Samuel C. Grant

A three-dimensional digital atlas database of the adult C57BL/6J mouse brain by magnetic resonance microscopy

A comprehensive three-dimensional digital atlas database of the C57BL/6J mouse brain was developed based on magnetic resonance microscopy images acquired on a 17.6-T superconducting magnet. By using both manual tracing and an atlas-based semi-automatic segmentation approach, T2-weighted magnetic resonance microscopy images of 10 adult male formalin-fixed, excised C57BL/6J mouse brains were segmented into 20 anatomical structures. These structures included the neocortex, hippocampus, amygdala, olfactory bulbs, basal forebrain and septum, caudate-putamen, globus pallidus, thalamus, hypothalamus, central gray, superior colliculi, inferior colliculi, the rest of midbrain, cerebellum, brainstem, corpus callosum/external capsule, internal capsule, anterior commissure, fimbria, and ventricles. The segmentation data were formatted and stored into a database containing three different atlas types: 10 single-specimen brain atlases, an average brain atlas and a probabilistic atlas. Additionally, quantitative group information, such as variations in structural volume, surface area, magnetic resonance microscopy image intensity and local geometry, were computed and stored as an integral part of the database. The database augments ongoing efforts with other high priority strains as defined by the Mouse Phenome Database focused on providing a quantitative framework for accurate mapping of functional, genetic and protein expression patterns acquired by a myriad of technologies and imaging modalities.

MR microscopy of multicomponent diffusion in single neurons.

This study examines multicomponent diffusion in isolated single neurons and discusses the implications of the results for macroscopic water diffusion in tissues. L7 Aplysia neurons were isolated and analyzed using a 600 MHz Bruker wide‐bore instrument with a magnetic susceptibility‐matched radiofrequency microcoil. Using a biexponential fit, the apparent diffusion coefficients (ADCs) from the cytoplasm (with relative fraction) were 0.48 ± 0.14 × 10−3 mm2s−1 (61 ± 11%) for the fast component, and 0.034 ± 0.017 × 10−3 mm2s−1 (32 ± 11%) for the slow component (N = 10). Diffusion in the nucleus appears to be primarily monoexponential, but with biexponential analysis it yields 1.31 ± 0.32 × 10−3 mm2s−1 (89 ± 6%) for the fast component and 0.057 ± 0.073 × 10−3 mm2s−1 (11 ± 6%) for the slow (N = 5). The slow component in the nucleus may be explained by cytoplasmic volume averaging. These data demonstrate that water diffusion in the cytoplasm of isolated single Aplysia neurons supports a multiexponential model. The ADCs are consistent with previous measurements in the cytoplasm of single neurons and with the slow ADC measurement in perfused brain slices. These distributions may explain the multiple compartments observed in tissues, greatly aiding the development of quantitative models of MRI in whole tissues. Magn Reson Med 46:1107–1112, 2001.

NMR spectroscopy of single neurons

The first spatially localized NMR spectra of osmolytes and metabolites from single isolated neurons have been obtained using a combination of high magnetic field strengths and NMR radio frequency (RF) microcoils. The proton spectra display peaks at high concentrations (100–300 mM) assigned to betaine and choline, and other metabolite resonances including lactate at lower concentrations in the order of 10s of millimoles. The volumes examined were approximately 10 nl, over two orders of magnitude less than previously possible. In these initial experiments; the cells were unperfused and the signal intensities of the osmolytes decrease with time, a phenomenon consistent with cell swelling. This work demonstrates the technical feasibility of NMR spectroscopy of single cells, further broadening the scope of NMR spectroscopy of living tissues from application to entire living organisms (man and animal models) and isolated tissues (perfused organs and cultured assemblies of cells) and now to single cells. Magn Reson Med 44:19–22, 2000.

Human erythrocyte ghosts: exploring the origins of multiexponential water diffusion in a model biological tissue with magnetic resonance.

