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PURPOSE: Traditional phase-contrast MRI is affected by displacement artifacts caused by non-synchronized spatial- and velocity-encoding time points. The resulting inaccurate velocity maps can affect the accuracy of derived hemodynamic parameters. This study proposes and characterizes a 3D radial phase-contrast UTE (PC-UTE) sequence to reduce displacement artifacts. Furthermore, it investigates the displacement of a standard Cartesian flow sequence by utilizing a displacement-free synchronized-single-point-imaging MR sequence (SYNC-SPI) that requires clinically prohibitively long acquisition times. METHODS: 3D flow data was acquired at 3T at three different constant flow rates and varying spatial resolutions in a stenotic aorta phantom using the proposed PC-UTE, a Cartesian flow sequence, and a SYNC-SPI sequence as reference. Expected displacement artifacts were calculated from gradient timing waveforms and compared to displacement values measured in the in vitro flow experiments. RESULTS: The PC-UTE sequence reduces displacement and intravoxel dephasing, leading to decreased geometric distortions and signal cancellations in magnitude images, and more spatially accurate velocity quantification compared to the Cartesian flow acquisitions; errors increase with velocity and higher spatial resolution. CONCLUSION: PC-UTE MRI can measure velocity vector fields with greater accuracy than Cartesian acquisitions (although pulsatile fields were not studied) and shorter scan times than SYNC-SPI. As such, this approach is superior to traditional Cartesian 3D and 4D flow MRI when spatial misrepresentations cannot be tolerated, for example, when computational fluid dynamics simulations are compared to or combined with in vitro or in vivo measurements, or regional parameters such as wall shear stress are of interest.
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Estenosis de la Válvula Aórtica , Imagen por Resonancia Magnética , Humanos , Imagen por Resonancia Magnética/métodos , Hemodinámica , Fantasmas de Imagen , Artefactos , Velocidad del Flujo Sanguíneo , Imagenología Tridimensional/métodosRESUMEN
The inhibitory axonless olfactory bulb granule cells form reciprocal dendrodendritic synapses with mitral and tufted cells via large spines, mediating recurrent and lateral inhibition. As a case in point for dendritic transmitter release, rat granule cell dendrites are highly excitable, featuring local Na+ spine spikes and global Ca2+- and Na+-spikes. To investigate the transition from local to global signaling, we performed holographic, simultaneous 2-photon uncaging of glutamate at up to 12 granule cell spines, along with whole-cell recording and dendritic 2-photon Ca2+ imaging in acute juvenile rat brain slices. Coactivation of less than 10 reciprocal spines was sufficient to generate diverse regenerative signals that included regional dendritic Ca2+-spikes and dendritic Na+-spikes (D-spikes). Global Na+-spikes could be triggered in one third of granule cells. Individual spines and dendritic segments sensed the respective signal transitions as increments in Ca2+ entry. Dendritic integration as monitored by the somatic membrane potential was mostly linear until a threshold number of spines was activated, at which often D-spikes along with supralinear summation set in. As to the mechanisms supporting active integration, NMDA receptors (NMDARs) strongly contributed to all aspects of supralinearity, followed by dendritic voltage-gated Na+- and Ca2+-channels, whereas local Na+ spine spikes, as well as morphological variables, barely mattered. Because of the low numbers of coactive spines required to trigger dendritic Ca2+ signals and thus possibly lateral release of GABA onto mitral and tufted cells, we predict that thresholds for granule cell-mediated bulbar lateral inhibition are low. Moreover, D-spikes could provide a plausible substrate for granule cell-mediated gamma oscillations.
