35

Lymphology 28 (1995) 35-40

Institute of Normal Anatomy (PP,AT), Institute of Histology and General Embryology (CM), and Department of Human Pathology (RS), University of Pavia, Italy

ABSTRACT light and electron microscopy (2,3). To clarify vesical lymph drainage, we now examined the After endoscopic transurethral biopsies of fine structure of small lymphatic vessels (SL V) normal human urinary bladder, an extensive and their distribution in the normal human network of small initial lymphatic vessels was urinary bladder. Particular attention was depicted by means of light and electron directed to the structure and composition of microscopy. Using light microscopy, lymphatic the connective matrix that surrounds the vessels were seen in the mucosa and lymphatic vessels in the different regions of submucosa and formed a complex network in the bladder wall. the detrusor muscular coat. These lymphatics were characterized by an irregular and MATERIALS AND METHODS attenuated wall and increased in number and size from the superficial to the deeper region of Ten male subjects (40 to 60 years of age) the bladder. Ultrastructurally, the lymphatic who had symptoms of bladder outlet wall was characterized by endothelial cells obstruction were utilized for this investigation. joined together end-to-end or by complicated Two endoscopic transurethral biopsies from interdigitations. Often intercellular channels the lateral wall of the normal urinary bladder and gaps between two contiguous endothelial were performed under a constant bladder cells were present. A broad network of elastic distention after introduction of physiological and collagen fibers joined the lymphatic saline at 50 cm H20 constant pressure. The endothelial wall to the neighboring connective biopsy trocar was regulated to obtain vesical tissue. Nevertheless, as far as the fibrillar specimens including both the bladder mucosa component was concerned, the vesical intra­ and the muscular coat. All samples were fixed muscular lymphatic endothelial wall lacked in a mixture of glutaraldehyde (2.5 %) and elastic fibers. These anatomic variations were paraformaldehyde (2%) in O.lM sodium examined in reference to lymph formation in cacodylate buffer at pH 7.4 for 4 hrs. at 4"C an organ (the urinary bladder) which under­ and postflxed by 1% OS04 in 0.2M collidine goes continual changes in volume and pressure. buffer at pH 7.4 for 2 hrs at 4"C. The speci­ mens were then dehydrated and embedded in In 1944, Powell (1) described a well­ Epoxy resin. Under light microscopy, small developed network of lymphatic capillaries in lymphatic vessels (SLV) were identified on the mucosa and muscular coat of the urinary semi thin sections stained with toluidine blue bladder using dye injection. Subsequently, as described previously (4,5). Approximately lymphatics of the vesical wall were depicted by 50 SL V were examined. At least 20 selected

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Fig. 1. Semithin sections from the human urinary bladder: A) A small lymphatic in the deeper region of the lamina propria between two large blood vessels (L=lymphatic) (500x); B) a lymphatic vessel in close proximity to the muscular layer (500x); C) a large lymphatic is seen in the muscular layer between smooth muscle cells (350x); D) a lymphatic of the subserosa is comparatively large with characteristics of a collecting vessel (200x).

lymphatics were observed on ultrathin TABLEt sections, contrasted with uranyl acetate and Quantitative Data on Small lead citrate, by electron microscope Zeiss Lymphatic Vessels (SLV) EMI09. of the Human Urinary Bladder The quantitative evaluation of SLV was performed with a computerized automatic Vesical Region SLV Diameter image analyzer (IBAS I and II Kontron-Zeiss) (n) (± SEM) as described previously (6). The morphometric analysis was performed at 3 levels: subepithe­ Subepithelial 10 30.2±O.82 lial region of the lamina propria; submucosa (deep region of the lamina propria); and Submucosal 17 48.3±1.35 detrusor muscle. Muscular 18 70.3±1.20 RESULTS Subserosal 5 185±4.21 Light Microscopy

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Fig. 2. A very thin endothelium characterizes this lymphatic vessel in which two contiguous endo­ thelial cells are joined end-to-end (F=fat cell). (17000x)

Fig. 3. In this mucosal lymphatic, two contiguous endothelial cells delimit a large channel. The lymphatic is surrounded by a thin fibrillar network of collagen fibers and anchoringfilaments (l7000x).

