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Ultrastructural identification of different subtypes of interstitial cells of Cajal in the chicken ileum
MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Ultrastructural identification of different subtypes of interstitial cells of Cajal in the chicken ileum P. Yang,* S. Wang,* J. A. Gandahi,*† X. Bian,* L. Wu,* Y. Liu,* L. Zhang,* Q. Zhang,* and Q. Chen*1 *Laboratory of Animal Cell Biology and Embryology, College of Veterinary Medicine, 210095 Nanjing Agricultural University, Nanjing, PR China; and †Department of Anatomy & Histology, Sindh Agriculture University, 70060 Tandojam, Pakistan nerves, but true gap junctions were not found between them. A new subtype of ICC located in the lamina propria mucosae has been discovered. Some of the ICC showed typical features of SMC, including a basal lamina, caveolae, and dense bodies. Lacking intermediate filaments and caveolae distinguished them from the fibroblast-like cells showing well-developed secretory organelles, including coated vesicles and a patchy basal lamina. The ultrastructural features and distribution of ICC in chicken intestine is similar to mammals. They may play similar key regulatory roles in gastrointestinal motility. The new subtype of ICC discovered in the lamina propria mucosae may play a role in the regulation of secretion and absorption.
Key words: chicken ileum, interstitial cells of Cajal, transmission electron microscopy 2012 Poultry Science 91:1936–1940 http://dx.doi.org/10.3382/ps.2011-02090
INTRODUCTION The interstitial cells of Cajal (ICC) are a special class of cells located between the autonomic nerve endings and smooth muscle cells (SMC) in the digestive tract. In 1893, Ramón y Cajal, a neuroanatomist, observed this special class of cells in the rabbit gastrointestinal tract by silver staining and methylene blue staining; these cells are thus named after him. The ICC have pacemaker activity; they initiate myoelectric slow waves, a basal electrical rhythm leading to contraction of the smooth muscle, for example, peristalsis and segmentation (Faussone-Pellegrini and Thuneberg, 1999; Ward et al., 2000; Daniel, 2001). A continuous network of ICC is required for the propagation of these slow waves. The recognition of the importance in motility led researchers to investigate ICC networks in different organs, such us the gastrointestinal tract, ureter (Oberritter et al., 2009), and fallopian tube of ©2012 Poultry Science Association Inc. Received December 10, 2011. Accepted March 31, 2012. 1 Corresponding author: [email protected]
different animals and humans (Popescu et al., 2007). Many motility features and disorders of the gastrointestinal tract have been found to be closely related to the constitution or abnormality of the ICC network (Lyford et al., 2002; Jain et al., 2003). Chicken has great economical and scientific notability. Chicken is an important source of meat, and it is considered the main representative animal of birds. Chicken together with quail have been widely used as experimental models to study the development of the enteric nervous system. Its major strength is the easy accessibility throughout all developmental stages which allows for embryologic manipulations not easily performed in other species. Although the ultrastructural identification of ICC was reported in the gizzard of the love bird (Imaizumi and Hama, 1969), in the digestive tract of turkeys (Reynhout and Duke, 1999), and the origin of ICC cells was investigated in chick embryos (Lecoin et al., 1996), a comprehensive study on the ultrastructural characteristics of ICC in the digestive system of the chicken is still lacking. Our aim was to provide further details on the identification and features of different ICC subtypes and
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ABSTRACT Ultrastructural characteristics of different subtypes of interstitial cells of Cajal (ICC) remain unclear in birds; however, birds have significant economical and scientific notability. Our aim was to describe and classify ICC in the chicken gut. The ileum of normal adult Three Yellow broiler chickens (n = 10) were studied by transmission electron microscopy (TEM). The ICC were spindle- or stellate-shaped with ramified cell processes. They had numerous mitochondria, abundant intermediate filaments, fusiform nuclei (oval or indented), with a dense band of peripheral heterochromatin, and formed close contacts by true gap junctions with each other and with neighboring smooth muscle cells (SMC). The ICC were in close contact with enteric
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examine their relationships with other ICC, nerves, and SMC in the ileum of the adult chicken.
MATERIALS AND METHODS
Figure 1. The ultrastructure of interstitial cells of Cajal (ICC) in chicken ileum. (a) A spindle-shaped ICC showing multiple tortuous slender cytoplasmic processes. There is a fibroblast (F). (b) Two ICC processes lying close together and making true gap junctions (arrowheads). The ultrastructure of ICC is evident from a and b. Abundant mitochondria (Mi), numerous caveolae (small arrows), sharp basal lamina (large arrows), rough endoplasmic reticulum (rER), scattered intermediate filaments (IF), and rare cytoplasmic dense bodies (d).