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Potenciales de Acción , Señalización del Calcio , Dendritas/metabolismo , Bulbo Olfatorio/metabolismo , Sodio/metabolismo , Animales , Calcio/metabolismo , Femenino , Holografía , Técnicas In Vitro , Masculino , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
PURPOSE: The purpose of this study was to develop and characterize beta-tricalcium phosphate (ß-TCP)/polycaprolactone (PCL) scaffolds, with 2 different ratios (50/50% and 65/35%), using 3-dimensionally (3D) printed dissolvable molds, and to evaluate cellular growth and osteogenic differentiation of both groups seeded with porcine bone marrow stem cells (pBMSCs) under dynamic culture in vitro. MATERIALS AND METHODS: Two different groups of scaffolds were produced: group 1 (n = 40) with a ratio (wt%) of 50/50% and group 2 (n = 40) with 65/35% of ß-TCP/PCL. Physicochemical, morphological, and mechanical characterization of the scaffolds were performed. Scaffolds were seeded with pBMSCs and differentiated osteogenically in dynamic culture. Cell density, distribution, and viability were assessed. Osteogenic differentiation was examined through alkaline phosphatase (ALP) staining, immunofluorescence, and photospectrometry. RESULTS: Osteogenic differentiated constructs showed homogenous and viable cell distribution. Cell density was significantly higher (P < .05) for 65/35% scaffolds at 10 days postseeding, whereas at 6 weeks, cell number equalized for both groups. ALP activity increased over time and was significantly higher (P < .05) for 65/35% scaffolds at 14 days postseeding. CONCLUSIONS: The mechanical properties of the developed 65/35% scaffolds were within the range of natural trabecular bone. Moreover, the 65/35% scaffolds showed biological advantages, such as higher cell growth and higher ALP activity.
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Osteogénesis , Andamios del Tejido , Animales , Huesos , Fosfatos de Calcio/farmacología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Humanos , Poliésteres/química , Porcinos , Andamios del Tejido/químicaRESUMEN
INTRODUCTION: In combined posterior-anterior stabilization of thoracolumbar burst fractures, the expandable vertebral body replacement device (VBRD) is typically placed bisegmentally for anterior column reconstruction (ACR). The aim of this study, however, was to assess feasibility, outcome and potential pitfalls of monosegmental ACR using a VBRD. In addition, clinical and radiological outcome of monosegmental ACR was related to that of bisegmental ACR using the same thoracoscopic technique. METHODS: Thirty-seven consecutive neurologically intact patients with burst fractures of the thoracolumbar junction (T11-L2) treated by combined posterior-anterior stabilization were included. Monosegmental ACR was performed in 18 and bisegmental ACR in 19 patients. Fracture type and extent of vertebral body comminution were determined on preoperative CT scans. Monosegmental and bisegmental kyphosis angles were analyzed preoperatively, postoperatively and at final radiological follow-up. Clinical outcome was assessed after a minimum of 2 years (74 ± 45 months; range 24-154; follow-up rate 89.2%) using VAS Spine Score, RMDQ, ODI and WHOQOL-BREF. RESULTS: Monosegmental ACR resulted in a mean monosegmental and bisegmental surgical correction of - 15.6 ± 7.7° and - 14.7 ± 8.1°, respectively. Postoperative monosegmental and bisegmental loss of correction averaged 2.7 ± 2.7° and 5.2 ± 3.7°, respectively. Two surgical pitfalls of monosegmental ACR were identified: VBRD positioning (1) onto the weak cancellous bone (too far cranially to the inferior endplate of the fractured vertebra) and (2) onto a significantly compromised inferior endplate with at least two (even subtle) fracture lines. Ignoring these pitfalls resulted in VBRD subsidence in five cases. When relating the clinical and radiological outcome of monosegmental ACR to that of bisegmental ACR, no significant differences were found, except for frequency of VBRD subsidence (5 vs. 0, P = 0.02) and bisegmental loss of correction (5.2 ± 3.7° vs. 2.6 ± 2.5°, P = 0.022). After exclusion of cases with VBRD subsidence, the latter did not reach significance anymore (4.9 ± 4.0° vs. 2.6 ± 2.5°, P = 0.084). CONCLUSIONS: This study indicates that monosegmental ACR using a VBRD is feasible in thoracolumbar burst fractures if the inferior endplate is intact (incomplete burst fractures) or features only a single simple split fracture line (burst-split fractures). If the two identified pitfalls are avoided, monosegmental ACR may be a viable alternative to bisegmental ACR in selected thoracolumbar burst fractures to spare a motion segment and to reduce the distance for bony fusion.
