Liver Pathology: Inflammation, Circulatory Disturbances, Fibrosis

1. Module 3a LIVER PATHOLOGY Prepared for the Australian Animal Pathology Standards Program by John Mackie (Vepalabs, Australia) & Roger Kelly (former Reader in Veterinary Pathology, University of Queensland). These modules are designed for primarily for candidates who are preparing for Australian College of Veterinary Scientists membership exams… …but they should be useful for undergraduate veterinary students as well.

2. Module 3 LIVER PATHOLOGY 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. Module 3 LIVER PATHOLOGY This is the 3rd of 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 • Fibrosis • Final common pathway of chronic liver disease • Viral infections • Module 4 • Bacterial infections • Parasitic infections • Acute hepatotoxicities • Chronic hepatotoxicities • Neoplasia

4. Module 3 INFLAMMATION Inflammation of the liver (hepatitis) may be classified in a number of ways, for example: • Focal hepatitis (including liver abscess) • Diffuse chronic hepatitis, including: • copper-associated hepatitis • idiopathic hepatitis of dogs (including “chronic active hepatitis”) • Zonal hepatitis • Cholangitis, cholangiohepatitis • Lymphocytic portal hepatitis • Non-specific reactive hepatitis • Capsular inflammation (e.g. FIP) • Portal vein thrombophlebitis • Cholecystitis, gall bladder mucocoele, gall bladder infarction

5. Module 3 Patterns of hepatic inflammation (hepatitis) Inflammation of the liver shares most features of inflammation in other tissues… … so hepatitis is sub-classified as being acute, subacute, chronic, suppurative, necrotising, etc. But hepatitis enjoys some special descriptive terms that have to do with distribution of inflammation… …thus we have focal hepatitis, cholangiohepatitis, perihepatitis, etc. Focal hepatitis we have more or less dealt with under focal necrosis (Module 2). We have also touched on the inflammatory features of single-cell necrosis

6. Module 3 Rabbit liver; salmonellosis Focal hepatitis involving liquefactive necrosis of hepatocytes Bovine liver; IBR focal necrosis/hepatitis (systemic herpesvirus infection) Focal hepatitis Much of what we said in module 2 about focal necrosis can be applied to small foci of hepatic parenchymal inflammation that are randomly distributed with respect to acinar zones. Focal hepatitis is most often caused by something that has been removed from sinusoidal blood by a Kupffer cell. More often than not this is an infectious agent. Hepatocytes themselves may be specifically involved in the infection, or they may simply be destroyed as “innocent bystanders”. There is usually more accompanying inflammation than there is with zonal necrosis, so in many cases there may be argument as to whether the process should be called focal necrosis or focal hepatitis. “Focal necrotising hepatitis” covers both aspects.

7. This TEM of perfusion-fixed avian liver reminds us how hard it is for particles in portal blood to get past the Kupffer cells Module 3 (Image courtesy M Ghoddusi) Focal hepatitis Probably every liver at any moment contains a few foci of inflammatory cells. These are usually randomly distributed with no zonal preference. Sinusoid These foci are presumably the result of trapping of microbes or other irritant particles by sinusoidal macrophages (Kupffer cells). Bile canaliculus Hepatocyte These particles must have been absorbed from the gut, although we don’t really know how they get across the mucosa. All we can be sure of is that such particles reach the liver and may cause significant problems. The classic example is necrotic hepatitis, which arises from spores of Clostridium novyi or C. haemolyticum that must have been ingested and reached the liver in the portal blood. Kupffer cell Another is liver abscessation in cattle, whose prevalence is related to the feeding of grain-rich diets. The causal bacteria mostly reach portal blood via unhealthy rumen mucosa

8. Module 3 A small focus of hepatitis in which several hepatocytes are being mugged by mononuclear inflammatory cells (probably T lymphocytes among them). This is an example of single-cell necrosis associated with cell-mediated immune response to something in Kupffer cells or in hepatocytes themselves. Every liver probably contains similar foci if you look for them. Focal hepatitis Not all causes of focal hepatitis are infectious. Some foci might be caused by immunological reactions to non-living compounds. These might have been sequestered in sinusoidal macrophages after removal from portal blood, or, alternatively, they might have been produced by hepatocytes as a by-product of metabolism of a nutrient or xenobiotic. Whatever its origin, if such material elicits an immune response (particularly if this is mediated by T-lymphocytes), a nasty little brawl takes place. Either the Kupffer cells and/or the hepatocytes that harbour the target compound are targeted. This is probably the mechanism behind the very common isolated foci of single-cell necrosis/focal hepatitis.

