Congenital Intrahepatic Vascular Anomaly in a Clinically Normal Laboratory Beagle

Abstract

Routine necropsy examination of a clinically normal, ten-month-old male beagle dog identified disruption of the dorsal aspect of the right median liver lobe by a multiloculated, pale tan-white, thick-walled mass, with diffuse hypertrophy of the left and caudate liver lobes. The nature of the lesion was investigated using a variety of immunohistochemical and histochemical techniques. Histologically, the lesion was characterized by marked, focally extensive vascular hyperplasia and ectasia with lobular atrophy, periportal fibrosis, and biliary hyperplasia. The endothelium of proliferating vessels was positive for von Willebrands factor, and proliferating bile ducts were positive for pancytokeratin. Based on the gross and histologic appearance, this lesion was identified as a vascular hamartoma with arteriovenous fistula. It is important to note that this spontaneous lesion may occur in the laboratory beagle in the absence of clinical signs.

Congenital anomalies of the liver occur infrequently in domestic species (McGavin and Henry 1972). Those reported in dogs are the consequences of portosystemic shunts, intrahepatic arteriovenous fistulae, vascular hamartomas, obstructions to the hepatic venous outflow, and hepatoportal microvascular dysplasia (Stalker and Hayes 2007; McGavin and Henry 1972).

A number of etiologies for intrahepatic arteriovenous fistulae have been described, including abdominal trauma, rupture of hepatic artery aneurysms, hepatic vein obstruction, cirrhosis (leading to portal hypertension), iatrogenic causes (secondary to surgery or percutaneous biopsy), and congenital defects (vascular hamartomas) (Stalker and Hayes 2007).

Vascular hamartomas and congenital intrahepatic arteriovenous fistulae are rare in dogs (Schermerhorn et al. 1997), and there has been only one previously reported case in a laboratory beagle (Yoshizawa et al. 1997). They have not been reported before in a clinically normal animal. Typically animals with these lesions exhibit ascites (secondary to portal hypertension) as well as gastrointestinal and neurological signs.

Given the small numbers of dogs per group in toxicity studies, it is particularly important that spontaneous lesions, especially those that are uncommon, are not interpreted as compound-related changes.

A ten-month-old, male beagle dog was sacrificed under deep anaesthesia at the conclusion of a one-month oral toxicity study. The animal was a vehicle control and received 2.5 mL/kg of a water, hydroxypropyl methylcellulose, and polysorbate 20 vehicle by oral gavage, once daily for twenty-eight days. The study was carried out in accordance with UK home office legislation (Animals [scientific procedures] Act 1986) and AstraZeneca’s institutional policies.

The dog had been clinically well, and routine hematology and serum biochemistry (during the course of the study) identified no abnormalities (data not shown). A full necropsy was performed at termination: external features were inspected; cranial, thoracic, and abdominal cavities and contents were examined; and a single femorotibial joint was opened and examined. Grossly, the caudate and left lateral hepatic lobes were severely, diffusely enlarged, and there was congestion of the surface vasculature on the gall bladder. The dorsal aspect of the right median lobe of the liver was severely disrupted by a multiloculated, pale tan-white, thick-walled, cystic structure. There were no other significant necropsy findings. A full Good Laboratory Practices (GLP) tissue list was collected for histological examination.

Sections of the cystic structure were taken for histopathology, along with routine sections of the gallbladder and sections of the left lateral, right median, and caudate liver lobes. In addition to routine hematoxylin and eosin (HE), periodic acid-Schiff (PAS), Masson’s trichrome, reticulin, and elastic van Gieson stains were performed (using standard methods) along with immunostaining for pancytokeratin, using monoclonal mouse anti-cytokeratin (pan), (neat; Invitrogen, Carlsbad, CA, USA); von Willebrands factor, using polyclonal rabbit anti-vWF (1:800; Dako, Glostrup, Denmark), and α-smooth muscle actin, using monoclonal mouse anti-α-smooth muscle actin (1:1000; Sigma, St. Louis, MO, USA) (Bancroft and Gamble 2007).

