Congenital and Acquired Portosystemic Shunts: Histological Features and Pathogenesis

Module 3b 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.

Module 3 REMEMBER THE NORMAL BLOOD SUPPLY TO THE LIVER? 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 vein Normal portal triad The hepatic artery supplies most of the O2 requirements of the liver.

CONGENITAL PORTOSYSTEMIC SHUNTS Hepatic arteriolesare increased in number and tortuosity, presumably to compensate for the reduced supply of portal blood Histological features Portal veinsare inconspicuous, small or absent

In shunted livers, hepatic arterioles are obvious because of their increased number and the “crowding” of their endothelial cells Hepatic arterioles in a normal triad are relatively inconspicuous, with a modest quota of endothelial cells

Module 3 Normal hepatic arteriole Hepatic arteriole in “shunted” liver Hepatic arterioles during life Hepatic arterioles after death CONGENITAL PORTOSYSTEMIC SHUNTS The histological appearance of the arterioles… can we explain that? (we’ll give it a try…) The prominence of the endothelial cells is probably the result of the arterioles in shunted livers being abnormally thin-walled and dilated Thin-walled arterioles need more endothelial cells to line them, relative to arterioles with smaller lumens Contraction of the vessel post mortem squeezes the endothelial cells together, hence the “busy” appearance of the arteriolar endothelium in livers with congenital or acquired porto-systemic shunts

Module 3 Hepatic venules and triads are crowded (small acini) and there are pigment deposits CONGENITAL PORTOSYSTEMIC SHUNTS Summary of expected histological features: Hepatic arterioles are increased in number and tortuosity, as the liver presumably attempts to compensate for the reduced supply of portal blood Portal veins are inconspicuous, small or absent Hepatocytes may appear atrophic and acini smaller Additional changes may include fatty granulomas with ceroid/haemosiderin pigmentation, and mild biliary hyperplasia Fibrosis is not expected

Module 3 Cuboidal mesothelium indicating ascites The apparent increase in bile ductules and fibrous tissue is due to condensation of original tissue ACQUIRED PORTOSYSTEMIC SHUNTS • Some histological features are the same as for congenital shunts: • Hepatic arterioles are increased in number and tortuosity • Portal veins are inconspicuous, small or absent • Hepatocytes may appear atrophic and acini smaller • Additional changes may include fatty granulomas with ceroid/haemosiderin pigmentation, and mild biliary hyperplasia But in acquired shunts excess fibrous tissue extends into parenchyma, and there is evidence of increased hepatocyte turnover, often with nodular hyperplasia. So ……. what’s the primary cause of the portal hypertension that’s causing these acquired shunts? Any chronic liver insult that causes liver fibrosis and/or reduces liver mass . So any chronic toxic or inflammatory process could eventually lead to restriction of portal blood flow. But some acquired shunts occur in young dogs in which there is no evidence for involvement of a toxic or inflammatory agent (see later).

Module 3 ACQUIRED PORTOSYSTEMIC SHUNTS Whatever the cause, the pathogenesis involves progressive failure to maintain liver mass, so that eventually there aren’t enough sinusoids to accept the full portal blood flow. The resulting portal hypertension is eventually sufficient to lead to the acquired shunts Once the acquired shunts have formed, the liver becomes progressively deprived of the hepatotrophic factors that are needed for maintenance of normal hepatic mass and function. So loss of hepatocytes is hastened further while their replacement is further compromised; a vicious cycle. The increased toughness of these livers is more due to condensation of pre-existing stroma than to de novo fibrosis. But it plays its part in progressive impedance of portal flow. Eventually, whatever the cause, these cases move onto the final common pathway of chronic liver failure and become essentially indistinguishable from cases of liver failure of whatever cause in older animals.

Module 3 ACQUIRED PORTOSYSTEMIC SHUNTS So, what’s the story with young dogs with acquired shunts? We don’t know. A popular theory is that these animals are born with hypoplastic portal venules. Alternatively, there may be an hepatic abiotrophy of unknown cause, with failure of maintenance of hepatic mass. Whatever the primary cause, acquired mesenteric shunting may develop once portal hypertension exceeds a certain level. Some better-perfused hepatocytes may attempt compensatory hyperplasia, thus producing the micronodular appearance often seen in these livers The term “non-cirrhotic portal hypertension” has been used to describe this condition. “Microvascular dysplasia” is used to describe cases that are clinically, biochemically and histologically consistent with portosystemic shunting, yet have neither a demonstrable macroscopic congenital shunt (admittedly hard to entirely rule out) nor acquired mesenteric shunting. “Microvascular hyperplasia” may be a mild variant of “non-cirrhotic portal hypertension” if portal hypertension is not severe enough to cause acquired mesenteric shunts and ascites.

