Extracellular vesicles produced by HIV-1 Nef-expressing cells induce myelin impairment and oligodendrocyte damage in the mouse central nervous system.

HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affect approximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy (ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlates with the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicated in HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo, ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injection of Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment. These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin maintenance in the pathogenesis of HAND.

Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination.

Regeneration of myelin is mediated by oligodendrocyte progenitor cells-an abundant stem cell population in the central nervous system (CNS) and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the CNS underlies a number of neurological diseases, including multiple sclerosis and diverse genetic diseases1-3. High-throughput chemical screening approaches have been used to identify small molecules that stimulate the formation of oligodendrocytes from oligodendrocyte progenitor cells and functionally enhance remyelination in vivo4-10. Here we show that a wide range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51, TM7SF2, or EBP, a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to oligodendrocyte progenitor cells in purified form whereas analogous sterols that lack this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism of action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics.

B cell depletion modulates glial responses and enhances blood vessel integrity in a model of multiple sclerosis.

Multiple sclerosis (MS) is characterized by a compromised blood-brain barrier (BBB) resulting in central nervous system (CNS) entry of peripheral lymphocytes, including T cells and B cells. While T cells have largely been considered the main contributors to neuroinflammation in MS, the success of B cell depletion therapies suggests an important role for B cells in MS pathology. Glial cells in the CNS are essential components in both disease progression and recovery, raising the possibility that they represent targets for B cell functions. Here, we examine astrocyte and microglia responses to B cell depleting treatments in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). B cell depleted EAE animals had markedly reduced disease severity and myelin damage accompanied by reduced microglia and astrocyte reactivity 20 days after symptom onset. To identify potential initial mechanisms mediating functional changes following B cell depletion, astrocyte and microglia transcriptomes were analyzed 3 days following B cell depletion. In control EAE animals, transcriptomic analysis revealed astrocytic inflammatory pathways were activated and microglial influence on neuronal function were inhibited. Following B cell depletion, initial functional recovery was associated with an activation of astrocytic pathways linked with restoration of neurovascular integrity and of microglial pathways associated with neuronal function. These studies reveal an important role for B cell depletion in influencing glial function and CNS vasculature in an animal model of MS.

Loss of optic nerve oligodendrocytes during maturation alters retinal organization.

The myelin sheath facilitates signal conduction along axons in white matter tracts, and when disrupted, can result in significant functional deficits. Demyelination, observed in diseases like multiple sclerosis and optic neuritis, are associated with neural degeneration, however the extent of this damage on upstream circuitry is not well understood. Here we use the MBP-iCP9 mouse model to induce selective oligodendrocyte ablation in the optic nerve at P14 via a chemical inducer of dimerization (CID), resulting in partial demyelination of retinal ganglion cell (RGC) axons with minimal inflammation after two weeks. Oligodendrocyte loss reduced axon diameter and altered compound action potential waveforms, blocking conduction in the slowest-conducting axon populations. Demyelination resulted in disruptions to the normal composition of the retina, including reduced density of RBPMS+, Brn3a+, and OFF-transient RGCs, thinning of the IPL, and reduced density of displaced amacrine cells. The INL and ONL were unaffected by oligodendrocyte loss, suggesting that demyelination-induced deficits in this model are specific to the IPL and GCL. These results show that a partial demyelination of a subpopulation of RGC axons disrupts optic nerve function and affects the organization of the retinal network. This study highlights the significance of myelination in maintaining upstream neural connectivity and provides support for targeting neuronal degeneration in treatments of demyelinating diseases.

Repurposing the cardiac glycoside digoxin to stimulate myelin regeneration in chemically-induced and immune-mediated mouse models of multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na+ /K+ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG35-55 (PLG-MOG35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.

Induction of myelinating oligodendrocytes in human cortical spheroids.

Cerebral organoids provide an accessible system for investigations of cellular composition, interactions, and organization but have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generated oligodendrocytes and myelin in 'oligocortical spheroids' derived from human pluripotent stem cells. Molecular features consistent with those of maturing oligodendrocytes and early myelin appeared by week 20 in culture, with further maturation and myelin compaction evident by week 30. Promyelinating drugs enhanced the rate and extent of oligodendrocyte generation and myelination, and spheroids generated from human subjects with a genetic myelin disorder recapitulated human disease phenotypes. Oligocortical spheroids provide a versatile platform for studies of myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development.

