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Identification of interstitial cells of Cajal in human urinary bladder: Concept of vesical pacemaker
BASIC SCIENCE
IDENTIFICATION OF INTERSTITIAL CELLS OF CAJAL IN HUMAN URINARY BLADDER: CONCEPT OF VESICAL PACEMAKER AHMED SHAFIK, OLFAT EL-SIBAI, ALI A. SHAFIK,
AND
ISMAIL SHAFIK
ABSTRACT Objectives. To determine whether, given the presence of interstitial cells of Cajal (ICCs) in organs that discharge slow waves, such as the gastrointestinal tract, such cells could also be present in the human urinary bladder (UB) and might exist in great numbers in the bladder dome, constituting the “primary pacemaker” from which slow waves spread to the body of the UB. This hypothesis was based on previous studies that had demonstrated the vesical electric waves starting in the dome and spreading caudad. Methods. Specimens (0.5 ⫻ 0.5 cm) from different vesical locations were obtained from the cystectomized UB of 18 patients (12 men, 6 women, mean age 42.6 ⫾ 3.8 years) with bladder cancer. Fixed sections were prepared and subjected to c-kit immunohistochemistry. Controls for the antiserum specificity consisted of incubation of the tissue with normal rabbit serum substituted for the primary antiserum. Results. Fusiform, c-kit positive, and ICC-like cells were detected in the vesical muscle layers; they had dendritic processes. They occurred separately or were connected through the dendritic processes to form a cellular network. An accumulation of ICCs surrounded by a connective tissue layer was detected in all the dome specimens. Mast cells occurred in the vesical mucosa and submucosa; they were c-kit positive but had a rounded body with no dendritic processes. Conclusions. We identified cells in the UB with morphologic and immunologic phenotypes similar to the ICCs of the gut. An accumulation of these cells, detected in the vesical dome, seems to constitute the “primary” vesical pacemaker that initiates the slow waves that spread to the other vesical walls. We believe that a deficiency or absence of these cells may be involved in vesical motility disorders. Additional studies are needed to delineate the role of these cells in UB physiology and pathologic conditions. UROLOGY 64: 809–813, 2004. © 2004 Elsevier Inc.
T
he smooth muscle cells of the urinary bladder (UB) appear to generate a resting vesical tone as evidenced by electrovesicography.1– 4 This tone seems to be myogenic in nature and associated with the generation of spontaneous transient depolarizations and regularly occurring slow waves (SWs).1– 4 This depolarizing current appears to be initiated by the spontaneous release of calcium from the intracellular stores, thereby setting off a calcium-activated chloride From the Department of Surgery and Experimental Research, Cairo University Faculty of Medicine, Cairo; and Department of Surgery, Menoufia University Faculty of Medicine, Shebin El-Kom, Egypt Reprint requests: Ahmed Shafik, M.D., Ph.D., Department of Surgery and Experimental Research, Cairo University Faculty of Medicine, 2 Talaat Harb Street, Cairo 11121, Egypt Submitted: March 3, 2004, accepted (with revisions): May 20, 2004 © 2004 ELSEVIER INC. ALL RIGHTS RESERVED
current that provides the depolarizing current responsible for the SWs.5,6 The exact source of the SWs recorded from the UB has not been determined. However, when it was demonstrated during mucosa-sparing vesical myotomies in a previous study that the SWs propagated caudad from the dome toward the trigone, we postulated that these SWs arise from a pacemaker located in the vesical dome.1 The discovery of the selective expression of the kit receptor in interstitial cells of Cajal (ICCs) within the gastrointestinal tract and its role in the normal development of the ICC system and pacemaker activity has produced a new tool for the investigation of these cells.7 The proto-oncogene c-kit is the cellular homologue of oncogene v-kit of HZ4 feline sarcoma virus.8 The c-kit encodes a transmembrane tyrosine kinase receptor and is located on human chromosome number 4. 0090-4295/04/$30.00 doi:10.1016/j.urology.2004.05.031 809
