COVID-19-Associated Myelitis Involving the Dorsal and Lateral White Matter Tracts: A Case Series and Review of the Literature.

Coronavirus disease 2019 (COVID-19) myelitis is a rare condition, most commonly presenting with nonenhancing central expansile cord T2 signal changes. A single case report has also described longitudinal involvement of the dorsal columns. We present 5 cases of COVID-19-associated myelitis with tract-specific involvement of the dorsal and lateral columns and discuss potential pathophysiologic pathways for this unique pattern.

C1 Posterior Arch Flare Point: A Useful Landmark for Fluoroscopically Guided C1-2 Puncture.

BACKGROUND AND PURPOSE: The C1-2 intrathecal puncture is routinely performed when lumbar puncture is not feasible. Usage has steadily decreased in part because of the perceived high risk of injury to the cervical cord. Up to this point, vague fluoroscopic guidelines have been used, creating uncertainty about the actual needle location relative to the spinal cord. We present a novel osseous landmark to aid in C1-2 intrathecal puncture, corresponding to the posterior spinal cord margin on lateral fluoroscopic views. This landmark, which we have termed the "flare point," represents the triangular "flaring" of the posterior C1 arch at its junction with the anterior arch. MATERIALS AND METHODS: Cervical spine CT myelograms were reviewed. High-resolution axial images were reformatted into the sagittal plane, and maximum-intensity-projection images were created to simulate a lateral fluoroscopic view. Tangential lines were drawn along the superior cortices of the anterior and posterior C1 arches, with the point of intersection used to approximate the flare point. Chart review was performed for all C1-2 punctures using the flare point technique in the past 3 years. RESULTS: Forty-two cervical myelograms were reviewed. The average flare point was 0.2 ± 0.5 mm posterior to the dorsal spinal cord margin. In 37/42 subjects, the flare point was localized posterior to the spinal cord. Targeting by means of the flare point was used in 16 C1-2 punctures without complications. CONCLUSIONS: The C1 posterior arch flare point accurately approximates the dorsal spinal cord margin on myelography. Targeting between the flare point and the spinolaminar line, at the mid-C1-2 interspace, allows safe and optimal needle positioning.

Association of Developmental Venous Anomalies with Demyelinating Lesions in Patients with Multiple Sclerosis.

We present 5 cases of demyelination in patients diagnosed with multiple sclerosis that are closely associated with a developmental venous anomaly. Although the presence of a central vein is a known phenomenon with multiple sclerosis plaques, demyelination occurring around developmental venous anomalies is an underreported phenomenon. Tumefactive demyelination can cause a diagnostic dilemma because of its overlapping imaging findings with central nervous system neoplasm. The relationship of a tumefactive plaque with a central vein can be diagnostically useful, and we suggest that if such a lesion is closely associated with a developmental venous anomaly, an inflammatory or demyelinating etiology should be a leading consideration.

Intranasal Esthesioneuroblastoma: CT Patterns Aid in Preventing Routine Nasal Polypectomy.

BACKGROUND AND PURPOSE: Esthesioneuroblastoma is a neuroectodermal tumor that commonly arises in the nasal cavity olfactory recess and, when isolated to the intranasal cavity, can be indistinguishable from benign processes. Because lesional aggressiveness requires a more invasive operation for resection than polypectomy, patients with isolated intranasal lesions were studied to define distinguishing CT characteristics. MATERIALS AND METHODS: Patients with intranasal esthesioneuroblastoma and controls without esthesioneuroblastoma with olfactory recess involvement were identified by using a report search tool. Studies demonstrating skull base invasion and/or intracranial extension were excluded. The imaging spectrum of these lesions was reviewed on both CT and MR imaging, and CT findings were compared with those of controls without esthesioneuroblastoma. Two blinded readers assessed subjects with esthesioneuroblastomas and controls without esthesioneuroblastoma and, using only CT criteria, rated their level of suspicion for esthesioneuroblastoma in each case. RESULTS: Eight histologically proved cases of intranasal esthesioneuroblastoma were reviewed. All cases had CT demonstrating 3 main findings: 1) an intranasal polypoid lesion with its epicenter in a unilateral olfactory recess, 2) causing asymmetric olfactory recess widening, and 3) extending to the cribriform plate. Twelve patients with non-esthesioneuroblastoma diseases involving the olfactory recess were used as controls. Using these 3 esthesioneuroblastoma CT criteria, 2 blinded readers evaluating patients with esthesioneuroblastoma and controls had good diagnostic accuracy (area under the curve = 0.85 for reader one, 0.81 for reader 2) for predicting esthesioneuroblastoma. CONCLUSIONS: Esthesioneuroblastoma can present as a well-marginated intranasal lesion that unilaterally widens the olfactory recess. CT patterns can help predict esthesioneuroblastoma, potentially preventing multiple operations by instigating the correct initial operative management.

