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Hepatic Hyaline Globules in an Eclectus Parrot (Eclectus roratus)
Kelli L. Boyd and Kenneth S. Latimer
Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA (USA) 30602-7388
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Abstract: Hepatic hyaline globules, similar to those observed in some human livers, were observed in liver tissue from an Eclectus parrot. The inclusions were PAS positive and diastase resistant. These cytoplasmic inclusions failed to stain by acid-fast or Congo red techniques. Ultrastructurally, the hepatic globules were composed of granular, amorphous material with small peripheral striations that extended into the cytoplasm.
Key Words: Liver, Bird, Parrot, Eclectus parrot, Eclectus roratus, Hepatic inclusions, Hyaline globules
Introduction
Cytoplasmic "inclusions" of noninfectious origin within hepatocytes of mammals most commonly include lipid, bile pigment, or hemosiderin.1 Less frequently, copper-rich granules may be observed. In birds, eosinophilic to basophilic inclusions may be observed in Kupffer cells following erythrophagocytosis. In addition, protoporphyrin accumulation has been observed within hepatocytes of a 6-week-old chicken.2,3 The present case report describes unique cytoplasmic inclusions in the liver of a psittaciform bird with chronic liver disease.
Case Report
A male Eclectus parrot (Eclectus roratus) was euthanized after prolonged treatment for liver disease. Necropsy was performed by the referring veterinarian. Samples of major organs and tissue were collected and preserved in 10% neutral buffered formalin and mailed to The University of Georgia College of Veterinary Medicine for histopathologic examination. These specimens were processed routinely, sectioned at 3µm, stained by hematoxylin and eosin, and examined by light microscopy.
Major histologic lesions were confined to the liver. Hepatic cords were atrophied and disorganized. Binucleate hepatocytes and megalocytes were common. Birefringent, round, amphophilic structures, ranging from 10-25µm in diameter, were present within hepatocyte cytoplasm, sinusoids and multinucleated giant cells (Figs. 1a & 1b). Numerous multinucleated giant cells were observed within sinusoids.
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| Fig 1a. Liver, Eclectus parrot, H&E stain, 400x. Section of liver with round, amphophilic inclusions within hepatocytes. | Fig 1b. Liver, Eclectus parrot, H&E stain, 400x. A multinucleated giant cell is present in the parenchyma surrounding an amphophilic inclusion body. |
Special stains were performed to further characterize the birefringent inclusions. The inclusions stained strongly positive with periodic-acid Schiff (PAS) technique (Figs. 2a & 2b). Digestion with diastase was performed, but the inclusions retained strong staining by the PAS method. The inclusions did not stain when an acid-fast or modified acid-fast stain was applied to additional sections of formalin-fixed liver. In addition, the inclusions failed to stain with Congo red dye.
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| Fig. 2a. Liver, Eclectus parrot, PAS technique with light green counterstain, 400x. Hyaline globules within hepatocytes are magenta. | Fig. 2b. Liver, Eclectus parrot, diastase digestion with PAS technique and light green counterstain, 400x. Hyaline globules within hepatocytes are resistant to diastase digestion and retain magenta color. |
Additional portions of the liver were cut into 1mm cubes, postfixed in osmium tetroxide, dehydrated in graded alcohols, and embedded in Spurr resin. Ultra thin tissue sections were placed on high-transmission nickel grids, stained with lead citrate and uranyl acetate, and examined by transmission electron microscopy. The inclusions usually were observed within hepatocyte cytoplasm. The inclusions consisted of granular, amorphous material and lacked internal structure (Fig. 3a). In addition, the inclusions were not bordered by a limiting membrane, but had small radial projections into the cytoplasm (Fig. 3b).
