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CASE I – 852731 (AFIP 2521964)
Signalment: 11-month-old, female fox terrier
History: This dog had a four-month history of abdominal enlargement with increasing dyspnea. Her appetite and bowel movements were normal. Clinically, she had hepatomegaly. Ophthalmoscopic examination revealed corneal arcus lipoides.
Gross Pathology: The liver was extremely large and yellow. The enlarged spleen was pulpy and bulged on cut surface. Mesenteric lymph nodes were enlarged and yellow.
Laboratory Results: Leukocytosis (25,100); serum enzymes: cholesterol (186 mg/dl), triglycerides (234 mg/dl), ALT (223 U/I), GLDH (35 U/L), and SAP (605 U/L).
Contributor’s Diagnoses and Comment: 1. Liver, interstice: Infiltration with lipid-laden macrophages, diffuse, severe.
2. Liver, hepatocytes: Fatty change.
3. Liver, macrophages and hepatocytes: Cholesterol crystal formation, diffuse, moderate, consistent with canine lipid storage disease (CLSD), fox terrier, canine.
4. Small intestine, mucosa and submucosa: Infiltration with lipid-laden macrophages, diffuse, severe, cholesterol crystal formation, moderate.
5. Spleen, lymph node, bone marrow: Infiltration with lipid-laden macrophages, diffuse, severe, and cholesterol crystal formation, moderate.
The principle histologic feature seen by light microscopy is the presence of lipid containing macrophages in the tissues. In liver, spleen, lymph nodes, and villi of the small intestine, the accumulation of many lipid-laden macrophages lead to a pronounced displacement of the parenchymal cells. In other tissues examined, single macrophages or small aggregates can be found within the interstitial connective tissue, except in the central nervous system (e.g., myocardium, trachea, esophagus, kidney, lung, choroid plexus, thyroid). In frozen sections under polarized light, a high percentage of fat-positive substance in the macrophages is lipid showing birefringence and a crystalline structure. There are also abundant fatty droplets lacking in birefringence.
The disease described in the fox terrier shares many clinical and morphological similarities with human diseases characterized by complete or incomplete deficiency of the lysosomal enzyme acid lipase (cholesterol ester hydrolase). In man, there are two distinct recognizable phenotypes of lipid storage disease. Wolman’s disease (WD) represents the severe, early-onset form; cholesterol ester storage disease (CESD) is the more benign late-onset type. Lipid storage takes place within lysosomes. The lysosomal enzyme deficiency in man is inherited as an autosomal recessive trait.
By genetic studies of both WD and CESD, a number of mutations have been found in the gene coding lysosomal acid lipase resulting in the two different phenotypes. In one case of WD, the insertion of a T residue after position 634 was combined with a T-to-C transition at nucleotide 638, resulting in a leucine-to-proline substitution at amino acid 179 that caused disruption of the alpha-helical structure. Reportedly, in CESD there are a number of different mutations that result in aberrant splicing, shifting of the reading frame, or single amino acid replacement.
In 1995, there were at least six cases of CLSD in fox terriers published in the German veterinary literature, and several other known examples of which both parents were half fox terriers. Pedigree analysis of four affected dogs revealed nothing to contradict the hypothesis of autosomal recessive inheritance in CLSD. Wolman’s disease in children is manifested by early onset in life, failure to gain weight, and increasing hepatosplenomegaly. The onset of clinical disease in the fox terrier at the age of approximately one-year-old is comparatively late. There might also exist varying degrees of cholesterol esterase deficiency in dogs, as there is in man. Similar to the rat serving as an animal model for CESD, CLSD may allow the dog to serve as an animal model for the study of WD.
AFIP Diagnoses: 1. Liver: Histiocytosis, diffuse, severe, with lipid vacuolation and cholesterol clefts, necrosis, and mineralization, fox terrier, canine.
2. Spleen; lymph node, mesenteric; small intestine: Histiocytosis, diffuse, severe, with lipid vacuolation and cholesterol clefts.