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Targeted Oligodendrocyte Apoptosis in Optic Nerve Leads to Persistent Demyelination.

The optic nerve represents one of the simplest regions of the CNS and has been useful in developing an understanding of glial development and myelination. While the visual system is frequently affected in demyelinating conditions, utilizing the optic nerve to model demyelination/remyelination studies has been difficult due to its accessibility, relatively small size, and dense nature that makes direct injections challenging. Taking advantage of the lack of oligodendrocytes and myelination in the mouse retina, we have developed a model in which the induction of apoptosis in mature oligodendrocytes allows for the selective, non-invasive generation of demyelinating lesions in optic nerve. Delivery of an inducer of oligodendrocyte apoptosis by intravitreous injection minimizes trauma to the optic nerve and allows for the assessment of oligodendrocyte death in the absence of injury related factors. Here we show that following induction of apoptosis, oligodendrocytes are lost within 3 days. The loss of oligodendrocytes is associated with limited microglial and astrocyte response, is patchy along the nerve, and results in localized myelin loss. Unlike in other regions of the murine CNS, where local demyelination stimulates activation of local oligodendrocyte precursors and remyelination, optic nerve demyelination induced by oligodendrocyte apoptosis fails to recover and results in persistent areas of myelin loss. Over time these chronic lesions change cellular composition and ultimately become devoid of GFAP+ astrocytes and OPCs. Why the optic nerve lesions fail to repair may reflect the lack of early immune responsiveness and provide a novel model of chronic demyelination.

The Activators of Cyclin-Dependent Kinase 5 p35 and p39 Are Essential for Oligodendrocyte Maturation, Process Formation, and Myelination.

The regulation of oligodendrocyte development and myelin formation in the CNS is poorly defined. Multiple signals influence the rate and extent of CNS myelination, including the noncanonical cyclin-dependent kinase 5 (Cdk5) whose functions are regulated by its activators p35 and p39. Here we show that selective loss of either p35 or p39 perturbed specific aspects of oligodendrocyte development, whereas loss of both p35 and p39 completely inhibited the development of mature oligodendrocytes and myelination. In the absence of p35, oligodendrocyte differentiation was delayed, process outgrowth was truncated in vitro, and the patterning and extent of myelination were perturbed in the CNS of p35(-/-) mice. In the absence of p39, oligodendrocyte maturation was transiently affected both in vitro and in vivo. However, loss of both p35 and p39 in oligodendrocyte lineage cells completely inhibited oligodendrocyte progenitor cell differentiation and myelination both in vitro and after transplantation into shiverer slice cultures. Loss of p35 and p39 had a more profound effect on oligodendrocyte development than simply the loss of Cdk5 and could not be rescued by Cdk5 overexpression. These data suggest p35 and p39 have specific and overlapping roles in oligodendrocyte development, some of which may be independent of Cdk5 activation.

Reliability testing of the Larsen and Sharp classifications for rheumatoid arthritis of the elbow.

BACKGROUND: Two popular systems for classifying rheumatoid arthritis affecting the elbow are the Larsen and Sharp schemes. To our knowledge, no study has investigated the reliability of these 2 systems. We compared the intraobserver and interobserver agreement of the 2 systems to determine whether one is more reliable than the other. METHODS: The radiographs of 45 patients diagnosed with rheumatoid arthritis affecting the elbow were evaluated. Anteroposterior and lateral radiographs were deidentified and distributed to 6 evaluators (4 fellowship-trained upper extremity surgeons and 2 orthopedic trainees). Each evaluator graded all 45 radiographs according to the Larsen and Sharp scoring methods on 2 occasions, at least 2 weeks apart. RESULTS: Overall intraobserver reliability was 0.93 (95% confidence interval [CI], 0.90-0.95) for the Larsen system and 0.92 (95% CI, 0.86-0.96) for the Sharp classification, both indicating substantial agreement. Overall interobserver reliability was 0.70 (95% CI, 0.60-0.80) for the Larsen classification and 0.68 (95% CI, 0.54-0.81) for the Sharp system, both indicating good agreement. There were no significant differences in the intraobserver or interobserver reliability of the systems overall and no significant differences in reliability between attending surgeons and trainees for either classification system. CONCLUSION: The Larsen and Sharp systems both show substantial intraobserver reliability and good interobserver agreement for the radiographic classification of rheumatoid arthritis affecting the elbow. Differences in training level did not result in substantial variances in reliability for either system. We conclude that both systems can be reliably used to evaluate rheumatoid arthritis of the elbow by observers of varying training levels.

