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Distribution of the interstitial Cajal-like cells in the gallbladder and extrahepatic biliary duct of the guinea-pig
ARTICLE IN PRESS Acta histochemica 111 (2009) 157—165
www.elsevier.de/acthis
Distribution of the interstitial Cajal-like cells in the gallbladder and extrahepatic biliary duct of the guinea-pig Yue Huanga, Feng Meia, Bin Yua, Hong-jun Zhanga, Juan Hana, Zhong-yong Jianga, De-shan Zhoua,b, a
Department of Histology and Embryology, Third Military Medical University, Chongqing 400038, China Department of Histology and Embryology, Capital University of Medical Sciences, Beijing 100054, China
b
Received 6 March 2008; received in revised form 30 April 2008; accepted 7 May 2008
KEYWORDS Interstitial Cajal-like cells; Ampulla; Gallbladder; Bile duct; Interstitial cells of Cajal
Summary It has been suggested that interstitial Cajal-like cells (ICLC) may be involved in the spontaneous rhythmic electrical activities of the extrahepatic bile duct system. The present study investigated the distribution and characteristics of ICLC, which are immunopositive for CD117/ Kit receptor tyrosine kinase, using immunohistochemistry employing a monoclonal antibody raised against CD117/Kit on whole-mount preparations. The Kit-positive ICLC were examined using confocal laser scanning microscopy or fluorescence microscopy. ICLC, immunoreactive for Kit, were pleiomorphic and/or spindle-shaped cells with a few bipolar processes and distributed in the smooth muscle layers of the gallbladder and bile duct system. They were scattered in the hepatic duct, cystic duct and gallbladder as well as in the upper part of the common bile duct. The ICLC gradually increased in number and formed a completed cellular network in the lower part of the common bile duct and ampulla. The numbers of ICLC in the ampulla were similar to that of the duodenum and significantly much greater in number than in the gallbladder and bile ducts. The density of the ICLC in the common bile duct was significantly higher than that of other bile ducts. Our results suggested that the ICLC might contribute to the regulation of the spontaneous rhythmic contraction and development of motility disorders of the bile duct system. & 2008 Elsevier GmbH. All rights reserved.
Corresponding author at: Department of Histology and Embryology, Capital University of Medical Sciences, Beijing 100054, China.
Fax: +86 23 6546 3259. E-mail address: [email protected] (D.-s. Zhou). 0065-1281/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2008.05.005
ARTICLE IN PRESS 158
Introduction In the gastrointestinal tract, a group of special interstitial cells, named interstitial cells of Cajal (ICCs), are the pacemaker cells that generate and propagate the slow waves (Kito et al., 2005) and play an important role in the regulation of gastrointestinal motility. They are responsible for the spontaneous rhythmic contraction of the gastrointestinal smooth muscle cells (SMCs) (Liu et al., 1998). Resembling the gastrointestinal tract, the bile duct system also shows spontaneous rhythmic motility, but the underlying mechanism is not clear. It has been reported that interstitial Cajal-like cells (ICLC) are present in some non-enteric organs such as pancreas (Popescu et al., 2005a), myometrium (Ciontea et al., 2005; Popescu et al., 2007), Fallopian tube (Popescu et al., 2005b), mammary gland (Radu et al., 2005) and human atrial (Hinescu et al., 2006) and ventricular myocardium (Lavoie et al., 2007). Like ICCs in the gastrointestinal tract, the ICLC are also believed to be responsible for spontaneous rhythmic electrical activities and play an important role in the regulation of the rhythmic motilities in these organs. The ICLC resemble the archetypal enteric ICCs also present in the human gallbladder (Hinescu et al., 2007) and some malignant human gallbladder tumors (Thomas, 2007); however, a detailed study of the features and distribution of ICLC in the mammalian bile duct system has not been performed. Many investigations have been carried out to understand the architecture of the musculature and neural distributions of the biliary system, including the gallbladder, cystic duct, hepatic duct, common bile duct and ampulla (Cai and Gabella, 1983a, b). The dysfunctions (e.g. pace-making, secretion) of the biliary system are thought to be associated with some pathologies that lead to a variety of diseases, such as pancreatitis, cholestasis, stone formation and dysfunction of the sphincters of Oddi. Therefore, investigation of the features of ICLC in mammalian bile ducts and gallbladder may help in the understanding of (dys) functions of the bile duct system.
Materials and methods Animals Ten adult guinea pigs (both males and females, weighing 250–350 g) were purchased from Animal Center of Third Military Medical University (Chongqing, China). All guinea pigs were housed in a local facility for laboratory animal care and fed
Y. Huang et al. with the special guinea pig feed. The experiments animals were performed in accordance with Health Guide for the Care and Use of Laboratory Animals of our University and the Institutional Animal Care Committee approved the work.
