Restoration of Foxp3+ Regulatory T Cells by HDAC-Dependent Epigenetic Modulation Plays a Pivotal Role in Resolving Pulmonary Arterial Hypertension Pathology.

Rationale: Immune dysregulation is a common feature of pulmonary arterial hypertension (PAH). Histone deacetylase (HDAC)-dependent transcriptional reprogramming epigenetically modulates immune homeostasis and is a novel disease-oriented approach in modern times. Objectives: To identify a novel functional link between HDAC and regulatory T cells (Tregs) in PAH, aiming to establish disease-modified biomarkers and therapeutic targets. Methods: Peripheral blood mononuclear cells were isolated from patients with idiopathic PAH (IPAH) and rodent models of pulmonary hypertension (PH): monocrotaline rats, Sugen5416-hypoxia rats, and Treg-depleted mice. HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) was used to examine the immune modulatory effects in vivo, ex vivo, and in vitro. Measurements and Main Results: Increased HDAC expression was associated with reduced Foxp3+ Tregs and increased PD-1 (programmed cell death-1) signaling in peripheral blood mononuclear cells from patients with IPAH. SAHA differentially modified a cluster of epigenetic-sensitive genes and induced Foxp3+ Treg conversion in IPAH T cells. Rodent models recapitulated these epigenetic aberrations and T-cell dysfunction. SAHA attenuated PH phenotypes and restored FOXP3 transcription and Tregs in PH rats; interestingly, the effects were more profound in female rats. Selective depletion of CD25+ Tregs in Sugen5416-hypoxia mice neutralized the effects of SAHA. Furthermore, SAHA inhibited endothelial cytokine/chemokine release upon stimulation and subsequent immune chemotaxis. Conclusions: Our results indicated HDAC aberration was associated with Foxp3+ Treg deficiency and demonstrated an epigenetic-mediated mechanism underlying immune dysfunction in PAH. Restoration of Foxp3+ Tregs by HDAC inhibitors is a promising approach to resolve pulmonary vascular pathology, highlighting the potential benefit of developing epigenetic therapies for PAH.

From biomechanics to pathology: predicting axonal injury from patterns of strain after traumatic brain injury.

The relationship between biomechanical forces and neuropathology is key to understanding traumatic brain injury. White matter tracts are damaged by high shear forces during impact, resulting in axonal injury, a key determinant of long-term clinical outcomes. However, the relationship between biomechanical forces and patterns of white matter injuries, associated with persistent diffusion MRI abnormalities, is poorly understood. This limits the ability to predict the severity of head injuries and the design of appropriate protection. Our previously developed human finite element model of head injury predicted the location of post-traumatic neurodegeneration. A similar rat model now allows us to experimentally test whether strain patterns calculated by the model predicts in vivo MRI and histology changes. Using a controlled cortical impact, mild and moderate injuries (1 and 2 mm) were performed. Focal and axonal injuries were quantified with volumetric and diffusion 9.4 T MRI at 2 weeks post injury. Detailed analysis of the corpus callosum was conducted using multi-shell diffusion MRI and histopathology. Microglia and astrocyte density, including process parameters, along with white matter structural integrity and neurofilament expression were determined by quantitative immunohistochemistry. Linear mixed effects regression analyses for strain and strain rate with the employed outcome measures were used to ascertain how well immediate biomechanics could explain MRI and histology changes. The spatial pattern of mechanical strain and strain rate in the injured cortex shows good agreement with the probability maps of focal lesions derived from volumetric MRI. Diffusion metrics showed abnormalities in the corpus callosum, indicating white matter changes in the segments subjected to high strain, as predicted by the model. The same segments also exhibited a severity-dependent increase in glia cell density, white matter thinning and reduced neurofilament expression. Linear mixed effects regression analyses showed that mechanical strain and strain rate were significant predictors of in vivo MRI and histology changes. Specifically, strain and strain rate respectively explained 33% and 28% of the reduction in fractional anisotropy, 51% and 29% of the change in neurofilament expression and 51% and 30% of microglia density changes. The work provides evidence that strain and strain rate in the first milliseconds after injury are important factors in determining patterns of glial and axonal injury and serve as experimental validators of our computational model of traumatic brain injury. Our results provide support for the use of this model in understanding the relationship of biomechanics and neuropathology and can guide the development of head protection systems, such as airbags and helmets.

