Pathologic classification of a late-onset peripheral neuropathy in a spontaneous Labrador retriever dog model.

Late-onset peripheral neuropathy (LPN) is a heritable canine neuropathy commonly found in Labrador retrievers and is characterized by laryngeal paralysis and pelvic limb paresis. Our objective was to establish canine LPN as a model for human hereditary peripheral neuropathy by classifying it as either an axonopathy or myelinopathy and evaluating length-dependent degeneration. We conducted a motor nerve conduction study of the sciatic and ulnar nerves, electromyography (EMG) of appendicular and epaxial musculature, and histologic analysis of sciatic and recurrent laryngeal nerves in LPN-affected and control dogs. LPN-affected dogs exhibited significant decreases in compound muscle action potential (CMAP) amplitude, CMAP area, and pelvic limb latencies. However, no differences were found in motor nerve conduction velocity, residual latencies, or CMAP duration. Distal limb musculature showed greater EMG changes in LPN-affected dogs. Histologically, LPN-affected dogs exhibited a reduction in the number of large-diameter axons, especially in distal nerve regions. In conclusion, LPN in Labrador retrievers is a common, spontaneous, length-dependent peripheral axonopathy that is a novel animal model of age-related peripheral neuropathy that could be used for fundamental research and clinical trials.

Wobbly hedgehog syndrome- a progressive neurodegenerative disease.

Wobbly hedgehog syndrome (WHS) has been long considered to be a myelin disease primarily affecting the four-toed hedgehog. In this study, we have shown for the first time that demyelination is accompanied by extensive remyelination in WHS. However, remyelination is not enough to compensate for the axonal degeneration and neuronal loss, resulting in a progressive neurodegenerative disease reminiscent of progressive forms of multiple sclerosis (MS) in humans. Thus, understanding the pathological features of WHS may shed light on the disease progression in progressive MS and ultimately help to develop therapeutic strategies for both diseases.

Wobbly hedgehog syndrome- a progressive neurodegenerative disease.

Wobbly hedgehog syndrome (WHS) has been long considered to be a myelin disease primarily affecting the four-toed hedgehog. In this study, we have shown for the first time that demyelination is accompanied by extensive remyelination in WHS. However, remyelination is not enough to compensate for the axonal degeneration and neuronal loss, resulting in a progressive neurodegenerative disease reminiscent of progressive forms of multiple sclerosis (MS) in humans. Thus, understanding the pathological features of WHS may shed light on the disease progression in progressive MS and ultimately help to develop therapeutic strategies for both diseases. Highlights: Wobbly hedgehog syndrome (WHS) is a progressive neurodegenerative disease.Spongy degeneration of the brain and spinal cord is the diagnostic feature of WHS.WHS affected brain and spinal cord show extensive demyelination and remyelination.Axonal degeneration is accompanied by loss of neurons in WHS.

Transmission Electron Microscopy and Morphometry of the CNS White Matter.

Transmission electron microscopy of central nervous system white matter has provided unparalleled access to the ultrastructural features of axons, their myelin sheaths, and the major cells of white matter; namely, oligodendrocytes, oligodendrocyte precursors, astrocytes, and microglia. In particular, it has been invaluable in elucidating pathological changes in axons and myelin following experimentally induced injury or genetic alteration, in animal models. While also of value in the examination of human white matter, the tissue is rarely fixed adequately for the types of detailed analyses that can be performed on well-preserved samples from animal models, perfusion fixed at the time of death. In this chapter we describe methods for obtaining, processing, and visualizing white matter samples using transmission electron microscopy of perfusion fixed tissue and for unbiased morphometry of white matter, with particular emphasis on axon and myelin pathology. Several advanced electron microscopy techniques are now available, but this method remains the most expedient and accessible for routine ultrastructural examination and morphometry.

Regulatory B Cells Normalize CNS Myeloid Cell Content in a Mouse Model of Multiple Sclerosis and Promote Oligodendrogenesis and Remyelination.

