25.06.2017

Malformations

Important fetal teratogenic virus infections in different species: Feline panleukopenia virus cat cerebellar hypoplasia, hydranencephaly

Introduction to Neuropathology – Part II Classical swine fever virus pig dysmyelinogenesis, cerebellar hypoplasia

Bovine virus diarrhea virus calf, lamb hydrocephalus. cerebellar hypo- and aplasia, prosencephaly, hypomyelination, porencephaly Malformations

Akabane, Cache valley, calf, lamb hydranencephaly, Prof. Dr. W. Baumgärtner and Dr. P. Wohlsein Schmallenberg virus arthrogryposis, Department of Pathology cerebellar hypoplasia, University of Veterinary Medicine porencephaly Hannover, Germany

Neurological disease spectrum in dogs Malformations

Important fetal teratogenic virus infections in different species: Canine parvovirus dog cerebellar hypoplasia, dysplasia

Bluetongue virus lamb, calf hydranencephaly

Chuzan virus calf hydranencephaly, cerebellar hypoplasia

Aino virus calf arthrogryposis, hydranencephaly, Introduction cerebellar hypoplasia Malformations

Border disease virus lamb porencephaly, hypomyelination

Wesselsbron virus calf hydranencephaly, Fluehmann et al., 2006; J Small Anim Pract; mod. porencephaly

Malformations Malformations

! frequent disorder in domestic animals (5% neonatal death) ! grossly or only microscopically visible Categories of CNS developmental defects: ! defects of neural tube closure Etiology: ! defects of forebrain induction ! primary: spontaneous or hereditary (point gene mutations, ! neuronal migration disorders and sulcation defects chromosomal abnormalities) ! disorders of proliferation and size ! secondary: infectious, toxic, physical, metabolic causes ! encephaloclastic defects ! in most cases no correlation with the causative agent ! congenital hydrocephalus and cysts possible (too long time period between impact [early ! cerebellar and spinal malformations and middle gestation period] and assessment of the disorder [peri-/postnatal period]) Malformations Malformations ! lesions with similar morphology may be caused by different noxes

Species-specific characteristics: Different morphological and functional maturity of the CNS at the time of birth!!

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Neural tube closure defects Disorders of proliferation and size

! anencephaly (complete lack of brain development) ! megalencephaly (↔ megalocephaly = enlargement ! cranium bifidum with meningocele of the head) ! cranium bifidum with meningoencephalocele ! microencephaly ! exencephaly Malformations Malformations

Defects of forebrain induction Encephaloclastic defects

! holoprosencephaly (failure of cerebral hemisphere Porencephaly: development) ! single, cystic fluid-filled cavity ! cerebral aplasia ! in the wall of cerebral hemispheres ! agenesis of corpus callosum (in conjunction with other malformations) Etiology: ! Orthobunya-viruses (Akabane, Schmallenberg-virus) ! Border disease ! sway back with in utero copper deficiency Malformations Malformations

Neuronal migration disorders/sulcation defects Schmallenberg virus (SBV) ! macro-/microgyria ! lissencephaly (agyria) [normal in rodents and rabbits] ! pachygyria (few, but widened gyri) ! polymicrogyria (excessive cortical folding) SBV in Europe Malformations Malformations

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Schmallenberg virus (SBV) Schmallenberg virus (SBV)

Cerebellar hypoplasia Hydranencephaly Porencephaly Internal Torticollis hydrocephalus Lordosis Micromyelia Kyphosis only cattle Scoliosis

sheep, goats, calves Brachygnathia inferior Malformations Malformations

Arthrogryposis

Inflammation and SBV protein in the CNS Herder et al., 2013

Schmallenberg virus (SBV) Schmallenberg virus (SBV)

SBV-Pathology in offspring ! malformations similar to Akabane virus ! arthrogryposis/Hydranencephaly-Syndrome ! multicystic encephalopathy ! meningoencephalomyelitis = rare event in naturally SBV-infected animals ! SBV-antigen associated with inflammation and malformation in the CNS ! Por-, and hydranencephaly probably virus-induced Malformations Malformations

micromyelia

Schmallenberg virus (SBV) Encephaloclastic defects

Hydranencephaly: ! total or subtotal destruction of cerebral hemispheres ! CSF-filled meninges ! brain stem and hippocampus normal ! particularly in calves and lambs

Herder et al., 2013 Etiology: ! Orthobunya-viruses (Akabane, Schmallenberg-virus, Cache valley virus, Aino virus) ! Bluetongue-virus Malformations Malformations ! Rift Valley Fever-virus ! Wesselsbron-Disease-virus ! Pestiviruses (CSF, BVD, BD) ! Chuzan virus (Orbivirus) ! feline parvovirus Brain regions with inflammation Malformations related to inflammation

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Cerebellar and spinal malformations Spinal malformations

! cerebellar agenesis (Simmental calves!) Spina bifida: ! cerebellar vermal agenesis Absence of one or more segments of the dorsal arch: ! Spina bifida aperta ! granule cell hypoplasia or aplasia associated with myelodysplasia ! cerebellar hypoplasia and dysplasia ! Spina bifida occulta ! cerebellar hypoplasia and hypomyelination Additional malformations: Etiology: ! Myeloschisis (dysraphia): incomplete closure of neural tube infectious causes ! Perosomus elumbis (partial spinal agenesis) ! virus infections ! Meningocele ! Meningomyelocele Malformations non-infectious causes: Malformations ! Diplomyelia, Diastematomyelia ! Hereditary/genetic disorder (autosomal-recessive; e.g. horse, cattle, dog) ! toxic (e.g. organophosphate in pregnant sows)