Conference Comment: The contributor has provided a concise review of CLSD. Corneal arcus lipoides, mentioned in the clinical history, is a circular arrangement of lipid deposits near the limbus. This lesion can be a valuable diagnostic feature of CLSD. Similar ring-shaped deposits of a cholesterol-triglyceride mixture near the corneal limbus are reported in human xanthomatosis, but not in Wolman’s disease. Arcus lipoides also can be induced in animals fed a high cholesterol diet.
Contributor: University of Munich, Department of Veterinary Pathology, Veterinärstrasse 13, 80539 Munich, Germany
References: 1. Anderson RA, Byrum RS, Coates PM, Sando GN: Mutations at the lysosomal acid cholesteryl ester hydrolase gene locus in Wolman’s disease. Proc Natl Acad Sci USA 91:2718-2722, 1994
2. Hänichen T, Brem G, Spiess C, Hermanns W: Canine lipid storage disease. Eur J Vet Pathol 1(2):37-44, 1995
3. Hänichen T, Breuer W, Hermanns W: Animal model of human disease: lipid storage disease. Comp Pathol Bull 28(2):3-6, 1996
4. Muntoni S, Wiebusch H, Funke H, Ros E, Seedorf U, Assmann G: Homozygosity for a splice junction mutation in exon 8 of the gene encoding lysosomal acid lipase in a Spanish kindred with cholesterol ester storage disease (CESD). Hum Genet 95:491-494, 1995
5. Yoshida H, Kuriyama M: Genetic lipid storage disease with lysosomal acid lipase deficiency in rats. Lab Anim Sci, 40(5):486-489, 1990
CASE II – 282M-721-8 or 9804282-7 (AFIP 2752924)
Signalment: Thirty-three-week-old, female Tg.AC mice, rodent
History: These two mice were part of the National Toxicology Program (NTP) subchronic toxicity/carcinogenicity study of Melphalan (282M-721-8) and DEHP (9804282-7).
Gross Pathology: Both mice had a hard mass in the mandible resulting in deformity of the lower jaw.
Laboratory Results: None.
Contributor’s Diagnosis and Comment: Tooth: Odontogenic tumor, Tg.AC mouse (Mus musculus).
Slides are submitted from two different mice with nearly identical features. The tissue is partially bordered by oral mucosa, and salivary glands are present in one set of slides. The submucosa contains an expansile, unencapsulated neoplastic mass that invades teeth and bone. The mass is composed of long anastomosing cords (two cells thick) and thin ribbons of cuboidal epithelial cells, closely associated with a loose, undifferentiated stroma of mesenchymal cells. The epithelial cells are arranged in a palisading fashion and in some areas they exhibit squamous differentiation. Small remnant islands of bone and portions of the tooth are present. Mitotic figures are more common in the stellate reticulum-like mesenchymal tissue.
A line of homozygous transgenic mice (Tg.AC) carrying a v-Ha-ras gene fused to the promoter of the zeta globin gene is currently being investigated for its potential usefulness as an alternative to long-term carcinogenicity testing. These transgenic mice develop a variety of mesenchymal and epithelial neoplasms including odontogenic tumors.
The submitted slides are from two different mice with nearly identical features (due to the limited tissue available from a single mouse). Although the submitted slides are from mice that had received the test chemical, the most common spontaneous tumors in control Tg.AC mice are odontogenic tumors. An increased incidence of these tumors has not been reported in any of the toxicity/carcinogenicity studies utilizing this strain.
The 1-year incidence of odontogenic tumor formation in Tg.AC mice is approximately 35%. Tumors form more often in the mandible than maxilla. These tumors generally exhibit one of three different morphologic patterns: 1) primarily mesenchymal cells in a dense fibrous-like matrix; 2) loose stroma surrounded by anastomosing cords of epithelial cells that exhibited squamous differentiation; or 3) odontomas forming mineralized tooth structures by well-differentiated odontoblasts and ameloblasts. The mineralized dentine and enamel in the odontoma is morphologically similar to those of normal murine teeth. Some tumors have areas with all three of these characteristics. Since a spectrum of morphologic patterns is evident in these tumors, “odontogenic tumor” is the preferred NTP consensus diagnosis for all dental tumors in this strain of mice. The Tg.AC mouse provides an excellent model for the study of odontogenic tumors and tooth development.
