Investigation of the role of infusate properties related to midline catheter failure in an ovine model.

PURPOSE: Infusate osmolarity, pH, and cytotoxicity were investigated as risk factors for midline catheter failure. METHODS: An experimental, randomized, controlled, blinded trial was conducted using an ovine model. Two 10-cm, 18-gauge single-lumen midline catheters were inserted into the cephalic veins of sheep. The animals were divided into 6 study arms and were administered solutions of vancomycin 4 mg/mL (a low-cytotoxicity infusate) or 10 mg/mL (a high-cytotoxicity infusate), doxycycline 1 mg/mL (an acidic infusate), or acyclovir 3.5 mg/mL (an alkaline infusate) and 0.9% sodium chloride injection; or 1 of 2 premixed Clinimix (amino acids in dextrose; Baxter International) products with respective osmolarities of 675 mOsm/L (a low-osmolarity infusate) and 930 mOsm/L (a mid-osmolarity infusate). Contralateral legs were infused with 0.9% sodium chloride injection for control purposes. Catheter failure was evaluated by assessment of adverse clinical symptoms (swelling, pain, leakage, and occlusion). A quantitative vessel injury score (VIS) was calculated by grading 4 histopathological features: inflammation, mural thrombus, necrosis, and perivascular reaction. RESULTS: Among 20 sheep included in the study, the overall catheter failure rate was 95% for test catheters (median time to failure, 7.5 days; range, 3-14 days), while 60% of the control catheters failed before or concurrently (median time to failure, 7 days; range, 4.5-14 days). Four of the 6 study arms (all but the Clinimix 675-mOsm/L and acyclovir 3.5-mg/mL arms) demonstrated an increase in mean VIS of ≥77% in test vs control legs (P ≤ 0.034). Both pain and swelling occurred at higher rates in test vs control legs: 65% vs 10% and 70% vs 50%, respectively. The mean difference in rates of occlusive pericatheter mural thrombus between the test and control arms was statistically significant for the vancomycin 10-mg/mL (P = 0.0476), Clinimix 930-mOsm/L (P = 0.0406), and doxycycline 1-mg/mL (P = 0.032) arms. CONCLUSION: Administration of infusates of varied pH, osmolarity, and cytotoxicity via midline catheter resulted in severe vascular injury and premature catheter failure; therefore, the tested infusates should not be infused via midline catheters.

Teriflunomide Does Not Change Dynamics of Nadph Oxidase Activation and Neuronal Dysfunction During Neuroinflammation.

The multiple sclerosis therapeutic teriflunomide is known to block the de novo synthesis of pyrimidine in mitochondria by inhibiting the enzyme dihydroorotate-dehydrogenase (DHODH). The metabolic processes of oxidative phosphorylation and glycolysis are further possible downstream targets. In healthy adult mice, high levels of dihydroorotate-dehydrogenase (DHODH) activity are measured in the central nervous system (CNS), and DHODH inhibition may cause indirect effects on reactive oxygen species production and NADPH oxidase (NOX) mediated oxidative stress, known to be key aspects of the inflammatory response of the CNS. However, little is known about the effect of teriflunomide on the dynamics of NOX activation in CNS cells and subsequent alterations of neuronal function in vivo. In this study, we employed fluorescence lifetime imaging (FLIM) and phasor analysis of the endogeneous fluorescence of NAD(P)H (nicotinamide adenine dinucleotide phosphate) in the brain stem of mice to visualize the effect of teriflunomide on cellular metabolism. Furthermore, we simultaneously studied neuronal Ca2+ signals in transgenic mice with a FRET-based Troponin C Ca2+ sensor based (CerTN L15) quantified using FRET-FLIM. Hence, we directly correlated neuronal (dys-)function indicated by steadily elevated calcium levels with metabolic activity in neurons and surrounding CNS tissue. Employing our intravital co-registered imaging approach, we could not detect any significant alteration of NOX activation after incubation of the tissue with teriflunomide. Furthermore, we could not detect any changes of the inflammatory induced neuronal dysfunction due to local treatment with teriflunomide. Concerning drug safety, we can confirm that teriflunomide has no metabolic effects on neuronal function in the CNS tissue during neuroinflammation at concentrations expected in orally treated patients. The combined endogenous FLIM and calcium imaging approach developed by us and employed here uniquely meets the need to monitor cellular metabolism as a basic mechanism of tissue functions in vivo.

Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging.

Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 µm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow.

Analyzing Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation in Aging and Vascular Amyloid Pathology.