9. Chronic canine indospicine poisoning. Severe mononuclear inflammatory cell infiltration associated with parenchymal collapse in the periacinar zones Module 3 Mononuclear inflammatory infiltrate and macrophage activity in canine copper-associated hepatopathy in a 6 year-old Labrador Chronic hepatitis Diffusely distributed hepatitis, regardless of its cause or initial pattern, tends to produce the same sort of chronic liver disease in its final stages. Chronic hepatotoxicities may produce chronic liver disease that is indistinguishable grossly and histologically from chronic hepatitis... …and to confuse things further, there are some chronic hepatoxicities in which chronic inflammation is a feature at all stages of the disease. Heritable copper poisoning of Bedlington terriers and other species, and canine indospicine poisoning are examples of such conditions. These features of chronic hepatitis include atrophy; fibrosis; nodular regeneration; portal hypertension leading to acquired portosystemic shunts and ascites. They are described in more detail under the heading of Final Common Pathways of Liver Disease. Severe canalicular cholestasis

10. Module 3 Chronic hepatitis There has always been a confusing welter of names used to designate the various types of chronic hepatitis in domestic animals. The confusion is compounded by the fact that the cause is very often unknown… …and by attempts to follow the nomenclature used in human medicine. There is now a push to simply use the term “chronic hepatitis” for persistent inflammatory liver disease, and to qualify it when the cause is known, and to grade it according to its severity. Thus there is a move away from such designations as “chronic active hepatitis” (see the introduction to Chronic Hepatitis by Stalker and Hayes in Jubb, Kennedy & Palmer’s Pathology of Domestic Animals, 5th edn, Vol 2. 2007). See also Piecemeal necrosis in Module 2.

11. Module 3 Canine adenovirus-1 hepatitis showing haemorrhagic necrosis in the periacinar zone. If not for the intranuclear inclusions, this would be difficult to distinguish from acute hepatotoxic insult. Periportal haemorrhagic necrosis in rabbit calicivirus hepatitis. There is little or no inflammatory response Zonal hepatitis We have seen examples of hepatic necrosis which are accompanied by inflammation of varying severity, and it can be difficult to decide which is the dominant process: degeneration (necrosis) or inflammation. It is usual for necrosis in any tissue to be accompanied by some reactive inflammation, provided the necrosis is of sufficient duration. But there are examples of liver disease that are designated as hepatitis, yet the inflammatory reaction is minimal. Good examples of this are the viral infections infectious canine hepatitis (CAV-1) and rabbit calicivirus infection (rabbit haemorrhagic disease). In peracute cases, there is very little inflammation. The severity of the necrosis is usually zonal. In CAV-1 infection, most necrosis occurs in the periacinar zone, while in the rabbit infection, the periportal zone is worst affected. We have no idea of the basis for the zonal distribution of the injury in either disease.

12. Module 3 Neutrophilic inflammatory infiltrate spilling out into periportal parenchyma in bacterial cholangiohepatitis, dog Cholangitis and cholangiohepatitis Whenever there are greater numbers of inflammatory cells in the portal triads than expected, the biliary apparatus may have become an inflammatory stimulus. This can be due to invasion of the ducts by bacteria or by parasites… …or some irritant may have been concentrated in them during excretion (see sporidesmin intoxication, Module 4.). If the inflammation is essentially limited to the ducts and portal stroma, it is referred to as cholangitis. If the inflammation appears to be centred on ducts but has spilled out and involves the periportal hepatocytes as well, it is called cholangiohepatitis. Minimal non-suppurative accumulations of inflammatory cells (particularly lymphocytes) in portal triads are very common. They are probably residual from prior episodes of irritation, and are of little or no diagnostic significance (and in birds they are usually normal). See also Non-specific reactive hepatitis.

13. Module 3 Atrophy and fibrosis of left lobe of bovine liver in severe chronic fascioliasis, with compensatory hypertrophy of right lobe. (remember that the falciform ligament demarcates right from left lobes in ruminants) Cholangitis and cholangiohepatitis This cat had pancreatitis, leading to common bile-duct obstruction, which in turn led to severe ascending bacterial infection of the biliary tree, with parenchymal extension (cholangiohepatitis). Intrahepatic bile ducts are markedly distended Bacterial biliary tract infection is sporadic and unpredictable in incidence, but is more likely to occur in animals in which biliary drainage is compromised. Whether or not the biliary irritation is infectious or toxic, the chronic effects are similar; i.e. portal fibrosis and atrophy of the more severely-affected parts of the liver. The most common cause of cholangiohepatitis in ruminants is probably fascioliasis. In ruminants, chronic cholangiohepatitis consistently causes more severe atrophy of the left lobe, while the right lobe undergoes compensatory hypertrophy. The reason for the asymmetry of this process is not known, but it may be because the path of biliary drainage is longer for the left lobe, and is therefore more easily interrupted.

