Ferumoxtran-10

22 rats were used for fMRI experiments, obtained in a total of 51 fMRI experimental runs. BOLD data were acquired from all 22 rats, and 5 of these rats were also used for CBV measurements after at least one successful BOLD run. Experiments with no seizures, continuous seizures resulting in lack of baseline EEG, poor systemic physiology, motion in fMRI images, or abortion of fMRI data acquisition due to technical reasons were excluded from analysis. BOLD fMRI experiments were performed without exogenous contrast administration. For CBV-weighted fMRI experiments a dose of 10–18 mg/kg iron oxide contrast agent (Combidex [ferumoxtran-10], Advanced Magnetics Inc., Cambridge, MA) was administered intravenously 10–15 minutes prior to image acquisition.
During MRI recordings, the rat was positioned prone in a specially designed plastic holder with the head fixed and bregma positioned at the center of the surface coil. The animals were then inserted into the magnet with its head positioned at the isocenter of the magnet. EEG signals were acquired simultaneously with fMRI using a pair of 1 mm diameter carbon-filament electrodes (WPI, Sarasota, FL). These carbon filaments were placed between the scalp and the upper surface of the skull in the frontal and occipital areas, with the exposed portion of each electrode running in the coronal plane crossing the midline (from left to right), and secured to the skin with tissue glue (3M Vetbond, 3M Animal Care Products, MN ) to minimize MRI signal distortion (Englot et al., 2008 (link)). The EEG signals were acquired in differential mode between the two carbon-filament electrodes, amplified (×100) and filtered (1–30 Hz) using a Model 79D Data Recording System (Grass Instruments Co., Quincy, MA). EEG signals were digitized and recorded (sampling rate 1000 Hz) using a CED Micro 1401 and Spike 2 software (Cambridge Electronic Design, Cambridge, UK).
All fMRI experiments were acquired on a 9.4 Tesla Bruker (Bruker Avance, Billerica, MA) horizontal bore (16-cm-internal diameter) spectrometer, equipped with passively shielded shim/gradient coils (47.5 G/cm) operating at 400.5 MHz for protons. The transceiver system consisted of a surface coil (15.18 mm diameter) for transmission of radio frequency pulses and receiving. To optimize the homogeneity of the static magnetic field, the system was shimmed before each experiment using global manual shimming.
Anatomical images for each animal were acquired with 5 or 11 interlaced slices in the coronal plane using the fast low angle shot (FLASH) sequence with repetition time (TR) 500 ms; echo time (TE) 6 ms; flip angle = 40–55°; field of view (FOV) 25 × 25 mm; 256 × 256 matrix size; in-plane resolution of 98 × 98 µm; and slice thickness 1000 µm, no gap. BOLD and CBV-weighted fMRI data were obtained in the same planes as anatomical images. We used single-shot spin echo, echo planar imaging (SE-EPI; 22 experimental runs in 16 animals) or gradient echo (GE-EPI; 25 experimental runs in 13 animals; 7 animals had both SE and GE acquisitions), and obtained similar results with both approaches. SE-EPI data were acquired with the following parameters: TR = 1000 ms, TE = 25 ms; excitation flip angle 90°; inversion flip angle 180°; FOV = 25 × 25 mm, 64 × 64 matrix size; in-plane resolution of 390 × 390 µm; and slice thickness 1000 µm. All SE-EPI experiments were acquired with 11 slices. The 11 slices were acquired over 1000 ms, followed by a 2s or 5s pause before the next image onset so that EEG could readily be interpreted during data acquisition; time between onset of consecutive image acquisitions was therefore 3s or 6s. We acquired 300 to 600 images per run, resulting in a total imaging time of 1800s for most experimental runs (a shorter imaging time of 900 or 1500 s was used for two runs). GE-EPI data were acquired with following parameters: TR = 1000 ms, TE = 13 ms, FOV = 25 × 25 mm, 64 × 64 matrix size; in-plane resolution of 390 × 390 µm. For most GE-EPI runs slice thickness was 2000 µm, and 5 slices were acquired over 1000 ms, followed by a 2s pause; time between consecutive image onsets was therefore 3s. We acquired 300 to 400 images per run, with a total imaging time of 900s for most runs. For two GE-EPI runs, slice thickness was 1000 µm (like in SE-EPI runs) resulting in 11 slices acquired every 3s, with 400 total images obtained over 1200 s. Images from the first 48s of each run were discarded from analysis.

At least 6 d prior to fMRI recordings, animals were stereotactically implanted with a MRI-compatible bipolar tungsten stimulating/recording electrode (∼0.1 MΩ resistance, Microprobe Inc., Gaithersburg, MD) in the dorsal hippocampus (same coordinates as above). To minimize hardware and susceptibility artifact in the vicinity of the fMRI measurements, the electrode was lowered at a 50° angle from the vertical using a posterior approach and fixed to the skull adjacent to lambda with 2-4 plastic screws and dental acrylic. Immediately prior to fMRI scans, animals were anesthetized with ketamine/xylazine (90/15 mg/kg, i.m., q1 hr), tracheotomized, and artificially ventilated (70% air and 30% O₂). To prevent any movements during the experiments and to facilitate artificial breathing, animals were paralyzed by repeated injections of D-tubocurarine chloride (0.5 mg/kg, i.v.) (Sigma-Aldrich, Inc., St. Louis, MO). A femoral artery was cannulated (Intramedic PE50 tubing, Becton Dickinson and Company, Sparks, MD) for continuous arterial blood pressure monitoring and periodic blood sampling for measurements of blood gases and pH using an ABL 5 blood gas analyzer (Radiometer Copenhagen, Copenhagen, Denmark). All physiological values (blood gases, mean arterial blood pressure [MABP], and pH) were maintained within physiological range throughout the experiments (Schridde et al., 2007 ). One femoral vein was cannulated and an IP line was placed (Intramedic PE10 tubing) for the injections of anesthetics and paralyzing agents. For flow-related cerebral blood volume (CBV) fMRI experiments, rats were also injected with iron oxide contrast agent (24-50 mg/kg) (Combidex [ferumoxtran-10], Advanced Magnetics Inc., Cambridge, MA). Body temperature was kept constant at ∼37 °C using a heating pad.