Abstract The gross and histopathological findings in thebrain and spinal cord of five Alaskan Husky dogs with anovel incapacitating and ultimately fatal familial and pre-sumed hereditary neurodegenerative disorder are de-scribed. Four dogs presented with neurological deficitsbefore the age of 1 year (7–11 months) and one animal at2.5 years old. Clinical signs in all dogs were of acute on-set and included ataxia, seizures, behavioral abnormali-ties, blindness, facial hypalgesia and difficulties in pre-hension of food. In animals allowed to survive, the dis-ease was static but with frequent recurrences. Pathologicalfindings were limited to the central nervous system.Grossly visible bilateral and symmetrical cavitated fociwere consistently present in the thalamus with variableextension into the caudal brain stem. Microscopic lesionswere more widespread and included foci of bilateral andsymmetrical degeneration in the basal nuclei, midbrain,pons and medulla, as well as multifocal lesions at the baseof sulci in the cerebral cortex and in the gray matter ofcerebellar folia in the ventral vermis. Neuronal loss withconcomitant neuronal sparing, spongiosis, vascular hyper-trophy and hyperplasia, gliosis, cavitation and transientmixed inflammatory infiltration were the main histo-pathological findings. In addition, a population of reactivegemistocytic astrocytes with prominent cytoplasmic vac-uolation was noted in the thalamus. Lesions of this naturein this distribution within the neuroaxis have not been re-ported in dogs. The neuropathological findings resembleLeigh’s disease/subacute necrotizing encephalomyelo-pathy of man. Neuronal sparing in conjunction with ap-parently transient astrocytic vacuolation point to the pos-
sible pathogenetic role of astrocytes in the evolution ofthese lesions. An inherited metabolic derangement of un-known nature is postulated as the cause of this breed-spe-cific disorder.
Key words Dog · Alaskan Husky · Metabolic encephalopathy · Leigh’s disease · Subacute necrotizing encephalomyelopathy
Introduction
Since its initial description in 1951 [25], the term Leigh’sdisease (LD) or, more appropriately, Leigh syndrome (LS)[12, 50] has been used in human neurology and neu-ropathology to designate patients with characteristic bilat-eral and symmetrical brain stem lesions that feature tissuedestruction, capillary proliferation, and neuronal sparing.The precise distribution of lesions within the brain stemand involvement of other parts of the central nervous sys-tem (CNS) vary [9, 16, 27]. Until the 1980s, variability inthe clinical presentation of LS allowed confident diagno-sis to be established only by postmortem examination [46,50]. In the last few years, clinical and neuroradiologicalfindings have been defined and permit antemortem pre-sumptive diagnosis [12, 21, 43, 45, 49]. Investigations ofLS have revealed an array of biochemical and genetic ab-normalities, clearly demonstrating that the characteristiccomplex of neuropathological features traditionally re-quired to make this diagnosis does not correlate to a singleand discrete disease entity. Rather, it has been proposedthat LS may be viewed as a paradigm in that it representsthe response of the developing CNS to energy deprivation[12]. Currently, some 75% of the cases in which the typi-cal phenotype of LS is found are known to be caused bydiverse defects of the mitochondrial respiratory chain.The cause of the remaining cases is unknown [12].
In 1992, one of us (A.D.) recognized a novel degener-ative disease affecting the CNS of juvenile AlaskanHusky dogs. Between 1992 and 1998 neurological andneuropathological studies were carried out at our institu-
Ori Brenner · Joseph J. Wakshlag ·Brian A. Summers · Alexander de Lahunta
Received: 6 August 1999 / Revised, accepted: 18 October 1999
REGULAR PAPER
O. Brenner · J. J. Wakshlag · B. A. Summers · A. de Lahunta (!)Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401, USAFax: +1-607-253-3541Present address:O. BrennerExperimental Animal Center, The Weizmann Institute of Science, Rehovot 76100, Israel
tion on five Alaskan Husky dogs (four females and onemale, from four litters) with this condition (Table 1).Pathological findings of the first cases (dogs 1 and 2 ofthis report) have been briefly reported [42]. Here we pre-sent the first comprehensive neuropathological descrip-tion of this disorder based on necropsy studies of the fiveaffected animals. We have designated this diseaseAlaskan Husky encephalopathy.
Materials and methods
Clinical evaluation and necropsy of five dogs (dogs 1–5) were per-formed at the College of Veterinary Medicine at Cornell Univer-sity. Specimens of brain, spinal cord, peripheral nerves and vis-ceral organs were fixed in 10% neutral buffered formalin, pro-cessed routinely in an automatic tissue processor, embedded inparaffin, sectioned at 5 µm, and stained with hematoxylin andeosin (H&E). Selected CNS sections were stained with Luxol-fastblue-cresyl Echt violet and Bielschowsky’s silver stain.
Immunohistochemical examination was performed on deparaf-finized sections processed by streptavidin-biotin-peroxidase com-plex procedure with diaminobenzidine as the chromogen. The pri-mary antibodies against glial fibrillary acidic protein (GFAP;Dako, polyclonal, 1 :300) and vimentin (Dako, monoclonal, 1 :40)were used. Before staining for vimentin, slides were microwavetreated.
