Neuronal Ceroid-Lipofuscinosis in Longhaired Chihuahuas: Clinical, Pathologic, and MRI Findings

Introduction

Neuronal ceroid-lipofuscinosis (NCL) is a rare group of inherited, neurodegenerative, lysosomal storage diseases characterized histopathologically by the abnormal accumulation of ceroid- or lipofuscin-like lipopigments in neurons and other cells throughout the body.^1–3 Compared with other lysosomal storage diseases, both the clinical manifestation and histopathologic characterization of NCL are variable in humans. NCL are classified mainly by the time of clinical onset into infantile, late infantile, early juvenile, juvenile, adult, and other variant types.^2,4,5 To date, mutations in at least 10 different genes have been elucidated as causative factors in the human forms of NCL.⁶

In veterinary medicine, NCL has been described in several domestic species and occurs most commonly in the dog.³ NCL has been reported in a number of canine breeds including English setters, dachshunds, salukis, border collies, cocker spaniels, American bulldogs, and Chihuahuas.^7–17 The causative mutations have been identified in English setters, border collies, American bulldogs, and miniature longhaired dachshunds.^18–21 The identified genetic mutations each reside in different genes, suggesting that NCL in the dog is also variable and attributable to a number of different genes.

The diagnosis of NCL is based on clinical signs, biochemical and genetic findings, and through pathologic identification of the stored ceroid- or lipofuscin-like lipopigments in affected tissues.²² Currently, the identification of canine NCL requires morphologic diagnosis despite progress in molecular genetics, mutation analysis, and enzymology. These latter techniques have not yet become common diagnostic procedures for NCL in domestic animals. Therefore, biomarkers for antemortum diagnosis are needed in NCL, especially in veterinary medicine.

MRI facilitates the early diagnosis of human NCL by the identification of characteristic MRI findings, including: cerebral and cerebellar atrophy; mild T2-hyperintensity of the cerebral white matter; and thinning of the cerebral cortex.^23,24 Recently, MRI has been used frequently for diagnosis or evaluation of cranial diseases even in veterinary medicine. Therefore, the characteristic MRI findings in each form or type of canine NCLs would be a useful diagnostic marker for this disease.

Recently, the authors diagnosed NCL based on the clinical, histopathologic, and pedigree analyses in three longhaired Chihuahuas, presenting with visual defects and behavioral abnormalities. Cranial MRI examinations were performed in all three cases and characteristic features were observed on the images. The present article describes the clinical, pathologic, and MRI findings of NCL in these Chihuahuas.

Materials and Methods

Case Report

Results

MRI Findings

In all cases, the MRI examinations demonstrated diffuse, severe dilation of the cerebral sulci, dilated fissures of the diencephalons, midbrain, and cerebellum, and lateral ventricular enlargement on transverse plane T2-weighted (Figure 1A), T1-weighted (Figure 1B), fluid attenuated inversion recovery, (Figure 1C), meglumine gadopentetate-T1-weighted (Figure 1D), and sagittal plane T2-weighted images (Figure 1E). The size of the brainstem seemed to be normal. The most unusual feature was the meningeal thickening observed over the entire cerebrum (Figure 1B) that was strongly enhanced by IV injection of meglumine gadopentetate (Figure 1D).

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Pathologic Findings

Grossly, the atrophic lesions in the cerebrum, cerebellum, and brainstem, and diffuse thickening of the meninges were observed in both cases 1 and 2. In case 2, there were no significant lesions in the visceral organs. Histopathological examinations demonstrated severe to moderate neuronal loss and astrogliosis throughout the brain, especially in the cerebral and cerebellar cortices. The remaining neuronal cells and occasionally infiltrated macrophages or activated microglia showed intracytoplasmic accumulation of pale to slightly yellow lipopigments mimicking ceroid or lipofuscin (Figures 2A–C). These store materials stained positively with Sudan black B on paraffin sections (Figure 2D). The thickened meninges consisted of the proliferation of connective tissues with abundant collagen fibers (Figure 2E) and mild infiltration of lipopigment-bearing macrophages, and a small number of lymphocytes and plasma cells, especially in the dura and arachnoid membranes (Figures 2E, F).

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Pedigree Study

The pedigrees of cases 1–3 were studied (Figure 3). Cases 2 and 3 were from different litters of the same sire and dam. There was no relation between case 1 and the two other dogs within three or four generations of each affected dog.

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Discussion

In the present article, the authors reported the clinical, pathologic, and MRI findings in two Chihuahuas diagnosed histopathologically with NCL and one Chihuahua in which NCL was strongly suspected based on the clinical features and pedigree analysis. These three Chihuahuas showed similar clinical and MRI findings, and the two dogs that underwent histopathologic examination showed similar findings. Therefore, these three Chihuahuas might have been affected by NCL attributable to the same molecular basis, although the pedigree lineage among these dogs could not be completely elucidated.

