Angioarchitecture of the rabbit extrahepatic bile ducts and gallbladder
Article first published online: 2 SEP 2005
Copyright © 2005 Wiley-Liss, Inc.
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology
Volume 286A, Issue 2, pages 974–981, October 2005
How to Cite
Jackowiak, H. and Lametschwandtner, A. (2005), Angioarchitecture of the rabbit extrahepatic bile ducts and gallbladder. Anat. Rec., 286A: 974–981. doi: 10.1002/ar.a.20240
- Issue published online: 21 SEP 2005
- Article first published online: 2 SEP 2005
- Manuscript Accepted: 6 JUL 2005
- Manuscript Received: 25 MAY 2005
- bile tract;
- corrosion cast;
- scanning electron microscopy
The angioarchitecture of extrahepatic bile ducts and gallbladder of the miniature rabbit was studied by scanning electron microscopy (SEM) of vascular corrosion casts. Light microscopy of Masson-stained, paraffin-embedded transverse tissue sections served to attribute cast vascular structures to defined layers of bile ducts and gallbladder. In all segments of the bile tract, a mucosal and a subserosal vascular network was found. In glandular segments, the mucosal network was composed of a meshwork of subepithelial and circumglandular capillaries, which serve the mucosal functions. Differences in the angioarchitectonic patterns existed only in the subserosal networks as hepatic ducts own one supplying arteriole only, while the common bile duct owns a well-defined rete arteriosum subserosum. A well-developed dense subserosus venous plexus was present throughout the bile tract. Vascular patterns of the gallbladder body resembled those of the bile duct, whereby the dense subserous venous plexus was located close to the mucosal capillary network. The subserosal network in the neck of the gallbladder resembled that of the cystic duct. Spatial changes of the mucosal vascular network during volume changes of the gallbladder were documented. Measurements from tissue sections revealed bile tract diameters of 220–400 μm (extrahepatic ducts), 500–650 μm (cystic duct), and 4–6 mm (common bile duct). Data gained from high-powered SEM micrographs of vascular corrosion casts revealed vessel diameters of 200 μm (cystic artery), 90–110 μm (cystic vein), 30–40 μm (feeding arterioles), and 25–110 μm (subserosal venules). Crypt diameters in the filled gallbladder were 300–1,500 μm; those in the contracted organ were 100–600 μm. © 2005 Wiley-Liss, Inc.
The rabbit bile tract is supplied by two branches of the common hepatic artery, namely, the left branch of the proper hepatic artery for the hepatic ducts, the gallbladder, and the cystic duct, and the pancreatic-duodenal artery, which gives off branches to the common bile duct and the duodenal papilla (Bednarova and Malinovsky, 1988). Presently, the microvasculature of the intrahepatic bile ducts of the rat (Ohtani and Murakami, 1978; Ohtani et al., 1983) and the rabbit (Ohtani, 1979; Cho and Lunderquist, 1983; Ohtani et al., 1983; Aharinejad et al., 1991; Cagiatti et al., 1992) is described. Ohtani and Murakami (1978), Ohtani (1979), Ohtani et al. (1983), and Cho and Lunderquist (1983) described the ramifications of the intrahepatic branches of the hepatic artery, which form peribiliary plexus and additionally give off arterioles to the liver sinusoids and/or Glisson sheaths, while Cagiatti et al. (1992) studied structure and angioarchitecture of the mucosal folds of the gallbladder, and Ohtani et al. (1997) referred to the extremely dense network of subepithelial capillaries of the guinea pig gallbladder, confirming previous findings reported by Aharinejad and Lametschwandtner (1992) and Caggiati et al. (1992). The authors reported two vascular plexuses within the wall of the gallbladder, namely, an outer subserosal plexus with supplying arterioles and draining venules and an inner mucosal plexus composed of the dense network of subepithelial capillaries.
The three-dimensional organization of the blood vessels of the bile tract is described in the rat, guinea pig, dog, and man (Lang, 1970; Pfoerringer, 1971; Ohtani, 1979; Aharinejad et al., 1991, 1992, 1994; Aharinejad and Lametschwandtner, 1992; Gaudio et al., 1993). These studies clearly showed that two vascular plexuses exist along the whole bile tract/bile ducts and that despite common vascular patterns, species-specific differences exist, e.g., in the distribution of the serosal blood vessel or the presence or absence of periglandular capillaries.
The aim of the present study is to analyze and correlate the light microscopical structure of selected segments of the extrahepatic bile ducts, namely, the extrahepatic ducts, the cystic duct, the common bile duct, and the gallbladder, with the architecture of the microvascular bed of these segments.
