Near-infrared branding efficiently correlates light and electron microscopy.

The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

In vivo imaging of dorsal root regeneration: rapid immobilization and presynaptic differentiation at the CNS/PNS border.

Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS. Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled, large-diameter DR axons in thy1-YFPH mice elongated through a DR crush site, but not a transection site, and grew along the root at >1.5 mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastructural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.

Lysosomal activity associated with developmental axon pruning.

Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, which generates considerable cellular debris, is synapse elimination, in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning.

The Association between the Change in Directly Measured Cardiorespiratory Fitness across Time and Mortality Risk.

BACKGROUND: The relationship between cardiorespiratory fitness (CRF) and mortality risk has typically been assessed using a single measurement, though some evidence suggests the change in CRF over time influences risk. This evidence is predominantly based on studies using estimated CRF (CRFe). The strength of this relationship using change in directly measured CRF over time in apparently healthy men and women is not well understood. PURPOSE: To examine the association of change in CRF over time, measured using cardiopulmonary exercise testing (CPX), with all-cause and disease-specific mortality and to compare baseline and subsequent CRF measurements as predictors of all-cause mortality. METHODS: Participants included 833 apparently healthy men and women (42.9 ±â€¯10.8 years) who underwent two maximal CPXs, the second CPX being ≥1 year following the baseline assessment (mean 8.6 years, range 1.0 to 40.3 years). Participants were followed for up to 17.7 (SD 11.8) years for all-cause-, cardiovascular disease- (CVD), and cancer mortality. Cox-proportional hazard models were performed to determine the association between the change in CRF, computed as visit 1 (CPX1) peak oxygen consumption (VO2peak [mL·kg-1·min-1]) - visit 2 (CPX2) VO2peak, and mortality outcomes. A Wald-Chi square test of equality was used to compare the strength of CPX1 to CPX2 VO2peak in predicting mortality. RESULTS: During follow-up, 172 participants died. Overall, the change in CPX-CRF was inversely related to all-cause, CVD, and cancer mortality (p < 0.05). Each 1 mL·kg-1·min-1 increase was associated with a ~11, 15, and 16% (all p < 0.001) reduction in all-cause, CVD, and cancer mortality, respectively. The inverse relationship between CRF and all-cause mortality was significant (p < 0.05) when men and women were examined independently, after adjusting for years since first CPX, baseline VO2peak, and age. Further, the Wald Chi-square test of equality found CPX2 VO2peak to be a significantly stronger predictor of all-cause mortality than CPX1 VO2peak (p < 0.05). CONCLUSION: The change in CRF over time was inversely related to mortality outcomes, and mortality was better predicted by CRF measured at subsequent test than CPX1 CRF. These findings emphasize the importance of adopting lifestyle behaviors that promote CRF, as well as support the need for routine assessment of CRF in clinical practice to better assess risk.

The Influence of Change in Cardiorespiratory Fitness With Short-Term Exercise Training on Mortality Risk From The Ball State Adult Fitness Longitudinal Lifestyle Study.

OBJECTIVE: To assess the influence of changes in cardiorespiratory fitness (CRF) after exercise training on mortality risk in a cohort of self-referred, apparently healthy adults. PATIENTS AND METHODS: A total of 683 participants (404 men, 279 women; mean age: 42.7±11.0 y) underwent two maximal cardiopulmonary exercise tests (CPX) between March 20, 1970, and December 11, 2012, to assess CRF at baseline (CPX1) and post-exercise training (CPX2). Participants were followed for an average of 29.8±10.7 years after their CPX2. Cox proportional hazards models were performed to determine the relationship of CRF change with mortality, with change in CRF as a continuous variable, as well as a categorical variable. A Wald chi-square test was used to compare the coefficients estimating the relationship of peak oxygen consumption (VO2peak) at CPX1 with VO2peak measured at CPX2 with time until death for all-cause mortality. RESULTS: During the follow-up period there were 180 deaths. When assessed independently, there were 20% (95% CI, 10-49%) and 38% (95% CI, 7-66%) lower mortality risks per 1 metabolic equivalent improvement in CRF (P<.01) in men and women, respectively, after multivariable adjustment. Those that remained unfit had ∼2-fold higher risk for all-cause mortality compared with those that remained fit and CRF at CPX2 was a stronger predictor of all-cause mortality than at CPX1 (P=.02). CONCLUSION: Improving CRF through exercise training lowers mortality risk. Clinicians should encourage individuals to participate in exercise training to improve CRF to lower risk of mortality.

