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Bob Thoolen, Robert R. Maronpot, Takanori Harada, Abraham Nyska, Colin Rousseaux, Thomas Nolte, David E. Malarkey, Wolfgang Kaufmann, Karin Kuttler, Ulrich Deschl, Dai Nakae, Richard Gregson, Michael P. Vinlove, Amy E. Brix, Bhanu Singh, Fiorella Belpoggi, and Jerrold M. Ward
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The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and differential diagnosis for classifying microscopic lesions observed in the hepatobiliary system of laboratory rats and mice, with color microphotographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available for society members electronically on the internet (http://goreni.org). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions of the hepatobiliary system in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Keywords

diagnostic pathology, hepatobiliary system, histopathology, liver, nomenclature, rodent pathology

I. General Introduction

The liver is a major target organ in safety assessment of preclinical toxicity and oncogenicity studies with rodents; hence, hepatic pathology is central to many toxicological pathology studies. As toxicologic pathologists sometimes experience difficulties in distinguishing the wide variety of liver lesions in the rodents for safety evaluation purposes, this document is a consensus of senior toxicologic pathologists regarding suggested nomenclature that should be used for specific lesions.

Standardized diagnostic criteria and nomenclature are essential to harmonize the classification and reporting of hepatic nonproliferative as well as proliferative lesions. This INHAND document serves as a framework that can be used for the harmonization of diagnostic criteria of hepatic lesions in laboratory rats and mice. These recommendations for diagnostic criteria and preferred terminology should not be considered mandatory; proper diagnoses are ultimately based on the discretion of the toxicologic study pathologist.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) initiative creates a framework for the harmonization of diagnostic nomenclature (classification of lesions using the same terminology) in different rodent organ systems. It is a joint initiative between Societies from the United States (STP), Great Britain (BSTP), Japan (JSTP), and European countries (ESTP).

This document is organized to provide introductory material that reviews comparative interspecies differences in anatomy and liver function, followed by a listing of liver lesions in a standardized format. The liver lesions descriptions include differential diagnoses to aid in distinguishing primary diagnoses from similar appearing lesions. Throughout the document, comparisons are made with respect to similar liver lesions that may occur in humans. It should be noted that the preferred diagnostic terminology for some lesions in this document might represent departures from traditional nomenclature schemes found in standard textbooks. Furthermore, illustrative photomicrographs for a given diagnostic entity may occasionally depict additional tissue changes as this reflects actual situations frequently observed in pathological evaluation of toxicity studies.

II. Anatomy

The liver occupies the cranial third of the abdominal cavity and is comprised of multiple lobes; however, the nomenclature for the liver lobes varies among authors. There are basically left, middle, right, and caudate lobes (Harada et al. 1999; Eustis et al. 1990). A thin connective tissue capsule that is externally lined by peritoneal mesothelial cells covers the parietal and visceral surfaces of the liver. The middle lobe has an incomplete fissure where the falciform ligament attaches. In mice the gallbladder is located in the middle lobe fissure, whereas the rat does not have a gallbladder. The right lobe has an anterior and posterior component and the small caudate lobe consists of two or more disclike sublobes (See Figure 1).

Nomenclature for liver lobes varies among species and sometimes among authors. A table showing differences in liver lobes between species is included based on current anatomic features (Table 1).

Figure 1. Gross appearance and tissue trimming recommendations for a normal rodent liver. Ref. to http://reni.item.fraunhofer.de/reni/trimming/index.php. Figure 2. Two-dimensional microarchitecture of the liver. Figure 3. Rat liver. Hepatodiaphragmatic nodule. Figure 4. Rat liver. Hepatodiaphragmatic nodule with intranuclear inclusions (chromatin). Higher magnification of Figure 3. Figure 5. Rat liver. Macrovesicular fatty change. Figure 6. Rat liver. Macrovesicular fatty change. Higher magnification of Figure 5.