The unmet medical need of patients with multiple sclerosis (MS) is the inexorable loss of CNS myelin and latterly neurons leading to permanent neurologic disability. Solicitation of endogenous oligodendrocytes progenitor cells, the precursor of oligodendrocytes, to remyelinate axons may abort the onset of disability. In female mice with experimental autoimmune encephalomyelitis (EAE), a murine model of MS, adoptive transfer of IL-10+ regulatory B cells (Bregs) has been shown to reverse EAE by promoting the expansion of peripheral and CNS-infiltrating IL-10+ T cells. Here, we examined whether Bregs treatment and its bystander effect on regulatory T cells are associated with CNS repair as reflected by oligodendrogenesis and remyelination. We have found that transfusion of Bregs reverses established clinical EAE and that clinical improvement is associated with a significant increase in spinal cord remyelination as reflected by g-ratio analysis within the thoracic and lumbar spine. We further observed in the spinal cords of EAE Bregs-treated mice that CNS resident CD11b/CD45intLy6C- microglia, and infiltrating CD11b+/CD45high monocytes/macrophages content reverts to normal and polarize to a M2-like CD206+ phenotype. Concurrently, there was a substantial increase in neo-oligodendrogenesis as manifest by an increase in CD45-/low CNS cells expressing A2B5, an early marker in oligodendrocytes progenitor cell differentiation as well as GalC+/O1+ premyelinating and myelin basic protein+/myelin oligodendrocyte glycoprotein+ mature oligodendrocytes with reciprocal downregulation of paired related homeobox protein 1. These results demonstrate that the clinical benefit of Bregs is associated with normalization of CNS immune milieu and concurrent activation of oligodendrocyte progenitor cells with subsequent remyelination.SIGNIFICANCE STATEMENT In multiple sclerosis patients, demyelination progresses with aging and disease course, leading to irreversible disability. In this study, we have discovered, using a mouse model of multiple sclerosis, that the transfusion of autologous regulatory B cells (Bregs) is able to ameliorate, cure, and sustain the durable remission of the disease. We show that the adoptive transfer of Bregs dramatically decreased the frequency of myeloid-derived cells, both infiltrating monocytes/macrophages and resident microglia, and converted their phenotype to an immunosuppressive-like phenotype. Moreover, we showed that CNS oligodendrocyte progenitor cells are activated following Bregs treatment and differentiate into myelinating oligodendrocytes, which results in neo-oligodendrogenesis and remyelination of spinal cords.

Feline irradiated diet-induced demyelination; a model of the neuropathology of sub-acute combined degeneration?

Irradiation of food at 50-55 kGy results in a profound, chronic demyelinating-remyelinating disease of the entire central nervous system (CNS) in cats, named Feline Irradiated Diet-Induced Demyelination (FIDID). This study examines the early stages of demyelination and long-term consequences of demyelination and remyelination on axon survival or loss. Myelin vacuolation is the primary defect leading to myelin breakdown, demyelination then prompt remyelination in the spinal cord and brain. There is no evidence of oligodendrocyte death. The spinal cord dorsal column is initially spared yet eventually becomes severely demyelinated with subsequent loss of axons in the core and then surface of the fasciculus gracilis. However remyelination of the sub-pial axons in the dorsal column results in their protection. While there was a lack of biochemical evidence of Vitamin B12 deficiency, the pathological similarities of FIDID with sub-acute combined degeneration (SCD) led us to explore treatment with Vitamin B12. Treatment led to recovery or improvement in some cats and neurologic relapse on cessation of B12 therapy. While the reason that irradiated food is myelinotoxic in the cat remains unresolved, nonetheless the neuropathological changes match exactly what is seen in SCD and its models and provide an ideal model to study the cellular and molecular basis of remyelination.

Evoked potentials as a biomarker of remyelination.

Multiple sclerosis (MS) is a common cause of neurologic disease in young adults that is primarily treated with disease-modifying therapies which target the immune and inflammatory responses. Promotion of remyelination has opened a new therapeutic avenue, but how best to determine efficacy of remyelinating drugs remains unresolved. Although prolongation and then shortening of visual evoked potential (VEP) latencies in optic neuritis in MS may identify demyelination and remyelination, this has not been directly confirmed. We recorded VEPs in a model in which there is complete demyelination of the optic nerve, with subsequent remyelination. We examined the optic nerves microscopically during active disease and recovery, and quantitated both demyelination and remyelination along the length of the nerves. Latencies of the main positive component of the control VEP demonstrated around 2-fold prolongation during active disease. VEP waveforms were nonrecordable in a few subjects or exhibited a broadened profile which precluded peak identification. As animals recovered neurologically, the VEP latencies decreased in association with complete remyelination of the optic nerve but remained prolonged relative to controls. Thus, it has been directly confirmed that VEP latencies reflect the myelin status of the optic nerve and will provide a surrogate marker in future remyelination clinical trials.

Conventional and quantitative MRI in a novel feline model of demyelination and endogenous remyelination.

