MRI Findings in a Rottweiler with Leukoencephalomyelopathy

CASE REPORTS

Joseph S. Eagleson, DVM, DACVIM (Neurology)*, Marc Kent, DVM, DACVIM (Neurology), Simon R. Platt, BVM&S, DECVN, DACVIM (Neurology), Raquel R. Rech, DVM, DACVP, PhDy, Elizabeth W. Howerth, DVM, DACVP, PhD

ABSTRACT A 22 mo old male rottweiler presented with a 1 mo progressive history of general proprioceptive ataxia and upper motor neuron tetraparesis. Neurologic examination was consistent with a lesion affecting the first through fifth cervical spinal cord segments. MRI disclosed bilaterally symmetric hyperintensities on T2-weighted (T2W) images in the crus cerebri and pyramidal tracts of the brain and the dorsal portion of the lateral funiculi of the cervical spinal cord. Fifty days after initial presentation, the dog was euthanized due to disease progression. Pathologic examination of the central nervous system (CNS) revealed a bilaterally symmetric chronic leukoencephalomyelopathy (LEM) consistent with previous reports of LEM in rottweilers. To the authors’ knowledge, this is the first report to describe the MRI characteristics of LEM in the rottweiler. The topography of the changes observed with MRI paralleled the pathologic changes, which were widespread loss of myelin, decreased axon numbers, and astroglial proliferation. Consequently, MRI of the CNS of affected rottweilers may aid in establishing a presumptive antemortem diagnosis of LEM. (JAmAnimHospAssoc2013; 49:255–261. DOI 10.5326/JAAHA-MS-5864)

Introduction antemortem diagnosis is based on exclusion of other diseases that Leukoencephalomyelopathy (LEM) in the rottweiler is a rare de- result in similar clinical signs, and diagnosis often entails ad- generative disorder that has been recognized since the early vanced imaging. To the authors’ knowledge, this is the ﬁrst report 1980s.1–3 The clinical syndrome manifests as a long-strided gait to describe the MRI characteristics of LEM in the rottweiler. In with the appearance of stiffness and overreaching as the limbs are the present case, the topography of the lesions observed with MRI advanced when walking, consistent with general proprioceptive exactly paralleled the pathologic changes observed on gross and ataxia and upper motor neuron tetraparesis that begins between microscopic examination of the central nervous system (CNS), 1.5 and 3.5 yr of age.3 Affected dogs initially develop abnormal- which consisted of severe myelin loss, decreased axonal numbers, ities in the thoracic limbs, and the thoracic limbs are often more astrogliosis, and astrocytosis. Consequently, MRI of the nervous severely affected than the pelvic limbs. Gender predilection has system may aid clinicians in establishing a presumptive ante- not been reported. Despite a familial relationship among affected mortem diagnosis of LEM in rottweilers. dogs, a mode of inheritance has not been determined.1–3 The etiology of LEM remains unknown. Progression of clinical signs Case Report occurs over months to up to a year, with most dogs being eu- A 22 mo old male rottweiler presented to the Veterinary Teaching thanized due to increased difﬁculty ambulating.2 Currently, the Hospital, University of Georgia with a 1 mo progressive history of

From the Department of Small Animal Medicine and Surgery (J.E., M.K., MBP myelin basic protein; CNS central nervous system; FLAIR fluid- S.P.) and Department of Pathology (R.R., E.H.), University of Georgia, attenuated inversion recovery; GFAP glial fibrillary acidic protein; LEM leuko- Athens, GA. encephalomyelopathy; NAD neuroaxonal dystrophy; T1W T1-weighted; T2W T2-weighted; T2*W T2*-weighted gradient echo Correspondence: [email protected] (M.K.) *J. Eagleson’s present affiliation is Veterinary Specialty & Emergency Center, Levittown, PA.

†R. Rech’s present afﬁlitation is Brazilian Agricultural Research Corp (EMBRAPA), Concordia, Brazil.

ª 2013 by American Animal Hospital Association JAAHA.ORG 255 an abnormal gait. The dog was current on vaccinations and was receiving heartworm preventative monthly. There was no history of other medical problems. Physical examination was normal. On neurologic examination, the dog displayed a longer than normal stride that was characterized by stiffness and overreaching, consistent with general proprioceptive ataxia and upper motor neuron paresis in all four limbs. The gait was characterized by hypermetria resulting in marked overreaching in the thoracic limbs. The dog scuffed the nails on all four feet while walking. Deficits in postural reactions (proprioceptive placing and hopping) were observed in all four limbs. Deficits were worse in the thoracic limbs. Addi- tionally, the left thoracic and pelvic limbs were more affected than the right thoracic and pelvic limbs. Spinal reflexes were normal in the thoracic limbs. In the pelvic limbs, the withdrawal reflexes were normal and patella reflexes were increased bilaterally. Muscle mass and muscular tone were both normal in all four limbs. Cranial nerve examination was normal. There was normal range of motion of the neck. The dog did not appear painful with either manip- FIGURE 1 Transverse plane T2-weighted (T2W) MRI of the ulation or palpation along the entire vertebral column. Neuro- second cervical vertebral (A) and fourth cervical vertebral (B) spinal fi anatomic diagnosis was consistent with a lesion affecting the rst cord segments, the medulla oblongata (C) and corresponding gross fi through fth cervical spinal cord segments. Differential diagnoses spinal cord specimen from the fourth cervical vertebra (D) from of included cervical vertebra(e) malformation/malarticulation, inter- a 22 mo old male rottweiler with general proprioceptive ataxia and fi vertebral disc disease, brotic stenosis, subarachnoid diverticula, upper motor neuron tetraparesis. There are bilaterally symmetrical, neoplasia, LEM, and neuroaxonal dystrophy (NAD). hyperintense lesions in the dorsolateral funiculi of the spinal cord Hematologic and serum biochemical examinations revealed (arrows in A and B). Images were acquired 50 days after initial 3 3 m – 3 3 m lymphocytosis (3.3 10 / L; reference range, 0 0.4 2.9 10 / L) presentation. A, B: Insets are from the MRI performed on initial 3 3 m – 3 3 m and eosinophilia (1.53 10 / L; reference range, 0 1.3 10 / L). presentation. In the caudal medulla oblongata, symmetrical hyper- Urinalysis was normal. Under anesthesia, MRI of the vertebral intensities also are observed in the pyramids of the medulla oblongata fi column from the rst cervical vertebra to the third thoracic (arrows in C). At the level of the fourth cervical vertebra, the a vertebra and the brain was performed using a 3.0T MR unit hyperintensities corresponded to bilaterally symmetric opaque foci and a multichannel phase array spine coil. The following pulse (arrows) on gross sections (D). sequences were obtained: T1-weighted fluid-attenuated inversion recovery (T1W FLAIR), T2-weighted (T2W), T2-weighted FLAIR (T2W FLAIR), and T2*-weighted gradient echo (T2*W) images. on T1W FLAIR images. Abnormal contrast enhancement was not Additionally, axial and sagittal plane T1W FLAIR images of the observed in the spinal cord and brain. vertebral column and the brain were obtained after IV adminis- Cytology and protein analysis of cerebrospinal fluid obtained tration (0.1 mmol/kg) of contrast agentb. from the cerebellomedullary cistern were normal. Based on the In comparison with unaffected white matter of the spinal signalment, history, the neurologic examination, and abnormal- cord, bilaterally symmetric intra-axial hyperintensities on T2W ities observed on MRI, a presumptive diagnosis of LEM was images were noted in the white matter of the dorsolateral fu- made; however, infiltrative disease, such as neoplasia or infectious/ niculi from the cervicomedullary junction extending contigu- noninfectious inflammatory myelitis, could not be excluded. ously to the level of the sixth to seventh cervical intervertebral Consequently, the dog was administered prednisonec (0.5 mg/kg disc (Figures 1A, B). In comparison with the unaffected areas of per os q 24 hr). After 10 days, no improvement was noted. Fifty brainstem, bilaterally symmetric intra-axial hyperintensities on days after initial presentation, the dog was presented for humane T2W images were noted in the pyramids (Figure 1C) and ventral euthanasia. The owner stated that the dog could no longer walk aspect of the crus cerebri of the brain. The lesions also were hy- without falling down; however, if allowed to either run or walk perintense on T2*W and T2W FLAIR images and were isointense fast, the dog would not fall as often.