Results
Clinical findings
A detailed description of the neurological findings is re-ported separately [48]. In brief, the onset of clinical signswas before 1 year of age (7–11 months) in four of the fivecases and at 2.5 years of age in other dog. The onset wasusually sudden with either ataxia (n =3) or seizures (n =2). In two dogs, both seizures and ataxia developed duringthe course of the disease. The ataxia included varying de-grees of cerebellar and vestibular signs with hypermetriaand balance loss. Gait abnormalities also included hyper-tonicity of all four limbs and proprioceptive deficits. Mostdogs had a disturbance of their behavior varying from ob-tundation to propulsive pacing and apparent visual deficits.Prehension of food was often abnormal. Decreased noci-ception, especially facial hypalgesia, was noted in someanimals. In most dogs the neuroanatomic diagnosis wasdiffuse involvement of the brain including cerebrum,brain stem and cerebellum. In dogs that were observed for
longer periods, the signs either remained static or improved,but recurrences were common and included both gait ab-normalities and seizures. One female dog (not included inthis report) died naturally at 4 years of age following adisease course lasting over 1 year. All other animals wereeuthanized between 2 and 7 months following onset ofclinical signs.
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Table 1 Signalment and clini-cal signs of five AlaskanHusky dogs with AlaskanHusky encephalopathy
a All dogs were euthanizedb Littermatesc An affected female littermatewas diagnosed by computedtomography at the age of 2.5years and died naturally at 4years old
Dog Gender No. Age at Age at Clinical signsaffected/ onset deathlitter size (months) (months)a
3 F 2/4b 8 10 Ataxia4 F 2/4b 11 18 Seizures, episodic ataxia, visual deficits5 F 2/6c 30 32 Episodic seizures, semicoma, ataxia,
propulsive behavior
Fig.1 A Bilateral and symmetrical oblique cavitation of the thal-amus. B In some dogs, the thalamic lesion extends caudally to thereticular formation in the medulla oblongata, where it retains itsoblique orientation (arrows). Note bilateral and symmetrical de-generation in the white matter of the reticulospinal and rubrospinaltracts (asterisks). Both lesions are seen at this magnification as re-duced myelin staining (LFB Luxol-fast blue). A, B Dog 1. LFBstaining; A × 3, B × 4.6
Pathology
Gross examination
The outer surface of the brain and spinal cord was normal.Transverse sections of the brain revealed bilateral andsymmetrical soft gray cavities in the thalamus, which ex-tended to the medulla in severely affected dogs (Fig.1). Inless profoundly affected dogs, the bilateral and symmetri-cal cavities were segmental rather than contiguousthroughout the brain stem. The thalamus was invariablythe most extensively affected region. Thalamic cavitarychanges were oriented along an oblique dorsolateral toventromedial axis, resulting in a V-shaped appearance,and involved approximately a third of the parenchyma,measuring on average 1.5 × 0.5 cm. More caudally, themalacic foci were markedly smaller but tended to retainan oblique orientation. In the cerebrum, the cortical rib-bon at the base of numerous sulci was attenuated andslightly brown tinged. Such cerebral foci were randomlydistributed, although concentrated in the parietal and tem-poral lobes. No gross abnormalities were detected in thespinal cord or outside the CNS.
Light microscopy
Distribution and classification of the lesions. All fivedogs had histopathological CNS lesions of similar natureand distribution but of variable severity. Brain lesionswere found in the cerebrum, brain stem and cerebellum,and occurred in two distribution patterns: (1) bilateral andsymmetrical degeneration within the basal nuclei, thala-mus, midbrain, pons and medulla oblongata and (2) mul-tifocally at the base of sulci in the cerebral cortex and inthe cortex of the ventral vermis of the cerebellum. In re-gions where gray and white matter are separated, neuro-parenchymal changes primarily affected the gray matter.In the brain stem, this predilection was less discernable.Lesions in the spinal cord were mild, inconsistent andlimited to the white matter. All brain lesions exhibitedneuronal depletion with variable neuronal sparing, vascu-lar prominence, spongiosis and gliosis. Distinction betweenactive degeneration and quiescent lesions was made. Thefollowing were considered indicative of ongoing degener-ation: marked vascular prominence, the presence of intactand ischemic neurons, glial necrosis, mild to moderategliosis and an occasional mixed infiltrate of inflammatorycells, thought to be secondary to tissue necrosis. Quies-cent foci were characterized by less prominent vascula-ture with more advanced gliosis and neuronal loss in theabsence of ischemic neurons. Spongiosis and cavitarychanges were observed in both types of lesions. In activedegeneration, such cavities occurred in a mildly to moder-ately gliotic neuropil and contained gitter cells with occa-sional lymphocytes. In quiescent ‘burnt out’ lesions, cav-ities were surrounded by a sclerotic neuropil and gittercells were absent. Both active and inactive lesions coex-isted in the same animals, and were sometimes juxtaposed
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Fig.2A–C An inactive lesion with well-demarcated boundaries(arrows) visible at this magnification because of associated myelinloss. The affected area is profoundly gliotic and the center has un-dergone cavitation. B Neuronal survival in a gliotic region. Tworeactive gemistocytic astrocytes are indicated by arrows. C Sur-vival of neurons with normal morphology (arrows) in a cavitatedarea. A, B Dog 2, C dog 1. A LFB, × 15; B H&E, × 152; C H&E, × 142
and many foci displayed features intermediate betweenthese two extremes.
