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The microvasculature of the gallbladder in adult Xenopus laevis(Amphibia, Anura): A scanning electron microscope study of vascularcorrosion casts and correlative light microscopyA. Lametschwandtnera; H. Bartela; B. Minnicha; S. Tholoa; U. Spornitzb

a Department of Organismic Biology, Division of Zoology and Functional Anatomy, Vessel and MuscleResearch Unit, University of Salzburg, Salzburg, Austria b Institute of Anatomy, University of Basel,Basel, Switzerland

Online publication date: 07 March 2011

To cite this Article Lametschwandtner, A. , Bartel, H. , Minnich, B. , Tholo, S. and Spornitz, U.(2011) 'The microvasculatureof the gallbladder in adult Xenopus laevis (Amphibia, Anura): A scanning electron microscope study of vascularcorrosion casts and correlative light microscopy', Italian Journal of Zoology, 78: 1, 27 — 34To link to this Article: DOI: 10.1080/11250003.2010.509137URL: http://dx.doi.org/10.1080/11250003.2010.509137

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ISSN 1125-0003 print/ISSN 1748-5851 online © 2011 Unione Zoologica ItalianaDOI: 10.1080/11250003.2010.509137

Italian Journal of Zoology, March 2011; 78(1): 27–34

TIZOThe microvasculature of the gallbladder in adult Xenopus laevis (Amphibia, Anura): A scanning electron microscope study of vascular corrosion casts and correlative light microscopy

Microvasculature of the gallbladder in adult XenopusA. LAMETSCHWANDTNER1*, H. BARTEL1, B. MINNICH1, S. THOLO1, & U. SPORNITZ2

1Department of Organismic Biology, Division of Zoology and Functional Anatomy, Vessel and Muscle Research Unit, University of Salzburg, Salzburg, Austria, and 2Institute of Anatomy, University of Basel, Basel, Switzerland

(Received 20 April 2010; accepted 9 July 2010)

AbstractMicrovasculature, anatomy and histomorphology of the gallbladder of the adult pipid frog, Xenopus laevis (Daudin) werestudied by light (LM) and scanning electron microscopy (SEM) of tissue preparations and vascular corrosion casts. Thethin wall of the gallbladder consisted of columnar epithelium, lamina propria, a fibromuscular layer, subserosa and serosa.A few diverticula but no epithelial folds were present. Up to five cystic arteries arose from the adjacent right hepatic arteryand approached the gallbladder via its neck region. One to two cystic veins departed from the bottom and drained into thenearby abdominal vein. Cystic arteries were of the pinnate-type and embraced the gallbladder with their branches. Termi-nal arterioles gave rise to wide subepithelial capillaries. Transition distances from arteries to veins were short. In general,three to four layers of blood vessels built the vascular bed of the gallbladder wall. The wide subepithelial capillaries estab-lished an interface well adapted (1) to allow capillary circulation over a wide range of filling states of the gallbladder and(2) to serve the exchange processes taking place between stored bile and the blood vascular system via the microvillouscolumnar epithelium.

Keywords: Xenopus, gallbladder, microvasculature, corrosion casting, scanning electron microscopy

Introduction

The gallbladder serves to concentrate and store bilebefore bile is discharged via the bile duct system intothe duodenum. Why a gallbladder is present in mostanimals but absent in others is still under discussion(Gilloteaux 1997; Oldham-Ott & Gilloteaux 1997).

Presently, the development of the gallbladder (Gil-bert 2003) along with the molecular basis of the dif-ferentiation of its epithelial lining from endoderm iswell known (Zorn & Wells 2007). Also well known inmammals and man is its macroscopic and micro-scopic anatomy (Schreiber 1939, 1942; Walraff 1969;Oldham-Ott & Gilloteaux 1997), innervation (Maweet al. 1997), mechanism of bile discharge (Schreiber1939), pathologies (Nakanuma et al. 1997), and itsblood vascular system (Gordon 1967; Ohtani 1981,1988; Polyzonis et al. 1989; Caggiatti et al. 1990,

1992; Aharinejad & Lametschwandtner 1992a;Nakanuma et al. 1997; Ohtani et al. 1997; Jackowiak& Lametschwandtner 2005; Jackowiak 2006; Jack-owiak et al. 2007).