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Prior to euthanasia and with owner consent, MRI of the cervical vertebral column and brain was performed to determine if the lesions had progressed. Imaging was performed as previously described with the same unit, with the exception that the brain was imaged with the dog in sternal recumbency using an extremity coil to provide improved image quality. The previously identified lesions were present, and new lesions were not identified. Sub- jectively, the lesions in the white matter of the dorsal aspect of the lateral funiculi were larger. The lesions in the brain were un- changed in size. Following MRI, the dog was euthanized and immediately necropsied. At necropsy, gross lesions were restricted to the cervical spinal cord and brainstem. Opaque white, well-demarcated, bilaterally symmetric foci were noted in the white matter in the dorsal aspect of the lateral funiculi of the cervical spinal cord FIGURE 2 Transverse gross and microscopic sections of the spi- (Figures 1D, 2A) and the pyramidal tracts of the medulla nal cord at the level of second cervical vertebra from the 22 mo old oblongata. The crus cerebri were grossly normal. Brain, spinal male rottweiler in Figure 1 reveal lesions involving the white matter. cord, and representative tissue samples of internal organs were A: Similar to Figure 1D, gross transverse section of the cervical spinal fi xed in 10% neutral buffered formalin, processed, embedded in cord at the level of the second cervical vertebra, bilaterally symmetric fi m paraf n, sectioned at 5 m, and stained with hematoxylin and white foci are also evident in the dorsal area of the lateral funiculi eosin. Selected sections of the brain and spinal cord were stained (arrows). B: On a low power magnification of a transverse section of with luxol fast blue. the cervical spinal cord, there are bilaterally symmetric areas of pallor Immunohistochemistry was performed with monoclonal in the lateral funiculi (arrows). Hematoxylin and eosin staining, d antibodies against neurofilament (1:8,0000), glial fibrillary acidic bar ¼ 2 mm. Inset of B: A transverse section of the cervical spinal e f protein ([GFAP], 1:8,000), myelin basic protein ([MBP], 1:2,000), cord from banked tissue from an age matched control dog. Bar ¼ g and canine distemper virus . Also samples from the affected 2 mm. Note the uniform staining of the white matter. C: The bi- fi dorsolateral region of the lateral funiculus of the rst cervical laterally symmetric areas of pallor in the dorsal areas of the lateral fi spinal cord segment were removed at necropsy and xed in 2% funiculi indicate loss of myelin (arrows). Luxol fast blue staining, (para)formaldehyde and 2% glutaraldehyde in 0.1 M phosphate bar ¼ 2 mm. Inset of C: A transverse section of the cervical spinal buffer for transmission electron microscopy. Following dehy- cord from banked tissue from an age matched control dog. Note the dration in graded alcohols, the tissues were embedded in epon- uniform staining of the white matter. Bar ¼ 2mm. D: The loss of araldite.Thinsectionsweremadeandstainedwithleadcitrate myelin is replaced by gliosis with numerous gemistocytic astrocytes and uranyl acetate. (arrows). In the affected areas, vessels are prominent with hyper- fi On low power magni cation, the white matter of the affected trophy of the endothelial cells (arrowheads). Hematoxylin and eosin areas observed on MRI showed a marked pallor consistent with loss staining, original magnification 3400, bar ¼ 40 mm. of myelin (Figures 2B, C). Microscopically, lesions were confined to the white matter of the brain and spinal cord. The most severe trigeminal nerve, and area of the optic tracts were most severely lesions were located in the cervical spinal cord, but lesions ex- affected. The white matter of the cerebellar folia was also multi- tended caudally into the thoracic spinal cord and rostrally into focally affected. The parenchymal portions of the oculomotor the brainstem. Within the cervical spinal cord, the lesion affected nerves in the mesencephalon were mildly affected. the white matter in the dorsal portion of the lateral funiculi in Within the affected white matter, myelin and axon loss was the area of the dorsal spinocerebellar, lateral corticospinal, retic- observed. The myelin and axonal loss was replaced by promi- ulospinal, and rubrospinal tracts. A subpial rim of normal white nent astrogliosis and astrocytosis with numerous GFAP-positive matter was always preserved except at the first cervical spinal cord astrocytic processes and gemistocytic astrocytes in the affected segment where the lesion extended to the pia. In the brain, the regions. Many normal appearing axons were present within the pyramidal tracts, crus cerebri, area of medial lemniscus, caudal area of severe demyelination. The degree of myelin loss exceeded cerebellar peduncle, trapezoid body, area of the spinal tract of the the loss of axons. Degenerative axonal changes were relatively mild,

