Assessment of clinical outcomes associated with mercury concentrations in harbor seal pups (Phoca vitulina richardii) in central California.

Monomethyl mercury (MeHg+) from the diet can cause mild to severe neurotoxicosis in fish-eating mammals. Chronic and low-level in utero exposure also can be neurotoxic, as documented in laboratory animal studies and epidemiologic investigations. In free-ranging animals, it is challenging to study low-level exposure related neurotoxicosis, and few studies have investigated the relationship between mercury (Hg) and adverse outcomes in wild populations. Relative to Hg concentrations on admission we evaluated different types of behaviors for 267 Pacific harbor seal (HS; Phoca vitulina richardii) pups at The Marine Mammal Center from 2015 to 2019 during rehabilitation after stranding and maternal separation. Admitted HS pups underwent a clinical exam; including sex and weight determination, and hair (partly lanugo grown in utero) and blood samples were collected for total Hg concentration ([THg]) determination. All pups were monitored weekly (behavior assessments included response to tactile stimulation, movement, swimming, interactions with other seals, hand feeding, and feeding independently), and days in rehabilitation and survival were recorded. There was a significant negative correlation between [THg] and responses to tactile stimulation and movements, measured in both hair and whole blood (p < 0.05). This relationship was found both during the intensive care unit (ICU) stage, and during the pool stage of rehabilitation. Additionally, there was a significant association between greater [THg] and number of days spent in rehabilitation, although there was no relationship between [THg] and survival. There was a significant sex difference, with greater [THg] in female pups, which contrasts with previously published findings in juvenile and adult harbor seals. Our findings support small, but significant associations between gestational THg exposure and clinical effects for tactile sensory response and movement, and longer rehabilitation durations for HS pups, although there was considerable variability among animals.

Evaluation of cell-free DNA as a diagnostic marker in cerebrospinal fluid of dogs.

OBJECTIVE: To determine whether cell-free DNA (cfDNA) was detectable in CSF samples from dogs, whether CSF sample volume impacted CSF cfDNA concentration measurement, and whether CSF cfDNA concentration was associated with CNS disease category or CSF RBC count (RBCC), nucleated cell count (NCC), or protein concentration, which could aid in the diagnosis of neurologic diseases in dogs. SAMPLE: 80 CSF samples collected from dogs with (n = 60) and without (20) clinical neurologic disease between February 2017 and May 2018. PROCEDURES: Results for CSF RBCC, NCC, protein concentration, and cfDNA concentration were compared across CSF groups established on the basis of whether they were obtained from dogs with (case groups) or without (control group) clinical signs of neurologic disease In addition, 5 paired CSF samples representing large (3.0-mL) and small (0.5-mL) volumes, were used to evaluate whether sample volume impacted measurement of CSF cfDNA concentration. RESULTS: cfDNA was detected in 76 of the 80 (95%) CSF samples used to evaluate parameters across disease categories and in all 5 of the paired samples used to evaluate whether sample volume impacted cfDNA quantification. There were no substantial differences in cfDNA concentrations identified between groups (on the basis of disease category or sample volume), and the CSF cfDNA concentration did not meaningfully correlate with CSF RBCC, NCC, or protein concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Although results indicated that the CSF cfDNA concentration could not be used to differentiate between categories of neurologic disease in dogs of the the present study, further investigation is warranted regarding the use of CSF analysis, including sequencing specific cfDNA mutations, for diagnosing and monitoring neurologic disease in dogs.

Functional duality of astrocytes in myelination.

Astrocytes undergo major phenotypic changes in response to injury and disease that directly influence repair in the CNS, but the mechanisms involved are poorly understood. Previously, we have shown that neurosphere-derived rat astrocytes plated on poly-L-lysine (PLL-astrocytes) support myelination in dissociated rat spinal cord cultures (myelinating cultures). It is hypothesized that astrocyte reactivity can affect myelination, so we have exploited this culture system to ascertain how two distinct astrocyte phenotypes influence myelination. Astrocytes plated on tenascin C (TnC-astrocytes), a method to induce quiescence, resulted in less myelinated fibers in the myelinating cultures when compared with PLL-astrocytes. In contrast, treatment of myelinating cultures plated on PLL-astrocytes with ciliary neurotrophic factor (CNTF), a cytokine known to induce an activated astrocyte phenotype, promoted myelination. CNTF could also reverse the effect of quiescent astrocytes on myelination. A combination of microarray gene expression analysis and quantitative real-time PCR identified CXCL10 as a potential candidate for the reduction in myelination in cultures on TnC-astrocytes. The effect of TnC-astrocytes on myelination was eliminated by neutralizing CXCL10 antibodies. Conversely, CXCL10 protein inhibited myelination on PLL-astrocytes. Furthermore, CXCL10 treatment of purified oligodendrocyte precursor cells did not affect proliferation, differentiation, or process extension compared with untreated controls, suggesting a role in glial/axonal ensheathment. These data demonstrate a direct correlation of astrocyte phenotypes with their ability to support myelination. This observation has important implications with respect to the development of therapeutic strategies to promote CNS remyelination in demyelinating diseases.