Little is known about the microvascular anatomy ofthe gallbladder in lower vertebrates. Presently weknow that in anuran amphibians the gallbladder issupplied by either one or two cystic arteries whichbranch off the right hepatic artery, and that one ortwo cystic veins drain it into the abdominal vein(Xenopus laevis Daudin; Millard 1941; Rana esculenta;Gaupp 1899). Only one short note is published onthe microvascularization of the gallbladder of theadult pipid frog, Xenopus laevis (Häring 2004).

In the present study we use scanning electron micro-scopy of vascular corrosion casts (Murakami 1971)and correlative light microscopy and demonstrate for

*Correspondence: Prof. Dr. Alois Lametschwandtner, Department of Organismic Biology, Division of Zoology and Functional Anatomy, Vessel and MuscleResearch Unit, University of Salzburg, Hellbrunnerstrasse 34, A-5020, Salzburg, Austria. Tel: +43 662 80445602. Fax: +43 662 80445698.Email: Alois.Lametschwandtner@sbg.ac.at

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the first time the microvasculature of an anuran gall-bladder in greater detail. Specifically, we show that thismicrovasculature by virtue of its architecture allows fora sufficiently high intrinsic blood circulation over awide range of filling states of the gallbladder.

Materials and methods

Stereomicroscopic dissection

Three adult males of the South African ClawedToad, Xenopus laevis Daudin (body weights: 75.0,78.0 and 108.7 g; snout–vent lengths (SVL): 80, 85and 105 mm) were killed by submersion into anoverdose of tricaine methansulfonate (MS 222;Sigma Chemicals, St. Louis, MO). The abdominalcavity was opened by a paramedian cut, and after abilateral anterior and posterior laterally directedincision the abdominal wall was flipped laterally andpinned down. Then gross arterial supply and venousdrainage of the gallbladder, its superficial microvas-culature, and the bile duct system were exposedunder stereomicroscopic control (StereomicrosopeWild M 651; Wild, Heerbrugg, Switzerland) anddocumented (digital camera system Nikon DS-5-L1).

The gallbladder was excised in the two smalleranimals, submerged in fixative (described below),and cut meridionally into two halves. While sub-merged, the fixed bile was carefully removed withforceps and the luminal surfaces of the halves of thegallbladder were cleaned by a gentle jet of fresh fixa-tive to allow for a detailled scanning electron micro-scope (SEM) inspection. Further details areprovided below.

Scanning electron microscopy of critical point dried gallbladders

After stereomicroscopical inspection and documen-tation the two halves of one gallbladder were fixedfurther by immersion in fresh 2.5% buffered glutaral-dehyde (0.15 M cacodylate; pH 7.4; 4°C, overnight),washed in 0.2 M cacodylate buffer (3 × 20 min),postfixed in buffered (0.1 M cacodylate) osmiumtextroxide (2%; 2 h; 20°C), washed in 0.2 Mcacodylate (3 × 20 min), and stored overnight inethanol (70%; 4°C). Finally, specimens were dehy-drated in a graded series of ethanol (80, 90, 96 and100%; three passages each), and critical point driedvia carbon dioxide. Then one of the two dried halvesof the gallbladder was fractured meridionally bypulling with two forceps. Finally, specimens weremounted to specimen stubs with colloidal silver, sput-tered with gold, and examined in the scanning electronmicroscope (XL-30; FEI-Company, Eindhoven,

Netherlands or Stereoscan 250, Cambridge, UK) at20 kV.

Histomorphology

Gallbladders of two adult animals (body weights: 46and 126 g; SVL: 55 and 110 mm) were studied.Animals were killed by immersion in an overdose ofan aqueous solution of MS 222 (0.5 %) and firstperfused via ventricle–arterial trunk with AmphibianRinger’s solution (20°C; for recipe see Adam & Czi-hak 1964) using manual pressure. After clear refluxfrom the opened atria 10 ml of Bouin’s solution(Adam & Czihak 1964) were injected for fixation ofthe animals. Then gallbladder, cystic duct and com-mon bile duct were excised and postfixed in freshfixative (20°C). After dehydration in a graded seriesof ethanol, specimens were embedded in paraplast,sectioned transversely and longitudinally (7 μm),and stained (Goldner’s trichrome stain; Romeis1989). Micrographs taken with a digital camera(Axiocam) were imported to Photoshop 7.0 (Adobe)and brightness and contrast were adjusted whennecessary.

