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

Abstract

We aimed to test the feasibility of detecting gliosis in living brains when the blood-brain barrier (BBB) is disrupted. We designed a novel magnetic resonance (MR) probe that contains superparamagnetic iron oxide nanoparticles (SPION, a T2 susceptibility contrast agent) linked to a short DNA sequence complementary to the cerebral mRNA of glial fibrillary acidic protein (GFAP) found in glia and astrocytes. As a control, we also used a sequence complementary to the mRNA of β-actin. Our objectives are to demonstrate that this new probe, SPION-gfap, could be delivered to the brain when administered by eyedrop solution to the conjunctival sac. We induced BBB leakage by puncture wound, global cerebral ischemia, and cortical spreading depression in C57BL6 mice; 1 day after probe delivery we acquired T2* MR images and R2* (R2*=1/T2*) maps using a transcription MRI technique in live mice. We found that the SPION-gfap probe reported foci with elevated signal in subtraction R2* maps and that these foci matched areas identified as having extensive glial network (gliosis) in postmortem immunohistochemistry. Similarly, animals administered the control probe exhibited foci of R2* elevation that matched β-actin-expressing endothelia in the vascular wall. We conclude that our modular MR probe, delivered in an eyedrop solution, effectively reports gliosis associated with acute neurological disorders in living animals. As BBB leakage is often observed in acute neurological disorders, this study also served to validate noninvasive delivery of MR probes to the brains of live animals after acute neurological disorders. Keywordsangiogenesis; antisense technology; blood-brain barrier; bulbar conjunctiva; gene expression; molecular imaging 1Correspondence: 149 Thirteenth St., Rm. 2410, Charles-town, MA 02129, USA. E-mail: philipl@nmr.mgh.harvard.edu. Note added in proof:We now have performed BBB leakage determination in mice using Evans blue extravasation (n=8) or Gd-MRI at 1 (n=6), 7 (n=10), and 24 (n=2) h after GCI of 60 min. We found BBB leakage in 4 mice, or 22%, within the first day of GCI. All mice that showed hyperintense DWI (with significant ADC drop) 1 day after GCI in the striatum developed striatal BBB leakage. Gliosis, the outgrowth of a fibrous network of glia in the central nervous system (CNS), is a permanent feature of many human neurological disorders and is especially prevalent in glioma, multiple sclerosis, viral encephalitis, traumatic brain injury, stroke, and cardiac arrest (1-5). Detection of gliosis or cells of abnormal growth has traditionally involved immunohistochemistry methods to detect elevated antigen of glial fibrillary acidic protein (GFAP) levels in postmortem tissue samples (6-8). However, brain biopsy to acquire tissue for these conventional assays is by itself invasive, and the procedure often results in the removal of important tissue. NIH Public AccessMRI is a powerful and noninvasive tool for in vivo imaging of soft tissue (9-12) but is somewhat limited by the unavailability of suitable p...

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