Light microscopy and ultrastructure of the liver of Astyanax altiparanae Garutti and Britski, 2000 (Teleostei, Characidae) - DOI: 10.4025/actascibiolsci.v30i1.1456

Livers of thirty specimens of Astyanax altiparanae obtained from a commercial fish farm were subjected to light and transmission electron microscopy, in order to describe the hepatic parenchyma and the intrahepatic exocrine pancreatic tissue. Anatomically, the liver showed only three hepatic lobes. Histological analysis demonstrated that the hepatocytes were spread out as anastomotic cords, arranged in two cellular layers and surrounded by sinusoids. The intrahepatic exocrine pancreatic tissue exhibited an acinar arrangement and was diffused in the hepatic parenchyma. Ultrastructural analysis showed that the hepatocytes had a rounded nucleus and a rough endoplasmatic reticulum, with a parallel disposition to the nuclear membrane. The exocrine pancreatic cells showed secretion granules at the apical portion, and the rough endosplasmatic reticulum was concentrically distributed

L L L Light microscopy and ultrastructure of the liver of ight microscopy and ultrastructure of the liver of ight microscopy and ultrastructure of the liver of ight microscopy and ultrastructure of the liver of A A A Astyanax styanax styanax styanax altiparanae altiparanae altiparanae altiparanae Garutti and Britski, 2000 (Teleostei, Characidae) Garutti ABSTRACT. Livers of thirty specimens of Astyanax altiparanae obtained from a commercial fish farm were subjected to light and transmission electron microscopy, in order to describe the hepatic parenchyma and the intrahepatic exocrine pancreatic tissue. Anatomically, the liver showed only three hepatic lobes. Histological analysis demonstrated that the hepatocytes were spread out as anastomotic cords, arranged in two cellular layers and surrounded by sinusoids. The intrahepatic exocrine pancreatic tissue exhibited an acinar arrangement and was diffused in the hepatic parenchyma. Ultrastructural analysis showed that the hepatocytes had a rounded nucleus and a rough endoplasmatic reticulum, with a parallel disposition to the nuclear membrane. The exocrine pancreatic cells showed secretion granules at the apical portion, and the rough endosplasmatic reticulum was concentrically distributed.

Introduction Introduction Introduction Introduction
Fish are especially susceptible to environmental variations and respond more sensitively to pollutants than numerous mammals. Their liver is then a very interesting model for the study of interactions between environmental factors and hepatic structures and functions (Bruslé and Anadon, 1996). Thus research on fish liver is important especially in the field of problems induced by aquaculture conditions and aquatic pollution (Gochfeld, 2003;Mela et al., 2007).
The liver in fish is a dense organ ventrally located in the cranial region of the general cavity. Its size, shape, and volume are adapted to the available space between other visceral organs. In many Teleostei species, the liver is divided into three lobes. However, lobulation was not found in some Teleostei (Bruslé and Anadon, 1996). The hepatic parenchyma in fish is made up of two cellular plates surrounded by sinusoids. Between two neighboring sinusoids, the hepatocytes are arranged as cords, generally two cells thick. The cords extend between central and portal zones (Hinton et al., 1972;Kendall and Hawkins, 1975;Hinton and Pool, 1976;Robertson and Bradley, 1992;Bruslé and Anadon, 1996).
Previous studies have indicated that in teleost fish, the pancreatic exocrine tissue develops around the portal vein during ontogenesis. It remains extrahepatic or penetrates somewhat deeply into the liver parenchyma depending on the species, such as Ictalurus punctatus (Kendall and Hawkins, 1975;Hinton and Pool, 1976), Pimelodus maculatus (Marconi Stipp et al., 1980), Micropogon undulatus (Eurell and Haensly, 1982), Serranus cabrilla (Gonzalez et al., 1993) and Oreochromis niloticus (Vicentini et al., 2005). Pancreatic tissue can be differentiated from hepatic tissue by its acinar arrangement. In addition, a thin septum of connective tissue separates the hepatocytes from the exocrine pancreatic cells (Bruslé and Anadon, 1996). The species Astyanax altiparanae is found in abundance in the Paraná river system, and it is appreciated in sport fishing for human consumption. It also plays an important role in the local food chain, especially for carnivorous fish (Smith, 2003), and presents potential to aquaculture (Godinho, 2007). Then, the present study was to describe the morphological characteristics of the liver and the intrahepatic exocrine pancreas in Astyanax altiparanae.

Material and methods Material and methods Material and methods Material and methods
Thirty specimens of Astyanax altiparanae -15 males (7.24 ± 0.7 cm) and 15 females (7.51 ± 0.6 cm), were obtained from a commercial fish farm in Bauru, São Paulo State, and transported to the Department of Biological Sciences, Faculty of Sciences, Unesp. The fishes were anaesthetized with 0.02% methanesulfonate and the peritoneal cavity was opened for liver exposure, which was then removed for light microscopy and transmission electron microscopy studies. For light microscopy, the liver samples were fixed in Karnovsky solution and embedded in historesin (Leica, Germany). The histological sections were stained with hematoxylin-eosin, toluidine blue and analyzed and documented photographically using an Olympus BX50 microscope (Japan).
For transmission electron microscopy, liver fragments were fixed in glutaraldehyde 2.5% in 0.1 M phosphate buffer, pH 7.2, for 3h, postfixed in 1% osmium tetroxide in phosphate buffer, washed in the same buffer, dehydrated in a growing acetone series and embedded in Araldite resin (Durcupan ACM, Fluka, Sigma-Aldrich, St. Louis, MO, USA). Resin polymerization was then completed in an oven at 60°C for 48 h. Ultrathin sections (60 and 80 nm) were cut and transported to copper grids, contrasted with uranyl acetate and lead citrate, analyzed and documented photographically using a Philips CEM 100 transmission electron microscope (TEM), at the Electron Microscopy Center of the Biosciences Institute of Botucatu, Unesp.

