Management of Cardiovascular Disease in Kidney Disease Study: Rationale and Design.

INTRODUCTION: Atherosclerosis, inflammation, and vascular stiffness are prominent interrelated risk factors contributing to the high incidence of cardiovascular disease (CVD) in patients with CKD. Conventional CVD management strategies in CKD largely target atherosclerotic CVD and have had a limited impact on the cardiovascular mortality in this population. Multiple in vivo and in vitro studies and epidemiological evidence from the rheumatologic cohorts have shown that low-dose hydroxychloroquine has beneficial effects on inflammation, endothelial function, insulin sensitivity, and metabolic syndrome. Our recent proof-of-concept animal study showed that hydroxychloroquine has marked protection against atherosclerosis and vascular stiffness. We hypothesize that hydroxychloroquine has the potential to provide significant cardiovascular benefits in patients with CKD. METHODS: The Management of Cardiovascular disease in Kidney disease study (NCT03636152) is a phase 2B, randomized, double-blind, placebo-controlled trial evaluating the effects of low-dose hydroxychloroquine therapy on the parameters of atherosclerosis, inflammation, and vascular stiffness in patients with CKD. The study plans to enroll 100 CKD patients estimated to be at high cardiovascular risk by a combination of low estimated glomerular filtration rate and albuminuria and treat them for 18 months with hydroxychloroquine or placebo in 1:1 allocation. RESULTS: The study will assess the change in the total carotid plaque volume as measured by serial noncontrast carotid MRI as the primary outcome and the serial changes in plasma inflammation markers, vascular stiffness, renal function, and the composition characteristics of the carotid plaque as secondary outcome measures. DISCUSSION/CONCLUSION: The results of this trial will provide the proof-of-applicability for hydroxychloroquine in the CVD in CKD. If positive, this trial should lead to phase-3 trials with clinical end points for this potentially transformative, novel, and inexpensive therapy for CVD in CKD.

On the Origins of Diffusion MRI Signal Changes in Stroke.

Magnetic resonance imaging (MRI) is a leading diagnostic technique especially for neurological studies. However, the physical origin of the hyperintense signal seen in MR images of stroke immediately after ischemic onset in the brain has been a matter of debate since it was first demonstrated in 1990. In this article, we hypothesize and provide evidence that changes in the glial cells, comprising roughly one-half of the brain's cells and therefore a significant share of its volume, accompanying ischemia, are the root cause of the MRI signal change. Indeed, a primary function of the glial cells is osmoregulation in order to maintain homeostasis in the neurons and nerve fibers for accurate and consistent function. This realization also impacts our understanding of signal changes in other tissues following ischemia. We anticipate that this paradigm shift will facilitate new and improved models of MRI signals in tissues, which will, in turn, impact clinical utility.

Visualization of live, mammalian neurons during Kainate-infusion using magnetic resonance microscopy.

Since its first description and development in the late 20th century, diffusion magnetic resonance imaging (dMRI) has proven useful in describing the microstructural details of biological tissues. Signal generated from the protons of water molecules undergoing Brownian motion produces contrast based on the varied diffusivity of tissue types. Images employing diffusion contrast were first used to describe the diffusion characteristics of tissues, later used to describe the fiber orientations of white matter through tractography, and most recently proposed as a functional contrast method capable of delineating neuronal firing in the active brain. Thanks to the molecular origins of its signal source, diffusion contrast is inherently useful at describing features of the microenvironment; however, limitations in achievable resolution in magnetic resonance imaging (MRI) scans precluded direct visualization of tissue microstructure for decades following MRI's inception as an imaging modality. Even after advancements in MRI hardware had permitted the visualization of mammalian cells, these specialized systems could only accommodate fixed specimens that prohibited the observation and characterization of physiological processes. The goal of the current study was to visualize cellular structure and investigate the subcellular origins of the functional diffusion contrast mechanism (DfMRI) in living, mammalian tissue explants. Using a combination of ultra-high field spectrometers, micro radio frequency (RF) coils, and an MRI-compatible superfusion device, we are able to report the first live, mammalian cells-α-motor neurons-visualized with magnetic resonance microscopy (MRM). We are also able to report changes in the apparent diffusion of the stratum oriens within the hippocampus-a layer comprised primarily of pyramidal cell axons and basal dendrites-and the spinal cord's ventral horn following exposure to kainate.

