MICROSCOPY RESEARCH AND TECHNIQUE 78:220–229 (2015)
Light Microscopy and Scanning Electron Microscopy Study on Microstructure of Gallbladder Mucosa in Pig EWELINA PROZOROWSKA,* AND HANNA JACKOWIAK Department of Histology and Embryology, Pozna n University of Life Sciences, Wojska Polskiego 71C, 60-625 Pozna n, Poland
KEY WORDS
gallbladder; mucosa; glands; domestic pig; LM; SEM; NaOH maceration
ABSTRACT The present light microscopy (LM) and scanning electron microscopy (SEM) studies on porcine gallbladder mucosa provide a description of the microstructures of great functional importance such as mucosal folds, the epithelium, glands, and lymphatic nodules. The results showed the regional structural differences of the porcine gallbladder wall. Depending on the part of the gallbladder, three types of mucosal structures were described: simple and branched folds and mucosal crypts. An important structural feature found in the mucosa is connected with the structural variety of type of mucosal folds, which change from simple located in the neck, to most composed, i.e., branched or joined, in the polygonal crypts toward the fundus of the gallbladder. The morphometric analysis showed statistically significantly differences in the form and size of the folds and between the fundus, body, and neck of the gallbladder. Differences in the size of mucosal epithelium are discussed in terms of processes of synthesis and secretion of glycoproteins. Regional, species-specific differences in morphology of mucosal subepithelial glands, i.e., their secretory units and openings, and intensity of mucus secretion were described. Our results on the pig gallbladder show adaptation and/or specialization in particular areas of the mucosa for (1) secretion of mucus in the neck or body of gallbladder and (2) for cyclic volume changes, especially in the fundus of gallbladder. The description of the microstructures of mucosa in the porcine gallbladder could be useful as reference data for numerous experiments on the bile tract in the pig. Microsc. Res. Tech. 78:220–229, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Studies on the microstructure of the mammalian gallbladder have focused so far on the characterization of the mucous membrane, including functional aspects of the ultrastructure of the absorptive epithelium with the brush border, as well as the description of the nutritional and functional microvasculature in the gallbladder wall. The studies take into account the aspect of cyclical changes in the volume of the organ, as well as changes in the gallbladder microstructure in frequent functional disturbances and in pathological processes. The microstructure of the gallbladder wall has been described in detail mainly in humans, rodents, dog, cat, and carnivores (Aharinejad and Lametschwwandtner, 1992; Caggiati et al., 1992; Hayward, 1962; Jackowiak, 2006; Jackowiak and Godynicki, 2006; Lamont et al., 1984; MacPherson et al., 1983; Wheeler, 1971). Most data emphasis the importance of the gallbladder epithelium, involved in processes of absorption and transport of water and electrolytes into subepithelial capillaries during modification of bile. Studies using light and scanning electron microscopy showed that due to changes of gallbladder size, the lamina propria mucosae forms species-specific simple folds or/and polygonal crypts, which are accumulations of the connective tissue undergoing progressive stretching of the mucosa during expansion of the gallbladder (Jackowiak and Lametschwwandtner, 2005; Jackowiak, 2006; Mueller et al., 1972; Pfuhl, 1932). Important structures C V
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in the lamina propria of the gallbladder mucosa are distributed subepithelially, tubular or tubulo-vesicular glands, which produce mucus. The distribution of these mucous glands is species specific and secretory units can be uniformly dispersed in the gallbladder mucosa or form glandular clusters, mainly in the body and/or the neck of the gallbladder (Jackowiak, 2006). Studies of mucus composition indicated different types of glycoproteins, which play protective functions again the action of bile (Madrid et al., 1997). Observations of the gallbladder in large farm animals, including also the pig, are rare. An inspiration for this study was provided by increasing interest in research on the digestive system in the pig, which nowadays is considered to be a model animal in observations of alimentary system diseases, e.g., in the course of mucosal inflammation or lesions connected with cholelithiasis or gallbladder cancers (Griffith et al., 1989; Kjaer et al., 2010; Meyerholz et al., 2010; Swindle et al., 2012). Our previous studies on the gallbladder in the domestic pig conducted on vascular micro-corrosion casts described the well-developed three-dimensional *Correspondence to: Ewelina Prozorowska; Department of Histology and Embryology, Pozna n University of Life Sciences, Wojska Polskiego 71C, 60-625 Pozna n, Poland. E-mail: [email protected] Received 17 October 2014; accepted in revised form 23 December 2014 Review Editor: Prof. George Perry DOI 10.1002/jemt.22465 Published online 20 January 2015 in Wiley Online Library (wileyonlinelibrary.com).
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system of blood vessels in the gallbladder wall and cyclic changes in the volume of the gallbladder. Scanning electron microscopic observations indicated a rich microvasculature of the gallbladder mucosa and numerous vascular loops as adaptations to volume changes (Prozorowska and Jackowiak, 2014). The aim of the present study is to describe the detailed microstructures of the mucosa lining the neck, body, and fundus of the gallbladder. Histological observations of the gallbladder are supplemented with the results of scanning electron observations of the gallbladder surface and also of tissue samples after NaOH maceration, facilitating visualization of the collagen scaffold in the lamina propria.
the gallbladder (Fig. 1). Tissues were fixed by immersion in 4% buffered formalin or Bouin’s solution for 24 h, after which the specimens were rinsed with distilled water and dehydrated in a series of ethanol dilutions (70–96%). Tissues were immersed in two changes of methyl benzoate and embedded in paraplast. The paraffin sections were 4.5 mm thick. Some sections were stained by the Masson’s-Goldner method (Romeis, 1989). Other sections were stained according to PAS and HID/AB methods to identify the substance in the mucus glycoproteins. Images were analyzed using a histological Axioscope plus light microscope (ZEISS) and a photographic camera (SONY SSC-DC 58AP Color Video Camera). Gallbladder specimens for observations of mucosal surfaces under a scanning electron microscope (SEM) were gently rinsed with saline to remove bile. After fixation in 4% formalin, the specimens of 0.5 cm were dehydrated in a series of ethanol with increasing concentrations (50–100%) and acetone (50 and 100%) and critical point dried using CO2 (Critical Point Dryer K850, EMITECH, England). The sections used to observe collagen structures were removed by maceration of cellular elements and intercellular substance according to Ohtani (1988). The fixed tissues were washed in distilled water and placed in 10% NaOH for 10–14 days at 20 C. After rinsing in distilled water and dehydratation of macerated samples in ethanol (50–100%) and acetone the tissues were critical point dried. Dried specimens were mounted on aluminum stubs covered with carbon tape using a silver paste and then gold sputtered (Sputter Coater S 150B, EDWARDS, England). Specimens were viewed and documented using a ZEISS 435 VP scanning electron microscope (Germany). Morphometric studies of mucosal pig gallbladder elements were performed on about 60 histological sections from all parts of the gallbladder, using the MULTISCAN 6.08 image analysis software (Scanning System Computer, Warsaw, Poland). For each section, 7–10 measurements were performed of thickness of the wall, height of the folds of the mucous membrane, the distance between folds, and the thickness of the epithelium covering the mucosa. Gallbladder wall thickness was measured between folds of the mucosa. Based on morphometric data, the mean values and standard deviations were calculated together with the R softminimum and maximum values. The StatisticaV ware was used to analyze the morphometric data. The significance of differences between the results was evaluated using Student’s t-test at P 0.05.