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive [Formula: see text] which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age-dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APPE693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein.

Synergistic Strategy for Multicolor Two-photon Microscopy: Application to the Analysis of Germinal Center Reactions In Vivo.

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method.

Tos-Nos-Mos: Synthesis of different aryl sulfonate precursors for the radiosynthesis of the alpha7 nicotinic acetylcholine receptor radioligand [(18)F]NS14490.

Radiopharmacological investigations of [(18)F]NS14490 have proven that this radiotracer could be a potential PET radiotracer for imaging of alpha7 nicotinic acetylcholine receptor particularly with regard to vulnerable plaques of diseased vessels. For further optimisation of the previously automated one-pot radiosynthesis of [(18)F]NS14490 using a tosylate precursor, precursors with other leaving groups (nosylate and mosylate) were synthesized and compared with the tosylate with respect to their reactivities towards [(18)F]fluoride. The use of these different precursors resulted in comparable labelling yields of [(18)F]NS14490. A novel mosylate precursor was synthesized and evaluated, which has revealed a higher stability during a storage period of five months compared to the corresponding tosylate and nosylate.

Development of a Novel Nonpeptidic (18)F-Labeled Radiotracer for in Vivo Imaging of Oxytocin Receptors with Positron Emission Tomography.

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.

Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer.

A hallmark of autoimmune retinal inflammation is the infiltration of the retina with cells of the innate and adaptive immune system, leading to detachment of the retinal layers and even to complete loss of the retinal photoreceptor layer. As the only optical system in the organism, the eye enables non-invasive longitudinal imaging studies of these local autoimmune processes and of their effects on the target tissue. Moreover, as a window to the central nervous system (CNS), the eye also reflects general neuroinflammatory processes taking place at various sites within the CNS. Histological studies in murine neuroinflammatory models, such as experimental autoimmune uveoretinitis (EAU) and experimental autoimmune encephalomyelitis, indicate that immune infiltration is initialized by effector CD4+ T cells, with the innate compartment (neutrophils, macrophages, and monocytes) contributing crucially to tissue degeneration that occurs at later phases of the disease. However, how the immune attack is orchestrated by various immune cell subsets in the retina and how the latter interact with the target tissue under in vivo conditions is still poorly understood. Our study addresses this gap with a novel approach for intravital two-photon microscopy, which enabled us to repeatedly track CD4+ T cells and LysM phagocytes during the entire course of EAU and to identify a specific radial infiltration pattern of these cells within the inflamed retina, starting from the optic nerve head. In contrast, highly motile [Formula: see text] cells display an opposite radial motility pattern, toward the optic nerve head. These inflammatory processes induce modifications of the microglial network toward an activated morphology, especially around the optic nerve head and main retinal blood vessels, but do not affect the neurons within the ganglion cell layer. Thanks to the new technology, non-invasive correlation of clinical scores of CNS-related pathologies with immune infiltrate behavior and subsequent tissue dysfunction is now possible. Hence, the new approach paves the way for deeper insights into the pathology of neuroinflammatory processes on a cellular basis, over the entire disease course.

Intravital FRET: Probing Cellular and Tissue Function in Vivo.

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo-ratiometrically and time-resolved by fluorescence lifetime imaging-and show their concrete application in the context of neuroinflammation in adult mice.

Diaryl-Substituted (Dihydro)pyrrolo[3,2,1-hi]indoles, a Class of Potent COX-2 Inhibitors with Tricyclic Core Structure.

A new compound class of diaryl-substituted heterocycles with tricyclic dihydropyrrolo[3,2,1-hi]indole and pyrrolo[3,2,1-hi]indole core structures has been designed and was synthesized by a modular sequence of Friedel-Crafts acylation, amide formation, and McMurry cyclization. This synthesis route represents a novel and versatile access toward dihydropyrrolo[3,2,1-hi]indoles and is characterized by good chemical yields and high modularity. From a set of 19 derivatives, 11 candidates were selected for determination of their COX inhibition potency and were found to be selective inhibitors with high affinity to COX-2 (IC50 ranging from 20-2500 nM and negligible inhibition of COX-1). The binding mode of the novel inhibitors in the active side of COX-2 was calculated in silico using the protein-ligand docking program GOLD by application of the molecular structures of two compounds derived from X-ray crystallography. Two novel compounds with high affinity to COX-2 (6k = 70 nM, 8e = 60 nM) have a fluoro substituent, making them promising candidates for the development of (18)F-radiolabeled COX-2 inhibitors for imaging purposes with positron emission tomography (PET).

