JPC SYSTEMIC PATHOLOGY

DIGESTIVE SYSTEM

September 2024

D-M17

 

Signalment (JPC #2384572): Female spayed Doberman pinscher

 

HISTORY: This dog presented with anorexia, icterus, and elevated ALT, ALP and total bilirubin.

 

SLIDE A:

HISTOPATHOLOGIC DESCIPTION: Liver: Markedly expanding portal areas and bridging adjacent portal areas are numerous macrophages, lymphocytes, neutrophils, fewer plasma cells, fibroblasts, scant to moderate amounts of variably mature collagen (fibrosis) and increased biliary profiles (biliary ductular reaction). Multifocally, inflammatory cells disrupt the limiting plate, extend into the adjacent periportal parenchyma, and separate, surround, or replace hepatocytes that are often degenerate with swollen vacuolated cytoplasm or are necrotic with shrunken, hypereosinophilic cytoplasm and karyolytic, pyknotic, or karyorrhectic nuclei (“piecemeal” necrosis). Central veins are indistinct and there is occasional degeneration and necrosis of centrilobular hepatocytes admixed with lymphocytes, neutrophils, and macrophages, and there are multifocal areas of bridging inflammation from the central vein to adjacent portal areas. Diffusely, lobules are decreased in size evidenced by decreased distance between portal areas. Hepatocytes often contain yellow-brown, intracytoplasmic pigment (copper, lipofuscin, or hemosiderin). The capsule is undulant with multifocal loss of subcapsular hepatocytes and infiltration of lymphocytes, plasma cells, neutrophils, and macrophages admixed with hemorrhage, fibrin, and edema. There are multifocal dilated lymphatics (edema), and scattered macrophages that contain golden brown cytoplasmic globules (hemosiderin). 

 

SLIDE B: Rhodanine: Liver: Diffusely, macrophages and hepatocytes, predominantly periportal but also midzonal and centrilobular, contain abundant red-brown, granular to globular pigment (copper).

 

MORPHOLOGIC DIAGNOSIS: Liver: Hepatitis, portal and periportal, histiocytic, lymphoplasmacytic, and neutrophilic, chronic, diffuse, moderate, with hepatocellular degeneration and necrosis, and abundant hepatocellular and histiocytic intracytoplasmic copper, Doberman pinscher, canine. 

 

CONDITION: Canine Chronic Hepatitis 

 

SYNONYMS: Canine chronic-active hepatitis, chronic progressive hepatitis

 

GENERAL DISCUSSION

• In dogs, chronic hepatitis (CH) is a relatively common, long term (≥6 months), necroinflammatory disease characterized by hepatocellular damage and loss with regeneration and fibrosis
• In dogs, etiology is most often idiopathic; it has been associated with various causes, most notably copper-associated hepatitis in certain breeds (see pathogenesis; see D-T05 for more on copper toxicity)
  • In other species, chronic hepatitis is commonly associated with ingestion of hepatotoxins
• Initial acute liver damage will not progress to fibrosis or cirrhosis unless inflammation and damage are protracted

 