Annexin A1 restores cerebrovascular integrity concomitant with reduced amyloid-ß and tau pathology.

Alzheimer's disease, characterized by brain deposits of amyloid-ß plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood-brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer's disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood-brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-ß levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood-brain barrier function and decreasing amyloid-ß and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood-brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-ß load, due to increased clearance and degradation of amyloid-ß by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood-brain barrier breakdown early in Alzheimer's disease can be restored by hrANXA1 as a potential therapeutic approach.

Type 2 MI induced by a single high dose of isoproterenol in C57BL/6J mice triggers a persistent adaptive immune response against the heart.

Heart failure is the common final pathway of several cardiovascular conditions and a major cause of morbidity and mortality worldwide. Aberrant activation of the adaptive immune system in response to myocardial necrosis has recently been implicated in the development of heart failure. The ß-adrenergic agonist isoproterenol hydrochloride is used for its cardiac effects in a variety of different dosing regimens with high doses causing acute cardiomyocyte necrosis. To assess whether isoproterenol-induced cardiomyocyte necrosis triggers an adaptive immune response against the heart, we treated C57BL/6J mice with a single intraperitoneal injection of isoproterenol. We confirmed tissue damage reminiscent of human type 2 myocardial infarction. This is followed by an adaptive immune response targeting the heart as demonstrated by the activation of T cells, the presence of anti-heart auto-antibodies in the serum as late as 12 weeks after initial challenge and IgG deposition in the myocardium. All of these are hallmark signs of an established autoimmune response. Adoptive transfer of splenocytes from isoproterenol-treated mice induces left ventricular dilation and impairs cardiac function in healthy recipients. In summary, a single administration of a high dose of isoproterenol is a suitable high-throughput model for future studies of the pathological mechanisms of anti-heart autoimmunity and to test potential immunomodulatory therapeutic approaches.

Accelerated point spread function mapping using signal modeling for accurate echo-planar imaging geometric distortion correction.

Echo-planar imaging is a fast and commonly used magnetic resonance imaging technique with applications in diffusion weighted and functional MRI. Fast data acquisition in echo-planar imaging is accomplished by the extended readout, which also introduces sensitivity to off-resonance effects such as amplitude of static (polarizing) field inhomogeneities and eddy-currents. These off-resonance effects produce geometric distortions in the corresponding echo-planar images. To correct for these distortions, an acceleration of point spread function (PSF) acquisition using a special sampling pattern is presented in this work. The proposed technique allows for reliable and fully automated distortion correction of echo-planar images at a field strength of 3 T. Additionally, a new approach to visualize and determine the distortions in a hybrid (x, y, kPSF) three-dimensional space is proposed. The accuracy and robustness of the proposed technique is demonstrated in phantom and in vivo experiments. The accuracy of the presented method here is compared to previous techniques for echo-planar imaging distortion correction such as PLACE.

Drug-based mobilisation of mesenchymal stem/stromal cells improves cardiac function post myocardial infarction.

There is an unmet need for treatments that prevent the progressive cardiac dysfunction following myocardial infarction. Mesenchymal stem/stromal cells (MSCs) are under investigation for cardiac repair; however, culture expansion prior to transplantation is hindering their homing and reparative abilities. Pharmacological mobilisation could be an alternative to MSC transplantation. Here, we report that endogenous MSCs mobilise into the circulation at day 5 post myocardial infarction in male Lewis rats. This mobilisation can be significantly increased by using a combination of the FDA-approved drugs mirabegron (ß3-adrenoceptor agonist) and AMD3100 (CXCR4 antagonist). Blinded cardiac magnetic resonance imaging analysis showed the treated group to have increased left ventricular ejection fraction and decreased end systolic volume at 5 weeks post myocardial infarction. The mobilised group had a significant decrease in plasma IL-6 and TNF-α levels, a decrease in interstitial fibrosis, and an increase in the border zone blood vessel density. Conditioned medium from blood-derived MSCs supported angiogenesis in vitro, as shown by tube formation and wound healing assays. Our data suggest a novel pharmacological strategy that enhances myocardial infarction-induced MSC mobilisation and improves cardiac function after myocardial infarction.

An in vitro comparison of three nucleus pulposus removal techniques for partial intervertebral disc replacement: An ultra-high resolution MRI study.

