MR contrast probes that trace gene transcripts for cerebral ischemia in live animals

Abstract

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;...

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“…An intriguing possibility is that the sets of genes identified in this study play not only a role in disease recognition but also as part of a bidirectional and functional communication between the CNS and a peripheral disease. This might have important clinical implications in the future, especially if gene expression levels in the brain can be detected [46] and eventually pharmacologically regulated [47].…”

Noninvasive delivery of gene targeting probes to live brains for transcription MRI

“…We performed a power calculation on the data gathered from the first set of animals, using inhouse software to calculate the number of animals required in each group to achieve 90% power for a P value of 0.01 (8,13). If the minimum number of animals calculated was greater than 15, we revised the hypothesis and the protocol.…”

“…We have designed modular MR probes capable of reporting specific cells based on gene transcription in living brains of ordinary subjects (8,13,14 …”

“…We induced BBB leakage by one of three methods: by cortical spreading depression, by inflicting a puncture wound and administering an i.c.v. injection of 2 μl saline in a Hamilton syringe with a 26S gauge needle to the left cerebral ventricle (14), or by performing bilateral carotid artery occlusion (BCAO) for 60 min (8,13). Another group of animals underwent a sham operation (the same surgical procedure without the puncture wound or vessel occlusion for cerebral ischemia) at the same time.…”

“…We have designed modular MR probes capable of reporting specific cells based on gene transcription in living brains of ordinary subjects (8,13,14). In several proof-of-concept studies, we have developed and validated the delivery and neuronal uptake of modular MR probes in the cerebrospinal fluid after intracerebroventricular (i.c.v.)…”