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition.

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes. While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue's specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

Simulated blast overpressure induces specific astrocyte injury in an ex vivo brain slice model.

Exposure to explosive blasts can produce functional debilitation in the absence of brain pathology detectable at the scale of current diagnostic imaging. Transient (ms) overpressure components of the primary blast wave are considered to be potentially damaging to the brain. Astrocytes participate in neuronal metabolic maintenance, blood-brain barrier, regulation of homeostatic environment, and tissue remodeling. Damage to astrocytes via direct physical forces has the potential to disrupt local and global functioning of neuronal tissue. Using an ex vivo brain slice model, we tested the hypothesis that viable astrocytes within the slice could be injured simply by transit of a single blast wave consisting of overpressure alone. A polymer split Hopkinson pressure bar (PSHPB) system was adapted to impart a single positive pressure transient with a comparable magnitude to those that might be present inside the head. A custom built test chamber housing the brain tissue slice incorporated revised design elements to reduce fluid space and promote transit of a uniform planar waveform. Confocal microscopy, stereology, and morphometry of glial fibrillary acidic protein (GFAP) immunoreactivity revealed that two distinct astrocyte injury profiles were identified across a 4 hr post-test survival interval: (a) presumed conventional astrogliosis characterized by enhanced GFAP immunofluorescence intensity without significant change in tissue area fraction and (b) a process comparable to clasmatodendrosis, an autophagic degradation of distal processes that has not been previously associated with blast induced neurotrauma. Analysis of astrocyte branching revealed early, sustained, and progressive differences distinct from the effects of slice incubation absent overpressure testing. Astrocyte vulnerability to overpressure transients indicates a potential for significant involvement in brain blast pathology and emergent dysfunction. The testing platform can isolate overpressure injury phenomena to provide novel insight on physical and biological mechanisms.

Magnetic Resonance Microscopy (MRM) of Single Mammalian Myofibers and Myonuclei.

Recently, the first magnetic resonance microscopy (MRM) images at the cellular level in isolated mammalian brain tissues were obtained using microsurface coils. These methods can elucidate the cellular origins of MR signals and describe how these signals change over the course of disease progression and therapy. In this work, we explore the capability of these microimaging techniques to visualize mouse muscle fibers and their nuclei. Isolated myofibers expressing lacZ were imaged with and without a stain for ß-galactosidase activity (S-Gal + ferric ammonium citrate) that produces both optical and MR contrast. We found that MRM can be used to image single myofibers with 6-µm resolution. The ability to image single myofibers will serve as a valuable tool to study MR properties attributed to healthy and myopathic cells. The ability to image nuclei tagged with MR/Optical gene markers may also find wide use in cell lineage MRI studies.

Diffusion tensor microscopy data (15.6 µm in-plane) of white matter tracts in the human, pig, and rat spinal cord with corresponding tissue histology.

The following article contains nine diffusion tensor imaging (DTI) datasets acquired with magnetic resonance microscopy (MRM, 15.6 µm in-plane). All data was collected in the region bordering the ventral horn and white matter of cross sections from the spinal cord enlargements along with each sample׳s corresponding tissue histology. These data are collected in fixed spinal cord sections of varying thicknesses taken from rat (2×21 direction DTI datasets), pig (1×21 direction DTI dataset), and human (5×21 direction DTI datasets + 1×6 direction DTI dataset) tissue sources. Following MRM acquisition, the sections were histologically processed using Nissl or Black-Gold II (Histo-Chem Inc., 1BGII) myelin stain and imaged again using light microscopy techniques. Methodological procedures are an amalgamation of protocol components described previously (doi:10.1016/j.neuroimage.2010.04.031 [1], doi:10.1016/j.neuroimage.2011.04.052 [2]).

