Pathology in Practice

Anna Gardini Neurology and Neurosurgery Department, Dick White Referrals, Six Mile Bottom, CB8 0UH, England.

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Simone de Brot Pathology Department, School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, LE12 5RD, England.

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Giunio Bruto Cherubini Neurology and Neurosurgery Department, Dick White Referrals, Six Mile Bottom, CB8 0UH, England.

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History

A 9-month-old 5-kg (11-lb) sexually intact female Jack Russell Terrier was evaluated because of lethargy and progressive 4-limb ataxia since birth. According to the owner, the other littermates did not have any abnormalities.

Clinical and Gross Findings

On evaluation, the dog had minimal responsiveness to the owner's verbal commands and had signs of extreme depression. On neurologic examination, absence of menace response, 4-limb ataxia, and severely diminished proprioception were recorded. Standard blood analyses and bile acids stimulation testing did not reveal notable abnormalities. Magnetic resonance imaging of the brain revealed asymmetrical dilation of the lateral ventricles (more pronounced in the left lateral ventricle) without any evidence of obstructive lesions and widened cerebral sulci. Analysis of a cisternal CSF sample revealed mononuclear pleocytosis (32 nucleated cells/μL; reference range, 0 to 6 nucleated cells/μL); protein concentration was within reference limits. The cells were a mix of large granular cells, monocytes, and lymphocytes. The granular cells had a medium-sized nucleus and abundant cytoplasm, which contained a variable number of purple granules when stained with Wright-Giemsa stain. In some cells, the granules were numerous and fine; in other cells, they were dark staining and quite large (Figure 1). These cells were identified as atypical monocytes. Examination of a blood smear preparation did not reveal similar cells in the circulation. Treatment with dexamethasone (0.2 mg/kg [0.09 mg/lb], PO, q 24 h) was attempted. One month after evaluation, the dog was euthanized by IV injection of pentobarbital because of worsening of the clinical signs, and a full necropsy was performed. Upon examination of the tissues, the only gross abnormality observed in the brain was a moderate asymmetrical bilateral dilation of the lateral ventricles. No other relevant gross abnormalities were present in any other examined tissues.

Figure 1—
Figure 1—

Photomicrographs of a cisternal CSF sample collected from a 9-month-old Jack Russell Terrier that was evaluated because of lethargy and progressive 4-limb ataxia since birth. The monocytes contain intracytoplasmic granular deposits. A—In the cell on the right, the granules are numerous and fine, whereas in the cell on the left, they are dark staining and quite large. Wright-Giemsa stain; bar = 10 μm. B—Both monocytes have dark staining granules, but of markedly different size. Wright-Giemsa stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 254, 3; 10.2460/javma.254.3.359

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→

Histopathologic Findings

Sections of brain, cervical portion of the spinal cord, sciatic nerve, femoral and tibial cranial muscles, liver, spleen, mesenteric lymph node, and kidneys were fixed in neutral-buffered 10% formalin and submitted for histologic evaluation. The brain was the only organ with relevant histologic changes. Within the forebrain, midbrain, hindbrain, and, to a minimal extent, the cervical portion of the spinal cord, low to moderate numbers of neurons with mild cytoplasmic vacuolation and mild intracytoplasmic finely granular pigment deposits were observed. The greatest number of affected neurons was seen bilaterally in the hippocampus within the pyramidal cell layer and in the cerebellum within the Purkinje cell layer. With H&E stain, the intraneuronal pigment had deep eosinophilic staining, occasionally with a slight brown tinge; with periodic acid-Schiff stain, the intraneuronal pigment appeared magenta (Figure 2). When unstained or H&E-stained tissue sections were examined with fluorescence microscopy, the pigment had green autofluorescence (Figure 3). In addition, mild multifocal gliosis and neuronal atrophy were present in sections of the brain and cervical portion of the spinal cord. For transmission electron microscopy, formalin-fixed hippocampal and cerebellar tissue samples were immersed in 2.5% glutaraldehyde, washed in buffer, and then postfixed in 1% osmium tetroxide with 1.5% potassium ferricyanide (wt/vol). After being washed in deionized water, the samples were further treated with 1% uranyl acetate (wt/vol), then dehydrated through an ethanol series before they were then embedded in resin. Ultrathin (70-nm) sections were then contrasted with lead citrate solution and scanned thoroughly. Numerous hippocampal pyramidal cells and Purkinje cells were markedly distended by intracytoplasmic osmiophilic inclusions. In all samples examined, the inclusions were of 1 type, which was characterized by lamellar stacks of membranes, compatible with fingerprint inclusions (Figure 4).

