell Tissue Research in case of dog. Changes in electrical activity within the circular muscle layer could be partially explained by changes in the structure of the muscle layer and changes in the distribution of ICCS in the rectoanal region of the GI tract [5]. ICCS generate electrical slow waves and also serve as intermediates in enteric motor neurotransmission in the GI tract [6,7]. Keeping in the view the immense role of gastric motility, the present study has been undertake to elucidate the normal distribution pattern of ICCS in the different segment of intestine of canine species. MATERIALS AND METHODS In the present investigation six healthy adult mongrel dogs with no apparent sign of illness were collected and observed critically. The animals were sacrificed by tranquilizing with xylazine. Incision was given in the linea alba in the ventrodorsal recombancy and abdomen was exposed. The intestinal components were separated out and the samples were collected. Then the samples were preserved in the deep freezer (-20 C) for immunohistochemistry. Experimental protocols were approved by the Institutional Animal Ethical Committee, F/O Vetinary and Animal Sciences, W.B.U.A.F.S. 31, K.B. Sarani, Kolkata. Immunohistochemistry: For immunohistochemical study the cryostat sections were obtained from frozen samples with 5μm thickness. Frozen sections were fixed in ice-cold paraformaledehyde (4% wt/vol in 0.1 M phosphate buffer) at 4 C for 25 minutes and then incubated for 30 minutes with 100μl of 0.3% H 2 O 2 in methanol at room temp to inhibit endogenous peroxidase activity. To check the non specific antigen tissues were incubated for 1hour with 1% BSA in 0.1 M PBS (PH-7.2) at room temperature. Rabbit polyclonal antibody raised against human Kit protein (CD117) used as primary antibody. ICCS expressed the proto oncogene C-Kit which encodes, C-Kit, a receptor tyrosine kinase. The C-Kit protein was used as a specific marker of ICCS. Immunoreactivity was detected after treating the samples with secondary antibody (Horseradish peroxidase conjugated goat anti -rabbit Ig 1:100). Microscopy and Micrometry: Microscopy and micrometry were performed using Leica Qwin Image Analyser software in Lecia DM 2000 Microscope. Statistical analysis: The data of the different characters were analyzed by Compare Mean Test with standard error by Duncan method (One Way ANOVA) and the significance (P value) of the test was recorded at 5% or 1% level and their interaction effects were further tested by LSD using Statistical Package for Social Scientists (SPSS, windows version 10.0). RESULTS The presence, distribution and morphology of interstitial cells of Cajal (ICCS) in the different segments of intestinal tract were identified using immunohistochemistry. The positive cells, classified according to their site of occurrence, were ICCS related to submucosal plexus or Meissner s plexus (ICCS-SMP), ICCS related to intramuscular region (ICCS-IM) and ICCS related to Auerbach s plexus or Myenteric plexus (ICCS-AP). Morphologically Explanation of Figures: Fig 1: Photomicrograph showing Kit- receptor immuno-histochemistry of Interstitial cells of cajal (ICCs) in the duodenum (x100). CM=Circular Muscle, DMP= Deep Muscular Plexus. Fig. 2: Photomicrograph showing Kit- receptor immuno-histochemistry of Interstitial cells of cajal (ICCs) in the duodenum (x100). CM=Circular Muscle Fig. 3: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (ICCs) in the duodenum (x100). CM=Circular Muscle, LM=Longitudinal Muscle, AP=Auerbach s Plexus. Fig. 4: Photomicrograph showing Kit- receptor immunohistochemistry of Interstitial cells of cajal (Arrow) in the jejunum (x100). LM=Longitudinal Muscle. Fig. 5: Photomicrograph showing Kit- receptor immuno-histochemistry of Interstitial cells of cajal (Arrow) in the jejunum(x100). CM=CircularMuscle, IM=Intramuscular. Fig. 6: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (ICCs) in the Ileum (x100). CM=Circular Muscle, LM=Longitudinal Muscle, AP=Auerbach s Plexus. Fig. 7: Photomicrograph showing Kit- receptor immuno-histochemistry of Interstitial cells of cajal (ICCs) in the Ileum (x100). CM=Circular Muscle. Fig. 8: Photomicrograph showing Kit- receptor immuno-histochemistry of Interstitial cells of cajal (ICCs) in the Ileo-colic junction (x100). AP=Auerbach s Plexus. 2866