Immunohistochemistry Animals were killed by an overdose of pentobarbital sodium (Nembutal 50 mg/g) and the gallbladder, cystic duct, hepatic duct, common bile duct, ampulla and duodenum were removed. After the luminal contents had been washed away with phosphate-buffered saline, the specimens were quickly frozen with liquid nitrogen in optimal cutting temperature compound. Around 6–8 mmthick sections were cut using a cryostat (Leica, 1850) and fixed with 100% acetone for 15 min at 4 1C. To make whole-mount preparations, the gallbladder, bile ducts and duodenum were inflated with acetone for 30 min and then the mucosa was removed and the smooth muscle layer was prepared under a dissection microscope. The immunolabeling procedures have been described previously (Mei et al., 2006). Briefly, the ICLC were identified by using a rat monoclonal antibody raised against CD117/Kit (ACK2, 5 mg/ml, eBioscience, San Diego, USA) and the specimens were incubated with this for 24 h at 4 1C. The immunoreactivity was detected by using a Cy3conjugated secondary antibody (anti-rat IgG, 1:100, Zymed, San Francisco, USA) or a peroxidase-conjugated secondary antibody (anti-rat IgG, 1:100, DAKO, Produktionsvej, Denmark) and were incubated with these reagents for 60 min at 4 1C. The SMCs were labeled using a mouse monoclonal antibody against a-smooth muscle actin (a-SMA, 1:100, Santa Cruz, USA) and were incubated with this reagent for 24 h at 4 1C. The immunoreactivity was labeled by an FITC-conjugated secondary antibody (anti-mouse IgG, 1:100, DAKO, Produktionsvej, Denmark) and was incubated for 60 min at 4 1C. The horseradish peroxidase reaction was developed in a solution containing 0.05% of 3,30 diaminobenzidine tetrahydrochloride (DAB, Sigma, Saint Louis, USA) in 0.05 M Tris–HCl buffer (pH 7.6) with 0.3% H2O2 for 10 min at 25 1C and then the cryosections were counterstained with hematoxylin, mounted with glycerol gelatin and photographed using a BX51 light microscope (Olympus Japan). The immunofluorescence labeled preparations, including the FITC or Cy3-labeled preparations, were directly mounted with glycerol gelatin and examined using a TCS SP5 confocal laser
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scanning microscope (Leica, Germany) or a BX51 fluorescence microscope (Olympus, Japan) with an excitation wavelength being appropriate for Cy3 (552 nm) or FITC (488 nm). The control specimens were labeled as described above but with omission of incubation with the primary antibody. In these controls, no labeling was seen.
Measurement and statistical analysis Sixteen sections were sampled from a series of 80 sections taken from each animal. This was done systematically with the first section being selected randomly, then every fifth section being taken thereafter. The Kit-immunopositive cells in the different locations were counted by using ImagePro Plus 5.0 (Media Cybernetics, Silver Spring, USA) on the sections and data were expressed as means7S.E.M. Difference in the data was evaluated by pairwise comparison analysis of variance, and Po0.05 was taken as a statistically significant difference.
Results In gross view, the whole extrahepatic bile duct system was composed of the pear-shaped gallbladder, cystic duct, left and right hepatic ducts, common bile duct and ampulla of Vater which was situated in the wall of duodenum.
Gallbladder In the gallbladder, the Kit-immunopositive ICLC, about 6.5–8.0 mm in diameter, were mainly distributed in the SMC bundles, which intermingled extensively and made various angles with each other. The ICLC were fusiform in shape, ran mainly parallel with the SMC and had 15–50 mm bipolar processes without branches on the whole-mount preparations (Figure 1A). The numbers of ICLC showed a gradual increase from the fundus to neck of the gallbladder. In addition, many mast cells, also immunolabeled positively for Kit, were observed in the mucosa of the biliary system. Most of them did not show obvious processes, but occasionally very short processes were identified (Figure 1B).
Hepatic duct and cystic duct In the cryo-sections and whole-mount preparations, the ICLC with one or two processes, 70–80 mm long, were usually observed in the muscular layer
Figure 1. Confocal photomicrographs showing Kit-immunopositive ICLC cells distributed in the gallbladder, hepatic duct and cystic duct of guinea pigs on wholemount preparations: (A) Kit-immunopositive ICLC in the smooth muscle layer of the gallbladder are characteristic with bipolar thick processes; (B) many mast cells with round cell bodies were mainly seen in the mucosa; (C) ICLC with one process observed in the hepatic duct; (D) in the cystic duct, ICLC are characterized by small cell bodies with slender processes. Scale bars; A ¼ 10 mm, B–D ¼ 25 mm.
of the hepatic duct and cystic duct. They were found dispersed in the muscular bundles (Figure 1C and D). Kit-immunopositive mast cells were also seen in the mucosa.
Common bile duct In the upper portion of the common bile duct, the SMC mainly ran in a circular direction and the ICLC were distributed in the muscular bundles. They possessed bipolar processes, about 50–100 mm in length, and had oval-shaped cell bodies 7.2–9.4 mm in diameter. They ran mainly parallel with the direction of the circular muscle but a few ICLC ran parallel to the longitudinal direction on the whole-mount preparations (Figure 2A and B). Near the beginning of the middle part of the common bile duct, longitudinal smooth muscle cells gradually appeared and the circular muscle coat became much thicker. In this portion, ICLC numbers were gradually increased and formed a cellular network (Figure 2C). These cells with extended
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Figure 2. Whole-mount preparations with immunolabeling showing ICLC in the common bile duct. The numbers of ICLC are gradually increased from the upper (A, B) to lower parts (E, F). A, The ICLC were scattered in a circular direction at the upper part of the common bile duct, and a few ICLC (arrowheads) began to appear in the longitudinal direction near the middle part (B). The ICLC increased in density (C) and were arranged in both longitudinal and circular directions in the middle part of the common bile duct (D). A large number of ICLC with long processes forming a cellular network are seen in the lower part of the common bile duct (E) and adjacent to the ampulla (F). Scale bars ¼ 100 mm.
processes measuring about 100–150 mm in length were arranged in both longitudinal and circular directions (Figure 2D). At the lower part of the common bile duct, a large number of ICLC with few processes formed a completed cellular network
between the longitudinal and circular smooth muscle layers. The bile duct opened into a chamber, the ampulla, situated within the duodenal wall where many ICLC were also seen in the smooth muscle layers (Figure 2F).
