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Abbreviations
Cho

choline

Cr

creatine

CSF

cerebrospinal fluid

FIESTA

fast imaging employing steady state acquisition

FLAIR

fluid attenuation inversion recovery

FSPGR

fast spoiled gradient echo FIESTA

Hex

hexosaminidase

mI

Myo-inositol

MRI

magnetic resonance imaging

MRS

magnetic resonance spectroscopy

NAA

N-acetylaspartate

PRESS

point resolved spectroscopy

T1 and T2W

T1 and T2 weighting

Case One

  1. Top of page
  2. Case One
  3. Case Two
  4. Acknowledgment
  5. References

A 15-month-old, male neutered Japanese Chin dog was presented for evaluation of blindness and an abnormal gait first observed at 11 months of age. On presentation, the dog was periodically obtunded and had cerebellar ataxia with spastic, hypermetric postural reactions. The dog had an absent menace response and ventro-lateral strabismus in both eyes.

A magnetic resonance imaging (MRI) examination was performed with a 3.0 T MRI unit1 and a receive-only quadrature knee coil, and consisted of turbo spin echo sequences with T2-weighting (T2W), and fluid attenuation inversion recovery T2- and T1-weighting (T2W FLAIR & T1W FLAIR) and gradient echo sequences (FSPGR, fast spoiled gradient echo; fast imaging employing steady state acquisition, FIESTA). The T1W FLAIR images were acquired before and after the IV administration of gadolinium-based MRI contrast (Magnevist,2 0.1 mmol/kg). The distinction between gray and white matter regions of the cerebrum and cerebellum was diminished to absent on T2W images (Fig 1). Bilaterally, small regions of more normal-appearing corona radiata were identified in the frontal lobes and rostral internal capsule. In the cranial cervical spinal cord, gray matter was indistinguishable from white matter. On the precontrast T1W FLAIR images, cerebral white matter was hyperintense to gray matter and had ill-defined margins. The cerebral sulci may have been mildly widened for the age of the patient, but the interthalamic adhesion and cerebellar folia were of the appropriate proportions (Fig 1). In addition, the lateral and third ventricles were subjectively dilated. Specifically, a focal enlargement of the caudodorsal aspect of the 3rd ventricle was noted, adjacent to a very small more caudal fluid accumulation, sometimes referred to as a supracollicular intra-arachnoid diverticulum. No abnormal enhancement was observed on postcontrast images.

image

Figure 1. Images A, C, and E are T2-weighted midline sagittal (A) and transverse images at the level of the frontal lobe (C) and thalamus (E) acquired from GM2-affected patient using 3.0 T MRI unit. Matched images (B, D, and F) are T2-weighted images from a neurologically normal 2-year-old Japanese Chin dog with a 1.5 T MRI unit. In the GM2-affected dog, there is generalized increased signal intensity in the cerebral white matter, with a small amount of normal-appearing white matter in the rostral internal capsule. Relatively lower white matter signal, compared with gray matter, is seen in the hippocampus and thalamus; the degree of contrast present is not as great as in the normal dog. The white matter regions of the cerebellum and hippocampus are also diminished in size.

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Spectroscopy3 was performed in the cerebellar white matter, by a point resolved spectroscopy (PRESS) pulse sequence (Fig 2) and showed a decrease in the N-acetylaspartate (NAA) peak and NAA/creatine (NAA/Cr) ratio in the cerebellar white matter, compared with the normal dog cerebellum. The choline/Cr (Cho/Cr) and the Ch/NAA ratios were higher in the affected dog.

image

Figure 2. Hydrogen proton MRS (H1-MRS) from the GM2-affected Japanese Chin in case 1 (A) and a juvenile Beagle dog (B), acquired with a 3.0 T MRI unit. The probes were placed within the cerebellar white matter. The NAA is diminished compared the normal brain and the CHO:Cr ratio is increased in normal brain. In Tay-Sachs patients, reduction in the NAA peak has correlated with neuronal damage and loss. The increase in the CHO : Cr ratio has been associated in human patients with demyelination.