Apoptosis of Oligodendrocytes during Early Development Delays Myelination and Impairs Subsequent Responses to Demyelination.

During mammalian development, myelin-forming oligodendrocytes are generated and axons ensheathed according to a tightly regulated sequence of events. Excess premyelinating oligodendrocytes are eliminated by apoptosis and the timing of the onset of myelination in any specific CNS region is highly reproducible. Although the developing CNS recovers more effectively than the adult CNS from similar insults, it is unknown whether early loss of oligodendrocyte lineage cells leads to long-term functional deficits. To directly assess whether the loss of oligodendrocytes during early postnatal spinal cord development impacted oligodendrogenesis, myelination, and remyelination, transgenic mouse lines were generated in which a modified caspase-9 molecule allowed spatial and temporal control of the apoptotic pathway specifically in mature, myelin basic protein expressing oligodendrocytes (MBP-iCP9). Activating apoptosis in MBP(+) cells of the developing spinal cord during the first postnatal week inhibited myelination. This inhibition was transient, and the levels of myelination largely returned to normal after 2 weeks. Despite robust developmental plasticity, MBP-iCP9-induced oligodendrocyte apoptosis compromised the rate and extent of adult remyelination. Remyelination failure correlated with a truncated proliferative response of oligodendrocyte progenitor cells, suggesting that depleting the oligodendrocyte pool during critical developmental periods compromises the regenerative response to subsequent demyelinating lesions. SIGNIFICANCE STATEMENT: This manuscript demonstrates that early insults leading to oligodendrocyte apoptosis result in the impairment of recovery from demyelinating diseases in the adult. These studies begin to provide an initial understanding of the potential failure of recovery in insults, such as periventricular leukomalacia and multiple sclerosis.

Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production.

PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal-dominant genetic condition underlying a subset of autism spectrum disorder (ASD) with macrocephaly. Caused by germline mutations in PTEN, PHTS also causes increased risks of multiple cancers via dysregulation of the PI3K and MAPK signaling pathways. Conditional knockout models have shown that neural Pten regulates social behavior, proliferation and cell size. Although much is known about how the intracellular localization of PTEN regulates signaling in cancer cell lines, we know little of how PTEN localization influences normal brain physiology and behavior. To address this, we generated a germline knock-in mouse model of cytoplasm-predominant Pten and characterized its behavioral and cellular phenotypes. The homozygous Pten(m3m4) mice have decreased total Pten levels including a specific drop in nuclear Pten and exhibit region-specific increases in brain weight. The Pten(m3m4) model displays sex-specific increases in social motivation, poor balance and normal recognition memory-a profile reminiscent of some individuals with high functioning ASD. The cytoplasm-predominant protein caused cellular hypertrophy limited to the soma and led to increased NG2 cell proliferation and accumulation of glia. The animals also exhibit significant astrogliosis and microglial activation, indicating a neuroinflammatory phenotype. At the signaling level, Pten(m3m4) mice show brain region-specific differences in Akt activation. These results demonstrate that differing alterations to the same autism-linked gene can cause distinct behavioral profiles. The Pten(m3m4) model is the first murine model of inappropriately elevated social motivation in the context of normal cognition and may expand the range of autism-related behaviors replicated in animal models.

Activated microglia enhance neurogenesis via trypsinogen secretion.

White matter neurons in multiple sclerosis brains are destroyed during demyelination and then replaced in some chronic multiple sclerosis lesions that exhibit a morphologically distinct population of activated microglia [Chang A, et al. (2008) Brain 131(Pt 9):2366-2375]. Here we investigated whether activated microglia secrete factors that promote the generation of neurons from white matter cells. Adult rat brain microglia (resting or activated with lipopolysaccharide) were isolated by flow cytometry and cocultured with neonatal rat optic nerve cells in separate but media-connected chambers. Optic nerve cells cocultured with activated microglia showed a significant increase in the number of cells of neuronal phenotype, identified by neuron-specific class III beta-tubulin (TUJ-1) labeling, compared with cultures with resting microglia. To investigate the possible source of the TUJ-1-positive cells, A2B5-positive oligodendrocyte progenitor cells and A2B5-negative cells were isolated and cocultured with resting and activated microglia. Significantly more TUJ-1-positive cells were generated from A2B5-negative cells (∼70%) than from A2B5-positive cells (~30%). Mass spectrometry analysis of microglia culture media identified protease serine 2 (PRSS2) as a factor secreted by activated, but not resting, microglia. When added to optic nerve cultures, PRSS2 significantly increased neurogenesis, whereas the serine protease inhibitor, secretory leukocyte protease inhibitor, decreased activated microglia-induced neurogenesis. Collectively our data provide evidence that activated microglia increase neurogenesis through secretion of PRSS2.