ARTICLE IN PRESS Distribution of the interstitial Cajal-like cells
Ampulla The ampulla consisted of both outer and inner walls (Figure 3A). The outer wall of the ampulla was difficult to distinguish from that of the
Figure 3. Kit immunoperoxidase labeling showing the ICLC in the ampulla (counterstain haematoxylin) in longitudinal sections: (A) The common bile duct opening into a chamber, the ampulla, situated in the wall of the duodenum consisted of an outer wall and an inner wall; a large number of ICLC distributed in and between the longitudinal and circular muscle layers of the outer wall (B); many ICLC also located in the inner wall (C). CML: circular muscle layer. LML: longitudinal muscle layer. Scale bars; A ¼ 1 mm, B ¼ 400 mm, C ¼ 200 mm.
161 duodenum and was also composed of longitudinal and circular smooth muscle layers. A large number of ICLC, running parallel with the SMCs, were often observed in the outer and inner walls of the ampulla (Figure 3B and C). They were spindle-like in shape with 2–3 slender processes, about 150–250 mm in length (Figure 4A). Some of them had oval or triangular cell bodies and projected more than three long processes between the longitudinal and circular smooth muscle layers. They were arranged into a cellular network which resembled the ICCs around the myenteric nervous plexus in the duodenum (Figure 4B). Many ICLC with enriched cytoplasm and long processes resembling the ICCs associated with the deep muscular plexus of the small intestine were seen in the inner side of the outer wall (Figure 4C). The inner wall of the ampulla had a rather complex muscular structure that mainly consisted of circular muscles. The aboral side of the common bile duct, where the ampulla opened into the duodenum, was surrounded by a distinct ring of muscle, the sphincter of Oddi (Figure 5A). Several longitudinal or oblique smooth muscle bundles were also seen in this region (Figure 5C and E). Immunohistochemical labeling showed that a large number of ICLC were mainly distributed in the smooth muscle layers (bundles) and lay parallel with them. In the ampulla, many ICLC were observed lying in circular and longitudinal or oblique directions. Most of them were characterized by 2–3 processes, 150–250 mm in length, but some ICLC had short branches and oval cell bodies of 7–9 mm in diameter (Figure 5B, D and F). They were arranged in parallel to the direction of the SMCs (Figure 6A). A few ICLC also possessed 3–5
Figure 4. Representative confocal images of Kit immunofluorescence in the ampulla on whole-mount preparations. The outer wall of the ampulla was difficult to distinguish from that of the duodenum. ICLC located in the smooth muscle layer are characterized by bipolar long process lying parallel to the smooth muscle cells (A); B, the ICLC arranged into a cellular network by their processes are observed between the longitudinal and circular muscle layers. A large number of ICLC with round cell bodies are seen in the inner side of the outer wall (C). Scale bars; A, B ¼ 250 mm, C ¼ 50 mm.
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Figure 5. Immunofluorescence labeling the distribution of a-SMA (A, C, E) and ICLC (B, D, F) in the inner wall of the ampulla on whole-mount preparations. The distinct ring of smooth muscle bundles around the common bile duct in the ampulla (A, arrows). A large number of ICLC within the muscle layer (B, arrows) are clearly visible; A thickened circular muscle layer (arrows) with several longitudinal or oblique smooth muscle bundles were seen in this area (C and E, arrowheads); D, the ICLC mainly run parallel with the direction of the circular muscle layer (arrows) and some ICLC lie parallel to the longitudinal muscle layer (arrowheads). Beside a large number of ICLC in the longitudinal and circular directions, some ICLC (arrowheads) are in oblique distribution in this location (F). Scale bars A–F ¼ 250 mm.
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Figure 6. Confocal images showing the ICLC within the smooth muscle layer of the inner wall of the ampulla on wholemount preparations. Most ICLC have bipolar long process (arrowheads) and lie parallel to the direction of smooth muscle cells (A); some ICLC having 3–5 processes with several branches (arrow) are also observed in this location (B); C showing a sparse cellular network of the ICLC connected by their cytoplasmic processes (arrowheads). Scale bars A–C ¼ 50 mm.
processes with few branches, about 100 mm in length, and formed a cellular network via their processes (Figure 6B and C).
Duodenum In the duodenum, at the level of the myenteric plexus between both longitudinal and circular muscle layers, the Kit-immunopositive cells (i.e. ICCs) were characterized by round or triangular cell bodies with 3–5 processes and formed a complete cellular network. The ICCs with enriched cytoplasm and projecting processes were also seen in the deep muscular plexus. Many intramuscular ICCs with bipolar processes lay parallel to the SMCs within the longitudinal and circular muscle layers.
Statistical analysis The numbers of ICLC in the ampulla were 15.773.94; fundus, body and neck of the gallbladder, 1.370.45, 2.670.18, 2.870.93, respectively; cystic duct, 3.270.98; hepatic duct, 1.470.39; common bile duct, 5.570.87; and duodenum, 12.472.07 (Figure 7). The statistical analysis revealed that the numbers of ICLC in the ampulla were similar to that of the duodenum and were significantly much greater in number than that of the gallbladder and bile ducts (Po0.05). The ICLC in the common bile duct were significantly different in number from those of the other bile ducts (Po0.05), while there was no significant difference between ICLC numbers between the gallbladder and other bile ducts.
Figure 7. The graph summarizes the mean numbers of ICLC in the whole extrahepatic bile duct system. Statistical analysis showed that the numbers of ICLC in the ampulla were greatest, followed by the duodenum. Both portions contained significantly larger numbers of ICLC than the gallbladder and bile ducts (Po0.05). The ICLC occurred in the common bile duct in significantly greater numbers than in the other bile ducts (Po0.05), while there was no significant difference between ICLC numbers between the gallbladder and other bile ducts.