A well-developed lymphatic vessel SLY were consistently present in the bladder network was found in the vesical wall. The submucosa. They were often adjacent to and distribution between mucosal and submucosal intermingled with arterioles and venules but SL V was difficult to distinguish because the were distinguishable from blood vessels by separation of these layers in human urinary their very thin, indented walls (Fig. 1A). In the bladder was unclear because of an irregularly deeper regions of the submucosa, lymphatics arranged muscularis mucosae (7). Accor­ abutted the detrusor muscular layer (Fig. 1B). dingly, we designated "subepithelial SLV" as Table 1 summarizes the quantitative data on the lymphatics lying immediately beneath the the number and size of SL V in the different epithelium and "submucosal SL V" as the regions of the vesical wall. In the detrusor lymphatics lying on the remaining lamina layer, wide lymphatics were seen in the loose propria up to the detrusor muscle. Lymphatic interstitial connective tissue which penetrated vessels were seen in all regions. They increased into the muscular bundles (Fig. 1 C). These in number and size from the mucosal layer to lymphatics were often difficult to recognize the detrusor muscular layer (Table 1). In the because the lumen was occluded over a long subepithelium, lymphatics were rare. Several stretch. In some specimens in which the

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Fig. 4. Closeup of lymphatic showing a gap between two contigu­ ous endothelial cells. Anchoring filaments are tightly joined to the abluminal side of the endothelial cells (arrows) 17000x).

Fig. 5. Bundles of elastic fibers (arrows) and some collagen fibers abut the lymphatic endothelium. (17000x).

subserosa was seen, the lymphatics were quite abluminal side of the endothelium. In the large and showed the characteristics of subepithelial and submucosal lymphatics, the collectors (Fig. lD). Their wall was supported contiguous endothelial cells were joined by neighboring tissue structures; consequently, together primarily by simple contact (Le., they had a winding appearance. "end-to-end") (Fig. 2) or by overlapping. Some endothelial gaps also occurred where the Electron Microscopy endothelial wall was prominently distended (Fig. 4). In the muscular and subserosal In all lymphatic vessels examined, the lymphatics the neighboring endothelial cells endothelial cells showed a very thin and were joined together by more complex typically irregular profile. They were interdigitations between two or more characterized by scanty cytoplasm; near the cytoplasmic edges. Here, the endothelium was nuclei, organelles such as mitochondria, markedly indented and intercellular channels ribosomes and Weibel-Palade bodies were were common (Fig. 3). seen. The micro pinocytotic vesicles were The loose connective tissue surrounding usually scarce both in the luminal and in the the lymphatic was filled with abundant