AFIP Diagnosis: Mandible, oral mucosa and subepithelial connective tissue, (per contributor): Odontogenic tumor, Tg.AC mouse (Mus musculus), rodent.
Conference Comment: The complex process of tooth formation involves elaborate signal-transduction mechanisms that stimulate both mesenchymal and ectodermal cell line proliferations. Many of the genes involved in odontogenesis have not been identified, but many of the human odontogenic tumors show over-expression of the ras proto-oncogene product, p21. Because Tg.AC mice carry the v-Ha-ras oncogene, they are genetically initiated for the development of certain tumors. Expression of the ras transgene can be triggered by local tissue injury, such as might occur in the oral mucosa with continuously gnawing animals. It is thought the ras transgene is activated and stimulates the formation of odontogenic tumors. Although the processes of cellular signalling and induction remain complicated and poorly understood, the Tg.AC mouse should serve as an excellent model for studying the role of the v-Ha-ras oncogene in tooth formation.
Contributor: Experimental Pathology Laboratories, Inc., P.O. Box 12766, Research Triangle Park, NC 27709
References: 1. Wright JT, Hansen L, Mahler J, Szczesniak
C, Spalding JW: Odontogenic tumours in the v-Ha-ras (Tg.AC) transgenic
mouse. Arch Oral Biol 40(7):631-638, 1995
2. Mahler JF, Flagler ND, Malarkey DE, Mann PC, Haseman JK, Eastin
W: Spontaneous and chemically induced proliferative lesions in
Tg.AC transgenic and p53-heterozygous mice. Toxicol Pathol
26(4):501-511, 1998
CASE III – WRAIR 00-58 (AFIP 2741011)
Signalment: 12-week-old, male Sprague-Dawley rat, Rattus norvegicus
History: This animal was surgically implanted with an intracerebroventricular (ICV) cannula and cortical screws five days prior to the experiment. On the day of the experiment, the animal received an ICV injection of 0.25 m g kainic acid (KA). Within fifteen minutes, the animal began exhibiting seizures, both behaviorally and on EEG; by one hour the animal was in status epilepticus. Twenty-four hours after KA injection the animal was euthanized and perfused with formalin. Tissues were submitted for histologic evaluation.
Gross Pathology: No gross lesions were apparent.
Laboratory Results: No laboratory tests were performed.
Contributor’s Diagnosis and Comment: Brain, hippocampus, thalamic nuclei, amygdala nuclei, pyriform lobe: Neuronal necrosis, multifocal, extensive, Sprague-Dawley rat, rodent.
Glutamate is a naturally occurring mammalian excitatory neurotransmitter. Ionotropic glutamate receptors include kainate, a -amino-3-hydroxy-5-methyl-isoxazole propionic acid (AMPA), and N-methyl-D-aspartate (NMDA). Activation of presynaptic KA receptors leads to the release of endogenous glutamate that, upon binding to post-synaptic receptors, causes membrane depolarization, excitotoxic action, and neuronal injury. There is evidence that KA can also block the re-uptake of glutamate, prolonging the excitatory stimulus. The cause of the neuronal injury that results from KA administration is multifactorial. The constant depolarization affects cytoplasmic membrane permeability and leads to overwhelming of energy-dependent ion pumps. There is convincing evidence that lipid peroxidation from free radical production is a significant indirect mechanism of damage. Secondary lesions may occur from seizure-induced hypoxia, hypoglycemia and edema.
The distribution of lesions following both intraventricular or intravenous administration of KA correlates with the distribution of high affinity KA receptors in the brain; lesions are most consistently found in the hippocampus (CA1 and CA3), amygdala, septum, entorhinal cortex, medial thalamus, pyriform cortex and midline hypothalamus. Excitatory stimulation of certain neuronal pathways likely leads to seizures and neuronal damage in distant neurons.
This animal was part of a study to evaluate the effects of several promising neuro-protective drugs on the behavioral, EEG and pathologic changes associated with kainic acid administration.