Thalamus. In all animals, the most extensive gross lesionwas a bilateral and symmetrical obliquely oriented cavita-tion situated approximately in the mid thalamus. Histo-logically, this corresponded to a well-demarcated focus ofsevere gray matter liquefaction with lesser degeneration
in the surrounding white matter (Fig.2). In the center ofthe lesion, the neuroparenchyma had undergone almostcomplete dissolution leaving an empty space traversed byinfrequent blood vessels, astrocytic processes and lownumbers of axons. At the margins of the cavities, the neu-ropil was replaced by an admixture of reactive gemisto-cytic astrocytes, gitter cells, proliferated capillaries andsurviving axons, some with focal swellings (spheroids). Inthis region, and less commonly in the more frankly cavi-tated center, there were variable numbers of survivingneurons, either isolated or in small groups within irregularislands of neuropil. Most surviving neurons appeared nor-mal but occasional swollen and chromatolytic forms werealso encountered. Despite a normal appearance in H&E-stained sections, the perikaryon of some neurons in af-fected foci stained black with a silver stain. Typically, thiswas observed in areas with features of long-standing de-generation containing low numbers of surviving neurons.In contrast, neurons in areas of active degeneration werenot argyrophilic.
Microgliosis and astrogliosis of variable intensity wereobserved in non-cavitated lesions. A GFAP preparationemphasized the focal nature of the gliosis and the sharpdelineation between affected and unaffected parenchyma,in which only scattered Wallerian degeneration was seen.
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Fig.3A–D Thalamus. A A discrete focus with both active andquiescent phases of degeneration. In this GFAP preparation, ex-tensive astrogliosis is seen as punctate dark structures scatteredthroughout much of the lesion. Two cavitated areas are present.Peripheral to the gliotic region are segments undergoing active de-generation (solid arrow and open arrow), one of which (open ar-row) is identified at this magnification by its lack of GFAP stain-ing. B Higher magnification of a region undergoing active degen-eration (indicated by an open arrow in A). The neuropil is edema-tous, partly dissolved and contains an admixture of proliferatedglial and mononuclear cells. There is conspicuous neuronal andaxonal preservation. C Focus of active degeneration (indicated bya solid arrow in A) with vascular hypertrophy and hyperplasia,mixed mononuclear and granulocytic infiltration, gliosis and rare-faction. D Higher magnification from the central inactive compo-nent of the lesion in A showing surviving neurons within a glioticneuropil (GFAP glial fibrillary acidic protein). A–D Dog 4. A GFAP, × 175; B Bielschowsky, × 175; C H&E, × 175; D GFAP× 350
In some animals, older sclerotic lesions coexisted with re-gions of active degeneration (Fig.3). In these cases, a cav-itated core with a gliotic rim containing surviving neuronswas in turn surrounded by zones of active degeneration.Vessels in areas undergoing active degeneration weremore conspicuous than those in adjacent sclerotic and qui-escent sites. This vascular prominence was due to hyper-
trophy and hyperplasia of endothelial cells and other cellular elements within vascular walls as well as due toliquefaction of the neuropil with exposure of the vascula-ture.
A striking component of the thalamic lesion was theoccurrence of numerous vacuolated gemistocytic astro-cytes interspersed among conventional reactive astrocytes(Fig.4). Vacuolated gemistocytic astrocytes contained sin-gle to numerous (average 5–6/cell but at times >20) ap-parently empty cytoplasmic vacuoles varying in size from<1–8 µm with an average of 4 µm The astrocytic cyto-plasmic vacuolation was evident in H&E-stained slidesbut was seen to advantage in GFAP preparations. Vi-mentin stained small numbers of these cells. Althoughgemistocytic astrocytes were a common element of le-sions at other sites, cytoplasmic vacuoles were not de-tected in astrocytes outside of the thalamus.
Non-thalamic bilateral and symmetrical lesions. Destruc-tive bilateral and symmetrical lesions of variable severitybut less extensive in comparison to the thalamus, werepresent in the dorsolateral caudate nucleus, dorsal puta-men, dorsal claustrum, caudal colliculi, midbrain tegmen-tum and the reticular formation in the medulla oblongata.All the lesions were morphologically similar to the thala-mic degeneration, except that vacuolated astrocytes werenot detected. Silver stains showed remarkable axonalpreservation within most affected regions. Mild bilateraland symmetrical as well as randomly scattered Walleriandegeneration in the reticular formation, tegmentum andthe reticulo-rubrospinal upper motor neuron (UMN) tractswas observed.
Cerebrum (Fig.5). Within the cerebral cortex there weremultiple, apparently random foci of minimal to profoundcortical attenuation associated with laminar necrosis, neu-ronal depletion, neuropil loss, gliosis, spongiosis andsometimes cavitation. As these neocortical lesions oc-curred most commonly at the base of sulci, they assumedan arcuate form with the most severely affected portion atthe base of the sulcus and variable extension into the ad-jacent gray matter of the cortex.
Although degenerative changes occurred in a laminarfashion, the neuronal layers affected were inconsistent. Insome foci, the superficial and middle cerebral laminaewere involved with relative sparing of deeper laminae,while in others the converse was observed.
Neuronal depletion varied from mild to profound. Ingeneral, neuronal loss was seen as zones of gliotic neu-ropil containing a diminished complement of neurons andsuch changes were a regular finding in all animals. In ac-tive lesions, the surrounding neuropil was sometimes onlyminimally gliotic, imparting the impression of neuronal‘drop out’. In more advanced lesions the neuropil was col-lapsed, the gliosis more extensive and the neuronal lossmore dramatic. Surviving neurons, both large and smalland mostly morphologically normal, were often malori-ented and haphazardly scattered within the attenuatedneuropil, presumably due to parenchymal collapse.
