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The sheath of Waldeyer is not a specific anatomical trait of the ureterovesical junction
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The sheath of Waldeyer is not a specific anatomical trait of the ureterovesical junction M.C. Rusu a,b,∗, F. Pop a,b, N. Petre c, M.A. Dobra a a
Division of anatomy, faculty of dental medicine, ‘‘Carol Davila’’ university of medicine and pharmacy, Bucharest, Romania b MEDCENTER—Center of excellence in laboratory medicine and pathology, Bucharest, Romania c ‘‘Carol Davila’’ Clinical Hospital of Nephrology, Bucharest, Romania
KEYWORDS Ureterovesical junction; Human ureter; Myoid markers; Immunohistochemistry; Telocytes
Summary The function of the ureterovesical junction depends upon a peculiar structure, the adventitial fibromuscular sheath of Waldeyer, which coats the distal end of the ureter. The origin of the smooth muscle of Waldeyer’s sheath (WS) is disputed. Evidence points more likely to an ureteral one. In this regard we hypothesized the WS is not specific to the distal ureter but is rather a common trait. We therefore aimed at exploring whether or not the proximal ureter is provided with a similar adventitial fibromuscular coat. We performed an immunohistochemical study on human samples of proximal ureter resulted after nephrectomies in ten patients. We applied myoid immunohistochemical markers: ␣-smooth muscle actin (␣-SMA), desmin, and heavy chain of smooth muscle myosin (SMM) which labeled additional adventitial smooth muscle bundles, a discontinuous inner circular one applied on the muscular coat, and outer longitudinal cords specifically located on one side of the ureter, as is the case for WS. Moreover, the lamina propria myoid deep layer showed isolated smooth muscle fibers and spindle-shaped stromal cells with telocyte morphology. Our results support the idea that WS may not be a specific structure of the distal ureter, instead being just a common anatomical characteristic of the ureter. © 2017 Elsevier Masson SAS. All rights reserved.
∗ Corresponding author at: ‘‘Carol Davila’’ University of Medicine and Pharmacy, 8 Eroilor Sanitari boulevard, RO-050474, Bucharest, Romania. E-mail address: [email protected] (M.C. Rusu).
https://doi.org/10.1016/j.morpho.2017.11.001 1286-0115/© 2017 Elsevier Masson SAS. All rights reserved.
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Introduction Waldeyer (1892, as quoted in [1]), introduced the term ‘‘Ureterscheide’’ to identify the fibromuscular sheath coating the distal end of the ureter (the juxtavesical ureter). This structure was previously reported by Krause (1876, quoted in [2,3]) as an extension of the bladder musculature around the distal part of ureter. It is considered that the Waldeyer’s sheath (WS) is the adventitia or outer fibrous coating of the distal 1-3 cm of the ureter, which is reinforced by longitudinal muscle bundles specifically located on one side of the ureter [1,4]. Elsewhere, the WS is presented as ‘‘an external layer of longitudinal smooth muscle’’, which ‘‘surrounds the ureter’’ [5]. The WS is also described as a non-striated ‘‘collar’’ [6], which leads to the perception of a circular muscle component. Such a fibromuscular sheath cannot be truly individualized over the transparietal segment of the ureter [7]. There are disputes on the ureteral, or bladder origin of the adventitial muscle layer of the WS. We therefore aimed at evaluating the muscular coats of the proximal end of the ureter, to check whether or not they include such an Ureterscheide. This was also suggested by our dissection studies in which the proximal ureter was seemingly provided with a muscularized adventitia, as was the distal ureter (Fig. 1).
