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Neuronal ceroid-lipofuscinosis and hydrocephalus in a chihuahua A two-year-old, female chihuahua presented with a six-month history of visual dysfunction. Computed tomography revealed dilation of the lateral ventricles in the central nervous system (CNS). The dog was

and Giesecke 1975). This report describes the clinical and pathological findings in a chihuahua suffering from NCL and hydrocephalus.

tentatively diagnosed as having hydrocephalus and a month later was euthanased at the owner’s request. The skull was expanded and dome-like in shape and an open fontanelle was observed on postmortem examination. Histologically, swollen neurons possessing yellowish pigment granules in the cytoplasm were observed throughout the CNS. These storage materials stained positively with periodic acid Schiff, Schmorl method for lipofuscin and oil red O for lipid, and showed autofluorescence under fluorescence microscopy. Ultrastructurally, the storage materials consisted of dense lamellar structures. This case was unique in having ceroid-lipofuscinosis in association with hydrocephalus. M. KUWAMURA, R. HATTORI, J. YAMATE, T. KOTANI AND K. SASAI* Journal of Small Animal Practice (2003) 44, 227-230

Laboratories of Veterinary Pathology and *Veterinary Internal Medicine, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan JOURNAL OF SMALL ANIMAL PRACTICE

INTRODUCTION Neuronal ceroid-lipofuscinosis (NCL) is a complex inherited neurodegenerative disorder of humans and animals, characterised by lipopigment deposition in the neurons and other cells of the body (Jolly and others 1994b). In humans, NCL has been classified mainly according to the clinical onset in infantile, late infantile, early juvenile, juvenile (Batten disease) and adult (Kuff ’s disease) types (Lake 1997). In the veterinary literature, NCL has been recorded in dogs, cats (Green and Little 1974, Nakayama and others 1993), sheep (Mayhew and other 1985, Jolly and others 1989, Edwards and others 1994), cattle (Harper and others 1988), goats (Fiske and Storts 1988) and primates (Jasty and others 1984). NCL has been encountered in many breeds of dogs including English setters (Koppang 1970), border collies (Taylor and Farrow 1988), cocker spaniels (Nimmo Wilkie and Hudson 1982, Jolly and others 1994a, Minatel and others 2000), dachshunds (Vandevelde and Fatzer 1980), salukis (Appleby and others 1982) and chihuahuas (Rac

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CASE HISTORY A two-year-old, female chihuahua presented to the Veterinary Teaching Hospital of Osaka Prefecture University with a sixmonth history of visual dysfunction. The dog had also developed a slight reduction in its sense of smell six months before presentation. When walking, the dog repeatedly curved to the right. The dog had also become aggressive. At the time of the first medical examination, loss of pupillary reaction to light was observed. Neurological and ophthalmoscopic examinations were otherwise normal. No abnormality was detected on haematological examination. Computed tomographic (CT) images showed dilation of the lateral ventricles in the brain (Fig 1) and hydrocephalus was tentatively diagnosed. Therapy with corticosteroids (0·25 mg dexamethasone, twice daily orally) and glycerol (1·2 ml of 50 per cent glycerin, twice daily orally) slightly improved the visual function; however, the dog was euthanased at the owner’s request on day 29 after presentation.

FIG 1. CT image in a case of concurrent ceroid-lipofuscinosis and hydrocephalus. Note the dilation of the ventricles

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FIG 3. Macroscopic appearance of the brain. Note the dilation of the lateral and third ventricles

FIG 2. Expanded, dome-like skull of the dog on gross postmortem examination

FIG 4. Swollen neurons in the cerebral cortex possessing yellowish storage material (arrow) in their cytoplasm. Haematoxylin and eosin (H&E) 220

The skull of the dog was expanded like a dome (Fig 2). On postmortem examination, an open fontanelle and partial defect of the skull in the left occipital region were observed. The lateral and third ventricles were markedly dilated; in particular dilation of the right lateral ventricle was prominent (Fig 3). No macroscopic abnormalities were found in the visceral organs. Histologically, swelling of neurons was observed throughout the central nervous system (CNS) and these neurons possessed yellowish pigment granules in the cytoplasm (Fig 4). Pigment deposition was marked in the neurons of the hippocampus and thalamus, and in the Purkinje cells in the cerebellum. Neurons in the retina also showed swelling with pigment storage. These pigments stained 228

