Alan P. Koretsky

High-field MRI of brain cortical substructure based on signal phase

The ability to detect brain anatomy and pathophysiology with MRI is limited by the contrast-to-noise ratio (CNR), which depends on the contrast mechanism used and the spatial resolution. In this work, we show that in MRI of the human brain, large improvements in contrast to noise in high-resolution images are possible by exploiting the MRI signal phase at high magnetic field strength. Using gradient-echo MRI at 7.0 tesla and a multichannel detector, a nominal voxel size of 0.24 × 0.24 × 1.0 mm3 (58 nl) was achieved. At this resolution, a strong phase contrast was observed both between as well as within gray matter (GM) and white matter (WM). In gradient-echo phase images obtained on normal volunteers at this high resolution, the CNR between GM and WM ranged from 3:1 to 20:1 over the cortex. This CNR is an almost 10-fold improvement over conventional MRI techniques that do not use image phase, and it is an ≈100-fold improvement when including the gains in resolution from high-field and multichannel detection. Within WM, phase contrast appeared to be associated with the major fiber bundles, whereas contrast within GM was suggestive of the underlying layer structure. The observed phase contrast is attributed to local variations in magnetic susceptibility, which, at least in part, appeared to originate from iron stores. The ability to detect cortical substructure from MRI phase contrast at high field is expected to greatly enhance the study of human brain anatomy in vivo.

In vivo detection of single cells by MRI

The use of high‐relaxivity, intracellular contrast agents has enabled MRI monitoring of cell migration through and homing to various tissues, such as brain, spinal cord, heart, and muscle. Here it is shown that MRI can detect single cells in vivo, homing to tissue, following cell labeling and transplantation. Primary mouse hepatocytes were double‐labeled with green fluorescent 1.63‐μm iron oxide particles and red fluorescent endosomal labeling dye, and injected into the spleens of recipient mice. This is a common hepatocyte transplantation paradigm in rodents whereby hepatocytes migrate from the spleen to the liver as single cells. One month later the animals underwent in vivo MRI and punctuated, dark contrast regions were detected scattered through the livers. MRI of perfused, fixed samples and labeled hepatocyte phantoms in combination with histological evaluation confirmed the presence of dispersed single hepatocytes grafted into the livers. Appropriate controls were used to determine whether the observed contrast could have been due to dead cells or free particles, and the results confirmed that the contrast was due to disperse, single cells. Detecting single cells in vivo opens the door to a number of experiments, such as monitoring rare cellular events, assessing the kinetics of stem cell homing, and achieving early detection of metastases. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

Detection of Single Mammalian Cells by High-Resolution Magnetic Resonance Imaging

This study reports the detection of single mammalian cells, specifically T cells (T lymphocytes) labeled with dextran-coated superparamagnetic iron oxide particles, using magnetic resonance microscopy. Size amplification due to sequestration of the superparamagnetic particles in vacuoles enhances contrast in localized areas in high-resolution magnetic resonance imaging. Magnetic resonance images of samples containing differing concentrations of T cells embedded in 3% gelatin show a number of dark regions due to the superparamagnetic iron oxide particles, consistent with the number predicted by transmission electron microscopy. Colabeling of T cell samples with a fluorescent dye leads to strong correlations between magnetic resonance and fluorescence microscopic images, showing the presence of the superparamagnetic iron oxide particles at the cell site. This result lays the foundation for our approach to tracking the movement of a specific cell type in live animals and humans.