A tissue model composed of erythrocyte ghosts was developed to study the effects of compartmentation on the MR signal acquired from biological tissues. This simple and flexible model offers control over the biophysical parameters that contribute to multicomponent signals arising from cellular systems. Cell density, size, intra‐ and extracellular composition, and membrane permeability can be independently altered. The effects of cell density and cell size on water diffusion properties were assessed. The data demonstrate non‐monoexponential water diffusion in ghost cell suspensions of 17–67% cell density. Data were analysed with the widely employed two‐compartment (biexponential) model, and with a two‐compartment model that accounted for exchange between compartments. Water exchange between the intra‐ and extracellular compartments appeared to be significant over the range of diffusion times studied (7–35 ms). The biexponential fit to the ghost data appeared to be underparameterised as the ADCs and relative fractions of the fast and slow components were dependent on the experimental acquisition parameters, specifically the diffusion time. However, both analysis methods proved effective at tracking changes in the ghost model when it was perturbed. This was demonstrated with cell density variation, cell swelling and shrinkage experiments, and reduction of membrane water permeability using a water channel blocker (pCMBS). We anticipate that this model system could be used to investigate compartmental diffusion effects to simulate a range of pathologies, especially ischemic stroke. Magn Reson Med 48:649–657, 2002.

NMR properties of alginate microbeads

Alginates are a family of unbranched polysaccharides with properties that vary widely depending on their composition. In the presence of multivalent cations (frequently Ca2+), alginates form a gel. Consequently, alginates have been used to encapsulate a variety of biological materials, including cells. In this study, we present NMR relaxation and diffusion data from alginate microbeads with similar size and properties to those used in the development of a bioartificial pancreas. Our data demonstrate that the transverse relaxation time (T2) of water within the gel depends on the guluronic acid content of the alginate, whereas the longitudinal relaxation time (T1) and the apparent diffusion coefficient of water do not. Our data further suggest that the diffusion of Ca2+ ions is hindered by the presence of a poly-L-lysine layer, a layer commonly added to provide mechanical support to the beads and immunoprotection to the encapsulated cells in the event of implantation. The impact of these data on our understanding of the role of alginate gels in the development of a bioartificial pancreas is discussed.

Noise analysis in magnetic resonance electrical impedance tomography at 3 and 11 T field strengths

In magnetic resonance electrical impedance tomography (MREIT), we measure the induced magnetic flux density inside an object subject to an externally injected current. This magnetic flux density is contaminated with noise, which ultimately limits the quality of reconstructed conductivity and current density images. By analysing and experimentally verifying the amount of noise in images gathered from two MREIT systems, we found that a carefully designed MREIT study will be able to reduce noise levels below 0.25 and 0.05 nT at main magnetic field strengths of 3 and 11 T, respectively, at a voxel size of 3 x 3 x 3 mm(3). Further noise level reductions can be achieved by optimizing MREIT pulse sequences and using signal averaging. We suggest two different methods to estimate magnetic flux noise levels, and the results are compared to validate the experimental setup of an MREIT system.

Beta-hydroxy-beta-methyl-butyrate blunts negative age-related changes in body composition, functionality and myofiber dimensions in rats

PurposeTo determine the effects of 16 wk. of beta-hydroxy-beta-methylbutyrate (HMB) administration on age-related changes in functionality and diffusion tensor imaging (DTI) determined myofiber dimensions.MethodsTwelve young (44 wk.), 6 middle-aged (60 wk.), 10 old (86 wk.), and 5 very old (102 wk.) male Fisher-344 rats body composition and grip strength were assessed at baseline. Following, 6 young, 6 middle-aged, 5 old and 5 very old rats were sacrificed for baseline myofiber dimensions and gene transcript factor expression in the soleus (SOL) and gastrocnemius (GAS). The remaining 6 young and 5 old rats were given HMB for 16 wk. and then sacrificed.ResultsFat mass increased in the middle-aged control condition (+49%) but not the middle-aged HMB condition. In addition, fat mass declined (-56%) in the old HMB condition but not the old control condition. Normalized strength declined and maintained respectively in the control and HMB conditions from 44 to 60 wk. and increased (+23%) (p < 0.05) from 86 to 102 wk. in only the HMB condition. Declines occurred in myofiber size in all muscles from 44 to 102 wk. in the control condition(-10 to -15%), but not HMB condition. Atrogin-1 mRNA expression in the SOL and GAS muscles was greater in the 102-wk control condition than all other conditions: SOL (+45%) and GAS (+100%). This elevation was blunted by HMB in the 102 wk. old SOL. There was a condition effect in the SOL for myogenin, which significantly increased (+40%) only in the 102-wk. HMB group relative to the 44-wk. group.ConclusionsHMB may blunt age-related losses of strength and myofiber dimensions, possibly through attenuating the rise in protein breakdown.