with neighboring SMC (Figure 2). Bundles of enteric nerve fibers lay close to ICC-MY (Figures 2c,d and 3a,b,c), although intimate relationships could not be observed. Most ICC-MY were stellate-shaped, often had 2 main processes, and often had long, slender secondary and tertiary ramifications. The processes of 2 to 5 cells were organized into bundles (Figure 2c), the surrounded nerves making close contact with them. A typical nerve-ICC-SMC structure (Figure 2c, 3a) was observed. The ICC-MY cells often surrounded blood vessels with long processes (Figure 2e). The ICC within the longitudinal and circular muscle layers contained many mitochondria, caveolae, and rough endoplasmic reticulum. These ICC were consid-
Results In all samples, the ICC were exactly identified. These ICC exhibited typical ultrastructural features of ICC already described in mammals. They showed numerous mitochondria and abundant intermediate filaments. The nuclei were fusiform, oval, or indented with a dense band of peripheral heterochromatin (Figure 1a,b). Morphologically, they were spindle- or stellate-shaped, with a different number of ramified cell processes. Some ICC exhibited typical features of SMC, including a basal lamina, caveolae, and dense bodies (Figure 1b). However, these ICC did not have thick myosin filaments like the SMC. In the myenteric plexus, ICC were easily identified surrounding the myenteric ganglia (Figure 2c, 3a). This subtype of ICC was named ICC-MY. The ICC-MY formed gap junctions with each other (Figure 1b) and
Figure 2. Myenteric interstitial cells of Cajal (ICC-MY) in the ileum of chicken. (a) A myenteric ICC forming gap junction with 2 smooth muscle cells (SMC). (b) Magnified view of the box shown in panel a. Note the gap junction between ICC and SMC (arrowheads), the SMC showing scattered mitochondria (Mi), and thick myosin filaments (TF). (c) Multiple ICC lying closer to the nerve fibers (NF) and SMC. (d) Higher magnification of the boxed area in panel c showing a gap junction (arrowheads) between ICC and SMC. (e) A spindleshaped bipolar ICC targeting the blood vessel (BV) and the vascular endothelium (VE). Another irregular-shaped ICC lying in the vicinity of the BV. Note a nucleated avian red blood cell (RBC).
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We used 10 normal adult Chinese Three Yellow broilers (English translation of the local Chinese chicken breed), between 7 and 9 wk of age, weighing 1.8 to 2 kg in current study. The feeding was done with commercially available feeds (New Hope, Chengdu, China) for adult broiler chickens. They were housed in temperature-controlled rooms (20 ± 1°C) with natural light (the natural light/dark cycle 12L:12D at the time of the study) and had free access to food and tap water. The chickens were killed by cervical dislocation, under ether anesthesia. The experiment has been approved by the Chinese Committee for Animal Use for Research and Education. Tissue samples were collected from the ileum and immersed in 2.5% glutaraldehyde fixative in 0.1 M PBS (4°C, pH 7.4) for one hour and washed in the same buffer overnight. The tissues were postfixed with a mixture of 1% osmiumtetroxide and 1.25% potassium ferrocyanide for 1.5 to 2 h, washed in the buffer, dehydrated in increasing concentrations of ethyl alcohol, infiltrated with a propylene oxide/Araldite mixture (Boster, Wuhan, China), and embedded in Araldite. Semithin sections of intestine were stained with 1% toluidine blue for light microscopic examination, and specific areas were chosen for ultrathin sectioning. Sections were cut using an ultramicrotome (Reichert-Jung, Wien, Austria). Ultrathin sections (50 nm) were collected on copper grids, counterstained for 10 min with 1% uranyl acetate and Reynold’s lead citrate, and then observed in a JEM-1200EX electron microscope (Jeol, Tokyo, Japan). All sections that contained ICC were analyzed and all ultrastructural characteristics were recorded. Semiquantification was done using – for absent; − + for occasionally present; and +, ++, +++ for constantly present with increasing richness.
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Figure 3. Typical muscle-interstitial cells of Cajal (ICC)-nerve fiber structure. (a) A stellate-shaped ICC forming multiple points of close contacts with smooth muscle cells (SMC) and nerve fibers (NF). (b) Magnified view of the upper box in a, showing the close contacts (arrowheads) with SMC. (c) The lower box in a magnified view. Note the close membrane associations between the ICC and NF.
Figure 4. Intramuscular interstitial cells of Cajal (ICC-IM) contacts with smooth muscle cells (SMC) and nerve fibers. (a) A stellateshaped ICC-IM with 2 long processes and many short processes, traversing between SMC. (b) The magnified view of small box in panel a. Note a big caveola (double arrowheads) at the end of the ICC process. (c) A magnified view of the ICC-IM in big box in panel a, showing the close contacts (arrowheads) with SMC. (d) A rounded ICC-IM lying between the SMC and nerve fibers (NF). It makes multiple points of close contacts (arrowheads) with the ICC and SMC.