Background: Nuclectomy, also known as nucleotomy, is a percutaneous surgical procedure performed to remove nucleus material from the center of the disc. Multiple techniques have been considered to perform a nuclectomy, however, the advantages and disadvantages of each are not well understood. Aims: This in vitro biomechanical investigation on human cadaveric specimens aimed to quantitatively compare three nuclectomy techniques performed using an automated shaver, rongeurs, and laser. Material & Methods: Comparisons were made in terms of mass, volume and location of material removal, changes in disc height, and stiffness. Fifteen vertebra-disc-vertebra lumbar specimens were acquired from six donors (40 ± 13 years) and split into three groups. Before and after nucleotomy axial mechanical tests were performed and T2-weighted 9.4T MRIs were acquired for each specimen. Results: When using the automated shaver and rongeurs similar volumes of disc material were removed (2.51 ± 1.10% and 2.76 ± 1.39% of the total disc volume, respectively), while considerably less material was removed using the laser (0.12 ± 0.07%). Nuclectomy using the automated shaver and rongeurs significantly reduced the toe-region stiffness (p = 0.036), while the reduction in the linear region stiffness was significant only for the rongeurs group (p = 0.011). Post-nuclectomy, 60% of the rongeurs group specimens showed changes in the endplate profile while 40% from the laser group showed subchondral marrow changes. Discussion: From the MRIs, homogeneous cavities were seen in the center of the disc when using the automated shaver. When using rongeurs, material was removed non-homogeneously both from the nucleus and annulus regions. Laser ablation formed small and localized cavities suggesting that the technique is not suitable to remove large volumes of material unless it is developed and optimized for this application. Conclusion: The results demonstrate that both rongeurs and automated shavers can be used to remove large volumes of NP material but the reduced risk of collateral damage to surrounding tissues suggests that the automated shaver may be more suitable.

Quantifying internal intervertebral disc strains to assess nucleus replacement device designs: a digital volume correlation and ultra-high-resolution MRI study.

Introduction: Nucleus replacement has been proposed as a treatment to restore biomechanics and relieve pain in degenerate intervertebral discs (IVDs). Multiple nucleus replacement devices (NRDs) have been developed, however, none are currently used routinely in clinic. A better understanding of the interactions between NRDs and surrounding tissues may provide insight into the causes of implant failure and provide target properties for future NRD designs. The aim of this study was to non-invasively quantify 3D strains within the IVD through three stages of nucleus replacement surgery: intact, post-nuclectomy, and post-treatment. Methods: Digital volume correlation (DVC) combined with 9.4T MRI was used to measure strains in seven human cadaveric specimens (42 ± 18 years) when axially compressed to 1 kN. Nucleus material was removed from each specimen creating a cavity that was filled with a hydrogel-based NRD. Results: Nucleus removal led to loss of disc height (12.6 ± 4.4%, p = 0.004) which was restored post-treatment (within 5.3 ± 3.1% of the intact state, p > 0.05). Nuclectomy led to increased circumferential strains in the lateral annulus region compared to the intact state (-4.0 ± 3.4% vs. 1.7 ± 6.0%, p = 0.013), and increased maximum shear strains in the posterior annulus region (14.6 ± 1.7% vs. 19.4 ± 2.6%, p = 0.021). In both cases, the NRD was able to restore these strain values to their intact levels (p ≥ 0.192). Discussion: The ability of the NRD to restore IVD biomechanics and some strain types to intact state levels supports nucleus replacement surgery as a viable treatment option. The DVC-MRI method used in the present study could serve as a useful tool to assess future NRD designs to help improve performance in future clinical trials.

Microcoil-based MR phase imaging and manganese enhanced microscopy of glial tumor neurospheres with direct optical correlation.

Susceptibility differences among tissues were recently used for highlighting complementary contrast in MRI different from the conventional T(1), T(2), or spin density contrasts. This method, based on the signal phase, previously showed improved image contrast of human or rodent neuroarchitecture in vivo, although direct MR phase imaging of cellular architecture was not available until recently. In this study, we present for the first time the ability of microcoil-based phase MRI to resolve the structure of human glioma neurospheres at significantly improved resolutions (10 × 10 µm(2)) with direct optical image correlation. The manganese chloride property to function as a T(1) contrast agent enabled a closer examination of cell physiology with MRI. Specifically the temporal changes of manganese chloride uptake, retention and release time within and from individual clusters were assessed. The optimal manganese chloride concentration for improved MR signal enhancement was determined while keeping the cellular viability unaffected. The presented results demonstrate the possibilities to reveal structural and functional observation of living glioblastoma human-derived cells. This was achieved through the combination of highly sensitive microcoils, high magnetic field, and methods designed to maximize contrast to noise ratio. The presented approach may provide a powerful multimodal tool that merges structural and functional information of submilimeter biological samples.