JAAHA.ORG 257 digestion chambers were rare, and few axonal spheroids were seen. processes (Figure 4A). Myelinated axons were some of the larger In affected areas, vessels were mildly thickened with prominent axons present and were typically irregularly shaped. The myelin endothelial cells and increased cellularity of the perivascular space sheaths had extensive splitting of the lamellae and lack of (Figure 2D). compaction. Schwann cells with similarly myelinated axons To better assess myelin content and axonal changes, banked were present near vessels indicating Schwann cell remyelination tissue samples of the cervical spinal cord from an age matched (Figure 4B). control dog free of neurologic disease were used for comparison. In the affected dog, immunohistochemistry for MBP confirmed Discussion partial to total loss of myelin in the white matter of affected areas The clinical and pathologic features of the dog described in this with patchy staining of the remaining white matter and naked report are consistent with a diagnosis of LEM as previously de- axons compared to the control dog. (Figures 3A, B) Immuno- scribed in rottweilers.1,3 Adefinitive diagnosis of LEM requires histochemistry for neurofilament demonstrated decreased num- histopathology. In this case, MRI precisely identified the affected bers of axons. (Figures 3C, D) Multiple axons had irregular areas, allowing representative transverse sections to be obtained profiles and many were smaller. Only a few axons were larger than for macroscopic, histopathologic, and ultrastructural examina- in the control dog. In the affected dog, immunohistochemistry for tion. In the case reported here, bilaterally symmetric, continuous GFAP revealed the abnormal neuroparenchyma to consist mostly of hyperintensities on T2W images were identified in the same to- gemistocytic astrocytes as well as astrocytic processes (Figures 3E, F) pography as reported for the most severe lesions in LEM in Immunohistochemistry for canine distemper virus was negative. rottweilers.1–3 Moreover, the lesions observed on MRI in the Ultrastructurally, affected spinal cord white matter contained present case correlated with the degenerative changes in the white oligodendroglia, a small number of small naked axons, small matter observed histologically, which included severe loss of numbers of myelinated axons, and hypertrophied astrocytic myelin and astrogliosis. Those findings were similar to findings

FIGURE 3 Microscopic sections of the demyelinated area of dorsolateral lateral funiculus of the first cervical vertebral spinal cord segment from the affected rottweiler compared with the white matter of a similar area of the cervical spinal cord from banked tissue samples from an age matched control dog free of neurologic disease using a variety of immunohistochemical stains. A: Immunohistochemical stain for myelin basic protein (MBP). In the bilaterally symmetric areas of pallor in the cervical spinal cord, there is severe myelin loss around axons with minimal punctuate positive remnants of myelin. MPB with fast red chromogen/hematoxylin counterstain, original magnification 31000. B: For comparison, similar location of the cervical spinal cord from a normal dog stained for MBP with fast red chromogen/hematoxylin counterstain, original magnification 31000. C: Immunohistochemical stain for neurofilament reveals decreased axon numbers. Di- aminobenzidine (DAB) chromogen/hematoxylin counterstain, original magnification 31000. D: For comparison, similar location of the cervical spinal cord from a normal dog stained for neurofilament as in panel C, original magnification 31000. E: Immunohistochemical stain for glial fibrillary acidic protein (GFAP). Myelin loss is replaced by astrocytosis and gemistocytic astrocytes. DAB chromagen/hematoxylin counterstain, original magnification 31000. F: For comparison, similar location of the cervical spinal cord from a normal dog stained for glial fibrillary acidic protein (GFAP) as in panel E. DAB chromagen/hematoxylin counterstain, original magnification 31000. Bar ¼ 15 mm.

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diagnoses for clinical signs referable to the first through fifth cervical spinal cord segments can be eliminated from consider- ation. Other important degenerative nervous system disorders may affect rottweilers. Those degenerative diseases must be considered in the differential diagnoses. NAD is a neurodegenerative disease that presents in young rottweilers as a chronic, progressive, general proprioceptive ataxia and upper motor neuron paresis of all four limbs.6,7 Pathologically, NAD results in axonal swellings (spheroids) throughout the CNS gray matter, except the cerebral cor- tex.6 The spheroids are primarily localized in the distal regions and axon terminals of afferent fibers entering sensory nuclei in the spinal cord, brainstem, and diencephalon.6 Cerebellar atrophy may also be appreciated in more chronic cases.6,7 Although not described in rottweilers with NAD, the MRI findings have been documented in one papillon with NAD.8 At 3 mo of age, no abnormalities were detected; however, at 6 mo of age, diffuse atrophy of the cerebrum, cerebellum, and brainstem were seen in the affected dog.8 In humans with NAD, the most consistent MRI ab- FIGURE 4 Electron micrographs from a demyelinated area of normality observed is a hyperintense cerebellum on T2W and T2 dorsolateral lateral funiculus of the first cervical vertebral spinal cord FLAIR images.9 To the authors’ knowledge, reports detailing im- segment reveal axons that are few in number and scattered among aging studies involving encephalomyelopathy and polyneuropathy oligodendroglial processes. A: Those axons present are irregularly in the rottweiler have not been published. Of the available MRI data shaped (A) with wavy decompacted myelin sheaths (M). Lead cit- for LEM and NAD in the dog, no similarities are seen. Encepha- rate/uranyl acetate staining, bar ¼ 1 mm. B: Schwann cell remye- lomyelopathy and polyneuropathy has also been reported in young lination is evident. Myelinated axons (A) are observed surrounded rottweilers.10 Signs consist of ataxia and paresis involving all four by Schwann cells (S) with basement membrane (arrow). Note the limbs as well as laryngeal and pharyngeal dysfunction.10 Although wavy, decompacted myelin (M) and adjacent hypertrophied astro- initial signs share similarities with LEM, dogs with encephalo- cytic process (As). Lead citrate/uranyl acetate staining, bar ¼ 1 mm. myelopathy and polyneuropathy are typically younger and, with disease progression, signs reflect neuromuscular dysfunction.10 previously described in rottweilers.3 Further, microscopic inves- MRI has been used in other white matter disorders. In spongy tigation in the present case using immunohistochemistry (i.e., degeneration of the CNS in Labrador retrievers, MRI disclosed antibodies against MBP, neurofilament, and GFAP) and electron symmetrical, hyperintense lesions on T2W images, which cor- microscopy corroborated severe myelin loss, astrogliosis/astrocytosis, related with degenerative white matter lesions detected during as well as demonstrating decreased numbers and degeneration of gross and histologic examination.11 Recently, a novel LEM in two axons. In addition, minimal abortive attempts of oligodendroglial leonbergers was described.12 As in the present case, the bilaterally and Schwann cell remyelination were present ultrastructurally. In- symmetric hyperintensities on T2W images were observed in the terestingly, Schwann cells have not be observed in the spinal cord in dorsolateral funiculi of the spinal cord; however, lesions were previously reported cases of LEM.1 Schwanncellinvasionintothe restricted to the second cervical spinal cord segment in one dog spinal cord can occur in a variety of conditions, including primary and to the second to fourth cervical spinal cord segments in the myelin disorders as well as focal compressive and concussive pro- other dog. Additionally, lesions were not noted on MRI of the cesses.4 Although typically excluded from the CNS, Schwann cell brain in affected leonbergers. MRI of globoid cell leukodystrophy invasion occurs at transition zones where the peripheral nervous of the West Highland white terrier revealed bilaterally sym- system interfaces with the CNS, such as the dorsal and ventral root metrical, hyperintense lesions on T2W images, which included entry zones and near blood vessels.5 Consistent with this, the corpus callosum, centrum semiovale, internal capsule, co- Schwann cells in the present case were observed near blood vessels. rona radiate, and cerebellar white matter. Symmetrical contrast Given the MRI characteristics and topography of the lesions enhancement was observed in the corpus callosum, internal observed in the dog reported here, many of the differential capsule, and corona radiata on T1W postcontrast images.13