glia of the submucosal plexus (Figure 6a). The ICC-SM surrounded perivascular cells of the blood vessels (Figure 6b). They also occasionally formed peg-and-socketlike junctions with neighboring SMC (Figure 6c). In addition, a new subtype of ICC was found in the lamina propria of the ileum and named ICC-LP according to their location. These cells had a stellate morphology with multiple branches. The branches made frequent close contacts with each other, forming a network and close contact with neighboring fibroblasts (Figure 7a). These ICC have abundant intermediate filaments and were easily distinguished from the neighboring fibroblasts (Figure 7b). In all samples, ICC were distinguished from the more numerous fibroblasts. However the fibroblasts had a close and constant association with collagen bundles, formed close contacts with neighboring SMC, blood vessels, and enteric nerves like the ICC. Fibroblasts were surrounded by elastic fibers, had oval-shaped nuclei with less peripheral heterochromatin, and a variable amount of perinuclear cytoplasm. Fibroblasts had numerous thin processes, well-developed secretory organelles (golgi, rER), including coated vesicles and
Figure 6. Interstitial cells of Cajal (ICC) found in the submucosal plexus (ICC-SMP). (a) ICC-SMP triplet uniting each other and surrounding the nerve fibers (NF). (b) Two ICC-SMP are seen targeting the blood vessel near the smooth muscle cell (SMC). Also note the vascular pericyte (PC), vascular endothelial cell (VE), and a nucleated red blood cell (RBC). (c) ICC-SM making a peg-and-socket-like junction with SMC (asterisk).
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ered intramuscular-ICC (ICC-IM). The ICC-IM displayed the same ultrastructural features, had mainly spindle shape with 2 long processes (Figure 4a), and stellate shape with 2 long processes and many short processes (Figure 4a). The endings of processes often formed a big caveola (Figure 4b). The ICC of the longitudinal layer formed distinct close contacts, not gap junctions, with neighboring ICC, with adjacent SMC (Figure 4c), and with enteric nerves (Figure 4d). Numerous ICC were found in the deep muscular plexus along the border of the circular muscle bundles, within the septa that separated the bundles. This subtype of ICC was named ICC-DMP. These cells aggregated to form small bundles containing 2 to 3 cells surrounding nerve endings (Figure 5a) making close contact with SMC (Figure 5b). These cells were stellate-shaped and often had slender secondary and tertiary processes. Many ICC were found along the submucosal surface of the circular muscle layer. They were identified as ICC-SM. This subtype of ICC contained numerous mitochondria, reduced number of caveolae, distinct basal lamina, and abundant rough and smooth endoplasmic reticula. The ICC-SM were arranged around the gan-
Figure 5. The interstitial cells of Cajal (ICC) located in deep muscular plexus (ICC-DMP). (a) Stellate-shaped ICC giving-off multiple slender processes to smooth muscle cells (SMC). (b) Higher magnification of the boxed area in panel a. Note the close contacts (arrowheads) of ICC with SMC, mitochondria (Mi), rough endoplasmic reticulum (rER), and basal lamina (large arrows) in the ICC process.
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Figure 7. The interstitial cells of Cajal (ICC) in lamina propria (ICC-LP). (a) A spindle-shaped bipolar ICC and an accompanying fibroblast (F) are seen. (b) A magnified view of the boxed area in panel a, showing the fibroblast (F) and the intermediate filaments (IF) scattered in the cytoplasm of ICC.
DISCUSSION Although chicken has a great economic and scientific impact, no extensive study has been performed on ICC in these birds. Chicken ICC have not been fully characterized, subtyped, and distinguished from fibroblast and SMC by transmission electron microscopy. Since Faussone-Pellegrini and Thuneberg (1999) provided the characteristics of typical ICC, electron microscopy became the most reliable method of identification of these cells. According to previous reports of Sanders (1996) and Faussone-Pellegrini and Thuneberg (1999), we have found the ultrastructure of the chicken ICC to be very similar to that of mammals. We were able to identify all subtypes of ICC described in mammals; furthermore, we report the first submucosal subtype (ICC-SM) in a bird. In early studies, Lecoin et al. (1996) and Reynhout and Duke (1999) described typical ICC in the digestive tract of birds, but no submucosal type had been identified. In addition, in this study, a completely new subtype of ICC located in the lamina propria mucosae (ICCLP) was discovered. Kunisawa and Komuro (2008) suggested ICC-SM may play a role in the regulation of
the secretion, absorption, and transportation of fluids in the mucosa in guinea pigs. The slender cytoplasmic processes of ICC-SM found in chicken closely surround the submucosal ganglia. This suggests a similar role as seen in guinea pigs. Studies have revealed that the primary targets of the nitrergic neurons are ICC located within the muscle bundles in intestine (Horiguchi et al., 2003; Cserni et al., 2009; Iino et al., 2009). The close contact of the myeneteric ICC with both the muscle bundles and the nerve plexus found in the chicken intestine indicates their active involvement in the regulation of motor activity. Imaizumi and Hama (1969) reported gap junctions between ICC and nerve bundles in love birds, but this has not been observed in our study in chickens. Although we discovered true gap junctions among ICC and between ICC and SMC in chickens, Reynhout and Duke (1999) found gap junctions only between ICC in turkey. It is interesting that gap junctions are common in mammals but do not exist in reptiles and amphibians (Junquera et al., 2001; Miyamoto-Kikuta and Komuro, 2007). This may be explained by evolution. It is less likely that differences in gap junctions in birds can be explained by evolution. More likely, this comes from technical reasons, different thickness of the specimens and limitation of the electron microscopy. The demonstration of close contacts between nerve and SMC suggests that the tunica muscularis of the chicken ileum has parallel pathways for direct and in-
Table 1. Ultrastructural features characterizing the interstitial cells of Cajal (ICC) at the various subtypes in the chicken ileum1,2 Item Rough endoplasmic reticulum Smooth endoplasmic reticulum Mitochondria Intermediate filaments Caveolae Basal lamina 1Symbols: 2MY
ICC-MY
ICC-IM
ICC-DMP
ICC-SM
ICC-LP
+ ++ +++ ++ ++ Discontinuous
+ ++ ++ + + Discontinuous
+ ++ ++ ++ ++ Discontinuous
++ ++ ++ + + Discontinuous
−+ + + +++ −+ Discontinuous
− + = occasionally present; +, ++, +++ = constantly present with increasing richness. = myenteric ganglia; IM = intramuscular; DMP = deep muscular plexus; SM = submucosal surface; LP = lamina propria.