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Fijación Interna de Fracturas/instrumentación , Fracturas Conminutas/cirugía , Vértebras Lumbares/lesiones , Fracturas de la Columna Vertebral/cirugía , Vértebras Torácicas/lesiones , Adulto , Hueso Esponjoso/cirugía , Diseño de Equipo , Femenino , Fijación Interna de Fracturas/métodos , Fracturas Conminutas/diagnóstico por imagen , Humanos , Cifosis/diagnóstico por imagen , Cifosis/cirugía , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/cirugía , Masculino , Persona de Mediana Edad , Postura , Radiografía , Fracturas de la Columna Vertebral/diagnóstico por imagen , Fusión Vertebral/instrumentación , Fusión Vertebral/métodos , Vértebras Torácicas/diagnóstico por imagen , Vértebras Torácicas/cirugía , Tomografía Computarizada por Rayos XRESUMEN
The "gut-brain axis" is emerging as an important target in Alzheimer's disease (AD). However, immunological mechanisms underlying this axis remain poorly understood. Using single-cell RNA sequencing of the colon immune compartment in the 5XFAD amyloid-ß (Aß) mouse model, we uncovered AD-associated changes in ribosomal activity, oxidative stress, and BCR/plasma cell activity. Strikingly, levels of colon CXCR4 + antibody secreting cells (ASCs) were significantly reduced. This corresponded with accumulating CXCR4 + B cells and gut-specific IgA + cells in the brain and dura mater, respectively. Consistently, a chemokine ligand for CXCR4, CXCL12, was expressed at higher levels in 5XFAD glial cells and in in silico analyzed human brain studies, supporting altered neuroimmune trafficking. An inulin prebiotic fiber diet attenuated AD markers including Aß plaques and overall frailty. These changes corresponded to an expansion of gut IgA + cells and rescued peripheral T regs levels. Our study points to a key glia-gut axis and potential targets against AD. Study Highlights: AD is associated with altered immune parameters in the gut of 5XFAD mice. 5 XFAD colon has reduced ASCs, including CXCR4 + cells with a migratory gene signature. 5XFAD brain gliosis includes increased CXCL12 expression. CXCR4 + B cells and gut-specific IgA + ASCs accumulate in the 5XFAD brain and/or dura mater. Inulin diet attenuates AD disease parameters while boosting IgA + cell and T reg levels.
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Objective: A specific type of mesenchymal stem/progenitor cells (MSPCs), CD105+ is reported to aid in cartilage regeneration through TGF-ß/Smad2-signalling. The purpose of this study was to identify and characterize CD105+ MSPCs in temporomandibular joint (TMJ) cartilage. Materials and Methods: MSPCs were isolated from mouse TMJ condyle explants and evaluated for their clonogenicity and pluripotential abilities. MSPC were examined for CD105 antigen using immunohistochemistry and flow cytometry. Results: Immunohistochemistry revealed presence of CD105+ MSPCs in the proliferative zone of condyle's cartilage. Only 0.2% of isolated MSPCs exhibited CD105, along with the stem cell surface markers CD44 and Sca-1. In CD105+ MSPCs, intracellular immunostaining revealed significantly higher (p < 0.05) protein levels of collagen type 1, 2, proteoglycan 4. Ability for chondrogenic differentiation was found to be significantly higher (p < 0.05) after 4 weeks compared to CD105- cells, using alcian blue staining. CD105+ cells were found to resemble an early MSPC subgroup with significantly higher gene expression of biglycan, proteoglycan 4, collagen type 2, Gli2, Sox5 (p < 0.001) and Sox9 (p < 0.05). In contrast, significantly lower levels of Runx2 (p < 0.05), Osterix, Trps1, Col10a1 (p < 0.01), Ihh (p < 0.001) related to chondrocyte senescence and commitment to osteogenic lineage, were observed compared to CD105- cells. Conclusion: The study showed the existence of a CD105+ MSPC subgroup within TMJ fibrocartilage that may be activated to aid in fibrocartilage repair.