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Fig.4A, B Thalamus. A GFAP stain of a gliotic focus demon-strates many reactive astrocytes with cytoplasmic vacuolation. Afew are indicated by arrows. B Cytoplasmic vacuoles within reac-tive astrocytes have sharp margins and are variably sized /thick ar-rows). Some vacuoles are minute, as may be barely seen in the cy-toplasm of the astrocyte at the bottom right corner (thin arrow). ADog 1, B dog 3. A GFAP, × 186, B GFAP, × 350
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Fig.5A–F Cerebrum. A An active lesion at the base of a sulcus.The lesion has an arcuate outline, identified at this magnification byvascular prominence in the affected segment. The vascular promi-nence is due to hypertrophy and hyperplasia of vascular cells as wellas mixed perivascular inflammatory infiltration. A more extensiveinfiltrate of similar composition is present in the overlyingmeninges. B Higher magnification of an area included in A. Themeningeal (arrow) and perivascular infiltrate is composed ofmononuclear cells and granulocytes, barely discernable at this mag-nification by their irregular nuclear contours. There is spongiosis ofthe neuropil with mild to moderate gliosis. C An active lesion withprominent vascular hyperplasia and hypertrophy in the superficialgray matter. There is mild spongiosis, gliosis and neuronal numbers
are decreased. A thick arrow indicates mild gliosis of the glia limi-tans at the base of the sulcus. Relatively normal gray matter is on thefar right. D An early, active lesion with a row of necrotic ‘ischemic’neurons (arrows). A few adjacent neurons are morphologically nor-mal (asterisks). E An inactive lesion at the base of a sulcus (thick ar-row points to vessels within the overlying meninges). The neuropil isshrunken with pronounced gliosis and neuronal loss. Note survivingneurons (some marked with thin arrows), unobtrusive vessels andspongiosis in a vaguely laminar pattern. F An inactive lesion withcavitation of the superficial gray matter extending to the overlyingmeninges. The cavitated area is traversed by gliovascular trabeculae.An asterisk indicates the base of the sulcus. A–D Dog 4, E dog 3, F dog 1. A–F H&E; A, F × 35; B × 175; C, E × 87.5; D × 350
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Fig.6A–D Cerebellum. A Well-demarcated segmental atrophy af-fecting contiguous folia in the vermis (asterisks). The cortex in themost dorsal folium (top) is normal. Note also relative sparing ofmore lateral gray matter (arrows). B Higher magnification of an areain the most ventral folium in A demonstrating sparing of Purkinjeneurons (arrows with p) within a moderately gliotic neuropil. Thereis subtotal atrophy of the granular cell layer with residual granulecells visible as dark dots. Thick arrows indicate the gray and whitematter junction. A few hypertrophied astrocytes (arrows with a) arepresent in the atrophic gray matter and in the underlying white mat-
ter. C A quiescent lesion with profound atrophy of all cortical layers.Advanced fibrillary gliosis affects the molecular layer (M) and thedepleted and attenuated granular layer (G) which are separated by aband of Bergmann’s gliosis (B). Thick arrows indicate the gray andwhite matter junction. D A focus of subacute degeneration with nu-merous necrotic granule cell neurons seen as dark dots scatteredthroughout a gliotic and edematous granular layer. There is loss ofall Purkinje neurons and Bergmann’s gliosis (B). Note a survivingGolgi neuron (arrow with g) (M molecular layer). A, B Dog 2; C, D dog 4. A, B LFB; C, D H&E; A × 87.5, B–D × 175
Spongiosis accompanied the changes described aboveand similarly followed a laminar pattern. It was composedof innumerable, mostly small vacuoles of indeterminatelocation within the neuropil as well as of enlarged emptyspaces surrounding ischemic neurons, presumably repre-senting swollen astrocytic foot processes. At some sites,spongiosis progressed to cavitation leaving opticallyempty spaces traversed by gliovascular trabeculae, attimes surrounded by histiocytes, gitter cells, lymphocytesand eosinophils. Microgliosis and astrogliosis of variableintensity were observed in affected neocortex. A strikingand selective decrease in GFAP-positive astrocytes withinthe affected laminae of active lesions resulted in a laminarpattern of immunoreactivity with increased numbers ofGFAP-positive reactive astrocytes above and below butnot within degenerate laminae. In contrast, quiescent le-sions contained GFAP-positive astrocytes throughout theentire width of the gray matter as well as in the underly-ing white matter. Similar observations were made inGFAP preparations of brain stem lesions.
There was moderate gliosis in the white matter subja-cent to affected gray matter, particularly at sites where thecortical lesion was severe. In the white matter furtherfrom such sites, there was often an impression of a morewidespread, milder gliotic process. Astrocytes were reac-tive with slightly enlarged nuclei and minimal expandedcytoplasm. They stained positively with GFAP and vi-mentin, the presence of the latter intermediate filamentconfirming their altered, reactive state [37]. Sporadicspheroids and modest Wallerian degeneration, most no-table in the white matter close to affected gray matter,were also seen.
Cerebellum (Fig.6). The cerebellar cortical lesion princi-pally involved the ventral portion of the vermis. As in theneocortex and brain stem, active and quiescent phases ofthe lesion were identifiable, often juxtaposed and clearlydemarcated from unaffected tissue. In active lesions, therewas partial depletion of granular neurons, the granule celllayer was spongiotic, expanded by edema and containedlarge amounts of pyknotic and karyorrhectic nuclear de-bris. These changes were accompanied by loss of Purkinjeneurons, astrocytic (Bergmann’s) gliosis, mild to moderategranular layer gliosis and mild hypertrophy of capillaryendothelial cells. Active lesions progressed through inter-mediate stages characterized generally by an increasingdegree of neuronal depletion and gliosis with decreasingamount of nuclear debris, edema and spongiosis. Segmen-tally in affected areas, Purkinje neurons of normal mor-phology were seen immediately adjacent to numerous py-knotic granular neurons, possibly implying that in somelocations, loss of granular neurons preceded depletion ofPurkinje neurons.
