To demonstrate the correlation between liver structures and phylogenic status, we observed 46 amphibian livers by light microscope, and subjected the data to phylogenic analyses. We focused on the architecture of hepatocyte-sinusoidal structures and hematopoietic tissue structures. Methods The present study was approved by the animal ethics committee of Shimane University, and carried out in strict accordance with the guidelines for the care and use of research animals

set by the committee. Sample collection PI3K Inhibitor Library 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 4EGI-1 2005 to 2010. Three to five specimens were sampled, respectively, except for Japanese giant salamander Gemcitabine cell line (Andrias japonicus) of which one sample was transported to our selleck chemical 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. Table 1 Summary of the phylogenetic

relationships in Amphibian Class Order Suborder Family Species number Gymnophiona   Typhlonectidae 1 Caudata Cryptobranchoidea Hynobiidae 10   Salamandroidea Cryptobranchidae 1     Salamandriae 2 Anura Archaeobatrachia Discoglossidae 1   Aglossa Pipidae 2   Neobatrachia Bufonidae 4     Hylidae 1     Ranidae 17     Rhacophoridae 6     Microhylidae 1 Table includes 13 urodeles, 1 caecilian, and 32 anuran species (Class: Amphibia; Subclass: Lissamphibia). Histology 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.