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patchy basal lamina, but lacked prominent intermediate filaments and caveolae. (Figure 8a,d). The ultrastructural characteristics of the different ICC subtypes are summarized in Table 1.
Figure 8. Fine ultrastructure of the fibroblasts (F) in the chicken ileum. (a) A fibroblast with oval nucleus and short processes. (b) A fibroblast at higher magnification, showing the coated vesicles (large arrows).
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ACKNOWLEDGMENTS This study was supported by grants (No. 30871833 and 31172282) from the National Science Foundation of China and Priority Academic Program Development of Jiangsu Higher Education Institutions.
REFERENCES Brooks, J. C. W., L. Zambreanu, A. Godinez, A. D. Craig, and I. Tracey. 2005. Somatotopic organisation of the human insula to painful heat studied with high resolution functional imaging. Neuroimage 27:201–209. Cserni, T., S. Paran, Z. Kanyari, A. M. O’Donnell, B. Kutasy, N. Nemeth, and P. Puri. 2009. New insights into the neuromuscular anatomy of the ileocecal valve. Anat. Rec. (Hoboken) 292:254– 261. Daniel, E. E. 2001. Physiology and pathophysiology of the interstitial cell of Cajal: From bench to bedside. III. Interaction of inter-
stitial cells of Cajal with neuromediators: An interim assessment. Am. J. Physiol.-Gast. L. 281:1329–1332. Daniel, E. E., Y. F. Wang, and F. S. Cayabyab. 1998. Role of gap junctions in structural arrangements of interstitial cells of Cajal and canine ileal smooth muscle. Am. J. Physiol.-Gast. L. 274:1125–1141. Faussone-Pellegrini, M. S., and L. Thuneberg. 1999. Guide to the identification of interstitial cells of Cajal. Microsc. Res. Tech. 47:248–266. Horiguchi, K., K. M. Sanders, and S. M. Ward. 2003. Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum. Cell Tissue Res. 311:299– 313. Iino, S., K. Horiguchi, Y. Nojyo, S. M. Ward, and K. M. Sanders. 2009. Interstitial cells of Cajal contain signalling molecules for transduction of nitrergic stimulation in guinea pig caecum. Neurogastroenterol. Motil. 21:542–550. Imaizumi, M., and K. Hama. 1969. An electron microscopic study on interstitial cells of gizzard in love-bird. Z. Zellforsch. 97:351. Jain, D., K. Moussa, M. Tandon, J. Culpepper-Morgan, and D. D. Proctor. 2003. Role of interstitial cells of Cajal in motility disorders of the bowel. Am. J. Gastroenterol. 98:618–624. Junquera, C., C. Martinez-Ciriano, T. Castiella, P. Serrano, J. Aisa, E. Calvo, and M. Lahoz. 2001. Enteric plexus and interstitial cells of Cajal: Interrelationship in the stomach of Podarcis hispanica (Reptilia). An ultrastructural study. Histol. Histopathol. 16:869–881. Komuro, T., K. Seki, and K. Horiguchi. 1999. Ultrastructural characterization of the interstitial cells of cajal. Arch. Histol. Cytol. 62:295–316. Kunisawa, Y., and T. Komuro. 2008. Interstitial cells of Cajal associated with the submucosal plexus of the Guinea-pig stomach. Neurosci. Lett. 434:273–276. Lecoin, L., G. Gabella, and N. LeDouarin. 1996. Origin of the ckit-positive interstitial cells in the avian bowel. Development 122:725–733. Lyford, G. L., C. L. He, E. Soffer, T. L. Hull, S. A. Strong, A. J. Senagore, L. J. Burgart, T. Young-Fadok, J. H. Szurszewski, and G. Farrugia. 2002. Pan-colonic decrease in interstitial cells of Cajal in patients with slow transit constipation. Gut 51:496–501. Mikkelsen, H. B. 2010. Interstitial cells of Cajal, macrophages and mast cells in the gut musculature: Morphology, distribution, spatial and possible functional interactions. J. Cell. Mol. Med. 14:818–832. Miyamoto-Kikuta, S., and T. Komuro. 2007. Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal. Cell Tissue Res. 328:271–279. Oberritter, Z., U. Rolle, Z. Juhasz, T. Cserni, and P. Puri. 2009. Altered expression of c-kit-positive cells in the ureterovesical junction after surgically created vesicoureteral reflux. Pediatr. Surg. Int. 25:1103–1107. Popescu, L. M., S. M. Ciontea, and D. Cretoiu. 2007. Interstitial Cajal-like cells in human uterus and fallopian tube. Ann. N. Y. Acad. Sci. 1101:139–165. Reynhout, J. K., and G. E. Duke. 1999. Identification of interstitial cells of Cajal in the digestive tract of turkeys (Meleagris gallopavo). J. Exp. Zool. 283:426–440. Rumessen, J. J., J. M. Vanderwinden, H. Rasmussen, A. Hansen, and T. Horn. 2009. Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon. Cell Tissue Res. 337:197–212. Sanders, K. M. 1996. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111:492–515. Thuneberg, L., and S. Peters. 2001. Toward a concept of stretchcoupling in smooth muscle. I. Anatomy of intestinal segmentation and sleeve contractions. Anat. Rec. 262:110–124. Wang, X. Y., K. M. Sanders, and S. M. Ward. 1999. Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine. Cell Tissue Res. 295:247–256. Ward, S. M., T. Ordog, S. D. Koh, S. Abu Baker, J. Y. Jun, G. Amberg, K. Monaghan, and K. M. Sanders. 2000. Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. J. Physiol. 525:355–361.