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The owner elected for euthanasia, and a necropsy was performed. Formalin and fresh frozen samples were collected. Using hematoxylin and eosin staining, the gray matter of the cerebrum and spinal cord was diffusely pale. Approximately 95% of the neurons throughout the cerebrum and dorsal and ventral horn of the spinal cord were up to 4 times larger than normal. These neurons contained abundant finely granular, foamy cytoplasm, which often displaced the nucleus and the Nissl substance to the periphery (Fig 3). Occasional swollen neurons contained dark basophilic nuclei consistent with neuronal degeneration. Many glial cells contained variably vacuolated cytoplasm. Primarily within the white matter of the spinal cord, axons were swollen with dilated myelin sheaths. Fragmented axons were replaced by eosinophilic granular necrotic debris and macrophages, consistent with digestion chambers. The white matter of the cerebrum was moderately hypercellular.

image

Figure 3. Intracytoplasmic storage material in the majority of the neurons of the trigeminal ganglion. Hematoxylin & Eosin, Bar = 50 μm.

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image

Figure 4. Electron microscopic image of the affected parenchyma. Neuronal cytoplasm filled with heterogeneous collection of lamellated membranous inclusions some of which contain central lipofuscin-like material that is heterogeneous (long arrow), homogeneously electron dense (medium arrow) or finely granular and less electron dense (short arrow). Rare dense bodies of lipofuscin-like material without membranes (L) are present. N, neuronal nucleus; C, neuronal cytoplasm; Bar = 500 nm. Lead citrate/uranyl acetate.

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By electron microscopy, swollen neurons contained numerous cytoplasmic inclusions resembling the membranous cytoplasmic inclusions seen in humans with GM2 gangliosidosis (B variant) and other gangliosidoses (Fig 4).[1] Axons were swollen and contained swollen mitochondria with ruptured cristae. Membranous inclusions were not noted in other cell types.

Case Two

  1. Top of page
  2. Case One
  3. Case Two
  4. Acknowledgment
  5. References

A 18-month-old, male neutered, Japanese Chin dog was presented for evaluation of progressive mental dullness of 2 months duration. On neurologic examination, the dog was ambulatory with moderate cerebellar ataxia and intention tremors. Menace response was absent OU.

MRI examination was performed on 0.3 T Vet-MR unit.4 On the T2W images, loss of distinction between the gray and white matter regions was noted throughout the cerebrum. The signal intensity of the white matter regions was subjectively increased, resulting in isointensity between these regions and the cortical gray matter. Some more normal-appearing white matter remained in the left corona radiata. The greatest change was noted in the gyral extension of the white matter. The thalamus and corpus callosum were not affected. Mild asymmetry of the lateral ventricles was noted.

Over a 5-month period, the dog became progressively more dull and disoriented and developed severe tremors and cerebellar ataxia. Euthanasia was elected, and a necropsy was performed with formalin and fresh frozen samples collected. Fixed tissues showed changes similar to those observed in case 1.

Increased amounts of GM2 gangliosides were identified in brain tissues of both dogs, and a missense mutation in HEXA, the gene that codes for the α subunit of Hex A was identified,[2] thus confirming a diagnosis of B variant of GM2 gangliosidosis.