Figure 1. Gross appearance and tissue trimming recommendations for a normal rodent liver. Ref. to http://reni.item.fraunhofer.de/reni/trimming/index.php.
Figure 2. Two-dimensional microarchitecture of the liver.
Figure 3. Rat liver. Hepatodiaphragmatic nodule.
Figure 4. Rat liver. Hepatodiaphragmatic nodule with intranuclear inclusions (chromatin). Higher magnification of Figure 3.
Figure 5. Rat liver. Macrovesicular fatty change.
Figure 6. Rat liver. Macrovesicular fatty change. Higher magnification of Figure 5.

table1-Proliferative and Nonproliferative Lesions of the Rat and Mouse Hepatobiliary System

III. Histomorphology

The two-dimensional microarchitecture of the liver has been categorized in at least three perspectives (Figure 2). The anatomic model is the classical lobule, a hexagonal structure divided into concentric centrilobular, midzonal, and periportal segments. The triangular portal lobule is based on bile flow and is centered on the portal triad (portal canal). The elliptical or diamond shaped liver acinus is a functional subunit of the liver. It incorporates blood flow and metabolic functions and is divided in zone 1 (periportal), zone 2 (transitional; midzonal), and zone 3 (centrilobular). Functionally, zone 1 hepatocytes are specialized for oxidative liver functions such as gluconeogenesis, b-oxidation of fatty acids, and cholesterol synthesis, while zone 3 cells are more important for glycolysis, lipogenesis, and cytochrome P-450–based drug detoxification.

1. Blood Supply and Bile Flow

The liver has a dual blood supply, the hepatic portal vein and the hepatic artery. The hepatic artery supplies oxygenated blood. Approximately 75% of the blood is delivered to the liver via the hepatic portal vein that drains the spleen, stomach, intestines, and pancreas. Branches of the hepatic artery and portal vein are seen in the portal triads along with bile ducts and are separated from the hepatic cords by a ‘‘limiting plate’’ of hepatocytes. The bile ducts join to form the hepatic duct leading to the small intestine in rats and to the gallbladder in mice. Blood flows from the portal areas to the central vein in the center of each lobule while bile flows from the center of the hepatic lobule to the portal areas and on to the hepatic duct.

2. Histology

The two most commonly used descriptions for the structural and functional units of the liver are the hepatic lobule (Kiernan 1883) and the acinus (Rappaport et al., 1954) (Figure 2). The structural unit, the hepatic lobule, is modeled on the blood flow within the liver and is commonly used for descriptive pathology and morphological diagnoses. The functional unit, the hepatic acinus, is modeled on blood flow and metabolism within the liver. More recently a parenchymal unit in the liver has been described as a cone-shaped three-dimensional structure comprised of approximately fourteen hepatic lobules supplied and drained by common vascular tributaries (Malarkey et al. 2005; Teutsch, Schuerfeld, and Groezinger 1999; Teutsch 2005). This parenchymal unit more closely explains the random size and shape distribution of the more classical hepatic lobule as seen in a conventional two-dimensional histology slide. It also provides a basis for understanding the heterogeneous response of various hepatic lobules to chemical insult.

In addition to hepatocytes, the liver is comprised of a variety of cell types, including biliary cells, endothelial cells, Kupffer cells, Ito cells (stellate cells), fat-storing cells, and pit cells in addition to hematopoietic cells in the sinusoids and blood vessels. Polyhedral hepatocytes comprise approximately 60% of the liver arranged in plates or cords that radiate from the central vein to the portal areas. In two-dimensional sections they are typically one cell layer thick and form anastomoses (Miyai 1991). On one surface they are separated from the sinusoidal wall by a peri-sinusoidal space, the space of Disse, where they are exposed to tissue fluids. On the opposite side of the hepatocyte bile canaliculi are formed with hepatocytes in an adjacent hepatic cord. Desmosomes, gap junctions, and stud-like protrusions connect contiguous hepatocytes within a cord. Biliary cells form bile ducts in the portal areas and constitute the portal triad with a hepatic artery and a portal vein. Fenestrated endothelial cells line the sinusoids and synthesize prostaglandins. Kupffer cells are a self-renewing fixed macrophage comprising approximately 10% of all liver cells (Eustis et al. 1990). Kupffer cells are phagocytic, secrete mediators of inflammation, and catabolize lipids and proteins. Ito cells (stellate cells) are peri-sinusoidal cells that store vitamin A and are also a major source of collagen in the liver. Pit cells are lymphocytes that have natural killer activity and are primarily located in periportal areas (Wright and Stacey 1991).