Figure 2—
Figure 2—

Photomicrographs of a section of the cerebellum from the dog in Figure 1. The Purkinje cells contain intracytoplasmic, finely granular, deeply eosinophilic deposits. H&E stain; bar = 20 μm. Insert—After periodic acid-Schiff staining, the intraneuronal deposits are more evident and magenta in color. Notice the shrunken Purkinje cell in the center. Periodic acid-Schiff stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 254, 3; 10.2460/javma.254.3.359

Figure 3—
Figure 3—

Fluorescence photomicrograph of a section of the cerebellum from the dog in Figure 1. Purkinje cells contain intracytoplasmic, green autofluorescent, finely granular deposits. Unstained tissue section; bar = 100 μm.

Citation: Journal of the American Veterinary Medical Association 254, 3; 10.2460/javma.254.3.359

Figure 4—
Figure 4—

Transmission electron photomicrograph of a section of the cerebellum from the dog in Figure 1. Purkinje cells contain numerous intracytoplasmic osmiophilic fingerprint inclusions. Osmium tetroxide and uranyl acetate staining; bar = 2 μm. Insert—At higher magnification, the closely packed lamellar inclusions are visible in greater detail. Osmium tetroxide and uranyl acetate staining; bar = 500 nm.

Citation: Journal of the American Veterinary Medical Association 254, 3; 10.2460/javma.254.3.359

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: intracytoplasmic autofluorescent neuronal inclusions mainly affecting the pyramidal cell layer of the hippocampus and the Purkinje cell layer of the cerebellum.

Case summary: neuronal ceroid lipofuscinosis (NCL) in a Jack Russell Terrier.

Comments

Neuronal ceroid lipofuscinoses are recessive inherited lysosomal storage diseases characterized by the accumulation of intracellular autofluorescent lipopigments within the CNS and peripheral tissues.1 Neuronal ceroid lipofuscinoses in humans and in a variety of domestic animals including dogs have been described.1–4 To our knowledge, NCL in Jack Russell Terriers has never been reported.

In humans and other animals, the major constituent of the intracytoplasmic inclusions is a lipidbinding protein that is a component (subunit C) of mitochondrial ATP synthase.5 Humans with the infantile form of NCL and 1 breed of dog—the Miniature Schnauzer—store a different protein, which has been identified as a sphingolipid activator protein (saposin).5–7

In dogs, clinical signs associated with NCLs include progressive cognitive decline, behavioral changes, ataxia, vision deficits, seizures, and variable degrees of motor and sensory dysfunction.1 The disease is variable in its rate of progression, irreversible, and fatal. Canine NCLs are clinically classified as prepubertal protracted disease, early adult-acute course disease, and adult-onset disease.1

Antemortem diagnosis of NCLs is challenging. However, DNA tests are available for several breeds (Border Collies,8 English Setters,9 American Bulldogs,10 Dachshunds,11 American Staffordshire Terriers,12 and Tibetan Terriers13) in which the spontaneous causative mutation has been identified.