BACKGROUND: The feeding of irradiated food to healthy adult cats results in widespread, noninflammatory demyelination of the central nervous system (CNS); a return to a normal diet results in endogenous remyelination with functional recovery. This recently discovered, reversible disease might provide a compelling clinical neuroimaging model system for the development and testing of myelin-directed MRI methods as well as future remyelination therapies. PURPOSE: Identify the noninvasive imaging characteristics of this new disease model and determine whether it features measurable changes on conventional and quantitative MRI. STUDY TYPE: Pilot study. ANIMAL MODEL: Ten adult cats at various stages of demyelinating disease induced by an irradiated diet (35-55 kGy), and during recovery following a return to a normal diet. FIELD STRENGTH/SEQUENCE: Conventional (T2 -weighted) and quantitative (diffusion tensor, magnetization transfer) at 3T. ASSESSMENT: MRI of the brain, optic nerves, and cervical spinal cord; a subset of diseased cats was euthanized for comparative histopathology. STATISTICAL TESTS: Descriptive statistics. RESULTS: Disease produced T2 prolongation, progressing from patchy to diffuse throughout most of the cerebral white matter (eventually involving U-fibers) and spinal cord (primarily dorsal columns, reminiscent of subacute combined degeneration but without evidence of B12 deficiency). Magnetization transfer parameters decreased by 50-53% in cerebral white matter and by 25-30% in optic nerves and spinal cord dorsal columns. Fractional diffusion anisotropy decreased by up to 20% in pyramidal tracts, primarily driven by increased radial diffusivity consistent with axon preservation. Histopathology showed scattered myelin vacuolation of major white matter tracts as well as many thin myelin sheaths consistent with remyelination in the recovery phase, which was detectable on magnetization transfer imaging. DATA CONCLUSION: Feline irradiated diet-induced demyelination features noninvasively imageable and quantifiable demyelination and remyelination of the CNS. It is therefore a compelling clinical neuroimaging model system. LEVEL OF EVIDENCE: 4 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;49:1304-1311.

The adult oligodendrocyte can participate in remyelination.

Endogenous remyelination of the CNS can be robust and restore function, yet in multiple sclerosis it becomes less complete with time. Promoting remyelination is a major therapeutic goal, both to restore function and to protect axons from degeneration. Remyelination is thought to depend on oligodendrocyte progenitor cells, giving rise to nascent remyelinating oligodendrocytes. Surviving, mature oligodendrocytes are largely regarded as being uninvolved. We have examined this question using two large animal models. In the first model, there is extensive demyelination and remyelination of the CNS, yet oligodendrocytes survive, and in recovered animals there is a mix of remyelinated axons interspersed between mature, thick myelin sheaths. Using 2D and 3D light and electron microscopy, we show that many oligodendrocytes are connected to mature and remyelinated myelin sheaths, which we conclude are cells that have reextended processes to contact demyelinated axons while maintaining mature myelin internodes. In the second model in vitamin B12-deficient nonhuman primates, we demonstrate that surviving mature oligodendrocytes extend processes and ensheath demyelinated axons. These data indicate that mature oligodendrocytes can participate in remyelination.

Thin myelin sheaths as the hallmark of remyelination persist over time and preserve axon function.

The presence of thin myelin sheaths in the adult CNS is recognized as a marker of remyelination, although the reason there is not a recovery from demyelination to normal myelin sheath thickness remains unknown. Remyelination is the default pathway after myelin loss in all mammalian species, in both naturally occurring and experimental disease. However, there remains uncertainty about whether these thin sheaths thicken with time and whether they remain viable for extended periods. We provide two lines of evidence here that thin myelin sheaths may persist indefinitely in long-lived animal models. In the first, we have followed thin myelin sheaths in a model of delayed myelination during a period of 13 years that we propose results in the same myelin sheath deficiencies as seen in remyelination; that is, thin myelin sheaths and short internodes. We show that the myelin sheaths remain thin and stable on many axons throughout this period with no detrimental effects on axons. In a second model system, in which there is widespread demyelination of the spinal cord and optic nerves, we also show that thinly remyelinated axons with short internodes persist for over the course of 2 y. These studies confirm the persistence and longevity of thin myelin sheaths and the importance of remyelination to the long-term health and function of the CNS.

A mutation in the Tubb4a gene leads to microtubule accumulation with hypomyelination and demyelination.