Given the presence of ICCs in organs that discharge spontaneous SWs, such as the gastrointestinal9 –12 and urinary tracts,13–15 we hypothesized that such cells also exist in the human UB and accumulate in the vesical dome, constituting the primary pacemaker from which the SWs spread to the remaining body of the UB. This hypothesis was investigated in the current study. MATERIAL AND METHODS We studied 0.5 ⫻ 0.5-cm vesical specimens obtained from the UB of 18 patients (12 men and 6 women, mean age 42.6 ⫾ 3.8 years, range 38 to 49) with bladder cancer after cystectomy. The specimens were taken from the dome, anterior and posterior walls, and the trigone of the UB away from the tumor sites. Patients with bilharzial bladder cancer were excluded from the study. The physical examination findings of the patients were normal. Laboratory work, including blood count, renal and hepatic function tests, and electrocardiography showed normal results. All patients provided informed written consent, and the Review Board and Ethics Committee of the Cairo University Faculty of Medicine approved the study. The specimens were placed in 2% buffered paraformaldehyde for 1 hour, cytoprotected in 30% sucrose overnight, and flash frozen in liquid nitrogen-cooled isopentane. Tissues were cut in 5-m frozen sections, mounted on polylysinecoated slides, and kept frozen until immunocytochemical processing. For immunocytochemical processing, the tissue sections were rehydrated in KPBS at room temperature for 20 minutes, blocked with 10% normal goat serum for 20 minutes, and incubated overnight with the primary antiserum, a rabbit polyclonal IgG antibody to the human c-kit protein (Oncogene Research Products, Cambridge, Mass), diluted 1:100 in KPBS, 0.05% goat serum, and 0.1% Triton X-100. The next day, the slides were rinsed with KPBS three times (each for 10 minutes) and then incubated with a Cy3-conjugated goat antirabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, Pa) at room temperature for 2 hours at a dilution of 1:800 in KPBS, 0.05% goat serum, and 0.1% Triton X-100. Because mast cells also contain the c-kit receptor and thus stain positive with c-kit antibodies, dual staining with fluorescein-avidin DCS (Vector Laboratories, Burlingame, Calif) diluted 1:200 for 2 hours was used to identify mast cell staining specifically. The slides were again washed three times with KPBS and overslipped. They were then imaged using an Olympus Fluoview 500 scanning confocal microscope. For control, prepared vesical sections were also treated with rabbit non-immune serum instead of the originally used primary antibody c-kit.
STATISTICAL ANALYSIS Statistical analysis was performed using analysis of variance, and values are given as the mean ⫾ standard deviation. Differences assumed statistical significance at P ⬍0.05.
RESULTS c-Kit positive cells could be identified in contrast to the negative control cells (Fig. 1). The immunopositive cells had a fusiform cell body with dendritic processes at each pole (bipolar) or along the side of the cell body (multipolar, Fig. 2). They had large oval nuclei with dispersed chromatin and a single nucleolus. The cytoplasm exhibited fine 810
FIGURE 1. Photomicrograph of section of vesical wall showing c-kit-positive cells (arrows). Cells appear elongated with dendritic processes. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
FIGURE 2. Photomicrograph of section of vesical wall showing c-kit-positive cells. Cells have dendritic processes at pole (bipolar) or along their side (multipolar). Top arrow points to the bipolar cell. Bottom arrow points to the mutipolar cell. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
granules that showed strong immunoreaction (Figs. 1 and 2). Because the anti-c-kit is a specific marker for ICCs, we referred to the labeled cells as ICCs. The ICCs were different sizes, ranging from 58 to 152 m (mean 106.4 ⫾ 41). They were separated from each other by connective tissue cells and fibers (Figs. 1 and 2). They commonly occurred as separate cells, but small groups of cells surrounded by concentric layers of connective tissue cells were also identified. The ICCs were located at the junctional area between the inner circular and outer longitudinal smooth muscle fibers. In the superior vesical wall, an accumulation of ICCs was identified (Fig. 3). It consisted of small groups of amassed ICCs surrounded by layers of connective tissue cells and fibers (Fig. 3). In all the studied specimens, this big mass of ICCs was detected in the superior vesical wall; the other vesical UROLOGY 64 (4), 2004
FIGURE 3. Photomicrograph of section of vesical dome showing c-kit-positive cells (arrow) accumulated to form mass surrounded by layers of connective tissue cells. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
FIGURE 5. Photomicrograph of section of vesical wall showing c-kit-positive cells (arrows) forming cellular network. Some cells show divided nuclei. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
The aforementioned findings of the ICCs contrasted with those of the surrounding smooth muscle cells. The latter had a typical spindle shape with more regular dimensions compared with the morphologic heterogeneity of the ICC. They were generally larger than the ICCs and had a size varying from 144 to 178 m (mean 162.4 ⫾ 18.7). The smooth muscle cells had no dendritic processes such as the ICCs and showed no immunoreactivity to c-kit antibodies (Figs. 1, 2, and 5). COMMENT FIGURE 4. Photomicrograph of section of vesical wall showing c-kit-positive cells (arrows) arranged in linear fashion. c-Kit-positive mast cell with rounded body without dendritic processes (arrow) also seen. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