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fascicles of collagen and elastic fibers that also regulated by vasomotion of adjacent joined the lymphatic endothelial wall. Elastic arterioles (15). This fibrillar network, however, fibers surrounded lymphatics mainly in the is lacking in the bladder muscle and subserosa. subepithelial and submucosal areas (Fig. 5). In This ultrastructural dichotomy suggests that fact, bundles of elastic fibers were tightly in the mucosal region of the human urinary joined to the abluminal side of the endothelial bladder anchoring elastic fiber filaments force cells by thin anchoring filaments. The expansion of the lymphatic vessel influx of endothelium of the lymphatics located in the interstitial fluid as lymph. On the other hand, muscular sheath generally lacked elastic in the deeper regions (muscle and subserosa) fibers. The lymphatic endothelium was of the bladder, contractions of the bladder wall surrounded by a thin network of collagen and adjacent arterial pulsations help propel fibers with anchoring filaments attached to the lymph onward. abluminal surface (Figs. 3,4). All the lymphatics examined lacked valves. Moreover, REFERENCES the larger lymphatics located in the muscular layer and in the submucosa did not have 1. Powell, TO: Studies on the lymphatics of the smooth muscle in the lymphatic wall. female urinary bladder. Surg. Gynec. Obstet. 78 (1944), 605-609. 2. Azzali, G, G Romita, R Gatti: Ultrastruttura DISCUSSION dei vasi linfatici della vescica orin aria. Acta Bio-Medica de I' Ateneo Parmense 54 (1983), Our findings on small lymphatic vessels 105-115. (SL V) of the human urinary bladder can be 3. Fawcett, DW: The urinary system. Textbook of Histology, 11th Ed., Saunders, Philadelphia, summarized as follows: 1) SLY are less PA, 1986,787-790. numerous and smaller in the mucosa than in 4. Poggi, P, C Marchetti, A Calligaro, et al: the detrusor muscular layer and in the Lymph vessels of the heart: A comparative subserosa; 2) SL V ultrastructural study of various techniques for light and electron microscopy. Anat. Anz. 162 (1986), characteristics are similar in each area 195-204. examined; 3) SLY in the detrusor muscle and 5. Marchetti, C, P Poggi, A Calligaro, et al: in the subserosa lack elastic fibers. Microscopic and ultrastructural study ofthe As to finding 1: The results agree with lymphatic system in the human parotid gland. those previously described (8,9) and suggest Acta Anat. 134 (1989), 106-112. 6. Poggi, P, C Marchetti, A Calligaro, et al: that SL V drains interstitial fluid from the Morphometric data on the lymph capillaries of more superficial to the deeper regions of the the ventricles of the rabbit heart. Acta Anat. bladder. 136 (1989),185-189. As to point 2: vesical SLV show the 7. Dixon, JS, JA Gosling: Histology and fine ultrastructural features of initial lymphatics, structure of the muscolaris mucosae of the human urinary bladder: J. Anat. 136 (1983), namely, a single endothelial lining but without 265-271. either smooth muscle or intraluminal valves. 8. Azzali, G, G Bucci, R Gatti, et al: Topography The presence of junctions along the and ultrastructure of kidney lymphatics in endothelial wall of bladder SL V suggest that some hibernating bats. Lymphology 21 (1988), they open during influx of interstitial fluid 212-223. 9. Ohtani, 0 : Three-dimensional organisation of into the lymphatic lumen, and close when lymphatics and its relationship to blood vessels lymph inflow closes (11). The opening and in rat small intestine. Cell Tissue Res. 248 closing of these junctions are linked to elastic (1987), 365-374. filaments that anchor the endothelial cells at 10. Schmid-Schonbein, GW: Mechanism causing initial lymphatics to expand and compress to the bladder mucosa to adjacent connective promote lymph flow. Arch. Histo!. Cyto!. 53 tissue (12-14). (1990),107-114. Besides this fibrillar network, SL V are 11. Castenholz, A: Morphological characteristics

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of initial lymphatics in the tongue as shown by 15. Skalak, TC, GW Schmid-Schonbein, BW scanning electron microscopy. Scanning Zweifach: New morphological evidence of a Electron Microscopy III (1984), 1343-1352. mechanism of lymph formation in skeletal 12. Leak, LV, JF Burke: Fine structure of the muscle. Microvasc. Res. 28 (1984), 95-112. lymphatic capillary and the adjoining connective tissue area. Am. J. Anat. 118 (1966),785-810. 13. Leak, LV, JF Burke: Ultrastructural studies on the lymphatic anchoring filaments. J. Cell Prof. Paola Poggi BioI. 36 (1968), 129-149. Instituto di Anatomia Umana normale 14. Casley-Smith, JR: Are the initial lymphatics normally pulled open by anchoring filaments? Via Forlanini 10 Lymphology 13 (1980), 120-129. 8-27100 Pavia, ITALY

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