Discussion The present study demonstrated that a large number of ICLC were located in the smooth muscle layers of the gallbladder and extrahepatic bile ducts and their features were similar to intramuscular ICC in the gastrointestinal tract. It has been reported that the intramuscular ICC often forms a close and synaptic-like connection with the
ARTICLE IN PRESS 164 varicosity of excitatory and inhibitory nerve ending in the small intestine (Ward and Sanders, 2001) and they may act as the pacemakers in the stomach (Beckett et al., 2003). Recently, the ICLC were identified in the gallbladder (Lavoie et al., 2007; Sun et al., 2006), urinary bladder (Shafik et al., 2004; Kubota et al., 2006), corporal tissue of penis (Hashitani and Suzuki, 2004) and portal vein (Harhun et al., 2004) and spontaneous electrical activity was also recorded in these tissues. Therefore, we propose that the ICLC in the bile duct system might be responsible for spontaneous rhythmic electrical activity and take part in the integration of nervous impulses involved in the regulation of the motility of the SMCs. It was noticeable that the ICLC appeared very variable in morphological appearance and numbers according to the portion of the biliary system in which they were located. The ICLC were scattered in the smooth muscle layers (bundles) of the gallbladder, cystic duct and hepatic duct and they gradually increased in density and number as the smooth muscle layers thickened. The number of ICLC in the common bile duct gradually increased from the upper part to the lower part and reached a maximum in number and density around the ampulla, particularly in the sphincter of Oddi. The ICLC were located within the smooth muscle layers in high density in the outer wall of the ampulla, and large numbers of ICLC were found in the inner wall of the ampulla, mainly in the circular muscle layer. These cells had longer processes with more branches and formed a completed cellular network in the ampulla where there was a thick, conspicuous circular muscle layer and some oblique or longitudinal smooth muscle cells. Moreover, the ICCs in the myenteric plexus and deep muscular plexus were also observed in the wall of the duodenum. These observations indicated that the ampulla possessed a more complex function and both the ICLC and the ICCs may synergistically control the storage and outflow of bile. Furthermore, the ICLC may also contribute to prevention of the reflux of bile or pancreatic juice through the pancreaticobiliary junction. Clinical studies have indicated that a reduction in the ICCs is closely associated with some gastrointestinal motility disorders, such as diabetic gastroparesis (Forster et al., 2005), slow transit constipation (Sabri et al., 2003), Crohn’s disease (Wang et al., 2007) and post-operational gut dysmotilities (Baig and Wexner, 2004). Sphincter of Oddi dysfunction often causes a chronic biliary duct pain or recurrent pancreatitis, but the underlying mechanism remains unclear. The contraction and relaxation of the sphincter of Oddi is believed
Y. Huang et al. to be controlled by nerves, hormones and local factors. The increased number and density of the ICLC in the ampulla and the lower part of the common bile duct strongly suggests that the ICLC could also contribute to the control of functions of the sphincter of Oddi and might be involved in the pathophysiologies of sphincter of Oddi dysfunction. Therefore, there is a need to further examine the relationship between the alterations of ICLC distribution and function and sphincter of Oddi dysfunction.
Acknowledgments This work was supported in parts by Grant nos. 30570983 and 06MA182 from the National Science Foundation of China (NSFC).
References Baig MK, Wexner SD. Postoperative ileus: a review. Dis Colon Rectum 2004;47:516–26. Beckett EA, McGeough CA, Sanders KM, Ward SM. Pacing of interstitial cells of Cajal in the murine gastric antrum: neurally mediated and direct stimulation. J Physiol 2003;553:545–59. Cai WQ, Gabella G. Innervation of the gall bladder and biliary pathways in the guinea-pig. J Anat 1983a;136: 97–109. Cai WQ, Gabella G. The musculature of the gall bladder and biliary pathways in the guinea-pig. J Anat 1983b; 136:237–50. Ciontea SM, Radu E, Regalia T, Ceafalan L, Cretoiu D, Gherghiceanu M, et al. C-kit immunopositive interstitial cells (Cajal-type) in human myometrium. J Cell Mol Med 2005;9:407–20. Forster J, Damjanov I, Lin Z, Sarosiek I, Wetzel P, McCallum RW. Absence of the interstitial cells of Cajal in patients with gastroparesis and correlation with clinical findings. J Gastrointest Surg 2005;9:102–8. Harhun MI, Gordienko DV, Povstyan OV, Moss RF, Bolton TB. Function of interstitial cells of Cajal in the rabbit portal vein. Circ Res 2004;95:619–26. Hashitani H, Suzuki H. Identification of interstitial cells of Cajal in corporal tissues of the guinea-pig penis. Br J Pharmacol 2004;141:199–204. Hinescu ME, Gherghiceanu M, Mandache E, Ciontea SM, Popescu LM. Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization. J Cell Mol Med 2006;10:243–57. Hinescu ME, Ardeleanu C, Gherghiceanu M, Popescu LM. Interstitial Cajal-like cells in human gallbladder. J Mol Hist 2007;38:275–84. Kito Y, Ward SM, Sanders KM. Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine. Am J Physiol Cell Physiol 2005;288:C710–20.