3 Choudhary et al. Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig

4 J. Cell Tissue Research ICCS-SMP were found in form of network, where as ICCS-IM spindle shaped cells present to the muscle fibre. The multipolar ICCS-AP were distributed in the form of the network. The branches of the cells generally extended into the smooth muscle fibres of the inner circular and outer longitudinal layers. Duodenum: In the duodenum intense immunoreactivity was observed in the ICCS. The cells were distributed near the Auerbach s plexus (AP) and within the intramuscular (IM) regions (Figs.1-3). The average number of ICCS was recorded as ± 7.74 /mm 2 (Table-1). The ICCS-IM were spindle shaped and found running parallel to the muscle fibres within the longitudinal muscle, where as the ICCS- SMP were located on the outer most aspect of the circular muscle bundle. At the Myenteric plexus or Auerbach s plexuses the ICCS-AP were also found. The ICCS-AP were morpholo-gically multipolar and was located by forming a network around the myenteric ganglia. The fibres were distributed on both the direction of inner circular and outer longitudinal muscle bundles. At the level of submucosal plexus (SMP) no immunopositive cells were detected. Jejunum: In the jejunum immunopositive ICCS were detected both in the AP and IM regions. High magnification of some sections showed ICCS as round to oval in shape located within the intramuscular region (Fig. 4,5). In other regions they were almost similar to that of duodenum. The average number of ICCS was ± 4.17/mm 2 (Table-1). Like that of the duodenum, no ICCS were detected at the submucosal plexus. Ileum: Immunohistochemical studies of ileum revealed distinct network of ICCS. These cells were distribution mostly at the level of Auerbach s plexus and intramuscular region (Figs. 6,7). Characteristically the ICCS-AP were multipolar and aggregated around the Auerbach s plexus ganglion and the processes were directed on both the faces of inner circular and outer longitudinal muscle bundle. On the contrary, the ICCS-IM were elongated to oval in appearance. The average number of ICCS was recorded as ± 6.60/mm 2 (Table-1). ICCS-SMP could not be detected in this region. Ileocolic junction: In ileocolic junction, immunopositive ICCS were found at the Auerbach s plexus and intramuscular regions (Figs. 8-10). The ICCS-IM were typical abundant and found running parallel to the fibre direction. The cells were also located on the outer aspect of circular muscle fibre. Some of the sections of this region revealed multiple aggregation of ICCS-IM in a space between the muscle bundles. ICCS-AP were observed around the myentric ganglion. The cells were multipolar and their processes were directed towards the muscle fibres. Some ICCS-SMP were also detected in ileocolic junction. The average number of ICCS was recorded as ± 10.02/mm 2 (Table 1). Cecocolic junction: Immunopositive cells of cecocolic junction were quite prominent (Figs. 11,12). All three variety of cells (IM, SMP and AP) were found in this region and morphologically they were similar to other regions cells. The average number of ICCS was ± 12.42/mm 2 (Table 1). Cecum: All three types immunoreactive cells were found in cecum and mostly distributed within the tunica muscularis (Figs. 13,14). Morphologically ICCS-SMP were branched and presented in the form of network towards the circular muscle fibrer. The Explanation of Figures: Fig. 9: Photomicrograph showing Kit- receptor immuno-histochemistry of Interstitial cells of cajal (ICCs) in the Ileo-colic junction (x100). LM=Longitudinal Muscle, DMP=Deep Muscular Plexus. Fig.10: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (Arrow) in