Material and method Sample tissues of the proximal 3—4 cm of ureter were obtained from human adult patients (6 males and 4 females) after nephrectomies for renal tumors. The age
of patients ranged from 57 to 63 years. Informed consent for research use of tissues was obtained prior to surgery. Approval for the present study was granted by the Institutional Committee. All experiments on human subjects were conducted in accordance with the Declaration of Helsinki (http://www.wma.net/en/30publications/10policies/b3/ index). The samples of ureter were oriented anatomically for transverse cuts and afterwards fixed for 24 hours in buffered formalin (8%) and processed with an automatic histoprocessor (Diapath, Martinengo, BG, Italy) with paraffin embedding. Sections (3 m) were manually cut and mounted ® on SuperFrost electrostatic slides for immunohistochemistry (Thermo Scientific, Menzel-Gläser, Braunschweig, Germany). Histological evaluations also used 3 m thick sections, stained with hematoxylin and eosin. Internal negative controls resulted when the primary antibodies were not applied on slides. We used primary antibodies for alpha-smooth muscle actin (␣-SMA, mouse monoclonal, clone D33, Biocare Medical, Concord, CA, USA, 1:50, 30 min, RT), desmin (mouse monoclonal, clone OV-TL 12/30, Biocare Medical, Concord, CA, USA, 1:100, 45 min, RT) and the smooth muscle myosin heavy chain (SMM, mouse monoclonal, clone S131, Novocastra-Leica, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, U.K., 1:100, 30 min, RT). Tissues were dewaxed and rehydrated, then endogenous peroxidase was blocked using Peroxidased 1 (Biocare Medical, Concord, CA, USA). For the heat-induced epitope retrieval the Decloaking Chamber (Biocare Medical, Concord, CA, USA) was used with a pH 6 retrieval solution (Biocare Medical, Concord, CA, USA). Background Blocker (Biocare Medical, Concord, CA, USA) was used to reduce
Figure 1 Dissections of the left kidney (A, posterior view) and the left hemipelvis (antero-superior view) in which the proximal end of the ureter (A, inset: magnified detail) as well as the distal end of the ureter (B, inset: magnified detail) seem provided with muscularized adventitial coats (arrows in A and B). 1. posterior surface of the left kidney; 2. left proximal ureter; 3. left renal vein; 4. urinary bladder, displaced antero-medially; 5. pubic symphysis; 6. left distal ureter.
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Figure 2 Successive transverse cuts of ureter labeled with myoid markers: (A) desmin, (B) SMM, (C and D) ␣-SMA. Arrows indicate adventitial smooth muscle longitudinal cords located on one side of the ureter, arrowheads point to an incomplete adventitial circular muscle layer. In (C) note the smooth muscle layers: (a) deep layer of lamina propria; (b) internal smooth muscle layer; (c) external smooth muscle layer.
non-specific background staining. The primary antibody was then applied. There the MACH 4 (Biocare Medical, Concord, CA, USA) detection system was used as a two-step (probe/polymer) universal HRP detection method. A HRPcompatible chromogen (DAB) was applied. Sections were counterstained with hematoxylin and rinsed with deionized water. A TBS solution, pH 7.6 for the wahsing steps.
Results On all slides the microanatomy of ureter was adequately identified and consisted of three histological layers, the mucous membrane, the muscular coat and the adventitia or the outer fibrous coat. The mucous membrane was composed of the urothelium and lamina propria, the latter appearing subdivided into a superficial (suburothelial) layer and a deep one. On histological slides, no age- or genderrelated differences were noticed. All three myoid markers were used (desmin, SMM and ␣SMA) gave a similar labelling. They labelled the muscular coat of the proximal ureter consisting of internal longitudinal and external circular bundles, which, occasionally, appeared intermingled. Unexpectedly, we found additional smooth muscle bundles within the adventitial coat (Fig. 2). One was applied over the muscular coat and consisted of thin circular bundles building a circumferentially incomplete layer. On one side of the ureter only were found additional myoid cords; these were embedded within a thickened adventitial layer and intermingled with vessels and nerves. Within these longitudinal adventitial cords, the muscle fibers appeared disorganized (Fig. 3), rather than being longitudinally oriented.
Figure 3 Desmin-positive adventitial longitudinal cord of proximal ureter, displaying a disorganized structure. bv: blood vessel.
Nevertheless, in 7 of the 10 ureteral samples we identified a myoid structure in the lamina propria deep layer, which was continuous (3/7 samples), discontinuous (2/7 samples) or dispersed (2/7 samples). This muscularis mucosa-like ureteral layer included isolated smooth muscle fibers and myoid stromal cells, which were positively labeled by all three markers we used. The respective stromal myoid cells were spindle-shaped and displayed long and extremely long prolongations, resembling a telocyte morphology (Fig. 4). Superficial and deep microvascular layers
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Figure 4 Transverse cuts of ureter labeled with myoid markers: (A) desmin, (B) SMM, (C) ␣-SMA. Stromal cells with telocyte morphologies (arrows) and isolated smooth muscle fibers (arrowheads) intermingle within the deep layer of the ureteral lamina propria. U: urothelium.
of lamina propria were observed, the latter ones being intermingled with the myoid cells when these were present.