MATERIALS AND METHODS Gallbladders were dissected after slaughter from five Polish Great Large White pigs aged 7–9 months (weight about 280–300 kg) and prepared for observations under a light microscope (LM) and a scanning electron microscope (SEM). The animals were not feed before slaughter, thus the organs at the time of dissection were physiologically shrunk. Specimens for light microscopy examinations were collected from the free and so-called hepatic surface of the gallbladder and also from the fundus and neck of
RESULTS The gallbladder wall in the pig consists of the wellfolded mucosa comprising subepithelial glands, the muscle layer, and serosa with a well-developed subserosa (Fig. 2). The morphometric results indicate that the thickest wall, up to 2,230 mm, is found in the area of the fundus of gallbladder (Tables 1 and 2). In the gallbladder body, the thickness of the wall does not exceed 1,814 mm, whereas the results of measurements in the free part of the body and in the hepatic part of the gallbladder body showed no significant differences at P 0.05 (Tables 1 and 2). The neck of the
Fig. 1. Macroscopic view of the pig’s gallbladder. (1) Fundus of the gallbladder; (2) Body of the gallbladder; (3) Neck of the gallbladder; (4) Liver. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Fig. 2. A cross-section of the wall of pig’s gallbladder. (1) Branched folds of mucosa; (2) Lamina propria of mucosa; (3) Muscular layer; (4) Subserosal layer. Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
gallbladder has a thin wall of max. 491 mm. Statistical analysis of the thickness of the gallbladder wall in the neck and fundus of the gallbladder showed significant differences at P 0.05. Similar results were obtained when analyzing the thickness of the fundus and body of the gallbladder wall and between body of the gallbladder and its neck. The maximum difference between the thinnest and thickest wall of the gallbladder of a pig is over 1,900 um (Table 1). The mucosa of the pig gallbladder is strongly undulated over the entire surface. A dense network of blood and lymph vessels and subepithelial glands is distributed in the connective tissue of the lamina propria of the mucosa (Figs. 13 and 15). Furthermore, single lymph nodules are distributed in the subepithelial and mucosal area in the neck and fundus of the gallbladder (Figs. 3a and 3b). The lamina propria of the mucosa is composed of loose connective tissue, rich in collagen bands of irregular array, and constitutes the basic element to establish the folds and form a fibrillar skeleton for blood vessels. In the pig gallbladder mucosa, two types of mucosal folds are distinguished, i.e., simple and branched folds (Figs. 4 and 5). The simple folds are lower, elongated undulations of connective tissue and outside the area of gallbladder’s neck are usually found between the branched folds (Figs. 4,5 and 10a–10c). The branched folds consist of a broad low base and fingerlike or conical branches (Figs. 5,7,8 and 9). The top of the branches is on average 2.5–4 times narrower than its base part.
Fig. 3. Magnification of pig’s gallbladder mucosa with a subepithelial (a) and mucosal (b) lymph nodules (asterisks). (a) Body of the gallbladder (part free); (b) Neck of the gallbladder. Staining: MassonGoldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
In the fundus and body of the gallbladder in the pig folds join and form polygonal structures, called mucosal crypts (Figs. 8a, 9a, and 9b). Secondary folds, i.e., low elevations of the lamina propria of the mucosa, were also observed both within the mucosal crypts and between simple and branched folds in all areas of the gallbladder. In the fundus of the gallbladder, the simple folds, branched folds, and crypts of the mucosa were observed in the lamina propria of the mucosa. The average height of the simple folds in fundus does not exceed 200 mm (Table 1). The branched folds are on average 2–3 times higher than the simple folds, and their height is ca. 542 mm (Tab.1). It was observed that in the fundus the branched folds usually join to form mucosal crypts. Irregular or polygonal crypts of mucosa are 150–1,500 lm in diameter. Single lymphatic nodules were visible in the fundus of gallbladder. In many cases, the subepithelially distributed nodules expand in the direction of gallbladder lumen. The presence of the subepithelial mucosal glands was not found either on histological slides or SEM crosssections of the mucosa of the gallbladder fundus. In the area of the gallbladder body fused with the surface of liver the mucosal folds are predominantly branched type of ca. 495 mm in height and they lie close Microscopy Research and Technique
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TABLE 1. Measurements from pig’s gallbladder wall and mucosa Part of the gallbladder
Thickness of the wall of the gallbladder High of compounded folds of the mucosa High of simple folds of the mucosa Distance between folds of the mucosa High of the epithelium
Fundus of the gallbladder
Body of the gallbladder part fused with liver
X
X
X
X
SD
SD
SD
SD
Min - Max
Min - Max
Min - Max
Min- Max
1697.3 283.1 1036.1–2230.8 542.6 86.4 410.9–707.6 196.5 68.2 104.1–320.1 282.0 77.2 152.9–413.9 26.4 3.1 20.3–32.1
1436.1 259.3 1024.1–1813.1 495.9 46.7 423.8–604.3 253.8 82.0 140.8–356.8 116.7 59.5 63.8–287.2 23.1 2.4 17.0–27.0
1326.9 132.7 1123.2–1590.5 400.4 55.1 297.7–507.7 136.9 25.3 85.6–190.3 103.8 48.8 41.5–211.3 27.6 4.2 20.0–36.0
391.1 69.4 307.1–491.0 138.6 34.7 94.1–191.1 162.9 57.0 96.2–293.0 22.7 4.4 17.8–26.6
Body of the gallbladder - part free Neck of the gallbladder
Results expressed as: X-arithmetical means, SD- standard deviation, Min- minimum value, Max- maximum value.
TABLE 2. Comparison of the thickness of the wall and the height of simple and branched folds of the mucosa in the fundus, the body, and the neck of the gallbladder in pig
to each other in rows (Figs. 7a and 7b). Between the branched folds, there are numerous openings of the subepithelial glands composed of tubulo-alveolar secretory units and short excretory duct (Figs. 7a, 13a, 14a, and 14b). Openings of mucosal glands lie often in small cavities surrounded by mucosal elevations and simple folds. SEM images of the surface of the socalled hepatic part of the gallbladder body often show one common hole, to which the excretory ducts of several secretory tubuli or acini anastomose (Fig. 13a). In the free surface of the gallbladder body, the branched folds with an average height of 400 mm Microscopy Research and Technique
combine at the basal sections and similarly as in the fundus of the gallbladder they form mucosal crypts (Fig. 8a). These structures in the gallbladder body have irregular shapes and their diameter is more than twice less than in the fundus of gallbladder. In the free part of the body of the gallbladder, few glands were observed on the mucosal surface and their diameter was smaller than in the hepatic area of gallbladder body (Figs. 8a and 8b). Similarly, as in the fundus of gallbladder, the lymphatic nodules bulge out into the lumen of this organ (Fig. 3a).