In the laboratory of the Department of Diagnostic Pathology at the Walter Reed Army Institute of Research, sections were stained with Fluoro-Jade for additional visualization of neuronal degeneration. Fluoro-Jade has increased technical simplicity and reliability when compared with suppressed silver staining.
Note: A photomicrograph of the thalamic nucleus area stained with Fluoro-jade was shown during conference, demonstrating fluorescence of degenerating neurons.
AFIP Diagnosis: Cerebral cortex, occipital and pyriform lobes; hippocampus: Neuronal necrosis, multifocal, Sprague-Dawley rat, rodent.
Conference Comment: Kainic acid is a potent natural neurotoxin originally isolated from the seaweed Digenea simplex. Structurally related to domoic acid, a marine biotoxin that has been involved in human and marine wildlife mortality, KA can cross the blood-brain barrier in a similar manner to cause neuronal death. Systemic administration of kainate causes a pattern of cerebral damage that approximates that seen following repeated temporal lobe seizures and in Alzheimer’s disease. Its ability to induce a syndrome characterized by an acute limbic status epilepticus and subsequent neuronal brain damage makes it a subject of considerable scientific interest for epilepsy research. The consistent secondary lesions, most notable in the hippocampus, pyriform cortex and the amygdala, led to the development of an animal model for the neuropathology associated with temporal lobe epilepsy.
Contributor: Walter Reed Army Institute of Research, Division of Pathology, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500
References: 1. MacGregor DG, Higgins MJ, Jones PA, Maxwell WL, Watson MW, Graham DI, Stone TW: Ascorbate attenuates the systemic kainate-induced neurotoxicity in the rat hippocampus. Brain Res 727:133-144, 1996
2. Miller RJ: The revenge of the kainate receptor. Trends Neurosci 14:477-486, 1991
3. Sandhya TL, Ong WY, Horrocks LA, Farooqui AA: A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase–2 in the hippocampus after kainate lesions. Brain Res 788:223-231, 1998
4. Schmued LC, Hopkins KJ: Fluoro-jade: Novel fluorochromes for detecting toxicant-induced neuronal degeneration. Tox Pathol 28:91-99, 2000
5. Scholin CA, Gulland F, Doucette GJ, Benson S, Busman M, Chavez P, Cordaro J, DeLong R, De Vogelaere A, Harvey J, Haulena M, Lefebvre K, Lipscomb T, Loscutoff S, Lowenstine LJ, Marin R, Miller PE, McLellan WA, Moeller PDR, Powell CL, Rowles T, Silvagni P, Silver M, Spraker T, Trainer V, Van Dolah FM: Mortality of sea lions along the central California coast linked to a toxic diatom bloom. Nature 403:80-84, 2000
6. Sperk G: Kainic acid seizures in the rat. Prog Neurobiol 42:1-32, 1994
CASE IV – 159642/58-46 (AFIP 2749921)
Signalment: One-year-old, male Min mouse
History: Moribund sacrifice.
Gross Pathology: Multiple nodular masses were present throughout the small intestines.
Laboratory Results: Not Applicable.
Contributor’s Diagnosis and Comment: Small intestine: Adenoma, multifocal.
The Swiss roll of small intestine has multiple adenomas that are polyploid, papillary or sessile.
Min (multiple intestinal neoplasia) is an ethylnitrosourea (ENU)-induced mutation in the murine Apc (adenomatous polyposis coli) gene. Mice carrying Min provide a model system for studying familial adenomatous polyposis and, in particular, for identifying genes that can modify the phenotype caused by the Apc mutation. Min encodes a nonsense mutation at codon 850, the human homolog of the Apc gene, resulting in premature truncation of the polypeptide. Analogous to humans with germ line mutations in Apc, Min/+ mice are predisposed to the development of intestinal adenomas along the duodenal-to-colonic axis. The number of adenomas is influenced by modifier loci carried by different inbred strains of mice. Min/+ C57BL/6J (B6) mice develop more than 50 adenomas throughout the intestinal tract. In contrast, Min/+ AKR/J (AKR) mice carry alleles that correlate with reduction in the number of neoplasms and have an average of 4 to 8 neoplasms in the intestinal tract. Reduction in the number of neoplasms has been attributed to a modifier locus, Mom1 (modifier of Min1), in the distal region of mouse chromosome 4. Mom1 is a semi-dominant modifier of both size and multiplicity of the neoplasms.