Large-scale self-assembled zirconium phosphate smectic layers via a simple spray-coating process.

The large-scale assembly of asymmetric colloidal particles is used in creating high-performance fibres. A similar concept is extended to the manufacturing of thin films of self-assembled two-dimensional crystal-type materials with enhanced and tunable properties. Here we present a spray-coating method to manufacture thin, flexible and transparent epoxy films containing zirconium phosphate nanoplatelets self-assembled into a lamellar arrangement aligned parallel to the substrate. The self-assembled mesophase of zirconium phosphate nanoplatelets is stabilized by epoxy pre-polymer and exhibits rheology favourable towards large-scale manufacturing. The thermally cured film forms a mechanically robust coating and shows excellent gas barrier properties at both low- and high humidity levels as a result of the highly aligned and overlapping arrangement of nanoplatelets. This work shows that the large-scale ordering of high aspect ratio nanoplatelets is easier to achieve than previously thought and may have implications in the technological applications for similar materials.

The Alzheimer pandemic: is paracetamol to blame?

HISTORICAL BACKGROUND: The clinical recognition of a form of dementia closely resembling Alzheimer's disease dates from around 1800. The role of analgesics derived from coal-tar in the spread of the pandemic is traced in terms of the introduction of phenacetin (PN) in 1887; its nephrotoxicity; the observation of lesions characteristic of the disease by Fischer and Alzheimer; the discovery of paracetamol (PA) as the major metabolite of PN; the linking of kidney injury and dementia with high PN usage; and the failure of PN replacement by PA to halt and reverse the exponential, inexorable rise in the incidence of Alzheimer-type dementia. Fischer observed his first case before Alzheimer; it is proposed to rename the syndrome Fischer-Alzheimer disease (F-AD). Disease development: PA-metabolising enzymes are localised in the synaptic areas of the frontal cortex and hippocampus, where F-AD lesions arise. The initiating chemical lesions in liver poisoning comprise covalent binding of a highly reactive product of PA metabolism to proteins; similar events are believed to occur in brain, where alterations in the antigenic profiles of cerebral proteins activate the microglia. ß-Amyloid forms, and, like PA itself, induces nitric oxide synthase. Peroxynitrite modifies cerebral proteins by nitrating tyrosine residues, further challenging the microglia and exacerbating the amyloid cascade. Spontaneous reinnervation, N-acetyl cysteine administration and tyrosine supplementation may attenuate the early stages of F-AD development. CONCLUSION: F-AD is primarily a man-made condition with PA as its principal risk factor.

Development of fluorinated CB(2) receptor agonists for PET studies.

A convergent strategy was followed to modify systematically carbazole based CB(2) receptor ligands. The length of the N-(fluoroalkyl) group (n in 7), the length of the alkanamide (m in 7) and the substitution pattern of the phenyl moiety (X and Y in 7) were varied systematically. The highest CB(2) affinity was found for the 2-fluoroethyl substituted carbazole derivative 20a (Ki=5.8nM) containing the propionamide and the 2-bromo-4-fluorophenyl moiety. According to docking studies 20a fits nicely into the binding pocket of the CB(2) receptor, but elongation of the fluoroethyl side chain leads to a different binding mode of the ligands. The high CB(2) affinity together with the high selectivity over the CB(2) subtype qualifies the fluoroethyl derivative 20a to be developed as a PET tracer.

Radiofluorination and biological evaluation of N-aryl-oxadiazolyl-propionamides as potential radioligands for PET imaging of cannabinoid CB2 receptors.