A Microperfusion and In-Bore Oxygenator System Designed for Magnetic Resonance Microscopy Studies on Living Tissue Explants.

Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges--the most evident of which are spatial limitations--to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and--being designed almost exclusively for light microscopy or electrophysiology studies--seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker's series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig's continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development.

Magnetic resonance microscopy of renal and biliary abnormalities in excised tissues from a mouse model of autosomal recessive polycystic kidney disease.

Polycystic kidney disease (PKD) is transmitted as either an autosomal dominant or recessive trait and is a major cause of renal failure and liver fibrosis. The cpk mouse model of autosomal recessive PKD (ARPKD) has been extensively characterized using standard histopathological techniques after euthanasia. In the current study, we sought to validate magnetic resonance microscopy (MRM) as a robust tool for assessing the ARPKD phenotype. We used MRM to evaluate the liver and kidney of wild-type and cpk animals at resolutions <100 µm and generated three-dimensional (3D) renderings for pathological evaluation. Our study demonstrates that MRM is an excellent method for evaluating the complex, 3D structural defects in this ARPKD mouse model. We found that MRM was equivalent to water displacement in assessing kidney volume. Additionally, using MRM we demonstrated for the first time that the cpk liver exhibits less extensive ductal arborization, that it was reduced in volume, and that the ductal volume was disproportionately smaller. Histopathology indicates that this is a consequence of bile duct malformation. With its reduced processing time, volumetric information, and 3D capabilities, MRM will be a useful tool for future in vivo and longitudinal studies of disease progression in ARPKD. In addition, MRM will provide a unique tool to determine whether the human disease shares the newly appreciated features of the murine biliary phenotype.

Investigation of the subcellular architecture of L7 neurons of Aplysia californica using magnetic resonance microscopy (MRM) at 7.8 microns.

Magnetic resonance microscopy (MRM) is a non-invasive diagnostic tool which is well-suited to directly resolve cellular structures in ex vivo and in vitro tissues without use of exogenous contrast agents. Recent advances in its capability to visualize mammalian cellular structure in intact tissues have reinvigorated analytical interest in aquatic cell models whose previous findings warrant up-to-date validation of subcellular components. Even if the sensitivity of MRM is less than other microscopic technologies, its strength lies in that it relies on the same image contrast mechanisms as clinical MRI which make it a unique tool for improving our ability to interpret human diagnostic imaging through high resolution studies of well-controlled biological model systems. Here, we investigate the subcellular MR signal characteristics of isolated cells of Aplysia californica at an in-plane resolution of 7.8 µm. In addition, direct correlation and positive identification of subcellular architecture in the cells is achieved through well-established histology. We hope this methodology will serve as the groundwork for studying pathophysiological changes through perturbation studies and allow for development of disease-specific cellular modeling tools. Such an approach promises to reveal the MR contrast changes underlying cellular mechanisms in various human diseases, for example in ischemic stroke.

Magnetic resonance microscopy of human and porcine neurons and cellular processes.