PATHOGENESIS 

• Chronic liver disease in canines is poorly understood and likely due to various causes including chronic bile duct obstruction, infection with infectious (e.g., leptospirosis), familial or hereditary metabolic diseases, or may be toxic (e.g., aflatoxin), drug-induced (e.g. phenobarbital, primidone, phenytoin, lomustine), or autoimmune; copper-accumulation is the best characterized
• Several breeds predisposed: Bedlington Terrier, Doberman Pinscher, West Highland White Terrier, Labrador Retriever, American and English Cocker Spaniel, Skye Terrier, Standard Poodle, Dalmatian, and English Springer Spaniel
• Copper-associated hepatitis
  • Hepatic copper accumulation can be primary (metabolic defect in hepatic copper metabolism), secondary (abnormal hepatic function with cholestasis and altered biliary copper excretion – e.g., cholestasis), or related to increased dietary copper / bioavailability
  • Hepatic copper concentrations >2,000 μg/g DW (dry weight) more likely associated with disease
  • Excess copper can lead to the production of reactive oxygen species and destructive lipid peroxidation
  • Increased malondialdehyde [MDA]) (a marker of oxidative stress) and active caspase-3 [casp-3] (a marker of apoptosis) were noted in samples with increased copper and corresponded with increased inflammation and fibrosis (Yamkate, J Comp Pathol. 2022)
  • Bedlington terriers are the only breed with a recognized disorder: a mutation in the COMMD1 gene (encodes a chaperone protein the ATP7B copper transporter involved in hepatocyte copper excretion into bile)
    • Bedlington Terriers accumulate copper continuously throughout life
  • Familial tendencies for increased hepatic copper also seen in other breeds (including most of the above breeds), however significance is unclear; it has also been noted less commonly in cats 
    • Doberman pinschers: Elevated hepatic copper (noted in some with chronic hepatitis) may be due to impaired copper excretion from decreased mRNA for metallothionein, ATP7A, ATP7B, and ceruloplasmin (proteins involved in copper binding, transport, and excretion)
      • Note: There was also decreased gene expression of components of the antioxidant defense system (SOD1, catalase) and reduced glutathione levels
• Immune-mediated hepatitis
  • Important in humans, role in canines is unclear 
  • Hepatocyte upregulation and presenting MHC-II-associated autoantigens have been suggested as targets for T-cell-mediated immune attack
  • Highly polymorphic DLA genes noted in Doberman pinschers and English Springer spaniels with chronic hepatitis
• Deficiency of alpha-1-antitrypsin (α1-AT), a hepatocyte-produced inhibitor of neutrophil elastase, may play a role (noted in some dog breeds, including English Cocker Spaniels)
• Hepatic fibrosis is associated with platelet-derived growth factors PDGFB and PDGFD, thrombospondin 1, transforming growth factors β1 and β2 (TGFB1 and TGFB2), matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinase 1, and the collagen genes COL1A1 and COL3A1.

 

TYPICAL CLINICAL FINDINGS 

• Most often middle-aged, female dogs
• Early signs: Nonspecific, may include anorexia, depression, weakness, fatigue, weight loss, vomiting
• Later: As the disease progresses, polydipsia, polyuria, icterus, ascites, and signs of hepatic encephalopathy (e.g. disorientation, behavioral changes, circling, head pressing, seizures, coma)
• Clinical pathology

1. Elevated ALT and ALP, hyperbilirubinemia, hypoproteinemia, (hypoalbuminemia), hypergammaglobulinemia, bilirubinuria; in advanced CH, liver enzyme activities can appear normal or below reference range 
2. Liver function tests: Usually abnormal; possibly prolonged coagulation times, but hemorrhagic diathesis/hemorrhage is unlikely 

 

TYPICAL GROSS FINDINGS: 

• Small liver with accentuated lobular pattern
• Severely affected livers have architectural distortion ranging from coarsely nodular texture to end stage liver

 

TYPICAL LIGHT MICROSCOPIC FINDINGS: 

• Chronic hepatitis is characterized by:
  1. Mononuclear or mixed inflammatory infiltrate (Lymphocytes and plasma cells) with necrosis of centrilobular / zone 3 hepatocytes
  2. Hepatocellular degeneration and necrosis
     • Periportal interface hepatitis (“piecemeal necrosis”) which initially destroys the limiting plate of periportal / zone 1 hepatocytes, and may continue to erode into the hepatic parenchyma, expanding portal areas
     • Bridging necrosis may develop between portal triads or between portal areas and central veins
  3. Biliary ductular reaction and degeneration
  4. Hepatocellular regeneration: 2-cell–thick hepatic plates and mitotic figures +/- regenerative nodules
  5. Fibrosis: Can become bridging and lead to cirrhosis with lobular collapse and fibrous tissue separating regenerative nodules
     • Deposition in the space of Disse > Capillarization of hepatic sinusoids; single or groups of hepatocytes may be isolated and entrapped in expanded portal areas
• Copper: Accumulation is initially evident in centrilobular hepatocytes but may progress to involve the whole lobule; its color ranges from orange to golden brown or gray-blue on H&E staining
• May also see intrahepatocyte bile pigment, intracanalicular bile stasis (especially in periportal zones) and/or iron accumulation in Kupffer cells/macrophages

 

ADDITIONAL DIAGNOSTIC TESTS: 