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In the rodent olfactory bulb the smooth dendrites of the principal glutamatergic mitral cells (MCs) form reciprocal dendrodendritic synapses with large spines on GABAergic granule cells (GC), where unitary release of glutamate can trigger postsynaptic local activation of voltage-gated Na+-channels (Navs), that is a spine spike. Can such single MC input evoke reciprocal release? We find that unitary-like activation via two-photon uncaging of glutamate causes GC spines to release GABA both synchronously and asynchronously onto MC dendrites. This release indeed requires activation of Navs and high-voltage-activated Ca2+-channels (HVACCs), but also of NMDA receptors (NMDAR). Simulations show temporally overlapping HVACC- and NMDAR-mediated Ca2+-currents during the spine spike, and ultrastructural data prove NMDAR presence within the GABAergic presynapse. This cooperative action of presynaptic NMDARs allows to implement synapse-specific, activity-dependent lateral inhibition, and thus could provide an efficient solution to combinatorial percept synthesis in a sensory system with many receptor channels.
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Células Dendríticas/fisiología , Neuronas/fisiología , Bulbo Olfatorio/citología , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/fisiología , Animales , Animales Modificados Genéticamente , Canales de Calcio , Estimulación Eléctrica , Femenino , Regulación de la Expresión Génica , Activación del Canal Iónico , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Canales de Sodio , Ácido gamma-Aminobutírico/genéticaRESUMEN
Patterned two-photon (2P) photolysis via holographic illumination is a powerful method to investigate neuronal function because of its capability to emulate multiple synaptic inputs in three dimensions (3D) simultaneously. However, like any optical system, holographic projectors have a finite space-bandwidth product that restricts the spatial range of patterned illumination or field-of-view (FOV) for a desired resolution. Such trade-off between holographic FOV and resolution restricts the coverage within a limited domain of the neuron's dendritic tree to perform highly resolved patterned 2P photolysis on individual spines. Here, we integrate a holographic projector into a commercial 2P galvanometer-based 2D scanning microscope with an uncaging unit and extend the accessible holographic FOV by using the galvanometer scanning mirrors to reposition the holographic FOV arbitrarily across the imaging FOV. The projector system utilizes the microscope's built-in imaging functions. Stimulation positions can be selected from within an acquired 3D image stack (the volume-of-interest, VOI) and the holographic projector then generates 3D illumination patterns with multiple uncaging foci. The imaging FOV of our system is 800×800 µm2 within which a holographic VOI of 70×70×70 µm3 can be chosen at arbitrary positions and also moved during experiments without moving the sample. We describe the design and alignment protocol as well as the custom software plugin that controls the 3D positioning of stimulation sites. We demonstrate the neurobiological application of the system by simultaneously uncaging glutamate at multiple spines within dendritic domains and consequently observing summation of postsynaptic potentials at the soma, eventually resulting in action potentials. At the same time, it is possible to perform two-photon Ca2+ imaging in 2D in the dendrite and thus to monitor synaptic Ca2+ entry in selected spines and also local regenerative events such as dendritic action potentials.
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Holografía/métodos , Imagenología Tridimensional , Estimulación Luminosa , Fotones , Animales , Ratas Wistar , Programas Informáticos , Sinapsis/fisiologíaRESUMEN
We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNaV1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNaV1.7 and hERG is described. Fast external solution exchange rates of <50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell-derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na+ and Ca2+ are shown.
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Automatización de Laboratorios/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Técnicas de Placa-Clamp/métodos , Animales , Línea Celular , Humanos , Canales Iónicos/análisis , Receptores de Superficie Celular/análisis , Robótica/métodosRESUMEN
INTRODUCTION AND AIMS: Frequent cannabis consumption can lead to severe physical and mental harm. As cannabis is often consumed in leisure time, this study aimed at identifying differences in leisure time activities that predicted the severity of cannabis use. DESIGN AND METHODS: In a prospective population-based survey on cannabis use a cohort of 5025 subjects aged 13-29 years were assessed by telephone interview and followed up 3 years later. Different leisure time activities and the persons (e.g. partner, friend, sibling) the activities were spent with were analysed for the initiation, progression and reduction/cessation of cannabis use over time using multinomial and ordered logistical regression models. RESULTS: The persons the leisure time was spent with at baseline led to a higher probability of initiation and progression of cannabis use over time than the type of leisure time activity. There also was a tendency for tobacco use to increase during the progression phase and to remain high after a reduction/cessation of cannabis use. DISCUSSION AND CONCLUSIONS: The influence of persons the leisure time was spent with was of higher relevance to most phases of cannabis use than the type of leisure time activity.