walls contained ICCs in a dispersed fashion or collected in small groups of a few cells. The ICCs were arranged in a linear fashion (Fig. 4) or formed a cellular network (Fig. 5). We could not identify ICCs in the vesical mucosa, lamina propria, or submucosa; mast cells were present in these areas. The distribution of the ICCs was identical in both sexes and at different ages. The mast cells were also c-kit positive; they existed mainly in the mucosa and submucosa but also occurred in the musculosa. They were round and had round nuclei but no dendritic processes (Fig. 4), unlike the fusiform cell body with dendritic processes and oval nuclei of the ICCs. In contrast to the ICCs, the mast cells stained positively for fluorescein-avidin DCS. These immunohistologic characteristics clearly characterized these cells as mast cells and differentiated them from the ICCs. UROLOGY 64 (4), 2004
The current study identified c-kit-positive cells in the UB. These cells displayed the morphologic characteristics of cells that had previously been defined as the ICCs or pacemaker cells in the gastrointestinal tract. It is known that ICCs function as pacemakers, signaling the smooth muscles to contract.16,17 They also seem to act as intermediaries in the transmission of nerve signals to the smooth muscle cells.18 –20 Electric activity has been recorded from the human UB at rest.1– 4 It was generally assumed that these electric waves originate from the smooth muscle cells of the UB and that no specialized pacemaker cells were necessary to initiate this activity. The current study demonstrated the presence of c-kit-positive cells in the UB similar to those found in the stomach and colon. Previous studies have revealed that the resting electric activity of the UB occurred in the form of SWs and action potentials.1– 4 The SW parameters increase significantly during bladder contraction.1– 4 These waves were demonstrated to spread caudad in the vesical wall, and we suggested that they originated from a pacemaker located in the vesical dome.1 The current study has shown that a big collection of ICCs was located in the superior 811
vesical wall, probably constituting the “primary pacemaker,” which initiates the SWs. The other vesical walls contained ICCs in a dispersed fashion or amassed in small groups. We hypothesized that the electric waves originate from the primary pacemaker in the vesical dome and are distributed to the rest of the vesical walls through the ICC. This was evidenced by the fact that the lidocaine blockage of the vesical dome effected complete suppression of the SWs in the UB.1 Furthermore, when mucosa-sparing vesical myotomy was performed just distal to the dome, no SWs were recorded distal to the myotomy.1 The existence of resting electric activity in the bladder musculature appears to denote that the smooth muscle cells of the UB are tonically contracted all the time. We postulated that the resting vesical electric activity is controlled by the constant firing of pacemaker cells, which are most probably the ICCs, the activity of which is conducted to the smooth muscle cells. We believe that the release of small amounts of excitatory transmitter appears to increase the firing of the ICCs, with a resulting smooth muscle contraction. Moreover, because the ICCs apparently mediate the response to nerves in the gastrointestinal tract,18 –20 such a pattern may also exist in the UB. Thus, it is suggested that the ICCs mediate the effects of neurally released nitric oxide, provided the ICCs are preferentially innervated. However, the association of ICCs with nerve endings needs to be studied by electron microscopy. A review of published reports revealed a few studies that evaluated c-kit immunoreactivity in the urinary tract.13–15 Lammie et al.17 found scattered c-kit-positive immunoreactivity in transitional epithelial cells and in the mast cells of the UB and in the gastrointestinal tract. The presence of interstitial cells in guinea pig and human bladders has previously been suggested using vimentin antibodies.21 The results of the current study indirectly support the role of the ICCs as mediators and/or initiators of UB smooth muscle contraction. Therefore, we believe that recognition of abnormalities of the ICC system in the UB may offer insight into the cause of some vesical motility disorders. A deficiency or absence of c-kit cells has been detected in human motility disorders of the gut such as Hirschsprung’s disease,22,23 intestinal pseudo-obstruction,24 and gastric motility dysfunction.25 CONCLUSIONS We have identified cells in the UB with morphologic and immunological phenotypes similar to the 812