FIG 5. Storage materials (arrows) in the Purkinje cells of the cerebellum showing positive staining with periodic acid Schiff 220

positively with periodic acid Schiff (Fig 5), Schmorl method for lipofuscin and oil red O for lipid. Fluorescence microscopy demonstrated yellow-green autofluores-

cent granules in the swollen cytoplasm of the affected neurons (Fig 6). Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed prominent astrocytosis

FIG 6. Autofluorescence under ultraviolet stimulation in the cerebellum. 220 JOURNAL OF SMALL ANIMAL PRACTICE

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FIG 7. Enhanced glial fibrillary acidic protein immunoreactivity in the Bergmann glia and astrocytes of the cerebellum. 180

FIG 8. Electron microscopy of the storage materials, showing dense lamellar structures in the cytoplasm of the neuron. 21,000

FIG 9. Infiltration of mononuclear cells around the ventricle in the cerebrum. H&E 110

consisting of gemistocytes throughout the CNS, and a laminar pattern of gliosis was often found in the cerebral cortex. Loss of Purkinje cells, Bergmann glial proliferation and an attenuated molecular layer were noted in the cerebellum. GFAP immunohistochemistry clearly demonstrated Bergmann gliosis in the molecular layer of the cerebellum (Fig 7). No obstructive lesions were apparent in the ventricular system. No storage material was apparent outside the CNS. Ultrastructurally, storage materials consisted of dense lamellar structures in the cytoplasm of the neurons (Fig 8). Occasionally, some lamellar structures were found in the cytoplasm of astrocytes. JOURNAL OF SMALL ANIMAL PRACTICE

The parenchyma of the cerebrum adjacent to the dilated ventricles was compressed, and diffuse astrocytosis was noted in the corna radiata of the cerebrum. Minute foci of mononuclear cell infiltration were found around the lateral ventricles with perivascular to diffuse infiltrating patterns (Fig 9). Most of these cells were lymphocytes; a few macrophages were also involved. There were no inflammatory lesions around the third and fourth ventricles.

DISCUSSION Frequent clinical signs of NCL include visual impairment, abnormal behaviour

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with aggression, tremor, ataxia and seizures (Jolly and others 1994b). Blindness was mostly reported as an early clinical change in dogs of several breeds affected with NCL (Jolly and others 1994b). The present case also showed visual dysfunction, which was similar to the previously reported cases of canine NCL in its onset and nature. Age at onset of canine NCL varies from six months to eight years; thus, it is clinically classified as prepubertal-protracted, early adult-acute course, and adult onset (Jolly and others 1994b). Reported cases of NCL in chihuahuas occur from 13 to 21 months of age (Rac and Giesecke 1975), similar to the present case. NCL affects the cerebral cortex, particularly the occipital pole. In addition, the cerebellum was severely involved in cases reported by Jolly and others (1994b), as in the present case. The storage materials showed similar staining and fluorescent properties to those of ceroid and lipofuscin. The major constituent of storage pigment has been described as a lipid-binding protein, which is a component (subunit c) of mitochondrial ATP synthase (Jolly and others 1994b). The other storage material was identified as sphingolipid activator protein (or saposins) in the human infantile form and in miniature schnauzers with NCL (Palmer and others 1997). The precise nature of the storage materials in the present case remains to be investigated. 229

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Generally, brain atrophy is the most common macroscopic finding in canine NCL (Jolly and others 1994b). Although slight dilation of the lateral ventricles associated with brain atrophy has been found, prominent hydrocephalus has not been recorded in previous cases of canine NCL (Jolly and others 1994b). Congenital hydrocephalus is more common in toy breeds of dogs, including the Maltese, Yorkshire terrier, English bulldog, chihuahua, lhasa apso, pug and Pekingese (Summers and others 1995). The present dog had an open fontanelle and a defect of the skull, suggesting that the hydrocephalus was congenital in nature. Focal inflammation was found exclusively

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around the lateral ventricles, indicating a reaction associated with degenerative changes caused by the increased intraventricular pressure. Neurological dysfunction would have resulted from ceroid-lipofuscin deposition in the neurons, and physical compression of the brain by the hydrocephalus. This dog may be unique in suffering from both NCL and hydrocephalus.