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Fig. 4. Magnification of the surface of the mucosa in the neck of gallbladder in pig; yellow arrows show simple folds of mucosa. NaOH maceration, SEM; Fig. 5. Magnification of the surface of the mucosa in the free part of body of the gallbladder in pig; yellow arrow shows fibrillar frameworks of simple fold of mucosa, black arrow shows fibrillar framework of branched fold of mucosa; (a) Apical part of
branched fold; (b) Basal part of branched fold. NaOH maceration, SEM; Fig. 6. A view of surface of mucosa in the body of the gallbladder in pig. (1) Folds of mucosa; (2) The mucus, SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 7. SEM and LM views of the pig’s gallbladder mucosa in the body of the gallbladder; part fused with liver. (a) A view of the surface of the mucosa. (1) Branched folds arranged in rows, arrows show the openings of subepithelial glands, NaOH maceration, SEM; (b) magni-
fication of the branched folds of mucosa, NaOH maceration, SEM; (c) a cross-section of mucosa with branched fold (1) and fingerlike fold (2), Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 8. SEM and LM views of the pig’s gallbladder mucosa in the body of the gallbladder; part free; (a) A view of the surface of the mucosa; (1) Branched and simple folds that surround irregular mucosal crypts, yellow arrows show the openings of subepithelial glands, NaOH maceration, SEM; (b) Magnification of the surface of mucosa
with opening of gland, NaOH maceration, SEM; (c) A cross-section of the mucosa with conical folds (1), Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Fig. 9. SEM and LM views of the pig’s gallbladder mucosa in the fundus of the gallbladder, (a) a view of the surface of the mucosa; (1) Wide folds forming a polygonal crypts, 2-compound folds (intensely folded area), NaOH maceration, SEM; (b) Magnification of the compound folds of mucosa, the arrow show the crypt of the mucosa,
NaOH maceration, SEM; (c) A cross-section of mucosa with conical fold (1) and digitate fold (2). Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 10. SEM and LM views of the pig’s gallbladder mucosa in the neck of the gallbladder. (a) A view of the surface of the mucosa; (1) Simple folds of the mucosa, (2) openings of the subepithelial glands, NaOH maceration, SEM, (b) magnification of the simple folds of mucosa (1), 2- opening of the subepithelial gland, NaOH maceration,
SEM; (c) A cross-section of the mucosa with simple folds (1) and subepithelial gland (2) Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
In the pig, the neck area of the gallbladder differs in the arrangement and size of the folds from the other parts of the gallbladder. Only low and wide simple and narrow pin-shaped folds may be observed on LM and SEM images of the gallbladder neck (Figs. 10a, 10b, and 10c). These folds lay parallel to each other or join and run circumferentially around the openings of mucosal glands (Figs. 10a and 10b). Numerous compound glands and single lymphatic nodules surrounded by loose connective tissue were observed in the mucosa of the neck of pig gallbladder (Fig. 3b). Significant differences were found in average height of the folds in individual parts of the gallbladder at a significance level P < 0.05. The greatest statistically significant differences were found in height of folds in the gallbladder fundus and neck (Tables 1 and 2). In turn, the least statistically significant differences were recorded between the folds located at the fundus of the gallbladder and the hepatic area of the gallbladder body. The largest branched folds, of up to 700 mm in height, are found in the fundus of the gallbladder, but the largest simple folds of ca. 365 mm are located in the
hepatic part of the body (Tables 1 and 2). The folds in the fundus of gallbladder lie at the greatest distance of about 282 lm, whereas the smallest distance between the folds, averaging 103 mm, was recorded in the mucosa of the free part of the gallbladder body (Table 1). Detailed metric measurements of gallbladder wall thickness, height of simple and branched mucosal folds, the distance between the folds and the thickness of the epithelium in porcine gallbladders are shown in Table 1. The entire surface of the gallbladder mucosa in the pig is covered with a single-layered cylindrical epithelium (Fig. 11). The high epithelial cells adhere tightly to each other, forming a polygonal configuration on the surface observed in SEM (Fig. 12). These cells have a characteristic arrangement for epithelial cell nuclei, because oval cell nuclei lie in the basal part of the cells and occupy one-third of their height (Figs. 11a–11d). Morphometric observations showed that the epithelium in different parts of the gallbladder reaches thickness from 17 to 36 mm (Table 1). SEM and histochemical observations of gallbladder mucosa identified the composition of porcine mucus produced by
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Fig. 11. A view of the cross-sections of monolayer cylindrical epithelium subjected to various staining in pig’s gallbladder; (a) Epithelial cells in free part of the body of gallbladder, Staining: MassonGoldner, LM. (b) Results of staining with HID/Alcian blue in epithelial cells in the fundus of gallbladder, arrow show a unicellular mucous gland, LM. (c) Results of staining with HID/ Alcian blue in epithelial cells in the body of the gallbladder; arrow show epithelial cells secreting the mucus. LM. (d) PAS positive reaction in epithelial cells in the body of the gallbladder; LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the epithelial cells (Fig. 6). Positive results of PAS and HID/AB staining in epithelial cells indicated the presence of mucus glycoproteins (Figs. 11b–11d). The purple color of the apical part of the epithelium as a positive result of the PAS reaction was observed throughout the gallbladder, wherein in different areas of the gallbladder different intensities of color reaction were found. The highest color intensity was recorded for the epithelium covering the gallbladder body and the smallest in gallbladder neck (Fig. 11d). The blue color in the apical parts of the epithelial cells in response to HID/AB staining indicated secretions containing acidic glycoprotein, i.e., sialomucins and sulfomucins. In the gallbladder body, an intense reaction was observed after HID/AB staining (Fig. 11c). In the fundus of the gallbladder, the blue coloration in response to alcian blue was observed only in isolated epithelial cells after HID/AB staining (Fig. 11b). DISCUSSION The present studies of the porcine gallbladder showed regional differences in the microstructure of the gallbladder mucosa. Microscopic and morphometric analyses revealed differences in the arrangement of mucosal folds, distribution and structure of subepithelial glands, and the distribution of lymphoid nodules between the studied regions of porcine gallbladders. The morphometric analysis showed that the thickness of the wall in the pig gallbladder varies in different areas. Significant differences were found between the fundus and the neck, whereas the wall of the neck was three times thinner than the walls of the body, and four times thinner than the fundus. The
Fig. 12. A magnification of the surface of polygonal epithelial cells in the body of the gallbladder in pig. SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
thickness of the wall in the body region of the gallbladder is similar to that in the fundus. The gallbladder mucosa, coating the entire surface of a single-layer cylindrical epithelium, is specialized for compaction and production of bile and the secretion of mucus. Previous studies of Prozorowska and Jackowiak (2014) on the microvascularization of the gallbladder in the pig conducted on vascular corrosion casts revealed that the mucosa is highly vascularized and possesses a dense network of subepithelial capillaries surrounding the secretory units. The morphometric data showed that the epithelial cells of the free part of gallbladder body and of the fundus of the gallbladder were on average 20% higher than the epithelial cells in the neck of the gallbladder and in the body of the gallbladder fused with liver. Histochemical observations of epithelial cells showed differences in the amount and intensity of mucus secretion. Lower epithelial cells in the gallbladder neck produced a weaker color reaction in PAS staining when compared with the higher epithelial cells in the body of the gallbladder. Similar results were obtained for HID/AB staining. The apical part of epithelial cells in the gallbladder body turned brighter alcian blue than the other parts of the epithelial cells of the gallbladder, indicating more intensive production of mucus in the gallbladder epithelium of the body of gallbladder. Microscopic studies performed, e.g., in carnivores and the rabbit showed that gallbladder epithelial cells are polarized and may change in size with synthesis processes (Evett et al., 1964; McPherson Microscopy Research and Technique
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Fig. 13. The views of subepithelial glands in pig gallbladder; (a) A magnification of opening of the subepithelial glands in the body of gallbladder in part fused with liver, 1- the main and common opening of the gland, 2- the openings of the individual secretory units; (b) The cross- section of the mucosa in the neck of gallbladder in pig after removing the epithelium and cells of connective tissue; 1- the secretory units of the subepithelial gland; 2- surface of the mucosa, NaOH maceration, SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