3. Immunohistochemistry

Immunohistochemistry (IHC), utilizing fluorescent or chromogen tagged antibodies, is a useful adjunct for identification of different cell types in the liver. Selected examples are provided in Table 2.

Use of IHC can be helpful for diagnostic purposes and is common in human pathology where panels of immunohistochemical stains are used for supporting diagnoses. Not all commercially available preparations of a given antibody will react the same way between different laboratories and between different species. Furthermore, expertise is required for tissue handling to unmask cellular antigens that may be cross-linked during tissue fixation. Diagnostic evaluation of immunostains typically requires inclusion of both positive and negative controls. The interpretations of IHC results are usually performed in conjunction with histopathological findings and sometimes also with consideration of gross findings and/or clinical pathology or other relevant study results.

table2

IV. Physiology

The liver is responsible for maintenance of many homeostatic and physiological functions. Liver size is governed both by genetic factors and by the rate of biochemical activity to maintain optimal functional mass. It is an organ system capable of rapid responses to a variety of noxious stimuli. Following loss of hepatocytes from stimuli such as transient toxic insult, infection, or partial hepatectomy, the liver is rapidly restored to its optimal mass to maintain normal function.

Liver functions are complex and diverse including endocrine and exocrine activity, metabolism, conjugation, detoxification, and hematopoiesis in early embryonic and fetal development (Harada et al. 1999). The liver is continuously exposed to all ingested substances absorbed through the intestinal tract via the portal vein and systemically via the arterial blood supply. A pivotal hepatic function in toxicologic pathology is xenobiotic biotransformation that leads to detoxification of materials absorbed in the intestinal tract. Xenobiotic metabolism by hepatocytes can occur by phase I (often the cytochrome oxidase series) and phase II reactions (often the formation of the water soluble glucuronide) (Graham and Lake 2008; Martignoni, Groothuis, and de Kanter 2006). Hepatic metabolic processes may also cause indirect toxicity by generating electrophilic species capable of reacting with proteins, nucleic acids, and other cytoplasmic organelles (Xu, Li, and Kong 2005). Intrinsic and induced enzymes responsible for hepatic function may be unevenly distributed throughout the hepatic lobule and between the different lobes (Greaves 2007).

The presence of background changes and undercurrent disease states affects the hepatic and biliary morphology, for example, caloric restriction diminishes hepatocellular size and can make interpretation of test-article–related changes more challenging. Other factors that influence the liver morphology are: body weight loss, blood flow, food intake, vascular and hemodynamic changes, timing and duration of exposure, withdrawal effects, and functional heterogeneity. Functional heterogeneity expresses itself via differences in metabolism, oxygen supply, b-oxidation, amino acid metabolism, gluconeogenesis, glycolysis, ureagenesis, liponeogenesis, and bile acid and bilirubin secretion. These factors can affect occurrence of nonproliferative as well as proliferative liver lesions in rodents.

V. Liver Necropsy and Trimming Protocol

At necropsy, rat and mouse liver may be weighed and individual liver lobes examined carefully for gross lesions. In conventional preclinical rodent studies, gross lesions must be correlated with the histopathological findings. Liver-specific trimming protocols (see Figure 1) according to standard operating procedures (SOPs) are used (e.g., see Ruehl-Fehlert et al. 2003). Dissected lobes and trimmed liver pieces can be fixed in 10% neutral buffered formalin (no more than 1 cm thick in 1:10 tissue: formalin).

VI. Grading Of Liver Lesions

Interpretation of hepatic lesions in safety assessment studies requires consideration of gross and microscopic findings, hematology, clinical chemistry, and liver weights in the concurrent control groups of animals and should take into account species and strain, age, caging, diet, and tissue sampling.