JAAHA.ORG 259 Demyelinating lesions have been found to correlate well with areas In the present case, MRI was helpful in establishing a pre- of hyperintensity on T2W images in canine distemper.14 Dysmye- sumptive antemortem diagnosis of LEM. Although unknown, it is linogenesis in the English springer spaniel and Portuguese water possible that either early in the course of the disease or if clinical dog secondary to GM1 gangliosidosis was correlated with a mild signs are mild, lesions may not be detected with MRI. The factors hyperintensity of the corona radiata on T2W images.15 that contribute to lesion conspicuity likely include the degree The etiology of LEM remains obscure, and the pathogenesis of myelin loss, number of affected axons (and their diameter), still remains to be determined. Bilaterally symmetric degenerative and number of glial cells in the white matter. Additionally, the lesions in the CNS are usually due to nutritional, metabolic, or sophistication of the MRI unit also may play a role. In the present toxic causes.16,17 It is possible that LEM is a result of an inborn error case, the lesions in the white matter of the dorsal aspect of the of metabolism.3 In humans, numerous primary disorders of myelin lateral funiculi were larger after 50 days. Therefore, a repeat MRI occur secondary to inborn errors of metabolism.18 In two dogs with should be recommended in cases where there is a high index of LEM, biochemical analysis of peripheral blood leukocytes for evi- suspicion yet lesions are not observed. dence of lysosomal storage defects was evaluated for activity of b-galactosidase, b-hexosaminidase, b-hexosaminidase A, aryl sul- Conclusion phatase A, acid phosphatase, b-glucuronidase, a-mannosidase, Understanding the correlation between lesion characteristics and a-fucosidase, b-glucocerebrosidase, b-galactocerebrosidase, and topography on MRI and histologic findings can refine the diag- sphingomyelinase). All evaluations were normal.2 nostic approach categorizing different morphologic changes ob- Based on immunohistochemical and ultrastructural studies in served in the CNS. The ability to establish an accurate presumptive those cases, the lesions are clearly demyelinating with simulta- antemortem diagnosis of LEM has many implications. Although neous, although inadequate and abnormal, remyelination. The not established, given the occurrence of LEM in a specific breed, question remains whether the defect in LEM involves either the rottweiler, a hereditary basis is suspected. The ability to identify a primary disorder of oligodendrocytes (primary demyelination) affected individuals and remove them from the breeding pool is or loss of myelin secondary to primary changes in the axons imperative. Also, owners of affected dogs could be provided an (axonopathy). Oevermann et al. (2008) hypothesized that LEM in accurate prognosis as rottweilers with LEM are typically euthanized the leonberger was a consequence of an alteration in the intimate within 1 yr of diagnosis. relationship between the oligodendrocyte and the neuron so myelin was produced, but not stable, and the topography of the FOOTNOTES a lesion reflected the population of neurons affected.12 In humans, Signa HDx; GE Healthcare, Milwaukee, WI b Magnevist; Berlex Laboratories, Wayne, NJ there is a close relation between myelin and axon, where axonal c Prednisone; West-Ward Pharmaceutical Corp., Eatontown, NJ pathology may precede demyelination.19–21 Interestingly, in this d Neurofilament antibody; Biogenex, San Ramon, CA case, not only was myelin loss observed, but also decreased axonal e Glial fibrillary acidic protein antibody; Abcam, Cambridge, MA fi diameters and irregularity in axonal shape. That nding could be f Myelin basic protein antibody; Abcam, Cambridge, MA related to either loss or disorganization of the neurofilaments, and g Canine distemper virus antibody; VMDR, Pullman WA thus, impaired cytoskeletal organization and axonal transport in dogs with LEM. Minimal attempts at remyelination were seen in REFERENCES this case, some of which was due to Schwann cells migrating into the 1. Gamble DA, Chrisman CL. A leukoencephalomyelopathy of rottweiler dogs. Vet Pathol 1984;21(3):274–80. spinal cord, suggesting disruption of the glia limitans formed 2. Slocombe RF, Mitten R, Mason TA. Leucoencephalomyelopathy in by astroglia, which, as a consequence of disruption, allowed Australian Rottweiler dogs. Aust Vet J 1989;66(5):147–50. Schwann cells to enter the CNS and promote remyelination.22 That 3. Wouda W, van Nes JJ. Progressive ataxia due to central demyelin- could indicate that the Schwann cells were compensating for some ation in Rottweiler dogs. 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JAAHA.ORG 261 Hirschvogel et al. BMC Veterinary Research 2013, 9:57 http://www.biomedcentral.com/1746-6148/9/57

CASE REPORT Open Access Magnetic resonance imaging and genetic investigation of a case of rottweiler leukoencephalomyelopathy Katrin Hirschvogel1, Kaspar Matiasek2, Katharina Flatz3, Michaela Drögemüller4, Cord Drögemüller4, Bärbel Reiner2 and Andrea Fischer1*

Abstract Background: Leukoencephalomyelopathy is an inherited neurodegenerative disorder that affects the white matter of the spinal cord and brain and is known to occur in the Rottweiler breed. Due to the lack of a genetic test for this disorder, post mortem neuropathological examinations are required to confirm the diagnosis. Leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate levels is a rare, autosomal recessive disorder in humans that was recently described to have clinical features and magnetic resonance imaging (MRI) findings that are similar to the histopathologic lesions that define leukoencephalomyelopathy in Rottweilers. Leukoencephalopathy with brain stem and spinal cord involvement is caused by mutations in the DARS2 gene, which encodes a mitochondrial aspartyl-tRNA synthetase. The objective of this case report is to present the results of MRI and candidate gene analysis of a case of Rottweiler leukoencephalomyelopathy to investigate the hypothesis that leukoencephalomyelopathy in Rottweilers could serve as an animal model of human leukoencephalopathy with brain stem and spinal cord involvement. Case presentation: A two-and-a-half-year-old male purebred Rottweiler was evaluated for generalised progressive ataxia with hypermetria that was most evident in the thoracic limbs. MRI (T2-weighted) demonstrated well- circumscribed hyperintense signals within both lateral funiculi that extended from the level of the first to the sixth cervical vertebral body. A neurodegenerative disorder was suspected based on the progressive clinical course and MRI findings, and Rottweiler leukoencephalomyelopathy was subsequently confirmed via histopathology. The DARS2 gene was investigated as a causative candidate, but a sequence analysis failed to identify any disease- associated variants in the DNA sequence. Conclusion: It was concluded that MRI may aid in the pre-mortem diagnosis of suspected cases of leukoencephalomyelopathy. Genes other than DARS2 may be involved in Rottweiler leukoencephalomyelopathy and may also be relevant in human leukoencephalopathy with brain stem and spinal cord involvement. Keywords: Rottweiler, DARS2, LBSL, White matter disease, Progressive ataxia