et al., 1983). Apical portion of the cells with microvilli increase the absorptive surface area, thereby contributing to the efficient compaction of bile. Our study show that differences in size of epithelial cells of porcine gallbladder mucosa may arise from the secretory activity of the epithelium. Subepithelial glands are important structures in the lamina propria mucosae. Types of glands, the complexity of their structure and their arrangement in mammalian gallbladders are species specific (Pfuhl, 1932). Mostly single secretory units of glands are straight or coil. In ruminants, there are many glands arranged close together. In bovines, they take the form of bands, whereas in the goat and sheep, they are more dispersed (Jurisch, 1909). In the rabbit, no glands are found on the gallbladder (Jackowiak and Lametschwwandtner, 2005). In humans as well as in the guinea pig, tubulo-vesicular glands are accumulated only in the region of the neck, whereas in carnivores, they are scattered across the mucosa (Bucher and Wartenberg, 1989; Lee, 1980; Laito and Nevalainen, 1975). Microscopy Research and Technique
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Fig. 14. The histological view of the compound subepithelial glands in pig gallbladder; (a) A magnification of the compound subepithelial gland with wide opening of the gland (arrow); asterisk indicate the tubular secretory units, b- alveolar (asterisk) and tubular secretory units in the body of gallbladder, Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
The highest density of glands in the pig gallbladder was found in the neck and in the part of the body fused with liver. In the free part of the gallbladder body, glands are rare, whereas in the fundus, they are absent. Our results also showed differences in types and size of secretory units of mucous glands. In the neck both small, single follicular glands are located, as well as large, complex glands of the coiled and alveolar secretory units, which extended up to the subserosal layer. Glands in the body of the gallbladder have rounded secretory units smaller than in the gallbladder neck and the secretory units take the form of elongated vesiculo-tubular glands. In the gallbladder body fused with liver, the glands are often located in the folds of the mucosa or near lymphatic nodules. SEM images showed the specificity of structure of gland openings on the mucosal surface in the body and in the neck of the gallbladder. Density of gland openings per 1 mm2 of mucosal surface also varies. In the neck, there are 0.6 outlets of glands per 1 mm2 of the mucosa, but in the body fused with liver, this value increases to 0.8 outlets per 1 mm2 of the mucosa. In
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Fig. 15. PAS staining of the mucosa in the neck of the gallbladder; (1) Secretory units of the subepithelial gland, (2) the mucosa layer, (3) lumen of the neck of the gallbladder filled with mucus, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the neck, the openings are wide and deep and they are evenly distributed over the entire surface, whereas in the gallbladder body the openings of several secretory units are compacted in a common, wide hole surrounded by an elevation of mucosa, which influenced the above-mentioned results concerning the density of openings. The single-layered epithelium lining the secretory units of glands is lower than the epithelium on the surface of mucosa. As it was shown by previous studies, in mammals the glandular cells, as well as surface epithelial cells of the gallbladder, produce and secrete sticky mucus with high contents of glycoproteins. Its function is to protect the surface against bile and microorganisms. Mucous also enriches the bile composition, so that bile flowing toward the duodenum has a thicker consistency (Madrid et al., 1997). Lymph nodules are the integral structural elements of the mucous membrane in the pig gallbladder. They are found in the gallbladder mucosa with the greatest density in the gallbladder body fused with liver. In this area, the subepithelially situated, oval-shaped lymph nodules accentuate over the mucosa, which makes these structures macroscopically visible. Single lym-
phatic nodules as structural components of the immune system are commonly found in the mucosa of the digestive, respiratory and urogenital tracts, where they serve defense functions. As it is presented in literature concerning microstructures of the gallbladder wall, they are not permanent structural elements of the mucosa. They are not observed in the gallbladder in humans and carnivores (Evett et al., 1964; Jackowiak, 2006). Their presence in the porcine gallbladder should be considered as a species-specific feature. This data could be important and useful as remark for pathologist. Folds of the mucosa are important features of gallbladder mucosa associated with the process of absorption. Many animal species possess regular folds, which join to form polygonal structures called mucosal crypts. Such a configuration is described in some carnivores, e.g. the dog, raccoon dog, foxes, American mink, primates, the rabbit and in the human (Castellucci and Caggiati, 1980; Caggiati et al., 1992; Jackowiak and Lametschwwandtner, 2005; Jackowiak 2006; Lang, 1970; Mueller et al., 1972; Pfuhl, 1932). Our observations in the pig allowed us to distinguish simple and branched folds, which are usually connected together by wide basal portions. Mucosal folds are connective tissue elevations of the lamina propria, composed primarily of the collagen fiber framework. The observations showed that each of the gallbladder areas has a unique arrangement of folds, which differ in shape and size. The wall of the fundus of gallbladder has the most corrugated surface and the folds of the mucosa are the highest, which was found in areas with greater amounts of connective tissue and indicates a greater potential for expansion of this ventral area of the gallbladder. It is important to remember that the folds in the fundus of pig gallbladder form polygonal crypts, indicating that during filling the gallbladder with bile they can distend, but they do not disappear. Such an observation was made previously on mucosal crypts in some carnivores (Jackowiak, 2006). The type, density and shape of the mucosal folds in the gallbladder change depending on the degree of filling of the organ with bile. Our studies in the contracted pig gallbladder showed structural differences related to particular areas of the gallbladder. It was concluded that wall thickness increases from the neck to the fundus of the gallbladder, similarly as it is with the height of mucosal folds. An important structural feature is connected with the structural variety of types of mucosal folds, which change from simple located in the neck, to most composed, i.e., branched or joined, in polygonal crypts toward the fundus of the gallbladder. Our results on the pig gallbladder show adaptation and/or specialization in particular areas of the gallbladder for (1) secretion of mucus in the neck or fundus, and (2) for cyclic volume changes especially in the gallbladder fundus. A description of the microstructures in the porcine gallbladder could be useful as reference data for numerous experiments on the bile tract in the pig. REFERENCES Aharinejad S, Lametschwwandtner A. 1992. Microangioarchitecture of the guinea pig and bile duct as studied by scanning electron microscopy of vascular corrosion casts. J Anat 181:89–100.
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MICROSTRUCTURE OF GALLBLADDER MUCOSA IN PIG Bucher O, Wartenberg H. 1989. Cytologie, Histologie und mikroskopische Anatomie des Menschen. Hans Huber, Bern. Caggiati A, Macchiarelli G, Nottola SA, Vizza E, Familiari G. 1992. Scanning electron microscopy of the rabbit gallbladder mucosal microvasculature. J Anat 180:275–280. Castellucci M, Caggiati A. 1980. Surface aspects of the rabbit gallbladder mucosa and their functional implications. J Submicrosc Cytol 12:375–390. Evett RD, Higgins JA, Brown AL. 1964. Fine structure of normal mucosa in human gallbladder. Gastroenterology 47:49–60. Griffith SL, Burney BT, Fry FJ, Franklin TD Jr. 1989. A large animal model (swine) to study the diagnosis and treatment of cholelithiasis. Invest Radiol 24:110–114. Hayward AF. 1962. Aspects of the fine structure of the gallbladder epithelium of the mouse. J Anat 96:227–236. Jackowiak H, 2006. Morphology and angioarchitecture of the gallbladder and extrahepatic bile tract in selected species of Carnivores. Monograph of Poznan University of Life Sciences No. 380, ISSN 1896–1894. Jackowiak H, Godynicki SZ. 2006. A comparative LM and SEM study of the structure of the mucosal glands of the gallbladder in two species of canids: The dog and the Chinese raccoon dog. Folia Morphol (Prague) 65:105–110. Jackowiak H, Lametschwwandtner A. 2005. Angioarchitecture of the rabbit extrahepatic bile duct and gallbladder. Anat Rec 286:974–981. Jurisch A. 1909. Beitrage zur mikroskopischen Anatomie und Histologie Gallenblase. Anat H 39:395–467. Kjaer F, Mortensen V, Mrller JK, Hamilton-Dutoit SJ, Funch-Jensen P. 2010. Internal gallbladder drainage prevents development of acute cholecystitis in a pig model: A randomized study. Ann Surg Innov Res 4:1–7. Laito M, Nevalainen T. 1975. Gland ultrastructure in human gallbladder. J Anat 120:105–112. Lamont TJ, Smith BF, Moore JR. 1984. Role of gallbladder mucin in pathophysiology of gallstones. Hepatology 4:51S–56S.