With its unparalleled ability to safely generate high-contrast images of soft tissues, magnetic resonance imaging (MRI) has remained at the forefront of diagnostic clinical medicine. Unfortunately due to resolution limitations, clinical scans are most useful for detecting macroscopic structural changes associated with a small number of pathologies. Moreover, due to a longstanding inability to directly observe magnetic resonance (MR) signal behavior at the cellular level, such information is poorly characterized and generally must be inferred. With the advent of the MR microscope in 1986 came the ability to measure MR signal properties of theretofore unobservable tissue structures. Recently, further improvements in hardware technology have made possible the ability to visualize mammalian cellular structure. In the current study, we expand upon previous work by imaging the neuronal cell bodies and processes of human and porcine α-motor neurons. Complimentary imaging studies are conducted in pig tissue in order to demonstrate qualitative similarities to human samples. Also, apparent diffusion coefficient (ADC) maps were generated inside porcine α-motor neuron cell bodies and portions of their largest processes (mean=1.7 ± 0.5 µm²/ms based on 53 pixels) as well as in areas containing a mixture of extracellular space, microvasculature, and neuropil (0.59 ± 0.37 µm²/ms based on 33 pixels). Three-dimensional reconstruction of MR images containing α-motor neurons shows the spatial arrangement of neuronal projections between adjacent cells. Such advancements in imaging portend the ability to construct accurate models of MR signal behavior based on direct observation and measurement of the components which comprise functional tissues. These tools would not only be useful for improving our interpretation of macroscopic MRI performed in the clinic, but they could potentially be used to develop new methods of differential diagnosis to aid in the early detection of a multitude of neuropathologies.

Diffusion tensor microscopy in human nervous tissue with quantitative correlation based on direct histological comparison.

Thanks to its proven utility in both clinical and research applications, diffusion tensor tractography (DTT) is regularly employed as a means of delineating white-matter tracts. While successful efforts have been made to validate tractographic predictions, comparative methods which would permit the validation of such predictions at microscopic resolutions in complex biological tissues have remained elusive. In a previous study, we attempted to validate for the first time such predictions at microscopic resolutions in rat and pig spinal cords using a semi-quantitative analysis method. In the current study, we report improved quantitative analysis methods that can be used to determine the accuracy of DTT through comparative histology and apply these techniques for the first time to human tissue (spinal cord) samples. Histological images are down-sampled to resolutions equivalent to our magnetic resonance microscopy (MRM) and converted to binary maps using an automated thresholding tool. These maps (n=3) are co-registered to the MRM allowing us to quantify the agreement based on the number of pixels which contain tracts common to both imaging datasets. In our experiments, we find that-on average-89% of imaging pixels predicted by DTT to contain in-plane white-matter tract structure correspond to physical tracts identified by histology. In addition, angular analysis comparing the orientation of fiber tracts measured in histology to their corresponding in-plane primary eigenvector components is presented. Thus, as well as demonstrating feasibility in human tissue, we report a robust agreement between imaging datasets taken at microscopic resolution and confirm the primary eigenvector's role as a fundamental parameter with clear physical correlates in the microscopic regime.

Cellular-level diffusion tensor microscopy and fiber tracking in mammalian nervous tissue with direct histological correlation.

Magnetic resonance imaging techniques have literally revolutionized neuroimaging with an unprecedented ability to explore tissue structure and function. Over the last three decades, the sensitivity and array of imaging techniques available have improved providing ever finer structural information and more sensitive functional techniques. Among these methods, diffusion imaging techniques have facilitated the generation of fiber-tract maps of the brain enabling an examination of issues related to brain structure and neural connectivity. Despite the potential utility of the techniques described, validation has not yet been achieved on biological samples. Recently, using newly developed surface microcoils on small samples at high magnetic fields, we demonstrated the ability of MR microscopy to image individual neurons in mammalian brain tissue. In the present work, we combine MR microscopy with the highest resolution (15microm) fiber tracking yet reported and demonstrate the accuracy of the fiber tract maps with direct histological validation. Thus it becomes possible to delineate fiber structure in tissues at the cellular level. A semi-quantitative approach was used to estimate the cell overlap fraction (cOF) and fiber tract overlap fraction (tOF), with cOFs of 94%, 92% and 100%, and tOFs of 84%, 86% and 100%, in rat cervical, rat lumbar, and pig spinal cord tissue, respectively. These methods provide a way to directly validate fiber tracking techniques with histology so that contemporary tracking techniques may be compared and refined using the microstructural details of a biological template as a ground truth.

