Altered expression of endogenous genes in the brain often accompanies neurological disorders. Genes or cells have been used to correct gene trancription so that brain can repair itself. Currently, most detection techniques of gene transcription require biopsy or autopsy samples. Invasive surgical procedures for removing tissue samples severely limit the benefits, especially to the cells we try to cure. Our goals are to develop methods to image and quantitatively compare endogenous gene expression at the transcript level using magnetic resonance (MR) in live animal or human subjects. We made a novel MR probe using short phosphorothioate-modified oligodeoxynucleotides (sODN) with SuperParamagnetic Iron Oxide Nanoparticles (SPION, an MR T2 agent). The work outlined in this application investigates the utility of this contrast probe as an biomarker for mRNA transcripts in live animals. We designed three probes for MRI: two are with sequence complementary to matrix metalloprotease-9 (sODN-mmp9) or beta- actin (sODN-bactin) mRNA and a randomized s-ODN (sODN-Ran) with no sequence complementary to mRNA. The SPION-Ran probes will serve as controls. We will evaluate conditions that allow optimal imaging endogenous gene expression using MR.The specific anims are to demonstrate:
Aim 1 : Maximize MR Contrast Enhancement using SPION-bactin in High-resolution MRI in Mouse Brains. The hypothesis is that sODN-linked SPION will be retained by brain cells for detection using MRI in live animals, and for validation using histology (iron oxide) and binding assay (SPION-bactin) in postmortem samples. We select beta-actin mRNA as a target because beta-actin mRNA is constant and is inert to stress. To support this hypothesis, we will: (a) select an optimal SPION-retention using MRI in live animals after infusion with various doses of SPION-bactin; We will demonstrate that the uptake of SPION in the brain is sODN-linkage dependent; (b) demonstrate the presence of intracellular iron oxide after infusion of SPION-bactin at the optimal dose, and (c) show that the internalized SPION-bactin binds to its target mRNA.
Aim 2 : Retention of cerebral SPION-mmp9 in live C57black6 mice predicts brain edema after stroke inducted by 60 or 90 minutes bilateral carotid occlusion. The hypothesis is that cerebral mmp-9 mRNA transcript reports MMP-9 expression. To support this hypothesis, we will demonstrate:(a) elevation of mmp-9 mRNA expression is positively correlated with cerebral edema after stroke, (b) retention of SPION-mmp9 is higher in the stroke-treated than in the sham-operated mice. Specifically, we will identify the hotspot of SPION-mmp9 retention in stroke-treated animals using subtraction of R2* map between stroke-treated and sham-operated animals. In addition, we will compare the hotspots of SPION-mmp9 retention to stroke-induced damage in the brain of wild type and mmp-9 knockout strains. R21NS05755601-A1 PI: Liu, Philip K. PhD Project Narratives: Brain edema is one neurological disorder that complicates the recovery of several diseases in the humans. Cardiac arrest in humans can induce brain edema despite restoration of circulation using various means (Roine et al. 1993; Fujioka et al. 1994; Xiao 2002); brain damage is predictive of a poor neurological outcome. Currently few models simulate brain edema from cardiac arrest for us to test therapeutic agents that can reduce neurological damage. To improve our understanding of cerebral injury caused by heart attack, we proposed this grant application a global cerebral ischemia-reperfusion model to investigate the pathophysiology in the brain of male C57black/6 mice simulating cardiac arrest (Liu et al. 1996). Our long-term goal is to establish new methods using MRI contrast agent to gain understanding on the pathological change in gene expression in live subjects, which can be saved for further evaluation on efficacy and toxicity of the therapy. The immediate goal is to show our probe can report the elevation of gene transcripts after cerebral ischemia. ? ? ?