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} Lang J. 1970. Uber die Kapillaren der Gallenblasenschleimhaut beim Hund. Acta Anat 75:566–577. Lee SP. 1980. The mechanism of mucous secretion by the gallbladder epithelium. Br J Exp Pathol 61:117–119. MacPherson BR, Scott GW, Lennon F. 1983. Morphology of canine gallbladder. Scanning electron microscope observations on the epithelium. Cell Tissue Res 233:161–174. Madrid JF, Hernandez F, Ballesta J. 1997. Charakterization of glycoproteins in the ephitelial cells of human and other mammalian gallbladder. A Rev Microsc Res Tech 38:616–630. Meyerholz DK, Stoltz DA, Pezzulo AA, Welsh MJ. 2010. Pathology of Gastrointestinal Organs in a Porcine Model of Cystic Fibrosis. Am J Pathol 176:1377–1389. Mueller JC, Jones AL, Long JA. 1972. Topographic and subcellular anatomy of the guinea pig gallbladder. Gastroenterology 63:856– 868. Ohtani O, Ushiki T, Taguchi T, Kikuta A. 1988. Collagen fibrillar networks as skeletal Farameworks: A demonstration by Cell-maceration/ Scanning Electron Microscope Method. Arch Histol Cytol 51: 249–261. Pfuhl W. 1932. Die Gallenblase und die extrahepatischen Galleng€ ange. W. In: Handbuch der mikroskopishen Anatomie des Menschen, Berlin: Springer. pp. 428–460. Prozorowska E, Jackowiak H. 2014. Angioarchitecture of gallbladder in pig: LM and SEM study on Vascular Microcorrosion Casts. Microsc Res Tech 77:675–683. Romeis B. 1989. Mikroskopische Technik. Urban and Schwarzenberg, M€ unchen. Swindle MM, Makin A, Herron AJ, Clubb FJ Jr, Frazier KS. 2012. Swine as Models in Biomedical Research and Toxicology Testing. Vet Pathol 49:738. Wheeler HO. 1971. Concentrating function of the gallbladder. Am J Med 51:588–595.
Light Microscopy and Scanning Electron Microscopy Study on Microstructure of Gallbladder Mucosa in Pig EWELINA PROZOROWSKA,* AND HANNA JACKOWIAK Department of Histology and Embryology, Pozna n University of Life Sciences, Wojska Polskiego 71C, 60-625 Pozna n, Poland
KEY WORDS
gallbladder; mucosa; glands; domestic pig; LM; SEM; NaOH maceration
ABSTRACT The present light microscopy (LM) and scanning electron microscopy (SEM) studies on porcine gallbladder mucosa provide a description of the microstructures of great functional importance such as mucosal folds, the epithelium, glands, and lymphatic nodules. The results showed the regional structural differences of the porcine gallbladder wall. Depending on the part of the gallbladder, three types of mucosal structures were described: simple and branched folds and mucosal crypts. An important structural feature found in the mucosa is connected with the structural variety of type of mucosal folds, which change from simple located in the neck, to most composed, i.e., branched or joined, in the polygonal crypts toward the fundus of the gallbladder. The morphometric analysis showed statistically significantly differences in the form and size of the folds and between the fundus, body, and neck of the gallbladder. Differences in the size of mucosal epithelium are discussed in terms of processes of synthesis and secretion of glycoproteins. Regional, species-specific differences in morphology of mucosal subepithelial glands, i.e., their secretory units and openings, and intensity of mucus secretion were described. Our results on the pig gallbladder show adaptation and/or specialization in particular areas of the mucosa for (1) secretion of mucus in the neck or body of gallbladder and (2) for cyclic volume changes, especially in the fundus of gallbladder. The description of the microstructures of mucosa in the porcine gallbladder could be useful as reference data for numerous experiments on the bile tract in the pig. Microsc. Res. Tech. 78:220–229, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Studies on the microstructure of the mammalian gallbladder have focused so far on the characterization of the mucous membrane, including functional aspects of the ultrastructure of the absorptive epithelium with the brush border, as well as the description of the nutritional and functional microvasculature in the gallbladder wall. The studies take into account the aspect of cyclical changes in the volume of the organ, as well as changes in the gallbladder microstructure in frequent functional disturbances and in pathological processes. The microstructure of the gallbladder wall has been described in detail mainly in humans, rodents, dog, cat, and carnivores (Aharinejad and Lametschwwandtner, 1992; Caggiati et al., 1992; Hayward, 1962; Jackowiak, 2006; Jackowiak and Godynicki, 2006; Lamont et al., 1984; MacPherson et al., 1983; Wheeler, 1971). Most data emphasis the importance of the gallbladder epithelium, involved in processes of absorption and transport of water and electrolytes into subepithelial capillaries during modification of bile. Studies using light and scanning electron microscopy showed that due to changes of gallbladder size, the lamina propria mucosae forms species-specific simple folds or/and polygonal crypts, which are accumulations of the connective tissue undergoing progressive stretching of the mucosa during expansion of the gallbladder (Jackowiak and Lametschwwandtner, 2005; Jackowiak, 2006; Mueller et al., 1972; Pfuhl, 1932). Important structures C V
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in the lamina propria of the gallbladder mucosa are distributed subepithelially, tubular or tubulo-vesicular glands, which produce mucus. The distribution of these mucous glands is species specific and secretory units can be uniformly dispersed in the gallbladder mucosa or form glandular clusters, mainly in the body and/or the neck of the gallbladder (Jackowiak, 2006). Studies of mucus composition indicated different types of glycoproteins, which play protective functions again the action of bile (Madrid et al., 1997). Observations of the gallbladder in large farm animals, including also the pig, are rare. An inspiration for this study was provided by increasing interest in research on the digestive system in the pig, which nowadays is considered to be a model animal in observations of alimentary system diseases, e.g., in the course of mucosal inflammation or lesions connected with cholelithiasis or gallbladder cancers (Griffith et al., 1989; Kjaer et al., 2010; Meyerholz et al., 2010; Swindle et al., 2012). Our previous studies on the gallbladder in the domestic pig conducted on vascular micro-corrosion casts described the well-developed three-dimensional *Correspondence to: Ewelina Prozorowska; Department of Histology and Embryology, Pozna n University of Life Sciences, Wojska Polskiego 71C, 60-625 Pozna n, Poland. E-mail: [email protected] Received 17 October 2014; accepted in revised form 23 December 2014 Review Editor: Prof. George Perry DOI 10.1002/jemt.22465 Published online 20 January 2015 in Wiley Online Library (wileyonlinelibrary.com).