ICCs of the gut. A big collection of these cells, detected in the dome, appears to constitute the “primary” vesical pacemaker that initiates the SWs that spread to the rest of the vesical wall. We predict that a deficiency or absence of these cells may be involved in vesical motility disorders. Thus, an abnormal distribution of the ICCs may be of diagnostic significance in UB motility disorders. However, additional studies are needed to delineate the role of these cells in UB physiology and pathologic conditions. ACKNOWLEDGMENT. To Margot Yehia who assisted in preparation of the manuscript. REFERENCES 1. Shafik A: Study of the electromechanical activity of the urinary bladder: experimental study. World J Urol 12: 316 – 318, 1994. 2. Shafik A: Electrovesicography in interstitial cystitis. Urol Int 54: 66 – 69, 1995. 3. Shafik A: Electrovesicogram in the neurogenic bladder. J Invest Surg 9: 481– 486, 1996. 4. Shafik A: Electrovesicogram in stress urinary incontinence: a preliminary study. Int Urogynecol J 7: 203–207, 1996. 5. Hashitani H, Van Helden DF, and Suzuki H: Properties of spontaneous depolarizations in circular smooth muscle cells of rabbit urethra. Br J Pharmacol 118: 1627–1632, 1996. 6. Hashitani H, and Edwards FR: Spontaneous and neurally-activated depolarizations in smooth muscle cells of guinea-pig urethra. J Physiol 514: 459 – 470, 1999. 7. Maeda H, Yamagata A, Nishikawa S, et al: Requirement of c-kit for development of intestinal pacemaker system. Development 116: 369 –375, 1992. 8. Chabot B, Stephenson DA, Chapman VM, et al: The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse (W) locus. Nature 335: 88 – 89, 1988. 9. Ward SM, and Sanders KM: Physiology and pathophysiology of the interstitial cells of Cajal: from bench to bed-side. 1. Functional development and plasticity of interstitial cells of Cajal networks. Am J Physiol 251: 602– 611, 2001. 10. Daniel EE, Thomas J, Ramnarain M, et al: Do gap junctions couple interstitial cells of Cajal pacing and neurotransmission to gastrointestinal smooth muscle? Neurogastroenterol Motil 13: 297–307, 2001. 11. Lui LWC, and Huizinga JD: Electrical coupling of circular muscle to longitudinal muscle and interstitial cells of Cajal in canine colon. J Physiol 470: 445– 451, 1993. 12. Rumessen JJ, Mikkelsen HB, and Thuneberg L: Ultrastructure of interstitial cells of Cajal associated with deep muscular plexus of human small intestine. Gastroenterology 102: 56 – 65, 1992. 13. Sergeant CP, Hollywood MA, McCloskey KD, et al: Specialized pacemaking cells in the rabbit urethra. J Physiol 526: 359 –366, 2000. 14. McCloskey KD, and Alison GM: Kit positive cells in the guinea pig bladder. J Urol 168: 832– 836, 2002. 15. Pezzone MA, Watkins SC, Alber SM, et al: Identification of c-kit-positive cells in the mouse ureter: the interstitial cells of Cajal of the urinary tract. Am J Physiol 284: 925–929, 2003. UROLOGY 64 (4), 2004
16. Hagger R, Finlayson C, and Jeffrey I: Role of interstitial cells of Cajal in the control of gut motility. Br J Surg 84: 445– 450, 1997. 17. Lammie A, Drobnjak M, Gerald W, et al: Expression of c-kit and kit ligand proteins in normal human tissues. J Histochem Cytochem 42: 1417–1425, 1994. 18. Vannucchi MG: Receptors in interstitial cells of Cajal: identification and possible physiological roles. Microsc Res Tech 47: 325–335, 1999. 19. Sanders KM: A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111: 492– 495, 1996. 20. Wang XY, Sanders KM, and Ward SM: Intimate relationship between interstitial cells of Cajal and enteric nerves in guinea pig small intestine. Cell Tiss Res 295: 247–252, 1999. 21. Smet PJ, Jonavicius J, Marshall VR, et al: Distribution of nitric oxide synthase-immunoreactive nerves and identifica-
UROLOGY 64 (4), 2004
tion of cellular targets of nitric oxide in guinea-pig and human urinary bladder by CGMP immunohistochemistry. Neuroscience 71: 337–348, 1996. 22. Vanderwinden JM, Rumessen JJ, Lui H, et al: Interstitial cells of Cajal in human colon and in Hirschsprung’s disease. Gastroenterology 111: 901–910, 1996. 23. Ward SM, Burn AJ, Torihashi S, et al: Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol 480: 91–97, 1994. 24. Isozaki K, Hirota S, Miyagawa JI, et al: Deficiency of c-kit cells in patients with a myopathic form of chronic idiopathic interstitial pseudo-obstruction. Am J Gastroenterol 92: 332–334, 1997. 25. Isozaki K, Hirota S, Nakama A, et al: Disturbed intestinal movement, bile reflux to the stomach and deficiency of c-kit expressing cells in Ws/Ws mutant rats. Gastroenterology 109: 456 – 464, 1995.