ARTICLE IN PRESS Distribution of the interstitial Cajal-like cells Kubota Y, Biers SM, Kohri K, Brading AF. Effects of imatinib mesylate (Glivec) as a c-kit tyrosine kinase inhibitor in the guinea-pig urinary bladder. Neurourol Urodyn 2006;25:205–10. Lavoie B, Balemba OB, Nelson MT, Ward SM, Mawe GM. Morphological and physiological evidence for interstitial cell of Cajal-like cells in the guinea pig gallbladder. J Physiol 2007;579:487–501. Liu LW, Thuneberg L, Huizinga JD. Development of pacemaker activity and interstitial cells of Cajal in the neonatal mouse small intestine. Dev Dyn 1998; 213:271–82. Mei F, Yu B, Ma H, Zhang HJ, Zhou DS. Interstitial cells of Cajal could regenerate and restore their normal distribution after disrupted by intestinal transection and anastomosis in the adult guinea pigs. Virchows Arch 2006;449(3):348–57. Popescu LM, Hinescu ME, Ionescu N, Ciontea SM, Cretoiu D, Ardeleanu C. Interstitial cells of Cajal in pancreas. J Cell Mol Med 2005a;9:169–90. Popescu LM, Ciontea SM, Cretoiu D, Hinescu ME, Radu E, Ionescu N, et al. Novel type of interstitial cell (Cajallike) in human fallopian tube. J Cell Mol Med 2005b;9:479–523. Popescu LM, Ciontea SM, Cretoiu D. Interstitial Cajal-like cells in human uterus and fallopian tube. Ann NY Acad Sci 2007;1101:139–65.
165 Radu E, Regalia T, Ceafalan L, Andrei F, Cretoiu D, Popescu LM. Cajal-type cells from human mammary gland stroma: phenotype characteristics in cell culture. J Cell Mol Med 2005;9:748–52. Sabri M, Barksdale E, Di LC. Constipation and lack of colonic interstitial cells of Cajal. Dig Dis Sci 2003;48: 849–53. Shafik A, El Sibai O, Shafik AA, Shafik I. Identification of interstitial cells of Cajal in human urinary bladder: concept of vesical pacemaker. Urology 2004;64: 809–13. Sun X, Yu B, Xu L, Dong W, Luo H. Interstitial cells of Cajal in the murine gallbladder. Scand J Gastroenterol 2006;41:1218–26. Thomas MB. Biological characteristics of cancers in the gallbladder and biliary tract and targeted therapy. Crit Rev Oncol Hematol 2007;61:44–51. Wang XY, Zarate N, Soderholm JD, Bourgeois JM, Liu LW, Huizinga JD. Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn’s disease. Neurogastroenterol Motil 2007;19: 349–64. Ward SM, Sanders KM. Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks. Am J Physiol Gastrointest Liver Physiol 2001;281:G602–11.
www.elsevier.de/acthis
Distribution of the interstitial Cajal-like cells in the gallbladder and extrahepatic biliary duct of the guinea-pig Yue Huanga, Feng Meia, Bin Yua, Hong-jun Zhanga, Juan Hana, Zhong-yong Jianga, De-shan Zhoua,b, a
Department of Histology and Embryology, Third Military Medical University, Chongqing 400038, China Department of Histology and Embryology, Capital University of Medical Sciences, Beijing 100054, China
b
Received 6 March 2008; received in revised form 30 April 2008; accepted 7 May 2008
KEYWORDS Interstitial Cajal-like cells; Ampulla; Gallbladder; Bile duct; Interstitial cells of Cajal
Summary It has been suggested that interstitial Cajal-like cells (ICLC) may be involved in the spontaneous rhythmic electrical activities of the extrahepatic bile duct system. The present study investigated the distribution and characteristics of ICLC, which are immunopositive for CD117/ Kit receptor tyrosine kinase, using immunohistochemistry employing a monoclonal antibody raised against CD117/Kit on whole-mount preparations. The Kit-positive ICLC were examined using confocal laser scanning microscopy or fluorescence microscopy. ICLC, immunoreactive for Kit, were pleiomorphic and/or spindle-shaped cells with a few bipolar processes and distributed in the smooth muscle layers of the gallbladder and bile duct system. They were scattered in the hepatic duct, cystic duct and gallbladder as well as in the upper part of the common bile duct. The ICLC gradually increased in number and formed a completed cellular network in the lower part of the common bile duct and ampulla. The numbers of ICLC in the ampulla were similar to that of the duodenum and significantly much greater in number than in the gallbladder and bile ducts. The density of the ICLC in the common bile duct was significantly higher than that of other bile ducts. Our results suggested that the ICLC might contribute to the regulation of the spontaneous rhythmic contraction and development of motility disorders of the bile duct system. & 2008 Elsevier GmbH. All rights reserved.
Corresponding author at: Department of Histology and Embryology, Capital University of Medical Sciences, Beijing 100054, China.
Fax: +86 23 6546 3259. E-mail address: [email protected] (D.-s. Zhou). 0065-1281/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2008.05.005
ARTICLE IN PRESS 158
Introduction In the gastrointestinal tract, a group of special interstitial cells, named interstitial cells of Cajal (ICCs), are the pacemaker cells that generate and propagate the slow waves (Kito et al., 2005) and play an important role in the regulation of gastrointestinal motility. They are responsible for the spontaneous rhythmic contraction of the gastrointestinal smooth muscle cells (SMCs) (Liu et al., 1998). Resembling the gastrointestinal tract, the bile duct system also shows spontaneous rhythmic motility, but the underlying mechanism is not clear. It has been reported that interstitial Cajal-like cells (ICLC) are present in some non-enteric organs such as pancreas (Popescu et al., 2005a), myometrium (Ciontea et al., 2005; Popescu et al., 2007), Fallopian tube (Popescu et al., 2005b), mammary gland (Radu et al., 2005) and human atrial (Hinescu et al., 2006) and ventricular myocardium (Lavoie et al., 2007). Like ICCs in the gastrointestinal tract, the ICLC are also believed to be responsible for spontaneous rhythmic electrical activities and play an important role in the regulation of the rhythmic motilities in these organs. The ICLC resemble the archetypal enteric ICCs also present in the human gallbladder (Hinescu et al., 2007) and some malignant human gallbladder tumors (Thomas, 2007); however, a detailed study of the features and distribution of ICLC in the mammalian bile duct system has not been performed. Many investigations have been carried out to understand the architecture of the musculature and neural distributions of the biliary system, including the gallbladder, cystic duct, hepatic duct, common bile duct and ampulla (Cai and Gabella, 1983a, b). The dysfunctions (e.g. pace-making, secretion) of the biliary system are thought to be associated with some pathologies that lead to a variety of diseases, such as pancreatitis, cholestasis, stone formation and dysfunction of the sphincters of Oddi. Therefore, investigation of the features of ICLC in mammalian bile ducts and gallbladder may help in the understanding of (dys) functions of the bile duct system.