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system of blood vessels in the gallbladder wall and cyclic changes in the volume of the gallbladder. Scanning electron microscopic observations indicated a rich microvasculature of the gallbladder mucosa and numerous vascular loops as adaptations to volume changes (Prozorowska and Jackowiak, 2014). The aim of the present study is to describe the detailed microstructures of the mucosa lining the neck, body, and fundus of the gallbladder. Histological observations of the gallbladder are supplemented with the results of scanning electron observations of the gallbladder surface and also of tissue samples after NaOH maceration, facilitating visualization of the collagen scaffold in the lamina propria.
the gallbladder (Fig. 1). Tissues were fixed by immersion in 4% buffered formalin or Bouin’s solution for 24 h, after which the specimens were rinsed with distilled water and dehydrated in a series of ethanol dilutions (70–96%). Tissues were immersed in two changes of methyl benzoate and embedded in paraplast. The paraffin sections were 4.5 mm thick. Some sections were stained by the Masson’s-Goldner method (Romeis, 1989). Other sections were stained according to PAS and HID/AB methods to identify the substance in the mucus glycoproteins. Images were analyzed using a histological Axioscope plus light microscope (ZEISS) and a photographic camera (SONY SSC-DC 58AP Color Video Camera). Gallbladder specimens for observations of mucosal surfaces under a scanning electron microscope (SEM) were gently rinsed with saline to remove bile. After fixation in 4% formalin, the specimens of 0.5 cm were dehydrated in a series of ethanol with increasing concentrations (50–100%) and acetone (50 and 100%) and critical point dried using CO2 (Critical Point Dryer K850, EMITECH, England). The sections used to observe collagen structures were removed by maceration of cellular elements and intercellular substance according to Ohtani (1988). The fixed tissues were washed in distilled water and placed in 10% NaOH for 10–14 days at 20 C. After rinsing in distilled water and dehydratation of macerated samples in ethanol (50–100%) and acetone the tissues were critical point dried. Dried specimens were mounted on aluminum stubs covered with carbon tape using a silver paste and then gold sputtered (Sputter Coater S 150B, EDWARDS, England). Specimens were viewed and documented using a ZEISS 435 VP scanning electron microscope (Germany). Morphometric studies of mucosal pig gallbladder elements were performed on about 60 histological sections from all parts of the gallbladder, using the MULTISCAN 6.08 image analysis software (Scanning System Computer, Warsaw, Poland). For each section, 7–10 measurements were performed of thickness of the wall, height of the folds of the mucous membrane, the distance between folds, and the thickness of the epithelium covering the mucosa. Gallbladder wall thickness was measured between folds of the mucosa. Based on morphometric data, the mean values and standard deviations were calculated together with the R softminimum and maximum values. The StatisticaV ware was used to analyze the morphometric data. The significance of differences between the results was evaluated using Student’s t-test at P 0.05.
MATERIALS AND METHODS Gallbladders were dissected after slaughter from five Polish Great Large White pigs aged 7–9 months (weight about 280–300 kg) and prepared for observations under a light microscope (LM) and a scanning electron microscope (SEM). The animals were not feed before slaughter, thus the organs at the time of dissection were physiologically shrunk. Specimens for light microscopy examinations were collected from the free and so-called hepatic surface of the gallbladder and also from the fundus and neck of
RESULTS The gallbladder wall in the pig consists of the wellfolded mucosa comprising subepithelial glands, the muscle layer, and serosa with a well-developed subserosa (Fig. 2). The morphometric results indicate that the thickest wall, up to 2,230 mm, is found in the area of the fundus of gallbladder (Tables 1 and 2). In the gallbladder body, the thickness of the wall does not exceed 1,814 mm, whereas the results of measurements in the free part of the body and in the hepatic part of the gallbladder body showed no significant differences at P 0.05 (Tables 1 and 2). The neck of the
Fig. 1. Macroscopic view of the pig’s gallbladder. (1) Fundus of the gallbladder; (2) Body of the gallbladder; (3) Neck of the gallbladder; (4) Liver. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Fig. 2. A cross-section of the wall of pig’s gallbladder. (1) Branched folds of mucosa; (2) Lamina propria of mucosa; (3) Muscular layer; (4) Subserosal layer. Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
gallbladder has a thin wall of max. 491 mm. Statistical analysis of the thickness of the gallbladder wall in the neck and fundus of the gallbladder showed significant differences at P 0.05. Similar results were obtained when analyzing the thickness of the fundus and body of the gallbladder wall and between body of the gallbladder and its neck. The maximum difference between the thinnest and thickest wall of the gallbladder of a pig is over 1,900 um (Table 1). The mucosa of the pig gallbladder is strongly undulated over the entire surface. A dense network of blood and lymph vessels and subepithelial glands is distributed in the connective tissue of the lamina propria of the mucosa (Figs. 13 and 15). Furthermore, single lymph nodules are distributed in the subepithelial and mucosal area in the neck and fundus of the gallbladder (Figs. 3a and 3b). The lamina propria of the mucosa is composed of loose connective tissue, rich in collagen bands of irregular array, and constitutes the basic element to establish the folds and form a fibrillar skeleton for blood vessels. In the pig gallbladder mucosa, two types of mucosal folds are distinguished, i.e., simple and branched folds (Figs. 4 and 5). The simple folds are lower, elongated undulations of connective tissue and outside the area of gallbladder’s neck are usually found between the branched folds (Figs. 4,5 and 10a–10c). The branched folds consist of a broad low base and fingerlike or conical branches (Figs. 5,7,8 and 9). The top of the branches is on average 2.5–4 times narrower than its base part.
Fig. 3. Magnification of pig’s gallbladder mucosa with a subepithelial (a) and mucosal (b) lymph nodules (asterisks). (a) Body of the gallbladder (part free); (b) Neck of the gallbladder. Staining: MassonGoldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
In the fundus and body of the gallbladder in the pig folds join and form polygonal structures, called mucosal crypts (Figs. 8a, 9a, and 9b). Secondary folds, i.e., low elevations of the lamina propria of the mucosa, were also observed both within the mucosal crypts and between simple and branched folds in all areas of the gallbladder. In the fundus of the gallbladder, the simple folds, branched folds, and crypts of the mucosa were observed in the lamina propria of the mucosa. The average height of the simple folds in fundus does not exceed 200 mm (Table 1). The branched folds are on average 2–3 times higher than the simple folds, and their height is ca. 542 mm (Tab.1). It was observed that in the fundus the branched folds usually join to form mucosal crypts. Irregular or polygonal crypts of mucosa are 150–1,500 lm in diameter. Single lymphatic nodules were visible in the fundus of gallbladder. In many cases, the subepithelially distributed nodules expand in the direction of gallbladder lumen. The presence of the subepithelial mucosal glands was not found either on histological slides or SEM crosssections of the mucosa of the gallbladder fundus. In the area of the gallbladder body fused with the surface of liver the mucosal folds are predominantly branched type of ca. 495 mm in height and they lie close Microscopy Research and Technique
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TABLE 1. Measurements from pig’s gallbladder wall and mucosa Part of the gallbladder
Thickness of the wall of the gallbladder High of compounded folds of the mucosa High of simple folds of the mucosa Distance between folds of the mucosa High of the epithelium
Fundus of the gallbladder
Body of the gallbladder part fused with liver
X
X
X
X
SD
SD
SD
SD
Min - Max
Min - Max
Min - Max
Min- Max
1697.3 283.1 1036.1–2230.8 542.6 86.4 410.9–707.6 196.5 68.2 104.1–320.1 282.0 77.2 152.9–413.9 26.4 3.1 20.3–32.1
1436.1 259.3 1024.1–1813.1 495.9 46.7 423.8–604.3 253.8 82.0 140.8–356.8 116.7 59.5 63.8–287.2 23.1 2.4 17.0–27.0
1326.9 132.7 1123.2–1590.5 400.4 55.1 297.7–507.7 136.9 25.3 85.6–190.3 103.8 48.8 41.5–211.3 27.6 4.2 20.0–36.0
391.1 69.4 307.1–491.0 138.6 34.7 94.1–191.1 162.9 57.0 96.2–293.0 22.7 4.4 17.8–26.6
Body of the gallbladder - part free Neck of the gallbladder
Results expressed as: X-arithmetical means, SD- standard deviation, Min- minimum value, Max- maximum value.