Cerebellar and spinal malformations Cerebro-spinal malformations Dandy-Walker syndrome ! rare midline defect of the cerebellum Congenital tremor in piglets ! foal, calf, lamb Type A: Myelin deficiency

! etiology: unknown AI Classical swine fever virus ! diagnostic trias: agenesis of cerebellar vermis AII Infectious – recently unknown, PCV2? cystic dilatation of fourth ventricle AIII Landrace/Landrace crossbred, Plp gene, X-linked recessive enlargement of caudal fossa AIV British saddleback breed, hypomyelination, autosomal recessive Occasionally concurrent polygyria, hydrocephalus AV Intoxication (trichlorfon)

Type B: No myelin loss, idiopathic, lacks structural or neurochemical defects Malformations Malformations

New AII-type! Atypical pestivirus

Cerebellar and spinal malformations Hydrocephalus Arnold Chiari syndrome Hydrocephalus (H.): ! calf, dog Accumulation of CSF in the head as a result of a ! shallow caudal fossa disturbance of CSF secretion, circulation or absorption ! elongation of the cerebellar vermis and the medulla ! their combined displacement into the cervical spinal canal Communicating hydrocephalus (H. communicans): ! in Cavalier King Charles Spaniels often combined with free passage of CSF from ventricles into subarachnoidal syringomyelia space (normal pressure hydrocephalus); usually bilateral and symmetric dilatation of the ventricular system without any detectable macro- or microscopic lesions

Malformations Malformations Occurrence: ! toy breeds (Yorkshire terrier Chiahuahua etc.) ! brachycephalic breeds (Boxer)

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Hydrocephalus Hydro- and syringomyelia Non-communicating hydrocephalus: Hydromyelia: result of partial or complete obstruction of CSF flow at any ! congenital or acquired of several critical stricture points within the intraventricular ! focal or diffuse dilatation of central canal pathways or in the subarachnoidal space ! may be associated with hydrocephalus

Critical points: Syringomyelia: ! interventricular foramina between the lateral and III. ! congenital or acquired ventricle (Foramina Monroi) ! cystic, fluid-filled tubular cavity with spinal cord ! mesencephalic aqueduct extending over several segments ! lateral foramina of the IV. ventricle (Foramen of Luschka) ! commonly associated wth neural tube defects ! median foramen of the IV. ventricle (Foramen of Magendii) Malformations Malformations ! acquired associated with hydromyelia and ependymal ! fusion of the mesencephalic colliculi rupture (Cavalier King Charles with Arnold-Chiari syndrome ! extraventricular: arachnoidal villi Syringobulbia: Causes: ! Fluid-filled slit-like cavity in the medulla oblongata Teratogenic noxes (e.g. viruses, protozoa)

Hydrocephalus Hydro- and syringomyelia

Pathogenesis of non-communicating hydrocephalus: Depending on time, site and degree of obstruction in the fetus " variable degree and manifestation of congenital hydrocephalus:

! fetal manifestation (" dystocia due to macrocephaly) ! postfetal manifestation (already closed bone sutures)

! Hydrocephalus internus (ventricular lumina; Malformations ventriculomegaly) Malformations ! Hydrocephalus externus (arachnoidal space)

Schematic diagram of syringomyelia Wünschmann et al., 1997

Hydrocephalus Cysts in the CNS

Acquired hydrocephalus: Cystic space-occupying lesion in the CNS: post natal non-communicating hydrocephalus ! often intra-spinal and extraneural (H. aresorptivus) ! different topography and histogenetic origin ! acquired or congenital Causes: Neoplasms, inflammation (leukoencephalitis, ependymitis, Types: ! chorioiditis, meningitis) ependymal cyst (extremely rare in animals) ! arachnoidal diverticulum/cyst Hydrocephalus ex vacuo: ! synovial cyst (synovial membrane of intervertebral joints) = compensatory hydrocephalus ! ganglion cyst (periarticular tissue of intervertebral joints) ! epidermoid cyst (inclusion of epithelial tissue elements)

Malformations ! congenital or post natal communicating hydrocephalus Malformations ! primary loss of neuroparenchyma (e.g. age-related) and ! dermoid cyst/sinus (insufficient separation of ectoderm secondary distension of of the ventricle and neural tube) ! discal cyst (in connection with intervertebral disc)

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Cysts in the CNS Pathophysiology of ischemia

! high glucose and oxygen demand in the CNS ! very limited storage capacity ! CNS is dependent on permanent arterial supply of ! quadrigeminal cyst (topographic term for cysts of variable oxygen and glucose histogenesis but located between cerebrum and cerebellum Vascular autoregulation: Diagnostic dilemma: " relatively constant cerebral blood flow Most cysts collapse at necropsy! " protection of the brain from ischemia up to a 60% reduction of blood flow!

Malformations Circulatory and respiratory disorders " neuronal damage

Vascular disorders = eosinophilic neuronal necrosis Adjacent rim with partial compromise of blood flow: penumbra!