the Ileo-colic junction (x100). LM=Longitudinal Muscle. Fig. 11: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (Arrow) in the Cecocolic junction (x40). LM=Longitudinal Muscle,CE=Cecum. Fig. 12: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (Arrow) in the Cecocolic junction (x100). LM=Longitudinal Muscle, CO=Colon, DMP=Deep Muscular Plexus, CE=Cecum. Fig. 13: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (Arrow) in the Cecum (x40). CM=Circular Muscle, LM=Longitudinal Muscle. Fig. 14: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (Arrow) in the Cecum (x100). IM=Intramuscular, AP=Auerbach s Plexus. Fig.15: Photomicrograph showing Kit-receptor immuno-histochemistry of Interstitial cells of cajal (Arrow) in the Colon (x100). IM=Intramuscular. 2868

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6 J. Cell Tissue Research Table 1: Mean (± SE) Values of No. of ICCS (NO/mm 2 ) in different segments of intestine. N.B. Mean Values bearing common superscripts in a row differ significantly. Where (P<0.05), P= Significant value. Segments of intestine Cell No./mm 2 Duodenum Jejunum Ileum bc ± bc ± bc ± 6.60 Ileocolic Junction c ± Cecocolic junction a ± Cecum Colon P-Value bc ± ab ± ICCS-AP were stellate shaped and less dense as compared to functional regions. Cells of intramuscular region (ICCS-IM) were seen running parallel to the longitudinal muscle fibres. The average number of ICCS recorded as ± 11.97/mm 2 (Table 1). Colon: In the colon the ICCS were distributed in the entire three distinct zones as described in cecum (Fig.15). Typical spindle shaped bipolar ICCS-IM were detected along the outer aspect of circular muscle fibres bundle. The average number of ICCS was recorded as ± 10.43/mm 2 (Table 1). DDISCUSSION Previously it was thought that myocyte, fibroblast around blood vessels, macrophages identified as ICCS, but immunohistochemistry with C-Kit protein isolated ICCS from the other tissues [8]. Mammalian ICCS were neither derived from the neural crest nor developmentally dependent on neurons. ICCS differentiation/survival required kit ligand, which could be provided by neurons or cells in a smooth muscle lineage. Neurons might be needed for development of Myenteric- ICCS and the mature ICCS network [9]. The ICCS were subtyped into three categories. All most similar sub typing was done by other scientists [10-12]. In present investigation, however all the subtypes were not present all along the intestinal tract rather it was region specific. ICCS-SMP were not detected in the small intestine. However it was present in the large intestine [10,11]. The ICCS population of duodenum was same as ileum, but less than colon. It was more than jejunum and cecum. The average number of immunopositive ICCS were significantly (P< 0.05) highest in cecocolic junction and least in ileocoloic junction. The density of ICCS in the jejunum was less as compare to duodenum, ileum, cecum and colon. However, in case of bovine ICCS were detected immunohistochemically in the jejunum, which was associated with AP [13]. Nevertheless, the presence of wide spread ICCS in longitudinal muscle layers of the jejunum differed from the previously described studies of other mammals. The ICCS population at cecocolic junction was highest as compared to the other segments of intestine. But lowest population was detected at ileocolic junction. No specific literatures were found in support of our findings. But it was observed that the sphincter had a dense innervations and a sparse population of interstitial cells of Cajal [4]. The ICCS population in the colon is more as compare to small intestine but less than the cecum. Similarly no supportive document can be cited in this regards. The density of ICCS was of the same magnitude along the extent of the colon about cells/mm² at Auerbach s plexus (AP) and cells/mm² at the submucosal plexus (SMP) in case of rat [14]. At the level of SMP, immunopositive cells were round in shaped and form a network. Intramuscular C-Kit protein