LIVER PATHOLOGY

1. LIVER PATHOLOGY Revision/update notes on liver disease Prepared for the Australian Animal Pathology Standards Program by John Mackie (Gribbles Veterinary Pathology, Australia) & Roger Kelly (former Reader in Veterinary Pathology, University of Queensland). These modules are designed for primarily for candidates who are preparing for ACVS membership exams… …but they should be useful for undergraduate veterinary students as well.

2. LIVER PATHOLOGY Revision/update notes on liver disease Images from various sources have been used in these presentations, and every effort has been made to acknowledge these when they have come from sources other than our own files. We apologise for any inadvertent omissions or errors in attribution, and would be pleased to make appropriate corrections should these errors be brought to our attention.

3. LIVER PATHOLOGY There are 4 modules: • Module 1 • Background, normal structure, developmental anomalies • Functions of the liver • Failure of function (including clinical chemistry) • Module 2 • Consequences of liver size • Sampling (including normal cytology) • General pathological reactions • Necrosis • Module 3 • Inflammation • Circulatory disturbances • Final common pathway of chronic liver disease, incl. fibrosis • Viral infections • Module 4 • Bacterial infections • Parasitic infections • Acute hepatotoxicities • Chronic hepatotoxicities • Neoplasia

4. Module 1. A quick refresher on features of hepatocytes: They are polyhedral cells with areas of specialisation on their surfaces They are arranged in one-cell-thick radiating plates (mammals) or cords (reptiles, birds), with blood-filled sinusoids on both sides, and bile canaliculi between adjoining surfaces. Avian liver (image courtesy Majid Ghoddusi

5. Module 1. Hepatocyte structure Desmosomes provide adhesion between cell surfaces. Tight junctions around the bile canaliculus provide a barrier to leakage of water and solutes. Gap junctions provide gated channels for communication between adjacent hepatocytes.

6. Hepatocyte ultrastructure (high magnification) Module 1. Discontinuous endothelial cytoplasm Space of Disse beneath endothelium is filled with microvilli mitochondria Kupffer cell Sinusoid Hepatocyte nucleus Avian liver (image courtesy Majid Ghoddusi

7. Module 1. LIVER SINUSOIDS DIFFER FROM MOST CAPILLARIES: Their endothelium is fenestrated, and their basement membrane is very delicate (may even be absent) So the fluid in Disses space is essentially plasma

8. Module 1. (Image courtesy Texas A&M University) LIVER SINUSOIDS DIFFER FROM MOST CAPILLARIES: And, unlike most capillaries, the sinusoidal endothelium bears numerous loosely-attached macrophages (Kupffer cells) This one has phagocytosed some erythrocytes Avian liver (image courtesy Majid Ghoddusi

9. Module 1. Setting the structural scene… acini and lobules: Normal livers seem to consist of lobules, and anatomists think of them that way… …but in disease, the lobular structure of liver often disappears… …and a pattern is delineated that reflects the liver’s embryonic origin as a compound acinar gland …and for that reason some pathologists prefer the acinar concept of liver microstructure

10. Module 1. Portal triad Sinusoids The acinar structure of liver can be likened to a bunch of grapes in which each grape corresponds to an acinus (or microcirculatory unit). The smallest stems represent the portal triads, and the spaces between the berries correspond to the hepatic venules (central veins in the conventional lobule) Hepatic venule (central vein) To complete the analogy, imagine that all the grape skins have been removed, and that the bunch has been compressed so that the periphery of the berries (the acini) now more or less merge, leaving some space for the hepatic venules between them.

11. Module 1. Portal triad Sinusoids Periacinar hepatocytes may lie next to larger branches of the portal triad tree. Hepatic venule This suggests that free intercommunication between adjacent acini can occur. Blood may enter the sinusoids of one acinus via its portal vein or artery and leave via the venule of another . Periacinar hepatocytes are those furthest from the smallest portal triads.