Cyclin-dependent kinase 5 mediates adult OPC maturation and myelin repair through modulation of Akt and GsK-3ß signaling.

Failure of remyelination in diseases, such as multiple sclerosis (MS), leads to permanent axonal damage and irreversible functional loss. The mechanisms controlling remyelination are currently poorly understood. Recent studies implicate the cyclin-dependent kinase 5 (Cdk5) in regulating oligodendrocyte (OL) development and myelination in CNS. In this study, we show that Cdk5 is also an important regulator of remyelination. Pharmacological inhibition of Cdk5 inhibits repair of lysolecithin lesions. This inhibition is a consequence of Cdk5 disruption in neural cells because remyelination in slice cultures is blocked by Cdk5 inhibitors, whereas specific deletion of Cdk5 in OLs inhibits myelin repair. In CNP-Cre;Cdk5(fl/fl) conditional knock-out mouse (Cdk5 cKO), myelin repair was delayed significantly in response to focal demyelinating lesions compared with wild-type animals. The lack of myelin repair was reflected in decreased expression of MBP and proteolipid protein and a reduction in the total number of myelinated axons in the lesion. The number of CC1(+) cells in the lesion sites was significantly reduced in Cdk5 cKO compared with wild-type animals although the total number of oligodendrocyte lineage cells (Olig2(+) cells) was increased, suggesting that Cdk5 loss perturbs the transition of early OL lineage cell into mature OL and subsequent remyelination. The failure of remyelination in Cdk5 cKO animals was associated with a reduction in signaling through the Akt pathway and an enhancement of Gsk-3ß signaling pathways. Together, these data suggest that Cdk5 is critical in regulating the transition of adult oligodendrocyte precursor cells to mature OLs that is essential for myelin repair in adult CNS.

Signaling through ERK1/2 controls myelin thickness during myelin repair in the adult central nervous system.

Oligodendrocytes, the myelin-forming cells of the CNS, exquisitely tailor the thickness of individual myelin sheaths to the diameter of their target axons to maximize the speed of action potential propagation, thus ensuring proper neuronal connectivity and function. Following demyelinating injuries to the adult CNS, newly formed oligodendrocytes frequently generate new myelin sheaths. Following episodes of demyelination such as those that occur in patients with multiple sclerosis, however, the matching of myelin thickness to axon diameter fails leaving remyelinated axons with thin myelin sheaths potentially compromising function and leaving axons vulnerable to damage. How oligodendrocytes determine the appropriate thickness of myelin for an axon of defined size during repair is unknown and identifying the signals that regulate myelin thickness has obvious therapeutic implications. Here, we show that sustained activation of extracellular-regulated kinases 1 and 2 (ERK1/2) in oligodendrocyte lineage cells results in accelerated myelin repair after injury, and is sufficient for the generation of thick myelin sheaths around remyelinated axons in the adult mouse spinal cord. Our findings suggest a model where ERK1/2 MAP kinase signaling acts as a myelin thickness rheostat that instructs oligodendrocytes to generate axon-appropriate quantities of myelin.

Cyclin dependent kinase 5 is required for the normal development of oligodendrocytes and myelin formation.

