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.2007 Jan 17;27(3):713-22.
doi: 10.1523/JNEUROSCI.4660-06.2007.

Imaging cerebral gene transcripts in live animals

Affiliations

Imaging cerebral gene transcripts in live animals

Christina H Liu et al. J Neurosci..

Abstract

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 beta-actin mRNA (SPION-cfos and SPION-beta-actin, respectively) (14-22 nm). The Stern-Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 x 10(6)/m. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (MRI) to monitor gene transcription, and demonstrated that these complexes at 40 mug of Fe per kilogram of body weight were retained at least 1 d after intracerebroventricular infusion into the left ventricle of C57Black6 mice. SPION retention measured by MRI as T(2)* or R(2)* maps (R(2)* = 1/T(2)*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T(2)* signal in the right cortex and striatum; retention of SPION-cfos and SPION-beta-actin positively correlated with c-fos and beta-actin mRNA maps obtained from in situ hybridization. Histological examination showed that intracellular iron oxide and FITC-sODN correlated positively with in vivo MR signal reduction. Furthermore, in animals that were administered SPION-cfos and amphetamine (4 mg/kg, i.p.), retention was significantly elevated in the nucleus accumbens, striatum, and medial prefrontal cortex of the forebrain. Control groups that received SPION-cfos and saline or that received a SPION conjugate with a random-sequence probe and amphetamine showed no retention. These results demonstrated that SPION-sODN conjugates can detect active transcriptions of specific mRNA species in living animals with MRI.