MATERIALS AND METHODS
Vascular Corrosion Casting
Six 4-month-old miniature New Zealand rabbits (four females, two males; body weight, 500–700 g) purchased from a local breeder were studied. Animals were injected intraperitoneally with an overdose of sodium pentobarbital and the blood vascular system was rinsed with saline via the thoracic aorta-celiac artery followed by the injection of 40–60 ml of Mercox CL-2B diluted with monomeric methylmethactylate (v:v = 4:1) containing 1.5 g catalyst (paste MA) per 20 ml monomeric methylmethactylate (Ladd Research, Burlington, VA) using manual pressure. Specimens were excised, placed into a water bath (12 hr, 60°C), macerated (15% NaOH, 12 hr, 35°C), rinsed in tap water, cleaned in 10% formic acid, washed in a series of distilled water, and air-dried. Some corrosion cast were frozen in the distilled water and cut into small pieces with a razor blade. All dry specimens were mounted with colloidal silver according to the method of Lametschwandtner et al. (1980). After sputter-coating with gold, specimens were examined in the scanning electron microscope LEO 435 VP (Zeiss, Oberkochen, Germany) at an accelerating voltage of 10–15 kV and a working distance of 4–10 mm. Micrographs were taken at magnifications ranging from 36 to 1,050×. Vessel diameters were measured in high-powered scanning electron microscopy (SEM) micrographs keeping image depth of focus low to minimize errors in length (diameter) measurements.
After a thorough SEM inspection of the serosal vascular patterns, selected specimens were demounted from specimen stubs, freed of conductive bridges, submerged in a container of suitable size filled with distilled water, frozen, mounted onto wooden support plates, and sectioned transversely by a mini-wheel-saw (Lametschwandtner and Lametschwandtner, 1992) in the frozen state for later comparison with histological transverse sections. Sectioned specimens were cleaned in several passages of distilled water and reprocessed as described above.
For light microscopical observations, three adult miniature rabbits were rinsed with phosphate-buffered saline via the thoracic artery. Then extrahepatic bile ducts and gallbladder were excised and fixed by immersion in Bouin's solution. Specimens were dehydrated, embedded in paraplast, sectioned (transverse section thickness: 3–4 μm), stained with Masson's trichrome, periodic acid Schiff (PAS), and orcein-resorcin methods according to Romeis (1989). Sections of representative segments of the bile tract and the gallbladder were documented by an Axioscope microscope (Zeiss, Jena, Germany) equipped with a digital camera, and diameters were measured.
In the rabbit, bile from the hepatic lobes drains via intrahepatic biliary canaliculi into four or five extrahepatic ducts, which individually join the cystic duct (Fig. 1). A common hepatic duct is missing. Diameters of hepatic ducts range from 220 μm (close to the liver) to 340–400 μm (close to cystic duct). Arteriolar branches from the left hepatic artery with an average diameter of 26 μm supply hepatic ducts (Fig. 2a and b). They lie in the subserosal tissue, run obliquely to the long axis of the ducts, and capillarize immediately below the ductal epithelium. The wall of a hepatic duct consists of a flat mucosal layer composed of a simple cuboidal epithelium with single goblet cells intermingled between and an outer subserosal connective tissue layer (Fig. 2a). Close to the cystic duct, epithelial cells become cylindrical and simple mucous glands appear in the subepithelial lamina propria. Subepithelial capillaries form elongated irregular meshes (Fig. 2d). Blood from the mucosa drains into venules, which form a distinct venous plexus (Fig. 2b and c). Diameters of subserosal venules range from 22 to 45 μm; those of the main collecting venules merging into the cystic vein average 70 μm.
The cystic duct of the miniature rabbit is 7–12 mm long; its diameter is 500–650 μm (Fig. 1). The cylindrical cells of the duct mucosa own elongated nuclei (Fig. 3a). Their apical cytoplasm is condensed and PAS-positive. Few simple alveolar mucous glands lie beneath the epithelium.
The subserosal vascular plexus of the cystic duct is very dense (Fig. 3b). Supplying arterioles arise from the left hepatic artery. In the subserosa, they give off branches with average diameters of 30 μm. Arterioles run helically along the cystic duct, branch off short precapillary vessels with diameters from 16 to 25 μm, which finally enter the mucosa (Fig. 3c). Here they form the subepithelial capillary network with irregular-shaped meshes (Fig. 3d). Mesh sizes range from 40 to 60 μm. Subepithelial capillaries immediately drain into the subserosal venous plexus formed by voluminous venules averaging 30–50 μm in diameter (Fig. 3b, c, and e–h). Between subserosal venules many interconnections exist (Fig. 3b and c). Venules located near the branch of the cystic vein, which collects blood from the cystic duct's own valves (Fig. 3f). The diameter of the cystic vein, which runs parallel to the cystic duct, ranges from 90 to 110 μm (Figs. 1 and 3b).
Common Bile Duct
The common bile duct is the longest and thickest segment of the bile tract. In the miniature rabbit, it is about 1.5 cm long and 4–6 mm thick. It opens on the duodenal papilla into the cranial duodenum some 2 cm distal to the pylorus.