12. Module 1. The dotted lines indicate margins of adjacent acini Portal triad Periportal zone Mid zone Periacinar zone Hepatic venule (central vein)

13. Module 1. In normal liver sections, hepatocytes are quite uniform in appearance across the acinus… Portal triad Periportal zone Mid zone Periacinar zone Hepatic venule (central vein)

14. Module 1. …but there are significant differences between their metabolic functions and regenerative potential… periacinar cells: high affinity for water-soluble toxins periportal cells: low affinity for water-soluble toxins Oxygen availability decreasing periportal cells: low levels of microsomal enzymes periacinar cells: high levels of microsomal enzymes Hepatic venule Portal triad …and in their susceptibility to damage

15. Module 1. H&E section, x4; bovine liver; congestive cardiomyopathy Hepatic venule Portal triad Periacinar parenchyma has been replaced by stagnant blood and condensed stroma (see later for discussion of passive congestion of liver). Periportal and midzonal parenchyma, while slightly fatty, survives as berry-like clumps. This example thus illustrates the acinar basis of hepatic microstructure.

16. Module 1. LIVER REGENERATIVE CAPACITY Stem cell characteristics Normal differentiated hepatocytes are long-lived and retain a capacity to replicate, which maintains normal liver mass in the face of slow turnover. They may also replicate after injury. If the injury is severe enough, or if it prevents the remaining hepatocytes from replicating, an additional population of cells is capable of restoring liver mass. Hepatic stem cells (progenitor cells) located around portal triads may be induced to proliferate and produce new hepatocytes or cholangiolar epithelial cells (i.e. they are bipotential). See also Oval cell hyperplasia; Biliary hyperplasia. This is a reflection of normal development whereby intrahepatic bile ducts and canals of Hering are derived from embryonic periportal hepatocytes.

17. Hepatic stem cells(include oval cells) Module 1. Capable of replication Differentiated hepatocyte Cholangiolar epithelial cell

18. Module 1. Extreme nodular regeneration of the liver (Image courtesy Dr J King, Cornell University REGENERATIVE CAPACITY Hepatocellular hyperplasia The liver retains remarkable regenerative capacity: If 75% of the liver is surgically removed, the remaining 25% can regenerate via hepatocellular hyperplasia to normal size in a few weeks. Regeneration may occur as either a diffuse process (maintaining the original shape) or a nodular process. Regeneration tends to occur as a nodular process if the normal framework of supporting connective tissue, blood flow and biliary drainage is lost or disrupted (e.g. because of fibrosis). The resulting tissue is not as functional as the original parenchyma.

19. Module 1. Look how briskly the stem cells respond after recent toxic midzonal necrosis (image courtesy of Lucy Anthenill) REGENERATIVE CAPACITY Hepatocellular hyperplasia The anatomic pattern and duration of injury influence the nature of the regenerative response. e.g. focal necrosis of hepatocytes is repaired by proliferation of adjacent differentiated cells. Random single cell necrosis is repaired by random hepatocyte division. On the other hand, after acute zonal necrosis, surviving periportal hepatocytes can rapidly replace lost cells after a delay of around 24 hours. There may also be biliary hyperplasia (see later)

20. Module 1. Marked biliary hyperplasia secondary to chronic extrahepatic biliary obstruction (cholelithiasis) in a horse Stem cell (bile ductule) hyperplasia Another powerful stimulant of portal stem cell proliferation is cholestasis (see below). If cholestasis of any cause persists long enough, there will be some degree of stem cell activation and proliferation. Simple irritation of tissues in the portal triad by parasitic or bacterial infection will likely also cause stem cells to proliferate. Under these circumstances, the proliferating stem cells are more likely to differentiate into bile ductules than hepatocytes. This reaction is more pronounced in some species than others. Camelids and sheep are particularly prone to over-react in this fashion.