In the fully developed ‘end stage’ lesion, which pre-dominated, cerebellar folia were markedly attenuated withall cortical layers atrophic and collapsed. There was wide-spread loss of Purkinje neurons, advanced Bergmann’sgliosis, complete depletion of the granule cell layer, se-vere gliosis of the depleted granule cell layer and milder
gliosis of the molecular layer. Of note was the presence ofsurviving Golgi neurons of normal morphology in the gli-otic granule cell layer and less commonly of a few Purk-inje neurons. In some of the animals, cerebellar corticaldegeneration was associated with mild to moderate gliosisof the fastigial and interposital cerebellar nuclei.
Cerebellar white matter changes resembled white mat-ter changes elsewhere in the neuroaxis.
Spinal cord. Two dogs had spinal cord lesions of signifi-cant severity. In these animals, there was a discrete bilat-eral and symmetrical C-shaped band of ongoing Walleriandegeneration and moderate astrogliosis situated in thedorsal half of the lateral funiculus. This well-demarcated,approximately 1-mm-wide band situated deep in the dor-solateral funiculus was evident throughout the entirelength of the spinal cord, but was most severe in the cer-vical portion. Anatomically, this distribution correspondsto descending UMN axons running within the reticulo-rubrospinal tract. A second bilateral and symmetrical fo-cus of moderate Wallerian degeneration accompanied bymild gliosis was present in the ventral funiculus flankingthe ventral sulcus. Also here, the cervical spinal cordshowed the greatest degree of involvement. In other dogs,spinal cord lesions were inconspicuous and consisted ofminimal to mild active Wallerian degeneration, most ofteninvolving the lateral funiculus in the cervical spinal cord.None of the dogs had lesions in the gray matter of thespinal cord.
Discussion
We describe a novel incapacitating and ultimately fatal fa-milial neurodegenerative disorder affecting Alaskanhusky dogs. Onset of neurological deficits was acute andoccurred in most cases (four of the five animals) before 1year of age. Neurological signs included ataxia, seizures,behavioral abnormalities, apparent blindness, facial hy-palgesia, loss of conscious proprioception, and difficultiesin the prehension of food. The neurological disorder wasepisodic. In dogs allowed to survive, gradual improve-ment after the acute deterioration of neurological functionwas observed. However, recurrences were common andled to euthanasia in most cases.
In addition to the five Alaskan Husky dogs included inthis study, one of us (A.D.) received in consultationhistopathological slides of autopsy material from six otherAlaskan Husky dogs with similar lesions. To date, wehave examined autopsy material from a total of 11 spon-taneous cases (5 males and 6 females) of this disorder inseven litters of Alaskan Husky dogs from five kennels inthe USA. The incidence of the disease is probably higher,as on several occasions littermates of affected dogs wereeuthanized following the onset of characteristic neurolog-ical deficits but pathological studies were not pursued.
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Neuroanatomical correlation
Some of the clinical signs in these dogs can be correlatedwith the location of structural lesions observed on grossand microscopic examination of the CNS. Neurologicalsigns may also reflect a functional disturbance of neuronalpopulations not revealed by light microscopy. This iscommon in metabolic disorders. The neocortical or thala-mic lesion could be the site of seizure initiation. Damageto thalamic relay nuclei may account for nasal hypalgesiaand loss of conscious proprioception. Lesions in the retic-ular formation could explain some of the other neurologi-cal deficits. Dysfunction of the pontine and medullarycomponent of the reticular formation may be responsiblefor the UMN deficits in the gait. Involvement of the as-cending component, specifically the ascending reticularactivating system, could contribute to the suppressed sen-sorium. Thalamic and reticular formation lesions mayhave led to the frequently observed difficulties in prehen-sion of food. The propulsive tendencies are difficult to lo-calize but often involve the motor basal nuclei, some ofwhich are affected in dogs with this encephalopathy (cau-date nucleus, putamen and claustrum). The structural ba-sis for the visual deficits is unknown. The clinical signssupport a central visual problem, but the cerebral lesion isunlikely to be related to these deficits, as it is segmentaland more prevalent in the parietal and temporal lobesrather than the occipital lobe. No consistent lesions werepresent in the retina, optic nerve, optic chiasm, optic tractor lateral geniculate nucleus in the thalamus. The cerebel-lar vestibular component of the gait disorder implies thepresence of further neuronal dysfunction than is evident inthe limited cortical degeneration of the ventral vermis.
Neuropathology
Thalamus and other bilateral and symmetrical lesions
In general, bilateral and symmetrical CNS lesions arethought to be due to either metabolic aberrations (neu-rodegenerative disorders and toxicoses) or are determinedby vascular anatomy, regardless of whether they involvethe white matter, gray matter or both. Consistent neuronaland axonal survival, a prominent feature of this canine en-cephalopathy, renders ischemia unlikely. Ischemia is ex-pected to cause non-selective destruction, or if less severe,to primarily involve neurons [24]. Intoxication by an ex-ogenous agent is unlikely, given the widely scattered ori-gin of the animals. We propose a metabolic derangement,presumably hereditary in this breed, as the cause of thisneurodegeneration. Initial pedigree studies and test mat-ing suggest an inherited basis with an autosomal recessivemode of inheritance (J.J.W., unpublished).