The development of oligodendrocytes, the myelinating cells of the vertebrate CNS, is regulated by a cohort of growth factors and transcription factors. Less is known about the signaling pathways that integrate extracellular signals with intracellular transcriptional regulators to control oligodendrocyte development. Cyclin dependent kinase 5 (Cdk5) and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Here we demonstrate a previously unrecognized function of Cdk5 in regulating oligodendrocyte maturation and myelination. During late embryonic development Cdk5 null animals displayed a reduction in the number of MBP+ cells in the spinal cord, but no difference in the number of OPCs. To determine whether the reduction of oligodendrocytes reflected a cell-intrinsic loss of Cdk5, it was selectively deleted from Olig1+ oligodendrocyte lineage cells. In Olig1(Cre/+); Cdk5(fl/fl) conditional mutants, reduced levels of expression of MBP and PLP mRNA were observed throughout the CNS and ultrastructural analyses demonstrated a significant reduction in the proportion of myelinated axons in the optic nerve and spinal cord. Pharmacological inhibition or RNAi knockdown of Cdk5 in vitro resulted in the reduction in oligodendrocyte maturation, but had no effect on OPC cell proliferation. Conversely, over-expression of Cdk5 promoted oligodendrocyte maturation and enhanced process outgrowth. Consistent with this data, Cdk5(-/-) oligodendrocytes developed significantly fewer primary processes and branches than control cells. Together, these findings suggest that Cdk5 function as a signaling integrator to regulate oligodendrocyte maturation and myelination.

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells.

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.

Longitudinal positron emission tomography imaging for monitoring myelin repair in the spinal cord.

OBJECTIVE: Novel therapeutic interventions aimed at myelin repair are now under development for neuroprotection as well as functional recovery of patients with multiple sclerosis. However, development of myelin repair therapy necessitates a noninvasive approach for measuring changes in myelin content in vivo in a quantitative fashion not yet possible using magnetic resonance imaging. For this reason, we developed a novel positron emission tomography (PET) probe, termed [11C]MeDAS, that is capable of longitudinally imaging central nervous system myelin content. METHODS: The binding properties of [11C]MeDAS for myelin were systematically evaluated by in vitro and in situ fluorescent staining of the spinal cord and the brain, and by in vivo competitive blocking studies. Longitudinal PET studies were conducted in 3 rat models involving acute focal neuroinflammation in the brain, lysophosphatidylcholine (LPC)-induced focal demyelination in the spinal cord, and experimental autoimmune encephalomyelitis (EAE). Image-guided myelin repair therapy was conducted in an LPC rat model using a mesenchymal stem cell-based hepatocyte growth factor (HGF). Biodistribution and acute toxicity studies of [11C]MeDAS were also conducted. RESULTS: MeDAS selectively stains myelin in the spinal cord and brain. Neuroinflammation did not affect [11C]MeDAS uptake in the brain as long as the myelin sheaths remained intact. Longitudinal PET studies in LPC and EAE rat models demonstrate that [11C]MeDAS uptake changes correlate with associated myelin loss in the spinal cord. Furthermore, using [11C]MeDAS-PET, the efficacy of myelin repair therapy with HGF was longitudinally monitored in vivo. INTERPRETATION: [11C]MeDAS-PET is a promising imaging marker for monitoring myelin pathology in vivo, future applications of which in humans should be achievable.

Accuracy and reliability testing of two methods to measure internal rotation of the glenohumeral joint.

BACKGROUND: We compared accuracy and reliability of a traditional method of measurement (most cephalad vertebral spinous process that can be reached by a patient with the extended thumb) to estimates made with the shoulder in abduction to determine if there were differences between the two methods. METHODS: Six physicians with fellowship training in sports medicine or shoulder surgery estimated measurements in 48 healthy volunteers. Three were randomly chosen to make estimates of both internal rotation measurements for each volunteer. An independent observer made objective measurements on lateral scoliosis films (spinous process method) or with a goniometer (abduction method). Examiners were blinded to objective measurements as well as to previous estimates. RESULTS: Intraclass coefficients for interobserver reliability for the traditional method averaged 0.75, indicating good agreement among observers. The difference in vertebral level estimated by the examiner and the actual radiographic level averaged 1.8 levels. The intraclass coefficient for interobserver reliability for the abduction method averaged 0.81 for all examiners, indicating near-perfect agreement. Confidence intervals indicated that estimates were an average of 8° different from the objective goniometer measurements. Pearson correlation coefficients of intraobserver reliability for the abduction method averaged 0.94, indicating near-perfect agreement within observers. Confidence intervals demonstrated repeated estimates between 5° and 10° of the original. CONCLUSIONS: Internal rotation estimates made with the shoulder abducted demonstrated interobserver reliability superior to that of spinous process estimates, and reproducibility was high. On the basis of this finding, we now take glenohumeral internal rotation measurements with the shoulder in abduction and use a goniometer to maximize accuracy and objectivity.