Simvastatin and ML141 Decrease Intracellular Streptococcus pyogenes Infection.

BACKGROUND: Recurrent pharyngotonsillitis due to Streptococcus pyogenes develops regardless of whether infecting strains are resistant or susceptible to first-line antimicrobials. Causation for recurrent infection is associated with the use of first-line antimicrobials that fail to penetrate deep tissue and host cell membranes, enabling intracellular S. pyogenes to survive throughout repeated rounds of antimicrobial therapy. OBJECTIVE: To determine whether simvastatin, a therapeutic approved for use in the treatment of hypercholesterolemia, and ML141, a first-in-class small molecule inhibitor with specificity for human CDC42, limit host cell invasion by S. pyogenes. METHODS: Assays to assess host cell invasion, bactericidal activity, host cell viability, actin depolymerization, and fibronectin binding were performed using the RAW 267.4 macrophage cell line and Human Umbilical Vein Endothelial Cells (HUVEC) infected with S. pyogenes (90-226) and treated with simvastatin, ML141, structural analogs of ML141, or vehicle control. RESULTS: Simvastatin and ML141 decreased intracellular infection by S. pyogenes in a dose-dependent manner. Inhibition by simvastatin persisted following 1 h washout whereas inhibition by ML141 was reversed. During S. pyogenes infection, actin stress fibers depolymerized in vehicle control treated cells, yet remained intact in simvastatin and in ML141 treated cells. Consistent with the previous characterization of ML141, simvastatin decreased host cell binding to fibronectin. Structural analogs of ML141, designated as the RSM series, decreased intracellular infection through non-cytotoxic, nonbactericidal mechanisms. CONCLUSION: Our findings demonstrate the potential of repurposing simvastatin and of developing CDC42-targeted therapeutics for eradicating intracellular S. pyogenes infection to break the cycle of recurrent infection through a host-directed approach.

Axon branch removal at developing synapses by axosome shedding.

In many parts of the developing nervous system, the number of axonal inputs to each postsynaptic cell is dramatically reduced. This synapse elimination has been extensively studied at the neuromuscular junction, but how axons are lost is unknown. Here, we combine time-lapse imaging of fluorescently labeled axons and serial electron microscopy to show that axons at neuromuscular junctions are removed by an unusual cellular mechanism. As axons disappear, they shed numerous membrane bound remnants. These "axosomes" contain a high density of synaptic organelles and are formed by engulfment of axon tips by Schwann cells. After this engulfment, the axosome's contents mix with the cytoplasm of the glial cell. Axosome shedding might underlie other forms of axon loss and may provide a pathway for interactions between axons and glia.

Simvastatin inhibits Staphylococcus aureus host cell invasion through modulation of isoprenoid intermediates.

Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis. Inhibition was due in part to depletion of isoprenoid intermediates within the cholesterol biosynthesis pathway and led to the cytosolic accumulation of the small GTPases CDC42, Rac, and RhoB. Actin stress fiber disassembly required for host invasion was attenuated by simvastatin and by the inhibition of phosphoinositide 3-kinase (PI3K) activity. PI3K relies on coupling to prenylated proteins, such as this subset of small GTPases, for access to membrane-bound phosphoinositide to mediate stress fiber disassembly. Therefore, we examined whether simvastatin restricts PI3K cellular localization. In response to simvastatin, the PI3K isoform p85, coupled to these small-GTPases, was sequestered within the cytosol. From these findings, we propose a mechanism whereby simvastatin restricts p85 localization, inhibiting the actin dynamics required for bacterial endocytosis. This approach may provide the basis for protection at the level of the host in invasive infections by S. aureus.

Cardiorespiratory Fitness and Mortality in Healthy Men and Women.

