Forebrain ischemia-reperfusion simulating cardiac arrest in mice induces edema and DNA fragmentation in the brain

Abstract

Brain injury affects one-third of persons who survive after heart attack, even with restoration of spontaneous circulation by cardiopulmonary resuscitation. We studied brain injury resulting from transient bilateral carotid artery occlusion (BCAO) and reperfusion by simulating heart attack and restoration of circulation, respectively, in live C57Black6 mice. This model is known to induce neuronal death in the hippocampus, striatum, and cortex. We report the appearance of edema after transient BCAO of 60 minutes and 1 day of reperfusion. Hyperintensity in diffusion-weighted magnetic resonance imaging (MRI) was detectable in the striatum, thalamus, and cortex but not in the hippocampus. To determine whether damage to the hippocampus can be detected in live animals, we infused a T(2) susceptibility magnetic resonance contrast agent (superparamagnetic iron oxide nanoparticles [SPIONs]) that was linked to single-stranded deoxyribonucleic acid (DNA) complementary in sequence to c-fos messenger ribonucleic acid (SPION-cfos); we acquired in vivo T(2)*-weighted MRI 3 days later. SPION retention was measured as T(2)* (milliseconds) signal reduction or R(2)* value (s(-1)) elevation. We found that animals treated with 60-minute BCAO and 7-day reperfusion exhibited significantly less SPION retention in the hippocampus and cortex than sham-operated animals. These findings suggest that brain injury induced by cardiac arrest can be detected in live animals.

Figure 1

A, Description of the oligo nucleic acid–based superparamagnetic iron oxide nanoparticle (SPION) probe. Linkage of SPION and single-stranded phosphorothioate-modified oligodeoxynucleotides is based on the avidin-biotin interaction. B, Cartoon depicting how living cells with normal cfos messenger ribonucleic acid (mRNA) transcription are able to retain SPION-cfos probe by complex formation based on complementary base pairing between the probe and the intracellular mRNA target. The presence of T₂* magnetic resonance contrast agent (SPION) can cause localized magnetic resonance signal reduction owing to cells that contain SPION. On the other hand, dead cells damaged by apoptosis exhibit fragmented deoxyribonucleic acid (DNA), and no new cfos mRNA can be transcribed owing to a lack of available DNA template. As a result, there is less or no uptake of SPION-cfos by the dead cell.

Figure 2

Detection of neuronal death using terminal UTP nick-end label (TUNEL) assay. A, Schematic of the experimental protocol. B–E and G, TUNEL staining of postmortem samples obtained 1 day after bilateral carotid artery occlusion (BCAO) of 60 minutes. Positive TUNEL stain (green) shows deoxyribonucleic acid (DNA) fragmentation in neuronal nuclei (C, D, E, and G); no TUNEL stain was observed in neurons of sham-operated animals (B) and in cells with astrocyte-specific glial fibrillary acidic protein (red in F). E and F show dual stains in the same tissue section in an animal treated with BCAO. Arrows show a reactive astrocyte near several dying neurons. G and H show dual stains as in E and F, except neurons were identified using antibodies against neuropeptide Y (NPY) in the same tissue section from the striatum. These panels show NPY-positive (arrowheads) and NPY-negative (long arrows) neurons, along with dying neurons with diminished NPY antigen (short arrows). ICV = intracerebroventricular; MRI = magnetic resonance imaging.

Figure 3

Comparison of diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) between bilateral carotid artery occlusion (BCAO) and sham-operated animals at two time points: DWIs and ADC were obtained from animals 1 (A) and 7 (B) days after treatment with BCAO of 60 minutes. The ADCs (mean ± SD) in sham-operated (baseline) animals were 0.57 ± 0.01 × 10⁻³ and 0.61 ± 0.01 × 10⁻³ for the cortex and the hippocampus, respectively. Regions with edema can be identified by comparing the ADC of the 1- and 7-day time points with baseline ADC (C). Edema, identified by visual inspection of DWI hyperintensity and reduced ADC, occurred in the striatum and septal nucleus in all animals but in the thalamus and hypothalamus of only four of nine animals.