Results of hematologic, biochemical, and CSF analyses are usually within reference intervals in dogs with NCL.14–16 Interestingly, in the dog of the present report, CSF mononuclear pleocytosis was identified; present were atypical monocytes that contained granular material in their cytoplasm, which had a similar appearance to the intraneuronal pigment present throughout the CNS. Therefore, these intracytoplasmic inclusions may have represented storage material in the CSF monocytes. On examination of a blood smear, no abnormalities were detected, which was consistent with what has been previously reported in veterinary medical literature. However, it is interesting to note that in childhood forms of NCL, circulating lymphocytes commonly contain lysosomal storage material with variable features.2

In agreement with descriptions14–20 of NCLs in dogs, we observed cerebral atrophy and widespread accumulation of autofluorescent material in neurons throughout the brain, especially in the hippocampus and cerebellum, of the dog of the present report. Ultrastructurally, the neuronal inclusions were of only 1 type, the fingerprint type. To our knowledge, this is the first case of canine NCL that had exclusively fingerprint-type inclusions. Fingerprint profiles in Border Collies, Dalmatians, and English Setters have been described but were always in combination with other types of profiles (curvilinear, lamellar, membranous, and crystalloid).3 In humans, there is a morphological correlation of the inclusions with NCL subtypes; in particular, fingerprint inclusions are associated mainly with juvenile NCL.4 Given that there are no reports of fingerprint profiles as the only lipopigment type in dogs with NCLs, it is possible that Jack Russell Terriers are affected by a specific form of NCL.

The case described in the present report has highlighted the need to include NCL as a differential diagnosis for young Jack Russell Terriers with progressive neurologic signs. The clinical, pathological, and ultrastructural characteristics of NCL in the Jack Russell Terrier of the present report, compared with those previously reported for affected dogs, have suggested that further investigation of NCL in this breed should be undertaken.

Acknowledgments

The authors thank Elizabeth Villiers from Dick White Referrals for performing CSF sample analysis and Natalie Allcock from Leicester University for performing the transmission electron microscopy.

References

  • 1. Jolly RD, Palmer DN, Studdert VP, et al. Canine ceroid-lipofuscinoses: a review and classification. J Small Anim Pract 1994;35:299306.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Anderson GW, Goebel HH, Simonati A. Human pathology in NCL. Biochim Biophys Acta 2013;1832:18071826.

  • 3. Palmer DN, Tammen I, Drogemuller C, et al. Chapter 18: large animal models. In: Mole SE, Williams RE, Goebel HH, eds. The neuronal ceroid lipofuscinosis (Batten diseases). 2nd ed. Oxford, England: Oxford University Press, 2011;284320.

    • Search Google Scholar
    • Export Citation
  • 4. Jadav RH, Sinha S, Yasha TC, et al. Clinical, electrophysiological, imaging, and ultrastructural description in 68 patients with neuronal ceroid lipofuscinoses and its subtypes. Pediatr Neurol 2014;50:8595.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Palmer DN, Jolly RD, van Mil HC, et al. Different patterns of hydrophobic protein storage in different forms of neuronal ceroid-lipofuscinosis (NCL, Batten disease). Neuropediatrics 1997;28:4548.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Tyynelä J, Suopanki J, Baumann M, et al. Sphingolipid activator proteins (SAPs) in neuronal ceroid lipofuscinoses (NCL). Neuropediatrics 1997;28:4952.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Palmer DN, Tyynelä J, van Mil HC, et al. Accumulation of sphingolipid activator proteins (SAPs) A and D in granular osmiophilic deposits in Miniature Schnauzer dogs with ceroid-lipofuscinosis. J Inherit Metab Dis 1997;20:7484.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Melville SA, Wilson CL, Chiang CS, et al. A mutation in canine CLN5 causes neuronal ceroid lipofuscinosis in Border Collie dogs. Genomics 2005;86:287294.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Katz ML, Khan S, Awano T, et al. A mutation in the CLN8 gene in English Setter dogs with neuronal ceroid-lipofuscinosis. Biochem Biophys Res Commun 2005;327:541547.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Awano T, Katz ML, O'Brien DP, et al. A mutation in the cathepsin D gene (CTSD) in American Bulldogs with neuronal ceroid lipofuscinosis. Mol Genet Metab 2006;87:341348.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Awano T, Katz ML, O'Brien DP, et al. A frame shift mutation in canine TPP1 (the ortholog of human CLN2) in a juvenile Dachshund with neuronal ceroid lipofuscinosis. Mol Genet Metab 2006;89:254260.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Abitbol M, Thibaud JL, Olby NJ, et al. A canine Arylsulfatase G (ARSG) mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis. Proc Natl Acad Sci U S A 2010;107:1477514780.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Farias FH, Zeng R, Johnson GS, et al. A truncating mutation in ATP13A2 is responsible for adult-onset neuronal ceroid lipofuscinosis in Tibetan Terriers. Neurobiol Dis 2011;42:468474.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Franks JN, Dewey CW, Walker MA, et al. Computed tomographic findings of ceroid lipofuscinosis in a dog. J Am Anim Hosp Assoc 1999;35:430435.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Rossmeisl JH, Duncan R, Fox J, et al. Neuronal ceroid-lipofuscinosis in a Labrador Retriever. J Vet Diagn Invest 2003;15:457460.