14. Module 3 Cholangitis and cholangiohepatitis The most common form of cholangiohepatitis in cats features a predominantly lymphocytic infiltrate, with bile duct inflammation and/or necrosis. It is called feline progressive lymphocytic cholangiohepatitis. If may also contain plasma cells and occasional neutrophils. Its cause is unknown, though some cases may represent a late chronic stage of neutrophilic cholangitis. Another feline hepatitis which lacks bile duct inflammation and necrosis has been termed lymphocytic portal hepatitis. Whether or not it is a variant of feline progressive lymphocytic cholangiohepatitis is debatable. It seems reasonable to assume that a continuous antigenic stimulus of some sort is emanating from the biliary tract in both of these conditions. Note that some cases of small cell lymphoma may be difficult to discriminate from lymphocytic cholangiohepatitis or lymphocytic portal hepatitis. PCR-based lymphocyte antigen receptor assays for clonality may assist in differentiating. Lymphocytic cholangitis, cat. There is expansion of portal areas by mainly lymphocytes and fewer plasma cells. The inflammation is centred around bile ducts with mild infiltration of the epithelium. Lymphocytic cholangiohepatitis, cat. There is expansion of portal areas by lymphocytes and fewer plasma cells, with disruption of biliary epithelium and extension into the parenchyma.

15. Module 3 Non-specific reactive hepatitis in a dog with pancreatitis. There is a mild inflammatory infiltrate in the stroma of the portal tract, consisting of mainly lymphocytes and plasma cells and fewer neutrophils. There is no hepatocellular necrosis or infiltration or hyperplasia of the bile duct epithelium. Non-specific reactive hepatitis Non-specific reactive hepatitis may occur as a response to systemic inflammation or to inflammation anywhere in the splanchnic bed (i.e. in the drainage area of the portal vein). It may also be residual to prior hepatic inflammation. It is characterised by a widespread but mild inflammatory infiltrate in the stroma of some portal tracts and hepatic venules, and possibly in the parenchyma. There is no associated necrosis. In acute disease the infiltrate is mainly neutrophilic. There may also be sinusoidal leucocytosis and/or Kupffer cell hypertrophy/hyperplasia. In chronic disease, or in the case of residual hepatic inflammation, mononuclear cells predominate (lymphocytes, plasma cells and macrophages, including pigmented macrophages). Morphologically, non-specific reactive hepatitis may overlap mild cases of focal hepatitis, cholangitis and lymphocytic portal hepatitis.

16. Module 3 Feline liver; capsular hepatitis. FIP. Capsular inflammation (perihepatitis) Inflammatory exudate may be deposited on the capsule of the liver in some systemic inflammatory diseases (e.g. feline infectious peritonitis). In this condition there may also be variable focal infiltrates within the liver capsule and the subcapsular parenchyma. The liver capsule will usually be involved in bacterial peritonitis (e.g. actinomycete infection in dogs; gastrointestinal perforation or translocation of enteric bacteria across compromised gut wall). Portal vein thrombophlebitis Necrotising vasculitis and thrombosis of intrahepatic branches of the portal vein may be seen in cattle secondary to necrotising fungal rumenitis.

17. Chronic cholecystitis, dog. The inflammatory infiltrate in the lamina propria consists of mainly lymphocytes and plasma cells. E coli was isolated in pure growth from bile. Module 3 Cholecystitis Inflammation of the gall bladder is termed cholecystitis. It is relatively uncommon in domestic animals. Causes include reflux of intestinal bacteria via the bile ducts (i.e. ascending infection) or haematogenous entry from adjacent hepatic circulation. Some bacteria (e.g. Salmonella sp) are excreted from the liver via the bile ducts (i.e. descending infection) and may cause cholecystitis. Cholecystitis may also be associated with cholelithiasis. Acute cholecystitis is usually neutrophilic, while in chronic stages the cellular infiltrate may be mixed or predominantly lymphoplasmacytic. Gall bladder infarction has been reported in dogs, and is characterised by transmural necrosis with intravascular thrombi. The cause is not known. It is not simply a sequel to cholecystitis. Other causes of transmural necrosis include gall bladder mucocoele. Chronic cholecystitis, dog. Note hyperplasia of gall bladder epithelium and infiltration of the lamina propria by mainly lymphocytes and plasma cells.

