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The neural control system underlying breathing is sexually dimorphic with males being more vulnerable to dysfunction. Microglia also display sex differences, and their role in the architecture of brainstem respiratory rhythm circuitry and modulation of cervical spinal cord respiratory plasticity is becoming better appreciated. To further understand the molecular underpinnings of these sex differences, we performed RNA sequencing of immunomagnetically isolated microglia from brainstem and cervical spinal cord of adult male and female rats. We used various bioinformatics tools (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, STRING, MAGICTRICKS) to functionally categorize identified gene sets, as well as to pinpoint common transcriptional gene drivers that may be responsible for the observed transcriptomic differences. We found few sex differences in the microglial transcriptomes derived from the brainstem, but several hundred genes were differentially expressed by sex in cervical spinal microglia. Comparing brainstem and spinal microglia within and between sexes, we found that the major factor guiding transcriptomic differences was central nervous system (CNS) location rather than sex. We further identified key transcriptional drivers that may be responsible for the transcriptomic differences observed between sexes and CNS regions; enhancer of zeste homolog 2 emerged as the predominant driver of the differentially downregulated genes. We suggest that functional gene alterations identified in metabolism, transcription, and intercellular communication underlie critical microglial heterogeneity and sex differences in CNS regions that contribute to respiratory disorders categorized by dysfunction in neural control. These data will also serve as an important resource data base to advance our understanding of innate immune cell contributions to sex differences and the field of respiratory neural control. SIGNIFICANCE STATEMENT: The contributions of central nervous system (CNS) innate immune cells to sexually dimorphic differences in the neural circuitry controlling breathing are poorly understood. We identify key transcriptomic differences, and their transcriptional drivers, in microglia derived from the brainstem and the C3-C6 cervical spinal cord of healthy adult male and female rats. Gene alterations identified in metabolism, gene transcription, and intercellular communication likely underlie critical microglial heterogeneity and sex differences in these key CNS regions that contribute to the neural control of breathing.

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 (β-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

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.

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.

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.

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 B(12)-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.

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.

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.

We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of theop/opoptic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic.In vivoandex vivoelectrophysiological examinations of P30op/opmice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.

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.