TABLE 2. Comparison of the thickness of the wall and the height of simple and branched folds of the mucosa in the fundus, the body, and the neck of the gallbladder in pig
to each other in rows (Figs. 7a and 7b). Between the branched folds, there are numerous openings of the subepithelial glands composed of tubulo-alveolar secretory units and short excretory duct (Figs. 7a, 13a, 14a, and 14b). Openings of mucosal glands lie often in small cavities surrounded by mucosal elevations and simple folds. SEM images of the surface of the socalled hepatic part of the gallbladder body often show one common hole, to which the excretory ducts of several secretory tubuli or acini anastomose (Fig. 13a). In the free surface of the gallbladder body, the branched folds with an average height of 400 mm Microscopy Research and Technique
combine at the basal sections and similarly as in the fundus of the gallbladder they form mucosal crypts (Fig. 8a). These structures in the gallbladder body have irregular shapes and their diameter is more than twice less than in the fundus of gallbladder. In the free part of the body of the gallbladder, few glands were observed on the mucosal surface and their diameter was smaller than in the hepatic area of gallbladder body (Figs. 8a and 8b). Similarly, as in the fundus of gallbladder, the lymphatic nodules bulge out into the lumen of this organ (Fig. 3a).
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Fig. 4. Magnification of the surface of the mucosa in the neck of gallbladder in pig; yellow arrows show simple folds of mucosa. NaOH maceration, SEM; Fig. 5. Magnification of the surface of the mucosa in the free part of body of the gallbladder in pig; yellow arrow shows fibrillar frameworks of simple fold of mucosa, black arrow shows fibrillar framework of branched fold of mucosa; (a) Apical part of
branched fold; (b) Basal part of branched fold. NaOH maceration, SEM; Fig. 6. A view of surface of mucosa in the body of the gallbladder in pig. (1) Folds of mucosa; (2) The mucus, SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 7. SEM and LM views of the pig’s gallbladder mucosa in the body of the gallbladder; part fused with liver. (a) A view of the surface of the mucosa. (1) Branched folds arranged in rows, arrows show the openings of subepithelial glands, NaOH maceration, SEM; (b) magni-
fication of the branched folds of mucosa, NaOH maceration, SEM; (c) a cross-section of mucosa with branched fold (1) and fingerlike fold (2), Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 8. SEM and LM views of the pig’s gallbladder mucosa in the body of the gallbladder; part free; (a) A view of the surface of the mucosa; (1) Branched and simple folds that surround irregular mucosal crypts, yellow arrows show the openings of subepithelial glands, NaOH maceration, SEM; (b) Magnification of the surface of mucosa
with opening of gland, NaOH maceration, SEM; (c) A cross-section of the mucosa with conical folds (1), Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Fig. 9. SEM and LM views of the pig’s gallbladder mucosa in the fundus of the gallbladder, (a) a view of the surface of the mucosa; (1) Wide folds forming a polygonal crypts, 2-compound folds (intensely folded area), NaOH maceration, SEM; (b) Magnification of the compound folds of mucosa, the arrow show the crypt of the mucosa,
NaOH maceration, SEM; (c) A cross-section of mucosa with conical fold (1) and digitate fold (2). Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 10. SEM and LM views of the pig’s gallbladder mucosa in the neck of the gallbladder. (a) A view of the surface of the mucosa; (1) Simple folds of the mucosa, (2) openings of the subepithelial glands, NaOH maceration, SEM, (b) magnification of the simple folds of mucosa (1), 2- opening of the subepithelial gland, NaOH maceration,
SEM; (c) A cross-section of the mucosa with simple folds (1) and subepithelial gland (2) Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
In the pig, the neck area of the gallbladder differs in the arrangement and size of the folds from the other parts of the gallbladder. Only low and wide simple and narrow pin-shaped folds may be observed on LM and SEM images of the gallbladder neck (Figs. 10a, 10b, and 10c). These folds lay parallel to each other or join and run circumferentially around the openings of mucosal glands (Figs. 10a and 10b). Numerous compound glands and single lymphatic nodules surrounded by loose connective tissue were observed in the mucosa of the neck of pig gallbladder (Fig. 3b). Significant differences were found in average height of the folds in individual parts of the gallbladder at a significance level P < 0.05. The greatest statistically significant differences were found in height of folds in the gallbladder fundus and neck (Tables 1 and 2). In turn, the least statistically significant differences were recorded between the folds located at the fundus of the gallbladder and the hepatic area of the gallbladder body. The largest branched folds, of up to 700 mm in height, are found in the fundus of the gallbladder, but the largest simple folds of ca. 365 mm are located in the
hepatic part of the body (Tables 1 and 2). The folds in the fundus of gallbladder lie at the greatest distance of about 282 lm, whereas the smallest distance between the folds, averaging 103 mm, was recorded in the mucosa of the free part of the gallbladder body (Table 1). Detailed metric measurements of gallbladder wall thickness, height of simple and branched mucosal folds, the distance between the folds and the thickness of the epithelium in porcine gallbladders are shown in Table 1. The entire surface of the gallbladder mucosa in the pig is covered with a single-layered cylindrical epithelium (Fig. 11). The high epithelial cells adhere tightly to each other, forming a polygonal configuration on the surface observed in SEM (Fig. 12). These cells have a characteristic arrangement for epithelial cell nuclei, because oval cell nuclei lie in the basal part of the cells and occupy one-third of their height (Figs. 11a–11d). Morphometric observations showed that the epithelium in different parts of the gallbladder reaches thickness from 17 to 36 mm (Table 1). SEM and histochemical observations of gallbladder mucosa identified the composition of porcine mucus produced by
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Fig. 11. A view of the cross-sections of monolayer cylindrical epithelium subjected to various staining in pig’s gallbladder; (a) Epithelial cells in free part of the body of gallbladder, Staining: MassonGoldner, LM. (b) Results of staining with HID/Alcian blue in epithelial cells in the fundus of gallbladder, arrow show a unicellular mucous gland, LM. (c) Results of staining with HID/ Alcian blue in epithelial cells in the body of the gallbladder; arrow show epithelial cells secreting the mucus. LM. (d) PAS positive reaction in epithelial cells in the body of the gallbladder; LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the epithelial cells (Fig. 6). Positive results of PAS and HID/AB staining in epithelial cells indicated the presence of mucus glycoproteins (Figs. 11b–11d). The purple color of the apical part of the epithelium as a positive result of the PAS reaction was observed throughout the gallbladder, wherein in different areas of the gallbladder different intensities of color reaction were found. The highest color intensity was recorded for the epithelium covering the gallbladder body and the smallest in gallbladder neck (Fig. 11d). The blue color in the apical parts of the epithelial cells in response to HID/AB staining indicated secretions containing acidic glycoprotein, i.e., sialomucins and sulfomucins. In the gallbladder body, an intense reaction was observed after HID/AB staining (Fig. 11c). In the fundus of the gallbladder, the blue coloration in response to alcian blue was observed only in isolated epithelial cells after HID/AB staining (Fig. 11b). DISCUSSION The present studies of the porcine gallbladder showed regional differences in the microstructure of the gallbladder mucosa. Microscopic and morphometric analyses revealed differences in the arrangement of mucosal folds, distribution and structure of subepithelial glands, and the distribution of lymphoid nodules between the studied regions of porcine gallbladders. The morphometric analysis showed that the thickness of the wall in the pig gallbladder varies in different areas. Significant differences were found between the fundus and the neck, whereas the wall of the neck was three times thinner than the walls of the body, and four times thinner than the fundus. The
Fig. 12. A magnification of the surface of polygonal epithelial cells in the body of the gallbladder in pig. SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