Congenital portal vein hypoplasia If you pointed out that this histology is essentially the same as that of a congenitally shunted liver, you’d be quite right So we shouldn’t kid ourselves that we know the pathogenesis of cases like this. (aka “idiopathic non-cirrhotic portal hypertension”) This case exhibited ascites and multiple, plexiform mesenteric venous shunts Acini are smaller (portal triads are closer together) Hepatic arteriolesare increased in number and tortuosity (compensation for reduced portal blood flow) Portal veinsare inconspicuous, small or absent

Module 3 CONGENITAL PORTAL VEIN HYPOPLASIA (“idiopathic non-cirrhotic portal hypertension”) Concept summary Grossly there may be ascites and multiple, plexiform mesenteric venous shunts. Ascites and/or other clinical signs usually delayed until 3-6 months of age. The liver may be small and may have a finely nodular capsule, or it may be smooth. The histology of these livers may very closely resemble that of congenitally shunted livers, or there may be variably severe condensation fibrosis and nodular remodelling and hepatocyte loss/single cell necrosis (none of which are features of congenital portosystemic shunts). The pathogenesis is obscure. The portal hypertension may be due to primary hypoplasia of the portal vein, or the underlying defect could be primary abiotrophy of the liver.

Module 3 HISTOPATHOLOGY GROSS PATHOLOGY Vascular anomalies of the liver share some microscopic features (blue) but differ in other respects (red) Portal veins Hepatic arterioles Fibrosis Ascites, plexiform mesenteric shunts CONGENITAL Portosystemic shunt - - Microvascular dysplasia a - - "Idiopathic non-cirrhotic portal hypertension" b Variable + Arteriovenous fistula c - + ACQUIRED Hepatopathy with secondary mesenteric shunts + + a, b The pathogenesis of these conditions remains uncertain. Microvascular dysplasia may be a mild variant of "idiopathic non-cirrhotic portal hypertension", and both may be due to primary hypoplasia of the portal vein or primary abiotrophy of the liver. c Congenital fistulae between hepatic artery and portal vein are a rare cause of portal hypertension.

Hepatic telangiectasis, bovine abattoir specimen. After exsanguination, blood drains from expanded sinusoids and the foci collapse Two foci of early telangiectasis, with associated hepatocyte disappearance. Bovine abattoir specimen. Module 3 Telangiectasis and Peliosis Hepatis These terms designate two liver changes that are characterised by expansion of sinusoids. We have seen how periacinar hepatic sinusoids become distended in passive venous congestion... ...but there are two other conditions that can produce sinusoidal expansion: The first, telangiectasis, is the result of focal loss of hepatocytes, so that the associated sinusoid/s expand to occupy the vacant space. For this to happen, the focal loss of hepatocytes has to be so subtle as not to stimulate local hepatocyte regeneration... ...nor to result in focal fibrosis or abscess formation. Telangiectasis is most commonly seen in older cattle that have had long-term access to dietary concentrates, but its pathogenesis is a complete mystery. It is most reliably seen in livers of old dairy cows, and in old cats.

Module 3 Telangiectasis and Peliosis Hepatis Field case of Pimelea poisoning exhibiting peliosis. Liver “like a huge blood-filled sponge”. Hydrothorax and ventral oedema are also characteristic of the disease Peliosis hepatis (Gr. “purple-red, livid liver”) also involves sinusoidal expansion, but is zonal in distribution (predominantly periportal), and the expansion is more pronounced in the subcapsular parenchyma than deeper in the liver. Peliosis is a response to generalised increase in blood volume, so it can occur in other tissues with a sinusoidal-type vasculature (adrenal cortex; bone marrow) It is an uncommon condition. Its first veterinary use was in descriptions of bovine Pimelea poisoning (“St George disease)”. Blood volumes can be doubled in cattle experimentally intoxicated with Pimelea, and the sinusoidal expansion in liver and other organs seems to be a response to this. Peliosis hepatis was first described as an incidental finding in humans with tuberculosis and following prolonged use of anabolic steroids. But in humans the cystic blood filled spaces are more focal than zonal and may be lined by or devoid of endothelium. This lesion might be better designated as telangiectasis. Thus the term “peliosis hepatitis” may have two slightly different meanings. Severe peliosis, bovine field case of Pimelea poisoning. The sinusoidal dilatation is most severe in the superficial (subcapsular) parenchyma, where stagnation thrombosis may occur. photo: M. Dodson