18. Module 3 GALL BLADDER MUCOCOELE Gall bladder mucocoele is the accumulation of a semisolid to immobile mucoid mass within the gall bladder. It may extend into the bile ducts, resulting in extrahepatic biliary obstruction. Some cases may progress to ischaemic pressure necrosis of the gall bladder wall with rupture resulting in bile-induced peritonitis. The aetiology is not known. Possible underlying factors include decreased gall bladder motility, bile stasis and altered bile composition and viscosity (“biliary sludge”). Gall bladder mucocoele is usually greenish-black. The absence of such pigment in some gall bladder mucocoeles suggests a variable pathogenesis and may reflect a dominant contribution of gall bladder mucin or impaired flow of bile through the cystic duct. Gall bladder mucocoele, dog. Note cystic hyperplasia of the gall bladder epithelium, usually with no inflammatory reaction. There is abnormal accumulation of eosinophilic mucus in lumen and glands. The gall bladder is distended with bile-stained mucus, which may become inspissated.

19. Module 3 CIRCULATORY DISTURBANCES OF THE LIVER Infarction Infarction is necrosis due to ischaemia (reduced blood supply). Peripheral lobar infarct; necrotic hepatitis, bovine Infarction is uncommon in the liver due to its dual blood supply (hepatic artery and portal vein). It tends to occur at the edge of the liver where the terminal divisions of the blood supply are found. Infarction of the liver may occur with torsion of a lobe or with portal thrombosis. It is classically associated with clostridial hepatitis (see Bacterial diseases) Infarcts are sharply demarcated and either dark red or pale. They consist of a central area of coagulative necrosis, bordered by a zone of variably intense inflammation and haemorrhage. Infarct ; ovine necrotic (clostridial) hepatitis.

20. Module 3 Sheep liver; torsion of accessory lobe CIRCULATORY DISTURBANCES OF THE LIVER Displacement and torsion Displacement of the liver may occur secondary to diaphragmatic or ventral hernias. Such hernias may be congenital or acquired. Displacements through hernias may cause circulatory disturbances such as congestion, which may proceed to rupture, or portal hypertension. Over time, displaced lobes may become firm and shrunken (fibrosed). Caudal displacement of the liver may occur due to enlargement of the liver, pleural effusion or a space occupying thoracic lesion. There is usually circulatory disturbance in such cases. Torsion of individual lobes of the liver is rare. It may occur secondary to neoplasia or trauma or may be idiopathic. It can result in infarction of affected lobes, or toxaemia due to vegetative growth of latent clostridial spores. (Image courtesy Dr J King, Cornell University “Nutmeg” pattern of passive venous congestion in liver lobes incarcerated through diaphragmatic hernia (cat)

21. Module 3 Periacinar hepatocytes undergo atrophy, fatty change or necrosis due to hypoxia. CIRCULATORY DISTURBANCES OF THE LIVER Passive venous congestion Chronic passive congestion may be a sequel to right-sided heart failure (or compression/obstruction of the caudal vena cava); in short, anything that reduces the pressure differential between the portal and the venular ends of the hepatic sinusoid. Reduction of this hydrostatic differential causes reduces the efficiency of perfusion of the parenchyma. This reduction will necessarily affect periacinar hepatocytes more severely than the periportal tissue So periacinar hepatocytes undergo atrophy, fatty change, and eventually die and are removed... ...and the periacinar ends of the sinusoids expand to take up the vacated space. Red cells may come to occupy and be trapped within spaces left by the absent periacinar hepatocytes, since the reticulin framework may remain intact.

22. Module 3 CIRCULATORY DISTURBANCES OF THE LIVER Passive venous congestion Intrauterine passive hepatic venous congestion; bovine foetus with cardiac anomaly Grossly, the liver is enlarged and dark. Depending on species and lobe, the edges may be either rounded, or thin and leaf-like. With increased sinusoidal blood pressure, accumulating hepatic lymph passes through the liver capsule, resulting in ascites. Eventually, if the animal survives long enough, fibrosis can occur around hepatic venules and isolate portal triads (so-called “cardiac fibrosis”).