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IDENTIFICATION OF INTERSTITIAL CELLS OF CAJAL IN HUMAN URINARY BLADDER: CONCEPT OF VESICAL PACEMAKER AHMED SHAFIK, OLFAT EL-SIBAI, ALI A. SHAFIK,
AND
ISMAIL SHAFIK
ABSTRACT Objectives. To determine whether, given the presence of interstitial cells of Cajal (ICCs) in organs that discharge slow waves, such as the gastrointestinal tract, such cells could also be present in the human urinary bladder (UB) and might exist in great numbers in the bladder dome, constituting the “primary pacemaker” from which slow waves spread to the body of the UB. This hypothesis was based on previous studies that had demonstrated the vesical electric waves starting in the dome and spreading caudad. Methods. Specimens (0.5 ⫻ 0.5 cm) from different vesical locations were obtained from the cystectomized UB of 18 patients (12 men, 6 women, mean age 42.6 ⫾ 3.8 years) with bladder cancer. Fixed sections were prepared and subjected to c-kit immunohistochemistry. Controls for the antiserum specificity consisted of incubation of the tissue with normal rabbit serum substituted for the primary antiserum. Results. Fusiform, c-kit positive, and ICC-like cells were detected in the vesical muscle layers; they had dendritic processes. They occurred separately or were connected through the dendritic processes to form a cellular network. An accumulation of ICCs surrounded by a connective tissue layer was detected in all the dome specimens. Mast cells occurred in the vesical mucosa and submucosa; they were c-kit positive but had a rounded body with no dendritic processes. Conclusions. We identified cells in the UB with morphologic and immunologic phenotypes similar to the ICCs of the gut. An accumulation of these cells, detected in the vesical dome, seems to constitute the “primary” vesical pacemaker that initiates the slow waves that spread to the other vesical walls. We believe that a deficiency or absence of these cells may be involved in vesical motility disorders. Additional studies are needed to delineate the role of these cells in UB physiology and pathologic conditions. UROLOGY 64: 809–813, 2004. © 2004 Elsevier Inc.
T
he smooth muscle cells of the urinary bladder (UB) appear to generate a resting vesical tone as evidenced by electrovesicography.1– 4 This tone seems to be myogenic in nature and associated with the generation of spontaneous transient depolarizations and regularly occurring slow waves (SWs).1– 4 This depolarizing current appears to be initiated by the spontaneous release of calcium from the intracellular stores, thereby setting off a calcium-activated chloride From the Department of Surgery and Experimental Research, Cairo University Faculty of Medicine, Cairo; and Department of Surgery, Menoufia University Faculty of Medicine, Shebin El-Kom, Egypt Reprint requests: Ahmed Shafik, M.D., Ph.D., Department of Surgery and Experimental Research, Cairo University Faculty of Medicine, 2 Talaat Harb Street, Cairo 11121, Egypt Submitted: March 3, 2004, accepted (with revisions): May 20, 2004 © 2004 ELSEVIER INC. ALL RIGHTS RESERVED