References APPLEBY, E. C., LONGSTAFFE, J. A. & BELL, F. R. (1982) Cereoid-lipofuscinosis in two Saluki dogs. Journal of Comparative Pathology 92, 375-380

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EDWARDS, J. F., STORTS, R. W., JOYCE, J. R., SHELTON, J. M. & MENZIES, C. S. (1994) Juvenile-onset neuronal ceroid-lipofuscinosis in Rambouillet sheep. Veterinary Pathology 31, 48-54 FISKE, R. A. & STORTS, R. W. (1988) Neuronal ceroid-lipofuscinosis in a Nubian goat. Veterinary Pathology 25, 171-173 GREEN, P. D. & LITTLE, P. B. (1974) Neuronal ceroid-lipofuscinosis in Siamese cats. Canadian Journal of Comparative Medicine 38, 207-212 HARPER, P. A. W., WALKER, K. H., HEALY, P. J., HARTLEY, W. J., GIBSON, A. J. & SMITH, J. S. (1988) Neurovisceral ceroid-lipofuscinosis in blind Devon cattle. Acta Neuropathologica 75, 632-636 JASTY, V., KOWALSKI, R. L., FONSECA, E. H., PORTER, M. C., CLEMENS, G. R., BARE, J. J. & HARTNAGEL, R. E. (1984) An unusual case of generalized ceroid-lipofuscinosis in a cynomolgus monkey. Veterinary Pathology 21, 46-50 JOLLY, R. D., HARTLEY, W. J., JONES, B. R., JOHNSTONE, A. C., PALMER, A. C. & BLAKEMOR, W. F. (1994a) Generalized ceroid-lipofuscinosis and brown bowel syndrome in cocker spaniel dogs. New Zealand Veterinary Journal 42, 236-239 JOLLY, R. D., PALER, D. N., STUDDERT, R. H., SUTTON, R. H., KELLY, W. R., KOPPANG, N., DAHME, G., HARTLEY, W. J., PATTERSON, J. S. & RIIS, R. C. (1994b) Canine ceroidlipofuscinoses: a review and classification. Journal of Small Animal Practice 35, 299-306 JOLLY, R. D., SHIMADA, A., DOPFMER, I., SLACK, P. M., BIRTLES, M. J. & PALMER, D. N. (1989) Ceroid-lipofuscinosis (Batten’s disease): pathogenesis and sequential neuropathological changes in the ovine model. Neuropathology and applied Neurobiology 15, 371-383 KOPPANG, N. (1970) Neuronal ceroid-lipofuscinosis in English setters. Journal of Small Animal Practice 10, 639-644 LAKE, B. D. (1997) Lysosomal and peroxisomal disorders. In: Greenfield’s Neuropathology, 6th edn. Eds D. I. Graham & P. L. Lantos. Arnold, London. pp 657-753 MAYHEW, I. G., JOLLY, R. D., PICKETT, B. T. & SLACK, P. M. (1985) Ceroid-lipofuscinosis (Batten’s disease): pathogenesis of blindness in the ovine model. Neuropathology and Applied Neurobiology 11, 273-290 MINATEL, L., UNDERWOOD, S. C. & CARFAGNINI, J. C. (2000) Ceroid-lipofuscinosis in a cocker spaniel dog. Veterinary Pathology 37, 488-490 NAKAYAMA, H., UCHIDA, K., SHOUDA, T., UETSUKA, K., SASAKI, N. & GOTO, N. (1993) Systemic ceroid-lipofuscinosis in a Japanese domestic cat. Journal of Veterinary Medical Science 55, 829-831 NIMMO WILKIE, J. S. & HUDSON, E. B. (1982) Neuronal and generalized ceroid-lipofuscinosis in a cocker spaniel. Veterinary Pathology 19, 623-628 PALMER, D. N., JOLLY, R. D., VAN MIL, H. C., TYYNELA, J. & WESTLAKE, V. J. (1997) Different patterns of hydrophobic protein storage in different forms of neuronal ceroid-lipofuscinosis (NCL, Batten disease). Neuropediatrics 28, 45-48 RAC, R. & GIESECKE, P. R. (1975) Lysosomal storage disease in chihuahuas. Australian Veterinary Journal 51, 403-404 SUMMERS, B. A., CUMMINGS, J. P. & DE LAHUNTA, A. (1995) Malformation of the central nervous system. In: Veterinary Neuropathology. Eds B. A. Summers, J. F. Cummings and A. de Lahunta. Mosby-Year Book, St. Louis. pp 68-94 TAYLOR, R. M. & FARROW, B. R. H. (1988) Ceroid-lipofuscinosis in border collie dogs. Acta Neuropathologica 75, 627-631 VANDEVELDE, M. & FATZER, R. (1980) Neuronal ceroid-lipofuscinosis in older dachshunds. Veterinary Pathology 17, 686-692

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