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| Fig. 3a. Liver, Eclectus parrot, electron micrograph. Cytoplasmic hyaline inclusion within a hepatocyte. Notice the granular, amorphous structure. | Fig. 3b. Liver, Eclectus parrot, electron micrograph. Radial projections extend from the periphery of the hyaline inclusion into the hepatocyte cytoplasm. A limiting membrane is not observed. |
Discussion
Inclusions of this nature have not been described previously in the liver of psittaciform birds, but have been presented as intracytoplasmic inclusions of undetermined significance in the liver of a turkey with cardiohepatic syndrome.2 The inclusions described in this manuscript are very similar to hyaline globules that have been reported in the livers of laboratory animals and human beings.4-6 The formation of these globules has been related to increased hepatic pressure. Initially, when pressure is increased, vacuolation of hepatocytes occurs. These vacuoles are commonly observed in humans with congestive right-sided heart failure. Hyaline globules may be observed in 10-15 per cent of human livers at autopsy. They are distinguished by their location in pericentral hepatocytes with pericentral congestion.
The progression from vacuole to hyaline globule has been reproduced experimentally in rats.4 The caudal vena cava was occluded to increase the portal pressure from a normal pressure of 20mm H2O to an increased pressure of 90-100mm H2O for fifteen minutes. The livers were biopsied immediately then several more times over a twenty-four hour period. Researchers discovered that vacuoles initially were present in the hepatocytes. The vacuoles either resolved relatively soon after the pressure was relieved or metamorphosed into hyaline globules. These hyaline globules stained positively with the PAS technique. Electron microscopic examination of the globules showed a structure within hepatocytes containing finely granular or flocculent material with a single limiting membrane. With the decrease in hepatic pressure, both the vacuoles and the globules decreased in number over a twenty-four hour period.
Vacuoles are formed by an invagination of the cell membrane following an acute increase in sinusoidal pressure. A decrease in pressure will result in the discharge of most of these vacuoles. The remaining vacuoles are thought to condense their contents, decrease in size, and merge with the cytoplasm to result in hyaline globules. Many investigators suggest that these globules are formed from protein in blood plasma.
The exact pathogenesis of hyaline globule formation is not clear; however, several hypotheses have been suggested. Hyaline globules may form as a result of damage to hepatocyte cell membrane, permeation of blood plasma protein into the hepatocyte due to increased sinusoidal pressure, or acceleration of phagocytosis by hepatocytes. It has been shown that increased portal pressure will result in the formation of vacuoles that metamorphose into hyaline globules.
In the case of this bird, the histologic changes in the liver suggest that there had been previous damage to hepatocytes. The electron microscopic findings differ slightly from previous reports in that a single limiting membrane was not observed. However, formalin fixation artifact may have precluded this observation. Otherwise, the internal structure of the globules is identical to that described in laboratory animals and human beings. Electron and light microscopic features of these globules, accompanied by strongly positive, diastase resistant PAS staining, lead us to believe that these globules are similar to those previously reported in humans and laboratory animals.
References
1. Kelly WR: The liver and biliary system. In: Jubb KVF, Kennedy PC, Palmer N (eds): Pathology of Domestic Animals, 4th ed, vol 2. Academic Press, Inc., San Diego, 1993, pp. 319-406.
2. Randall CJ, Reece RL: Color Atlas of Avian Histopathology. Mosby-Wolfe, London, 1996, pp. 75-100.
3. Hafner S, Bounous D, Kelley LC, Campagnoli R: Hepatic protoporphyrin accumulation in a broiler chicken. Avian Dis 37:900-904, 1993.
4. Shibayama Y, Yahara M, Nakata K: The fate of vacuolation in liver cells with special reference to hyaline globules. J Pathol 162:335-340, 1990.
5. Shibayama Y, Hashimoto K, Nakata K: Role of sinusoidal pressure in the development of vacuolation in liver cells. J Pathol 162:77-81, 1990.
6. An T, Ghatak N, Kastner R, Kay S, Lee HM: Hyaline globules and intracellular lumina in a hepatocellular carcinoma. Am J Clin Pathol 79:392-396, 1983.