Histologically, the right median lobe was largely effaced by a poorly defined, cavernous proliferation of well-differentiated vessels, infiltrating and dissecting the hepatic parenchyma and supported by a prominent fibrous stroma (Figure 1). Vascular walls were irregularly thickened, with mild to moderate, diffuse expansion of the tunica intima, extensive subintimal fibromusclar proliferation, and smooth muscle hyperplasia within the tunica media (Figure 2). Multifocally, smooth muscle fibers within the subintimal area assumed a longitudinal orientation. Vascular lumina were frequently either ectatic (predominantly venules) or narrowed (in some cases almost to the point of occlusion—predominantly arterioles). Partial elastolysis, splitting, and reduplication of elastic fibers were apparent within many arterioles, and increased deposition of elastic fibers was present in many venules, highlighted in elastic van Gieson-stained sections; however, complete elastic lamina were not apparent in venules (Figure 2). Prominent vasa vasorum were sporadically observed in association with large veins. Definitive differentiation of arteries and veins was difficult in places owing to the nature of the extensive changes in vascular walls. Moderate amounts of multifocal intimal and subintimal degeneration (often mucinous) and occasional foci of fibrinoid necrosis were observed within a proportion of vessels.

Within the lesion, the hepatic parenchyma was effaced, exhibiting marked parenchymal atrophy and fibrosis lymphangiectasia and biliary hyperplasia (Figure 3), with extensive disruption of the hepatic plates and sinusoidal dilation, highlighted in sections stained with Masson’s trichrome and Reticulin techniques (not shown). There was marked, diffuse hypertrophy of the caudate and left hepatic lobes and moderate amounts of glycogen (confirmed by PAS, not shown). No other microscopic findings were apparent.

Immunohistochemical techniques further validated the findings. There was positive staining of the endothelium of the anomalous vessels for von Willebrand’s factor (Figures 4A and 4B), and proliferating bile ducts were highlighted by antibodies to pancytokeratin (Figures 4C and 4D). Rarely, large bile ducts within portal triads also demonstrated some intracytoplasmic staining for von Willebrands factor. Staining produced by α-smooth muscle actin was less specific, and although the tunica media of the vessels did appear to be highlighted, more background staining was apparent (not shown).

Discussion

The histopathological findings in this case were focally extensive, marked vascular hyperplasia and ectasia, with lobular atrophy, periportal fibrosis, and biliary hyperplasia, suggestive of a congenital vascular anomaly, most likely a hepatic vascular hamartoma with arteriovenous fistulae (McGavin and Henry 1972; Moore and Whiting 1986). Vascular changes, in addition to the presence of multiple intermingling, dilated, and abnormal vessels—including elastolysis, splitting and reduplication of the internal elastic lamina, intimal hyperplasia, mucinous degeneration of the subintima and media, and smooth muscle hyperplasia—have been reported by numerous authors, including Moore and Whiting (1986), Easley and Carpenter (1975), Schaeffer et al. (2001), and Yoshizawa et al. (1997). All authors also report fibrosis, parenchymal atrophy, and biliary hyperplasia. Although Moore and Whiting (1986), Easley and Carpenter (1975), and Schaffer et al. (2001) were grossly able to recognize fistulae, McGavin and Henry (1972) and Schermerhorn et al. (1997) were unable to definitively identify arteriovenous anastomoses (as in this case). Thrombosis and recanalization, described as a feature of cases reported by Moore and Whiting (1986), were not seen in this case.

The age of this animal and the absence of obvious prior trauma suggest that this lesion was most probably a congenital form of vascular hamartoma. The diffuse nature of the lesion within the right median lobe in this case is also indicative of congenital origin (Van Way et al. 1971).