OBJECTIVE: Our goal was to define the genetic cause of the profound hypomyelination in the taiep rat model and determine its relevance to human white matter disease. METHODS: Based on previous localization of the taiep mutation to rat chromosome 9, we tested whether the mutation resided within the Tubb4a (ß-tubulin 4A) gene, because mutations in the TUBB4A gene have been described in patients with central nervous system hypomyelination. To determine whether accumulation of microtubules led to progressive demyelination, we analyzed the spinal cord and optic nerves of 2-year-old rats by light and electron microscopy. Cerebral white matter from a patient with TUBB4A Asn414Lys mutation and magnetic resonance imaging evidence of severe hypomyelination were studied similarly. RESULTS: As the taiep rat ages, there is progressive loss of myelin in the brain and dorsal column of the spinal cord associated with increased oligodendrocyte numbers with accumulation of microtubules. This accumulation involved the entire cell body and distal processes of oligodendrocytes, but there was no accumulation of microtubules in axons. A single point mutation in Tubb4a (p.Ala302Thr) was found in homozygous taiep samples. A similar hypomyelination associated with increased oligodendrocyte numbers and arrays of microtubules in oligodendrocytes was demonstrated in the human patient sample. INTERPRETATION: The taiep rat is the first animal model of TUBB4 mutations in humans and a novel system in which to test the mechanism of microtubule accumulation. The finding of microtubule accumulation in a patient with a TUBB4A mutation and leukodystrophy confirms the usefulness of taiep as a model of the human disease. Ann Neurol 2017;81:690-702.

Modeling the Chronic Loss of Optic Nerve Axons and the Effects on the Retinal Nerve Fiber Layer Structure in Primary Disorder of Myelin.

PURPOSE: We determined whether the chronic lack of optic nerve myelination and subsequent axon loss is associated with optical coherence tomography (OCT) changes in the retinal nerve fiber layer (RNFL), and whether this models what occurs in multiple sclerosis (MS) and confers its use as a surrogate marker for axon degeneration. METHODS: Using an animal model of Pelizaeus-Merzbacher disease (shp) bilateral longitudinal measurements of the peripapillary RNFL (spectral-domain OCT), electroretinograms (ERG), and visual evoked potentials (VEP) were performed in affected and control animals from 5 months to 2 years and in individual animals at single time points. Light and electron microscopy of the optic nerve and retina and histomorphometric measurements of the RNFL were compared to OCT data. RESULTS: Of the shp animals, 17% had an average reduction of OCT RNFL thickness on the superior retinal quadrant compared to controls (P < 0.05). Electroretinograms showed normal photopic A- and B-waves but flash VEPs were disorganized in shp animals. Morphologically, the shp retinas and optic nerves revealed significant RNFL thinning (P < 0.001) without retinal ganglion cell (RGC) loss, decrease total and relative retinal axonal area, and loss of optic nerve axons. There was strong positive correlation between OCT and morphometric RNFL thickness measurements (r = 0.878, P = 0.004). CONCLUSION: The loss of optic nerve axons demonstrated in the shp model resulted in moderate thinning of the RNFL confirmed by OCT and histology. These results indicate that OCT-derived RNFL measurement can be a useful surrogate biomarker of optic nerve axon loss and potentially disease progression in demyelinating diseases.

Myelin repair by transplantation of myelin-forming cells in globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a devastating demyelinating disease that affects both the central and peripheral nervous systems. It is caused by genetic deficiency in the activity of a lysosomal enzyme, galactocerebrosidase (GALC), which is necessary for the maintenance of myelin. Hematopoietic stem cell transplantation (HSCT) including umbilical cord stem cell transplantation is the only effective therapy available to date. HSCT significantly prolongs the life span of patients with GLD when performed before disease onset, although it is not curative. In HSCT, infiltrating donor-derived macrophages are thought to indirectly supply the enzyme (called "cross-correction") to the host's myelinating cells. Given the limitation in treating GLD, it is hypothesized that remyelinating demyelinated axons with GALC-competent myelinating cells by transplantation will result in more stable myelination than endogenous myelin repair supported by GALC cross-correction. Transplantation of myelin-forming cells in a variety of animal models of dysmyelinating and demyelinating disorders suggests that this approach is promising in restoring saltatory conduction and protecting neurons by providing new healthy myelin. However, GLD is one of the most challenging diseases in terms of the aggressiveness of the disease and widespread pathology. Experimental transplantation of myelin-forming cells in the brain of a mouse model of GLD has been only modestly effective to date. Thus, a practical strategy for myelin repair in GLD would be to combine the rapid and widespread cross-correction of GALC by HSCT with the robust, stable myelination provided by transplanted GALC-producing myelin-forming cells. This short review will discuss such possibilities. © 2016 Wiley Periodicals, Inc.