  • 16. Evans J, Katz ML, Levesque D, et al. A variant form of neuronal ceroid lipofuscinosis in American Bulldogs. J Vet Intern Med 2005;19:4451.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Kuwamura M, Hattori R, Yamate J, et al. Neuronal ceroid-lipofuscinosis and hydrocephalus in a Chihuahua. J Small Anim Pract 2003;44:227230.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Koie H, Shibuya H, Sato T, et al. Magnetic resonance imaging of neuronal ceroid lipofuscinosis in a Border Collie. J Vet Med Sci 2004;66:14531456.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. O'Brien DP, Katz ML. Neuronal ceroid lipofuscinosis in 3 Australian Shepherd littermates. J Vet Intern Med 2008;22:472475.

  • 20. Asakawa MG, MacKillop E, Olby NJ, et al. Imaging diagnosis-neuronal ceroid lipofuscinosis with a chronic subdural hematoma. Vet Radiol Ultrasound 2010;51:155158.

    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Photomicrographs of a cisternal CSF sample collected from a 9-month-old Jack Russell Terrier that was evaluated because of lethargy and progressive 4-limb ataxia since birth. The monocytes contain intracytoplasmic granular deposits. A—In the cell on the right, the granules are numerous and fine, whereas in the cell on the left, they are dark staining and quite large. Wright-Giemsa stain; bar = 10 μm. B—Both monocytes have dark staining granules, but of markedly different size. Wright-Giemsa stain; bar = 10 μm.

  • Figure 2—

    Photomicrographs of a section of the cerebellum from the dog in Figure 1. The Purkinje cells contain intracytoplasmic, finely granular, deeply eosinophilic deposits. H&E stain; bar = 20 μm. Insert—After periodic acid-Schiff staining, the intraneuronal deposits are more evident and magenta in color. Notice the shrunken Purkinje cell in the center. Periodic acid-Schiff stain; bar = 20 μm.

  • Figure 3—

    Fluorescence photomicrograph of a section of the cerebellum from the dog in Figure 1. Purkinje cells contain intracytoplasmic, green autofluorescent, finely granular deposits. Unstained tissue section; bar = 100 μm.

  • Figure 4—

    Transmission electron photomicrograph of a section of the cerebellum from the dog in Figure 1. Purkinje cells contain numerous intracytoplasmic osmiophilic fingerprint inclusions. Osmium tetroxide and uranyl acetate staining; bar = 2 μm. Insert—At higher magnification, the closely packed lamellar inclusions are visible in greater detail. Osmium tetroxide and uranyl acetate staining; bar = 500 nm.

  • 1. Jolly RD, Palmer DN, Studdert VP, et al. Canine ceroid-lipofuscinoses: a review and classification. J Small Anim Pract 1994;35:299306.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Anderson GW, Goebel HH, Simonati A. Human pathology in NCL. Biochim Biophys Acta 2013;1832:18071826.