Materials and methods Animals Ten adult guinea pigs (both males and females, weighing 250–350 g) were purchased from Animal Center of Third Military Medical University (Chongqing, China). All guinea pigs were housed in a local facility for laboratory animal care and fed
Y. Huang et al. with the special guinea pig feed. The experiments animals were performed in accordance with Health Guide for the Care and Use of Laboratory Animals of our University and the Institutional Animal Care Committee approved the work.
Immunohistochemistry Animals were killed by an overdose of pentobarbital sodium (Nembutal 50 mg/g) and the gallbladder, cystic duct, hepatic duct, common bile duct, ampulla and duodenum were removed. After the luminal contents had been washed away with phosphate-buffered saline, the specimens were quickly frozen with liquid nitrogen in optimal cutting temperature compound. Around 6–8 mmthick sections were cut using a cryostat (Leica, 1850) and fixed with 100% acetone for 15 min at 4 1C. To make whole-mount preparations, the gallbladder, bile ducts and duodenum were inflated with acetone for 30 min and then the mucosa was removed and the smooth muscle layer was prepared under a dissection microscope. The immunolabeling procedures have been described previously (Mei et al., 2006). Briefly, the ICLC were identified by using a rat monoclonal antibody raised against CD117/Kit (ACK2, 5 mg/ml, eBioscience, San Diego, USA) and the specimens were incubated with this for 24 h at 4 1C. The immunoreactivity was detected by using a Cy3conjugated secondary antibody (anti-rat IgG, 1:100, Zymed, San Francisco, USA) or a peroxidase-conjugated secondary antibody (anti-rat IgG, 1:100, DAKO, Produktionsvej, Denmark) and were incubated with these reagents for 60 min at 4 1C. The SMCs were labeled using a mouse monoclonal antibody against a-smooth muscle actin (a-SMA, 1:100, Santa Cruz, USA) and were incubated with this reagent for 24 h at 4 1C. The immunoreactivity was labeled by an FITC-conjugated secondary antibody (anti-mouse IgG, 1:100, DAKO, Produktionsvej, Denmark) and was incubated for 60 min at 4 1C. The horseradish peroxidase reaction was developed in a solution containing 0.05% of 3,30 diaminobenzidine tetrahydrochloride (DAB, Sigma, Saint Louis, USA) in 0.05 M Tris–HCl buffer (pH 7.6) with 0.3% H2O2 for 10 min at 25 1C and then the cryosections were counterstained with hematoxylin, mounted with glycerol gelatin and photographed using a BX51 light microscope (Olympus Japan). The immunofluorescence labeled preparations, including the FITC or Cy3-labeled preparations, were directly mounted with glycerol gelatin and examined using a TCS SP5 confocal laser
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scanning microscope (Leica, Germany) or a BX51 fluorescence microscope (Olympus, Japan) with an excitation wavelength being appropriate for Cy3 (552 nm) or FITC (488 nm). The control specimens were labeled as described above but with omission of incubation with the primary antibody. In these controls, no labeling was seen.
Measurement and statistical analysis Sixteen sections were sampled from a series of 80 sections taken from each animal. This was done systematically with the first section being selected randomly, then every fifth section being taken thereafter. The Kit-immunopositive cells in the different locations were counted by using ImagePro Plus 5.0 (Media Cybernetics, Silver Spring, USA) on the sections and data were expressed as means7S.E.M. Difference in the data was evaluated by pairwise comparison analysis of variance, and Po0.05 was taken as a statistically significant difference.
Results In gross view, the whole extrahepatic bile duct system was composed of the pear-shaped gallbladder, cystic duct, left and right hepatic ducts, common bile duct and ampulla of Vater which was situated in the wall of duodenum.
Gallbladder In the gallbladder, the Kit-immunopositive ICLC, about 6.5–8.0 mm in diameter, were mainly distributed in the SMC bundles, which intermingled extensively and made various angles with each other. The ICLC were fusiform in shape, ran mainly parallel with the SMC and had 15–50 mm bipolar processes without branches on the whole-mount preparations (Figure 1A). The numbers of ICLC showed a gradual increase from the fundus to neck of the gallbladder. In addition, many mast cells, also immunolabeled positively for Kit, were observed in the mucosa of the biliary system. Most of them did not show obvious processes, but occasionally very short processes were identified (Figure 1B).