Lysosomal storage diseases are hereditary degenerative conditions seen in humans and domestic animals.[3, 4] Deficient activity of lysosomal hydrolases results in accumulation of substrates within membrane bound lysosomes ultimately leading to cellular dysfunction, cell death or both.[5, 6] GM2 ganglioside, a component of the cellular membrane, is catabolized within the lysosomes by hexosaminidase. The two isoforms, hexosaminidase A (Hex A) and hexosaminidase B (Hex B), are composed of 2 subunits: α β in Hex A and β β in Hex B. Catabolism is facilitated by the presence of cofactors, particularly GM2 activator protein.[7-9],[10] A defect in the α subunit results in deficiency of Hex A. This deficiency is known as Tay-Sachs disease (B variant) in people. A different variant of Tay-Sachs disease (B1 variant) has been recognized in which a stable but enzymatically inactive α subunit is produced. Sandhoff's disease (O variant) in people is caused by deficiency in the β subunit affecting both Hex A and B. This variant has been described in a Golden Retriever dog,[11] toy Poodles, and in cats.[9, 12-14] With deficiencies in the activator protein, there is normal to increased activity of both Hex A and B isoenzymes for the artificial substrates used in the assays, but not for the natural GM2 ganglioside.[7] A deficiency in the GM2 activator protein, AB variant of Tay-Sachs disease, has been described in cats[15] and Japanese Chin dogs,[7, 16] but recent data suggest the defect in the Japanese Chin is caused by a mutation in the gene that codes for Hex A (B variant).[2]

The use of imaging techniques such as MRI has been evaluated in the diagnosis of lysosomal storage diseases in humans.[16, 17] There are reports describing MRI changes in dogs with histopathologically confirmed GM2 gangliosidosis (Sandoff type or O variant) but currently none describing the B variant.[17, 18]

Ante-mortem methods of diagnosis include measurement of Hex activity in leukocytes, serum, and cerebrospinal fluid.[19] Measurement of Hex activity is not helpful in the diagnosis of the AB variant, which is characterized by normal to increased Hex A activity.[19] Increased GM2 ganglioside concentrations have been detected in dogs with GM2 gangliosidosis type O (Sandhoff disease).[11, 19] Electron microscopy of skin biopsies can detect the fine lamellated membrane structures called “fingerprints” or “zebra bodies” characteristic of the stored material.[20] In addition, fibroblasts from skin biopsies can be biochemically analyzed for Hex A and B activity and activator protein concentrations.[20]

Although the aforementioned tests allow possible antemortem confirmation of GM2 gangliosidosis, the clinician must decide when to run these tests. Differential diagnosis for a young patient presenting with progressive intracranial signs is not limited to lysosomal storage diseases and includes anomalous diseases (eg, hydrocephalus, supracollicular intra-arachnoid diverticula), inflammatory diseases, and less commonly neoplasia. MRI allows noninvasive evaluation of the brain and, as shown in this report, can increase the index of suspicion for lysosomal storage diseases.

GM2 gangliosidosis does not result in pathognomonic MRI changes. The MRI appearance, as well as spectroscopic changes, of Tay-Sachs (B variant) and Sandoff (O variant) have been reported in affected human patients, because macroscopic lesions can be identified.[21] However, these, as well as other congenital encephalopathies, have similar MRI characteristics. A case report of a human affected with the AB variant documented normal intracranial structures using MRI.[21] The MRI changes identified in the infantile B1 variant in people are similar to changes seen with early onset Tay-Sachs and Sandhoff. These changes included diffuse symmetrical hyperintensities in the basal nuclei and thalamus consistent with severe demyelination.[22] MRI changes reported in the juvenile B1 variant consist of diffuse hypomyelination, marked brain atrophy, and ventricular enlargement.[22]

The MRI appearance of Sandhoff disease has been described in cats,[17] and a Golden Retriever dog.[18] Cats with Sandhoff disease had diffuse white matter hyperintensity on T2W images and hypointensity on T1W images.[17] These findings correlated with histologic white matter changes attributed to a defect in myelin formation secondary to neuronal injury.[17] The Golden Retriever dog had bilaterally symmetrical MRI lesions within the caudate nucleus. These lesions were hyperintense to the surrounding brain on T2W and hypointense on T1W images. The lesions did not enhance after administration of gadolinium.[18] In addition, mild atrophy of the cerebral cortex, particularly in the temporal lobes, was observed.[18]

