BIHAREAN BIOLOGIST 12 (2): 102-105 ©Biharean Biologist, Oradea, Romania, 2018 Article No.: e181303 http://biozoojournals.ro/bihbiol/index.html

Histological characteristics of the pancreas and spleen of Pelophylax bedriagae (Anura: Ranidae)

Esra AKAT

Ege University, Science Faculty, Biology Department, Zoology Section 35100 Bornova-Izmir/Turkey,

Tel: +902323112404; Fax: +902323881036, E-mail: esra.akat@ege.edu.tr

Received: 02. December 2017 / Accepted: 28. February 2018 / Available online: 28. February 2018 / Printed: December 2018

Abstract. The histological characteristics of the spleen and pancreas of Pelophylax bedriagae were examined using light microscopy. The spleen was a highly vascular organ and composed of two functional compartments, the red pulp and the white pulp. The spleen was surrounded by connective tissue and trabeculae were not observed in the parenchyma of the spleen. The pancreas was composed of small clusters of glandular epithelial cells. Some of the cell clusters were arranged into pancreatic islets of Langerhans and corresponded to the endocrine portion. The remaining pancreatic epithelial cells were organized into clusters called acini and constituted the exocrine portion. Additionally, melanomacrophages were observed in both spleen parenchyma and pancreas parenchyma.

Key words: Histology, spleen, pancreas, amphibian, Pelophylax bedriagae.

Introduction The spleen is the biggest and the most important secondary lymphoid organ and is closely associated with the circula-tory system. It is responsible for initiating immune reactions, filtering the blood of foreign material, removing old or dam-aged red blood cells and reutilization of hemoglobin iron (Mebius & Kraal 2005, Junqueira & Carneiro 2006).

The pancreas is a mixed gland which has exocrine and endocrine functions. The pancreas of vertebrates is divided into two functional parts, the exocrine portion and the endo-crine portion. The exocrine cells secrete enzymes into the alimentary canal, and the endocrine cells secrete hormones into the circulation (Slack 1995, Junqueira & Carneiro 2006).

Pelophylax bedriagae has a widespread distribution and tolerates a broad range of conditions. P. bedriagae is widely distributed in the eastern Mediterranean and in Turkey this species is found along the Aegean coast and in the southern part of the Anatolian highlands. It is also present on the Is-land of Rhodes in Greece and is in much of Cyprus. P. bedriagae is an aquatic frog and the dorsum is typically greenish or brownish with large dark spots over the body. The females lay their eggs in water and the eggs hatch into tadpoles. Although the tadpoles are herbivores, the adults feed on terrestrial arthropods, molluscs and small verte-brates such as fish, amphibians (Çiçek & Mermer 2007, Bu-dak & Göçmen 2008, AmphibiaWeb 2017).

There are no available reports on the anatomical organi-sation, or histochemistry of the pancreas/spleen in P. bedriagae. Therefore, the aim of the present study was to re-veal the histological characteristics of the spleen and pan-creas of P. bedriagae, and to compare it with the histological features of the spleen and pancreas of other vertebrates. Materials and Methods Four adult specimens (two males and two females) of P. bedriagae were obtained from Bornova-İzmir/Turkey (N 38°50 and E 23°29). They were anaesthetized with ether, and euthanized by decapitation. Immediately afterwards the spleen and pancreas were removed. Tis-sue samples were fixed in Bouin’s fluid for 48 h. Thereafter, the sam-ples were processed using standard histological procedures for par-

affin embedding. Five micrometer thick sections of paraffin-embedded tissues were stained with Gill’s hematoxylin-eosin and Mallory’s trichrome to demonstrate the general morphology of the tissue. Additionally, Herlant’s tetrachrome and Heidenhain’s azan trichrome were carried out to distinguish endocrine cells from pan-creatic exocrine cells. The sections were photographed using a Zeiss Axioscope light microscope that was equipped with an AxioCam Erc5S digital camera. Results The spleen of P. bedriagae was a soft, dark red and globular shaped organ which was connected to the mesentery of the small intestine (Fig. 1). According to light microscopic ob-servations, it was encapsulated by connective tissue. Trabe-cular connective tissue of the capsule entering into the organ was not observed in P. bedriagae.

The spleen was a highly vascular organ and composed of two functional compartments, the red pulp and the white

Figure 1. Anatomy diagram of Anura, ventral view. Modified from “Free and Printable Frog Diagram (2018)”.

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Figure 2. Light microscopic view of spleen in P. bedriagae. A: The splenic artery divided into small arterioles which constituted cen-tral arterioles. The spleen was composed of two functionally compartments, the red pulp (RP) and the white pulp (WP), hema-toxylin-eosin staining. B: The spleen was encapsulated by a connective tissue capsule (black arrow). Melanin-containing cells (white arrow) were localised in the spleen parenchyma, Mallory’s trichrome staining.