Vascular casting

For vascular casting, 13 adult animals (bodyweights: 46–126 g; SVL: 55–110 mm) were used.Animals were killed by immersion in an overdose ofan aqueous solution of MS 222 (0.5 %) and firstperfused via ventricle–arterial trunk with AmphibianRinger’s solution (20°C; for recipe see Adam & Czi-hak 1964) using manual pressure. After clear refluxfrom the opened atria 10 ml Mercox CL-2B (LaddResearch Inc.; Burlington, VT, USA) diluted withmonomeric methylmethacrylate (4 + 1, v + v) wereinjected with manual pressure. After polymerizationof the injected resin, whole animals were tempered(water bath; 60°C, 12 h), macerated (KOH; 7.5%,12–24 h), rinsed (tap water), cleaned (5% formicacid; 10 min), rinsed (distilled water), frozen (dis-tilled water), and freeze-dried (Lyovac GT2; LeyboldHeraeus, Cologne, Germany). In dry specimens,gallbladders were exposed by removal of the overlay-ing right lobe of the liver, and specimens weremounted by the ‘conductive bridge method’(Lametschwandtner et al. 1980). Mounted speci-mens were evaporated with carbon and gold, sput-tered with a thin layer of gold and examined in thescanning electron microscope (Stereoscan 250,Cambridge Ltd, UK; accelerating voltage: 10 kV).After the microvascular anatomy of the gallbladdershad been documented in-situ, gallbladders wereremoved, mounted onto new stubs, coated, and

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Microvasculature of the gallbladder in adult Xenopus 29

previously hidden areas were documented. Finally,gallbladders were removed from the specimen stubs,frozen in distilled water, and sectioned transversely,longitudinally, tangentially or horizontally with amini wheel-saw placed in the chamber of a cryo-microtome (Lametschwandtner & Lametschwandt-ner 1992). Sectioned ice-embedded specimens wereallowed to thaw in distilled water, cleaned, andrefrozen in bidistilled water for subsequent freeze-drying (Lyovac GT 1, Leybold-Hereaus; Cologne,Germany). Further steps in specimen preparationwere as described above. For further details on SEMand vascular corrosion casting see Aharinejad andLametschwandtner (1992b), Motta et al. (1992),and Lametschwandtner et al. (2006).

Results

Anatomy

In adult Xenopus laevis, the gallbladder is located atthe medial border of the right lobe of the liver closeto the base of the heart ventricle. Depending on howfull it is, the gallbladder is ball-, ovoid- or club-shaped and 4–5 mm in diameter (Figure 1). A bot-tom, a body, and a neck region can be discerned(Figures 1 and 2). The cystic duct is joined first bythe right hepatic duct, then by the median and lefthepatic ducts (Figures 1 and 2).

Gross arterial supply and venous drainage

The right hepatic artery supplies the gallbladder.When this artery passes the gallbladder neck region

it abuts one to five cystic arteries towards the gall-bladder (Figures 1–3). Cystic arteries often bifurcatewhen they approach the serosal side of the neckregion (Figure 2). One to two cystic veins leave thebottom region and drain the gallbladder into thenearby abdominal vein.

Histomorphology

The wall of the gallbladder of adult Xenopus laevisconsists (from inside to outside) of epithelium, lam-ina propria, fibromuscular layer, subserosa, andserosa (Figure 4). Wide vessels locate in the laminapropria while the capillary bed lies directly beneaththe epithelium (Figure 4). The epithelium consistsof a few roundish basal cells with a round nucleus

Figure 1. Gallbladder and bile duct system in adult male Xenopuslaevis Daudin. Stereomicroscopy. Ventral view. Note the righthepatic artery (rha) which abuts the cystic arteries. cbd commonbile duct, cd cystic duct, dd duodenum, gb gallbladder, hdhepatic duct, ml median lobe of liver, rl right lobe of liver.

Figure 2. Arterial supply of the gallbladder (gb). Note the cysticarteries (arrows) arising from the right hepatic artery (rha). cdcystic duct, hd hepatic duct.