In man, bilateral and symmetrical brain stem lesionswith tissue destruction, vascular proliferation and variableneuronal survival may be seen either in LS or Wernicke’sencephalopathy. These conditions can be differentiatedaccording to neuropathological [27] and clinical [35] crite-
ria. Similar neuropathological findings are described inseveral spontaneous (see below) and experimental [36]disorders of animals. In dogs, thiamine deficiency causeswell-demarcated bilateral and symmetrical spongy changeand necrosis of many brain stem nuclei. The caudal col-liculus is the most severely affected structure and thala-mic cavitation is lacking. Microscopically, lesions exhibithypertrophy and hyperplasia of endothelial and adventi-tial cells, gliosis, frequent hemorrhages and variable neu-ronal preservation [34]. The encephalopathy induced bythiamine deficiency shares many similarities with theAlaskan Husky encephalopathy but differs in several as-pects. In Alaskan Husky encephalopathy, involvement ofthe caudal colliculus is infrequent and mild, cerebral andcerebellar cortical lesions are characteristic and hemor-rhage is not a feature. Canine disorders of unknown etiol-ogy but possibly inherited, which are characterized bysymmetrical gray matter rarefaction with neuronal pre-servation, include a neurodegenerative condition in Aus-tralian Cattle Dogs [6] and familial cerebellar ataxia withhydrocephalus in Bull Mastiffs [8]. The polioencephalo-myelopathy of the Australian Cattle Dog differs from theAlaskan Husky encephalopathy in its extensive spinalcord lesions, more discrete targeting of gray matter nucleiin the brain stem and more remarkable neuronal preserva-tion. The lesions in the Bull Mastiffs are spongiotic ratherthan frankly cavitary, their distribution is different andthey are accompanied by hydrocephalus. Lesions withsimilar histopathological findings are also recognized infarm animals. In pigs, focal symmetrical poliomalacia dueto selenium poisoning [41] or of unknown cause [52] iswell documented. Identical lesions can be produced inpigs by the experimental administration of 6-aminonico-tinamide (6-AN) [30, 53], an antimetabolite of niacin witha selective gliotoxic effect [26]. Several outbreaks of aneurological disorder of unknown etiology with lesions ofsimilar morphology in the spinal cord, brain stem andcerebellum are documented in sheep in Africa [2] and incattle (multifocal subacute necrotizing encephalomyelo-pathy in Simmental calves [40] and focal symmetrical po-liomalacia of the spinal cord in Ayrshire calves [31]).
A striking element of the thalamic lesion in AlaskanHusky encephalopathy is the presence of vacuolated reac-tive gemistocytic astrocytes. Astrocytes with cytoplasmicvacuolation are an unusual finding both in veterinary andhuman neuropathology. In the polioencephalomyelopathyof the Australian Cattle Dogs, vacuolated gemistocytic as-trocytes were observed [6]. More recently, vacuolated as-trocytes and perineuronal satellite cells in ganglia were ob-served in dogs following prolonged, low-level experimen-tal administration of 6-AN [22]. Rarely, vacuolated astro-cytes may be seen admixed among conventional reactiveastrocytes in areas of advanced gliosis in the dog (O.B.personal observation).
Ultrastructural studies are required to identify the mor-phological basis of the astrocytic cytoplasmic vacuolationin Alaskan Husky encephalopathy. In MELAS (mitochon-drial encephalopathy, lactic acidosis and stroke-like epi-sodes), smooth muscle cells and to a lesser degree endo-
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thelial cells of blood vessels in the brain contain smallvacuoles which have been shown by ultrastructural exam-ination to correspond to proliferated and swollen mito-chondria [10, 39]. Why vacuolated reactive astrocytes areobserved only in the thalamus and not in other bilateraland symmetrical lesions in Alaskan Husky encephalopa-thy is unknown. It may relate to the fact that degenerativechanges are most severe at this location.
Cerebrum
Cerebrocortical lesions in this encephalopathy bear someresemblance to cerebrocortical necrosis (CCN)/polioen-cephalomalacia as encountered in the dog, but differ intheir distribution within the cortical mantle. CCN in dogsis seen sporadically, either alone or in conjunction with le-sions elsewhere in the brain. In some cases, the underly-ing cause is known, e.g., intraoperative cardiac arrest[32], cyanide poisoning [18], thiamine deficiency [34],lead poisoning [54] or hypoglycemia [23]. Sometimes the cause is undetermined [3, 18]. In other cases, CCN oc-curs with coexistent conditions such as meningitis, throm-boembolic disease, atherosclerosis [3], canine distemperencephalitis, infectious canine hepatitis [18], or gastroen-teritis [13], but the relationship between the two is un-clear. Whereas cerebrocortical lesions in Alaskan Huskyencephalopathy occur exclusively at the base of sulci, asimilar predilection has not been noted in canine CCN. Inman, the tendency of a circulatory disturbance to involvegray matter at the base of sulci rather than at their crest iswell recognized [7, 19]. In contrast, this predilection hasnot been well documented in veterinary neuropathology.
Occasionally in the Alaskan Husky encephalopathy, aninflammatory infiltrate comprising histiocytes as well asneutrophils and eosinophils is observed in acutely com-promised regions and is interpreted as secondary to tissuenecrosis. The occasional eosinophilic component is un-usual but is well documented in cerebrocortical necrosis(‘salt poisoning’) and in focal symmetrical poliomyelo-malacia [52] in pigs. It could be suggested that the corti-cal lesion is secondary to seizure activity. Althoughseizures are common in Alaskan Husky encephalopathy,they were not present in three of the five dogs in this re-port. Further, the association between seizure disordersand brain injury in domestic animals is much less clearlyestablished than in human subjects. In dogs with idio-pathic epilepsy, ischemic neuronal change rarely occurs[42]. Cortical necrosis, interpreted as seizure induced, isseen in some cases of canine distemper encephalitis. Thepyriform lobe and the hippocampus are selectively af-fected [3].