23. Module 3 This acinar pattern is supposed to resemble the cut surface of a nutmeg, hence the pathologist’s shorthand “nutmeg liver” for chronic passive hepatic congestion CHRONIC PASSIVE CONGESTION – CANINE HEARTWORM DISEASE This dog had severe right-sided heart failure. The pallor of the surviving central parenchyma is due to (1) fatty change and (2) loss of cytochromes in the distressed hepatocytes The subcapsular parenchyma contains very few hepatocytes. It consists mostly of stagnant blood mixed with collapsed stroma.

24. Module 3 PORTAL HYPERTENSION Definition: elevation in the normally low pressure in the portal vein, usually due to increased resistance to portal blood flow. The anatomic site of this resistance may be: Pre-hepatic prehepatic portal vein thrombosis, or compression by neoplasia, etc. Intrahepatic fibrosis due to any chronic hepatopathy Post-hepatic congestive (right-sided) heart failure hepatic vein or caudal vena caval thrombosis or compression

25. Module 3 PORTAL HYPERTENSION Consequences Ascites due to altered abdominal and/or hepatic lymph production/drainage. Note that ascites may also be the result of hypoalbuminaemia of any cause. Hypoalbuminaemic ascites, however, is rarely as voluminous as ascites caused by portal hypertension. • Acquired (secondary) portosystemic shunts • with non-cardiac causes of portal hypertension, venous pressure in the portal system is higher than in the caudal vena cava and its branches, so attempts to provide collateral drainages begin. • with portal hypertension of cardiac origin, the elevation in central venous pressure does not increase the pressure gradient between portal and caudal vena caval veins, so shunts do not develop.

26. Module 3 A case of acquired porto-systemic shunting due to portal hypertension. Small, distorted (tough) liver, ascites and multiple anomalous tortuous thin-walled veins coursing between duodenum and various branches of the posterior vena cava. ACQUIRED PORTOSYSTEMIC SHUNTS These develop secondary to portal hypertension, and are usually accompanied by distension of the portal vein and by copious ascites They appear as connections between mesenteric veins and the caudal vena cava or its branches (e.g. renal or gonadal veins). The new vessels are multiple, thin-walled and tortuous veins (thus distinguishing them from congenital shunts, which may also be indirect but are usually single or at the most double) Hepatic encephalopathy may develop, as portal blood rich in ammonia and other neurotoxins bypasses the liver. These anomalous vessels are very easy to miss at necropsy by inexperienced operators, who tend to inadvertently destroy them.

27. Module 3 Liver small; normal surface and texture., no ascites or prehepatic shunts. Persistent ductus venosus Probe in vena cava. Yellow arrow shows direct connection between portal vein and vena cava CONGENITAL PORTOSYSTEMIC SHUNTS These are vein-to-vein shunts with a variable anatomic pattern; by definition they are present at birth. Large breed dogs typically have a single large intrahepatic shunt, usually a persistent ductus venosus.

28. Module 3 CONGENITAL PORTOSYSTEMIC SHUNTS Important in dogs and cats These are vein-to-vein shunts with a variable anatomic pattern. Small breed dogs and cats typically have single large extrahepatic shunts between the portal vein and the caudal vena cava or azygous vein. Congenital shunts should produce no portal hypertension (at least in the early stages), so there should be no ascites, nor any plexiform prehepatic collaterals

29. Module 3 CONGENITAL PORTOSYSTEMIC SHUNTS Consequences of portal blood bypassing the liver may include • Microhepatica (small liver), stunted growth • due to failure of nutrients and hepatotrophic factors to reach the liver • Elevated plasma ammonia, bile acids • due to insufficient extraction by hepatocytes • NH4 biurate crystalluria or urolithiasis; urinary tract infections • due to high systemic blood levels of ammonia • Polyuria/polydypsia • due to reduced hepatic synthesis of urea (renal medullary tonicity) • Hepatic encephalopathy • due to high systemic blood levels of ammonia and other gut-derived neurotoxins

30. Module 3 WHAT’S YOUR INTERPRETATION? 6-month-old Yorkshire terrier dog with a history of ill-thrift, post-prandial neurological signs, polyuria/polydypsia and sporadic vomiting. There is evidence of hepatic insufficiency (bile acids, glucose, urea, albumin, cholesterol) with only very mild increases in liver enzymes (ALP, AST, ALT) and normal bilirubin. Microcytosis may reflect altered iron metabolism. The dog had a congenital extrahepatic portosystemic shunt. Elevated bile acids are typical. Other changes on routine biochemistry and haematology are much more variable (and these parameters may be normal in some cases).