Sizing it up: Cellular MRI using micron‐sized iron oxide particles

There is rapidly increasing interest in the use of MRI to track cell migration in intact animals. Currently, cell labeling is usually accomplished by endocytosis of nanometer‐sized, dextran‐coated iron oxide particles. The limitations of using nanometer‐sized particles, however, are that millions of particles are required to achieve sufficient contrast, the label can be diluted beyond observability by cell division, and the label is biodegradable. These problems make it difficult to label cells other than macrophages in vivo, and to conduct long‐term engraftment studies. It was recently demonstrated that micron‐sized iron oxide particles (MPIOs) can be taken up by a number of cell types. In this study we examined the MRI properties of single MPIOs with sizes of 0.96, 1.63, 2.79, 4.50, and 5.80 μm. Furthermore, the capacity of cells to endocytose these MPIOs was investigated, and the MRI properties of the labeled cells at 7.0 and 11.7 Tesla were measured as a function of image resolution and echo time (TE). Cells labeled with MPIOs generally contained iron levels of ∼100 pg, which is approximately threefold higher than those obtained with the best strategies to label cells using nanometer‐sized particles. On occasion, some cells had levels as high as ∼400 pg. We demonstrate that these large particles and the cells labeled with them can be detected by spin echo (SE)‐based imaging methods. These measurements indicate that MPIOs should be useful for improving cell tracking by MRI. Magn Reson Med 53:329–338, 2005. Published 2005 Wiley‐Liss, Inc.

The role of creatine kinase in inhibition of mitochondrial permeability transition.

Cyclosporin A sensitive swelling of mitochondria isolated from control mouse livers and from the livers of transgenic mice expressing human ubiquitous mitochondrial creatine kinase occurred in the presence of both 40 μM calcium and 5 μM atractyloside which was accompanied by a 2.5‐fold increase over state 4 respiration rates. Creatine and cyclocreatine inhibited the latter only in transgenic liver mitochondria. Protein complexes isolated from detergent solubilised rat brain extracts, containing octameric mitochondrial creatine kinase, porin and the adenine nucleotide translocator, were reconstituted into malate loaded lipid vesicles. Dimerisation of creatine kinase in the complexes and exposure of the reconstituted complexes to 200 μM calcium induced a cyclosporin A sensitive malate release. No malate release occurred with complexes containing octameric creatine kinase under the same conditions.

In vivo detection of neuroarchitecture in the rodent brain using manganese-enhanced MRI

Visualizing brain anatomy in vivo could provide insight into normal and pathophysiology. Here it is demonstrated that neuroarchitecture can be detected in the rodent brain using MRI after systemic MnCl2. Administration of MnCl2 leads to rapid T1 enhancement in the choroid plexus and circumventricular organs, which spreads to the CSF space in ventricles and periventricular tissue. After 1 day, there was MRI enhancement throughout the brain with high intensity in the pituitary, olfactory bulb, cortex, basal forebrain, hippocampus, basal ganglia, hypothalamus, amygdala, and cerebellum. Contrast obtained enabled visualization of specific features of neuroarchitecture. The arrowhead structure of the dentate gyrus as well as the CA1-CA3 region of the hippocampus and layers in cortex, cerebellum, as well as the olfactory bulb could be readily observed. Preliminary assignments of olfactory bulb layers, cortical layers in frontal and somatosensory cortex, and cerebellum were made. Systemic MnCl2 leads to MRI visualization of neuroarchitecture nondestructively.

Tracing odor-induced activation in the olfactory bulbs of mice using manganese-enhanced magnetic resonance imaging.

Ithas previously been demonstrated that it is possible to map active regions of the brain using MRI relying on the fact that Mn(2+) ion enters excitable cells through voltage-gated calcium channels and is an excellent relaxation agent. In addition, Mn(2+) has been shown to trace neuronal connections in the mouse olfactory and visual systems, enabling MRI neuronal tract tracing. The purpose of the present studies was to determine if these two properties could be combined to trace Mn(2+) from sites of activation in the olfactory epithelium to the olfactory bulb thereby localizing regions within the olfactory bulb that respond to a particular odor. Mice were exposed to an aerosolized solution containing either a high pheromone content odor (male mouse urine) or amyl acetate plus MnCl(2). In both cases the odors caused a localized T(1) MRI enhancement in the olfactory epithelium and bulb that was dependent upon the presence of Mn(2+). The high pheromone containing solution caused enhancement in the anatomically correct location of the accessory olfactory bulb. Amyl acetate also caused T(1)-weighted MRI enhancement in specific regions of the olfactory bulb. These areas showing activation agree well with previous 2-deoxyglucose and BOLD fMRI results in the rat. Using manganese-enhanced MRI (MEMRI) it should be possible to rapidly map a variety of odors. Furthermore, since the effects of activation are imaged after the activation protocol it should be possible to take the time to obtain very high resolution images and make MEMRI maps from awake behaving animals.