Microcoil-based MRI: feasibility study and cell culture applications using a conventional animal system.

OBJECT: The aim of this study was to demonstrate the feasibility of MR microimaging on a conventional 9.4 T horizontal animal MRI system using commercial available microcoils in combination with only minor modifications to the system, thereby opening this field to a larger community. MATERIALS AND METHODS: Commercially available RF microcoils designed for high-resolution NMR spectrometers were used in combination with a custom-made probehead. For this purpose, changes within the transmit chain and modifications to the adjustment routines and image acquisition sequences were made, all without requiring expensive hardware. To investigate the extent to which routine operation and high-resolution imaging is possible, the quality of phantom images was analysed. Surface and solenoidal microcoils were characterized with regard to their sensitive volume and signal-to-noise ratio. In addition, the feasibility of using planar microcoils to achieve high-resolution images of living glioma cells labelled with MnCl(2) was investigated. RESULTS: The setup presented in this work allows routine acquisition of high-quality images with high SNR and isotropic resolutions up to 10 µm within an acceptable measurement time. CONCLUSION: This study demonstrates that MR microscopy can be applied at low cost on animal MR imaging systems, which are in widespread use. The successful imaging of living glioma cells indicates that the technique promises to be a useful tool in biomedical research.

Sensitivity of Intervertebral Disc Finite Element Models to Internal Geometric and Non-geometric Parameters.

Finite element models are useful for investigating internal intervertebral disc (IVD) behaviours without using disruptive experimental techniques. Simplified geometries are commonly used to reduce computational time or because internal geometries cannot be acquired from CT scans. This study aimed to (1) investigate the effect of altered geometries both at endplates and the nucleus-anulus boundary on model response, and (2) to investigate model sensitivity to material and geometric inputs, and different modelling approaches (graduated or consistent fibre bundle angles and glued or cohesive inter-lamellar contact). Six models were developed from 9.4 T MRIs of bovine IVDs. Models had two variations of endplate geometry (a simple curved profile from the centre of the disc to the periphery, and precise geometry segmented from MRIs), and three variations of NP-AF boundary (linear, curved, and segmented). Models were subjected to axial compressive loading (to 0.86 mm at a strain rate of 0.1/s) and the effect on stiffness and strain distributions, and the sensitivity to modelling approaches was investigated. The model with the most complex geometry (segmented endplates, curved NP-AF boundary) was 3.1 times stiffer than the model with the simplest geometry (curved endplates, linear NP-AF boundary), although this difference may be exaggerated since segmenting the endplates in the complex geometry models resulted in a shorter average disc height. Peak strains were close to the endplates at locations of high curvature in the segmented endplate models which were not captured in the curved endplate models. Differences were also seen in sensitivity to material properties, graduated fibre angles, cohesive rather than glued inter-lamellar contact, and NP:AF ratios. These results show that FE modellers must take care to ensure geometries are realistic so that load is distributed and passes through IVDs accurately.

Multiscale modelling of cerebrovascular injury reveals the role of vascular anatomy and parenchymal shear stresses.

Neurovascular injury is often observed in traumatic brain injury (TBI). However, the relationship between mechanical forces and vascular injury is still unclear. A key question is whether the complex anatomy of vasculature plays a role in increasing forces in cerebral vessels and producing damage. We developed a high-fidelity multiscale finite element model of the rat brain featuring a detailed definition of the angioarchitecture. Controlled cortical impacts were performed experimentally and in-silico. The model was able to predict the pattern of blood-brain barrier damage. We found strong correlation between the area of fibrinogen extravasation and the brain area where axial strain in vessels exceeds 0.14. Our results showed that adjacent vessels can sustain profoundly different axial stresses depending on their alignment with the principal direction of stress in parenchyma, with a better alignment leading to larger stresses in vessels. We also found a strong correlation between axial stress in vessels and the shearing component of the stress wave in parenchyma. Our multiscale computational approach explains the unrecognised role of the vascular anatomy and shear stresses in producing distinct distribution of large forces in vasculature. This new understanding can contribute to improving TBI diagnosis and prevention.