The common bile duct mucosa is slightly folded and owns alveolar and partly tubular PAS-positive mucous glands (Fig. 4a). Toward its junction with the duodenal wall, mucosal folds and mucous glands increase in number.
The blood supply to the common bile duct is via short arterioles, which arise about every 0.5 mm from the nearby pancreatic-duodenal artery. Arterioles have diameters around 40 μm, bifurcate, and obliquely embrace the duct (Fig. 4b). Vessels frequently interconnect and form a subserosal arterial network (= rete arteriosum subserosum). Subserosal venules (diameters: 80–110 μm) also interconnect frequently and form a dense venous network (= plexus venosus subserosum; Fig. 4b), whereby distances between collecting venules are larger than in the hepatic and the cystic duct. Locally, arteries and veins run side by side (Fig. 4b and c). A characteristic feature of the subserosal layer is the abundance of capillaries in the connective tissue (Fig. 4c). In the mucosa, precapillary arterioles give rise to a very dense subepithelial capillary network with 30–60 μm wide, rather polygonal meshes (Fig. 4d). Mucosal glands of the alveolar type located in the lamina propria (Fig. 4a) open into the duct lumen (Fig. 4d). A distinct capillary ring encircles the 50–120 μm wide glandular openings (Fig. 4d).
Gallbladders dissected from two dead miniature rabbits contained 0.6 ml in a small contracted organ and 1.0–1.2 ml in a fully filled distended gallbladder, suggesting a decrease in bile volume up to 50% during gallbladder contraction. If filled, the miniature rabbit gallbladder reveals an elongated 3 cm long body and a narrow neck of approximately 5 mm in diameter.
The wall of the gallbladder consists of a mucosal layer and a thin muscular layer, which is covered by a well-developed outer subserosal connective tissue layer. The muscular layer is made up by an obliquely or circularly running thin band of muscle cells. The prominent structures of the gallbladder mucosa are the connective tissue folds delineating the polygonal crypts. The height of the folds and the size of the crypts change with the grade of filling of the gallbladder. While crypt diameters in the filled state gain 300–1,500 μm, their diameters decrease to 100–600 μm and the mucosal folds become taller when the gallbladder contracts.
The muscular cystic artery is the main supply of the gallbladder. It arises from the left hepatic artery, has an average diameter of 200 μm, and owns a well-developed internal elastic membrane (Fig. 1). Along the neck and the body of the gallbladder, the artery runs parallel to the cystic vein. In the neck region, it gives off 2–3 arterioles, which obliquely traverse the dense subserosal venous plexus. This venous plexus closely resembles that of the cystic duct (Fig. 5). In the body region, the cystic artery gives off symmetrical arterioles, which further branch to form the arterial subserosal rete (= rete arteriosum subserosum; Fig. 6a). After crossing two-thirds of the length of the body of the gallbladder, the cystic artery bifurcates into two terminal branches directed toward the apex of the gallbladder. Terminal arterioles give off capillaries both to the subserosal connective tissue and the muscular layer, before they finally divide into short precapillary arterioles running to the base of mucosal folds (Fig. 6b). The mucosa shows a delicate subepithelial capillary network with 20–40 μm wide meshes, which closely follow the mucosal folds (Fig. 6c and d). Blood from the subepithelial capillaries drains both into venules running at the base of mucosal folds and into venules located at the bottom of the crypts (Fig. 6c, e, and f). In the full gallbladder, collecting venules are straight and run slightly obliquely from the tip toward the base of the mucosal folds (Fig. 6c). The diameter of collecting venules is about 25 μm. During contractions of the gallbladder, the regular patterns of polygonal mucosal crypts change so that venules in the then higher folds run almost perpendicular to the muscular layer (Fig. 6d). Additionally, capillaries and collecting venules in the mucosal folds become more tortuous. The venules draining blood from several crypts form the subserosal venous plexus (= plexus venosus subserosum). This plexus locates next to the muscular layer, i.e., beneath the superficial network of subserosal arterioles (Fig. 6c). Interestingly, venous valves were observed only in the cystic vein at the level of the gallbladder neck.