General aspects Vascular lesions of the brain

Disturbance of blood circulation in the CNS! Ischemic brain infarct: Main lesions: ! thrombembolism (e.g. cardiac disease) ! Hemorrhage (due to vascular injury) ! tumor embolism ! Infarction: focal vascular obstruction: ! accidental intracarotid injection - septic or bland ! vasculitis with associated thrombus formation - fibro-cartilaginous embolism ! vascular degeneration (e.g. atherosclerosis) rare in animals - atherosclerosis - intravascular lymphoma global ischemia: Feline ischemic encephalopathy - severe blood loss ! adult cats in the USA - cardio-respiratory arrest ! unilateral extensive infarction of cerebral hemispheres

Vascular disorders - heart disease Vascular disorders! cause: parasitic migration of Cuterebra larvae " malacia (softening and necrosis of CNS tissue)

Differential diagnoses of Hemorrhages Vascular lesions of the brain

Hemorrhage/hematoma: ! trauma ! toxic-metabolic causes ! inflammatory vascular injury ! following myelography (also spinal subarachnoidal hemorrhages)

Post-anesthetic cerebral necrosis ! adult horses; 2-7 days after anesthesia ! patchy areas of cerebral necrosis Vascular disorders Vascular disorders! likely cause: circulatory disturbance

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Vascular lesions of the spinal cord Pathogenesis

Fibrocartilagenous embolism (FCE) After depletion of anti-oxidative defense mechanisms ! dogs (large breed!); other domestic animals seldom " oxygen radicals induce peroxidation of membranes of ! emboli histochemically identical to nucleus pulposus neurons and vascular structures " necrosis ! exact pathogenesis unclear; ! depending o the caliber of occluded blood vessels Altered tissue environment with high levels of inflammatory " leuko- and/or poliomyelomalacia mediators " vasospasm " additional damage

Membrane damage at the level of the blood-brain barrier Hematomyelia " vasogenic and cytotoxic brain edema " due to the ! dogs with steroid-responsive meningitis-arteritis confined space within the skull " further compromise of tissue perfusion Post-anesthetic hemorrhagic myelopathy Vascular disorders ! young horses; clinical signs immediately after anesthesia Traumatic changes Brain swelling, midline shift, dislocation of brain structures ! anesthesia in dorsal recumbency " lethal brain stem compression with petechial hemorrhages ! hemorrhagic poliomyelopathy (Duret hemorrhages)

Traumatic changes of the CNS Mechanics of brain trauma

Topography: Syn.: traumatic brain injury (TBI) ! Lesions immediately below the site of impact (coup) and/or Pathogenesis: ! On the opposite site of impact (contre-coup) ! direct mechanical impact (with variable severity, speed and duration of the distortion) Pathology: ! sudden acceleration/deceleration Coup lesions are more severe when a stationary but mobile " collision of CNS with bony structures head is hit by a moving object.

Sequelae: Contre-coup lesions are more severe when a moving head ! mechanical disruption of tissue (laceration) strikes a stationary object. ! damage to blood vessels " ischemia, hemorrhage with Traumatic changes secondary space-occupying damage Traumatic changes Pathogenesis: Inertia of the brain that is freely movable in the CSF Impact of a range of forces

Pathogenesis Mechanics of brain trauma

mechanical distortion " lesions of the microvasculature " leakage of blood and plasma

Primary brain damage: immediate effects of sheer forces with disruption of neurons and other cells Secondary brain damage: within hours and days aggravation of damage

! disruption of cerebral blood flow autoregulation ! increase of blood pressure due to rise in catecholamines ! life-threatening brain edema ! pressure-induced ischemia Traumatic changes Traumatic changes ! break-down of energy-dependent ion pumps ! depolarization of membranes ! excessive release of excitatory amino acids " excitotoxicity Coup and contre coup lesions Vandevelde et al., 2012

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Traumatic changes of the brain Traumatic changes of the spinal cord

Concussion: ! mildest form of head injury Contusion: ! temporary impairment of neurological functions ! following impact and due to inertia the spinal cord ! edema collides with the bony wall ! hemorrhage ! at the site of impact: damage to microvasculature ! axonal/neuronal damage " intramedullary hemorrhage (more pronounced in grey than in white matter, which is less vascularized) In contrast to man, data on animals are lacking! " secondary lesions (edema, ischemia, necrosis) " progressive hemorrhagic myelomalacia (ascending and descending from the site of impact) Traumatic changes Traumatic changes

Traumatic changes of the brain Traumatic changes of the spinal cord

Hemorrhage/Hematoma: Compression: ! diffuse or focal ! due to narrowing of the spinal canal ! mass hemorrhage = hematoma ! important: dynamic factor, ! epidural, subdural, subarachnoidal (leptomeningeal) i.e. the speed with which the compression develops or parenchymal Contusion: Acute compression: within seconds (similar to contusion) ! following blunt trauma Chronic compression: within hours, days or weeks ! arachnoidal/parenchymal hemorrhages " hemodynamic changes with perfusion failure Laceration: " decompression surgery!! ! following blunt trauma with skull fracture ! arachnoidal/parenchmyal hemorrhages and disruption Traumatic changes of CNS tissue Traumatic changes Iatrogenic trauma: ! following CSF puncture or surgery

Traumatic changes of the spinal cord Traumatic changes of the spinal cord

Causes: ! vertebral fracture Pathology: ! vertrebral luxation ! edema of white matter (spongy status) ! vertebral subluxation ! reactive changes (demyelination, axonal disruption, ! intervertebral disc herniation spheroids, neuronal loss) ! tail avulsion ! mobile resorption (myelinophagia, vascular proliferation, ! exogenous physical forces (e.g. bullets) gliosis) ! Wallerian degeneration Even without mechanical deformation a sudden blow may ! dilatation of central canal (hydromyelia) lead to spinal cord lesions. ! rupture of ependymal lining " interstitial edema and formation of syringomyelia