21. Module 1. The hepatic artery supplies most of the O2 requirements of the liver. HEPATIC BLOOD SUPPLY The portal vein drains the intestines, stomach, pancreas and spleen. It accounts for 70% of the blood supply to the liver, bringing in O2-poor but nutrient-rich blood. Portal blood flow is streamlined, so that different lobes of the liver preferentially receive blood from different viscera. This may lead to asymmetric distribution of lesions caused by toxins, infections or metastatic neoplasms. Portal vein

22. Module 1. In some species (e.g. the dog) there are also lymphatics around sublobular veins. Coagulated lymph between lobes of an acutely congested liver BLOOD AND LYMPHATIC DRAINAGE FROM THE LIVER Blood supplied by branches of the portal vein and the hepatic artery passes through the liver sinusoids and then into hepatic venules (central veins)  sublobular veins  hepatic vein  caudal vena cava. Hepatic lymph originates in the space of Disse and (because of the porous endothelium) is almost like plasma. It is coagulable, unlike lymph from other tissues. Hepatic lymph normally flows towards the portal triad to enter lymphatics in the connective tissue of portal tracts. These flow via the portal hilus to the hepatic lymph nodes. In some species (e.g. the dog) there are also lymphatics around sublobular veins. Under abnormal conditions (e.g. congestion) hepatic lymph may flow towards central veins and the capsule.

23. Module 1. FOETAL BLOOD SUPPLY The hepatic sinusoidal plexus initially receives blood from vitelline veins which drain the wall of the yolk sac. As the liver grows, the hepatic sinusoidal plexus also receives blood from the umbilical veins which carry oxygenated and nutrient-rich blood from the placenta. The left umbilical vein becomes the major blood supply to the liver during foetal development. The vitelline veins then give way to the portal vein, which communicates with the distal part of the umbilical vein. The ductus venosus develops by enlargement of some pre-existing hepatic sinusoidal channels. It directs most blood from the umbilical and portal veins into the caudal vena cava, thus bypassing the hepatic sinusoids. The hepatic artery appears relatively late in foetal development. • At birth: • the ductus venosus closes (and becomes the ligamentum venosum) flow ceases in the umbilical vein (which becomes the ligamentum teres).

24. Module 1. FOETAL BLOOD SUPPLY Congenital vascular anomalies • The ductus venosus may persist post-natally as a congenital intrahepatic portosystemic shunt (mostly in large breed dogs). • Persistence of the terminal branches of the vitelline veins post-natally has been speculated to result in congenital microvascular dysplasia. • The effects of these anomalies on the liver will be described later under the heading of Modes of Circulatory Disturbance

25. Module 1. DEVELOPMENTAL ANOMALIES Obstructive jaundice and marked bile duct distension due to atresia in the common bile duct of an adult cat hypoplasia of gall bladder duplicated gall bladder You must remember the species (equine, elephant, rats, deer and some birds) that have no gall bladder, otherwise you can make embarrassing mistakes… Feline livers (Images courtesy Dr J King, Cornell University In addition to congenital vascular anomalies, other developmental anomalies include congenital cysts, malformations of the gall bladder and biliary atresia. Congenital cysts may be single or multiple. Those attached to the capsule may be serosal inclusion cysts or may be of biliary origin. Those within the parenchyma are most likely of biliary origin. Congenital hepatic cysts need to be distinguished from parasitic cysts (such as those caused by larval cestodes).

26. Module 1. DEVELOPMENTAL ANOMALIES Biliary cysts may be solitary or there may be widespread bile duct hyperplasia and ectasia, associated with variable degrees of fibrosis. The latter condition is also referred to as congenital hepatic fibrosis, congenital fibrocystic disease and ductal plate malformation. The ductal plate is an embryologic structure where bipotential liver progenitor cells (which form embryonic bile ducts) interact with mesenchyme of the portal vein. Congenital hepatic cysts may be associated with polycystic kidney anomalies. Cysts may also be found in pancreas and other organs.