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Figures

Figure 1.
Figure 1.
A illustrates our probe design, andB illustrates a theoretical diagram for cellular mRNA targeting by SPION-sODN. FITC-sODN-biotin is conjugated to SPION-NA to generate SPION-sODN. The sODN can be labeled with an MR susceptibility agent, SPION, and/or with other imaging dye, such as FITC.
Figure 2.
Figure 2.
Linkage of SPION and sODN shows mobility shift during gel electrophoresis.A shows the migration of SPION-NA with (lanes 1–3) or without (lane 4) sODN, as acquired with an ordinary light source after gel electrophoresis at room temperature (900 V · h; 40 V/h). The arrow shows the direction of sODN migration.B shows FITC-sODN after electrophoresis (300 V · h; 150 V/h) under a 302/365 nm light source (exposure time, 8 s). The distance of elution from the well for each gel slice is shown in centimeters. SPION-NA of batch A is 17 months of age, and that of batch B is freshly prepared (2 months). The quenching effect of SPION reduces the fluorescence of the FITC-sODN on SPION (saturation binding). The rows on the top of panels show the concentrations of components in each sample applied to the agarose gel.C shows T2 relaxation times in gel slices fromA andB.
Figure 3.
Figure 3.
Quenching effect of SPION reduces fluorescence emission of the FITC-sODN on SPION.A shows decreasing fluorescence emission of FITC-sODN-biotin on binding to SPION-NA. Error bars indicate SEM.B shows a Stern–Volmer plot constructed for SPION-sODN.Kq is the Stern–Volmer constant calculated from the equationF0/Fq = 1 +Kq[Q], whereF0 andFq are fluorescence emission at zero (F0) and quencher concentration (Fq) of SPION-NA, andQ is the concentration of SPION-NA (Murphy et al., 2004; Li et al., 2005).
Figure 4.
Figure 4.
SPION-cfos probe provides an extended window for MRI in live mice. Images acquired at TR/TE = 500/6 ms are shown (N = 4 each). Images were acquired from one representative animal from each probe group. Images include brain regions posterior to bregma at −6.0 mm, at −0.4 mm (the infusion site), and 2 mm anterior to bregma.A shows the preinfusion (baseline) MR images.B and insetb show MR images after SPION-cfos (1 μg of Fe per mouse). LH, Left hippocampus; RH, right hippocampus.C andCC show MR images of two representative animals after unconjugated SPION; insetsa andb show MR brain slices from the posterior to the infusion site at −0.9 mm (bregma) in the same animal as shown inA andB, respectively.D shows MR images after infusion of unconjugated SPION, or mixture of SPION and sODN without NA and biotin linkage.
Figure 5.
Figure 5.
Colocalization of sODN-cfos and iron oxide in mouse brains 1 d after infusion of SPION-cfos. Animals were given an infusion of SPION-cfos-FITC (A–C, 1 μg of Fe per mouse) or unconjugated SPION (D; 5 μg of Fe per mouse), all with lipofectin (N = 4 in each group). Brain tissue was obtained 1 d later. PB stain and NFR counterstain are shown inA,B, andD (10× objective) and in the insetsa,b,bb, andd (100×).A andC show adjacent tissue slices.C andE–I show fluorescent micrographs.E shows FITC-sODN-cfos in brain samples from animals that were given infusion of SPION-cfos-FITC (1 d after infusion);F shows the baseline control. Tissue inG was from the same group of animals as inC andE but was treated with antibodies against glial fibrillary acidic protein (GFAP) and DAB stains.H andI show sODN uptake in the contralateral hemisphere of the animals infused with SPION-cfos-dig (2 μg of Fe per mouse) with (H) and without (I) lipofectin (N = 3 each). Presence of sODN-dig 1 d after infusion was detected using FITC-IgG against dig (Cui et al., 1999).J andK show SPION uptake in the hippocampus and the cerebellum of the same animal as inI. Purkinje neurons are located in the Purkinje layer as aligned, stacked cells with a large nucleus (K). The asterisks show stain on the surface of ventricular wall. (J). All images except those inE andF were acquired from tissue sections treated with xylene.E andF show non-dehydrated tissue. Tissue thickness was 20 μm (A–D,H,I) or 100 μm (E–G,J,K), which allows us to observe axons (long arrows;E) and vascular wall; (E,J). The brackets inE–G point to the CA1 neuronal formation in the hippocampus. DG, Dentate gyrus.
Figure 6.
Figure 6.
Temporal profiles of SPION retention in R2* maps.A shows animals infused with SPION-cfos, SPION-βactin, or unconjugated SPION (1 μg of Fe per mouse;N ≥ 4 each). Multiple cerebral MRIs were acquired as GE MRI in live animals at indicated time points. Preinfusion data serve as baseline (see examples in Fig. 5 for images or saline groups in Fig. 7 for R2* maps). We calculated mean R2* values in an ROI in contralateral SSC to determine SPION retention. No significant differences in R2* values were observed in baseline and animals that received SPION. Thep values denote mean R2* values that are significantly different from baseline (*) or significant differences between SPION-cfos and SPION-βactin (**), calculated using at test. Based on the mean and SEM from 7 h after infusion, and because power calculation reveals that four animals per group are needed to obtain a statistically significant difference for the effect sizes seen here with 95% confidence andp = 0.01, we measured five mice in each group. The ROIs are shown at right.B andC show mRNA maps of c-fos (B) and β-actin (C) gene transcripts in a normal mouse (no infusion) usingin situ hybridization and a32P-labeled antisense RNA probe (Cui et al., 1999a) (N = 4).D shows the profile of SPION retention in the contralateral striatum. Regional SPION retention of SPION-cfos and SPION-Ran (1 μg of Fe per mouse) is shown in the contralateral striatum for ROIs depicted to the right. Error bars indicate SEM.
Figure 7.
Figure 7.
Specific elevation of c-fos mRNA in R2* maps after acute amphetamine exposure. SPION-Ran (N = 5 pairs;A) or SPION-cfos (N = 6 pairs;B) was infused as in Figure 5, except that amphetamine (4 mg/kg body weight) or saline (10 ml/kg) was administered intraperitoneally 4 h later, and MRI was acquired 3 h thereafter. Representative R2* maps (0–200 s−1 R2* values are shown in the scale bar) are from four MR slices (same as Fig. 6D) in one randomly selected animal from each group. Schematic diagrams of anatomic features are within 0.25 mm of each MR slice (Paxinos and Franklin, 2001).C shows profiles of SPION retention in ROIs from R2* maps (see Fig. 6).mPFC, Medial prefrontal cortex; NAc, nucleus accumbens; CPu, caudate/putamen.
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