Traumatic changes Additionally, narrowing of the spinal canal " continuous Traumatic changes ! chronic compression " severe attenuation of spinal cord pressure on spinal cord! due to atrophy

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Traumatic changes of the spinal cord Wobbler syndrome/cervical spondylomyelopathy

Pathogenesis: Static or dynamic stenosis of the cervical spinal canal; Genetic and dietary factors as well as congenital anomalies

Horses: ! degenerative changes of the facet joints ! synovial cysts secondary to degenerative joint disease ! hypertrophy of the ligamentum flavum Traumatic changes Traumatic changes

Vandevelde et al., 2012

Avulsion Intervertebral disc disease (IVDD)

! avulsion of brachial plexus traumatic ! avulsion of cauda equina nerves spinal cord injury

intrinsic extrinsic

luxation herniation of fracture intervertebral disc

extrusion chondrodystrophic breeds, Traumatic changes Traumatic changes frequently

protrusion non-chondrodystrophic breeds, frequently

Wobbler syndrome/cervical spondylomyelopathy Intervertebral disc disease (IVDD)

Spinal cord inury (SCI) Occurrence: Chondrodystrophic breeds: ! horse (stallion), rapid growth, large body size ! degeneration of intervertebral discs ! degradation and loss of proteoglycans static stenosis of spinal canal: C5-C7; 1-4 years ! change from collagen II to I dynamic stenosis, decreased stability, ventro-flexion: ! proliferation of chondrocyte-like cells C3-C5; 8-18 months ! loss of notochordal cells ! mineralization of the nucleus pulposus ! dogs, large breed (Dobermann, Great Dane, Irish Setter, Non-Chondrodystrophic breeds: Rhodesian, ) ! atrophy of intervertebral discs spondylolisthesis; C5-C7; 8-12 months ! degeneration and weakening of anulus fibrosus Traumatic changes Traumatic changes

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Intervertebral disc disease (IVDD) Intervertebral disc disease (IVDD)

Samples Extrusion = Hansen type I herniation ! 13 spinal cord samples = disc material in extradural ! Formalin-fixed, paraffin-embedded space Methods 9 7.5 ! HE 6 4.5 → semiquantitatively (0 / + / ++ / +++) 3 # hemorrhages 1.5 Protrusion 0 epicenter = Hansen type II herniation # necrosis -1.5 -3 Traumatic changes = prolaps of anulus fibrosus Traumatic changes # spheroids -4.5 and disc material # myelinophagia -6 -7.5 # inflammation -9 cm

Intervertebral disc disease (IVDD) IVDD - morphological in vivo findings

Therapeutic Hemorrhages Necrosis intervention Vascular effects Seconds to Hemorrhage minutes Thrombosis

Biochemical processes Minutes to Lipid peroxydation hours Free radicals/fatty acids

Primary injury 100µm Secondary injury 3 days 1-2 days 100µm Mechanical impact Inflammation Hours to Macrophages, neutrophils, T cells, weeks immediately astroglia, microglia Apoptosis mean semiquantitative score semiquantitative mean mean semiquantitative score semiquantitative mean Fibre tract pathology

Traumatic changes Focal destruction Traumatic changes Weeks to Wallerian degeneration of neural tissue months Demyelination ABCD ABCDE ABCDE ABCD ABCDE ABCDE control acute subacute control acute subacute Bareyre and Schwab, 2003; 1-4 days >4 days 1-4 days >4 days Trends Neurosci; mod. p < 0.05 (Mann-Whitney test) p < 0.05 (Mann-Whitney test)

Intervertebral disc disease (IVDD) IVDD - morphological in vivo findings

acute 17 Study dogs Spheroids ! 3.5 – 12 y (mean 7 y), male & female ! naturally occuring spinal cord injury caused by intervertebral disc disease

100µm acute subacute n = 9 n = 8 subacute

0 1 2 3 4 5 6 7 10 days mean semiquantitative score semiquantitative mean

Traumatic changes 10 Control dogs Traumatic changes ABCD ABCDE ABCDE ! without neurologic disease control acute subacute 1-4 days >4 days 100µm p < 0.05 (Mann-Whitney test)

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IVDD - morphological in vivo findings IVDD - morphological in vivo findings

subacute Myelinophagia spatial extension of axonal swellings

acute SCI subacute SCI

100µm

subacute mean semiquantitative score semiquantitative mean Traumatic changes Traumatic changes

ABCD ABCDE ABCDE control acute subacute 1-4 days >4 days 100µm Bock, Spitzbarth et al., 2012; Brain Pathol Spitzbarth et al., 2011; p < 0.05 (Mann-Whitney test) J Neuropathol Exp Neurol

IVDD - morphological in vivo findings IVDD - morphological in vivo findings

Axonopathy control Normal axon MHC class II 2 Axonal reaction Axonal degeneration (retraction ball)

100µm

subacute SCI MHC-II positive cells per 0.063 mm 0.063 per cells positive MHC-II Axonal regeneration = neurofilament Group 1 = controls = mitochondria Traumatic changes Group 2 = acute (1 – 4 days post SCI) Traumatic changes Group 3 = subacute (5 – 10 days post SCI) = dense bodies 100µm * = p < 0.05 Spitzbarth et al., 2011; Kruskal Wallis Test and pair-wise = vesicles J Neuropathol Exp Neurol Mann Whitney U Tests