Figure 3. Same as Figure 2, but gallbladder (gb) flipped over.Note five cystic arteries (arrows). cd cystic duct, hd hepatic duct,rha right hepatic artery.

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30 A. Lametschwandtner et al.

and slender columnar epithelial cells with a longishnucleus located in the basal half of the cell (Figure 5).Cell nuclei of columnar cells show several nucleoli.Subepithelial capillaries have a conspicuous flat pro-file and locate directly beneath the basal lamina(Figure 5).

Luminal surface structure

Medium-powered scanning electron micrographs ofcritical point dried fractured gallbladders confirmthe histomorphological findings and show that thelateral surfaces of the columnar epithelial cells are

strongly ruffled (Figure 6). Apical cell surfaces areslightly convex, polygonal in shape and have diame-ters around 3.3 μm (Figure 6). They usually have adense coating with ∼20 nm thick microvilli. Locally,a varying number of longer microvilli with diametersaround 16 nm or even one or two centred longmicrovilli can be found (Figure 6, arrows). Con-spicious epithelial folds are missing.

Microvascular architecture

One to five cystic arteries approach via the neckregion and give off branches bilaterally on theircourse towards body and bottom (Figures 7 and 8).Branches embrace the gallbladder from ventral anddorsal (Figures 7 and 8). When daughter vesselshave a similar diameter, flow dividers can be found(Figure 7, inset a, arrows), whereas intimal cushionsare present if a branch is much smaller than the par-ent artery (Figure 7, inset b, arrow). At the terminalportion, cystic arteries give off several terminal arte-rioles within a short distance (Figures 7 and 9). Ter-minal arterioles either directly approach thesubepithelial capillary bed (Figure 9, large arrows)or they give off branches which supply the laminapropria adjacent to the fibromuscular layer (Figure 9,small arrows). Locally, arteriovenous transition dis-tances are short (Figure 7, inset b; Figure 10). Someof the arteriovenous transition vessels have deepimpressions at places where they branch off from theparent vessel (Figure 7, inset b) while others lackthese impressions (Figure 10).

Figure 4. Histomorphology of the gallbladder wall. Light micros-copy. Transverse section (10 μm; Goldner staining). Note the flatcapillaries beneath the columnar epithelium (arrows) and a diver-ticulum (di) extending into the wall. e columnar epithelium, fmfibromuscular layer, lu lumen, lp lamina propria, s serosa, ss sub-serosa, v vein, vv venule.

Figure 5. Histomorphology of the columnar epithelium (e). Notethe flat profiles of capillaries (c) beneath the basal lamina. lp lam-ina propria.

Figure 6. Columnar epithelium of the gallbladder of adult maleXenopus laevis Daudin. Scanning electron micrograph of a criticalpoint dried fractured gallbladder. Note the strong ruffling of thelateral surfaces of the columnar epithelial cells (e). Inset. Polygo-nal apical surface of a columnar epithelial cell with a dense coat ofshort microvilli and two long central microvilli (arrows). bl basallamina, c capillary, lp lamina propria, lu lumen of gallbladder.

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The subepithelial vascular network is made up ofrelatively wide flat capillaries, which form a widemeshwork with venules interposed between (Figure 11).Occasionally, outbulgings of the capillary bed arepresent outlining diverticula extending into the gall-bladder wall (Figure 10; compare with Figure 4).Subepithelial capillaries drain either via adjacentvenules or directly into postcapillary venules locatedsomewhat deeper in the lamina propria. Some castvenules have holes of varying sizes and shapes,which can be interpreted as signs of ongoing intus-suceptive microvascular growth (Figures 12–14).

Vascular sprouts imposing as blind-ending vesselswith fine tapering endings are few (Figures 14 and 15).They arise from subepithelial venules. Venules ofthe lamina propria join at the bottom of the gallblad-der and form one or two cystic veins (Figure 8) anddrain into the nearby abdominal vein.

Throughout the wall of the gallbladder three tofour layers of blood vessels are present (Figure 16).