Laminar specificity of functional MRI onset times during somatosensory stimulation in rat

The blood oxygenation level-dependent (BOLD) response to somatosensory stimulation was measured in α-chloralose-anesthetized rats. BOLD fMRI was obtained at 40-ms temporal resolution and spatial resolution of 200 × 200 × 2,000 μm3 by using a gated activation paradigm in an 11.7 T MRI. Results show a consistent heterogeneity of fMRI onset times and amplitudes. The earliest onset time (0.59 ± 0.17 s, n = 9) corresponded anatomically to layer IV, with superficial and deeper layers starting significantly later (1.27 ± 0.43 s in layers I–III, and 1.11 ± 0.45 s in layer VI). The amplitude of BOLD signal changes also varied with the cortical depth from the pial surface. Changes in the supragranular layers (8.3%) were 44% bigger than changes in the intermediate layers (5.5%), located only ≈700 μm below, and 144% larger than the bottom layer (3.5%), located ≈1.4 mm below the pial surface. The data presented demonstrate that BOLD signal changes have distinct amplitude and temporal characteristics, which vary spatially across cortical layers.

Transcranial amelioration of inflammation and cell death after brain injury

Traumatic brain injury (TBI) is increasingly appreciated to be highly prevalent and deleterious to neurological function. At present, no effective treatment options are available, and little is known about the complex cellular response to TBI during its acute phase. To gain insights into TBI pathogenesis, we developed a novel murine closed-skull brain injury model that mirrors some pathological features associated with mild TBI in humans and used long-term intravital microscopy to study the dynamics of the injury response from its inception. Here we demonstrate that acute brain injury induces vascular damage, meningeal cell death, and the generation of reactive oxygen species (ROS) that ultimately breach the glial limitans and promote spread of the injury into the parenchyma. In response, the brain elicits a neuroprotective, purinergic-receptor-dependent inflammatory response characterized by meningeal neutrophil swarming and microglial reconstitution of the damaged glial limitans. We also show that the skull bone is permeable to small-molecular-weight compounds, and use this delivery route to modulate inflammation and therapeutically ameliorate brain injury through transcranial administration of the ROS scavenger, glutathione. Our results shed light on the acute cellular response to TBI and provide a means to locally deliver therapeutic compounds to the site of injury.

Functional Reactivity of Cerebral Capillaries

The spatiotemporal evolution of cerebral microcirculatory adjustments to functional brain stimulation is the fundamental determinant of the functional specificity of hemodynamically weighted neuroimaging signals. Very little data, however, exist on the functional reactivity of capillaries, the vessels most proximal to the activated neuronal population. Here, we used two-photon laser scanning microscopy, in combination with intracranial electrophysiology and intravital video microscopy, to explore the changes in cortical hemodynamics, at the level of individual capillaries, in response to steady-state forepaw stimulation in an anesthetized rodent model. Overall, the microcirculatory response to functional stimulation was characterized by a pronounced decrease in vascular transit times (20% ± 8%), a dilatation of the capillary bed (10.9% ± 1.2%), and significant increases in red blood cell speed (33.0% ± 7.7%) and flux (19.5% ± 6.2%). Capillaries dilated more than the medium-caliber vessels, indicating a decreased heterogeneity in vessel volumes and increased blood flow-carrying capacity during neuronal activation relative to baseline. Capillary dilatation accounted for an estimated ˜18% of the total change in the focal cerebral blood volume. In support of a capacity for focal redistribution of microvascular flow and volume, significant, though less frequent, local stimulation-induced decreases in capillary volume and erythrocyte speed and flux also occurred. The present findings provide further evidence of a strong functional reactivity of cerebral capillaries and underscore the importance of changes in the capillary geometry in the hemodynamic response to neuronal activation.