Initial in vivo rodent sodium and proton MR imaging at 21.1 T

The first in vivo sodium and proton magnetic resonance (MR) images and localized spectra of rodents were attained using the wide bore (105 mm) high resolution 21.1-T magnet, built and operated at the National High Magnetic Field Laboratory (Tallahassee, FL, USA). Head images of normal mice (C57BL/6J) and Fisher rats (approximately 250 g) were acquired with custom designed radiofrequency probes at frequencies of 237/900 MHz for sodium and proton, respectively. Sodium MR imaging resolutions of approximately 0.125 microl for mouse and rat heads were achieved by using a 3D back-projection pulse sequence. A gain in SNR of approximately 3 for sodium and approximately 2 times for proton were found relative to corresponding MR images acquired at 9.4 T. 3D Fast Low Angle Shot (FLASH) proton mouse images (50x50x50 microm(3)) were acquired in 90 min and corresponding rat images (100x100x100 microm(3)) within a total time of 120 min. Both in vivo large rodent MR imaging and localized spectroscopy at the extremely high field of 21.1 T are feasible and demonstrate improved resolution and sensitivity valuable for structural and functional brain analysis.

Quantitative measurement of neurodegeneration in an ALS–PDC model using MR microscopy

Exposure to cycad (Cycas micronesica K.D. Hill) toxins via diet has been shown to induce neurodegeneration in vivo that mimics the progressive neurological disease, amyotrophic lateral sclerosis--parkinsonism dementia complex (ALS--PDC). In previous studies, specific cortical and subcortical cell loss was measured with conventional stained sections. In the present study, magnetic resonance (MR) microscopy was used to examine neurodegeneration in three dimensions (3D) in isolated intact brains and spinal cords. Mice were fed washed cycad for 2 months and showed progressive motor deficits resembling human ALS--PDC. CNS tissue was imaged at 17.6 T. T2* scans were acquired on both spinal cord and brain samples with an isotropic resolution of 41 microm. Through MR volumetrics, cycad-fed mice showed significantly decreased volumes in lumbar spinal cord gray matter, substantia nigra, striatum, basal nucleus/internal capsule, and olfactory bulb. Cortical measurements of conventionally stained sections revealed that cycad-fed mice also showed decreased cortical thickness. These results show that MR microscopy (MRM) is sensitive enough to measure degeneration in this early stage model of a progressive neurological disease with strong correlations to behavioral deficits and histological results and may be applicable in vivo to the same model. Similar analysis may be used in the future as a diagnostic aid in tracking the early progression of neurological disorders in preclinical human subjects.

Anatomical and functional phenotyping of mice models of Alzheimer's disease by MR microscopy

Abstract:  The wide variety of transgenic mouse models of Alzheimers disease (AD) reflects the search for specific genes that influence AD pathology and the drive to create a clinically relevant animal model. An ideal AD mouse model must display hallmark AD pathology such as amyloid plaques, neurofibrillary tangles, reactive gliosis, dystrophic neurites, neuron and synapse loss, and brain atrophy and in parallel behaviorally mimic the cognitive decline observed in humans. Magnetic resonance (MR) microscopy (MRM) can detect amyloid plaque load, development of brain atrophy, and acute neurodegeneration . MRM examples of AD pathology will be presented and discussed. What has lagged behind in preclinical research using transgenic AD mouse models is functional phenotyping of the brain; in other words, the ability to correlate a specific genotype with potential aberrant brain activation patterns. This lack of information is caused by the technical challenges involved in performing functional MRI (fMRI) in mice including the effects of anesthetic agents and the lack of relevant “cognitive” paradigms. An alternative approach to classical fMRI using external stimuli as triggers of brain activation in rodents is to electrically or pharmacologically stimulate regions directly while simultaneously locally tracking the activated interconnected regions of rodents using, for example, the manganese‐enhanced MRI (MEMRI) technique. Finally, transgenic mouse models, MRM, and future AD research would be strengthened by the ability to screen for AD‐like pathology in other non‐AD transgenic mouse models. For example, molecular biologists may focus on cardiac or pulmonary pathologies in transgenic mice models and as an incidental finding discover behavioral AD phenotypes. We will present MRM data of brain and cardiac phenotyping in transgenic mouse models with behavioral deficits.