Vascular hamartomas and congenital intrahepatic arteriovenous fistulae are rare in dogs (Schermerhorn et al. 1997), and there has been only one previously reported case in a laboratory beagle (Yoshizawa et al, 1997). They arise from focal failure of differentiation of the common embryologic anlage into capillary, arterial, or venous structures and represent communications between the hepatic arteries and portal venous radicals, which leads to a retrograde flow of blood within the portal vein (Moore and Whiting 1986). Acquired portosystemic shunts develop (via opening of fetal vessels) to counteract the portal hypertension. With time, an increasing number of vessels are recruited to the fistula, which increases the fraction of blood shunted into the venous circulation without perfusing the liver (Tappin and Rizzo 2007). There is a certain degree of compliance within the portal vein, so some adaptation is thought to occur. Clinical signs develop when the animal can no longer compensate for the volume overload. Increasing portal hypertension leads to the development of ascites, splenomegaly, and gastrointestinal varices, and there is insufficient detoxification of the portal blood by the liver (owing to development of an acquired portosystemic shunt), which can lead to hyperammonemia and encephelopathic signs. Clinical signs vary in intensity and are most likely proportional to the caliber of affected vessels and the proportion of shunted blood (Cullen 2007).

Although definitive identification of fistulae was not possible in this case, the closely opposed, tortuous, and intertwined nature of the vessels, along with changes in vessel wall structure, suggest a significant degree of vascular overload. Substantial subintimal fibromuscular proliferation and deposition of elastin fibers (arterialization) was apparent in many venules, representing a response to increased intravascular hydrostatic pressure. Similar histologic changes are reported in experimentally induced arteriovenous fistulae (Gomes and Bernatz 1970). The histologic morphology of the lesion was identical to that of hepatic arteriovenous fistula described previously (Easley and Carpenter 1975; Yoshizawa et al. 1997; Moore and Whiting 1986; McGavin and Henry 1972). Hepatic parenchyma adjacent to the fistula was characterized by marked biliary hyperplasia, hepatocyte atrophy, and periportal fibrosis. Yoshisawa et al. (1997) suggest that these lesions occur as a consequence of abnormal blood supply (and concurrent lack of hepatotrophic factors) to the affected lobules. The dystrophic changes apparent in the walls of some vessels occur as a result of disruption to the vasa vasorum of the arterial wall and high-velocity, turbulent blood flow (Hosgood 1989). Changes are reported to occur initially within the tunica media as an accumulation of mucopolysaccharides, secondary to a reduction in oxidative processes, and are followed by focal areas of fibrinoid necrosis (Hosgood 1989). With time there is extracellular fibrosis and connective tissue substitution of muscle and elastic fibers (Hosgood 1989).

All other reported cases of intrahepatic arteriovenous fistula in dogs have presented with acute onset neurological and gastrointestinal signs, ascites, and secondary (acquired) portocaval shunts from two to eighteen months of age (Easley and Carpenter 1975; Koide et al. 2004; McGavin and Henry 1972; Moore and Whiting 1986; Schaeffer et al. 2001; Schermerhorn et al. 1997; Yoshizawa et al. 1997). Although no clinical or biochemical abnormalities were apparent in this case, we hypothesize that as the lesion was limited to one liver lobe (the right median), which accounts for less than 25% of the normal liver, and considering the marked hypertrophy of the left and caudate lobes, the animal was able to compensate. Arterioportal fistulae in humans may remain asymptomatic for long periods (Gomes and Bernatz 1970). As recruitment of local vessels into intrahepatic arteriovenous fistulae has been demonstrated to occur over time (Tappin and Rizzo 2007), it is thought likely that this lesion would have become more extensive and led to the development of clinical signs with time.

The clinical history of this animal, along with the histological appearance of this lesion, suggest that the fistula was arterioportal in nature. Anastamoses of the hepatic artery and hepatic vein have been reported in humans (Creech et al. 1965) and are associated with rapid, malignant cardiac failure (secondary to overload), jaundice, and hepatomegaly. With hepatic arterioportal fistulae, the interposition of the hepatic sinusoids may act to dampen blood flow, thereby attenuating portal pressure and buffering hemodynamic effects on the heart. This is only the second report of an intrahepatic vascular hamartoma (presumptively intrahepatic arteriovenous fistulae) in a laboratory beagle and is the first documentation of a case in an animal that was clinically normal.