27. Module 1. METABOLIC FUNCTIONS, RESERVE CAPACITY The liver has a vast number of metabolic functions (several hundred, depending on how you count them) • These metabolic functions may be categorised broadly as: • Synthetic (i.e. anabolic) • Catabolic • Secretory • Excretory • Detoxifying • Storage There is overlap between categories (e.g. detoxification may be achieved by any of the other categories). The liver has tremendous reserve capacity for almost everything it does, including its synthetic functions.

28. Module 1. SYNTHESIS The liver has a large number and variety of synthetic functions From the perspective of clinical liver disease, synthesis of the following substances is of the greatest significance: • Albumin & other proteins, including lipoproteins • Coagulation factors • Glucose • Cholesterol • Urea • Bile acids The concentration of these substances may also be influenced by numerous other factors, which can complicate their interpretation.

29. Module 1. CONVERSIONS The metabolic conversions carried out by the liver may be synthetic or catabolic and essentially involve two classes of compounds: endogenous and exogenous. Endogenous compounds converted by the liver include glycogen, ammonia, lipids, arachidonic acid metabolites and steroid hormones. Exogenous compounds are also known as xenobiotics. Examples include drugs, toxins and environmental chemicals. Hepatic metabolism/conversion renders these compounds harmless and facilitates their excretion from the body (see also Acute hepatotoxicities - general considerations). In some instances, the same hepatocyte enzyme systems (e.g. cytochrome P450 enzymes) may be used for conversion of endogenous substrates (e.g. arachidonic acid metabolites and steroid hormones) and exogenous substrates (e.g. phenobarbital).

30. Metabolic conversions Module 1. Endogenous Exogenous Glycogen  Glucose Phenobarbital  harmless metabolite dysfunction dysfunction hypogycaemia Drug effect increased Ammonia  Urea dysfunction Hepatic encephalopathy

31. Module 1. (Image courtesy http://www.colorado.edu/intphys/Class/IPHY3430-200/image/figure16e.jpg) SECRETION AND EXCRETION The liver tends to use common pathways for secretion (removal of useful compounds) and excretion (removal of waste compounds), including for detoxification. (remember that hepatocytes in different zones of the acinus have different capacities for metabolic conversions). Some compounds such as bile salts are both useful (for emulsification of fat globules prior to absorption in the intestine) and a waste product (of cholesterol catabolism). Steps in the hepatic uptake, conjugation, intracellular transport and canalicular transport of bile acids are also used by a number of other endogenous and exogenous compounds (see also cyanobacterial toxicity). Another example of shared secretory and excretory pathways involves membrane-associated P-glycoproteins Some substances may be excreted virtually unchanged in bile (e.g. heavy metals).

32. Module 1. LIVER STORAGE FUNCTIONS Hepatocytes plays a central role in metabolism and storage of glycogen, lipids and many other macro- and micronutrients (see Chemical changes). Ito cells are present in the space of Disse. They are specialised mesenchymal cells (hepatic stellate cells) that store vitamin A and some fat, and are thought to play a role in the control of sinusoidal blood flow. Following injury, hepatic stellate cells can become myofibroblasts and play a role in hepatic fibrosis. Ito cells Liver is the main storage site of vitamin A. It also has a role in storage of vitamin B12 and activation of vitamin D3. It requires vitamin K to synthesise several of the clotting factors. Chronic hepatic insufficiency may result in deficiencies of fat-soluble and water-soluble vitamins.

33. Module 1. SINUSOIDAL CLEARANCE OF PARTICLES The liver acts a barrier, or sentinel filter, between the non-sterile intestine and the systemic circulation. Endotoxins, bacteria and other particulates are phagocytosed by Kupffer cells. Impaired Kupffer cell function (which may occur in a wide variety of liver diseases) can result in endotoxaemia or septicaemia.