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direct innervation of the smooth muscle, as reported in the digestive tract of mammals (Daniel et al., 1998; Brooks et al., 2005; Rumessen et al., 2009). The features of the structure reveal that the ICC adapt as the intermediary of the nervous system and SMC that can control gastrointestinal function. The close connection revealed between nerves and ICC supports the hypothesis reported by Wang et al. (1999), Iino et al. (2009), and Mikkelsen (2010), that nerves might have a functional effect on ICC in regulating the gastrointestinal smooth muscles via neurotransmitters. Thuneberg and Peters (2001) have proposed that peg-and socket-like junctions play a crucial role in the coordination of smooth muscle and pacemaking in mice. Peg-and socket-like junctions were revealed in chicken intestine as well between ICC and SMC. The close contacts between the ICC and SMC vascular wall in our study have not been reported in birds. This new finding indicates ICC regulate intestinal microcirculation. The ICC, SMC, and fibroblasts may look very similar; there is no single feature that identifies ICC properly. In our study, several ultrastructural criteria helped us to separate ICC from fibroblasts and SMC. We have found the presence of intermediate filaments, caveolae, and basal lamina most reliable to distinguish an ICC from a fibroblast and the absence of thick filaments to differentiate ICC from SMC. The ICC may be differentiated from fibroblasts based on the presence of gap junctions and the connections with nerve bundles. However, Komuro et al. (1999) noted that fibroblastlike cells may have gap junctions with SMC. In our study, we first identified the various subtypes of ICC in chicken ileum based on ultrastructural criteria. A new subtype in the lamina propria mucosae has been identified. The location and the connections between the neighboring ICC, SMC, nerve bundles, and vessels indicate they play a similar role in gastrointestinal motility as has been reported in mammals.
Key words: chicken ileum, interstitial cells of Cajal, transmission electron microscopy 2012 Poultry Science 91:1936–1940 http://dx.doi.org/10.3382/ps.2011-02090
INTRODUCTION The interstitial cells of Cajal (ICC) are a special class of cells located between the autonomic nerve endings and smooth muscle cells (SMC) in the digestive tract. In 1893, Ramón y Cajal, a neuroanatomist, observed this special class of cells in the rabbit gastrointestinal tract by silver staining and methylene blue staining; these cells are thus named after him. The ICC have pacemaker activity; they initiate myoelectric slow waves, a basal electrical rhythm leading to contraction of the smooth muscle, for example, peristalsis and segmentation (Faussone-Pellegrini and Thuneberg, 1999; Ward et al., 2000; Daniel, 2001). A continuous network of ICC is required for the propagation of these slow waves. The recognition of the importance in motility led researchers to investigate ICC networks in different organs, such us the gastrointestinal tract, ureter (Oberritter et al., 2009), and fallopian tube of ©2012 Poultry Science Association Inc. Received December 10, 2011. Accepted March 31, 2012. 1 Corresponding author: [email protected]
different animals and humans (Popescu et al., 2007). Many motility features and disorders of the gastrointestinal tract have been found to be closely related to the constitution or abnormality of the ICC network (Lyford et al., 2002; Jain et al., 2003). Chicken has great economical and scientific notability. Chicken is an important source of meat, and it is considered the main representative animal of birds. Chicken together with quail have been widely used as experimental models to study the development of the enteric nervous system. Its major strength is the easy accessibility throughout all developmental stages which allows for embryologic manipulations not easily performed in other species. Although the ultrastructural identification of ICC was reported in the gizzard of the love bird (Imaizumi and Hama, 1969), in the digestive tract of turkeys (Reynhout and Duke, 1999), and the origin of ICC cells was investigated in chick embryos (Lecoin et al., 1996), a comprehensive study on the ultrastructural characteristics of ICC in the digestive system of the chicken is still lacking. Our aim was to provide further details on the identification and features of different ICC subtypes and
1936
Downloaded from http://ps.oxfordjournals.org/ by guest on March 18, 2015
ABSTRACT Ultrastructural characteristics of different subtypes of interstitial cells of Cajal (ICC) remain unclear in birds; however, birds have significant economical and scientific notability. Our aim was to describe and classify ICC in the chicken gut. The ileum of normal adult Three Yellow broiler chickens (n = 10) were studied by transmission electron microscopy (TEM). The ICC were spindle- or stellate-shaped with ramified cell processes. They had numerous mitochondria, abundant intermediate filaments, fusiform nuclei (oval or indented), with a dense band of peripheral heterochromatin, and formed close contacts by true gap junctions with each other and with neighboring smooth muscle cells (SMC). The ICC were in close contact with enteric
SUBTYPES OF INTERSTITIAL CELLS OF CAJAL
1937
examine their relationships with other ICC, nerves, and SMC in the ileum of the adult chicken.