Discussion During embryogenesis, the Wolffian duct opens into the cloaca and the ureteral bud emerges on its dorsal aspect above the junction with the cloaca [8]. Further, the ureteral bud achieves an independent opening into the cloaca, dorsolateral to the opening of the Wolffian duct [8]. The former will migrate upward and laterally and the later migrates downward and medially, thus becoming separated by mesodermal tissue, which forms the bladder trigone [8]. So, all the mesodermal structures arise from the Wolffian duct in contrast to the bladder which is endodermal in nature [8]. These include the smooth muscle coat of the ureter and makes logical for the adventitial muscle cords to build a WS-like in ureteral segments different of the distal ureter. However, although a WS-like structure was found here in the proximal ureter, which supports our findings in dissections (Fig. 1), further microscopic studies should evaluate the intermediate segments of the ureter for the presence, or absence, of such a fibromuscular sheath. Roshani et al. (1996) discussed that subtly different descriptions on the ureterovesical junction smooth muscle anatomy have been reported since Waldeyer’s initial description of the ‘‘Ureterscheide’’; the respective differences being related to the muscles contributing to the WS [2]. These authors brought evidence that the muscular coat of the ureter and the bladder musculature are not connected [2]. This evidence is supported by experiments,
which proved that the ureteral muscle tone, and not the bladder muscle, prevents the vesicouretral reflux [9]. The trigonal bladder zone is located between the bladder neck and the ureterovesical junction being intimately related to both of these [10]. Although the trigone is essentially of mesodermal origin, it is contributed also by the endodermal urogenital sinus [10], which fuses with the ureter by day 37 of fetal development [11]. The two layers of the trigone were considered a direct continuation of the lower ureter and its WS [10]. Waldeyer described the ‘‘Ureterscheide’’ as deriving from the vesical musculature [3]. Indeed, it is discussed that the deep trigone arises from the Wolffian duct and continues cranially as the WS and caudally as middle circular layer of urethra [8]. However, there are studies that found no separate ureteric sheath, and few or even absent muscular connections between the ureter and the bladder musculature [12]. Moreover, it seems that instead of being bi-layered, the muscular coat of the ureter, which is able to generate spontaneous contractions, is rather composed of longitudinal and circular fascicles, which blend into one another [13], as we also found here. The first one who denied a layering of the ureter musculature was Sappey (1889, quoted in [13]). We could not find any previous studies indicating a myoid composition of the deep layer of the ureteral lamina propria (ureteral muscularis mucosa), although its bi-layered structure is described [4]. On the other hand, it was previously shown that the lamina propria of the urinary bladder can also be muscularized, as muscularis mucosa with variable consistency was reported in 94/100 samples [14]. Another studies identified a bladder muscularis mucosa in 48/54 cases [15], or showed its absence from the trigone
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The sheath of Waldeyer in the proximal ureter [12]. The bladder muscularis mucosa embeds microvessels [14,15], such as we found in ureter. The histologic similarity of the muscularis mucosa in the urinary bladder and ureter determine us to speculate a common origin of the bladder and ureter. However, being known that subsets of telocytes could play progenitor roles [16—18], the muscularis mucosa of ureter could be equally regarded as an occasional regenerative layer. The regenerative potential of the ureteral muscularis mucosa in adult mirrors the acquisition of ␣-SMA expressing smooth muscle cells during morphogenesis. The first situs of ␣-SMA expression in embryos is subserosal, at the level of the urogenital sinus, in this stage the epithelial bud of the ureter being surrounded only by ␣-SMA-negative spindleshaped cells [19]. In further stages ␣-SMA will be positively expressed in