Cerebellum
The cerebellar cortical lesion, which mainly involves theventral vermis, shares many similarities with degenerativechanges seen in other areas, but progression to cavitation
does not occur. Quiescent ‘burnt out’ lesions involving allcortical layers are the most frequent finding and resemblelong-standing lesions of cerebellar cortical abiotrophy, asseen in dogs, However, survival of isolated Golgi neuronsand rarely Purkinje neurons would be unusual in a cere-bellar abiotrophy or a hypoxic lesion, to which Purkinjeneurons are particularly susceptible [16]. Gliosis of thefastigial and interposital cerebellar nuclei is probably a re-flection of trans-synaptic degeneration following loss ofPurkinje neurons in the vermis. In animals, selective in-volvement of the ventral vermis was reported in five ofsix dogs with thiamine deficiency [34] and in three of fiveanimals with cardiac arrest [32].
White matter
Degenerative changes in this encephalopathy preferen-tially affect the gray matter but the white matter is not en-tirely spared. In brain stem lesions for example, the de-structive process frequently involves surrounding whitematter. In the cerebrum, cerebellum and spinal cord, whitematter lesions are either necrotizing, undergoing Wal-lerian degeneration, pure gliosis, or a mixture. While de-generative white matter changes are clearly accentuated inthe vicinity of gray matter lesions, they are not limited to these regions. Some of the Wallerian degeneration inthe midbrain, medulla and spinal cord is bilateral andsymmetrical and anatomically consistent with UMN de-generation of the reticulo- and rubrospinal tracts, possiblyreflecting the bilateral and symmetrical lesion in the retic-ular formation. Widespread gliosis may reflect cerebraledema which is prone to occur in white matter. Such puregliosis, perhaps the most pervasive white matter lesion inthis condition, is widespread and often mild and thus dif-ficult to delineate.
Nature and distribution of the lesions
Irrespective of site in the neuroaxis, affected regions shareseveral histopathological similarities. They primarily in-volve the gray matter, tissue destruction and neuronal lossis seen concomitant with variable neuronal sparing, thereis striking hypertrophy and hyperplasia of capillaries,spongiosis and gliosis are prominent, and active and inac-tive phases of the degenerative process are discernable.White matter changes seem to be largely reactive. Theclassification of lesions into active, quiescent and inter-mediate stages based on morphological features, is basedon the definitions of Cavanagh and Harding [9] whoanalysed a series of cases with LS. It seems possible thatareas of tissue destruction with partial neuronal sparing,as seen in this canine encephalopathy, could be the resultof a primary gliopathic process with neuronal loss a sec-ondary event. Studies with glial toxins such as 6-AN,which produce lesions of similar morphology, are sup-portive of this contention. It remains to be determinedwhether the transient vacuolation of astrocytes in Alaskan
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Husky encephalopathy is a reflection of a gliocentric de-generative process or a nonspecific event. The topographyof Alaskan Husky encephalopathy lesions in the neu-roaxis is unexplained, as is often the case with neurode-generative diseases of animals and man. There is someoverlap in the distribution of extracortical lesions betweenAlaskan Husky encephalopathy and canine CCN due tovarious causes [3]. As some cases of canine CCN arecaused by energy deprivation (thiamine deficiency,cyanide poisoning), an overlap is not surprising.
Comparison of Alaskan Husky encephalopathy and LD
Since its initial description in 1951 [25], LS/LD subacutenecrotizing encephalomyelopathy has been recognized inman as a neuropathological syndrome [9, 27, 45, 46, 50].In the last decade, numerous investigations have shedlight on the clinical recognition [21, 49], neuroimagingfindings [43], enzymatic deficiencies, genetic mutationsand inheritance patterns [1, 12, 15, 45] of this heteroge-neous disorder. The pathological diagnosis of LS restsupon the demonstration of characteristic lesions in the
brain stem and lateral walls of the third ventricle, primar-ily in its caudal part. Brain lesions are usually bilateraland symmetrical and show a tendency to be non-contigu-ous. They do not respect gray and white matter bound-aries, especially in the brain stem [27]. Characteristic fea-tures of acute lesions are loosening and spongiosis of theneuropil followed by necrosis. There is capillary prolifer-ation, macrophage infiltration, gliosis and occasionalperivascular cuffs. An important feature is the relativepreservation of neurons. Findings may vary in differentregions of the same case; while some areas are ‘end stage’lesions, others show florid changes [16]. The destructiveprocess is episodic and total tissue damage is cumulative[9]. In human autopsy material, lesions with quiescentfeatures predominate [9]. Astrocytic vacuolation is not de-scribed in LS.