  • 3. Palmer DN, Tammen I, Drogemuller C, et al. Chapter 18: large animal models. In: Mole SE, Williams RE, Goebel HH, eds. The neuronal ceroid lipofuscinosis (Batten diseases). 2nd ed. Oxford, England: Oxford University Press, 2011;284320.

    • Search Google Scholar
    • Export Citation
  • 4. Jadav RH, Sinha S, Yasha TC, et al. Clinical, electrophysiological, imaging, and ultrastructural description in 68 patients with neuronal ceroid lipofuscinoses and its subtypes. Pediatr Neurol 2014;50:8595.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Palmer DN, Jolly RD, van Mil HC, et al. Different patterns of hydrophobic protein storage in different forms of neuronal ceroid-lipofuscinosis (NCL, Batten disease). Neuropediatrics 1997;28:4548.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Tyynelä J, Suopanki J, Baumann M, et al. Sphingolipid activator proteins (SAPs) in neuronal ceroid lipofuscinoses (NCL). Neuropediatrics 1997;28:4952.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Palmer DN, Tyynelä J, van Mil HC, et al. Accumulation of sphingolipid activator proteins (SAPs) A and D in granular osmiophilic deposits in Miniature Schnauzer dogs with ceroid-lipofuscinosis. J Inherit Metab Dis 1997;20:7484.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Melville SA, Wilson CL, Chiang CS, et al. A mutation in canine CLN5 causes neuronal ceroid lipofuscinosis in Border Collie dogs. Genomics 2005;86:287294.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Katz ML, Khan S, Awano T, et al. A mutation in the CLN8 gene in English Setter dogs with neuronal ceroid-lipofuscinosis. Biochem Biophys Res Commun 2005;327:541547.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Awano T, Katz ML, O'Brien DP, et al. A mutation in the cathepsin D gene (CTSD) in American Bulldogs with neuronal ceroid lipofuscinosis. Mol Genet Metab 2006;87:341348.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Awano T, Katz ML, O'Brien DP, et al. A frame shift mutation in canine TPP1 (the ortholog of human CLN2) in a juvenile Dachshund with neuronal ceroid lipofuscinosis. Mol Genet Metab 2006;89:254260.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Abitbol M, Thibaud JL, Olby NJ, et al. A canine Arylsulfatase G (ARSG) mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis. Proc Natl Acad Sci U S A 2010;107:1477514780.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Farias FH, Zeng R, Johnson GS, et al. A truncating mutation in ATP13A2 is responsible for adult-onset neuronal ceroid lipofuscinosis in Tibetan Terriers. Neurobiol Dis 2011;42:468474.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Franks JN, Dewey CW, Walker MA, et al. Computed tomographic findings of ceroid lipofuscinosis in a dog. J Am Anim Hosp Assoc 1999;35:430435.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Rossmeisl JH, Duncan R, Fox J, et al. Neuronal ceroid-lipofuscinosis in a Labrador Retriever. J Vet Diagn Invest 2003;15:457460.

  • 16. Evans J, Katz ML, Levesque D, et al. A variant form of neuronal ceroid lipofuscinosis in American Bulldogs. J Vet Intern Med 2005;19:4451.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Kuwamura M, Hattori R, Yamate J, et al. Neuronal ceroid-lipofuscinosis and hydrocephalus in a Chihuahua. J Small Anim Pract 2003;44:227230.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Koie H, Shibuya H, Sato T, et al. Magnetic resonance imaging of neuronal ceroid lipofuscinosis in a Border Collie. J Vet Med Sci 2004;66:14531456.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. O'Brien DP, Katz ML. Neuronal ceroid lipofuscinosis in 3 Australian Shepherd littermates. J Vet Intern Med 2008;22:472475.

  • 20. Asakawa MG, MacKillop E, Olby NJ, et al. Imaging diagnosis-neuronal ceroid lipofuscinosis with a chronic subdural hematoma. Vet Radiol Ultrasound 2010;51:155158.

    • Search Google Scholar
    • Export Citation

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