Modified according to Lampert, 1967 and Coleman, 2005

IVDD - morphological in vivo findings IVDD - morphological in vivo findings

Swollen axons Semiquantitave evaluation of spheroids in Histology the white matter

Transmission electron Ultrastructural analysis of axons microscopy Swollenaxons [mean score]

β-amyoid precursor protein (β-APP) 50 µm Immunohistology Phosphorylated and non-phosphorylated Neurofilament (nNF, pNF) Traumatic changes Traumatic changes subacute SCI Myelin basic protein (MBP)

Bock, Spitzbarth et al., 2012; Brain Pathol

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IVDD - morphological in vivo findings IVDD - morphological in vivo findings

Group 1: control animal Semiquantitative evaluation of spheroids Myelin basic protein expression

100 3,0 100 µm

2,0 Group 2: acute trauma 90

1,5 Spheroids

1,0 80

100 µm

0,5 [percentage] MBP- area positive 100 µm Group 3: subacute trauma 70 Traumatic changes Traumatic changes = p < 0.05 Group 1 = control animals Group 2 = acute spinal cord lesions (1-4 days) Group 1 = control animals Group 3 = subacute spinal cord lesions ( > 4 days) Group 2 = dogs with acute spinal cord lesions (1-4 days) 100 µm Group 3 = dogs with subacute spinal cord lesions (> 4 days)

IVDD - morphological in vivo findings IVDD - morphological in vivo findings

Group 1: control animal β-amyloid precursor protein expression Summary: Axonopathy and myelin expression

1,0 100 µm

0,8 Group 2: acute trauma

p = 0.06 0,6

0,4

100 µm 0,2 -APP positive area [percentage] area positive -APP β

Group 3: subacute trauma 0,0 Traumatic changes Traumatic changes = p < 0.05 ! Early appearance of axonal degeneration Group 1 = control animals ! Delayed myelin loss Group 2 = dogs with acute spinal cord lesions (1 – 4 days) ! Ultrastructural indication for axonal regeneration 100 µm Group 3 = dogs with subacute spinal cord lesions (> 4 days)

IVDD - morphological in vivo findings IVDD - morphological in vivo findings pNF nNF Group 1: control animal Group 1: control animal Spatial progression of axonal pathology

200

150 100 µm 100 µm + 9 cm + 3 cm epicen -3 cm n-NF ter Group 2: acute trauma 100 Group 2: acute trauma acute SCI β-APP

pNF/nNF50 ratio GAP-43 0

100 µm 100 µm Group 1 = control animals Group 2 = dogs with acute + 9 cm + 3 cm epicenter -3 cm Group 3: subacute trauma Group 3: subacute trauma

Traumatic changes spinal cord lesions (1-4 days) Traumatic changes n-NF Group 3 = dogs with subacute spinal cord lesions (> 4 days) β-APP subacute SCI Bock, Spitzbarth et al., 2012; Brain Pathol GAP-43 100 µm 100 µm

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IVDD - morphological in vivo findings IVDD – proposed pathogenesis in vivo Summary of results subacute canine SCI

! morphology ?? 1 – 4 days > 4 days Cytokines MHC class II IL-8 9 cm expression ! site & dimension ?? TGF-β

β-APP β-APP β-APP epi- ! spatio-temporal center n-NF n-NF n-NF Myelin development ?? phagocytosis

9 cm

cranial β-APP β-APP n-NF n-NF

epicenter Traumatic changes Traumatic changes

Axonal GAP-43 GAP-43 caudal regeneration attempts?

adapted from DeLahunta (Saunders 1983) „Veterinary Neuroanatomy and Clinical Neurology“

IVDD – proposed pathogenesis IVDD – proposed pathogenesis

acute canine SCI

Neutrophilic granulocytes Macrophages

Lymphocytes

Astrocytes

Microglia IL-5 IL-17 IL-1rA IL-2 IL-4 TGF-β IFN-γ TNF IL-8 IL-13 IL-10 IL-1 IL-12 IL-6 Primary injury: IVD herniation Traumatic changes Traumatic changes

Neurodegeneration Regeneration Disease exacerbation Repair

According to Spitzbarth et al., 2011; Vet Immunol Immunopathol

IVDD – proposed pathogenesis IVDD – therapeutic options

acute canine SCI Transplantation of BO-1 cells into myelin-deficient mice

Cytokines IL-6 IL-8 TNF

β-APP n-NF n-NF

Primary injury: IVD herniation # Day 17 post BO-1 cell transplantation: lethargy, weakness, tremor; euthanized! # Necropsy: infiltrative tumor growth from transplanted site to the meninges

Traumatic changes Axonal Traumatic changes GAP-43 regeneration and normal brain parenchyma. attempts? # Histological and histochemical analyses: giant cell glioblastoma. BO-1 cells not suitable for transplantation!

Hansmann et al., Cell Transplantation, 1161–1175, 2012

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IVDD – therapeutic options General aspects Olfactory ensheathing cells (OECs): non-myelinating cells present in PNS/CNS Compilation of disorders with selective degeneration and loss of cells or cell components (e.g. axons, myelin sheaths) in a bilaterally symmetric fashion!

! usually slowly progressive ! commonly start at young age ! suspected or confirmed genetic origin

Proposed classification: ! neuronal degenerations ! axonal degenerations !