Module 3 FIBROSIS • Fibrosis is the deposition of new extracellular matrix (mainly collagen) by either: • hepatic stellate cells/Ito cells (which differentiate into myofibroblasts), or • fibroblasts in connective tissue in portal areas or around hepatic venules (central veins). It is a sequel to hepatocellular necrosis and subsequent generation of inflammatory cytokines (especially by activated Kupffer cells). Fibrosis is a common manifestation of chronic liver injury. It often occurs in combination with other reactions such as hyperplasia of hepatocytes or stem cells/oval cells/biliary epithelium (see Biliary hyperplasia and Oval cell hyperplasia in Module 2). The combination of fibrosis and hepatocellular hyperplasia may impart a coarse or fine nodularity to the liver. Within acini, collagen is initially laid down in the perisinusoidal space of Disse, where it causes obstruction of the gaps between sinusoidal endothelial cells (“capillarisation of sinusoids”) and loss of microvilli on hepatocytes. The resulting hypoperfusion, hypoxia, malnutrition and deprivation of hepatotrophic factors all contribute to atrophy and death of adjacent hepatocytes, thus perpetuating liver injury.

Module 3 PATTERNS OF FIBROSIS Focal fibrosis (“milk spots”), pig liver; ascariasis Focal or multifocal hepatic fibrosis (randomly distributed) - e.g. migrating nematode larvae. Portal (biliary) fibrosis Periacinar (centrilobular) fibrosis Diffuse hepatic fibrosis; also called “dissecting” fibrosis (involves both portal and perivenular stroma) Pseudo-fibrosis, or stromal condensation after loss of larger groups of hepatocytes (these patterns are demonstrated in the next series of slides). When fibrosis is severe/diffuse, it is often impossible to determine either its initial pattern or its cause. Mild fibrosis may be reversible by degradation of collagen by proteases. More severe fibrosis is essentially irreversible and is associated with progressive liver disease/failure, often accompanied by nodular hyperplasia of hepatocytes. The term “end-stage liver” is sometimes used. See also Final Common Pathway of Chronic Liver Disease.

Module 3 PERIPORTAL (BILIARY) FIBROSIS If inflammation is centred on portal triads, the fibrosis may remain largely confined to these areas. In this case, the fibrosis is usually accompanied by hyperplasia of bile duct epithelium. Examples include chronic cholangitis (e.g. of cats or associated with fluke infection in ruminants), chronic extrahepatic biliary obstruction and chronic toxic injury such as sporidesmin poisoning. The fibrosis may become extensive enough to link adjacent portal triads. Bridging biliary fibrosis associated with chronic fascioliasis in a cow

Module 3 PERIACINAR FIBROSIS Fibrosis may develop around hepatic venules when the primary injury is in that zone. This pattern of fibrosis may occur due to chronic toxic injury (e.g. pyrrolizidine alkaloid poisoning in ruminants). This pattern of fibrosis may also be a response to passive venous congestion of the liver, including chronic right sided heart failure. This may develop into bridging fibrosis, which links hepatic venules. Periacinar hepatic (“cardiac”) fibrosis; chronic passive venous congestion in heart failure, dog

Module 3 DIFFUSE HEPATIC FIBROSIS Diffuse hepatic fibrosis affects all zones of the acinus and is present throughout the liver. It is the outcome of chronic, slowly progressive infiltration of the parenchyma or repeated episodes of parenchymal injury (e.g. zonal necrosis). The fibrosis is generated slowly and eventually links portal areas and hepatic venules, intersecting the classic lobules to produce pseudolobulation. Bridging fibrosis (central-central, portal-portal or portal-central) is more likely to impair hepatic function than other forms of fibrosis. This pattern of fibrosis is particularly likely to produce portal hypertension and secondary (acquired) portosystemic shunting of blood (see Circulatory disturbances).

Module 3 PSEUDO-FIBROSIS (STROMAL CONDENSATION) It is important to differentiate fibrosis (an absolute increase in the extracellular matrix in the liver) from a relative increase in connective tissue due to loss of hepatocytes and subsequent collapse and condensation of pre-existing stroma (the reticulin framework of the sinusoids). Stromal collapse and condensation may occur following zonal necrosis and loss of hepatocytes. It is also a feature of idiopathic hepatopathy of older dogs characterised by vacuolation and loss of hepatocytes accompanied by parenchymal collapse (see Hepatocutaneous syndrome, Module 1) The fibrous tissue in this tough, nodular liver (Doberman hepatopathy) is the result of loss of parenchyma between the regenerative nodules. It is not de novo fibrosis. (Image courtesy Dr R Sutton)