BACKGROUND: There is a well-established inverse relationship between cardiorespiratory fitness (CRF) and mortality. However, this relationship has almost exclusively been studied using estimated CRF. OBJECTIVES: This study aimed to assess the association of directly measured CRF, obtained using cardiopulmonary exercise (CPX) testing with all-cause, cardiovascular disease (CVD), and cancer mortality in apparently healthy men and women. METHODS: Participants included 4,137 self-referred apparently healthy adults (2,326 men, 1,811 women; mean age: 42.8 ± 12.2 years) who underwent CPX testing to determine baseline CRF. Participants were followed for 24.2 ± 11.7 years (1.1 to 49.3 years) for mortality. Cox-proportional hazard models were performed to determine the relationship of CRF (ml·kg-1·min-1) and CRF level (low, moderate, and high) with mortality outcomes. RESULTS: During follow-up, 727 participants died (524 men, 203 women). CPX-derived CRF was inversely related to all-cause, CVD, and cancer mortality. Low CRF was associated with higher risk for all-cause (hazard ratio [HR]: 1.73; 95% confidence interval [CI]: 1.20 to 3.50), CVD (HR: 2.27; 95% CI: 1.20 to 3.49), and cancer (HR: 2.07; 95% CI: 1.18 to 3.36) mortality compared with high CRF. Further, each metabolic equivalent increment increase in CRF was associated with a 11.6%, 16.1%, and 14.0% reductions in all-cause, CVD, and cancer mortality, respectively. CONCLUSIONS: Given the prognostic ability of CPX-derived CRF for all-cause and disease-specific mortality outcomes, its use should be highly considered for apparently healthy populations as it may help to improve the efficacy of the individualized patient risk assessment and guide clinical decisions.

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination.

Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.

Myelinosome formation represents an early stage of oligodendrocyte damage in multiple sclerosis and its animal model.

Oligodendrocyte damage is a central event in the pathogenesis of the common neuroinflammatory condition, multiple sclerosis (MS). Where and how oligodendrocyte damage is initiated in MS is not completely understood. Here, we use a combination of light and electron microscopy techniques to provide a dynamic and highly resolved view of oligodendrocyte damage in neuroinflammatory lesions. We show that both in MS and in its animal model structural damage is initiated at the myelin sheaths and only later spreads to the oligodendrocyte cell body. Early myelin damage itself is characterized by the formation of local myelin out-foldings-'myelinosomes'-, which are surrounded by phagocyte processes and promoted in their formation by anti-myelin antibodies and complement. The presence of myelinosomes in actively demyelinating MS lesions suggests that oligodendrocyte damage follows a similar pattern in the human disease, where targeting demyelination by therapeutic interventions remains a major open challenge.

The use of a laser for correlating light and electron microscopic images in thick tissue specimens.

Correlating images between light and electron microscopy is difficult especially in tissue specimens with a substantial z-dimension. To facilitate correlated light and electron microscopy (CLEM) in thick tissue specimens, we describe a basic method of using a femto-pulsed near-infrared laser to burn precise three-dimensional fiducial markers that circumscribe cells or regions of interest for easy identification between imaging methods. This rapid and reliable approach permits traditional fixation and avoids the use of electron-dense labeling methods that can obscure ultrastructural details. The versatility of the technique permits CLEM in a variety of tissue specimens to allow interpretation of highly resolved ultrastructural data in the more macroscopic and potentially dynamic context of light microscopy.

Pervasive axonal transport deficits in multiple sclerosis models.

Impaired axonal transport can contribute to axon degeneration and has been described in many neurodegenerative diseases. Multiple sclerosis (MS) is a common neuroinflammatory disease, which is characterized by progressive axon degeneration-whether, when, and how axonal transport is affected in this condition is unknown. Here we used in vivo two-photon imaging to directly assay transport of organelles and the stability of microtubule tracks in individual spinal axons in mouse models of MS. We found widespread transport deficits, which preceded structural alterations of axons, cargos, or microtubules and could be reversed by acute anti-inflammatory interventions or redox scavenging. Our study shows that acute neuroinflammation induces a pervasive state of reversible axonal dysfunction, which coincides with acute disease symptoms. Moreover, perpetuated transport dysfunction, as we found in a model of progressive MS, led to reduced distal organelle supply and could thus contribute to axonal dystrophy in advanced stages of the disease.

Small Molecule Inhibitors Limit Endothelial Cell Invasion by Staphylococcus aureus.

