Relative to common clinical magnetic field strengths, higher fields benefit functional brain imaging both by providing additional signal for high-resolution applications and by improving the sensitivity of endogenous contrast due to the blood oxygen level dependent (BOLD) mechanism, which has limited detection power at low magnetic fields relative to the use of exogenous contrast agent. This study evaluates the utility of iron oxide contrast agent for gradient echo functional MRI at 9.4 T in rodents using cocaine and methylphenidate as stimuli. Relative to the BOLD method, the use of high iron doses and short echo times provided a roughly twofold global increase in functional sensitivity, while also suppressing large vessel signal and reducing susceptibility artifacts. MRI is widely used to assess brain function in humans and animals due to a powerful combination of capabilities, including high spatiotemporal resolution, volumetric coverage, and the potential for noninvasive, longitudinal studies. Many of the target applications for fMRI in animal models are inherently challenging in terms of sensitivity. For instance, functional signals often are attenuated in disease or recovery states, such as the evolution of neuronal plasticity during recovery from stroke (1-3). Pharmacological stimuli can produce widespread, graded, dosedependent changes in local brain function; low-field blood oxygen level dependent (BOLD) signal is simply inadequate for detecting changes in many brain regions without averaging results from a very large number of animals (4,5).High magnetic field strengths provide numerous advantages for fMRI, as well as challenges (6). Sample polarization increases with magnetic field, providing additional signal that can be traded for higher spatial resolution. Functional changes in the BOLD relaxation rate also increase with field strength (7), making BOLD detection power more competitive with that provided by an exogenous agent (8). Moreover, paramagnetic deoxyhemoglobin shortens blood relaxation times at high field strengths, which should decrease spatially nonspecific signal associated with draining vessels. However, the time scale for relaxation of transverse magnetization using gradient echoes (T 2 *) becomes progressively shorter and more heterogeneous, especially in regions near magnetic susceptibility interfaces that arise at air-tissue and bone-tissue interfaces. Signal dropout and image distortion reduce some of the theoretical advantages of BOLD fMRI at high fields by forcing a choice between increased image artifacts or the reduced sensitivity that accompanies short gradient echo times or spin echo methods.Because of the limitations of BOLD sensitivity, many fMRI applications in animal models have employed exogenous contrast agents (1-3,9 -11), which experimentally have been shown to markedly improve fMRI sensitivity at magnetic field strengths up to 4.7 T (4,5,8,12-14). The use of exogenous agents with very long blood half lives for fMRI has been termed IRON fMRI (5), to denote the increas...

To circumvent the limitations of using postmortem brain in molecular assays, we used avidin-biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15-20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and ␤-actin mRNA (SPION-cfos and SPION-␤actin, respectively) (14 -22 nm). The Stern-Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 ϫ 10 6 /M. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (

The aim of this research was to validate transcription magnetic resonance (MR) imaging (MRI) for gene transcript targeting in acute neurological disorders in live subjects. We delivered three MR probe variants with superparamagnetic iron oxide nanoparticles (SPION, a T 2 susceptibility agent) linked to a phosphorothioate-modified oligodeoxynucleotide (sODN) complementary to c-fos mRNA (SPION-cfos) or β-actin mRNA (SPION-β-actin) and to sODN with random sequence (SPION-Ran). Each probe (1 μg Fe in 2 μl) was delivered via intracerebroventricular infusion to the left cerebral ventricle of male C57Black6 mice. We demonstrated SPION retention, measured as decreased T 2 * signal or increased R 2 * value (R 2 *=1/T 2 *). Animals that received the SPION-β-actin probe exhibited the highest R 2 * values, followed (in descending order) by SPION-cfos and SPIONRan. SPION-cfos retention was localized in brain regions where SPION-cfos was present and where hybrids of SPION-cfos and its target c-fos mRNA were detected by in situ reverse transcription PCR. In animals that experienced cerebral ischemia, SPION-cfos retention was significantly increased in locations where c-fos mRNA increased in response to the ischemic insult; these elevations were not observed for SPION-β-actin and SPION-Ran. This study should enable MR detection of mRNA alteration in disease models of the central nervous system. Keywords cardiac arrest; immediate early genes; oxidative stress; nanotechnology; signal transduction Brain damage resulting from cardiac arrest and stroke is a major cause of mortality and disability in the United States. Although the brain may repair itself, elevation of immediate early genes has been known to be positively related to expression of matrix metalloproteinase-9, a precursor of brain edema (1-5), a major cause of stroke-induced death in humans. The ability to detect alterations in endogenous gene transcription in live subjects can be pivotal for early intervention after an ischemic episode. Contrast-enhanced magnetic resonance (MR) imaging (MRI) permits real-time imaging, tracking of pathophysiological changes, and longitudinal studies at both the cellular and molecular levels (6-8). The goal of this study is to establish whether MR contrast agents function as suitable labels for nucleic acid probes to report gene transcription in the brains of live animals (Fig. 1B, C). The target transcript in this study is c-fos mRNA, an immediate early gene transcript that encodes the Fos peptide, an essential component of activator protein-1 and a neuronal transcription regulator that activates the expression of many genes including nerve growth factor (17,19,20). In normal resting neurons, c-fos mRNA is minimally expressed (is less abundant), but neuronal activities, stress, and cerebral ischemia can elevate its expression to levels at least 1 to 2 orders of magnitude higher than resting levels (21, 22). Conversely, β-actin mRNA is constitutively expressed at high levels and is not significantly elevated by cerebral ischemia;...

Brain responses to external stimuli after permanent and transient ischemic insults have been documented using cerebral blood volume weighted (CBVw) functional magnetic resonance imaging (fMRI) in correlation with tissue damage and neurological recovery. Here, we extend our previous studies of stroke recovery in rat models of focal cerebral ischemia by comparing blood oxygen level-dependent (BOLD) and cerebral blood volume (CBV) changes. Responses to forepaw stimulation were measured in normal rats (n=5) and stroke rats subjected to 2 h of middle cerebral artery occlusion (n=6). Functional magnetic resonance imaging was performed 2 weeks after stroke to evaluate the recovery process. After stroke, animals showed variable degrees of fMRI activation in ipsilesional cortex, the extent of which did not correlate with structural damages as measured using apparent diffusion coefficient, fractional anisotropy, blood volume, and vessel size index. While the contralesional cortex showed good overlap between BOLD and CBV-activated regions, the ipsilesional cortex showed low covariance between significantly activated voxels by BOLD and CBVw techniques. In particular, the relative activation during contralateral stimuli in the ipsilesional somatosensory cortex was significantly higher for CBVw responses than BOLD, which might be due to stroke-related alterations in fMRI hemodynamic coupling. Aberrant subcortical activations were also observed. When unaffected forelimbs were stimulated, strong bilateral responses were observed. However, little thalamic responses accompanied stimulation of affected forelimbs despite significant activation in the ipsilesional somatosensory cortex. These results suggest that stroke affects not only local hemodynamics and coupling but also other factors including neural connectivity.