Module 3 Post-necrotic scarring, bovine: broad irregular fibrous bands and intervening nodular regeneration POST-NECROTIC SCARRING A single event of widespread hepatocellular necrosis, in which large areas of the parenchyma are destroyed, may result in grossly discernible broad bands of dense fibrous connective tissue. This is referred to as post-necrotic scarring. The underlying pattern of necrosis is usually either massive or severe periacinar necrosis. Post-necrotic scarring usually includes both fibrosis and collapse and condensation of the reticulin network, so that surviving portal areas come to lie adjacent to each other. Image courtesy Jubb KVF, Kennedy PC & Palmer N. Pathology of domestic animals. 4th edn. 1993

Module 3 CIRRHOSIS • Most definitions of cirrhosis emphasise: • a diffuse process throughout the entire liver, characterised by • (bridging) fibrosis accompanied by nodularity (usually due to hyperplasia of hepatocytes) • significant disruption of the normal acinar architecture. Grossly, these changes impart a coarse nodularity to the liver, with nodules of regenerating parenchyma separated by fibrous bands. The fibrosis may be fundamentally portal, periacinar, or both (with portal-central bridging). Cirrhosis may lead to portal hypertension with secondary (acquired) portosystemic shunts. The term “cirrhosis” is sometimes also used imprecisely to refer to the gross appearance of livers that are tough due to post-necrotic collapse and condensation rather than de novo collagen synthesis, or to livers distorted by fibrosis rather than nodular regeneration. Because of such imprecision, some authors would prefer to avoid the term “cirrhosis”, and instead use the various designations of fibrosis, combined with the addition of nodular regeneration where appropriate.

Module 3 FINAL COMMON PATHWAY OF CHRONIC LIVER DISEASE Regardless of the initial insult, a number of secondary effects may perpetuate injury to hepatocytes, leading to progressive (irreversible) liver disease : • Fibrosis deprives hepatocytes of oxygen and nutrients • directly • indirectly, by causing portal hypertension and secondary (acquired) portosystemic shunting of blood. Accumulation of constituents of bile, particularly salts of bile acids, which are hepatotoxic at high concentrations. Accumulation of metals, including copper and iron, which catalyse formation of reactive oxygen species leading to oxidative stress. Iron also directly stimulates hepatic fibrosis. Sustained or repetitive hepatocyte death increases the severity of each of these factors, which leads to further hepatocyte death. This self-perpetuating cycle is a feature of chronic progressive liver disease.

Module 3 Final common pathways of chronic liver disease Responses to hepatocyte death Inflammation Cholestasis, bile salt accumulation • Hyperplasia • hepatocyte • oval cell • biliary Cu, Fe accumulation Fibrosis Portosystemic shunting Pathways coloured red may lead to further hepatocyte death

Module 3 Final common pathways of chronic liver disease Major determinants of the final outcome of liver injury include: Whether hepatocellular injury is sustained or repetitive. This is particularly relevant during exposure to toxins. The severity and extent of fibrosis. • How much of the liver’s framework remains on which regeneration can take place. Elements of this framework include: • the original fibrous and reticulin scaffold • an adequate supply of blood • free drainage of bile.

Module 3 Final common pathways of chronic liver disease What are the gross and microscopic features of chronic liver disease? Hepatocellular loss, resulting in a small liver. Hepatocellular hyperplasia. This begins in scattered islands of less severely affected hepatocytes; the result is randomly distributed regenerative nodules Oval cell and biliary hyperplasia. Fibrosis, which is both an effect of chronic liver disease and a perpetuating factor. It results in a small, firm liver and contributes to the nodularity, and to portal hypertension, leading to acquired portosystemic shunts and ascites. Variable pigmentation, due to accumulation of bile, ceroid/lipofuscin and iron. Variable fatty change, due to disturbances in liver lipid metabolism. Variable inflammation, which may occur in response to the original aetiologic agent, hepatocyte necrosis, cholestasis or accumulation of metals.

Module 3 VIRAL INFECTIONS While the list of viruses that may cause liver lesions in animals is relatively long, most changes are fairly stereotypical (and are covered in the next few slides). The liver is a major target organ for a few viruses that cause substantial hepatic disease, sometimes culminating in liver failure. Examples include infectious canine hepatitis, Rift Valley fever and Wesselsbron disease. Other viral diseases may involve the liver, but the hepatic involvement either does not occur invariably or it may be just one manifestation of a systemic process. Examples of the latter include herpesviruses and adenoviruses (which cause similar lesions in a variety of species), rabbit calicivirus, porcine circovirus 2, feline coronavirus infection (feline infectious peritonitis) and equine infectious anaemia.