ureter, but with regional differences, the number of myoid cells being greatest in the distal ureter but least in the proximal ureter [19]. While the smooth muscle formation of the bladder is subserosal, it is subepithelial in ureter, and occurs in an ascending fashion from the bladder towards the kidney [19]. Moreover, in postnatal ureters expression of ␣-SMA intensifies within the periepithelial spindle-shaped cells [19], thus configuring the ureteral muscularis mucosa built-up by subepithelial myoid telocytes. The heterogeneous and variable composition of the deep layer of the lamina propria, of muscle fibers and myoid telocytes, indicates it as a possible supplier of the muscular coat, which, on one hand, allows a certain turnover of the muscular coat and, on other hand, could impede on a layered pattern of the muscular coat. On other hand, it is known that the ureter propels urine towards the urinary bladder through peristaltic contractions generated by interstitial Cajal cells located in the upper urinary tract [20]. Telocytes, initially regarded as interstitial Cajal-like cells [21], were found also in the urinary tract [22—27]. It was demonstrated that telocytes are activated in inflammatory and proliferative stages, when they eventually gain a myoid phenotype [28]. In these regards, further studies of ureteral telocytes should use larger panels of markers for their better characterization. Although controversies may persist among researchers in what concerns the functional anatomy of the distal end of the ureter and the ureterovesical junction, our results support the idea that the WS may not be a specific structure of the distal ureter. Histological studies of the entire length of ureter should further indicate whether the WS is or not a common anatomical feature of ureter on its entire length. Noteworthy, it appears that not only longitudinal, but also circular muscle bundles can be found within the ureteral adventitia, thus they might equally support shortening and constriction of the ureter.
Disclosure of interest The authors declare that they have no competing interest.
Acknowledgements All authors have equally contributed to this study.
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References [1] Noordzij JW, Dabhoiwala NF. A view on the anatomy of the ureterovesical junction. Scand J Urol Nephrol 1993;27(3):371—80. [2] Roshani H, Dabhoiwala NF, Verbeek FJ, Lamers WH. Functional anatomy of the human ureterovesical junction. Anat Rec A Discov Mol Cell Evol Biol 1996;245(4):645—51. [3] Elbadawi A. Anatomy and function of the ureteral sheath. J Urol 1972;107(2):224—9. [4] Velardo JT. Histology of the ureter. The ureter. Springer; 1981. p. 13—54. [5] Tanagho EA, Nguyen HT. Vesicoureteral reflux. In: Tanagho EA, McAninch JW, editors. Smith’s general urology. New York: Lange Medical Books/McGraw-Hill; 2008. p. 179. [6] Standring S. Gray’s anatomy: the anatomical basis of clinical practice. Elsevier Health Sciences; 2015. [7] Juskiewenski S, Vaysse P, Moscovici J, de Graeve P, Guitard J. The ureterovesical junction. Anat Clin 1984;5(4):251—9. [8] Hutch JA. The mesodermal component: its embryology, anatomy, physiology and role in prevention of vesicoureteral reflux. J Urol 1972;108(3):406—10. [9] King PA, Stephens FD. Ureteral muscle tone in prevention of vesicoureteral reflux. Invest Urol 1977;14(6):488—91. [10] Tanagho EA, Meyers FH, Smith DR. The trigone: anatomical and physiological considerations. I. In relation to the ureterovesical junction. J Urol 1968;100(5):623—32. [11] Radmayr C, Schwentner C, Lunacek A, Karatzas A, Oswald J. Embryology and anatomy of the vesicoureteric junction with special reference to the etiology of vesicoureteral reflux. Ther Adv Urol 2009;1(5):243—50. [12] Thomson AS, Dabhoiwala NF, Verbeek FJ, Lamers WH. The functional anatomy of the ureterovesical junction. Br J Urol 1994;73(3):284—91. [13] Woodburne RT. The ureter, ureterovesical junction, and vesical trigone. Anat Rec A Discov Mol Cell Evol Biol 1965;151:243—9. [14] Ro JY, Ayala AG, el-Naggar A. Muscularis mucosa of urinary bladder. Importance for staging and treatment. Am J Surg Pathol 1987;11(9):668—73. [15] Aydin A, Ucak R, Karakok M, Guldur ME, Kocer NE. Vascular plexus is a differentation