Comparative histological study of hepatic architecture in the three orders amphibian livers

For this comparative morphological study, the livers of 46 different amphibian species were used. Using hand nets, we collected 21 species from ponds and streams in Shimane Prefecture, 8 species in Iriomote Ishigaki and Miyako Islands in Okinawa Prefecture, 4 species in Amami-oosihma Islands in Kagoshima Prefecture, 2 species in Hokkaidou, 2 species in Aomori Prefecture, 1 species in Oita Prefecture, 1 species in Miyazaki Prefecture, 1 species in Nagasaki Prefecture, 1 species in Gifu Prefecture, and 1 species in Hyogo Prefecture Cayenne caecilians (Typhlonectes sp), Oriental fire-bellied toads (Bombina orientalis) and African clawed frogs (Xenopus laevis and Xenopus tropicalis) were reared in the Biological Fresh Water Laboratory, Shimane University. In order to eliminate the influence of seasonal changes or growth, all specimens were both male and female in the adult stage, anurans were caught from April to October, and urodeles were caught from December to March in each locality from 2005 to 2010. Three to five specimens were sampled, respectively, except for Japanese giant salamander (Andrias japonicus) of which one sample was transported to our laboratory by accident. Animals were anesthetized by immersion in an ice water bath in 2 ml/L aqueous ethylene glycol monophenyl ether (Merck). After deep anesthetization, liver was taken from the animal. The phylogenetic relationships of Amphibian Class, comprising three orders of amphibian: 13 urodeles, 1 caecilian, and 32 anurans species, is shown in Table 1.

The livers were perfusion-fixed via the heart with 4% paraformaldehyde buffered at pH 7.4 with 0.1 M phosphate for 15 min, cut into small pieces, and immersed in the same solution for 3 days at 4°C. The specimens were rinsed, dehydrated and embedded in paraffin. Serial 4 μm sections were obtained, and some were stained with both hematoxylin and eosin, and others were stained with Azan for morphometric analysis.

Morphometry is the evaluation of forms and in histology describes measurements made from two-dimensional sections. Liver sections stained with Azan for hepatic cells were subjected to morphometric analysis. We used a computerized image analysis system comprised of a photomicroscope (Olympus BX51) and digital camera (Olympus DP70) and the Lumina Vision software (Mitani Corporation, Tokyo, Japan). Lumina Vision is a Microsoft Windows XP application of semi-automated quantitative analysis of fixed histological sections. The software performs automatic measurement of areas defined using an interactive threshold editing functions. The latter results in colored overlay that marks which pixels in the image are to be measured. In the current study, the percentage extension of the sinusoidal areas was quantified in two different zones: periportal zone (PZ) in around the portal triad and pericentral zone (CZ) in around the central vein in hepatic lobules. At least three areas, 6 random fields in each section were captured on digitalized images at a final magnification of 200X. The analysis in each species was made from 18 randomly chosen zones in three specimens.

Following cardiac perfusion fixation, hepatic sinusoids were cleared of blood cells and the definition of hepatocyte-sinusoidal structures was enhanced. Depending on the percentage of hepatic sinusoids per unit area, measured by morphometry, hepatocyte-sinusoidal structures of amphibian livers were divided into three classes as follows: class I (percentage 5 to < 15), class II (percentage 15 to < 25) and class III (percentage ≥ 25).

Histologically, in hepatocyte-sinusoidal structures, class I showed the several-cell-thick plate type, the major part of the hepatocyte lining was multi-layered. The hepatic sinusoids were narrow and short tortuous capillaries. The hepatocytes were rounded and had a rounded large nucleus (Figure 1c). In class II, hepatocyte-sinusoidal structures were observed in the two-cell-thick plate type, the majority of the hepatocyte lining was double-layered. The sinusoidal capillaries were narrow with irregularly shaped sinusoids appearing throughout the interstice between the hepatic plates. Three to four hepatocytes surrounded a sinusoidal capillary. The hepatocytes were polyhedral or rounded, and had a large rounded nucleus (Figure 1d). Class III showed the one-cell-thick plate type, the majority of the hepatocyte lining was simple-layered. The hepatic sinusoids were enlarged with straight capillaries connecting through the perilobular to the centrolobular vessels. The hepatocytes were polyhedral, with a small rounded nucleus (Figure 1e).

In the order Caudata, the hepatocytes were rounded, and had a large rounded nucleus. The sinusoidal capillaries were narrow with short tortuous capillaries. The parenchyma arrangements of some genus Hynobius (nebulosus, dunni, and naevius) were of the combined several- and two-cell-thick plate types (Figure 1f), but other genus Hynobius groups, genus Andrias and the Salamandridae family were of the combined one- and two-cell-thick plate type (Figure 1g). A few urodeles, (Hynobius retardatus, Onnychodactylus japonicus, and Cynops pyrrhogaster), are shown as the one-cell-thick plate type. In the order Gymnophiona, the hepatocytes were square, and had a large rounded nucleus. The sinusoidal capillaries were enlarged. The parenchyma arrangement was the one-cell-thick plate type (Figure 1h). In the order Anura, the hepatocytes were square and polyhedral, and had a small rounded nucleus. The sinusoidal capillaries were enlarged, and the parenchyma arrangement was the one-cell-thick plate type (Figure 1i).