Hepatic duct and cystic duct In the cryo-sections and whole-mount preparations, the ICLC with one or two processes, 70–80 mm long, were usually observed in the muscular layer
Figure 1. Confocal photomicrographs showing Kit-immunopositive ICLC cells distributed in the gallbladder, hepatic duct and cystic duct of guinea pigs on wholemount preparations: (A) Kit-immunopositive ICLC in the smooth muscle layer of the gallbladder are characteristic with bipolar thick processes; (B) many mast cells with round cell bodies were mainly seen in the mucosa; (C) ICLC with one process observed in the hepatic duct; (D) in the cystic duct, ICLC are characterized by small cell bodies with slender processes. Scale bars; A ¼ 10 mm, B–D ¼ 25 mm.
of the hepatic duct and cystic duct. They were found dispersed in the muscular bundles (Figure 1C and D). Kit-immunopositive mast cells were also seen in the mucosa.
Common bile duct In the upper portion of the common bile duct, the SMC mainly ran in a circular direction and the ICLC were distributed in the muscular bundles. They possessed bipolar processes, about 50–100 mm in length, and had oval-shaped cell bodies 7.2–9.4 mm in diameter. They ran mainly parallel with the direction of the circular muscle but a few ICLC ran parallel to the longitudinal direction on the whole-mount preparations (Figure 2A and B). Near the beginning of the middle part of the common bile duct, longitudinal smooth muscle cells gradually appeared and the circular muscle coat became much thicker. In this portion, ICLC numbers were gradually increased and formed a cellular network (Figure 2C). These cells with extended
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Figure 2. Whole-mount preparations with immunolabeling showing ICLC in the common bile duct. The numbers of ICLC are gradually increased from the upper (A, B) to lower parts (E, F). A, The ICLC were scattered in a circular direction at the upper part of the common bile duct, and a few ICLC (arrowheads) began to appear in the longitudinal direction near the middle part (B). The ICLC increased in density (C) and were arranged in both longitudinal and circular directions in the middle part of the common bile duct (D). A large number of ICLC with long processes forming a cellular network are seen in the lower part of the common bile duct (E) and adjacent to the ampulla (F). Scale bars ¼ 100 mm.
processes measuring about 100–150 mm in length were arranged in both longitudinal and circular directions (Figure 2D). At the lower part of the common bile duct, a large number of ICLC with few processes formed a completed cellular network
between the longitudinal and circular smooth muscle layers. The bile duct opened into a chamber, the ampulla, situated within the duodenal wall where many ICLC were also seen in the smooth muscle layers (Figure 2F).
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Ampulla The ampulla consisted of both outer and inner walls (Figure 3A). The outer wall of the ampulla was difficult to distinguish from that of the
Figure 3. Kit immunoperoxidase labeling showing the ICLC in the ampulla (counterstain haematoxylin) in longitudinal sections: (A) The common bile duct opening into a chamber, the ampulla, situated in the wall of the duodenum consisted of an outer wall and an inner wall; a large number of ICLC distributed in and between the longitudinal and circular muscle layers of the outer wall (B); many ICLC also located in the inner wall (C). CML: circular muscle layer. LML: longitudinal muscle layer. Scale bars; A ¼ 1 mm, B ¼ 400 mm, C ¼ 200 mm.
161 duodenum and was also composed of longitudinal and circular smooth muscle layers. A large number of ICLC, running parallel with the SMCs, were often observed in the outer and inner walls of the ampulla (Figure 3B and C). They were spindle-like in shape with 2–3 slender processes, about 150–250 mm in length (Figure 4A). Some of them had oval or triangular cell bodies and projected more than three long processes between the longitudinal and circular smooth muscle layers. They were arranged into a cellular network which resembled the ICCs around the myenteric nervous plexus in the duodenum (Figure 4B). Many ICLC with enriched cytoplasm and long processes resembling the ICCs associated with the deep muscular plexus of the small intestine were seen in the inner side of the outer wall (Figure 4C). The inner wall of the ampulla had a rather complex muscular structure that mainly consisted of circular muscles. The aboral side of the common bile duct, where the ampulla opened into the duodenum, was surrounded by a distinct ring of muscle, the sphincter of Oddi (Figure 5A). Several longitudinal or oblique smooth muscle bundles were also seen in this region (Figure 5C and E). Immunohistochemical labeling showed that a large number of ICLC were mainly distributed in the smooth muscle layers (bundles) and lay parallel with them. In the ampulla, many ICLC were observed lying in circular and longitudinal or oblique directions. Most of them were characterized by 2–3 processes, 150–250 mm in length, but some ICLC had short branches and oval cell bodies of 7–9 mm in diameter (Figure 5B, D and F). They were arranged in parallel to the direction of the SMCs (Figure 6A). A few ICLC also possessed 3–5
Figure 4. Representative confocal images of Kit immunofluorescence in the ampulla on whole-mount preparations. The outer wall of the ampulla was difficult to distinguish from that of the duodenum. ICLC located in the smooth muscle layer are characterized by bipolar long process lying parallel to the smooth muscle cells (A); B, the ICLC arranged into a cellular network by their processes are observed between the longitudinal and circular muscle layers. A large number of ICLC with round cell bodies are seen in the inner side of the outer wall (C). Scale bars; A, B ¼ 250 mm, C ¼ 50 mm.
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Figure 5. Immunofluorescence labeling the distribution of a-SMA (A, C, E) and ICLC (B, D, F) in the inner wall of the ampulla on whole-mount preparations. The distinct ring of smooth muscle bundles around the common bile duct in the ampulla (A, arrows). A large number of ICLC within the muscle layer (B, arrows) are clearly visible; A thickened circular muscle layer (arrows) with several longitudinal or oblique smooth muscle bundles were seen in this area (C and E, arrowheads); D, the ICLC mainly run parallel with the direction of the circular muscle layer (arrows) and some ICLC lie parallel to the longitudinal muscle layer (arrowheads). Beside a large number of ICLC in the longitudinal and circular directions, some ICLC (arrowheads) are in oblique distribution in this location (F). Scale bars A–F ¼ 250 mm.