Macroscopic intracranial changes were identified on the MRI examinations of the affected dogs in this case series. Similar to infants with the B1 variant of Tay-Sachs, the primary MRI abnormality in these dogs was white matter changes consistent with decreased myelin content. In both dogs, the predominant imaging feature was mild (case 2) to severely (case 1) increased signal intensity of cerebral white matter on T2W images, resulting in the white matter regions being isointense to the cerebral gray matter. The hyperintensity in white matter regions, as compared with gray matter regions on T2W images, has been referred to as hypomyelination.[23, 24] Myelination is disrupted or halted as a result of abnormal cellular metabolism. This finding is seen only in the early onset forms of GM2 gangliosidosis in human patients.[24] Similar to GM2 gangliosidosis-affected human patients, these dogs maintained normal subjective signal intensity in the corpus callosum.[24]

The hyperintensity of cerebral white matter to the cerebral gray matter within the canine brain, on T1W and T2W images, normally is established by 16 weeks of age. The structure of the corpus callosum and lower relative signal intensity on T2W images develops by 8 weeks of age.[25] An inversion of the relative signal intensities on T2W images indicates pathology related to myelin, but the relative signal intensity on the T1W images tends to be more specific for the nature of myelin pathology, according to human leukodystrophy disease algorithms.[24] The T1W signal intensity of white matter is proportional to the amount of myelin present. The higher signal intensity of the white matter on T1W images in case 1, in comparison to normal, would suggest that there is more myelin. The signal changes also could be secondary to gliosis, because lysosomal storage stimulates glial cell proliferation. Finally, abnormal storage of product within lysosomes can contribute to signal intensity alteration.[26]

Although 1 dog was imaged with a 3.0 T MRI unit and the other with a low-field magnet (0.3 T), similar MRI features were identified. This finding is in accordance with the human medical literature, which generally supports the fact that although higher field strength offers improved signal-to-noise ratios and a measurably greater enhancement of lesions with gadolinium for a given dose of contrast medium than lower field strength magnets, there is no clinically relevant difference in lesion detection.[27]

Magnetic resonance spectroscopy (MRS) evaluating at the hydrogen proton also has been used to characterize GM2 gangliosidosis in human patients. In Tay-Sachs patients, a progressive decrease in NAA concentration with a progressive decrease in NAA/Cr ratio has been seen in serial MRS studies.[28] The spectra are indicative of neuronal damage and loss. Inflammation also has been documented in GM2 gangliosidosis variant B and corresponds to Myo-inositol (mI) peak denoting glial cell activity. Finally, demyelination was seen as an increase in the Cho/Cr ratio. In a patient with Sandoff's disease, a new resonance on proton MRS was found in a patient, located at 2.07 ppm. It was ascribed to N-acetylhexosamine, which was found to be highest in white matter and thalamus.[29] In one of our cases, a decrease in the NAA peak and NAA/Cr ratio was present in the cerebellar white matter, compared with the normal dog cerebellum. However, the Cho/Cr ratio was lower in the affected dog. The clinical relevance of the MRS results is difficult to critically assess, because it presents a single measurement at a single time point in this one dog.

In conclusion, we report the first MRI description of the B variant of GM2 gangliosidosis in 2 Japanese Chin dogs, which was confirmed by the detection of normal to increased Hex A and B activity and with histopathology and ultrastructural findings. This description provides useful information about the MRI appearance of this lysosomal storage disease that may be used in the ante-mortem diagnosis of this condition.

Acknowledgment

  1. Top of page
  2. Case One
  3. Case Two
  4. Acknowledgment
  5. References

Conflict of Interest: Authors disclose no conflict of interest.

Footnotes
  1. 1

    GE 3.0 T Signa HDx; GE Healthcare, Milwaukee, WI

  2. 2

    Gadopentetate dimeglumine, Magnevist®, Bayer HealthCare Pharmaceuticals, Wayne, NJ

  3. 3

    N-acetylaspartate (NAA), Creatine (Cr), Choline (Cho), and Myo-inositol (mI)

  4. 4

    Vet MRI 0.3 T Esaote SpA, Genoa, Italy

References

  1. Top of page
  2. Case One
  3. Case Two
  4. Acknowledgment
  5. References