* Correspondence: [email protected] 1Department of Veterinary Clinical Sciences Ludwig-Maximilians-Universitaet, Neurology Service, Clinic of Small Animal Medicine, Munich, Germany Full list of author information is available at the end of the article

© 2013 Hirschvogel et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hirschvogel et al. BMC Veterinary Research 2013, 9:57 Page 2 of 8 http://www.biomedcentral.com/1746-6148/9/57

Background Case presentation Rottweiler leukoencephalomyelopathy (LEM) was initially A two-and-a-half-year-old male purebred Rottweiler was recognised in the US as a cause of chronic progressive referred for further investigation of progressive ataxia. The ataxia with insidious onset in Rottweilers between 1.5 and dog had been placed in an animal shelter 8 weeks prior to 4 years of age [1]. The clinical and pathological character- the study. Unfortunately, no pedigree data were available, istics of this disease entity were further defined in subse- and we were unable to ascertain whether inbreeding had quent reports originating from Australia, the Netherlands occurred. At the time of shelter placement, the dog had and the UK, which described 16 pathologically confirmed already been ataxic, and the ataxia progressed during the cases (of 22 total cases described in the literature) and subsequent 8 weeks. Haematologic and serum biochem- suggested an autosomal recessive pattern of inheritance ical analyses, thoracic and abdominal radiographs, and [2-6]. In these reports, Rottweiler LEM presented as a dis- echocardiography had been performed prior to referral, tinctive neurodegenerative disorder restricted to the lat- and the findings were unremarkable. eral and dorsal funiculi of the cervical spinal cord and Physical examination showed excessive wearing of the spinal tracts of the trigeminal nerve, pyramids, caudal nails on all four limbs, particularly of the thoracic limbs. cerebellar peduncles, cerebellar medulla and optic tracts A neurological examination showed severe generalised that showed a sharp demarcation between abnormal and ataxia with hypermetria of the thoracic (prolonged pro- normal white matter and occasional microcavitation in traction and overreaching action with limb extension) the centre of the lesion. Clinically, affected dogs exhibit and pelvic limbs. Additionally, difficulties in rising, inter- progressive ataxia with hypermetria and subtle postural mittent crossing of the thoracic limbs, and a wide-based reaction deficits. Thus far, the ante mortem diagnosis of stance of all limbs were observed. The postural reactions LEM in Rottweilers has been based on clinical suspicion (wheelbarrowing with and without neck extension, hop- and the exclusion of other diseases of the cervical spinal ping, and proprioceptive positioning) were delayed, and cord, e.g., compression/instability, neoplasia and inflam- the thoracic limbs were more severely affected than the mation. To date, there have been no magnetic resonance pelvic limbs. A supplemental movie file shows these imaging (MRI) studies or genetic investigations of this dis- findings in more detail (see Additional file 1). The spinal ease entity. reflexes (extensor carpi radialis, thoracic and pelvic limb Leukoencephalopathy with brain stem and spinal cord flexor, patellar, cranial tibial, gastrocnemius, cutaneous involvement and lactate elevation (LBSL) is a neurodegen- trunci, and perineal) were all normal. The mentation erative disease in humans with clinical features and MRI and cranial nerve function, including vision, were unim- findings that are surprisingly similar to the histopathologic paired, but an inconsistent menace response was ob- lesions of LEM in Rottweilers. Specifically, these patients served; this was attributed to the lack of cooperation by exhibit slow progressive spasticity and ataxia, MRI find- the dog but could also indicate a cerebellar lesion. Palpa- ings of selective involvement of the brain stem and spinal tion of the head and spine and neck flexion and exten- tracts in both lateral funiculi and dorsal columns and sion did not elicit any signs of pain. There was no changes in the cerebral and cerebellar white matter. Spinal evidence of tremor or uncoordinated movements of the cord involvement with MR signal intensity changes has head. The findings of the neurological examination were also been reported in other leukodystrophies in humans, most consistent with a cervical myelopathy (C1-C5 e.g., adult onset autosomal dominant leukodystrophy with spinal cord segments) involving the spinocerebellar autonomic features, Alexander’s disease, vitamin B12 defi- tracts, although a cerebellar lesion could not be ruled ciency myelopathy and sporadic cases of adult onset lyso- out completely. The differential diagnoses included several somal leukodystrophies [7-11]; however, a very distinct breed-related neurodegenerative disorders: neuronal vacu- and well-demarcated pattern of signal intensity change is olation and spinocerebellar degeneration, neuroaxonal dys- considered to be most characteristic of LBSL. In LBSL, trophy, LEM, cervical spondylomyelopathy and arachnoid high levels of lactate are frequently demonstrated in brain diverticula [16-20]. lesions using magnetic resonance (MR) spectroscopy; this A follow-up laboratory examination revealed mild eo- finding suggests a respiratory chain defect, but lactate is sinophilia (1.51 ×103 eosinophilic granulocytes/μl; reference rarely elevated in the blood or cerebrospinal fluid (CSF) range: 0.04 - 0.6 ×103/μl) and unremarkable serum bio- [12,13]. To date, all human cases of LBSL have been found chemical results. The dog was subsequently anesthetised to be caused by mutations in the DARS2 gene, which en- for further examination of the cervical spine and brain codes mitochondrial aspartyl-tRNA synthetase [14,15]. using MRI and CSF analysis. Electrodiagnostic examination To investigate the hypothesis that LEM in Rottweilers was scheduled as a supplementalexaminationtoinvestigate could represent a possible animal model of LBSL, MRI the presence of additional lesions in the peripheral nerves. results and DARS2 gene integrity were investigated in a Magnetic resonance imaging was performed using a 1.5 T single, affected dog. magnetic resonance unit. The brain imaging protocol Hirschvogel et al. BMC Veterinary Research 2013, 9:57 Page 3 of 8 http://www.biomedcentral.com/1746-6148/9/57