NCL in Chihuahuas was reported first in Australia in 1975, and a similar disease was recognized in New Zealand in the 1970s.^15,16 In addition, one Chihuahua with NCL was found in Japan in 2003.¹⁷ In the Australian report,¹⁵ two Chihuahuas without a clear blood relationship showed a clinical history of visual defects, motor disorders, and behavioral abnormalities between 21 mo and 24 mo of age. A review of canine NCL noted that the clinical course of these Chihuahuas affected with NCL in the southern hemisphere runs from 13 mo to 24 mo of age.³ The histopathologic examination of the two dogs in Australia demonstrated the presence of eosinophilic granular material in the cytoplasm of neurons and glial cells throughout the central nervous system, which stained weakly positive with acid-fast, periodic acid-Schiff, and Sudan black stains. The report in Japan in 2003 described a 2 yr old, cream, shorthaired Chihuahua with a 6 mo history of visual defect, motor disorder, and behavioral abnormalities.¹⁷ Computed tomographic (CT) images showed dilation of the lateral ventricles in the brain. NCL was finally diagnosed based on the histopathologic features (i.e., swollen neurons with slightly yellow pigment granules in the cytoplasm, which could be stained positively with periodic acid-Schiff, Schmorl, and oil red O stains). In addition, ultrastructural examination of the brain demonstrated that the storage materials consisted of dense lamellar structures.

The present article is only the fourth report of a form of NCL in Chihuahuas over a 34 yr period. For the first time, pedigree data including two unrelated families were provided, which supports the expectation that NCL in Chihuahuas is inherited as an autosomal recessive trait; however, the pedigree provides insufficient information to definitively conclude this assertion. The pathogenic mutation needs to be identified.

A number of different clinical forms of NCL have been described in dogs, as well as in humans.^2–5,25 NCL in dogs are classified by the age of onset and the course of the disease (i.e, prepubertal-protracted, early adult acute, and adult onset). NCL in Chihuahuas reported in the 1970s was categorized into the early adult acute form together with NCL found in English setters, border collies, and salukis.³ In the longhaired Chihuahuas described in the present article, the clinical symptoms, including visual defects and behavioral abnormalities, started between 16 mo and 18 mo of age, and the dogs finally died subsequent to neurologic deterioration (motor disorders, feeding difficulty, and convulsions) between 23 mo and 24 mo of age. This finding suggests that this clinical form of NCL closely resembles those of previously reported Chihuahuas in Australia and Japan. The clinical similarity between the past and present reports of NCL in Chihuahuas probably reflects the same genetic mutation(s) as in other cases.

In the present article, ultrastructural examination and specific staining methods, except Sudan black B, were not available to correctly classify the disease pathologically. Nonetheless, the microscopic appearance and staining reaction of Sudan black B agreed with the common pathologic findings of both canine and human NCLs. Further morphologic and biochemical analyses may provide a clue to elucidate the molecular aspects of this disease.

Characteristic MRI findings of NCL in the three Chihuahuas in the present article included: diffuse severe dilation of the cerebral sulci; dilated fissures of the diencephalons, midbrain and cerebellum; and lateral ventricular enlargement, suggesting atrophy of the forebrain. These MRI features were similar to common MRI features in both human NCL and those of canine NCL reported in other breeds.^11,25,26 In addition, these MRI features were (in part) consistent with those observed on CT images previously reported in the Chihuahua.¹⁷ The common features on MRI and CT images suggesting atrophy of the brain could be a key to diagnosing NCL in dogs with progressive neurologic symptoms. It should be noted that lateral ventricular enlargement is often observed in congenital hydrocephalus, which is much more common than NCL in toy breeds of dogs, especially Chihuahuas. One Chihuahua with NCL reported in Japan in 2003 was affected simultaneously with both NCL and congenital hydrocephalus.¹⁷ Veterinary clinicians should be aware that NCL is possible, as well as hydrocephalus, in Chihuahuas showing lateral ventricular enlargement.

The most unusual feature of the MRI images in the three dogs described in this article was diffuse meningeal thickening observed over the entire cerebrum that was strongly enhanced on contrast-enhanced T1-weighted images. Histopathologically, fibrin deposit and infiltration of macrophages and plasma cells were observed in the thickened meninges, suggesting an inflammatory response to some type of material. To the authors' knowledge, meningeal thickening has not previously been reported in NCL in either humans or domestic animals; however, the relationship between NCL and the neuroimmune response has been described in some previous reports.^27–29 Human juvenile NCL is one of the lysosomal storage diseases that are predisposed toward immune system hyperactivity.²⁷ Serum obtained from juvenile NCL patients includes autoantibodies that recognize a variety of proteins in the central nervous system and α-fetoprotein has been identified as one of the autoantigens in this disease.^28–30 Nonetheless, it has not been completely elucidated what precipitates this autoimmune response, and it has not been determined to what extent this altered immune response contributes to the pathology of NCL.²⁷ A similar type of abnormal neuroimmune response might occur in the brain of Chihuahuas affected with NCL resulting in meningeal thickening. Further studies are needed to clarify this pathologic mechanism.

Conclusion

The pathologic and MRI examinations demonstrated that these three Chihuahuas were affected with NCL characterized by atrophy of the forebrain and neuroimmune hyperactivity around the meninges. MRI findings such as meningeal thickening may be a useful diagnostic marker in this variant form of canine NCL.