criterion for muscularis mucosa from muscularis propria in small biopsies and transurethral resection materials of urinary bladder? Int Urol Nephrol 2002;34(3):315—9. [16] Rusu MC, Hostiuc S, Vrapciu AD, Mogoanta L, Manoiu VS, Grigoriu F. Subsets of telocytes: myocardial telocytes. Ann Anat 2017;209:37—44. [17] Rusu MC, Cretoiu D, Vrapciu AD, Hostiuc S, Dermengiu D, Manoiu VS, et al. Telocytes of the human adult trigeminal ganglion. Cell Biol Toxicol 2016;32(3):199—207. [18] Grigoriu F, Hostiuc S, Vrapciu AD, Rusu MC. Subsets of telocytes: the progenitor cells in the human endocardial niche. Rom J Morphol Embryol 2016;57(2 Suppl.):767—74. [19] Baker LA, Gomez RA. Embryonic development of the ureter and bladder: acquisition of smooth muscle. J Urol 1998;160(2):545—50. [20] Prisca RA, Loghin A, Gozar HG, Moldovan C, Moso T, Derzsi Z, et al. Morphological aspects and distribution of interstitial cells of Cajal in the human upper urinary tract. Turk Patoloji Derg 2014;30(2):100—4. [21] Popescu LM, Faussone-Pellegrini MS. Telocytes —– a case of serendipity: the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to Telocytes. J Cell Mol Med 2010;14(4):729—40. [22] Vannucchi MG, Traini C, Guasti D, Del Popolo G, FaussonePellegrini MS. Telocytes subtypes in human urinary bladder. J Cell Mol Med 2014;18(10):2000—8.
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[23] Gevaert T, De Vos R, Van Der Aa F, Joniau S, van den Oord J, Roskams T, et al. Identification of telocytes in the upper lamina propria of the human urinary tract. J Cell Mol Med 2012;16(9):2085—93. [24] Rusu MC, Folescu R, Manoiu VS, Didilescu AC. Suburothelial interstitial cells. Cells Tissues Organs 2014;199(1): 59—72. [25] Vannucchi MG, Faussone-Pellegrini MS. The telocyte subtypes. Adv Exp Med Biol 2016;913:115—26.
[26] Wolnicki M, Aleksandrovych V, Gil K. Interstitial cells of Cajal and telocytes in the urinary system: facts and distribution. Folia Med Cracov 2016;56(4):81—9. [27] Zheng Y, Zhu T, Lin M, Wu D, Wang X. Telocytes in the urinary system. J Transl Med 2012;10:188. [28] Diaz-Flores L, Gutierrez R, Garcia MP, Gonzalez M, Saez FJ, Aparicio F, et al. Human resident CD34+ stromal cells/telocytes have progenitor capacity and are a source of alphaSMA+ cells during repair. Histol Histopathol 2015;30(5):615—27.
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ORIGINAL ARTICLE
The sheath of Waldeyer is not a specific anatomical trait of the ureterovesical junction M.C. Rusu a,b,∗, F. Pop a,b, N. Petre c, M.A. Dobra a a
Division of anatomy, faculty of dental medicine, ‘‘Carol Davila’’ university of medicine and pharmacy, Bucharest, Romania b MEDCENTER—Center of excellence in laboratory medicine and pathology, Bucharest, Romania c ‘‘Carol Davila’’ Clinical Hospital of Nephrology, Bucharest, Romania
KEYWORDS Ureterovesical junction; Human ureter; Myoid markers; Immunohistochemistry; Telocytes
Summary The function of the ureterovesical junction depends upon a peculiar structure, the adventitial fibromuscular sheath of Waldeyer, which coats the distal end of the ureter. The origin of the smooth muscle of Waldeyer’s sheath (WS) is disputed. Evidence points more likely to an ureteral one. In this regard we hypothesized the WS is not specific to the distal ureter but is rather a common trait. We therefore aimed at exploring whether or not the proximal ureter is provided with a similar adventitial fibromuscular coat. We performed an immunohistochemical study on human samples of proximal ureter resulted after nephrectomies in ten patients. We applied myoid immunohistochemical markers: ␣-smooth muscle actin (␣-SMA), desmin, and heavy chain of smooth muscle myosin (SMM) which labeled additional adventitial smooth muscle bundles, a discontinuous inner circular one applied on the muscular coat, and outer longitudinal cords specifically located on one side of the ureter, as is the case for WS. Moreover, the lamina propria myoid deep layer showed isolated smooth muscle fibers and spindle-shaped stromal cells with telocyte morphology. Our results support the idea that WS may not be a specific structure of the distal ureter, instead being just a common anatomical characteristic of the ureter. © 2017 Elsevier Masson SAS. All rights reserved.