Ventricular fibrillation mechanism and global fibrillatory organization are determined by gap junction coupling and fibrosis pattern.

AIMS: Conflicting data exist supporting differing mechanisms for sustaining ventricular fibrillation (VF), ranging from disorganized multiple-wavelet activation to organized rotational activities (RAs). Abnormal gap junction (GJ) coupling and fibrosis are important in initiation and maintenance of VF. We investigated whether differing ventricular fibrosis patterns and the degree of GJ coupling affected the underlying VF mechanism. METHODS AND RESULTS: Optical mapping of 65 Langendorff-perfused rat hearts was performed to study VF mechanisms in control hearts with acute GJ modulation, and separately in three differing chronic ventricular fibrosis models; compact fibrosis (CF), diffuse fibrosis (DiF), and patchy fibrosis (PF). VF dynamics were quantified with phase mapping and frequency dominance index (FDI) analysis, a power ratio of the highest amplitude dominant frequency in the cardiac frequency spectrum. Enhanced GJ coupling with rotigaptide (n = 10) progressively organized fibrillation in a concentration-dependent manner; increasing FDI (0 nM: 0.53 ± 0.04, 80 nM: 0.78 ± 0.03, P < 0.001), increasing RA-sustained VF time (0 nM: 44 ± 6%, 80 nM: 94 ± 2%, P < 0.001), and stabilized RAs (maximum rotations for an RA; 0 nM: 5.4 ± 0.5, 80 nM: 48.2 ± 12.3, P < 0.001). GJ uncoupling with carbenoxolone progressively disorganized VF; the FDI decreased (0 µM: 0.60 ± 0.05, 50 µM: 0.17 ± 0.03, P < 0.001) and RA-sustained VF time decreased (0 µM: 61 ± 9%, 50 µM: 3 ± 2%, P < 0.001). In CF, VF activity was disorganized and the RA-sustained VF time was the lowest (CF: 27 ± 7% vs. PF: 75 ± 5%, P < 0.001). Global fibrillatory organization measured by FDI was highest in PF (PF: 0.67 ± 0.05 vs. CF: 0.33 ± 0.03, P < 0.001). PF harboured the longest duration and most spatially stable RAs (patchy: 1411 ± 266 ms vs. compact: 354 ± 38 ms, P < 0.001). DiF (n = 11) exhibited an intermediately organized VF pattern, sustained by a combination of multiple-wavelets and short-lived RAs. CONCLUSION: The degree of GJ coupling and pattern of fibrosis influences the mechanism sustaining VF. There is a continuous spectrum of organization in VF, ranging between globally organized fibrillation sustained by stable RAs and disorganized, possibly multiple-wavelet driven fibrillation with no RAs.

On-chip three dimensional microcoils for MRI at the microscale.

We present for the first time a fully MEMS-integrated technology to manufacture 3D geometrically perfect solenoidal microcoils for microscale MRI applications. We report 25 microm isotropic resolution MR images of a copper sulfate aqueous phantom. These images are acquired using microcoils with 5 windings of insulated 25 microm diameter Au wire and with quality factors as high as 46 at the operating frequency (400 MHz).

Hybrid MPI-MRI System for Dual-Modal In Situ Cardiovascular Assessments of Real-Time 3D Blood Flow Quantification-A Pre-Clinical In Vivo Feasibility Investigation.

Non-invasive quantification of functional parameters of the cardiovascular system, in particular the heart, remains very challenging with current imaging techniques. This aspect is mainly due to the fact, that the spatio-temporal resolution of current imaging methods, such as Magnetic Resonance Imaging (MRI) or Positron Emission Tomography (PET), does not offer the desired data repetition rates in the context of real-time data acquisition and thus, can cause artifacts and misinterpretations in accelerated data acquisition approaches. We present a fast non-invasive and quantitative dual-modal in situ cardiovascular assessment using a hybrid imaging system which combines the new imaging modality Magnetic Particle Imaging (MPI) and MRI. This pre-clinical hybrid imaging system provides either a 0.5 T homogeneous B0 field for MRI or a 2.2 T/m gradient field featuring a Field-Free-Point for MPI. A comprehensive coil system allows in both imaging modes for spatial encoding, signal excitation and reception. In this work, 3-dimensional anatomical information acquired with MRI is combined with in situ sequentially acquired time-resolved 3D (i.e. 3D + t) MPI bolus tracking of superparamagnetic iron oxide nanoparticles. MPI data were acquired during a 21 [Formula: see text] (40 µ mol(Fe)/kgBW) bolus tail vein injection under free-breathing with an ungated and non-triggered MPI scan with a repetition rate of 46 volumes per seconds. We successfully determined quantitative hemodynamics as 3D + t velocity vector estimations of a beating rat's heart by analyzing 3 seconds of 3D + t MPI image data. The used hybrid system allows for MR-based MPI Field-of-View planning and cardiac cross-sectional anatomy analysis, precise co-registration of dual-modal datasets, as well as for MPI-based hemodynamic functional analysis using an optical flow technique. We present the first in-vivo results of a new methodology, allowing for fast, non-invasive, quantitative and in situ hybrid cardiovascular assessment, showing its potential for future clinical applications.