current that provides the depolarizing current responsible for the SWs.5,6 The exact source of the SWs recorded from the UB has not been determined. However, when it was demonstrated during mucosa-sparing vesical myotomies in a previous study that the SWs propagated caudad from the dome toward the trigone, we postulated that these SWs arise from a pacemaker located in the vesical dome.1 The discovery of the selective expression of the kit receptor in interstitial cells of Cajal (ICCs) within the gastrointestinal tract and its role in the normal development of the ICC system and pacemaker activity has produced a new tool for the investigation of these cells.7 The proto-oncogene c-kit is the cellular homologue of oncogene v-kit of HZ4 feline sarcoma virus.8 The c-kit encodes a transmembrane tyrosine kinase receptor and is located on human chromosome number 4. 0090-4295/04/$30.00 doi:10.1016/j.urology.2004.05.031 809
Given the presence of ICCs in organs that discharge spontaneous SWs, such as the gastrointestinal9 –12 and urinary tracts,13–15 we hypothesized that such cells also exist in the human UB and accumulate in the vesical dome, constituting the primary pacemaker from which the SWs spread to the remaining body of the UB. This hypothesis was investigated in the current study. MATERIAL AND METHODS We studied 0.5 ⫻ 0.5-cm vesical specimens obtained from the UB of 18 patients (12 men and 6 women, mean age 42.6 ⫾ 3.8 years, range 38 to 49) with bladder cancer after cystectomy. The specimens were taken from the dome, anterior and posterior walls, and the trigone of the UB away from the tumor sites. Patients with bilharzial bladder cancer were excluded from the study. The physical examination findings of the patients were normal. Laboratory work, including blood count, renal and hepatic function tests, and electrocardiography showed normal results. All patients provided informed written consent, and the Review Board and Ethics Committee of the Cairo University Faculty of Medicine approved the study. The specimens were placed in 2% buffered paraformaldehyde for 1 hour, cytoprotected in 30% sucrose overnight, and flash frozen in liquid nitrogen-cooled isopentane. Tissues were cut in 5-m frozen sections, mounted on polylysinecoated slides, and kept frozen until immunocytochemical processing. For immunocytochemical processing, the tissue sections were rehydrated in KPBS at room temperature for 20 minutes, blocked with 10% normal goat serum for 20 minutes, and incubated overnight with the primary antiserum, a rabbit polyclonal IgG antibody to the human c-kit protein (Oncogene Research Products, Cambridge, Mass), diluted 1:100 in KPBS, 0.05% goat serum, and 0.1% Triton X-100. The next day, the slides were rinsed with KPBS three times (each for 10 minutes) and then incubated with a Cy3-conjugated goat antirabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, Pa) at room temperature for 2 hours at a dilution of 1:800 in KPBS, 0.05% goat serum, and 0.1% Triton X-100. Because mast cells also contain the c-kit receptor and thus stain positive with c-kit antibodies, dual staining with fluorescein-avidin DCS (Vector Laboratories, Burlingame, Calif) diluted 1:200 for 2 hours was used to identify mast cell staining specifically. The slides were again washed three times with KPBS and overslipped. They were then imaged using an Olympus Fluoview 500 scanning confocal microscope. For control, prepared vesical sections were also treated with rabbit non-immune serum instead of the originally used primary antibody c-kit.
STATISTICAL ANALYSIS Statistical analysis was performed using analysis of variance, and values are given as the mean ⫾ standard deviation. Differences assumed statistical significance at P ⬍0.05.