Many pathologists use a grading system to document lesion severity. In toxicological pathology, the generation of ordinal data using a scoring system allows statistical analysis for effects and trends (Gad and Rousseaux 2002). However, not all grading systems are the same and may differ in how they incorporate distribution, stage, and extent of lesions. The problem of harmonization as it relates to lesion severity has been recognized and discussed in some detail (Hardisty and Eustis 1990; World Health Organization 1978).

Most toxicologic pathologists use a common grading scale such as marginal or minimal, slight, moderate, marked, and severe for inflammatory, necrotizing, or other degenerative and responsive lesions. Tissue-specific locators are often used, such as portal, periportal, midzonal, centrilobular, hilar, ductal, periductal, peri-canalicular, or subcapsular to indicate the lesion distribution within the liver. Focal, multifocal, and diffuse are commonly used modifiers in the morphological diagnosis for distribution parameters. Based on the formal definition, a focal lesion refers to one specific area, or focus, whereas multifocal refers to more than one focus (foci). However, some pathologists use focal for both focal and multifocal, referring to the nature of the lesion rather than its actual distribution and using grading to reflect the extent of the multifocality. Schemes for scoring lesion severity vary widely and no single system is likely to be accepted by all pathologists. While a sample grading scheme for focal and multifocal liver lesions is provided in Table 3, this should not be regarded as a universal or specific INHAND-recommended grading scheme.

table3

A. Congenital Lesions

Introduction

Developmental anomalies occasionally occur in the liver of rodents. These malformations might be expressed in different forms and be of different origin. They mostly occur as isolated effects and are considered by the pathologist in distinguishing background hepatic lesions versus xenobiotic-induced lesions that occur in rodent preclinical toxicity studies.

Hepatodiaphragmatic Nodule (Figures 3 and 4)

Pathogenesis: Developmental alteration.

Diagnostic features:

  • Visible grossly and tinctorially similar to normal hepatic parenchyma.

  • Rounded extensions usually of the medial lobe(s).

  • Increased mitoses, cytological alterations, and nuclear alterations may be present.

  • Linear chromatin structures with small lateral projections are pathognostic.

Differential diagnosis:

  • Hepatocellular focus of cellular alteration—tinctorial variation from normal parenchyma and does not protrude into the diaphragm.

  • Hepatocellular neoplasia—when visible grossly does not protrude into the thoracic cavity.

  • Regenerative hyperplastic nodule (nodular hyperplasia)—typically involves multiple nodules of hyperplasia separated by proliferative bands of oval cells or connective tissue.

Comment

Hepatodiaphragmatic nodules can be seen in rats at any age and their occurrence in fetuses is considered presumptive evidence of a congenital origin. While they appear to be protruding through the diaphragm and extending into the thoracic cavity, they actually are attached to and covered by a thin fibrous portion of the diaphragm (Eustis et al. 1990).

An incidence ranging from 1% to 11% has been reported for hepatodiaphragmatic nodules in Fischer 344 rats (Eustis et al. 1990), with few cases reported in other rat stocks and strains. Mice do not develop such nodules but may have focal lesions similar to those in rat hepatodiaphragmatic nodules and with large nuclei with large central nucleoli-like basophilic bodies.

B. Hepatocellular Responses, Cellular Degeneration, Injury, and Death

Introduction

The function and structure of most liver cells are relatively constrained by their genetic programs of metabolism, differentiation, and specialization. While the cells of the hepatic parenchyma have the flexibility to adapt to changing physiological demands with reversible functional and morphological alterations, sufficient stress, or noxious stimuli may lead to inability to maintain homeostasis and adverse cellular adaptations. The morphological response to injurious stimuli depends on the nature of the injury and its severity and duration. Often at high doses, targeted cells go through a sequence of cellular degeneration followed by cell death, but at lower doses degenerative changes do not necessarily lead to cell death. Consequentially, cellular changes that do not lead to cell death or death of the animal may be called “adaptive” changes that can be considered either adverse or not adverse reactions, depending on the nature of the change. There are cellular adaptations involving metabolic or functional alterations that lead to increases in cellular organelles and intracell