Min mice have been maintained by crossing Min/+ males with B6 females. B6-Min+ mice develop anemia by 60 days of age and seldom live beyond 120 days. The average total number of neoplasms in the duodenal-to-colonic axis is 29 ± 10. The small intestine is the predominant site for tumors in mice whereas, in humans, the colon is the primary site of involvement. Min mice also develop lesions in other tissues, including mammary and desmoid neoplasms and epidermoid cysts. Min/+ female mice are predisposed to mammary neoplasms.
AFIP Diagnosis: Small intestine: Adenomas, multiple, Min mouse, rodent.
Conference Comment: The histologic differentiation of intestinal mucosal hyperplasia, adenoma and carcinoma was discussed. Hyperplastic polyps are composed of well-formed glands and crypts, most of which show differentiation into mature goblet or absorptive cells. Cytologic atypia and lack of differentiation into specialized cell types characterize adenomas. The additional features of anaplasia and invasion typify carcinomas. Lesions with anaplasia that have not invaded through the basement membrane are carcinoma in situ.
Spontaneous colorectal cancer is one of the leading causes of cancer death in industrialized countries. It is primarily a disease of the elderly. Human familial adenomatous polyposis (FAP) is an inherited autosomal dominant disease that is probably best defined in its relationship to the development of colorectal cancer, and is due to a defect in one copy of the tumor suppressor gene adenomatous polyposis coli (APC). A somatic mutation in the remaining copy of the APC gene (wild-type allele) is a critical event in the carcinogenic process that results in the loss of heterozygosity (LOH) and initiates tumor growth. This early mutation appears necessary for development of most adenomas and carcinomas.
APC protein normally increases in epithelial cells as they migrate from the colonic crypts to the mucosal surface, and appears to play a role in cell migration control, apoptosis, and possibly proliferation. An accumulation of mutations, with loss of tumor suppressor genes, such as p53, and activation of oncogenes, such as K-ras, are also required for carcinogenesis. APC mutations can result in accumulation of b -catenin in intestinal epithelial cell cytoplasm. Overexpression of b -catenin protein, whether due to APC mutations or mutations in the b -catenin gene itself, can induce intestinal epithelial cell proliferation and inhibit migration of these cells out of crypts, which has been linked to early development of intestinal adenomas. Other genes appear to be responsible for invasion and metastasis. Loss of E-cadherin, a key adhesion molecule of epithelial cells that contributes to formation of the zonula adherens and is important in regulation of normal epithelial cell morphology and migration, has been shown to contribute to malignant progression in a variety of organs.
The Min mouse was the first animal model for FAP. Additional strains of mice, such as those described by the contributor, have been developed through gene-targeting techniques that have specific mutations in the murine Apc gene, the mouse homolog of the human APC gene. So far, no animal model replicates human colorectal cancer in its entirety, but available models approximate many of the characteristics of colonic carcinogenesis and metastasis.
Contributor: National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709
References: 1. McEntee MF, Brenneman KA: Dysregulation of b -catenin is common in canine sporadic colorectal tumors. Vet Pathol 36(3):228-236, 1999
2. Moser AR, Luongo C, Gould KA, McNeley MK, Shoemaker AR, Dove WF: ApcMin: a mouse model for intestinal and mammary tumorigenesis. Eur J Cancer 31(A):1061-1064, 1995
3. Moser AR, Mattes EM, Dove WF, Lindstrom MJ, Haag JD, Gould MN: ApcMin, a mutation in the murine Apc gene, predisposes to mammary carcinomas and focal alveolar hyperplasias. Proc Natl Acad Sci 90:8977-8981, 1993
4. Moser AR, Pitot HC, Dove WF: A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247:322-324, 1990
5. Thompson MB: The Min mouse: a genetic model for intestinal
carcinogenesis. Toxicol Pathol 25:329-332, 1997
*Sponsored by the American Veterinary Medical Association, the American College of Veterinary Pathologists and the C. L. Davis Foundation.
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