MATERIALS AND METHODS
Figure 1. The ultrastructure of interstitial cells of Cajal (ICC) in chicken ileum. (a) A spindle-shaped ICC showing multiple tortuous slender cytoplasmic processes. There is a fibroblast (F). (b) Two ICC processes lying close together and making true gap junctions (arrowheads). The ultrastructure of ICC is evident from a and b. Abundant mitochondria (Mi), numerous caveolae (small arrows), sharp basal lamina (large arrows), rough endoplasmic reticulum (rER), scattered intermediate filaments (IF), and rare cytoplasmic dense bodies (d).
with neighboring SMC (Figure 2). Bundles of enteric nerve fibers lay close to ICC-MY (Figures 2c,d and 3a,b,c), although intimate relationships could not be observed. Most ICC-MY were stellate-shaped, often had 2 main processes, and often had long, slender secondary and tertiary ramifications. The processes of 2 to 5 cells were organized into bundles (Figure 2c), the surrounded nerves making close contact with them. A typical nerve-ICC-SMC structure (Figure 2c, 3a) was observed. The ICC-MY cells often surrounded blood vessels with long processes (Figure 2e). The ICC within the longitudinal and circular muscle layers contained many mitochondria, caveolae, and rough endoplasmic reticulum. These ICC were consid-
Results In all samples, the ICC were exactly identified. These ICC exhibited typical ultrastructural features of ICC already described in mammals. They showed numerous mitochondria and abundant intermediate filaments. The nuclei were fusiform, oval, or indented with a dense band of peripheral heterochromatin (Figure 1a,b). Morphologically, they were spindle- or stellate-shaped, with a different number of ramified cell processes. Some ICC exhibited typical features of SMC, including a basal lamina, caveolae, and dense bodies (Figure 1b). However, these ICC did not have thick myosin filaments like the SMC. In the myenteric plexus, ICC were easily identified surrounding the myenteric ganglia (Figure 2c, 3a). This subtype of ICC was named ICC-MY. The ICC-MY formed gap junctions with each other (Figure 1b) and
Figure 2. Myenteric interstitial cells of Cajal (ICC-MY) in the ileum of chicken. (a) A myenteric ICC forming gap junction with 2 smooth muscle cells (SMC). (b) Magnified view of the box shown in panel a. Note the gap junction between ICC and SMC (arrowheads), the SMC showing scattered mitochondria (Mi), and thick myosin filaments (TF). (c) Multiple ICC lying closer to the nerve fibers (NF) and SMC. (d) Higher magnification of the boxed area in panel c showing a gap junction (arrowheads) between ICC and SMC. (e) A spindleshaped bipolar ICC targeting the blood vessel (BV) and the vascular endothelium (VE). Another irregular-shaped ICC lying in the vicinity of the BV. Note a nucleated avian red blood cell (RBC).
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We used 10 normal adult Chinese Three Yellow broilers (English translation of the local Chinese chicken breed), between 7 and 9 wk of age, weighing 1.8 to 2 kg in current study. The feeding was done with commercially available feeds (New Hope, Chengdu, China) for adult broiler chickens. They were housed in temperature-controlled rooms (20 ± 1°C) with natural light (the natural light/dark cycle 12L:12D at the time of the study) and had free access to food and tap water. The chickens were killed by cervical dislocation, under ether anesthesia. The experiment has been approved by the Chinese Committee for Animal Use for Research and Education. Tissue samples were collected from the ileum and immersed in 2.5% glutaraldehyde fixative in 0.1 M PBS (4°C, pH 7.4) for one hour and washed in the same buffer overnight. The tissues were postfixed with a mixture of 1% osmiumtetroxide and 1.25% potassium ferrocyanide for 1.5 to 2 h, washed in the buffer, dehydrated in increasing concentrations of ethyl alcohol, infiltrated with a propylene oxide/Araldite mixture (Boster, Wuhan, China), and embedded in Araldite. Semithin sections of intestine were stained with 1% toluidine blue for light microscopic examination, and specific areas were chosen for ultrathin sectioning. Sections were cut using an ultramicrotome (Reichert-Jung, Wien, Austria). Ultrathin sections (50 nm) were collected on copper grids, counterstained for 10 min with 1% uranyl acetate and Reynold’s lead citrate, and then observed in a JEM-1200EX electron microscope (Jeol, Tokyo, Japan). All sections that contained ICC were analyzed and all ultrastructural characteristics were recorded. Semiquantification was done using – for absent; − + for occasionally present; and +, ++, +++ for constantly present with increasing richness.