Inherited and acquired disorders of myelin: The underlying myelin pathology.

Remyelination is a major therapeutic goal in human myelin disorders, serving to restore function to demyelinated axons and providing neuroprotection. The target disorders that might be amenable to the promotion of this repair process are diverse and increasing in number. They range primarily from those of genetic, inflammatory to toxic origin. In order to apply remyelinating strategies to these disorders, it is essential to know whether the myelin damage results from a primary attack on myelin or the oligodendrocyte or both, and whether indeed these lead to myelin breakdown and demyelination. In some disorders, myelin sheath abnormalities are prominent but demyelination does not occur. This review explores the range of human and animal disorders where myelin pathology exists and focusses on defining the myelin changes in each and their cause, to help define whether they are targets for myelin repair therapy.

Myelin and oligodendrocyte development in the canine spinal cord.

We studied the developmental pattern of oligodendrocyte differentiation and myelin formation in the fetal canine spinal cord from E40 to P0. The pattern of development matches what has been described in the spinal cord of humans, rodents, and many other species. Oligodendrocytes were first found at E40, close to the central canal, with their spread in a tangential manner to the ventral and then lateral columns. Myelin development followed the same pattern but was not seen until E46. A clear subpial zone lacking glial cells and myelin was seen in the lateral column in early development, suggesting that there may also be a radial component of migration of oligodendrocyte progenitor cells (OPCs) from a ventral site. This spatial-temporal developmental pattern seen in wild type matches a delay in myelination of the superficial tracts of the spinal cord seen in a canine myelin mutant, suggesting that the mutation prevents the distribution and differentiation of OPCs at an early, but narrow, window of time during fetal development.

Modeling the natural history of Pelizaeus-Merzbacher disease.

Major gaps in our understanding of the leukodystrophies result from their rarity and the lack of tissue for the interdisciplinary studies required to extend our knowledge of the pathophysiology of the diseases. This study details the natural evolution of changes in the CNS of the shaking pup (shp), a model of the classical form of the X-linked disorder Pelizaeus-Merzbacher disease, in particular in glia, myelin, and axons, which is likely representative of what occurs over time in the human disease. The mutation in the proteolipid protein gene, PLP1, leads to a delay in differentiation, increased cell death, and a marked distension of the rough endoplasmic reticulum in oligodendrocytes. However, over time, more oligodendrocytes differentiate and survive in the spinal cord leading to an almost total recovery of myelination, In contrast, the brain remains persistently hypomyelinated. These data suggest that shp oligodendrocytes may be more functional than previously realized and that their early recruitment could have therapeutic value.

A mutation in the canine gene encoding folliculin-interacting protein 2 (FNIP2) associated with a unique disruption in spinal cord myelination.

Novel mutations in myelin and myelin-associated genes have provided important information on oligodendrocytes and myelin and the effects of their disruption on the normal developmental process of myelination of the central nervous system (CNS). We report here a mutation in the folliculin-interacting protein 2 (FNIP2) gene in the Weimaraner dog that results in hypomyelination of the brain and a tract-specific myelin defect in the spinal cord. This myelination disruption results in a notable tremor syndrome from which affected dogs recover with time. In the peripheral tracts of the lateral and ventral columns of the spinal cord, there is a lack of mature oligodendrocytes. A genome-wide association study of DNA from three groups of dogs mapped the gene to canine chromosome 15. Sequencing of all the genes in the candidate region identified a frameshift mutation in the FNIP2 gene that segregated with the phenotype. While the functional role of FNIP2 is not known, our data would suggest that production of truncated protein results in a delay or failure of maturation of a subpopulation of oligodendrocytes.

Modality-based organization of ascending somatosensory axons in the direct dorsal column pathway.

The long-standing doctrine regarding the functional organization of the direct dorsal column (DDC) pathway is the "somatotopic map" model, which suggests that somatosensory afferents are primarily organized by receptive field instead of modality. Using modality-specific genetic tracing, here we show that ascending mechanosensory and proprioceptive axons, two main types of the DDC afferents, are largely segregated into a medial-lateral pattern in the mouse dorsal column and medulla. In addition, we found that this modality-based organization is likely to be conserved in other mammalian species, including human. Furthermore, we identified key morphological differences between these two types of afferents, which explains how modality segregation is formed and why a rough "somatotopic map" was previously detected. Collectively, our results establish a new functional organization model for the mammalian direct dorsal column pathway and provide insight into how somatotopic and modality-based organization coexist in the central somatosensory pathway.