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Figure 6. Confocal images showing the ICLC within the smooth muscle layer of the inner wall of the ampulla on wholemount preparations. Most ICLC have bipolar long process (arrowheads) and lie parallel to the direction of smooth muscle cells (A); some ICLC having 3–5 processes with several branches (arrow) are also observed in this location (B); C showing a sparse cellular network of the ICLC connected by their cytoplasmic processes (arrowheads). Scale bars A–C ¼ 50 mm.
processes with few branches, about 100 mm in length, and formed a cellular network via their processes (Figure 6B and C).
Duodenum In the duodenum, at the level of the myenteric plexus between both longitudinal and circular muscle layers, the Kit-immunopositive cells (i.e. ICCs) were characterized by round or triangular cell bodies with 3–5 processes and formed a complete cellular network. The ICCs with enriched cytoplasm and projecting processes were also seen in the deep muscular plexus. Many intramuscular ICCs with bipolar processes lay parallel to the SMCs within the longitudinal and circular muscle layers.
Statistical analysis The numbers of ICLC in the ampulla were 15.773.94; fundus, body and neck of the gallbladder, 1.370.45, 2.670.18, 2.870.93, respectively; cystic duct, 3.270.98; hepatic duct, 1.470.39; common bile duct, 5.570.87; and duodenum, 12.472.07 (Figure 7). The statistical analysis revealed that the numbers of ICLC in the ampulla were similar to that of the duodenum and were significantly much greater in number than that of the gallbladder and bile ducts (Po0.05). The ICLC in the common bile duct were significantly different in number from those of the other bile ducts (Po0.05), while there was no significant difference between ICLC numbers between the gallbladder and other bile ducts.
Figure 7. The graph summarizes the mean numbers of ICLC in the whole extrahepatic bile duct system. Statistical analysis showed that the numbers of ICLC in the ampulla were greatest, followed by the duodenum. Both portions contained significantly larger numbers of ICLC than the gallbladder and bile ducts (Po0.05). The ICLC occurred in the common bile duct in significantly greater numbers than in the other bile ducts (Po0.05), while there was no significant difference between ICLC numbers between the gallbladder and other bile ducts.
Discussion The present study demonstrated that a large number of ICLC were located in the smooth muscle layers of the gallbladder and extrahepatic bile ducts and their features were similar to intramuscular ICC in the gastrointestinal tract. It has been reported that the intramuscular ICC often forms a close and synaptic-like connection with the
ARTICLE IN PRESS 164 varicosity of excitatory and inhibitory nerve ending in the small intestine (Ward and Sanders, 2001) and they may act as the pacemakers in the stomach (Beckett et al., 2003). Recently, the ICLC were identified in the gallbladder (Lavoie et al., 2007; Sun et al., 2006), urinary bladder (Shafik et al., 2004; Kubota et al., 2006), corporal tissue of penis (Hashitani and Suzuki, 2004) and portal vein (Harhun et al., 2004) and spontaneous electrical activity was also recorded in these tissues. Therefore, we propose that the ICLC in the bile duct system might be responsible for spontaneous rhythmic electrical activity and take part in the integration of nervous impulses involved in the regulation of the motility of the SMCs. It was noticeable that the ICLC appeared very variable in morphological appearance and numbers according to the portion of the biliary system in which they were located. The ICLC were scattered in the smooth muscle layers (bundles) of the gallbladder, cystic duct and hepatic duct and they gradually increased in density and number as the smooth muscle layers thickened. The number of ICLC in the common bile duct gradually increased from the upper part to the lower part and reached a maximum in number and density around the ampulla, particularly in the sphincter of Oddi. The ICLC were located within the smooth muscle layers in high density in the outer wall of the ampulla, and large numbers of ICLC were found in the inner wall of the ampulla, mainly in the circular muscle layer. These cells had longer processes with more branches and formed a completed cellular network in the ampulla where there was a thick, conspicuous circular muscle layer and some oblique or longitudinal smooth muscle cells. Moreover, the ICCs in the myenteric plexus and deep muscular plexus were also observed in the wall of the duodenum. These observations indicated that the ampulla possessed a more complex function and both the ICLC and the ICCs may synergistically control the storage and outflow of bile. Furthermore, the ICLC may also contribute to prevention of the reflux of bile or pancreatic juice through the pancreaticobiliary junction. Clinical studies have indicated that a reduction in the ICCs is closely associated with some gastrointestinal motility disorders, such as diabetic gastroparesis (Forster et al., 2005), slow transit constipation (Sabri et al., 2003), Crohn’s disease (Wang et al., 2007) and post-operational gut dysmotilities (Baig and Wexner, 2004). Sphincter of Oddi dysfunction often causes a chronic biliary duct pain or recurrent pancreatitis, but the underlying mechanism remains unclear. The contraction and relaxation of the sphincter of Oddi is believed
Y. Huang et al. to be controlled by nerves, hormones and local factors. The increased number and density of the ICLC in the ampulla and the lower part of the common bile duct strongly suggests that the ICLC could also contribute to the control of functions of the sphincter of Oddi and might be involved in the pathophysiologies of sphincter of Oddi dysfunction. Therefore, there is a need to further examine the relationship between the alterations of ICLC distribution and function and sphincter of Oddi dysfunction.
Acknowledgments This work was supported in parts by Grant nos. 30570983 and 06MA182 from the National Science Foundation of China (NSFC).
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