thickness of the wall in the body region of the gallbladder is similar to that in the fundus. The gallbladder mucosa, coating the entire surface of a single-layer cylindrical epithelium, is specialized for compaction and production of bile and the secretion of mucus. Previous studies of Prozorowska and Jackowiak (2014) on the microvascularization of the gallbladder in the pig conducted on vascular corrosion casts revealed that the mucosa is highly vascularized and possesses a dense network of subepithelial capillaries surrounding the secretory units. The morphometric data showed that the epithelial cells of the free part of gallbladder body and of the fundus of the gallbladder were on average 20% higher than the epithelial cells in the neck of the gallbladder and in the body of the gallbladder fused with liver. Histochemical observations of epithelial cells showed differences in the amount and intensity of mucus secretion. Lower epithelial cells in the gallbladder neck produced a weaker color reaction in PAS staining when compared with the higher epithelial cells in the body of the gallbladder. Similar results were obtained for HID/AB staining. The apical part of epithelial cells in the gallbladder body turned brighter alcian blue than the other parts of the epithelial cells of the gallbladder, indicating more intensive production of mucus in the gallbladder epithelium of the body of gallbladder. Microscopic studies performed, e.g., in carnivores and the rabbit showed that gallbladder epithelial cells are polarized and may change in size with synthesis processes (Evett et al., 1964; McPherson Microscopy Research and Technique
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Fig. 13. The views of subepithelial glands in pig gallbladder; (a) A magnification of opening of the subepithelial glands in the body of gallbladder in part fused with liver, 1- the main and common opening of the gland, 2- the openings of the individual secretory units; (b) The cross- section of the mucosa in the neck of gallbladder in pig after removing the epithelium and cells of connective tissue; 1- the secretory units of the subepithelial gland; 2- surface of the mucosa, NaOH maceration, SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
et al., 1983). Apical portion of the cells with microvilli increase the absorptive surface area, thereby contributing to the efficient compaction of bile. Our study show that differences in size of epithelial cells of porcine gallbladder mucosa may arise from the secretory activity of the epithelium. Subepithelial glands are important structures in the lamina propria mucosae. Types of glands, the complexity of their structure and their arrangement in mammalian gallbladders are species specific (Pfuhl, 1932). Mostly single secretory units of glands are straight or coil. In ruminants, there are many glands arranged close together. In bovines, they take the form of bands, whereas in the goat and sheep, they are more dispersed (Jurisch, 1909). In the rabbit, no glands are found on the gallbladder (Jackowiak and Lametschwwandtner, 2005). In humans as well as in the guinea pig, tubulo-vesicular glands are accumulated only in the region of the neck, whereas in carnivores, they are scattered across the mucosa (Bucher and Wartenberg, 1989; Lee, 1980; Laito and Nevalainen, 1975). Microscopy Research and Technique
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Fig. 14. The histological view of the compound subepithelial glands in pig gallbladder; (a) A magnification of the compound subepithelial gland with wide opening of the gland (arrow); asterisk indicate the tubular secretory units, b- alveolar (asterisk) and tubular secretory units in the body of gallbladder, Staining: Masson-Goldner, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
The highest density of glands in the pig gallbladder was found in the neck and in the part of the body fused with liver. In the free part of the gallbladder body, glands are rare, whereas in the fundus, they are absent. Our results also showed differences in types and size of secretory units of mucous glands. In the neck both small, single follicular glands are located, as well as large, complex glands of the coiled and alveolar secretory units, which extended up to the subserosal layer. Glands in the body of the gallbladder have rounded secretory units smaller than in the gallbladder neck and the secretory units take the form of elongated vesiculo-tubular glands. In the gallbladder body fused with liver, the glands are often located in the folds of the mucosa or near lymphatic nodules. SEM images showed the specificity of structure of gland openings on the mucosal surface in the body and in the neck of the gallbladder. Density of gland openings per 1 mm2 of mucosal surface also varies. In the neck, there are 0.6 outlets of glands per 1 mm2 of the mucosa, but in the body fused with liver, this value increases to 0.8 outlets per 1 mm2 of the mucosa. In
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Fig. 15. PAS staining of the mucosa in the neck of the gallbladder; (1) Secretory units of the subepithelial gland, (2) the mucosa layer, (3) lumen of the neck of the gallbladder filled with mucus, LM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the neck, the openings are wide and deep and they are evenly distributed over the entire surface, whereas in the gallbladder body the openings of several secretory units are compacted in a common, wide hole surrounded by an elevation of mucosa, which influenced the above-mentioned results concerning the density of openings. The single-layered epithelium lining the secretory units of glands is lower than the epithelium on the surface of mucosa. As it was shown by previous studies, in mammals the glandular cells, as well as surface epithelial cells of the gallbladder, produce and secrete sticky mucus with high contents of glycoproteins. Its function is to protect the surface against bile and microorganisms. Mucous also enriches the bile composition, so that bile flowing toward the duodenum has a thicker consistency (Madrid et al., 1997). Lymph nodules are the integral structural elements of the mucous membrane in the pig gallbladder. They are found in the gallbladder mucosa with the greatest density in the gallbladder body fused with liver. In this area, the subepithelially situated, oval-shaped lymph nodules accentuate over the mucosa, which makes these structures macroscopically visible. Single lym-
phatic nodules as structural components of the immune system are commonly found in the mucosa of the digestive, respiratory and urogenital tracts, where they serve defense functions. As it is presented in literature concerning microstructures of the gallbladder wall, they are not permanent structural elements of the mucosa. They are not observed in the gallbladder in humans and carnivores (Evett et al., 1964; Jackowiak, 2006). Their presence in the porcine gallbladder should be considered as a species-specific feature. This data could be important and useful as remark for pathologist. Folds of the mucosa are important features of gallbladder mucosa associated with the process of absorption. Many animal species possess regular folds, which join to form polygonal structures called mucosal crypts. Such a configuration is described in some carnivores, e.g. the dog, raccoon dog, foxes, American mink, primates, the rabbit and in the human (Castellucci and Caggiati, 1980; Caggiati et al., 1992; Jackowiak and Lametschwwandtner, 2005; Jackowiak 2006; Lang, 1970; Mueller et al., 1972; Pfuhl, 1932). Our observations in the pig allowed us to distinguish simple and branched folds, which are usually connected together by wide basal portions. Mucosal folds are connective tissue elevations of the lamina propria, composed primarily of the collagen fiber framework. The observations showed that each of the gallbladder areas has a unique arrangement of folds, which differ in shape and size. The wall of the fundus of gallbladder has the most corrugated surface and the folds of the mucosa are the highest, which was found in areas with greater amounts of connective tissue and indicates a greater potential for expansion of this ventral area of the gallbladder. It is important to remember that the folds in the fundus of pig gallbladder form polygonal crypts, indicating that during filling the gallbladder with bile they can distend, but they do not disappear. Such an observation was made previously on mucosal crypts in some carnivores (Jackowiak, 2006). The type, density and shape of the mucosal folds in the gallbladder change depending on the degree of filling of the organ with bile. Our studies in the contracted pig gallbladder showed structural differences related to particular areas of the gallbladder. It was concluded that wall thickness increases from the neck to the fundus of the gallbladder, similarly as it is with the height of mucosal folds. An important structural feature is connected with the structural variety of types of mucosal folds, which change from simple located in the neck, to most composed, i.e., branched or joined, in polygonal crypts toward the fundus of the gallbladder. Our results on the pig gallbladder show adaptation and/or specialization in particular areas of the gallbladder for (1) secretion of mucus in the neck or fundus, and (2) for cyclic volume changes especially in the gallbladder fundus. A description of the microstructures in the porcine gallbladder could be useful as reference data for numerous experiments on the bile tract in the pig. REFERENCES Aharinejad S, Lametschwwandtner A. 1992. Microangioarchitecture of the guinea pig and bile duct as studied by scanning electron microscopy of vascular corrosion casts. J Anat 181:89–100.
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