Localizing the L5 Vertebra Using Nerve Morphology on MRI: An Accurate and Reliable Technique.

BACKGROUND AND PURPOSE: Multiple methods have been used to determine the lumbar vertebral level on MR imaging, particularly when full spine imaging is unavailable. Because postmortem studies show 95% accuracy of numbering the lumbar vertebral bodies by counting the lumbar nerve roots, attention to lumbar nerve morphology on axial MR imaging can provide numbering clues. We sought to determine whether the L5 vertebra could be accurately localized by using nerve morphology on MR imaging. MATERIALS AND METHODS: One hundred eight cases with full spine MR imaging were numbered from the C2 vertebral body to the sacrum with note of thoracolumbar and lumbosacral transitional states. The origin level of the L5 nerve and iliolumbar ligament were documented in all cases. The reference standard of numbering by full spine imaging was compared with the nerve morphology numbering method. Five blinded raters evaluated all lumbar MRIs with nerve morphology technique twice. Prevalence and bias-adjusted κ were used to measure interrater and intrarater reliability. RESULTS: The L5 nerve arose from the 24th presacral vertebra (L5) in 106/108 cases. The percentage of perfect agreement with the reference standard was 98.1% (95% CI, 93.5%-99.8%), which was preserved in transitional and numeric variation states. The iliolumbar ligament localization method showed 83.3% (95% CI, 74.9%-89.8%) perfect agreement with the reference standard. Inter- and intrarater reliability when using the nerve morphology method was strong. CONCLUSIONS: The exiting L5 nerve can allow accurate localization of the corresponding vertebrae, which is essential for preprocedure planning in cases where full spine imaging is not available. This neuroanatomic method displays higher agreement with the reference standard compared with previously described methods, with strong inter- and intrarater reliability.

Engineering a multi-nucleated myotube, the role of the actin cytoskeleton.

Myoblasts in vitro form characteristic arrays of bipolar-shaped cells prior to fusion. We have shown that the actin cytoskeleton re-organizes in these fusing cells and that the interaction of non-muscle myosin 2A with actin at the plasma membrane helps to generate the bipolar shape of myoblasts, which is key for fusion. Here we discuss how fusion occurs, and in particular how the actin cytoskeleton is involved. Myoblast fusion is essential to form the multi-nucleated muscle fibres that make up the skeletal muscle. Skeletal muscle fibres contain many nuclei, roughly one nucleus to every 15 sarcomeres (35 microm) in adult muscle, although this varies with muscle type (Bruusgaard et al., 2006). Thus a muscle fibre 30 cm long contains about 8000 nuclei and is formed by the fusion of about 8000 cells during development. The formation of multi-nucleated myotubes has been intensively studied for many years using a number of different systems. Many different proteins have been identified using Drosophila as a model system (e.g. see reviews by Taylor, 2000, 2002) that have given an insight into what happens in mammals. However, the process of fusion of mammalian cells is less well understood, and this paper will cover some of the aspects of mammalian myoblast fusion, with a particular focus on the role of the actin cytoskeleton.

Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro.

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

Cell biochemistry studied by single-molecule imaging.

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

Imaging myosin 10 in cells.

Cellular motors (kinesin, dynein and myosin) are ubiquitous. A major task in cell biology is to determine how they function in cells. Here we focus on myosin 10, an intrafilopodial motor, and show how imaging green fluorescent protein fused to myosin 10 or its tail domains can help us understand the function of this myosin.

Microtubule assembly in cultured myoblasts and myotubes following nocodazole induced microtubule depolymerisation.

When myoblasts fuse into myotubes, the organisation of the cytoskeleton changes dramatically. For example, microtubules emanate in a radial array form the centrosome in myoblasts, but form linear arrays not linked to a centrosome in myotubes. It is not clear how these linear arrays are formed and nucleated. They could arise in a number of ways: by nucleation and release from centrosomal like structures, cytoplasmic assembly, breakage/severing or nucleation from non-centrosomal sites. To test which of the above mechanisms or combination of mechanisms are responsible we investigated the re-formation of microtubules after depolymerisation by nocodazole, using antibodies against pericentrin, gamma-tubulin, EB1, and tyrosinated alpha-tubulin. In myoblasts, we found that when microtubules were allowed to recover after complete depolymerisation with nocodazole, microtubule recovery began within 1 min and was complete after 5 min. Microtubules grew out from the centrosome, which was positively stained for gamma-tubulin or pericentrin. In untreated myotubes, microtubules were arranged in linear arrays, with EB1 at their ends. The pericentriolar protein, pericentrin was arranged in a band around the nucleus as well as discrete spots in the cytoplasm. In contrast, the microtubule nucleating protein gamma-tubulin was not found in a band around the nucleus, but was found in several punctuate spots throughout the cytoplasm. Further, when microtubules were allowed to recover, after complete depolymerisation with nocodazole, recovery was not as rapid as that seen in myoblasts, and we found that regrowth began with the formation of short microtubule fragments throughout the cytoplasm. Gamma-tubulin was associated with these fragments. These results suggest that in myotubes, nucleation of microtubules can be non-centrosomal.

Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy.

Over the past 10 years, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. Here, we describe methods based on total internal reflection fluorescence microscopy (TIRFM) that we have developed to study the behavior of individual protein molecules within living mammalian cells. We have used cultured myoblasts that were transiently transfected with DNA plasmids encoding a target protein fused to green fluorescent protein (GFP). Expression levels were quantified from confocal images of control dilutions of GFP and cells with 1-100 nM GFP were then examined using TIRFM. An evanescent field was produced by a totally internally reflected, argon ion laser beam that illuminated a shallow region (50-100 nm deep) at the glass-water interface. Individual GFP-tagged proteins that entered the evanescent field appeared as individual, diffraction-limited spots of light, which were clearly resolved from background fluorescence. Molecules that bound to the basal cell membrane remained fixed in position for many seconds, whereas those diffusing freely in the cytoplasm disappeared within a few milliseconds. We developed automated detection and tracking methods to recognize and characterize the behavior of single molecules in recorded video sequences. This enabled us to measure the kinetics of photobleaching and lateral diffusion of membrane-bound molecules.

Different regional effects of voluntary exercise on the mechanical and electrical properties of rat ventricular myocytes.

Short-term (6 weeks) voluntary wheel running exercise in young female rats that were in an active growth phase resulted in whole-heart hypertrophy and myocyte concentric hypertrophy, when compared to sedentary controls. The cross-sectional area of ventricular myocytes from trained rats was significantly greater than for those isolated from sedentary rats, with the greatest change in morphology seen in sub-endocardial cells. There was no statistically significant effect of training on cell shortening in the absence of external mechanical loading, in [Ca2+](i) transients, or in myofilament Ca2+ sensitivity (assessed during re-lengthening following tetanic stimulation). Under the external mechanical load of carbon fibres, absolute force developed in myocytes from trained rats was significantly greater than in those from sedentary rats. This suggests that increased myocyte cross-sectional area is a major contractile adaptation to exercise in this model. Training did not alter the passive mechanical properties of myocytes or the relative distribution of titin isomers, which was exclusively of the short, N2B form. However, training did increase the steepness of the active tension-sarcomere length relationship, suggesting an exercise-induced modulation of the Frank-Starling mechanism. This effect would be expected to enhance cardiac contractility. Training lengthened the action potential duration of sub-epicardial myocytes, reducing the transmural gradient in action potential duration. This observation may be important in understanding the cellular causes of T-wave abnormalities found in the electrocardiograms of some athletes. Our study shows that voluntary exercise modulates the morphological, mechanical and electrical properties of cardiac myocytes, and that this modulation is dependent upon the regional origin of the myocytes.

Alignment of myoblasts on ultrafine gratings inhibits fusion in vitro.

During development, skeletal muscle precursor cells fuse to form multi-nucleated myotubes. However, it is unclear how this fusion is regulated such that linear myotubes are produced. In a previous study, we found that linear arrays of myoblasts cultured on micropatterns of laminin fused to form linear myotubes of a constant diameter, independent of the width of the laminin track. This suggested that a mechanism exists to prevent myoblasts from fusing laterally [Exp. Cell Res. 230 (1997) 275]. In this study, we have investigated this further by culturing myoblasts on ultrafine grooved surfaces previously shown to align fibroblasts and epithelial cells. We found that all the individual myoblasts were highly aligned along the groove axis, and time-lapse recordings showed that motility was mostly restricted to a direction parallel to the grooves. In contrast to the previous study, however, there was a strong tendency for early differentiating cells to form aggregates either at an angle of approximately 45 degrees or perpendicular to the groove axis. Nevertheless, we rarely saw myotubes formed at those angles, supporting our earlier idea that the ability of cells to fuse laterally is prohibited. Our data strongly suggest that myoblasts are most likely to fuse in an end-to-end configuration, and it is this that enables them to form linear, rather than irregular myotubes.

Specific changes to the mechanism of cell locomotion induced by overexpression of beta-actin.