Identification of Tmem10/Opalin as an oligodendrocyte enriched gene using expression profiling combined with genetic cell ablation.

Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, which allows efficient and rapid propagation of action potentials. However, little is known about the molecular mechanisms operating at the onset of myelination and during maintenance of the myelin sheath in the adult. Here we use a genetic cell ablation approach combined with Affymetrix GeneChip microarrays to identify a number of oligodendrocyte-enriched genes that may play a key role in myelination. One of the "oligogenes" we cloned using this approach is Tmem10/Opalin, which encodes for a novel transmembrane glycoprotein. In situ hybridization and RT-PCR analysis revealed that Tmem10 is selectively expressed by oligodendrocytes and that its expression is induced during their differentiation. Developmental immunofluorescence analysis demonstrated that Tmem10 starts to be expressed in the white matter tracks of the cerebellum and the corpus callosum at the onset of myelination after the appearance of other myelin genes such as MBP. In contrast to the spinal cord and brain, Tmem10 was not detected in myelinating Schwann cells, indicating that it is a CNS-specific myelin protein. In mature oligodendrocytes, Tmem10 was present at the cell soma and processes, as well as along myelinated internodes, where it was occasionally concentrated at the paranodes. In myelinating spinal cord cultures, Tmem10 was detected in MBP-positive cellular processes that were aligned with underlying axons before myelination commenced. These results suggest a possible role of Tmem10 in oligodendrocyte differentiation and CNS myelination.

Distribution of mitochondria along small-diameter myelinated central nervous system axons.

Small-diameter myelinated CNS axons are preferentially affected in multiple sclerosis (MS) and in the hereditary spastic paraplegias (HSP), in which the distal axon degenerates. Mitochondrial dysfunction has been implicated in the pathogenesis of these and other disorders involving axonal degeneration. The aim of this study was to determine whether the frequency of axonal mitochondria changes along the length of small-diameter fibers and whether there is a preferential localization to the region of the node of Ranvier. We find that mitochondrial numbers do not change along the length of a myelinated small-diameter fiber, and, in contrast to the peripheral nervous system, there is no tendency for mitochondrial numbers to increase at the node.

Murine spinal cord explants: a model for evaluating axonal growth and myelination in vitro.

In vitro models of myelinating central nervous system axons have mainly been of two types, organotypic or dissociated. In organotypic cultures, the tissue fragment is thick and usually requires sectioning (physically or optically) before visual examination. In dissociated cultures, tissue is dispersed across the culture surface, making it difficult to measure the extent of myelinated fiber growth. We aimed to develop a method of culturing myelinated CNS fibers in defined medium that could be 1) studied by standard immunofluorescence microscopy (i.e., monolayer type culture), 2) used to measure axonal growth, and 3) used to evaluate the effect of substrate and media components on axonal growth and myelination. We used 120-micro m slices of embryonic murine spinal cord as a focal source of CNS tissue from which myelinated axons could extend in a virtual monolayer. Explants were cultured on both poly-L-lysine and astrocytes. The latter were used because they are the scaffold on which axonal growth and myelination occurs during normal development. Outgrowth from the explant and myelination of axons was poor on poly-L-lysine but was promoted by an astrocyte bed layer. The best myelin formation occurred in defined media based on DMEM using N2 mix; it was not promoted by Sato mix or Neurobasal medium with B27 supplement. Neuronal survival was poor in serum-containing medium. This tissue culture model should facilitate the study of factors involved in promoting outgrowth of CNS axons and their myelination. As such it is relevant to studies on myelination and spinal cord repair.

Processing of PLP in a model of Pelizaeus-Merzbacher disease/SPG2 due to the rumpshaker mutation.

The rumpshaker mutation of the X-linked myelin proteolipid protein (PLP1) gene causes spastic paraplegia type 2 or a mild form of Pelizaeus-Merzbacher disease in man. The identical mutation occurs spontaneously in mice. Both human and murine diseases are associated with dysmyelination. Using the mouse model, we show that the low steady state levels of PLP result from accelerated proteasomal degradation rather than decreased synthesis. The T(1/2) for degradation of rumpshaker PLP is 11 h compared with 23 h for wild type. A minority of newly synthesized PLP is incorporated into myelin in the correct orientation but at a reduced rate compared with wild type. However, inhibition of proteasomal degradation does not increase the level of PLP incorporated into myelin. As Plp null mice do not have a similar myelin deficiency, it is unlikely that the reduced PLP levels are the main cause of the dysmyelination. Rumpshaker oligodendrocytes also have a reduced level of other myelin proteins, such as MBP, although the mechanisms are not yet defined but are likely to operate at a translational or post-translational level.