BACKGROUND: The level of expression of cannabinoid receptor type 2 (CB2R) in healthy and diseased brain has not been fully elucidated. Therefore, there is a growing interest to assess the regional expression of CB2R in the brain. Positron emission tomography (PET) is an imaging technique, which allows quantitative monitoring of very low amounts of radiolabelled compounds in living organisms at high temporal and spatial resolution and, thus, has been widely used as a diagnostic tool in nuclear medicine. Here, we report on the radiofluorination of N-aryl-oxadiazolyl-propionamides at two different positions in the lead structure and on the biological evaluation of the potential of the two tracers [18F]1 and [18F]2 as CB2 receptor PET imaging agents. RESULTS: High binding affinity and specificity towards CB2 receptors of the lead structure remained unaffected by the structural changes such as the insertion of the aliphatic and aromatic fluorine in the selected labelling sites of 1 and 2. Aliphatic and aromatic radiofluorinations were optimized, and [18F]1 and [18F]2 were achieved in radiochemical yields of ≥30% with radiochemical purities of ≥98% and specific activities of 250 to 450 GBq/µmol. Organ distribution studies in female CD1 mice revealed that both radiotracers cross the blood-brain barrier (BBB) but undergo strong peripheral metabolism. At 30 min after injection, unmetabolized [18F]1 and [18F]2 accounted for 60% and 2% as well as 68% and 88% of the total activity in the plasma and brain, respectively. The main radiometabolite of [18F]2 could be identified as the free acid [18F]10, which has no affinity towards the CB1 and CB2 receptors but can cross the BBB. CONCLUSIONS: N-aryl-oxadiazolyl-propionamides can successfully be radiolabelled with 18F at different positions. Fluorine substitution at these positions did not affect affinity and specificity towards CB2R. Despite a promising in vitro behavior, a rather rapid peripheral metabolism of [18F]1 and [18F]2 in mice and the generation of brain permeable radiometabolites hamper the application of these radiotracers in vivo. However, it is expected that future synthetic modification aiming at a replacement of metabolically susceptible structural elements of [18F]1 and [18F]2 will help to elucidate the potential of this class of compounds for CB2R PET studies.

A fluoro versus a nitro derivative-a high-performance liquid chromatography study of two basic analytes with different reversed phases and silica phases as basis for the separation of a positron emission tomography radiotracer.

To develop a basis for the separation of a (18)F-labeled PET radiotracer from its nitro precursor, we performed an analytical HPLC study using the unlabeled reference compound and the corresponding nitro precursor. Aim of the study was to find a separation in which the fluoro derivative elutes in front of the nitro precursor with appropriate separation parameters. Several RP phases as well as a bare silica column were investigated with ACN and MeOH as organic modifiers and aqueous NH4OAc because of the basic character of the analytes. Four types of separation were observed based on different interaction mechanisms. When ACN/20mM NH4OAc aq. was used mainly cationic-exchange and hydrophobic interactions contributed to the retention. A reversal of elution order could be observed starting from 95% ACN and subsequent increasing of the water content. This phenomenon was observed for all RP phases and seems to be independent of the different spacers bound to the silica. By contrast, using MeOH/20mM NH4OAc aq. the elution order depends on the phase material. Two columns with the potential to perform π-π interactions showed different separation behavior compared to the other RP phases.

High-resolution intravital microscopy.

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy--the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.

Cannabinoid receptor type 2 (CB2)-selective N-aryl-oxadiazolyl-propionamides: synthesis, radiolabelling, molecular modelling and biological evaluation.

BACKGROUND: The endocannabinoid system is involved in many physiological and pathological processes. Two receptors (cannabinoid receptor type 1 (CB1) and type 2 (CB2)) are known so far. Many unwanted psychotic side effects of inhibitors of this system can be addressed to the interaction with CB1. While CB1 is one of the most abundant neuroreceptors, CB2 is expressed in the brain only at very low levels. Thus, highly potent and selective compounds for CB2 are desired. N-aryl-((hetero)aromatic)-oxadiazolyl-propionamides represent a promising class of such selective ligands for the human CB2. Here, a library of various derivatives is studied for suitable routes for labelling with 18F. Such 18F-labelled compounds can then be employed as CB2-selective radiotracers for molecular imaging studies employing positron emission tomography (PET). RESULTS: By varying the N-arylamide substructure, we explored the binding pocket of the human CB2 receptor and identified 9-ethyl-9H-carbazole amide as the group with optimal size. Radioligand replacement experiments revealed that the modification of the (hetero)aromatic moiety in 3-position of the 1,2,4-oxadiazoles shows only moderate impact on affinity to CB2 but high impact on selectivity towards CB2 with respect to CB1. Further, we could show by autoradiography studies that the most promising compounds bind selectively on CB2 receptors in mouse spleen tissue. Molecular docking studies based on a novel three-dimensional structural model of the human CB2 receptor in its activated form indicate that the compounds bind with the N-arylamide substructure in the binding pocket. 18F labelling at the (hetero)aromatic moiety at the opposite site of the compounds via radiochemistry was carried out. CONCLUSIONS: The synthesized CB2-selective compounds have high affinity towards CB2 and good selectivity against CB1. The introduction of labelling groups at the (hetero)aromatic moiety shows only moderate impact on CB2 affinity, indicating the introduction of potential labelling groups at this position as a promising approach to develop CB2-selective ligands suitable for molecular imaging with PET. The high affinity for human CB2 and selectivity against human CB1 of the herein presented compounds renders them as suitable candidates for molecular imaging studies.