The lesions of LS bear a considerable resemblance intheir distribution and quality to Alaskan Husky en-cephalopathy. There are differences in the topography oflesions between the canine and human disorders. In LS,the most frequent involved regions are the midbraintegmentum and substantia nigra [9, 27], the pontinetegmentum [27] and the medullary tegmentum [9, 27]. In-
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Table 2 Suspecteda spontaneous mitochondrial diseases in domestic animals (EM electron microscopy)
Species No. Organ affected and main Features suggesting mitochondrial involvement Referencecases clinical signs
Irish Terrier 1 Skeletal muscle; stiff gait, Degenerative myopathic changes by histology, [51]difficulty in swallowing, abnormal enzyme distribution by histochemistry,muscle atrophy with high tone metabolic defect in isolated mitochondria
Simmental and > 30 CNS; pelvic limb ataxia, Bilateral and symmetrical malacic lesions in brain [14, 17, 40]Simmental cross caudal paresis, sudden death stem (olivary nucleus most consistent) and in somecalves cases spinal cord with hypertrophied capillaries and
Sheepdogs weakness of mitochondria and glycogen accumulation inskeletal myofibers. One dog had scattered raggedred fibers
Arabian 1 Skeletal muscle; profound Lactic acidosis, a few ragged red fibers; EM: [44]horsea exercise intolerance aggregates of large mitochondria with bizarre
cristae, deficiency of Complex I respiratory chainenzyme documented
Jack Russell 1 Skeletal muscle; progressive Lactic acidosis, ragged red fibers; EM: large [29]Terrier exercise intolerance subsarcolemmal accumulations of normal
mitochondriaSwaledale Not CNS Increased CSF lactate, bilateral and symmetrical [28]
lambs given brain stem lesions with neuronal sparingperiaqueductal gray matter, olives and thalamus
Australian 3 CNS; seizures with progression Bilateral and symmetrical cavitating lesions in the [6]Cattle dogs to spastic tetraparesis brain stem and spinal cord with neuronal sparing;
EM: increased numbers of morphologically normalmitochondria in astrocytes
English 1 CNS; ataxia, mild behavioral Marked atrophy of optic nerves and tract, bilateral [5]Springer abnormalities and symmetrical spongiosis in the brain stem; EM: Spaniel mitochondria with abnormal morphology in neurons
Dogs 25 Skeletal muscle; myalgia, Resting lactic acidosis, abnormal accumulation of [38]weakness, muscle atrophy lipid primarily in type 1 fibers
a Enzyme deficiency demonstrated
volvement of the neuroaxis outside the brain stem (e.g.,basal nuclei, corona radiata, optic nerves, cerebellum,spinal cord) is variable. In Alaskan Husky encephalopathy,the thalamus is the most severely affected region but is in-volved only in half [27] or less [9] of LS cases. Involve-ment of cerebral gray matter is a consistent feature ofAlaskan Husky encephalopathy. Although recognized inLD [47], it is very uncommon and seen in only 10% [27]or less [9] of cases. A more detailed comparison of thedistribution of lesions in LS and Alaskan Husky en-cephalopathy is unwarranted at this stage, in part becausethe distribution of LS is very variable [27, 33, 46]. Pre-liminary electron microscopic examination of brain tissueand conventional light microscopic examination of skele-tal muscle in these dogs failed to detect anything but non-specific degenerative changes, which may not be surpris-ing. In LS, ultrastructural mitochondrial abnormalities inthe brain have beens described in very few cases [33, 50]and are considered to be of minor diagnostic significance[49]. Ragged red fibers are generally absent in LS [21,49].
Proposed similarities between Alaskan Husky en-cephalopathy and LS do not rest solely on pathologicalfindings but also on the clinical presentation. Both aremostly diseases of juvenile, or less commonly youngadult to adult onset with episodic and cumulative deterio-ration. This pattern is well documented in human patientsand is suggested by the history in the canine cases.
LS may be viewed as a designation for a typical con-stellation of brain lesions which develop in patients with abiochemically heterogeneous group of abnormalities [12,50]. A diagnosis of LS does not necessarily imply a mito-chondrial disorder [12]. It has been suggested that LS is theneuropathological paradigm caused by impaired oxidativemetabolism in the developing brain, irrespective of spe-cific biochemical defects [11, 12]. At this time, the patho-genesis of this disorder is unknown. Biochemical studiesaimed at defining the role of mitochondria in this disorderare currently underway. In domestic animals, a number ofencephalopathies and myopathies have been suspected tobe due to primary mitochondrial dysfunction (Table 2).With a few exceptions, none of these tentative diagnosesis supported by demonstration of enzymatic deficiencies.
In conclusion, this is a unique encephalopathy of un-known pathogenesis and undetermined mode of inheri-tance which affects juvenile and less commonly adultAlaskan Husky dogs. Onset of neurological signs is acuteand the course is static with multiple recurrences. Patho-logically, the disorder is characterized by a degenerationwith distinctive bilateral and symmetrical as well as mul-tifocal distribution in the neuroaxis and by the coexistenceof lesions of different ages. Neuronal sparing in conjunc-tion with apparently transient astrocytic vacuolation pointto a possible pathogenetic role of glial abnormality.Recognition of this canine disorder is important both froma differential diagnostic point of view to veterinary clini-cians and pathologists as well as because of its potentialuse as an animal model, should mitochondria be demon-strated to play a primary role in the degenerative process.
Acknowledgements This study was supported by the Zipporah S.Fleisher Fund for Canine Neurologic Research. The authors thankDr. Roger P. Pitts of Duluth, Minnesota for initiating this study,Dr. Susan Morgello of the Division of Neuropathology, Depart-ment of Pathology, The Mount Sinai Medical Center for her infor-mative discussions of comparative neuropathology, Dr. Victor L.F. Friedrich of the Brookdale Center for Molecular and Develop-mental Biology, The Mount Sinai Medical Center, New York, NYfor his assistance with ultrastructural studies and Dr. T. Robinsonwho wrote the first detailed description of this condition in his fi-nal year at the College of Veterinary Medicine, Cornell University.The expert technical support of Ms. Joy Cramer, Ms. Tina Smithand Ms. Alexis Wenski-Roberts is greatly appreciated.
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Note added in proof Through the curtesy of Dr. T. van Winkle,University of Pennsylvania, we have examined the brain sectionsof an 8-month-old Yorkshire Terrier with identical lesions to theAlaskan Husky encephalopathy. Bilateral thalamic cavitation wasevident on MRI examination of this Yorkshire Terrier.