∗ Corresponding author at: ‘‘Carol Davila’’ University of Medicine and Pharmacy, 8 Eroilor Sanitari boulevard, RO-050474, Bucharest, Romania. E-mail address: [email protected] (M.C. Rusu).
https://doi.org/10.1016/j.morpho.2017.11.001 1286-0115/© 2017 Elsevier Masson SAS. All rights reserved.
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Introduction Waldeyer (1892, as quoted in [1]), introduced the term ‘‘Ureterscheide’’ to identify the fibromuscular sheath coating the distal end of the ureter (the juxtavesical ureter). This structure was previously reported by Krause (1876, quoted in [2,3]) as an extension of the bladder musculature around the distal part of ureter. It is considered that the Waldeyer’s sheath (WS) is the adventitia or outer fibrous coating of the distal 1-3 cm of the ureter, which is reinforced by longitudinal muscle bundles specifically located on one side of the ureter [1,4]. Elsewhere, the WS is presented as ‘‘an external layer of longitudinal smooth muscle’’, which ‘‘surrounds the ureter’’ [5]. The WS is also described as a non-striated ‘‘collar’’ [6], which leads to the perception of a circular muscle component. Such a fibromuscular sheath cannot be truly individualized over the transparietal segment of the ureter [7]. There are disputes on the ureteral, or bladder origin of the adventitial muscle layer of the WS. We therefore aimed at evaluating the muscular coats of the proximal end of the ureter, to check whether or not they include such an Ureterscheide. This was also suggested by our dissection studies in which the proximal ureter was seemingly provided with a muscularized adventitia, as was the distal ureter (Fig. 1).
Material and method Sample tissues of the proximal 3—4 cm of ureter were obtained from human adult patients (6 males and 4 females) after nephrectomies for renal tumors. The age
of patients ranged from 57 to 63 years. Informed consent for research use of tissues was obtained prior to surgery. Approval for the present study was granted by the Institutional Committee. All experiments on human subjects were conducted in accordance with the Declaration of Helsinki (http://www.wma.net/en/30publications/10policies/b3/ index). The samples of ureter were oriented anatomically for transverse cuts and afterwards fixed for 24 hours in buffered formalin (8%) and processed with an automatic histoprocessor (Diapath, Martinengo, BG, Italy) with paraffin embedding. Sections (3 m) were manually cut and mounted ® on SuperFrost electrostatic slides for immunohistochemistry (Thermo Scientific, Menzel-Gläser, Braunschweig, Germany). Histological evaluations also used 3 m thick sections, stained with hematoxylin and eosin. Internal negative controls resulted when the primary antibodies were not applied on slides. We used primary antibodies for alpha-smooth muscle actin (␣-SMA, mouse monoclonal, clone D33, Biocare Medical, Concord, CA, USA, 1:50, 30 min, RT), desmin (mouse monoclonal, clone OV-TL 12/30, Biocare Medical, Concord, CA, USA, 1:100, 45 min, RT) and the smooth muscle myosin heavy chain (SMM, mouse monoclonal, clone S131, Novocastra-Leica, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, U.K., 1:100, 30 min, RT). Tissues were dewaxed and rehydrated, then endogenous peroxidase was blocked using Peroxidased 1 (Biocare Medical, Concord, CA, USA). For the heat-induced epitope retrieval the Decloaking Chamber (Biocare Medical, Concord, CA, USA) was used with a pH 6 retrieval solution (Biocare Medical, Concord, CA, USA). Background Blocker (Biocare Medical, Concord, CA, USA) was used to reduce
Figure 1 Dissections of the left kidney (A, posterior view) and the left hemipelvis (antero-superior view) in which the proximal end of the ureter (A, inset: magnified detail) as well as the distal end of the ureter (B, inset: magnified detail) seem provided with muscularized adventitial coats (arrows in A and B). 1. posterior surface of the left kidney; 2. left proximal ureter; 3. left renal vein; 4. urinary bladder, displaced antero-medially; 5. pubic symphysis; 6. left distal ureter.