utilised sagittal, dorsal and transverse T2-weighted (TR/TE 5190/108 ms) and T1-weighted (TR/TE 386/13 ms) sequences and transverse FLAIR (TR/TE/TI 9110/122/ 2500 ms) and gradient echo (TR/TE 1000/28 ms) sequences. The spinal imaging protocol included sagittal and dorsal T2-weighted (TR/TE 2880/111 ms) and T1- weighted (TR/TE 623/1 ms), transverse T2-weighted (TR/ TE 3290/99 ms) and T1-weighted (TR/TE 651/12 ms) and sagittal STIR (TR/TE/TI 3310/61/140 ms) sequences. The sagittal and dorsal spinal sequences were performed from C1 to T3 (vertebral body), and the transverse sequences used C1 to C7 (vertebral body). Gadolinium (0.1 mmol/kg; 0.045 mmol/lb) was administered intravenously, and postcontrast transverse T1-weighted sequences of the brain and dorsal and sagittal T1-weighted sequences of the spine were acquired. Descriptions of intensity referred to normal appearance of grey matter. The spinal MRI studies showed bilateral symmetrical hyperintensities in the region of both lateral funiculi on transverse T2-weighted images (Figure 1). The lesions were most visible on the transverse sections; they appeared well demarcated and ovoid and extended from the level of the first to the sixth cervical vertebral body (Figure 2). In T1-weighted plain images, the lesions were isointense, and no contrast enhancement was observed. MRI studies of the brain failed to reveal any abnormalities. Routine CSF analysis (cisterna cerebellomedullaris) with leukocyte (0/μl; reference range 0- 5/μl) and erythrocyte counts (4/μl), CSF cytology and protein measurements (0.18 g/l; reference range 0–0.3 g/l) were unremarkable, as were the lactate concentrations in the CSF (1.6 mmol/l; reference range 0.2-3.1 mmol/l [21,22]) and serum (1.0 mmol/l; reference range 1.1-

Figure 2 Dorsal T2-weighted MR images of the cervical spinal Figure 1 Transverse T2-weighted MR images of the cervical cord. The image shows linear, hyperintense signals (arrow) spinal cord at the level of the C4-C5 intervertebral disc space. corresponding to the lesions in Figure 1 that extend from the level The images show well-demarcated, ovoid, hyperintense signals with of the first to the sixth cervical vertebral body in a bilateral, a bilateral, symmetrical appearance in the region of the lateral symmetrical fashion. The line denotes the C4-C5 intervertebral funiculi (arrow). disk space. Hirschvogel et al. BMC Veterinary Research 2013, 9:57 Page 4 of 8 http://www.biomedcentral.com/1746-6148/9/57

3.3 mmol/l [22]). No abnormal spontaneous activity pat- examination. Histologically, the cervical spinal cord (from tern was observed during electromyographic recordings C2 to C6 (vertebral body)) exhibited severe, bilaterally sym- using a concentric needle electrode in the anesthetised metrical funicular disruption of the inner dorsal part of the dog. The tibial and ulnar motor nerve conduction vel- lateral funiculus, including the rubrospinal tract, the inner- ocity, tibial nerve F-waves and repetitive nerve stimula- most layer of the dorsal spinocerebellar tract and the dorsal tion were within established laboratory reference ranges. aspects of the lateral fasciculus proprius. Upon low-power Considering the progressive clinical course and the inspection, the lesion was characterised by a severe loss of MRI lesion pattern, a neurodegenerative disorder pre- myelin staining; at high-power, the lesion resembled a dominantly involving the cervical spinal cord white mat- dense core of non-myelinated white matter with extensive ter with a bilateral and symmetrical distribution was astrocytosis and astrogliosis with the occasional observa- suspected. Due to the existing severe neurological signs, tion of bizarre cells surrounded by a rim of spongiotic the progressive deterioration and the poor prognosis, the white matter with fibre degeneration, resorptive lesions, dog was euthanised. vascular prominence and mild-to-marked angiocentric A complete necropsy was performed, and it confirmed lymphohistiocytic infiltration. The adjacent cervical grey Rottweiler LEM. Significant lesions were confined to the matter appeared hypoplastic in both the ventral and dorsal central nervous system. Macroscopic examination revealed horns, but there were no further histomorphological bilateral, symmetrical lesions restricted to the dorsal aspect changes. Another severe white matter lesion identified in of the lateral funiculi of the cervical spinal cord segments. the cerebellar roof showed focal, bilaterally symmetric tis- In transverse sections, these lesions appeared as well- sue necrosis, macrospongiosis due to interlamellar myelin demarcated, whitish, opaque discoloured areas (Figure 3). sheath oedema (ballooning) and severe intralesional No gross changes were observed in the brain. Formalin- astrogliosis and astrocytosis accompanied by fibrillary fixed and paraffin-embedded tissue samples of the brain astrogliosis and gemistocytes at the margins. A moderate and spinal cord were sectioned at 5 μm and stained using vascular prominence with endothelial hyperplasia was haematoxylin-eosin and Luxol Fast Blue for histological again observed both in the intra- and perilesional areas.

Figure 3 Pathological lesions in the brain (A, B) & spinal cord (C, D). The most severe white matter lesions were observed in the cerebellum (A: asterisk) and cervical spinal cord (C: framed area). Macroscopic examination revealed bilateral, symmetrical lesions in the lateral funiculi of the cervical cord segments only. In transverse sections, these lesions appeared as well-demarcated, whitish, opaque discoloured areas (C: framed area). The cerebellar lesions spared the fibres adjacent to the roof nuclei (A: arrow). Nuclear degeneration was most severe in the raphe nuclei (B) and medial vestibular nuclei (not shown). Note the extensive juxtaneuronal vacuolisation (B: asterisk). The affected spinal cord segments show demyelination, astrogliosis and astrocytosis (D: white arrowhead) with gemistocytes (D: black arrowheads). Within the grey matter, hypoplasia of the dorsal and ventral horn (C: black arrow) is evident. Scale bars: A: 1.5 cm; B: 100 μm; C: 2 mm; D: 35 μm. Hirschvogel et al. BMC Veterinary Research 2013, 9:57 Page 5 of 8 http://www.biomedcentral.com/1746-6148/9/57