• Rubeanic acid and rhodanine histochemical stains to detect copper in hepatocytes as well as Kupffer cells and macrophages
• Quantitative copper analysis: Fresh or formalin-fixed tissues
  1. Hepatocytes in regenerative nodules often have lower copper levels and intervening areas of parenchymal collapse may contain high copper; because of this regional variation, small samples, (e.g. from needle biopsies), are often inaccurate, and larger samples are required
• Cytology: May suggest the presence of hepatic fibrosis (pink fibrillar extracellular matrix material and spindled mesenchymal cells); copper within hepatocytes stains as pale blue, coarse, crystalline granules with routine cytochemical stains
• Disease grading and staging
  1. Grade correlated with increasing hepatocellular apoptosis, proliferation, expression of nitric oxide synthase (NOS) isoforms, and total hepatic iron
  2. Stage correlated with expression of genes associated with fibrosis (see Pathogenesis) and degree of fibrosis 
• Serum protein electrophoresis: May be useful to monitor canine chronic hepatitis; may see hypoalbuminemia, increased globulins (α1,γ, α2, β2, and β1), and decreased albumin:globulin (A:G) ratios (Gori, J Vet Diagn Invest. 2022) 
• A hepatic fibrosis score calculator using serum biochemical markers (ALT, ALP, total bilirubin, potassium, and gamma-glutamyl transferase) is available (Menard, J Vet Diagn Invest. 2019).

 

DIFFERENTIAL DIAGNOSIS: 

• Hemosiderosis/hemochromatosis (D-M20): Excessive accumulation of hepatocellular iron; periportal and perivenular bridging fibrosis with nodular regeneration; common in birds and lemurs, otherwise considered rare
• Cholangitis / chronic biliary obstruction: May see loss of bile ducts with pigment laden macrophages and mixed inflammation including neutrophils; fibrosis with chronicity; biliary hyperplasia; +/- histologic evidence of cholestasis; may be associated with chronic bacterial cholangitis/cholangiohepatitis

• Infectious agents 
  • Leptospirosis: Hepatocellular dissociation and single-cell necrosis; spiral bacteria, seen with Warthin-Starry stains

1. Infectious canine hepatitis virus (Canine adenovirus type 1) – basophilic intranuclear inclusion bodies may be seen in the first ten days of infection

  

COMPARATIVE PATHOLOGY: 

• In horses and ruminants, particularly cattle, end-stage liver is assumed to be associated with ingestion of hepatotoxins (e.g., pyrrolizidine alkaloids) or consequence of prolonged administration of hepatotoxic therapeutics
• Lobular dissecting hepatitis: Described in young dogs with ascites and acquired portosystemic shunts; sinusoidal inflammation and dissection of lobular parenchyma by reticulin + collagen leading to individual and small groups of hepatocytes; unknown etiology; in one study of chronic hepatitis in American Cocker Spaniels (7 of 13 dogs had this pattern in addition to typical cirrhosis)
• Copper-associated chronic hepatitis is seen in a variety of species, including
  1. Sheep (U-T10): Sensitive to exquisitely susceptible to copper toxicity due to dietary levels +/- molybdenum as well as other hepatotoxins; characterized by centrilobular and midzonal necrosis, paroxysmal intravascular hemolysis and liver failure; kidneys deep red brown to black kidney; acute tubular injury (hemoglobinuric nephrosis) due to of hemoglobinuria with oxidation to methemoglobin and concurrent icterus
  2. Feline: Copper-associated chronic hepatitis and cirrhosis is rare
  3. Rabbits: Sensitive to dietary copper, associated with hemolytic anemia with intravascular hemolysis, splenic erythrophagocytosis centrilobular to midzonal hepatic necrosis and later fibrosis, and hematuria with tubular casts.
  4. Avians: Hepatic, proventricular, ventricular degeneration and necrosis as well as rickets disease associated with copper toxicity
  5. Invertebrates: Wide-ranging effects of copper toxicity is also reported in numerous invertebrate species (corals, gastropods, sea hares, oysters, clams, crabs)
  6. In humans, chronic hepatitis is usually the result of chronic infection with hepatotropic viruses, and, less commonly, autoimmune, drug-induced, or associated with inherited metabolic diseases, such as copper in Wilson disease
• Chronic hepatitis with portal fibrosis, biliary hyperplasia, and focal nodular dysplasia found in aged hamsters infected with a Helicobacter spp. (see D-B18)

 

REFERENCES: 

 

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