Traumatic changes myelin disorders ! storage diseases Degenerative diseases Degenerative ! spongiform encephalopathies

Lempp et al., Viruses, 2014 ! spongy degenerations

IVDD – therapeutic options Degeneration of neurons

Canine glial cells used for cell transplantation in rats Morphological hallmark: Degeneration and loss of neurons in specific anatomical Focal Bestrahlung Ethidium bromide injection Cell transplantation, 0.5µl 0.3mg/ml lesion site (glass micro structures electodes) Depths of 0,7 72hrs 72hrs TH10/11 and 0,5 mm, Cause: each 0,5 µl cell suspension or 30 Intrinsic inherited biochemical defects (e.g. transcriptional 000 donor cells regulation defects, aggregation of proteins, activation of pro-apoptotic pathways etc.)

Pathology: ! restriction to particular neuronal types ! affection of several functionally different neuronal Traumatic changes populations Degenerative diseases Degenerative ! simultaneously (= multisystem neuronal degeneration) ! occasionally CNS + PNS + ANS affected • daily injections (i.p.) of cyclosporine A (15 mg/kg) beginning 1 day before cell transplantation and continued throughout the survival period

IVDD – therapeutic options Degeneration of neurons

Canine glial cells used for cell transplantation in rats Examples for selected neuronal degenerations/losses

Motor neurons hereditary canine spinal muscular atrophy

Purkinje cells cerebellocortical atrophy in Arabian foals Remyelination was observed after transplantation of each of the three glial cell types. Granule cells cerebellar granule cell High power (100x oil) images of the dorsal funiculus after transplantation of degeneration in Collie dogs SCs (A), OB-OECs (B), and OM-OECs (C) showing remyelination for all

Traumatic changes three cell types with characteristic peripheral–like myelination with large nuclei and cytoplasma. Other neurons multisystem neuronal Degenerative diseases Degenerative degeneration in Cocker spaniels Scale bar in C is 12 µm and pertains to A-C.

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Motor neuron diseases (MND) Cerebellar degenerations

Definition: Purkinje cell degenerations Degenerative disorder affecting motor neurons of spinal cord, = cerebellar cortical abiotrophies (CCA) brainstem and motor cortex most common type of neurodegeneration in domestic animals

Comparable disorder in man: Secondary transneuronal retrograde degeneration and loss Amyotrophic lateral sclerosis (ALS) of granule cells! = most common human MND familial ALS: mutation in superoxide dismutase (SOD) 1 gene CCA in dogs Scottish Terrier, Chow chow " neurons might die due to oxidative stress CCA in horses Arabian, Gotland horse CCA in bovines Holstein, Shorthorn Due to severe neurogenic muscle atrophy these disorders CCA in pigs Yorkshire are also termed spinal muscle atrophies (SMA). CCA in sheep Merino, Coriedale

Degenerative diseases Degenerative diseases Degenerative Olivo-ponto-cerebellar atrophy cats striato-nigral and cerebello- olivary degeneration Kerry blue Terrier

Motor neuron diseases (MND) Cerebellar degenerations

Hereditary canine musclar atrophy Brittany Spaniel Hereditary progressive neurogenic Pathogenesis muscular atrophy Pointer Cerebellar abiotrophy in Arabian horses: Familial motor neuron disease Rottweiler Mutation affecting a DNA-repairing gene (MUTYH) Asymmetrical spinal muscular atrophy German Shepherd dog Stockard´s paralysis Great dane Hereditary progressive neurogenic muscular atrophy English Pointer Hereditary neuronal abiotrophy Swedish Lapland dog Multisystem axonopathy and neuronopathy Golden Retriever Multisyst. chromatolytic neuronal degeneration Cairn Terrier Inherited motor neuron disease Domestic cats Bovine SMA Brown Swiss, Holstein Hereditary porcine neuronal system degeneration pigs

Spontaneous porcine motor diseases Degenerative Degenerative diseases Degenerative neuron disease pigs Lower motor neuron disease with neurofilamentous accumulation York-/Hampshire pigs

Motor neuron diseases (MND) Cerebellar degenerations

Pathological findings: Cerebellar granule cell degeneration Motor neuron changes: ! swelling of perikaryon ! familial/hereditary condition ! chromatolysis ! small number of canine breeds (e.g. Lagotto Romagnolo) ! peripheral eccentric neuronal nuclei ! primary granule cell degeneration ! pyknosis of the nucleus ! secondary astrogliosis ! neuronophagia ! in some cases T-lymphocytic infiltration (immune-mediated ! some disorders with accumulation of p-NF in response against granule cells?) degenerated motor neurons (= lower MND with neurofibrillary accumulation)

Additional findings: ! gliosis Degenerative diseases Degenerative ! Wallerian degeneration of peripheral motor nerves diseases Degenerative ! neurogenic muscle atrophy

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Other neuronal degenerations Axonal degenerations

Axonopathies with Wallerian-like degeneration Multisystem neuronal degeneration in Cocker Spaniels ! Hereditary ataxia Smooth-haired Fox and Jack - inherited disorder Russell Terrier -ataxia, mental deterioration ! Sensoric ataxic neuropathy Golden Retriever - diffuse neuronal loss in various brain nuclei ! Labrador Retriever axonopathy Labrador Retriever -additional axonal degeneration and demyelination ! Peripheral and central axonopathy Birman cat ! Degenerative myelopathy of large breed dogs German shepherd, Boxer, a.o. Neuronal vacuolation and spinocerebellar degeneration ! Degenerative myelopathy Pembroke Welsh Corgi in dogs ! Bovine progressive degenerative - various breeds (Rottweiler, Boxer, Husky a.o.) myeloencephalopathy Brown Swiss cattle - ataxia, tetraparesis, laryngeal paralysis ! Degenerative axonopathy in neonatal calves Friesian Holstein cattle - cerebellar nuclei, thalamus, spinal grey matter, ! Degenerative axonopathy Tyrolean cattle Degenerative diseases Degenerative autonomic ganglia: diseases Degenerative ! Familial degenerative neuro- neuronal vacuolation, spongiosis muscular disease Gelbvieh cattle