Results Results Results
The liver of A. altiparanae is a dense organ located in the cranial region of the general cavity. It is divided into three lobes: the right lobe, the left dorsal lobe and the left ventral lobe ( Figure  1A). The gallbladder is well developed and has an elongated shape.
The hepatic parenchyma is made of hepatocytes spread out as anastomotic cords arranged in two cellular layers surrounding the sinusoids ( Figure 1B). Light microscope observations showed that it is not possible to find hexagonal subdivisions of hepatic parenchyma or hepatic lobules. The bile ducts are usually found near the portal vein, and are lined by a simple cuboidal epithelium ( Figure 1C). A concentric layer of collagen and muscular fibers were observed under the epithelium.
Ultrastructurally, the hepatocytes show a single rounded nucleus, usually centrally located. The chromatin is granular, with more condensed heterochromatin located at the periphery of the nucleus. The nucleolus is more homogenous and presents high electronic density. The rough endoplasmic reticulum is often arranged in an array parallel to the nuclear membrane. The shape of the mitochondria varies from round to elongated, and are associated to the rough endoplasmic reticulum (Figures 1D and E).
The bile canaliculi between hepatocytes typically lie in apical cell regions, toward the center of tubules or cords, although a few perisinusoidal canaliculi do occur. Microvilli from parenchymal cells often completely occlude the canalicular lumen. Desmosomal junctions commonly occur between hepatocytes near bile canaliculi, contributing to complete pericanalicular junctional complexes ( Figure 1F).
Microscopic observations allowed the identification of the intrahepatic exocrine pancreatic tissue as a result of its acinar arrangement and its diffused distribution in the hepatic parenchyma ( Figure 1G). The exocrine pancreatic cells were differentiated ultrastructurally from the hepatocytes by the presence of secretion granules, usually located at the apical portion of the cell. The nucleus of the pancreatic cells is generally round with a central nucleolus and it is located at the basal portion of Acta Sci. Biol. Sci.

Discussion Discussion Discussion
The study of the light microscopy and ultrastructure of the liver of A. altiparanae showed a typical structural organization, which is similar in many teleosts. The hepatocytes are arranged in cords, usually two cellular layers, and surrounding the sinusoids (Kendall and Hawkins, 1975;Hinton and Pool, 1976;Gonzalez et al., 1993;Bruslé and Anadon, 1996;Vicentini et al., 2005). The absence of division into hepatic lobules and the lack of portal triads are features of A. altiparanae, as evidenced in many teleosts (Hampton et al., 1985;Gonzalez et al., 1993;Vicentini et al., 2005). However, some triads are found in Caranx spp. and Lutjanus bohar (Gonzalez, 1992).
The organization of the biliary tree in fish is similar to that observed in vertebrates. It originates in many Teleostei as an intercellular canaliculus, formed by the close apposition of two hepatocytes. The cell membrane projects numerous microvilli into the canalicular lumen and desmosomes, and tight junctions assure the cohesion (Bruslé and Anadon, 1996). According to Robertson and Bradley (1992), the canaliculi of Salmo salar are formed by 3 to 4 hepatocytes, and these cells do not feature filaments in the pericanalicular cytoplasm, as noted in some other teleosts (Weis, 1972;Hampton et al., 1985). The structural organization of the intercellular canaliculus in A. altiparanae is in accordance with that reviewed in the literature (Weis, 1972;Hampton et al., 1985;Robertson and Bradley, 1992;Bruslé and Anadon, 1996).
Observations by light microscopy also evidenced intrahepatic exocrine pancreatic tissue in A. altiparanae, associated to afferent vessels. In some species, the pancreatic tissue was identified as diffused, surrounding the digestive tract (Beccaria et al., 1992;Marconi Stipp et al., 1980). The pancreas in Pimelodus maculatus is compact, enclosed by a thin layer of conjunctive tissue and is attached to the stomach and intestine wall as small masses of glandular tissue (Marconi Stipp et al., 1980). The ultrastructure of exocrine pancreatic cells of A. altiparanae exhibits similar characteristics to that of other teleosts (Kendall and Hawkins, 1975;Hinton and Pool, 1976;Marconi Stipp et al., 1980;Beccaria et al., 1992;Vicentini et al., 2005).
Sea bass (Beccaria et al., 1992) subjected to long fasting showed narrower lumen of excretory ducts and reduced cellular activity, demonstrated by the scarcity of zymogen granules. On the other hand, intensively fed fish were seen to have increased cellular activity and greater quantity of zymogen granules. Electron-dense zymogen granules were abundant in A. altiparanae. They were generally located in the apical portion of the cell. The rough endoplasmic reticulum was well developed and showed an organized pattern.

Conclusion Conclusion Conclusion Conclusion
The liver of A. altiparanae showed three hepatic lobes. Histological analysis demonstrated that the hepatocytes were spread out as anastomotic cords, arranged in two cellular layers and surrounded by sinusoids, similar to those found in other Teleostei. However, no hepatic lobules or portal triads were observed. Another characteristic observed in A. altiparanae was the intrahepatic exocrine pancreatic tissue associated to afferent vessels. Previous studies in teleost fish have indicated that the pancreatic tissue can remain extrahepatic or penetrate somewhat deeply into the liver parenchyma, depending on the species.