Necrotic areas exhibited macrophage-mediated resorption changes is very helpful in defining disease because it re- (Figure 3). veals the distribution of histopathologic changes [24,25]. Similar demyelinating lesions were observed in the pyra- At present, there are few case reports describing the use mids and caudal cerebellar peduncles and – to a lesser of MRI for the diagnosis of canine and feline neurodegen- extent – in the medial lemniscus, optic tracts, crura erative diseases. T2-weighted hyperintensities of white cerebri and subcortical white matter. Lesions in the cen- brain matter were evident in cats with GM2 gangliosidosis tral visual pathways projected to the optic nerves and [26,27] and in a West Highland white terrier with globoid manifested as the degeneration of multiple fibres. Further cell leukodystrophy [28]. Increased signal was also evident brain stem changes included macrovacuolar degeneration in T2-weighted images of the spinal cord of Leonberger of the raphe nuclei and medial vestibular nuclei associated dogs with leukoencephalomyelopathy [29]. Dogs with with mild gliosis and axonal spheroids. Immunohisto- GM2 gangliosidosis displayed T2-weighted hyperintensities chemical staining for canine distemper virus was negative. in the region of the caudate nucleus and atrophy of the A mild diffuse endoneurial hypercellularity was observed cerebrum and cerebellum [30,31]. MRI of Papillon dogs in the preganglionic aspects of the dorsal roots of the cer- with neuroaxonal dystrophy [32] and Scottish Terriers with vical spine. Both the radial and common peroneal nerve hereditary cerebellar degeneration demonstrated atrophy presented with a mild dropout of myelinated fibres, as de- only and failed to detect changes in white matter [33]. noted by enlarged subperineurial spaces with myxoid re- MRI of the cervical spine may be used to support the placement oedema, reduced endoneurial area and clinical diagnosis of LEM in Rottweilers. A similar MRI decreased myelinated nerve fibre density that was associ- pattern has been described in Leonberger dogs with ated with a mild expansion of the endoneurial collagenous LEM [29]. Interestingly, however, brain lesions were not matrix. Residual large A (alpha)-type myelinated fibres detected using MRI in the Leonberger dogs or in the showed myelin ovoids, consistent with stage II – III case reported here, although histological analyses Wallerian degeneration, and abundant internodal and showed that the optic tracts and particularly the cerebel- paranodal inner and outer myelin loops due to the moder- lar medulla were significantly affected in both breeds ate axonal atrophy of the respective fibres. [2,29]. It is possible that the white matter lesions in the Due to the phenotypic similarities between human brain were less advanced than those in the spinal cord at LBSL patients and LEM-affected Rottweilers, the DARS2 the time of imaging, and improved imaging protocols gene was investigated as a candidate for canine LEM. may be required for the visualisation of brain lesions. Genomic DNA was extracted from blood collected in These protocols may include smaller slice thicknesses tubes containing EDTA using DNeasy blood spin col- and the application of sequences other than conven- umns (Qiagen). For the DARS2 mutation analysis, suit- tional T1- and T2-weighted imaging, e.g., diffusion ten- able PCR products were amplified using AmpliTaq Gold sor imaging, magnetisation transfer imaging or MR 360 (Life Technologies). The PCR products were spectroscopy [34]. It is also questionable whether the resequenced after rAPid alkaline phosphatase (Roche) pathological changes in these regions have sufficiently al- and exonuclease I (New England Biolabs) treatment tered the physics of the tissue to induce changes visible using both PCR primers and the ABI BigDye Terminator with a 1.5 T clinical scanner. Sequencing Kit 3.1 (Life Technologies) in an ABI 3730 Many inherited white matter diseases and associated sequencer (see Additional file 2: Table S1). The sequence genetic defects have been described in humans [35]. data were analysed using Sequencer 4.9 software Leukoencephalopathies may be characterised as lysosomal (GeneCodes). The sequences of all 17 coding exons and or peroxisomal disorders, mitochondrial disorders, methy- flanking intron sequences of the DARS2 gene from the lation cycle disorders, organic acidaemias or amino acid affected Rottweiler were identical to a canine reference disorders or as leukoencephalopathy associated with calci- genome sequence (CanFam3 assembly; http://genome. fication, hypomyelination, abnormal lipid metabolism, ucsc.edu). vasculopathy or muscular dystrophy. Many distinct en- tities, e.g., Alexander’s disease, adult onset autosomal dom- Conclusion inant leukoencephalopathy, vanishing white matter disease Magnetic resonance imaging has become the primary and adult polyglucosan encephalopathy, have also been tool for the ante mortem diagnosis of white matter dis- recognised. A vast number of genetic defects are currently ease in humans due to its high sensitivity for detecting associated with these conditions, and many more remain changes in white matter. Decreased myelin and elevated to be elucidated; the molecular cause remains unknown water content is revealed by increased T1 and T2 relax- in ~50% of affected humans [35]. The lesion distribution ation times, with a consequent reduction in signal inten- and MRI appearance of LBSL are considered unique and sity in T1-weighted images and increased signal intensity diagnostic in humans; consequently, only a single candi- in T2-weighted images [23]. Thus, the pattern of MRI date gene was examined in the present study [36]. Hirschvogel et al. BMC Veterinary Research 2013, 9:57 Page 6 of 8 http://www.biomedcentral.com/1746-6148/9/57

Leukoencephalopathy with brain stem and spinal cord Further limitations of our case report include the lack involvement is a rare, autosomal recessive disorder that of pedigree analysis and brain lactate MR spectroscopy typically manifests in childhood or adolescence. The measurements and the failure of MR to demonstrate the diagnosis of LBSL in humans is based on clinical presen- involvement of the cerebrum despite the pathology ob- tation and is characterised by a slowly progressive cere- served in histological sections. Another limitation is that bellar ataxia, spasticity, dorsal column dysfunction and a the comparison of the pathologies of these diseases in highly characteristic pattern of abnormalities observed dogs and humans is limited by the paucity of case data using MRI and spectroscopy. Typical MRI findings in- from both. To date, there is only one short description clude a combination of high T2-weighted signal changes of the pathology of LBSL in humans which has shown in the cerebral white matter accompanied by the select- spongy white matter degeneration, rarefaction of the ive involvement of the brain stem and spinal cord tracts neuropil, macrophage infiltration and an increased num- (the entire length of the pyramidal tracts with the add- ber of astrocytes in the white matter of the brain and itional involvement of cerebellar connections and the axonal degeneration of the peripheral nerves [25]. Spinal intraparenchymal and mesencephalic parts of the tri- cord changes have not been noted, but it is unknown geminal nerve) [13,37]. MR spectroscopy demonstrates whether this part of the CNS was sampled and investi- an elevation in lactate levels in the abnormal white mat- gated. In dogs with LBSL-like changes, the neuroana- ter of almost all of the affected human patients. These tomical mapping of CNS lesions is more precise [2,29]. findings led researchers to assume that the disease was a Clinical and pathological findings emphasise cerebellar mitochondrial disorder, which was subsequently con- and spinal changes, although the microscopic white mat- firmed by the discovery of various mutations in the ter damage is far more widespread and extends from the DARS2 gene, which encodes mitochondrial aspartyl- lower brain stem to the subcortical white matter. Con- tRNA synthetase [14,38]. As demonstrated by multiple sistent with the fibres affected, Gamble et al. discovered case reports of LBSL in humans, normal CSF and blood secondary grey matter changes in connected brain stem lactate concentrations, as were noted in the case nuclei, such as the accessory cuneate nucleus, nucleus reported herein, do not exclude a mitochondrial disorder gracilis, nucleus cuneatus and nucleus of the dorsal as the underlying cause of leukoencephalomyelopathy. spinocerebellar tract [1]. However, the involvement of Thus, further investigations should utilise MR spectros- multiple independent centres and tracts is compatible copy to investigate the possible mitochondrial origin of with multisystemic degeneration, as has been shown in Rottweiler LEM. Leonbergers and Rottweilers (discussed above) [2,29]. The diagnosis of mitochondrial disorders faces spe- We also discovered macrovacuolar nuclear degeneration cific difficulties due to the complex genetics of these in the Rottweiler, which has not previously been de- conditions. Mitochondrial disorders may occur due to scribed in dogs. Vacuole formation in LBSL patients was mutations in mitochondrial genes or mutations in nu- predominantly perineuronal and was therefore dissimilar clear proteins, with mitochondrial tRNA representing a to the neuronal vacuolation and spinocerebellar degener- hot spot for mutations. Heteroplasmy, i.e., the simul- ation observed in Rottweiler dogs [16]. This degener- taneous presence of mutated and normal RNA/DNA in ation merits further examination to investigate the the cell, is a characteristic feature of mitochondrial dis- relationship between neurons and astrocytes in the sub- orders. The degree of heteroplasmy varies between tis- cortical grey matter. It also remains to be established sues in the same organism, which is considered a whether this manifestation causes the white matter path- critical factor in the manifestation of mitochondrial dis- ology or whether it is an additional, co-existing disorder ease in specific tissues [36,39]. Finally, we investigated that is distinct from the breed-specific neurodegenera- the coding region of the canine DARS2 gene as a candi- tive disorders described above. date causative gene for LEM, and no mutation was In summary, magnetic resonance imaging revealed found. At this time, we cannot rule out the possibility leukodystrophic lesions in the lateral funiculi of the cer- of variants in the promoter or intronic regions that vical spinal cord; these findings will assist in the ante could affect DARS2 expression. More comprehensive mortem diagnosis of future cases of suspected LEM in DNA sequencing approaches, such as the use of next- Rottweilers. Further investigations should utilise MR generation technologies for whole-exome or whole- spectroscopy to investigate the possible mitochondrial genome resequencing, may enable the identification of origin of Rottweiler LEM. Although LEM is similar to the causative mutation of Rottweiler LEM. A recent LBSL based on its clinical features and imaging results, study identified the causative mutation of canine neo- we were unable to identify a coding or splice site muta- natal cerebellar cortical degeneration in SPTBN2 (gen- tion in the canine DARS2 gene in our case, suggesting ome-wide mRNA sequencing) using only a single case that other genes may be involved in Rottweiler LEM and of this neurodegenerative disease [40]. potentially also in human LBSL. Hirschvogel et al. BMC Veterinary Research 2013, 9:57 Page 7 of 8 http://www.biomedcentral.com/1746-6148/9/57