Magnetic resonance microscopy of mammalian neurons.

Magnetic resonance imaging (MRI) is now a leading diagnostic technique. As technology has improved, so has the spatial resolution achievable. In 1986 MR microscopy (MRM) was demonstrated with resolutions in the tens of micrometers, and is now an established subset of MRI with broad utility in biological and non-biological applications. To date, only large cells from plants or aquatic animals have been imaged with MRM limiting its applicability. Using newly developed microsurface coils and an improved slice preparation technique for correlative histology, we report here for the first time direct visualization of single neurons in the mammalian central nervous system (CNS) using native MR signal at a resolution of 4-8 microm. Thus MRM has matured into a viable complementary cellular imaging technique in mammalian tissues.

Diffusion weighted magnetic resonance imaging of neuronal activity in the hippocampal slice model.

Functional magnetic resonance imaging (fMRI) has become the leading modality for studying the working brain. Being based on measuring the haemodynamic changes after enhanced mass neuronal activity the spatiotemporal resolution of the method is somewhat limited. Alternative MR-based methods for detection of brain activity have been proposed and investigated and studies have reported functional imaging based on diffusion weighted (DW) MRI. The basis for such DW fMRI is believed to be the sensitivity of diffusion weighted MRI to changes in tissue micro-structure. However, it remains unclear whether signal changes observed with these methods reflect cell swelling related to neural activation, residual vascular effects, or a combination of both. Here we present evidence of a detectable, activity-related change in the diffusion weighted MR-signal from the cellular level in live hippocampal slices in the absence of vasculature. Slices are exposed to substances which evoke or inhibit neural activity and the effects are evaluated and compared. The results are also compared to earlier DW fMRI studies in humans.

Postmortem interval alters the water relaxation and diffusion properties of rat nervous tissue--implications for MRI studies of human autopsy samples.

High-resolution imaging of human autopsy tissues may improve our understanding of in vivo MRI findings, but interpretation is complicated because samples are obtained by immersion fixation following a postmortem interval (PMI). This study tested the hypotheses that immersion fixation and PMI's from 0-24 h would alter the water relaxation and diffusion properties in rat cortical slice and spinal cord models of human nervous tissue. Diffusion data collected from rat cortical slices at multiple diffusion times (10-60 ms) and b-values (7-15,000 s/mm(2)) were analyzed using a two-compartment model with exchange. Rat spinal cords were characterized with standard diffusion tensor imaging (21 directions, b=1250 s/mm(2)). Switching from perfusion- to immersion-fixation at 0 h PMI altered most MRI properties of rat cortical slices and spinal cords, including a 22% decrease in fractional anisotropy (P<0.001). After 4 h PMI, cortical slice T(1) and T(2) increased 22% and 65% respectively (P<0.001), transmembrane water exchange decreased 23% (P<0.001) and intracellular proton fraction increased 25% (P=0.002). After 6 h PMI, spinal cord white matter fractional anisotropy had decreased 38% (P<0.001). MRI property changes were observed for PMIs up to 24 h. The MRI changes correlated with protease activity and histopathological signs of autolysis. Thus, immersion fixation and/or even short PMIs (4-6 h) altered the MRI properties of rat nervous tissue. This suggests comparisons between in vivo clinical MRI and MRI data from human autopsy tissues should be interpreted with caution.

Non-invasive evaluation of alginate/poly-l-lysine/alginate microcapsules by magnetic resonance microscopy.

In this report, we present data to demonstrate the utility of (1)H MR microscopy to non-invasively examine alginate/poly-l-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly-l-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6+/-6.2 microm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 min). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T(2) relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T(2) over the duration of the experiment, while ADC was unaffected. This decrease in T(2) is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T(2) was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T(2) or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions.

Accumulation of calpain and caspase-3 proteolytic fragments of brain-derived alphaII-spectrin in cerebral spinal fluid after middle cerebral artery occlusion in rats.

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.