The Effect of Degeneration on Internal Strains and the Mechanism of Failure in Human Intervertebral Discs Analyzed Using Digital Volume Correlation (DVC) and Ultra-High Field MRI.

The intervertebral disc (IVD) plays a main role in absorbing and transmitting loads within the spinal column. Degeneration alters the structural integrity of the IVDs and causes pain, especially in the lumbar region. The objective of this study was to investigate non-invasively the effect of degeneration on human 3D lumbar IVD strains (n = 8) and the mechanism of spinal failure (n = 10) under pure axial compression using digital volume correlation (DVC) and 9.4 Tesla magnetic resonance imaging (MRI). Degenerate IVDs had higher (p < 0.05) axial strains (58% higher), maximum 3D compressive strains (43% higher), and maximum 3D shear strains (41% higher), in comparison to the non-degenerate IVDs, particularly in the lateral and posterior annulus. In both degenerate and non-degenerate IVDs, peak tensile and shear strains were observed close to the endplates. Inward bulging of the inner annulus was observed in all degenerate IVDs causing an increase in the AF compressive, tensile, and shear strains at the site of inward bulge, which may predispose it to circumferential tears (delamination). The endplate is the spine's "weak link" in pure axial compression, and the mechanism of human vertebral fracture is associated with disc degeneration. In non-degenerate IVDs the locations of failure were close to the endplate centroid, whereas in degenerate IVDs they were in peripheral regions. These findings advance the state of knowledge on mechanical changes during degeneration of the IVD, which help reduce the risk of injury, optimize treatments, and improve spinal implant designs. Additionally, these new data can be used to validate computational models.

Neuron labeling with rhodamine-conjugated Gd-based MRI contrast agents delivered to the brain via focused ultrasound.

Gadolinium-based magnetic resonance imaging contrast agents can provide information regarding neuronal function, provided that these agents can cross the neuronal cell membrane. Such contrast agents are normally restricted to extracellular domains, however, by attaching cationic fluorescent dyes, they can be made cell-permeable and allow for both optical and magnetic resonance detection. To reach neurons, these agents also need to cross the blood-brain barrier. Focused ultrasound combined with microbubbles has been shown to enhance the permeability of this barrier, allowing molecules into the brain non-invasively, locally and transiently. The goal of this study was to investigate whether combining fluorescent rhodamine with a gadolinium complex would form a dual-modal contrast agent that could label neurons in vivo when delivered to the mouse brain with focused ultrasound and microbubbles. Methods: Gadolinium complexes were combined with a fluorescent, cationic rhodamine unit to form probes with fluorescence and relaxivity properties suitable for in vivo applications. The left hemisphere of female C57bl/6 mice (8-10 weeks old; 19.07 ± 1.56 g; n = 16) was treated with ultrasound (centre frequency: 1 MHz, peak-negative pressure: 0.35 MPa, pulse length: 10 ms, repetition frequency: 0.5 Hz) while intravenously injecting SonoVue microbubbles and either the 1 kDa Gd(rhodamine-pip-DO3A) complex or a conventionally-used lysine-fixable Texas Red® 3 kDa dextran. The opposite right hemisphere was used as a non-treated control region. Brains were then extracted and either sectioned and imaged via fluorescence or confocal microscopy or imaged using a 9.4 T magnetic resonance imaging scanner. Brain slices were stained for neurons (NeuN), microglia (Iba1) and astrocytes (GFAP) to investigate the cellular localization of the probes. Results: Rhodamine fluorescence was detected in the left hemisphere of all ultrasound treated mice, while none was detected in the right control hemisphere. Cellular uptake of Gd(rhodamine-pip-DO3A) was observed in all the treated regions with a uniform distribution (coefficient of variation = 0.4 ± 0.05). Uptake was confirmed within neurons, whereas the probe did not co-localize with microglia and astrocytes. Compared to the dextran molecule, Gd(rhodamine-pip-DO3A) distributed more homogeneously and was less concentrated around blood vessels. Furthermore, the dextran molecule was found to accumulate unselectively in microglia as well as neurons, whereas our probe was only taken up by neurons. Gd(rhodamine-pip-DO3A) was detected via magnetic resonance imaging ex vivo in similar regions to where fluorescence was detected. Conclusion: We have introduced a method to image neurons with a dual-modal imaging agent delivered non-invasively and locally to the brain using focused ultrasound and microbubbles. When delivered to the mouse brain, the agent distributed homogeneously and was only uptaken by neurons; in contrast, conventionally used dextran distributed heterogeneously and was uptaken by microglia as well as neurons. This result indicates that our probe labels neurons without microglial involvement and in addition the probe was found to be detectable via both ex vivo MRI and fluorescence. Labeling neurons with such dual-modal agents could facilitate the study of neuronal morphology and physiology using the advantages of both imaging modalities.