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The sheath of Waldeyer in the proximal ureter
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Figure 2 Successive transverse cuts of ureter labeled with myoid markers: (A) desmin, (B) SMM, (C and D) ␣-SMA. Arrows indicate adventitial smooth muscle longitudinal cords located on one side of the ureter, arrowheads point to an incomplete adventitial circular muscle layer. In (C) note the smooth muscle layers: (a) deep layer of lamina propria; (b) internal smooth muscle layer; (c) external smooth muscle layer.
non-specific background staining. The primary antibody was then applied. There the MACH 4 (Biocare Medical, Concord, CA, USA) detection system was used as a two-step (probe/polymer) universal HRP detection method. A HRPcompatible chromogen (DAB) was applied. Sections were counterstained with hematoxylin and rinsed with deionized water. A TBS solution, pH 7.6 for the wahsing steps.
Results On all slides the microanatomy of ureter was adequately identified and consisted of three histological layers, the mucous membrane, the muscular coat and the adventitia or the outer fibrous coat. The mucous membrane was composed of the urothelium and lamina propria, the latter appearing subdivided into a superficial (suburothelial) layer and a deep one. On histological slides, no age- or genderrelated differences were noticed. All three myoid markers were used (desmin, SMM and ␣SMA) gave a similar labelling. They labelled the muscular coat of the proximal ureter consisting of internal longitudinal and external circular bundles, which, occasionally, appeared intermingled. Unexpectedly, we found additional smooth muscle bundles within the adventitial coat (Fig. 2). One was applied over the muscular coat and consisted of thin circular bundles building a circumferentially incomplete layer. On one side of the ureter only were found additional myoid cords; these were embedded within a thickened adventitial layer and intermingled with vessels and nerves. Within these longitudinal adventitial cords, the muscle fibers appeared disorganized (Fig. 3), rather than being longitudinally oriented.
Figure 3 Desmin-positive adventitial longitudinal cord of proximal ureter, displaying a disorganized structure. bv: blood vessel.
Nevertheless, in 7 of the 10 ureteral samples we identified a myoid structure in the lamina propria deep layer, which was continuous (3/7 samples), discontinuous (2/7 samples) or dispersed (2/7 samples). This muscularis mucosa-like ureteral layer included isolated smooth muscle fibers and myoid stromal cells, which were positively labeled by all three markers we used. The respective stromal myoid cells were spindle-shaped and displayed long and extremely long prolongations, resembling a telocyte morphology (Fig. 4). Superficial and deep microvascular layers
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Figure 4 Transverse cuts of ureter labeled with myoid markers: (A) desmin, (B) SMM, (C) ␣-SMA. Stromal cells with telocyte morphologies (arrows) and isolated smooth muscle fibers (arrowheads) intermingle within the deep layer of the ureteral lamina propria. U: urothelium.
of lamina propria were observed, the latter ones being intermingled with the myoid cells when these were present.
Discussion During embryogenesis, the Wolffian duct opens into the cloaca and the ureteral bud emerges on its dorsal aspect above the junction with the cloaca [8]. Further, the ureteral bud achieves an independent opening into the cloaca, dorsolateral to the opening of the Wolffian duct [8]. The former will migrate upward and laterally and the later migrates downward and medially, thus becoming separated by mesodermal tissue, which forms the bladder trigone [8]. So, all the mesodermal structures arise from the Wolffian duct in contrast to the bladder which is endodermal in nature [8]. These include the smooth muscle coat of the ureter and makes logical for the adventitial muscle cords to build a WS-like in ureteral segments different of the distal ureter. However, although a WS-like structure was found here in the proximal ureter, which supports our findings in dissections (Fig. 1), further microscopic studies should evaluate the intermediate segments of the ureter for the presence, or absence, of such a fibromuscular sheath. Roshani et al. (1996) discussed that subtly different descriptions on the ureterovesical junction smooth muscle anatomy have been reported since Waldeyer’s initial description of the ‘‘Ureterscheide’’; the respective differences being related to the muscles contributing to the WS [2]. These authors brought evidence that the muscular coat of the ureter and the bladder musculature are not connected [2]. This evidence is supported by experiments,