RESULTS c-Kit positive cells could be identified in contrast to the negative control cells (Fig. 1). The immunopositive cells had a fusiform cell body with dendritic processes at each pole (bipolar) or along the side of the cell body (multipolar, Fig. 2). They had large oval nuclei with dispersed chromatin and a single nucleolus. The cytoplasm exhibited fine 810
FIGURE 1. Photomicrograph of section of vesical wall showing c-kit-positive cells (arrows). Cells appear elongated with dendritic processes. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
FIGURE 2. Photomicrograph of section of vesical wall showing c-kit-positive cells. Cells have dendritic processes at pole (bipolar) or along their side (multipolar). Top arrow points to the bipolar cell. Bottom arrow points to the mutipolar cell. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
granules that showed strong immunoreaction (Figs. 1 and 2). Because the anti-c-kit is a specific marker for ICCs, we referred to the labeled cells as ICCs. The ICCs were different sizes, ranging from 58 to 152 m (mean 106.4 ⫾ 41). They were separated from each other by connective tissue cells and fibers (Figs. 1 and 2). They commonly occurred as separate cells, but small groups of cells surrounded by concentric layers of connective tissue cells were also identified. The ICCs were located at the junctional area between the inner circular and outer longitudinal smooth muscle fibers. In the superior vesical wall, an accumulation of ICCs was identified (Fig. 3). It consisted of small groups of amassed ICCs surrounded by layers of connective tissue cells and fibers (Fig. 3). In all the studied specimens, this big mass of ICCs was detected in the superior vesical wall; the other vesical UROLOGY 64 (4), 2004
FIGURE 3. Photomicrograph of section of vesical dome showing c-kit-positive cells (arrow) accumulated to form mass surrounded by layers of connective tissue cells. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
FIGURE 5. Photomicrograph of section of vesical wall showing c-kit-positive cells (arrows) forming cellular network. Some cells show divided nuclei. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
The aforementioned findings of the ICCs contrasted with those of the surrounding smooth muscle cells. The latter had a typical spindle shape with more regular dimensions compared with the morphologic heterogeneity of the ICC. They were generally larger than the ICCs and had a size varying from 144 to 178 m (mean 162.4 ⫾ 18.7). The smooth muscle cells had no dendritic processes such as the ICCs and showed no immunoreactivity to c-kit antibodies (Figs. 1, 2, and 5). COMMENT FIGURE 4. Photomicrograph of section of vesical wall showing c-kit-positive cells (arrows) arranged in linear fashion. c-Kit-positive mast cell with rounded body without dendritic processes (arrow) also seen. c-Kit immunostain counterstained with hematoxylin, original magnification ⫻400.
walls contained ICCs in a dispersed fashion or collected in small groups of a few cells. The ICCs were arranged in a linear fashion (Fig. 4) or formed a cellular network (Fig. 5). We could not identify ICCs in the vesical mucosa, lamina propria, or submucosa; mast cells were present in these areas. The distribution of the ICCs was identical in both sexes and at different ages. The mast cells were also c-kit positive; they existed mainly in the mucosa and submucosa but also occurred in the musculosa. They were round and had round nuclei but no dendritic processes (Fig. 4), unlike the fusiform cell body with dendritic processes and oval nuclei of the ICCs. In contrast to the ICCs, the mast cells stained positively for fluorescein-avidin DCS. These immunohistologic characteristics clearly characterized these cells as mast cells and differentiated them from the ICCs. UROLOGY 64 (4), 2004
The current study identified c-kit-positive cells in the UB. These cells displayed the morphologic characteristics of cells that had previously been defined as the ICCs or pacemaker cells in the gastrointestinal tract. It is known that ICCs function as pacemakers, signaling the smooth muscles to contract.16,17 They also seem to act as intermediaries in the transmission of nerve signals to the smooth muscle cells.18 –20 Electric activity has been recorded from the human UB at rest.1– 4 It was generally assumed that these electric waves originate from the smooth muscle cells of the UB and that no specialized pacemaker cells were necessary to initiate this activity. The current study demonstrated the presence of c-kit-positive cells in the UB similar to those found in the stomach and colon. Previous studies have revealed that the resting electric activity of the UB occurred in the form of SWs and action potentials.1– 4 The SW parameters increase significantly during bladder contraction.1– 4 These waves were demonstrated to spread caudad in the vesical wall, and we suggested that they originated from a pacemaker located in the vesical dome.1 The current study has shown that a big collection of ICCs was located in the superior 811