Hematopoietic tissue structures were observed in the three regions: (a) portal triad region (PTR), (b) perihepatic subcapsular region (PSR), and (c) inter-hepatic lobular nodule (Figures 2a-c). In PTR, numerous hematopoietic cells were observed in the connective tissue (Figure 2a). The PSR, usually two to six cell layers thick, almost completely enveloped the hepatic parenchyma, with occasional sites where hepatic parenchymal cells and visceral peritoneum adjoined. This tissue contained neutrophils and eoshinophils (Figure 2b). In the hepatic lobule, hematopoietic nodules were observed in the sinusoidal capillaries with involvement in the Kupffer cells (Figure 2c).

In the order Caudata, the liver consisted of several incompletely separated lobes of parenchymal tissue, each of which was covered by a PSR of hematopoietic tissue. Hematopoietic tissue was also shown in both the portal triads, and was also observed in the inter-hepatic nodule. In the order Gymnophiona, the liver also consisted of several incompletely separated lobes of parenchymal tissue, each of which was covered by a PSR of hematopoietic tissue. Hematopoietic tissue was also shown in both the portal triads and inter-hepatic nodules. In the order Anura, the liver of most anurans was not observed in the hematopoietic tissue structure, but the liver of the genus Bombina and Xenopus was observed in the PSR. Hematopoietic nodules were observed in the hepatic lobule in most anuran amphibians.

All ingested materials are absorbed via the intestines, and reach the liver through the portal vein. Blood flows from the portal veins at the portal triads through the sinusoid and between the hepatic plates to the central vein. The hepatocyte-sinusoidal structure is physiologically important, not only because hepatocytes take up large molecules (e.g., amino acids, glucose, and vitamins) from the sinusoid, but also because a large number of macromolecules (e.g., lipoproteins and albumin) are secreted into the sinusoid [1]. In mammalian livers, hepatocytes are closely contacted with sinusoidal capillaries that form a dense network [2]. In teleosts, hepatocyte-sinusoidal structures are shown as a rough network [1‐3, 13].

This study has shown that the hepatocyte-sinusoidal structures of amphibian livers can be classified into three different types: (I) several-cell-thick plate type, (II) two-cell-thick plate type, and (III) one-cell-thick plate type. This classification is based on the investigation of Elias and Bengelsdorf in several vertebrate animals [2]. Previous studies described that some fish had a similar structure to normal humans, while others were modified in a more primitive form [3, 21]. Our study of 46 species showed that the primitive form was a combination of several-cell-thick plate and two-cell-thick plate types in the genus Hynobius. The traditional form was the combined two- and one-cell-thick plate type, and was observed in another genus, the Hynobius group, genus Andrias and the Salamandridae family. The mammalian form was the one-cell-thick plate type, and was observed in the order Anura, order Gymnophiona and part of the order Caudata. It is well known that the phylogenetic relationships in amphibians is clearly categorized (Table 1). Anura is the sister group of Caudata to the exclusion of Gymnophiona [18].

In this study, we revealed that anuran livers had structures identical to the mammalian arrangement, which possess higher metabolic functions. In contrast, urodeles livers had sinusoids of a primitive form, which were narrow with an undeveloped network, identical to teleosts. As phylogenetic relationships are branched from urodeles to anurans, the parenchyma arrangement progressed from the combined several- or two-cell-thick plate to the one-cell-thick plate type, and the hepatocytes changed from round to square and polyhedral cells. We speculated that these structural changes reflect the route of hepatic ontogenesis, and are essential for the acquisition of higher hepatic function.

The liver develops as a hematopoietic organ at the fetal stage in the mammalian liver, prior to bone marrow development [8]. In amphibians, the liver is an immunocompetent organ, and hepatic hematopoiesis is initiated in urodele sites. It is well known that the thymus, spleen and liver are the three primary sites of hematopoiesis in the adult newt [5, 7, 22]. Previous investigations indicate that the thymus is lymphopoietic, the spleen is lymphopoietic thrombopoietic and erythropoietic [23, 24], and the liver is granulopoietic with small lymphocyte-like cells in the perihepatic subcapsular region (PSR) which might be granulocyte precursors [7, 23]. The newt liver possesses immunologic capabilities due to the presence of lymphocytes in the PSR of the liver [4].

This study has shown that the hematopoietic tissue structures of amphibian livers were observed in three regions: (a) the perihepatic subcapsular region (PSR), (b) portal triads region (PTR), and (c) inter-hepatic lobular nodule. Our study of 46 species showed that hematopoietic tissue structures were observed in both PSR and PTR in both Caudata and Gymnophiona orders, but in the order Anura, hematopoietic tissue was not observed in either PSR or PTR. Inter-hepatic lobular nodules were observed in all amphibian livers. In this study, we revealed that anuran livers did not have hematopoietic tissue structures, as did mammal liver. In contrast, urodele and caecilian livers had hematopoietic tissue structures with hepatic initial sites of hematopoiesis.