A workflow for 3D-CLEM investigating liver tissue.

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. LAY DESCRIPTION: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10-9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail.

Development of conventional dendritic cells: from common bone marrow progenitors to multiple subsets in peripheral tissues.

Our understanding of conventional dendritic cell (cDC) development and the functional specializations of distinct subsets in the peripheral tissues has increased greatly in recent years. Here, we review cDC development from the distinct progenitors in the bone marrow through to the distinct cDC subsets found in barrier tissues, providing an overview of the different subsets described in each location. In addition, we detail the transcription factors and local signals that have been proposed to control this developmental process. Importantly, despite these significant advances, numerous questions remain to be answered regarding cDC development. For example, it remains unclear whether the different subsets described, such as the CD103+CD11b+ and CD103-CD11b+ cDCs in the intestines, truly represent different populations or rather distinct developmental or activation stages. Furthermore, whether distinct progenitors exist for these cDC subsets remains to be determined. Thus in the last part of this review we discuss what we believe will be the main questions facing the field for the coming years.

CCR2(+)CD103(-) intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells.

The identification of intestinal macrophages (mφs) and dendritic cells (DCs) is a matter of intense debate. Although CD103(+) mononuclear phagocytes (MPs) appear to be genuine DCs, the nature and origins of CD103(-) MPs remain controversial. We show here that intestinal CD103(-)CD11b(+) MPs can be separated clearly into DCs and mφs based on phenotype, gene profile, and kinetics. CD64(-)CD103(-)CD11b(+) MPs are classical DCs, being derived from Flt3 ligand-dependent, DC-committed precursors, not Ly6C(hi) monocytes. Surprisingly, a significant proportion of these CD103(-)CD11b(+) DCs express CCR2 and there is a selective decrease in CD103(-)CD11b(+) DCs in mice lacking this chemokine receptor. CCR2(+)CD103(-) DCs are present in both the murine and human intestine, drive interleukin (IL)-17a production by T cells in vitro, and show constitutive expression of IL-12/IL-23p40. These data highlight the heterogeneity of intestinal DCs and reveal a bona fide population of CCR2(+) DCs that is involved in priming mucosal T helper type 17 (Th17) responses.

Division of labor between lung dendritic cells and macrophages in the defense against pulmonary infections.

The lung is highly exposed to the external environment. For this reason, the lung needs to handle a number of potential threats present in inhaled air such as viruses or bacteria. Dendritic cells (DCs) and macrophages (MFs) play an important role in orchestrating the immune responses to these challenges. The severe lung inflammation caused by some pathogens poses a unique challenge to the immune system: the potential insult must be eliminated rapidly whereas tissue inflammation must be controlled in order to avoid collateral damages that can lead to acute respiratory failure. Immune responses to infectious agents are initiated and controlled by various populations of antigen-presenting cells with specialized functions, which include conventional DCs (cDCs), monocyte-derived DCs (moDCs), plasmacytoid DCs (pDCs), and alveolar MFs (AMFs). This review will discuss the role of these different cells in responses to pulmonary infections, with a focus on influenza virus and Mycobacterium tuberculosis.

Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors.

Macrophages (mφ) are essential for intestinal homeostasis and the pathology of inflammatory bowel disease (IBD), but it is unclear whether discrete mφ populations carry out these distinct functions or if resident mφ change during inflammation. We show here that most resident mφ in resting mouse colon express very high levels of CX3CR1, are avidly phagocytic and MHCII(hi), but are resistant to Toll-like receptor (TLR) stimulation, produce interleukin 10 constitutively, and express CD163 and CD206. A smaller population of CX3CR1(int) cells is present in resting colon and it expands during experimental colitis. Ly6C(hi)CCR2(+) monocytes can give rise to all mφ subsets in both healthy and inflamed colon and we show that the CX3CR1(int) pool represents a continuum in which newly arrived, recently divided monocytes develop into resident CX3CR1(hi) mφ. This process is arrested during experimental colitis, resulting in the accumulation of TLR-responsive pro-inflammatory mφ. Phenotypic analysis of human intestinal mφ indicates that analogous processes occur in the normal and Crohn's disease ileum. These studies show for the first time that resident and inflammatory mφ in the intestine represent alternative differentiation outcomes of the same precursor and targeting these events could offer routes for therapeutic intervention in IBD.

Sensorimotor reconditioning during and after spaceflight.

Exposure to microgravity drives adaptive changes in healthy individuals reconditioned for abnormal gravity states. These changes are maladaptive for return to earth's gravity. The intersubject variability of sensorimotor decrements is striking, although poorly understood. Multisensory integration, which is important for resolving sensory ambiguity on earth, is a critical mechanism for sensorimotor adaptation during and following space flight. The removal of gravitational loading also has profound effects that both negatively impact sensorimotor function and reduce capacity to overcome sensorimotor deficits. Countermeasure strategies include preflight training to facilitate transition to microgravity, pharmaceuticals and restriction of some activities early on orbit, and inflight exercise to minimize deconditioning during longer duration missions. Active motion is important to promote reconditioning upon return to earth's gravity. A supervised reconditioning program utilizes exercises that challenge multisensory integration with an increasing level of difficulty customized to the individual's state of recovery. This program also serves to increase crew self-awareness of fall risk. New resistive and aerobic exercise capabilities onboard the International Space Station contribute to improved postflight mobility. Lessons learned from inflight and postflight reconditioning programs have implications for future exploration crews that will operate more autonomously, as well as rehabilitation in clinical populations on earth.