Axonal degenerations Axonal degenerations

Neuronal transport mechanisms: Axonopathies with Wallerian-like degeneration ! existence of effective axonal transport mechanisms ! axonal necrosis ! directions: away from the cell body = anterograde ! segmentation of myelin towards the cell body = retrograde ! removal of myelin and axonal debris by blood-borne MΦ ! molecular motor proteins (kinesin, dynein) ! astrogliosis ! transport tracks provided by cytoskeleton ! status spongiosus ! highly complex system " huge variety of disorders Axonal damage and secondary demyelination in ascending Axonal degenerations: or/and descending fibre tracts usually involving multiple ! typically young animals affected anatomical systems " ! often onset at hind limbs multisystem axonal degeneration (MAD) ! proprioceptive deficits Degenerative diseases Degenerative ! preservation of nocireception diseases Degenerative

Axonal degenerations Axonal degenerations

Morphological differentiation: Axonopathies with axonal swelling: ! axonopathies with Wallerian-like degeneration (white matter lesion!) ! Neuroaxonal dystrophy Rottweiler dog, Papillon, ! axonopathies with focal axonal swelling (axonal spheroids) Jack Russell Terrier, dom. (white matter lesion, except lesions are located at the shorthair cat, Suffolk sheep distal end of the axon where they synapse to their target ! Equine degenerative myelo- neuron) encephalopathy Morgan, Mongolian, others

! Giant axonal neuropathy German shepherd dog ! Central axonopathy Scottish Terrier ! Progressive axonopathy Boxer ! Degenerative myelopathy Pembroke Welsh Corgi ! Segmental axonopathy Merino sheep Degenerative diseases Degenerative diseases Degenerative

„digestion chambers“ Spheroid formation

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Axonal degenerations Myelin disorders

Axonopathy with prominent axonal swelling Leukodystrophy manifestations: Neuroaxonal dystrophy (NAD) = morphological alteration of axons leading to swelling, ! Cavitating leukodystrophy Dalmation dog, Labrador atrophy and/or degeneration: Retriever Axonal spheroid = localized swelling of the axon with distal ! Necrotizing myelopathy Afghan dog, Kooiker dog atrophy and secondary myelin degeneration ! Leukomyeloencephalopathy Rottweiler, Leonberger ! Globoid cell leukodystrophy West Highland White and Axonal changes start at the preterminal portions of the axon Cain Terrier, Kelpie and in the synaptic terminals " dystrophic axons found in ! Leukodystrophy Crossbred Maltese dog nuclei of the gray matter and spinal cord! ! Fibrinoid leukodystrophy In addition: dying back of neuronal cell bodies (Alexander´s disease) Labrador Retriever, Merino ! Progressive ataxia Charolais cattle ! Oligodendroglial dysplasia Bull Mastiff Degenerative diseases Degenerative diseases Degenerative

Axonal degenerations Myelin disorders

Fibrinoid leukodystrophy (Alexander´s disease)

Perro de Agua Español: mutation of TECPR2 = Tectonin beta-propeller repeat containing protein 2

! involved in protein degradation during autophagia " disturbance of neuronal autophagia as a possible cause of Myelin lesions associated neurodegnerative disease! with formation of Rosenthal fibers = string-like depositions of brightly eosinophilc amorphous In humans: ovoid bodies (perivascularly, Mutation in TECPR2 reveals a role for autophagy in hereditary spastic paraparesis below the pia and ependyma) Oz-Levi D et al., Am J Hum Genet. 91(6):1065-72. (2012) Degenerative diseases Degenerative diseases Degenerative

Myelin disorders Myelin disorders

" Leukodystrophies " Myelin dysgenesis Myelin dysgenesis Leukodystrophy (= myelinolytic diseases): (= hypomyelinogenesis or retarded myelinogenesis) Outstanding clinical sign: tremor of the whole body! ! disorder of myelin synthesis and maintenance ! bilateral symmetric affection of white matter ! hereditary or suspected hereditary forms ! destruction of myelin, eventually of axons ! defect of structural myelin proteins and/or ! infiltration of MΦ (myelinophages) metabolic defects of the oligodendrocyte Degenerative diseases Degenerative diseases Degenerative

17 25.06.2017

Myelin disorders Storage disorders

Neuronal ceroid lipofuscinosis (NCL): Myelin dysgenesis manifestations: = group of lysosomal storage disorders characterized by intracellular accumulation of autofluorescent lipofuscin, a ! Dysmyelination Chow Chow, Weimaraner, pigment-like granular brownish storage material. Lurcher dog, Siamese cat " inhibition of the fusion between autophagosomes ! Shaking pups Springer Spaniel and lysosomes leading to reduced autophagy ! CNS hypomyelination Rat Terrier ! Congenital tremor Jersey, Holstein-Friesian, Angus, Shorthorn cattle American Staffordshire Terrier: ! Congenital tremor type AIII Landrace pigs Defect of the arylsulfatase G gene (late onset!) ! Congenital tremor type AIV Saddleback pig ! Congenital bovine spinal Pathology: dysmyelination Brown Swiss cattle ! degeneration and loss of mainly Purkinje cells ! rarefication of the granular layer Degenerative diseases Degenerative diseases Degenerative ! storage of a fluorescent lipopigment in affected neurons