Synthesis and in vivo behaviour of an exendin-4-based MRI probe capable of ß-cell-dependent contrast enhancement in the pancreas.

Global rates of diabetes mellitus are increasing, and treatment of the disease consumes a growing proportion of healthcare spending across the world. Pancreatic ß-cells, responsible for insulin production, decline in mass in type 1 and, to a more limited degree, in type 2 diabetes. However, the extent and rate of loss in both diseases differs between patients resulting in the need for the development of novel diagnostic tools, which could quantitatively assess changes in mass of ß-cells over time and potentially lead to earlier diagnosis and improved treatments. Exendin-4, a potent analogue of glucagon-like-peptide 1 (GLP-1), binds to the receptor GLP-1R, whose expression is enriched in ß-cells. GLP-1R has thus been used in the past as a means of targeting probes for a wide variety of imaging modalities to the endocrine pancreas. However, exendin-4 conjugates designed specifically for MRI contrast agents are an under-explored area. In the present work, the synthesis and characterization of an exendin-4-dota(ga)-Gd(iii) complex, GdEx, is reported, along with its in vivo behaviour in healthy and in ß-cell-depleted C57BL/6J mice. Compared to the ubiquitous probe, [Gd(dota)]-, GdEx shows selective uptake by the pancreas with a marked decrease in accumulation observed after the loss of ß-cells elicited by deleting the microRNA processing enzyme, DICER. These results open up pathways towards the development of other targeted MRI contrast agents based on similar chemistry methodology.

Tracing Nutrient Flux Following Monocarboxylate Transporter-1 Inhibition with AZD3965.

The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism and inhibition of MCT1 with AZD3965 is undergoing clinical trials. We aimed to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 to identify possible biomarkers of drug action. We synthesized an 18F-labeled lactate analogue, [18F]-S-fluorolactate ([18F]-S-FL), that was used alongside [18F]fluorodeoxyglucose ([18F]FDG), and 13C-labeled glucose and lactate, to investigate the modulation of metabolism with AZD3965 in diffuse large B-cell lymphoma models in NOD/SCID mice. Comparative analysis of glucose and lactate-based probes showed a preference for glycolytic metabolism in vitro, whereas in vivo, both glucose and lactate were used as metabolic fuel. While intratumoral L-[1-13C]lactate and [18F]-S-FL were unchanged or lower at early (5 or 30 min) timepoints, these variables were higher compared to vehicle controls at 4 h following treatment with AZD3965, which indicates that inhibition of MCT1-mediated lactate import is reversed over time. Nonetheless, AZD3965 treatment impaired DLBCL tumor growth in mice. This was hypothesized to be a consequence of metabolic strain, as AZD3965 treatment showed a reduction in glycolytic intermediates and inhibition of the TCA cycle likely due to downregulated PDH activity. Glucose ([18F]FDG and D-[13C6]glucose) and lactate-based probes ([18F]-S-FL and L-[1-13C]lactate) can be successfully used as biomarkers for AZD3965 treatment.