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Figure 3. Typical muscle-interstitial cells of Cajal (ICC)-nerve fiber structure. (a) A stellate-shaped ICC forming multiple points of close contacts with smooth muscle cells (SMC) and nerve fibers (NF). (b) Magnified view of the upper box in a, showing the close contacts (arrowheads) with SMC. (c) The lower box in a magnified view. Note the close membrane associations between the ICC and NF.
Figure 4. Intramuscular interstitial cells of Cajal (ICC-IM) contacts with smooth muscle cells (SMC) and nerve fibers. (a) A stellateshaped ICC-IM with 2 long processes and many short processes, traversing between SMC. (b) The magnified view of small box in panel a. Note a big caveola (double arrowheads) at the end of the ICC process. (c) A magnified view of the ICC-IM in big box in panel a, showing the close contacts (arrowheads) with SMC. (d) A rounded ICC-IM lying between the SMC and nerve fibers (NF). It makes multiple points of close contacts (arrowheads) with the ICC and SMC.
glia of the submucosal plexus (Figure 6a). The ICC-SM surrounded perivascular cells of the blood vessels (Figure 6b). They also occasionally formed peg-and-socketlike junctions with neighboring SMC (Figure 6c). In addition, a new subtype of ICC was found in the lamina propria of the ileum and named ICC-LP according to their location. These cells had a stellate morphology with multiple branches. The branches made frequent close contacts with each other, forming a network and close contact with neighboring fibroblasts (Figure 7a). These ICC have abundant intermediate filaments and were easily distinguished from the neighboring fibroblasts (Figure 7b). In all samples, ICC were distinguished from the more numerous fibroblasts. However the fibroblasts had a close and constant association with collagen bundles, formed close contacts with neighboring SMC, blood vessels, and enteric nerves like the ICC. Fibroblasts were surrounded by elastic fibers, had oval-shaped nuclei with less peripheral heterochromatin, and a variable amount of perinuclear cytoplasm. Fibroblasts had numerous thin processes, well-developed secretory organelles (golgi, rER), including coated vesicles and
Figure 6. Interstitial cells of Cajal (ICC) found in the submucosal plexus (ICC-SMP). (a) ICC-SMP triplet uniting each other and surrounding the nerve fibers (NF). (b) Two ICC-SMP are seen targeting the blood vessel near the smooth muscle cell (SMC). Also note the vascular pericyte (PC), vascular endothelial cell (VE), and a nucleated red blood cell (RBC). (c) ICC-SM making a peg-and-socket-like junction with SMC (asterisk).
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ered intramuscular-ICC (ICC-IM). The ICC-IM displayed the same ultrastructural features, had mainly spindle shape with 2 long processes (Figure 4a), and stellate shape with 2 long processes and many short processes (Figure 4a). The endings of processes often formed a big caveola (Figure 4b). The ICC of the longitudinal layer formed distinct close contacts, not gap junctions, with neighboring ICC, with adjacent SMC (Figure 4c), and with enteric nerves (Figure 4d). Numerous ICC were found in the deep muscular plexus along the border of the circular muscle bundles, within the septa that separated the bundles. This subtype of ICC was named ICC-DMP. These cells aggregated to form small bundles containing 2 to 3 cells surrounding nerve endings (Figure 5a) making close contact with SMC (Figure 5b). These cells were stellate-shaped and often had slender secondary and tertiary processes. Many ICC were found along the submucosal surface of the circular muscle layer. They were identified as ICC-SM. This subtype of ICC contained numerous mitochondria, reduced number of caveolae, distinct basal lamina, and abundant rough and smooth endoplasmic reticula. The ICC-SM were arranged around the gan-
Figure 5. The interstitial cells of Cajal (ICC) located in deep muscular plexus (ICC-DMP). (a) Stellate-shaped ICC giving-off multiple slender processes to smooth muscle cells (SMC). (b) Higher magnification of the boxed area in panel a. Note the close contacts (arrowheads) of ICC with SMC, mitochondria (Mi), rough endoplasmic reticulum (rER), and basal lamina (large arrows) in the ICC process.
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SUBTYPES OF INTERSTITIAL CELLS OF CAJAL
Figure 7. The interstitial cells of Cajal (ICC) in lamina propria (ICC-LP). (a) A spindle-shaped bipolar ICC and an accompanying fibroblast (F) are seen. (b) A magnified view of the boxed area in panel a, showing the fibroblast (F) and the intermediate filaments (IF) scattered in the cytoplasm of ICC.