Staphylococcus aureus is a leading causative agent in sepsis, endocarditis, and pneumonia. An emerging concept is that prognosis worsens when the infecting S. aureus strain has the capacity to not only colonize tissue as an extracellular pathogen, but to invade host cells and establish intracellular bacterial populations. In previous work, we identified host CDC42 as a central regulator of endothelial cell invasion by S. aureus. In the current work, we report that ML 141, a first-in-class CDC42 inhibitor, decreases invasion and resultant pathogenesis in a dose-dependent and reversible manner. Inhibition was found to be due in part to decreased remodeling of actin that potentially drives endocytic uptake of bacteria/fibronectin/integrin complexes. ML 141 decreased binding to fibronectin at these complexes, thereby limiting a key pathogenic mechanism used by S. aureus to invade. Structural analogs of ML 141 were synthesized (designated as the RSM series) and a subset identified that inhibit invasion through non-cytotoxic and non-bactericidal mechanisms. Our results support the development of adjunctive therapeutics targeting host CDC42 for mitigating invasive infection at the level of the host.

An assay to image neuronal microtubule dynamics in mice.

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.

Glial imaging during synapse remodeling at the neuromuscular junction.

Glia are an indispensable structural and functional component of the synapse. They modulate synaptic transmission and also play important roles in synapse formation and maintenance. The vertebrate neuromuscular junction (NMJ) is a classic model synapse. Due to its large size, simplicity and accessibility, the NMJ has contributed greatly to our understanding of synapse development and organization. In the past decade, the NMJ has also emerged as an effective model for studying glia-synapse interactions, in part due to the development of various labeling techniques that permit NMJs and associated Schwann cells (the glia at NMJs) to be visualized in vitro and in vivo. These approaches have demonstrated that Schwann cells are actively involved in synapse remodeling both during early development and in post-injury reinnervation. In vivo imaging has also recently been combined with serial section transmission electron microscopic (ssTEM) reconstruction to directly examine the ultrastructural organization of remodeling NMJs. In this review, we focus on the anatomical studies of Schwann cell dynamics and their roles in formation, maturation and remodeling of vertebrate NMJs using the highest temporal and spatial resolution methods currently available.

Ectopic endplates induce localized changes in skeletal muscle ultrastructure.

To investigate the processes by which motoneurons control protein synthesis, and thus the ultrastructure of the muscle fibers they innervate, ectopic endplates were induced to form on adult mouse skeletal muscle fibers by transplantation of a foreign nerve onto the muscle. In the dually innervated muscle fibers thus created, we examined two ultrastructural parameters that correlate with the expression of distinct isoforms of the myofibrillar proteins alpha-actinin and titin, specifically, Z-line width and sarcomere length. It was found that Z-lines were significantly thinner (98 vs. 128 nm) and sarcomeres were significantly shorter (1.69 vs. 2.06 microm) near the ectopic than near the original endplates. Thus, ectopic endplate formation on adult skeletal muscle fibers induces a localized alteration in myofibrillar morphology. These results may help to elucidate the role played by motoneurons in the determination and maintenance of muscle fiber properties and the processes that occur following muscle reinnervation after nerve injury.

Age-associated synapse elimination in mouse parasympathetic ganglia.

Little is known about the effects of aging on synapses in the mammalian nervous system. We examined the innervation of individual mouse submandibular ganglion (SMG) neurons for evidence of age-related changes in synapse efficacy and number. For approximately 85% of adult life expectancy (30 months) the efficacy of synaptic transmission, as determined by excitatory postsynaptic potential (EPSP) amplitudes, remains constant. Similarly, the number of synapses contacting individual SMG neurons is also unchanged. After 30 months of age, however, some neurons (23%) dramatically lose synaptic input exhibiting both smaller EPSP amplitude and fewer synaptic boutons. Attenuation of both the amplitude and frequency of miniature EPSPs was also observed in neurons from aged animals. Electron micrographs revealed that, although there were many vesicle-laden preganglionic axonal processes in the vicinity of the postsynaptic membrane, the number of synaptic contacts was significantly lower in old animals. These results demonstrate primary, age-associated synapse elimination with functional consequences that cannot be explained by pre- or postsynaptic cell death.