Overexpression of beta-actin is known to alter cell morphology, though its effect on cell motility has not been documented previously. Here we show that overexpressing beta-actin in myoblasts has striking effects on motility, increasing cell speed to almost double that of control cells. This occurs by increasing the areas of protrusion and retraction and is accompanied by raised levels of beta-actin in the newly protruded regions. These regions of the cell margin, however, show decreased levels of polymerised actin, indicating that protrusion can outpace the rate of actin polymerisation in these cells. Moreover, the expression of beta*-actin (a G244D mutant, which shows defective polymerisation in vitro) is equally effective at increasing speed and protrusion. Concomitant changes in actin binding proteins show no evidence of a consistent mechanism for increasing the rate of actin polymerisation in these actin overexpressing cells. The increase in motility is confined to poorly spread cells in both cases and the excess motility can be abolished by blocking myosin function with butanedione monoxime (BDM). Our observations on normal myoblasts are consistent with the view that they protrude by the assembly and cross linking of actin filaments. In contrast, the additional motility shown by cells overexpressing beta-actin appears not to result from an increase in the rate of actin polymerisation but to depend on myosin function. This suggests that the additional protrusion arises from a different mechanism. We discuss the possibility that it is related to retraction-induced protrusion in fibroblasts. In this phenomenon, a wave of increased protrusion follows a sudden collapse in cell spreading. This view could explain why it is only the additional motility that depends on spreading, and has implications for understanding the differences in locomotion that distinguish tissue cells from highly invasive cell types such as leucocytes and malignant cells.

Evidence for differential post-translational modifications of slow myosin heavy chain during murine skeletal muscle development.

The contractile properties of muscle fibres are, in part, determined by the myosin heavy chain (MyHC) isoforms they express. Using monoclonal antibodies, we show that at least three forms of slow twitch MyHC accumulate sequentially during mouse fetal development and that slow MyHC maturation in slow fibres occurs before expression of the adult fast MyHCs in fast fibres. Expression of deletion derivatives of beta-cardiac MyHC cDNA shows that the slow MyHC epitopes that are detected in adult but not in young animals are located near the N-terminus. The same N-terminal region of various fast MyHC molecules contains a conserved epitope that can, on occasions, be observed when slow MyHC cDNA is expressed in non-muscle cells. The results raise the possibility that the N-terminal epitopes result from post-translational modification of the MyHC and that a sequence of slow and fast MyHC isoform post-translational modifications plays a significant role during development of murine muscle fibres.

Abdominal relapse in stage 1 nonseminomatous germ cell tumours of the testis managed by surveillance or with adjuvant chemotherapy.

OBJECTIVE: To assess the impact on patterns of recurrence of adjuvant chemotherapy in patients with stage 1 nonseminomatous germ cell tumours (NSGCT) of the testis, who have a high likelihood of relapse on surveillance if certain risk factors are identified in the orchidectomy specimen, and thus the theoretical need for retroperitoneal lymph node dissection (RPLND). PATIENTS AND METHODS: The incidence of abdominal relapse was recorded in 417 men presenting with stage 1 NSGCT over the past 18 years. Up to 1986, 161 men were managed by surveillance alone, and abdominal relapse occurred in 26. From 1986 onwards, men with positive risk factors in the orchidectomy specimen were offered two courses of chemotherapy; 60 accepted and one relapsed in the abdomen, and 196 underwent surveillance and 19 relapsed in the abdomen. RESULTS: Abdominal relapse was significantly reduced from 16% before 1986 to 8% afterward (P = 0.014). Mortality from testicular tumour or treatment toxicity remained low, at 0.6% before 1986 and 2.0% since then. CONCLUSION: The need for RPLND in stage 1 NSGCT remains highly doubtful.

Forced MyHCIIB expression following targeted genetic manipulation of conditionally immortalized muscle precursor cells.

The ability to carry out gene targeting in somatic stem cells while maintaining their stem cell characteristics would have important implications for gene therapy and for the analysis of gene function. Using mouse myoblasts, we have explored this possibility by attempting to alter the promoter of a myosin heavy chain gene (MyHCIIB) characteristic of physiologically "fast" muscle so as to force its unscheduled expression in physiologically "slow" muscle fibers. Conditionally immortalized muscle precursor cells were transfected with a gene targeting construct designed to replace the MyHCIIB promoter with that for the carbonic anhydrase III gene (CAIII), which is highly expressed in slow muscle. A potentially targeted clone was isolated and differentiated in culture to form myotubes which expressed MyHCIIB. Cells from the same clone were injected into both slow and fast muscle of host mice, where they contributed to fiber formation. In slow muscle, the fibers derived from this clone did not express MyHCIIB; this may reflect an instability of the targeted MyHCIIB locus and/or a failure of the hybrid promoter to function in slow fibers in vivo. Nonetheless, we have demonstrated that a "promoter knock-in" gene targeting procedure can be used to generate unique MyHCIIB-expressing myotubes in culture and that conditionally immortalized myoblasts can be subjected to extensive passaging and genetic manipulation without losing their ability to form fibers in culture and in vivo.