Module 3 VIRAL INFECTIONS Canine adenovirus 1 (infectious canine hepatitis; ICH) Most viral infections of the liver are species-specific. They characteristically cause multifocal random hepatocellular necrosis. The inflammatory response may be minimal, especially in young animals. Infectious canine hepatitis virus is tropic for hepatocytes, endothelial cells and mesothelial cells, with intranuclear inclusions. The disease is now rare, thanks to vaccination. In some viral infections, zonal (periacinar) necrosis may be superimposed on random necrosis. This is often seen in cases of ICH. It may also be a feature of exotic viral infections such as Rift Valley fever. Intranuclear inclusion bodies (arrows) within and around a focus of hepatocellular necrosis Distinctly zonal (periacinar) distribution of necrosis in CAV1 hepatitis

Module 3 Equine herpesvirus 1 infection, foetus. Grossly there may be miliary grey foci on capsular and cut surfaces of the liver. VIRAL INFECTIONS Systemic herpes virus infections Generalised (systemic) herpesvirus infections occur in many species, often causing abortion or neonatal death. Equine herpesvirus-1 (and occasionally EHV-4) infects equine foetuses in late gestation, causing necrosis in liver, lung and other organs including thymus and spleen. Histologically there is multifocal random necrosis, often with minimal inflammatory cell reaction. Intranuclear inclusion bodies are present in hepatocytes, often with margination of chromatin. Canine herpesvirus infection occurs in utero or soon after birth. Foci of necrosis and intranuclear inclusion bodies are seen in many tissues including the liver. (Image courtesy Texas A&M University) (Image courtesy Dr K Credille, Texas A&M University)

Module 3 hepatic venule VIRAL INFECTIONS Rabbit calicivirus Some viruses such as rabbit calicivirus (= rabbit haemorrhagic disease) cause mainly periportal necrosis of hepatocytes. Grossly there is bloody nasal discharge, multifocal haemorrhage, hepatomegaly with accentuated acinar pattern, pulmonary congestion and oedema and splenomegaly. The key histological feature is periportal hepatocellular necrosis, which may extend to the midzone. Inflammation is minimal. Other histological findings include splenic and lympho-follicular necrosis, and capillary thrombi in multiple tissues including lung and kidney (disseminated intravascular coagulation). Histologically there is widespread periportal to midzonal hepatocellular necrosis with replacement haemorrhage and minimal inflammatory response. portal triad

Module 3 Normal liver on left; “dishrag liver” on right EQUINE SERUM HEPATITIS a.k.a Theiler’s disease A sporadic form of acute liver failure in horses, in which there is often a preceding history of injection (usually 30-90 days beforehand) with a biological product of equine origin, generally serum (e.g. tetanus antitoxin or pregnant mare serum) or occasionally a vaccine grown on equine tissue culture... ...but in some cases there is no prior history of administration of such a product. The aetiology has not been established, though infection with a novel flavivirus has recently been reported in one outbreak. It remains possible other viral infections and immune-mediated mechanisms may be involved. This disease appears to be only rarely reported in Australia. The clinical course is usually short and characterised by icterus and hepatic encephalopathy. The mortality rate is high. In the more rapidly fatal cases there is periacinar to submassive hepatocellullar vacuolation, haemorrhagic and lytic necrosis leading to profound loss of parenchyma. So, grossly, the liver is typically small, flabby (“dishrag liver”) and may be discoloured greenish brown. (Image courtesy Dr J King, Cornell University

In the most dramatic variant of the condition, virtually all hepatocytes undergo lytic necrosis and are replaced by pooled blood. A light mononuclear inflammatory infiltrate usually persists in the stroma. Module 3 EQUINE SERUM HEPATITIS a.k.a Theiler’s disease In peracute cases nearly all hepatocytes disintegrate and are replaced by pooled blood. But even in these there are often small stromal accumulations of mononuclear inflammatory cells. In less fulminating cases the hepatocellular damage is more zonal (periacinar), and hydropic and fatty vacuolation and canalicular cholestasis are prominent features. Additional changes include early regenerative hyperplasia of biliary epithelium and/or hepatocytes In these cases non-suppurative stromal inflammatory infiltrates may be quite prominent. A proportion of cases may also have had recent intravascular haemolysis, as evidenced by mild haemoglobinuric nephrosis

Module 3 LIVER PATHOLOGY Module 4 follows: • 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

Congenital and Acquired Portosystemic Shunts: Histological Features and Pathogenesis