Hepatic fat is superior to BMI, visceral and pancreatic fat as a potential risk biomarker for neurodegenerative disease.

OBJECTIVES: Prior studies relating body mass index (BMI) to brain volumes suggest an overall inverse association. However, BMI might not be an ideal marker, as it disregards different fat compartments, which carry different metabolic risks. Therefore, we analyzed MR-based fat depots and their association with gray matter (GM) volumes of brain structures, which show volumetric changes in neurodegenerative diseases. METHODS: Warp-based automated brain segmentation of 3D FLAIR sequences was obtained in a population-based study cohort. Associations of temporal lobe, cingulate gyrus, and hippocampus GM volume with BMI and MR-based quantification of visceral adipose tissue (VAT), as well as hepatic and pancreatic proton density fat fraction (PDFFhepatic and PDFFpanc, respectively), were assessed by linear regression. RESULTS: In a sample of 152 women (age 56.2 ± 9.0 years) and 199 men (age 56.1 ± 9.1 years), we observed a significant inverse association of PDFFhepatic and cingulate gyrus volume (p < 0.05) as well as of PDFFhepatic and hippocampus volume (p < 0.05), when adjusting for age and sex. This inverse association was further enhanced for cingulate gyrus volume after additionally adjusting for hypertension, smoking, BMI, LDL, and total cholesterol (p < 0.01) and also alcohol (p < 0.01). No significant association was observed between PDFFhepatic and temporal lobe and between temporal lobe, cingulate gyrus, or hippocampus volume and BMI, VAT, and PDFFpanc. CONCLUSIONS: We observed a significant inverse, independent association of cingulate gyrus and hippocampus GM volume with hepatic fat, but not with other obesity measures. Increased hepatic fat could therefore serve as a marker of high-risk fat distribution. KEY POINTS: • Obesity is associated with neurodegenerative processes. • In a population-based study cohort, hepatic fat was superior to BMI and visceral and pancreatic fat as a risk biomarker for decreased brain volume of cingulate gyrus and hippocampus. • Increased hepatic fat could serve as a marker of high-risk fat distribution.

The role of visceral and subcutaneous adipose tissue measurements and their ratio by magnetic resonance imaging in subjects with prediabetes, diabetes and healthy controls from a general population without cardiovascular disease.

OBJECTIVE: To study the relationship of area- and volumetric-based visceral and subcutaneous adipose tissue (VAT and SAT) by MRI and their ratio in subjects with impaired glucose metabolism from the general population. METHODS: Subjects from a population-based cohort with established prediabetes, diabetes and healthy controls without prior cardiovascular diseases underwent 3 T MRI. VAT and SAT were assessed as total volume and area on a single slice, and their ratio (VAT/SAT) was calculated. Clinical covariates and cardiovascular risk factors, such as hypertension and glycemic state were assessed in standardized fashion. Univariate and adjusted analyses were conducted. RESULTS: Among 384 subjects (age: 56.2 ± 9.2 years, 58.1% male) with complete MRI data available, volumetric and single-slice VAT, SAT and VAT/SAT ratio were strongly correlated (all >r = 0.89). Similarly, VAT/SATvolume ratio was strongly correlated with VATvolume but not with SAT (r = 0.72 and r = -0.21, respectively). Significant higher levels of VAT, SAT and VAT/SAT ratio were found in subjects with impaired glucose metabolism (all p ≤ 0.01). After adjustment for potential cardiovascular confounders, VATvolume and VAT/SATvolume ratio remained significantly higher in subjects with impaired glucose metabolism (VATvolume = 6.9 ± 2.5 l and 3.4 ± 2.3 l; VAT/SATvolume ratio = 0.82 ± 0.34 l and 0.49 ± 0.29 l in patients with diabetes and controls, respectively, all p < 0.02), whereas the association for SATvolume attenuated. Additionally, there was a decreasing effect of glycemic status on VAT/SATvolume ratio with increasing body mass index and waist circumference (p < 0.05). CONCLUSIONS: VATvolume and VAT/SATvolume ratio are associated with impaired glucose metabolism, independent of cardiovascular risk factors or MRI-based quantification technique, with a decreasing effect of VAT/SATvolume ratio in obese subjects. Advances in knowledge: Quantification of VATvolume and VAT/SATvolume ratio by MRI represents a reproducable biomarker associated with cardiometabolic risk factors in subjects with impaired glucose metabolism, while the association of VAT/SATvolume ratio with glycemic state is attenuated in obese subjects.