Figure 4

Colocalization of single-stranded phosphorothioate-modified oligodeoxynucleotide (sODN)-cfos and iron oxide in the ipsilateral hippocampus of a mouse 1 day after infusion of SPION-cfos. Animals were infused with SPION-cfos-digoxigenin (Fe = 0.08 mg/kg, n = 3). A to C show T₂*-weighted images of preinfusion baseline, SPION-cfos-digoxigenin, and SPION-infused brains 1 day after infusion in live animals. Long arrows (pointed upward) point to the tissue-air interface from the trachea and ears. Images were produced using a 9.4 T magnetic resonance imaging (MRI) system. Histology of SPION uptake was acquired in postmortem brain samples after MRI. These brain sections were 10 μm in thickness. D shows fluorescein isothiocyanate (FITC)-antidigoxigenin antibodies (white fluorescent staining) on cfos-digoxigenin in the dentate gyrus (DG). Iron oxide was detected in a similar area of the adjacent brain section using Prussian blue (PB) stain and nuclear fast red (NFR) counterstain (E–H). F shows the PB stains of the SPION-infused brain sample. Short arrows (pointed down) indicate colocalization of iron oxide detected by PB stain. G and H show the cortices. ICV = intracerebroventricular; IPSI = ipsilateral to the infusion site.

Figure 5

In vivo R₂* profile in two contralateral brain regions of the brain. Regions of interest (ROI) were outlined in the panels to the left (hippocampus) and right (somatosensory [SS] cortex) of the bar graph. Progressive elevation of R₂* in both brain regions was observed for 3 days. Hippocampal regions reached plateau at 3 days (p = .01), whereas the SS cortex exhibited significant elevations at 1, 2, and 3 days (p ≤ .03). R₂* values in both regions decreased to the baseline on day 6, suggesting washout (clearance) of the probe. SEM = standard error of measurement.

Figure 6

Apoptotic hot spots as determined by in vivo R₂* maps. A shows a RARE image (rapid acquisition with relaxation enhancement; TR/TE = 7,000/26 ms, RARE factor = 8, number of averaging = 2) that depicts the anatomic reference from which T₂* images, T₂* and R₂* maps of animal brains are shown to its right. B, A subtraction map shows percent decreases in R₂* values in R₂* maps of cerebral ischemia (CI)-treated animals compared with sham-operated (SO) animals. A computer-generated intensity scale for percent change is shown at the left. The outline of the hippocampus was referenced from the anatomic images in A and superimposed on subtraction R₂* maps in B. The number of animals for subtraction R₂* maps in B is listed in Figure 7.

Figure 7

Profiles of superparamagnetic iron oxide nanoparticle (SPION) retention in R₂* maps of three groups of animals. Probe retention in the ischemia-treated group is significantly lower than in the sham-operated group (t-test). No significant difference in retention was observed between baseline animals without SPION-cfos infusion and cerebral ischemia–treated animals with SPION-cfos infusion. The number of animals is given below the bar graph. The outlines of the hippocampus and cortex were referenced from the anatomic images and superimposed on individual R₂* maps as regions of interest (ROI); R₂* values within two ROIs of each animal were extracted from individual R₂* maps as ROI-hippocampus and ROI-cortex for statistical analysis. Mean R₂* and standard error (SEM) are shown. RARE = rapid acquisition with relaxation enhancement.

Figure 8

Less cerebral superparamagnetic iron oxide nanoparticle (SPION) retention in animals with stroke cerebral ischemia. Three-dimensional T₂*-weighted images of postmortem brains collected 3 days after intracerebroventricular infusion of SPION-cfos and treatment with either sham operation (A) or bilateral carotid occlusion (B) for 60 minutes 1 week before. The hippocampus of the contralateral hemisphere is shown from one representative mouse in each group. Short arrows show the dentate gyrus (DG) and pyramidal cell layer (CA) neuronal formation in the hippocampus. C shows an animal brain without infusion. The signal void in the upper-left corner of B (thin arrow) was caused by missing tissue during brain sample handling. The number of animals is listed in Figure 7.