Storage disorders Storage disorders

Pathogenesis I: hereditary defects of lysosomal hydrolase enzyme:

Kyöstilä et al., 2015 Lagotto Romagnolo dog: ! missense change in the autophagy-related ATG4D gene ! progressive cerebellar ataxia, sometimes accompanied by Degenerative diseases Degenerative Degenerative diseases Degenerative episodic nystagmus and behavioral changes ! vacuolization of the neuronal cytoplasm ! Purkinje cell loss and granular cell depletion

Storage disorders Storage disorders

Pathogenesis II: Types: ! acquired disorders of lysosomal hydrolase enzyme ! glycoproteinoses (fucosidosis, mannosidosis, ! herbivorous species galactosialidosis) ! USA, South America, Australia, Africa ! sphingolipidoses (GM1, GM2 gangliosidosis, globoid ! plant toxins (Swainsona, Astralagus, Oxytropis, cell leukodystrophy [Morbus KRABBE], Gauchers Ipomea a.o.) containing lysosomal glycosidase inhibitors disease, Niemann-Pick disease) (alkaloids) ! mucopolysaccharidoses (MPS I – VII) " lysosomal accumulation of substrates leading to ! glycogenoses (Oligosaccharidoses) neuronal vacuolation mimicking inherited defects ! proteinoses (ceroid-lipofuscinosis) ! Lafora´s disease (polyglucosan accumulation) Degenerative diseases Degenerative diseases Degenerative

18 25.06.2017

Transmissible spongiform encephalopathies Transmissible spongiform encephalopathies

Neuropathology of TSE ! progressive lethal diseases 1. Vacuolation of the neuropil (vacuoles in neuronal ! very long incubation periods (years) cell processes) ! degenerative, symmetrical spongy vacuolation Always bilaterally symmetrical ! no inflammatory changes Neuroanatomical localization depends on the species ! conformational change of a cellular protein and TSE strain (prion protein = PrPC " PrPsc ) ! accumulation of PrPsc " amyloid fibrils DDx: = scrapie-associated fibrils ! spongy degeneration ! accumulated PrPsc " disease transmission, ! brain edema because prions are capable of initiating its own replication ! postmortem changes ! causes: spontaneous, hereditary, infectious ! histotechnological artifacts 2. Vacuolation of neuronal cytoplasm Degenerative diseases Degenerative diseases Degenerative

DDx: Neuronal vacuoles in midbrain of normal ruminants

Transmissible spongiform encephalopathies Transmissible spongiform encephalopathies

TSE in man and animals Scrapie Man Creutzfeldt-Jakob disease (CJD), sporadic, ! sheep, goats familial ! natural transmission Kuru, food borne (cannibalism) ! 20 different strains with different incubation periods and variant CJD, food borne infection (BSE) lesion profiles Gerstmann-Sträußler-Scheinker, hereditary ! genetic disposition familial fatal insomnia ! brain stem predominantly affected ! replication in lymphatic tissue and placenta Small ruminants classical scrapie, natural infection atypical scrapie, transmission mode unknown Atypical Scrapie ! first detected in Norwegian sheep („Nor98“) Cattle classical BSE, food borne infection ! affected animals older as in scrapie-infected sheep atypical BSE, transmission mode unknown ! ataxia! No pruritus or wool loss!

Degenerative diseases Degenerative diseases Degenerative ! no natural transmission ! cerebellum predominantly affected

Transmissible spongiform encephalopathies Transmissible spongiform encephalopathies

TSE in man and animals (continued) BSE ! cattle (> 200.000 cases since 1985) Mink transmissible mink encephalopathy, ! transmission by animal protein supplements food borne infection (scrapie?) (meat and bone meal) Zoo ungulates BSE, food borne infection (BSE) ! spread to more than 10 other species (antelopes, felids, man) Domestic cats, zoo felids feline spongiform encephalopathy, food borne infection (BSE) Atypical BSE ! L- (lower) and H-type (higher molecular mass than American mule deer/elk chronic wasting disease (CWD), PrPsc of typical BSE) natural infection ! affected animals older than in classical BSE ! no reliable data on neuropathology Degenerative diseases Degenerative diseases Degenerative

19 25.06.2017

Spongy degeneration

! spongy state of gray and/or white matter ! cause: cytotoxic edema ! fluid accumulation in astrocytes ! sharply delineated vacuoles ! often neither degeneration of neurons/axons nor of the white matter ! long lasting cases: reactive changes resulting from compression (neuronal, axonal and myelin degeneration, infiltration of MΦ, gliosis) Degenerative diseases Degenerative

Spongy degeneration Degenerative diseases Degenerative

(Vandevelde et al., 2012)

Selective symmetrical encephalomalacias SSE: ! sharply defined areas of encephalomalacia ! smilarity to Leigh syndrome in children ! bilaterally symmetrical fashion ! selective anatomical areas of brain and spinal cord ! mostly juvenile animals affected ! lesion vary from spongiosis to cavitation

Mitochondrial hereditary polioencephalopathy Austral. Cattle dog (possibly) Alaskan Husky encephalopathy Alaskan Husky Subacute necrotizing encephalo- pathy Yorkshire Terrier multifocal symmetrical necrotizing encephalomyelopathy Angus cattle Degenerative diseases Degenerative multifocal subacute necrotizing encephalomyelopathy Simmental cattle Simmental/crosses

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