Dexamethasone-conjugated DNA nanotubes as anti-inflammatory agents in vivo.

The biopolymer DNA allows to create nanoscale, biocompatible structures, which can be designed in a target-specific and stimuli-responsive manner. DNA carrier systems with these characteristics hold a great potential for nanomedical applications, such as for the treatment of inflammatory diseases. Here we used a DNA-based drug carrier system for the pH-dependent delivery of the glucocorticoid dexamethasone into macrophages, a cell type with a key role in the regulation of inflammation. Dexamethasone (Dex) nanotubes were internalized within minutes by MH-S macrophages in vitro and by tissue resident macrophages in the mouse cremaster muscle in vivo and localized in their endosomes. Treatment with Dex nanotubes in vitro significantly reduced the LPS-induced TNF secretion by macrophages, as compared to equivalent amounts of free dexamethasone without affecting cell viability. Microinjection of Dex nanotubes into postischemic muscle tissue of anesthetized mice resulted in a marked reduction of ischemia-reperfusion-elicited leukocyte transmigration and diminished vascular expression of the endothelial adhesion molecules VCAM-1 and ICAM-1. Taken together, our results demonstrate that DNA nanotubes can be used as a platform for the targeted delivery of glucocorticoids and could thus foster the development of nanomedical therapeutics with reduced off-target effects.

Microglial CX3CR1 promotes adult neurogenesis by inhibiting Sirt 1/p65 signaling independent of CX3CL1.

Homo and heterozygote cx3cr1 mutant mice, which harbor a green fluorescent protein (EGFP) in their cx3cr1 loci, represent a widely used animal model to study microglia and peripheral myeloid cells. Here we report that microglia in the dentate gyrus (DG) of cx3cr1 (-/-) mice displayed elevated microglial sirtuin 1 (SIRT1) expression levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 activation, despite unaltered morphology when compared to cx3cr1 (+/-) or cx3cr1 (+/+) controls. This phenotype was restricted to the DG and accompanied by reduced adult neurogenesis in cx3cr1 (-/-) mice. Remarkably, adult neurogenesis was not affected by the lack of the CX3CR1-ligand, fractalkine (CX3CL1). Mechanistically, pharmacological activation of SIRT1 improved adult neurogenesis in the DG together with an enhanced performance of cx3cr1 (-/-) mice in a hippocampus-dependent learning and memory task. The reverse condition was induced when SIRT1 was inhibited in cx3cr1 (-/-) mice, causing reduced adult neurogenesis and lowered hippocampal cognitive abilities. In conclusion, our data indicate that deletion of CX3CR1 from microglia under resting conditions modifies brain areas with elevated cellular turnover independent of CX3CL1.

Multiphoton Microscopy of Nonfluorescent Nanoparticles In Vitro and In Vivo.

Nanotechnology holds great promise for a plethora of potential applications. The interaction of engineered nanomaterials with living cells, tissues, and organisms is, however, only partly understood. Microscopic investigations of nano-bio interactions are mostly performed with a few model nanoparticles (NPs) which are easy to visualize, such as fluorescent quantum dots. Here the possibility to visualize nonfluorescent NPs with multiphoton excitation is investigated. Signals from silver (Ag), titanium dioxide (TiO2 ), and silica (SiO2 ) NPs in nonbiological environments are characterized to determine signal dependency on excitation wavelength and intensity as well as their signal stability over time. Ag NPs generate plasmon-induced luminescence decaying over time. TiO2 NPs induce photoluminescent signals of variable intensities and in addition strong third harmonic generation (THG). Optimal settings for microscopic detection are determined and then applied for visualization of these two particle types in living cells, in murine muscle tissue, and in the murine blood stream. Silica NPs produce a THG signal, but in living cells it cannot be discriminated sufficiently from endogenous cellular structures. It is concluded that multiphoton excitation is a viable option for studies of nano-bio interactions not only for fluorescent but also for some types of nonfluorescent NPs.

Influence of Surface Modifications on the Spatiotemporal Microdistribution of Quantum Dots In Vivo.