cells were spindle shaped and ran parallel to the muscle fibre. The ICCS-IM were found in the cecum and colon along the outer aspects of the circular muscle bundles in case of dog [5]. Keeping in view of the histoarchitecture of the junctional complex the ICCS population was comparatively higher in cecocolic junction. The morphological studies showed ICCS lining the submucosal surface of the circular layer and the septal structures that separate the circular layers into bundles. Electrical measurements suggested that the slow wave might propagate into the thickness of circular muscle in a regenerative manner along the surface of the septa [15]. However, intramuscular septae were smaller at this junction, therefore, a delay electrical coupling may occur across this region. Functional studies of the intestinal tract by the various workers indicate that there are significant difference in the motility pattern and electrical activity of the intestine. The present study indicates the functional differences among the different regions are 2870

7 accompanied by difference in both structural arrangement of smooth muscle cells and as well as distribution of ICCS in different segments of intestine. Due to lack of ICCS-SMP in the small intestine, the question comes how and where the slow wave is generated. The structural relationships between ICCS, varicose nerve fibres and smooth muscle cells in the gastrointestinal tract of pig had led to the suggestion that ICCS might mediate enteric neurotransmission. However, it was evident that different subclasses of ICCS might play a role in neurotransmission depending on the area of the gastrointestinal tract [16,17]. These results are helpful for better understanding of canine gastric motility and at the sametime canine model can be used for experimental purpose. Abbreviation used: SMP = submucosal plexus; AP = Auerbach s plexus; ICCS= interstitial cells of Cajal; ICCS-SMP = cell in submucosal plexus or Meissner s plexus; ICCS-IM = cells belonging to intramuscular region; ICCS-AP = cell located in Auerbach s plexus or Myenteric plexus; C-Kit proteins = a receptor tyrosine kinase; ACKNOWLEDGEMENT We acknowledge the financial support rendered by the authority of West Bengal University of Animal and Fishery Sciences Kolkata for undertaking the research project. REFERENCES [1] Daniel, E.E and Posey-Daniel, V.: Am. J. Physiol., 246: (1984). [2] Hudson, N.P.H., Pearson, G.T., Kitamura, N. and Mayhew, I.G.: Res. Veterinary Sci., 66(3): (1999). [3] Vanderwinden, J.M., Rumessen, J.J., Bernex, F., Schiffmann, S.N. and Panthier, J.J.: Cell Tissue Research., 302: (2000). [4] Daniel, E.E., Berezin, I., Allescher, H.D., Manaka, H. and Posey-Daniel, V.: Canadian J. Physiol. Pharmacol, 67(12): (1989). [5] Horiguchi, K., Keef, K.D. and Ward, S.M.: Am. J. Physiol., 284(5(1)): G756-G767 (2003). [6] Christensen, J.: J. Auton. Nerv. Syst., 37: (1992). [7] Sander, K.M.: Gastroenterology, 111: (1996). [8] Torihashi, S., Kobayashi, S.. Gerthoffer, W.T. and Sanders, K.M.: Am. J. Physiol. Gastrointestinal Liver Physiol., 265(4): (1993). Choudhary et al. [9] Jun, J., Taube, Wu., Rothman, P. and Michael Gershon, D.: J. Neurosci. Res., 59: (2000). [10] Rumessen, J.J., Peters, S. and Thuneberg, L.: Lab. Invest., 68: (1993). [11] Smith T.K., Reed, J.B. and Sanders, K.M..: Amer. J. Physiol. Cell Physiol., 252: (1987). [12] Thuneberg, L.: Advance. Anatomy. Embryol.. Cell Biol., 71: (1982). [13] Marquez, S.G., Galotta, J.M., Portiansky, E.L. and Barbeito, C.G.: Veterinary Res. Communications., 30(3): (2006). [14] Elena Alberti i Martizez de Velasco.: Bellaterra, novembre de., pp 41 (2004). [15] Ward S.M. and Sanders K.M.: Amer. J. Physiol. Gastrointestinal Liver Physiol., 259(2): (1990). [16] Hert, T.: Regulation of Gastrointestinal Function in Cunningham. In: Text book of Veterinary Physiology W.B. Sanders Company, Philadelphia, pp (1997). [17]Toma, H., Nakamura, K., Emson, P. C. and Kawabuchi, M.: Journal. autonomus. Nervous. System., 75:93-99 (1999). 2871