Additional files neural pathways or vascular territories. AJR Am J Roentgenol 1995, 165(3): 515–523. 11. van der Knaap MS, Ramesh V, Schiffmann R, Blaser S, Kyllerman M, Gholkar Additional file 1: Movie of a Rottweiler with confirmed A, Ellison DW, van der Voorn JP, van Dooren SJ, Jakobs C, et al: Alexander leukoencephalomyelopathy. The movie shows the severe generalised disease: ventricular garlands and abnormalities of the medulla and ataxia with hypermetria of the thoracic (with prolonged protraction, spinal cord. Neurology 2006, 66(4):494–498. overreaching action and limb extension) and pelvic limbs. The postural reactions were delayed, and the thoracic limbs were more severely 12. Miyake N, Yamashita S, Kurosawa K, Miyatake S, Tsurusaki Y, Doi H, Saitsu H, affected than the pelvic limbs. Matsumoto N: A novel homozygous mutation of DARS2 may cause a severe LBSL variant. Clin Genet 2011, 80(3):293–296. Additional file 2: Sequencing methods and primers. 13. van der Knaap MS, van der Voorn P, Barkhof F, Van Coster R, Krageloh-Mann I, Feigenbaum A, Blaser S, Vles JS, Rieckmann P, Pouwels PJ: A new leukoencephalopathy with brainstem and spinal cord involvement and Abbreviations high lactate. Ann Neurol 2003, 53(2):252–258. CSF: Cerebrospinal Fluid; FLAIR: Fluid-Attenuated Inversion Recovery; 14. Scheper GC, van der Klok T, van Andel RJ, van Berkel CG, Sissler M, Smet J, LBSL: Leukoencephalopathy with Brain Stem and Spinal Cord Involvement; Muravina TI, Serkov SV, Uziel G, Bugiani M, et al: Mitochondrial aspartyl- LEM: Leukoencephalomyelopathy; MR: Magnetic Resonance; MRI: Magnetic tRNA synthetase deficiency causes leukoencephalopathy with brain Resonance Imaging; Ms: Millisecond; TE: Time to Echo; TR: Time to stem and spinal cord involvement and lactate elevation. Nat Genet 2007, Repetition. 39(4):534–539. 15. van Berge L, Dooves S, van Berkel CG, Polder E, van der Knaap MS, Scheper Competing interests GC: Leukoencephalopathy with brain stem and spinal cord involvement The authors declare that they have no competing interests. and lactate elevation is associated with cell-type-dependent splicing of mtAspRS mRNA. Biochem J 2012, 441(3):955–962. Authors’ contributions 16. Kortz GD, Meier WA, Higgins RJ, French RA, McKiernan BC, Fatzer R, Zachary KH was responsible for data collection and interpretation and for drafting JF: Neuronal vacuolation and spinocerebellar degeneration in young the manuscript. KM performed the necropsy and histopathology and rottweiler dogs. Vet Pathol 1997, 34(4):296–302. provided histopathology images. KF performed all diagnostic imaging 17. Cherrone KL, Dewey CW, Coates JR, Bergman RL: A retrospective procedures and selected the appropriate images. MD and CD conducted the comparison of cervical intervertebral disk disease in genetic study. BR contributed substantially to the acquisition of data used in nonchondrodystrophic large dogs versus small dogs. J Am Anim Hosp the manuscript. AF contributed to data collection, helped draft the Assoc 2004, 40(4):316–320. manuscript and finalised the version to be published. All authors have 18. Jurina K, Grevel V: Spinal arachnoid pseudocysts in 10 rottweilers. J Small approved the final manuscript. Anim Pract 2004, 45(1):9–15. 19. Skeen TM, Olby NJ, Munana KR, Sharp NJ: Spinal arachnoid cysts in 17 Author details dogs. J Am Anim Hosp Assoc 2003, 39(3):271–282. 1Department of Veterinary Clinical Sciences Ludwig-Maximilians-Universitaet, 20. Cork LC, Troncoso JC, Price DL, Stanley EF, Griffin JW: Canine neuroaxonal Neurology Service, Clinic of Small Animal Medicine, Munich, Germany. dystrophy. 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doi:10.1186/1746-6148-9-57 Cite this article as: Hirschvogel et al.: Magnetic resonance imaging and genetic investigation of a case of rottweiler leukoencephalomyelopathy. BMC Veterinary Research 2013 9:57.

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