In the miniature rabbit, the extrahepatic bile tract becomes more complex and thicker the closer the tract approaches the duodenum. So diameters increase from approximately 400 μm (hepatic duct) to approximately 3–4 mm (common bile duct). Light microscopically, the hepatic ducts show a thin mucosa with an inner single-layered cuboidal epithelium and a thin outer subserosal connective tissue layer. In contrast, cystic duct, common bile duct, and gallbladder reveal a mucosa composed of a cylindrical epithelium with distinct microvilli. Moreover, the mucosa of the gallbladder forms folds, mucous glands appear in the connective tissue of the lamina propria, and the subserosal layer gets significantly thicker. These structures indicate not only that extrahepatic bile ducts, along with the gallbladder, serve the transport of liver metabolites, but that water and mineral salts are actively removed from the hepatic bile and that mucous components are supplemented to the gallbladder bile (Strombeck, 1979). The vascular network in the wall of the bile tract, and in particular the rather dense, volumenous subserosal venous plexus demonstrated so clearly by vascular casts, is strongly involved in these processes. It is interesting to note that in the miniature rabbit, separate branches of the left hepatic artery supply blood to extrahepatic ducts and to the cystic duct, whereas short arteriolar branches of the cranial pancreatic-duodenal artery supply the common bile duct and its intraduodenal segment in the duodenal papilla. The cystic artery, as a large branch of the left hepatic artery, supplies the gallbladder only. According to the classification of Gordon (1967), the dividing/branching of the cystic artery on the gallbladder body is defined as bipinnate.
Previous studies on the microvascularization of the walls of the bile tract in the rabbit, rat, and guinea pig described a subserosal and a mucosal plexus (Cho and Lunderquist, 1983; Aharinejad and Lametschwandtner, 1992; Aharinejad et al., 1992; Gaudio et al., 1993). We found a similar system of blood vessels in the miniature rabbit, where supplying arterioles and draining venules lie in the subserosal layer. Short arterioles arise from the subserosal arterial plexus and pass to the mucosa, where they form a subepithelial capillary network with small meshes making an intimate contact with epithelial cells and subepithelial mucosal glands. Blood from the mucosal capillary plexus drains via short postcapillary venules into the well-developed venous network of the subserosal layer.
Although the general structure of the layers is similar all over the whole bile duct, architectonic patterns of blood vessels differ in certain segments of the duct. The differences concern the system of supplying subserosal arterioles, which in the hepatic and cystic ducts impose as single vessels twisted spirally around the ducts, whereas they interconnect in the common bile duct and form an arterial network (rete), which according to the Nomina Anatomica Veterinaria (NAV) nomenclature (1994) may be defined as rete arteriosum subserosum. In all the above-mentioned segments of the bile tract, subserosal venules form a well-developed dense plexus venosus subserosus.
Interestingly, in the widest end segment of the bile tract, blood vessels run parallel in the subserosal layer, which resembles the situation given in the stomach and the intestines. The change in the vascular pattern found in the widest portion of the rabbit bile duct might be considered a feature characteristic for larger animals as a similar system is reported in the bile tract of the cat (data not shown). In the guinea pig, diameters of all the ducts of the bile tract remain constantly 1 mm thick and the vascular system is similar throughout its length (Aharinejad et al., 1992).
The second difference of the vascular system in the wall of the above-mentioned segments of the bile ducts is the mucosal capillary network composed of 30–60 μm wide polygonal meshes. As in the rat (Gaudio et al., 1993) and the guinea pig (Aharinejad et al., 1994), a dense subepithelial rete is present in the cystic and the common bile duct. This rete surrounds mucosal glands located in the lamina propria of the mucosa.
The patterns of subserosal and mucosal vascular networks are similar in the wall of the gallbladder and the bile duct system. Differences in the subserosal vascular network exist between the short neck and the body of the gallbladder. The more superficial, dense subserosal venous plexus in the neck resembles that in the cystic duct, whereas in the body the large veins lie under the superficial network of the subserosal arterioles close to the muscular coat. The microvascularization of the mucosal network of the gallbladder was the subject of research based on the vascular corrosion casts in the guinea pig and rabbit (Aharinejad and Lametschwandtner, 1992; Cagiatti et al., 1992). The present observations in the miniature rabbit made it possible to correlate vascular patterns with volume changes of the gallbladder. In the filled gallbladder, these postcapillary venules descend obliquely from the apex of the crypt folds to their base and further to the muscular coat; the pattern of these vessels is straight. In the contracted gallbladder, folds of the mucosa elongate with the simultaneous narrowing of the crypts and the mucosal venules become elevated in such a way that they become vertical blood vessels. Moreover, the pattern of venules and capillaries is more tortuous than in the bile tract segments. Contrary to the results obtained in the guinea pig, dog, and cat (Lang, 1970; Aharinejad and Lametschwandtner, 1992; Jackowiak and Godynicki, 2003), the rabbit has no special structures defined as vascular plicae or glomerula, constituting a kind of a reservoir, i.e., a set of replacement vessels observed as projections extending toward the lumen in the contracted gallbladder.
In conclusion, we consider the dense subserosal venous plexus found over the entire length of the bile tract and the rete arteriosum subserosum being present in the common bile duct only as vascular beds with high transport capacities for the delivery of substances to and/or the removal of others from the bile and thus play an important functional role for bile modification as well as for supplying nutrients and oxygen to the bile tract tissues.
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