For biomedical applications of nanoconstructs, it is a general prerequisite to efficiently reach the desired target site. In this regard, it is crucial to determine the spatiotemporal distribution of nanomaterials at the microscopic tissue level. Therefore, the effect of different surface modifications on the distribution of microinjected quantum dots (QDs) in mouse skeletal muscle tissue has been investigated. In vivo real-time fluorescence microscopy and particle tracking reveal that carboxyl QDs preferentially attach to components of the extracellular matrix (ECM), whereas QDs coated with polyethylene glycol (PEG) show little interaction with tissue constituents. Transmission electron microscopy elucidates that carboxyl QDs adhere to collagen fibers as well as basement membranes, a type of ECM located on the basolateral side of blood vessel walls. Moreover, carboxyl QDs have been found in endothelial junctions as well as in caveolae of endothelial cells, enabling them to translocate into the vessel lumen. The in vivo QD distribution is confirmed by in vitro experiments. The data suggest that ECM components act as a selective barrier depending on QD surface modification. For future biomedical applications, such as targeting of blood vessel walls, the findings of this study offer design criteria for nanoconstructs that meet the requirements of the respective application.

Intercellular Transport of Nanomaterials is Mediated by Membrane Nanotubes In Vivo.

So-called membrane nanotubes are cellular protrusions between cells whose functions include cell communication, environmental sampling, and protein transfer. It has been previously reported that systemically administered carboxyl-modified quantum dots (cQDs) are rapidly taken up by perivascular macrophages in skeletal muscle of healthy mice. Expanding these studies, it is found, by means of in vivo fluorescence microscopy on the mouse cremaster muscle, rapid uptake of cQDs not only by perivascular macrophages but also by tissue-resident cells, which are localized more than 100 µm distant from the closest vessel. Confocal microscopy on muscle tissue, immunostained for the membrane dye DiI, reveals the presence of continuous membranous structures between MHC-II-positive, F4/80-positive cells. These structures contain microtubules, components of the cytoskeleton, which clearly colocalize with cQDs. The cQDs are exclusively found inside endosomal vesicles. Most importantly, by using in vivo fluorescence microscopy, this study detected fast (0.8 µm s(-1) , mean velocity), bidirectional movement of cQDs in such structures, indicating transport of cQD-containing vesicles along microtubule tracks by the action of molecular motors. The findings are the first to demonstrate membrane nanotube function in vivo and they suggest a previously unknown route for the distribution of nanomaterials in tissue.

A microfluidics approach to study the accumulation of molecules at basal lamina interfaces.

For an efficient distribution of drugs and drug carriers through biological barriers such as the vascular system, the size and surface properties of nanoparticles and molecules play a key role. To screen for important parameters which determine the ability of drugs or drug carriers to translocate through complex biological barriers, an in vitro assay which correctly predicts the behavior of those objects in vivo would be highly desirable. Here, we present a microfluidic setup to probe the diffusive spreading of molecules with different net charges and molecular weights through a basal lamina interface - a biopolymer system which contributes to the barrier function of the vascular system and the skin. From our data, we find a charge dependent accumulation of molecules at the gel interface which is consistent with transient binding of those molecules to the gel constituents. We also observe a similar charge-dependent accumulation of molecules in living mice where the test molecules colocalize with collagen IV, a key component of the basal lamina. Our assay may serve as a platform to perform penetration experiments with even more complex interfaces combining cellular barriers with biopolymer coatings.

DNA nanotubes as intracellular delivery vehicles in vivo.

DNA-based nanoconstructs possess great potential for biomedical applications. However, the in vivo behavior of such constructs at the microscopic tissue/cell level as well as their inflammatory potential is largely unknown. Unmethylated CpG sequences of DNA are recognized by Toll-like receptor 9 (TLR9), and thus initiate an innate immune response. In this study, we investigated the use of DNA-based nanotubes as carrier systems for CpG delivery and their effect on immune cells in vivo and in real time. DNA nanotubes were microinjected into skeletal muscle of anesthetized mice. Using in vivo microscopy, we observed that the DNA tubes were internalized within minutes by tissue-resident macrophages and localized in their endosomes. Only microinjection of CpG-decorated DNA nanotubes but not of plain DNA nanotubes or CpG oligonucleotides induced a significant recruitment of leukocytes into the muscle tissue as well as activation of the NF-ĸB pathway in surrounding cells. These results suggest that DNA nanotubes are promising delivery vehicles to target tissue macrophages, whereupon the immunogenic potential depends on the decoration with CpG oligonucleotides.