which proved that the ureteral muscle tone, and not the bladder muscle, prevents the vesicouretral reflux [9]. The trigonal bladder zone is located between the bladder neck and the ureterovesical junction being intimately related to both of these [10]. Although the trigone is essentially of mesodermal origin, it is contributed also by the endodermal urogenital sinus [10], which fuses with the ureter by day 37 of fetal development [11]. The two layers of the trigone were considered a direct continuation of the lower ureter and its WS [10]. Waldeyer described the ‘‘Ureterscheide’’ as deriving from the vesical musculature [3]. Indeed, it is discussed that the deep trigone arises from the Wolffian duct and continues cranially as the WS and caudally as middle circular layer of urethra [8]. However, there are studies that found no separate ureteric sheath, and few or even absent muscular connections between the ureter and the bladder musculature [12]. Moreover, it seems that instead of being bi-layered, the muscular coat of the ureter, which is able to generate spontaneous contractions, is rather composed of longitudinal and circular fascicles, which blend into one another [13], as we also found here. The first one who denied a layering of the ureter musculature was Sappey (1889, quoted in [13]). We could not find any previous studies indicating a myoid composition of the deep layer of the ureteral lamina propria (ureteral muscularis mucosa), although its bi-layered structure is described [4]. On the other hand, it was previously shown that the lamina propria of the urinary bladder can also be muscularized, as muscularis mucosa with variable consistency was reported in 94/100 samples [14]. Another studies identified a bladder muscularis mucosa in 48/54 cases [15], or showed its absence from the trigone
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The sheath of Waldeyer in the proximal ureter [12]. The bladder muscularis mucosa embeds microvessels [14,15], such as we found in ureter. The histologic similarity of the muscularis mucosa in the urinary bladder and ureter determine us to speculate a common origin of the bladder and ureter. However, being known that subsets of telocytes could play progenitor roles [16—18], the muscularis mucosa of ureter could be equally regarded as an occasional regenerative layer. The regenerative potential of the ureteral muscularis mucosa in adult mirrors the acquisition of ␣-SMA expressing smooth muscle cells during morphogenesis. The first situs of ␣-SMA expression in embryos is subserosal, at the level of the urogenital sinus, in this stage the epithelial bud of the ureter being surrounded only by ␣-SMA-negative spindleshaped cells [19]. In further stages ␣-SMA will be positively expressed in ureter, but with regional differences, the number of myoid cells being greatest in the distal ureter but least in the proximal ureter [19]. While the smooth muscle formation of the bladder is subserosal, it is subepithelial in ureter, and occurs in an ascending fashion from the bladder towards the kidney [19]. Moreover, in postnatal ureters expression of ␣-SMA intensifies within the periepithelial spindle-shaped cells [19], thus configuring the ureteral muscularis mucosa built-up by subepithelial myoid telocytes. The heterogeneous and variable composition of the deep layer of the lamina propria, of muscle fibers and myoid telocytes, indicates it as a possible supplier of the muscular coat, which, on one hand, allows a certain turnover of the muscular coat and, on other hand, could impede on a layered pattern of the muscular coat. On other hand, it is known that the ureter propels urine towards the urinary bladder through peristaltic contractions generated by interstitial Cajal cells located in the upper urinary tract [20]. Telocytes, initially regarded as interstitial Cajal-like cells [21], were found also in the urinary tract [22—27]. It was demonstrated that telocytes are activated in inflammatory and proliferative stages, when they eventually gain a myoid phenotype [28]. In these regards, further studies of ureteral telocytes should use larger panels of markers for their better characterization. Although controversies may persist among researchers in what concerns the functional anatomy of the distal end of the ureter and the ureterovesical junction, our results support the idea that the WS may not be a specific structure of the distal ureter. Histological studies of the entire length of ureter should further indicate whether the WS is or not a common anatomical feature of ureter on its entire length. Noteworthy, it appears that not only longitudinal, but also circular muscle bundles can be found within the ureteral adventitia, thus they might equally support shortening and constriction of the ureter.
Disclosure of interest The authors declare that they have no competing interest.
Acknowledgements All authors have equally contributed to this study.
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Please cite this article in press as: Rusu MC, et al. The sheath of Waldeyer is not a specific anatomical trait of the ureterovesical junction. Morphologie (2017), https://doi.org/10.1016/j.morpho.2017.11.001