vesical wall, probably constituting the “primary pacemaker,” which initiates the SWs. The other vesical walls contained ICCs in a dispersed fashion or amassed in small groups. We hypothesized that the electric waves originate from the primary pacemaker in the vesical dome and are distributed to the rest of the vesical walls through the ICC. This was evidenced by the fact that the lidocaine blockage of the vesical dome effected complete suppression of the SWs in the UB.1 Furthermore, when mucosa-sparing vesical myotomy was performed just distal to the dome, no SWs were recorded distal to the myotomy.1 The existence of resting electric activity in the bladder musculature appears to denote that the smooth muscle cells of the UB are tonically contracted all the time. We postulated that the resting vesical electric activity is controlled by the constant firing of pacemaker cells, which are most probably the ICCs, the activity of which is conducted to the smooth muscle cells. We believe that the release of small amounts of excitatory transmitter appears to increase the firing of the ICCs, with a resulting smooth muscle contraction. Moreover, because the ICCs apparently mediate the response to nerves in the gastrointestinal tract,18 –20 such a pattern may also exist in the UB. Thus, it is suggested that the ICCs mediate the effects of neurally released nitric oxide, provided the ICCs are preferentially innervated. However, the association of ICCs with nerve endings needs to be studied by electron microscopy. A review of published reports revealed a few studies that evaluated c-kit immunoreactivity in the urinary tract.13–15 Lammie et al.17 found scattered c-kit-positive immunoreactivity in transitional epithelial cells and in the mast cells of the UB and in the gastrointestinal tract. The presence of interstitial cells in guinea pig and human bladders has previously been suggested using vimentin antibodies.21 The results of the current study indirectly support the role of the ICCs as mediators and/or initiators of UB smooth muscle contraction. Therefore, we believe that recognition of abnormalities of the ICC system in the UB may offer insight into the cause of some vesical motility disorders. A deficiency or absence of c-kit cells has been detected in human motility disorders of the gut such as Hirschsprung’s disease,22,23 intestinal pseudo-obstruction,24 and gastric motility dysfunction.25 CONCLUSIONS We have identified cells in the UB with morphologic and immunological phenotypes similar to the 812
ICCs of the gut. A big collection of these cells, detected in the dome, appears to constitute the “primary” vesical pacemaker that initiates the SWs that spread to the rest of the vesical wall. We predict that a deficiency or absence of these cells may be involved in vesical motility disorders. Thus, an abnormal distribution of the ICCs may be of diagnostic significance in UB motility disorders. However, additional studies are needed to delineate the role of these cells in UB physiology and pathologic conditions. ACKNOWLEDGMENT. To Margot Yehia who assisted in preparation of the manuscript. REFERENCES 1. Shafik A: Study of the electromechanical activity of the urinary bladder: experimental study. World J Urol 12: 316 – 318, 1994. 2. Shafik A: Electrovesicography in interstitial cystitis. Urol Int 54: 66 – 69, 1995. 3. Shafik A: Electrovesicogram in the neurogenic bladder. J Invest Surg 9: 481– 486, 1996. 4. Shafik A: Electrovesicogram in stress urinary incontinence: a preliminary study. Int Urogynecol J 7: 203–207, 1996. 5. Hashitani H, Van Helden DF, and Suzuki H: Properties of spontaneous depolarizations in circular smooth muscle cells of rabbit urethra. Br J Pharmacol 118: 1627–1632, 1996. 6. Hashitani H, and Edwards FR: Spontaneous and neurally-activated depolarizations in smooth muscle cells of guinea-pig urethra. J Physiol 514: 459 – 470, 1999. 7. Maeda H, Yamagata A, Nishikawa S, et al: Requirement of c-kit for development of intestinal pacemaker system. Development 116: 369 –375, 1992. 8. Chabot B, Stephenson DA, Chapman VM, et al: The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse (W) locus. Nature 335: 88 – 89, 1988. 9. Ward SM, and Sanders KM: Physiology and pathophysiology of the interstitial cells of Cajal: from bench to bed-side. 1. Functional development and plasticity of interstitial cells of Cajal networks. Am J Physiol 251: 602– 611, 2001. 10. Daniel EE, Thomas J, Ramnarain M, et al: Do gap junctions couple interstitial cells of Cajal pacing and neurotransmission to gastrointestinal smooth muscle? Neurogastroenterol Motil 13: 297–307, 2001. 11. Lui LWC, and Huizinga JD: Electrical coupling of circular muscle to longitudinal muscle and interstitial cells of Cajal in canine colon. J Physiol 470: 445– 451, 1993. 12. Rumessen JJ, Mikkelsen HB, and Thuneberg L: Ultrastructure of interstitial cells of Cajal associated with deep muscular plexus of human small intestine. Gastroenterology 102: 56 – 65, 1992. 13. Sergeant CP, Hollywood MA, McCloskey KD, et al: Specialized pacemaking cells in the rabbit urethra. J Physiol 526: 359 –366, 2000. 14. McCloskey KD, and Alison GM: Kit positive cells in the guinea pig bladder. J Urol 168: 832– 836, 2002. 15. Pezzone MA, Watkins SC, Alber SM, et al: Identification of c-kit-positive cells in the mouse ureter: the interstitial cells of Cajal of the urinary tract. Am J Physiol 284: 925–929, 2003. UROLOGY 64 (4), 2004
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