DISCUSSION Although chicken has a great economic and scientific impact, no extensive study has been performed on ICC in these birds. Chicken ICC have not been fully characterized, subtyped, and distinguished from fibroblast and SMC by transmission electron microscopy. Since Faussone-Pellegrini and Thuneberg (1999) provided the characteristics of typical ICC, electron microscopy became the most reliable method of identification of these cells. According to previous reports of Sanders (1996) and Faussone-Pellegrini and Thuneberg (1999), we have found the ultrastructure of the chicken ICC to be very similar to that of mammals. We were able to identify all subtypes of ICC described in mammals; furthermore, we report the first submucosal subtype (ICC-SM) in a bird. In early studies, Lecoin et al. (1996) and Reynhout and Duke (1999) described typical ICC in the digestive tract of birds, but no submucosal type had been identified. In addition, in this study, a completely new subtype of ICC located in the lamina propria mucosae (ICCLP) was discovered. Kunisawa and Komuro (2008) suggested ICC-SM may play a role in the regulation of
the secretion, absorption, and transportation of fluids in the mucosa in guinea pigs. The slender cytoplasmic processes of ICC-SM found in chicken closely surround the submucosal ganglia. This suggests a similar role as seen in guinea pigs. Studies have revealed that the primary targets of the nitrergic neurons are ICC located within the muscle bundles in intestine (Horiguchi et al., 2003; Cserni et al., 2009; Iino et al., 2009). The close contact of the myeneteric ICC with both the muscle bundles and the nerve plexus found in the chicken intestine indicates their active involvement in the regulation of motor activity. Imaizumi and Hama (1969) reported gap junctions between ICC and nerve bundles in love birds, but this has not been observed in our study in chickens. Although we discovered true gap junctions among ICC and between ICC and SMC in chickens, Reynhout and Duke (1999) found gap junctions only between ICC in turkey. It is interesting that gap junctions are common in mammals but do not exist in reptiles and amphibians (Junquera et al., 2001; Miyamoto-Kikuta and Komuro, 2007). This may be explained by evolution. It is less likely that differences in gap junctions in birds can be explained by evolution. More likely, this comes from technical reasons, different thickness of the specimens and limitation of the electron microscopy. The demonstration of close contacts between nerve and SMC suggests that the tunica muscularis of the chicken ileum has parallel pathways for direct and in-
Table 1. Ultrastructural features characterizing the interstitial cells of Cajal (ICC) at the various subtypes in the chicken ileum1,2 Item Rough endoplasmic reticulum Smooth endoplasmic reticulum Mitochondria Intermediate filaments Caveolae Basal lamina 1Symbols: 2MY
ICC-MY
ICC-IM
ICC-DMP
ICC-SM
ICC-LP
+ ++ +++ ++ ++ Discontinuous
+ ++ ++ + + Discontinuous
+ ++ ++ ++ ++ Discontinuous
++ ++ ++ + + Discontinuous
−+ + + +++ −+ Discontinuous
− + = occasionally present; +, ++, +++ = constantly present with increasing richness. = myenteric ganglia; IM = intramuscular; DMP = deep muscular plexus; SM = submucosal surface; LP = lamina propria.
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patchy basal lamina, but lacked prominent intermediate filaments and caveolae. (Figure 8a,d). The ultrastructural characteristics of the different ICC subtypes are summarized in Table 1.
Figure 8. Fine ultrastructure of the fibroblasts (F) in the chicken ileum. (a) A fibroblast with oval nucleus and short processes. (b) A fibroblast at higher magnification, showing the coated vesicles (large arrows).
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ACKNOWLEDGMENTS This study was supported by grants (No. 30871833 and 31172282) from the National Science Foundation of China and Priority Academic Program Development of Jiangsu Higher Education Institutions.
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direct innervation of the smooth muscle, as reported in the digestive tract of mammals (Daniel et al., 1998; Brooks et al., 2005; Rumessen et al., 2009). The features of the structure reveal that the ICC adapt as the intermediary of the nervous system and SMC that can control gastrointestinal function. The close connection revealed between nerves and ICC supports the hypothesis reported by Wang et al. (1999), Iino et al. (2009), and Mikkelsen (2010), that nerves might have a functional effect on ICC in regulating the gastrointestinal smooth muscles via neurotransmitters. Thuneberg and Peters (2001) have proposed that peg-and socket-like junctions play a crucial role in the coordination of smooth muscle and pacemaking in mice. Peg-and socket-like junctions were revealed in chicken intestine as well between ICC and SMC. The close contacts between the ICC and SMC vascular wall in our study have not been reported in birds. This new finding indicates ICC regulate intestinal microcirculation. The ICC, SMC, and fibroblasts may look very similar; there is no single feature that identifies ICC properly. In our study, several ultrastructural criteria helped us to separate ICC from fibroblasts and SMC. We have found the presence of intermediate filaments, caveolae, and basal lamina most reliable to distinguish an ICC from a fibroblast and the absence of thick filaments to differentiate ICC from SMC. The ICC may be differentiated from fibroblasts based on the presence of gap junctions and the connections with nerve bundles. However, Komuro et al. (1999) noted that fibroblastlike cells may have gap junctions with SMC. In our study, we first identified the various subtypes of ICC in chicken ileum based on ultrastructural criteria. A new subtype in the lamina propria mucosae has been identified. The location and the connections between the neighboring ICC, SMC, nerve bundles, and vessels indicate they play a similar role in gastrointestinal motility as has been reported in mammals.