Vetflurano

Two or 6 weeks after BDL, animals from each group were euthanized with an excess of isoflurane (Vetflurano ® ; Virbac, São Paulo, Brazil). Liver samples were taken from the major left and median lobes (that comprise approximately 70% of the liver), fixed in 10% neutralbuffered formalin (Synth, São Paulo, Brazil) for 24 h, and embedded in paraffin (Paraplast; Oxford, St. Louis, MO, USA). At least four 4-µm-thick sections were used from each rat and stained with Sirius Red (EasyPath, São Paulo, Brazil) for fibrosis and collagen analysis, according to the manufacturer's instructions or placed on polysine-Lcoated slides (Menzel-Glaser, Braunsch, Germany) for immunohistochemical stainings.

Six weeks after Bdl, the rats from each experimental group were euthanized with an excess of isoflurane (Vetflurano®; Virbac, São Paulo, Brazil), and liver samples were collected from the medial lobe and right lobe. The specimens were fixed in 10% buffered formalin for 48 h and embedded in Paraplast (SigmaAldrich, Germany). Four-µm-thick sections were obtained with a semiautomatic microtome, and stained with masson's trichrome for fibrosis and collagen analysis, Bras Cienc (2019) 91(3) e20190220 4 | 14 according to the manufacturer's instructions or placed on polysinine-l coated slides for immunohistochemical assessment.
QUAlITATIVe ANAlYSIS OF THe FIBROSIS deGRee
The degree of liver fibrosis was qualitatively assessed using a Nikon eclipse e200 optical microscope at 100x magnification by the Knodell scoring system, which classified the histopathological alterations, as follows (Brunt 2000) : score 0: no fi brosis; score 1: fi brous portal expansion; score 3: bridging fi brosis (portal-portal or portal-central); score 4: cirrhosis. The average score taken from 10 non-overlapping random fi elds per section was used to generate a single score for each specimen, in each experimental group (Sant'anna et al. 2011a ).