Discussion

Scanning electron microscopy of the luminal surfaceof the gallbladder of Xenopus laevis demonstrated

Figure 7. Microvascular anatomy of the gallbladder in adultXenopus. Vascular corrosion cast. Ventral aspect. SEM micro-graph. Anterior is to the left, medial is on top. Arteries are red,veins are blue. av abdominal vein, ca cystic artery, cv cystic vein,dd duodenum, gb gallbladder, hd hepatic duct, ml median lobe ofliver, pv portal vein. Inset a. Cystic artery (ca) with side branch.Note the imprints of a flow divider (arrows). Inset b. Cysticartery (ca) with small side branch representing a terminal arteriole(ta). Note the narrowing at the origin of the terminal arteriolecaused by an intimal cushion (arrow).

Figure 8. Same as Figure 6, but gallbladder rotated 180° anti-clockwise along its long axis to show its dorsal aspect. b bottom,bo body, ca cystic artery, cv cystic vein, ne neck. Asterisks mark‘conductive bridges’.

Figure 9. Transition zone of three terminal arterioles (ta) to post-capillary venules (vv)(dashed lines). Serosal view. Arteries arered. Note that interposed capillaries (c) are few and short.Locally, terminal arterioles (large arrows) seem to continuedirectly as venules. lu lumen of gallbladder.

Figure 10. Short terminal arteriole (ta) without narrowing at theorigin. Arteries are red. c capillary, ca cystic artery, lu lumen ofgallbladder, vv venule.

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that (1) the epithelium lacked prominent folds, and(2) epithelial cells had one or two long microvilliwith diameters of ∼16 nm. Up to now, such micro-villi, which protrude above the dense short microvillicoat of the gallbladder epithelial cells, were neitherreported in anuran amphibians (Grzycki & Nowakowski1967; Azanza et al. 1989; Oldham-Ott & Gilloteaux1997) nor in urodelian ones (Oldham-Ott & Gilloteaux1997). Interestingly, a similar, extremely tall micro-villus per epithelial cell was demonstrated sofar inthe Armadillo lizard only (Oldham-Ott & Gilloteaux

1997). The functional importance of the tall micro-villus remains open.

The pattern of the arterial supply with the cysticarteries coming from the hepatic artery and thecystic arteries entering via the neck region clearlyreflects the development of the gallbladder as aderivative of the draining duct of the liver diverticu-lum (Gilbert 2003). This pattern obviously ensuresthat the entire gallbladder is well supplied with oxy-genated blood while the venous drainage from thebottom region via the cystic veins into the abdomi-nal vein facilitates reabsorbed substances to betransported via the left branch of the abdominal vein

Figure 11. Microvascular pattern of the subepithelial vascularbed at the bottom of the gallbladder. Luminal view. Cystic artery(ca) and its branches are red, part of cystic vein (cv) is blue. Notethe venules (vv) interposed between capillaries (c) and venulesforming kind of ‘venular ring’ (outlined in blue). Asterisks mark‘conductive bridges’.

Figure 12. Venule (vv) of the lamina propria with a small hole(arrow) indicating ongoing intussusceptive microvascular growth.Serosal view. c capillary, lu lumen of gallbladder.

Figure 13. Same as Figure 12, but intussusception (arrows) in amore advanced state. c capillary, lu lumen of gallbladder, vv venule.

Figure 14. Vascular sprouts (sp) and ongoing intussusception(arrows) at a venule (vv) in the lamina propria. Serosal view. lulumen of gallbladder.

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directly to the left liver lobe and via the right branchof the abdominal vein into the portal which drainsinto the right liver lobe.

The microvascular patterns of the subepithelialcapillary bed of the gallbladder of adult Xenopusshow three striking differences if compared withthose in mammals. In mammals, (1) capillariesare much thinner, (2) capillary mesh sizes are muchsmaller, and (3) venules always stay beneath

capillaries and are never interposed between capil-laries like they are in adult Xenopus. As a conse-quence the surface of the subepithelial vascular bedof the gallbladder, which serves the exchange of sol-utes and electrolytes between luminal bile andblood, is much smaller in Xenopus than it is inmammals.

Acknowledgements

This work was supported by the Fonds zurFörderung der wissenschaftlichen Forschung, ProjektP-19050 and the Stiftungs- und Förderungsgesells-chaft der Paris-Lodron-Universität Salzburg (to A.L.).The authors thank OR Dr. W. D. Krautgartner forproviding excellent working conditions in the SEMfacility.

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