Figure 3. Light microscopic view of pancreas in P. bedriagae. A: The pancreas was composed of small cluster of glandular epithelial cells. Some of the cells were organized into clusters called pancreatic islets (islets of Langerhans) which were the endocrine portion (*), hematoxylin-eosin staining. B: Easily distinguished endocrine cells from pancreatic exocrine cells (*) by Herlant’s tetrachrome staining, melanin-containing cells (white arrow). C: Pancreatic endocrine cells (*) were stained a red-orange colour by Heiden-hain’s azan trichrome. D: The exocrine acinar cells had apical eosinophilic zymogen granules, hematoxylin-eosin staining.

pulp. The majority of the organ was composed of the red pulp, which was not clearly distinguishable from the white pulp in P. bedriagae. In the splenic parenchyma, the splenic artery divided into small arterioles which constituted the central arterioles. The arterioles were surrounded by lym-phoid tissue which formed white pulp, which was sur-rounded by the red pulp (Fig. 2A). Melanomacrophages, melanin containing cells, were observed in the spleen paren-

chyma (Fig. 2B). The pancreas of P. bedriagae was an elongated gland that

was located posterior to the greater curvature of stomach and was connected to the small intestine (Fig. 1). In P. bedriagae the pancreas was composed of small clusters of glandular epithelial cells. Some of the cells were arranged into clusters called pancreatic islets of Langerhans and corre-sponded to the endocrine portion of the pancreas (Fig. 3A).

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After Herlant tetrachrome staining endocrine cells were readily distinguished from pancreatic exocrine cells (Fig. 3B). The islet cells of langerhans were stained a red-orange col-our by Heidenhain’s azan trichrome (Fig. 3C). The remain-ing epithelial cells were organized into clusters called acini and constituted the exocrine portion of the pancreas. The exocrine cells had a dark basophilic cytoplasm, and apical eosinophilic zymogen granules (Fig. 3D). Additionally, melanin containing cells were present in the pancreatic pa-renchyma (Fig. 3B). Discussion The present study was carried out to describe the histologi-cal characteristics of the spleen and pancreas of P. bedriagae. The spleen was surrounded by connective tissue. Trabecular connective tissue of the capsule entering into the organ was not examined in P. bedriagae in common with reports in the amphibia, Physalaemus nattereri, and in the turtle, Testudo graeca (Kassab et al. 2009, Franco-Belussi & de Oliveira 2016). In mammalian species, the spleen is encapsulated by a cap-sule of dense connective tissue from which emerge trabecu-lae, which pass inwards to the splenic tissue (Connolly et al. 1999, Mebius & Kraal 2005). Cesta (2006) reported that the spleen of rat was surrounded by a capsule composed of dense fibrous tissue and smooth muscle. The trabeculae in mammals were composed of smooth muscle and bundles of connective tissue emanated from the capsule into the splenic parenchyma. Valli et al. (2002) reported that the capsule and trabeculae of dogs contained more smooth muscle than that of mice and rats, and suggested that for this reason the spleens of rodents does not contract as rapidly as dog. It was reported that trabeculae gave rise to possibility for contrac-tion of the spleen and therefore the organ has a role as a blood volume regulator (Barcroft 1925). However, the spleen of P. bedriagae was covered by a thin fibrous capsule with lit-tle evidence of contractile ability.

The spleen was composed of the red and white pulp. The majority of the organ consisted of the red pulp, which was not clearly distinguishable from the white pulp in P. bedriagae. The splenic artery divided into small arterioles which constituted the central arterioles in the splenic paren-chyma. The arterioles were surrounded by lymphoid tissue which formed white pulp, which was surrounded by the red pulp. The same basic histological structures as in higher ver-tebrates were typically present in the spleen; blood vessels, and the red and white pulp. However there is no white pulp in caecilians (e.g. Ichthyophis kohtaoensis), and Rana perezi, which have scattered lymphoid cells spread within the red pulp (Cooper & Wright 1976, Zapata et al. 1982, Alvarez 1990). Additionally, in some fish species, an ellipsoid capil-lary branch was also reported as well as white pulp and red pulp. The splenic ellipsoids are special vascular terminations which have a specialized function in plasma filtration and trapping of antigens or antigen-antibody complexes an im-portant step for the immune response (Fänge & Nilsson 1985, Espenes et al. 1995). Ellipsoid capillary branches were not observed in P. bedriagae spleen.

Melanomacrophages were examined in the spleen pa-renchyma. These melanin containing cells originate from

hematopoietic stem cells and the melanin has bactericidal and cytoprotective activity against free radicals (Franco-Belussi & de Oliveira 2016). The splenic macrophages of the red pulp are important for iron recycling, and they are also involved in the removal of bacteria from blood (Mebius & Kraal 2005).

The pancreas of P. bedriagae was an elongated gland that was located posterior to the greater curvature of stomach and was connected to the small intestine. In many fish, some amphibians and some reptiles, the pancreas adheres to the spleen or liver. This conjoined organ is termed the splenopancreas or hepatopancreas, respectively (Aughey & Frye 2001). In P. bedriagae the pancreas was composed of small clusters of glandular epithelial cells. Some of the cells were organized into clusters called pancreatic islets (islets of Langerhans). The exocrine pancreas is a diffuse organ in teleosts and in some species, the exocrine pancreas sur-rounding the portal vessels extends into the liver and occa-sionally the spleen. The islets of Langerhans are usually situ-ated within the bounds of the exocrine pancreas, but not within the hepatopancreas (Mahabady et al. 2012). The his-tology of the pancreas in P. bedriagae was similar to that of other tetrapod species.

In conclusion, since amphibians have a prominent role as a standard model for the research into many biological proc-esses (Feder 1992), studies related to the biology of amphibi-ans makes relevant contribution to the areas; morphology, histology, embryology, ecology, endocrinology, genetics and public health. Compared with the studies published to date, our results add information in relation to the histological characterization of the pancreas and spleen in amphibians. References Alvarez, R. (1990): An ultrastructural study of the spleen of the ranid frog Rana

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