Strength gains following different combined concentric and eccentric exercise regimens.

BACKGROUND: Loss of muscle strength and cross-sectional area is a well-recognized consequence of spaceflight. Existing countermeasures have not been fully effective in preventing muscle weakness and atrophy in microgravity. Resistance exercise programs that consist of both eccentric and concentric actions have resulted in strength and muscle mass gains in ground-based studies. HYPOTHESES: 1) A concentric/eccentric combination exercise regimen (with a bias of either concentric or eccentric exercise) will result in a greater strength gain than concentric exercise alone; and 2) an eccentrically biased regimen will result in the greatest strength gain of all. METHODS: The 31 subjects were randomly assigned to one of three isokinetic exercise groups (CON-ECC: 75% concentric and 25% eccentric; ECC-CON: 75% eccentric and 25% concentric; CON: 100% concentric); each subject trained the right leg 3 d per week for 5 wk. Pre- and post-training isokinetic concentric/ eccentric strength tests and DEXA scans assessed changes in muscle strength and/or mass. RESULTS: All three groups showed an increase in eccentric muscle strength with the CON group showing the smallest gain (10.1%). Significantly larger gains were noted in the two combination groups (19.5%, 18.1%; p < 0.042), with the largest gains in eccentric strength. No significant change was noted in muscle mass. CONCLUSIONS: A resistance exercise protocol which includes eccentric as well as concentric exercise, particularly when the eccentric exercise is emphasized, appears to result in greater strength gains than concentric exercise alone. Findings suggest eccentric exercise may be an important component of the in-flight resistance exercise protocol for long-duration spaceflight.

Carbon dioxide accumulation, walking performance, and metabolic cost in the NASA launch and entry suit.

BACKGROUND: In the event of an emergency on landing, Space Shuttle crewmembers while wearing the Launch and Entry Suit (LES) must stand, move to the hatch, exit the spacecraft with the helmet visor closed breathing 100% O2, and walk or run unassisted to a distance of 380 m upwind from the vehicle. The purpose of this study was to characterize the inspired CO2 and metabolic requirements during a simulated unaided egress from the Space Shuttle in healthy subjects wearing the LES. METHODS: As a simulation of a Shuttle landing with an unaided egress, 12 male subjects completed a 6-min seated pre-breathe with 100% O2 followed by a 2-min stand and 5-min walking at 1.56 m x s(-1) (5.6 km x h(-1), 3.5 mph) with the helmet visor closed. During walks with four different G-suit pressures (0.0, 0.5, 1.0, 1.5 psi; 3.4, 6.9, 10.3 kPa), inspired CO2 and walking time were measured. After a 10-min seated recovery, subjects repeated the 5-min walk with the same G-suit pressure and the helmet visor open for the measurement of metabolic rate (VO2). RESULTS: When G-suit inflation levels were 1.0 or 1.5 psi, only one-third of our subjects were able to complete the 5-min visor-closed walk after a 6-min pre-breathe. Inspired CO2 levels measured at the mouth were routinely greater than 4% (30 mmHg) during walking. The metabolic cost at the 1.5 psi G-suit inflation was over 135% of the metabolic cost at 0.0 psi inflation. CONCLUSION: During unaided egress, G-suit inflation pressures of 1.0 and 1.5 psi resulted in elevated CO2 in the LES helmet and increased metabolic cost of walking, both of which may impact unaided egress performance. Neither the LES, the LES helmet, nor the G-suit were designed for ambulation. Data from this investigation suggests that adapting flight equipment for uses other than those for which it was originally designed can result in unforeseen problems.

Resistance exercise prevents plantar flexor deconditioning during bed rest.

Because resistance exercise (REX) and unloading induce opposing neuromuscular adaptations, we tested the efficacy of REX against the effects of 14 d of bed rest unloading (BRU) on the plantar flexor muscle group. Sixteen men were randomly assigned to no exercise (NOE, N = 8) or REX (N = 8). REX performed 5 sets x 6-10 repetitions to failure of constant resistance concentric/eccentric plantar flexion every other day during BRU. One-repetition maximum (1RM) strength was tested on the training device. The angle-specific torque-velocity relationship across 5 velocities (0, 0.52, 1.05, 1.75, and 2.97 rad.s-1) and the full range-of-motion power-velocity relationship were assessed on a dynamometer. Torque-position analyses identified strength changes at shortened, neutral, and stretched muscle lengths. Concentric and eccentric contractile work were measured across ten repetitions at 1.05 rad.s-1. Maximal neural activation was measured by surface electromyography (EMG). 1RM decreased 9% in NOE and improved 11% in REX (P < 0.05). Concentric (0.52 and 1.05 rad.s-1), eccentric (0.52 and 2.97 rad.s-1), and isometric angle-specific torques decreased (P < 0.05) in NOE, averaging 18%, 17%, and 13%, respectively. Power dropped (P < 0.05) in NOE at three eccentric (21%) and two concentric (14%) velocities. REX protected angle-specific torque and average power at all velocities. Concentric and eccentric strength decreased at stretched (16%) and neutral (17%) muscle lengths (P < 0.05) in NOE while REX maintained or improved strength at all joint positions. Concentric (15%) and eccentric (11%) contractile work fell in NOE (P < 0.05) but not in REX. Maximal plantar flexor EMG did not change in either group. In summary, constant resistance concentric/eccentric REX completely prevented plantar flexor performance deconditioning induced by BRU. The reported benefits of REX should prove useful in prescribing exercise for astronauts in microgravity and for patients susceptible to functional decline during bed- or chair-bound hospital stays.