Materials and methods was prepared by an investigator not involved in image

302 H demonstrated the ability of lamotrigine to attenuate EAA neck for the later production of cerebral circulatory arrest. accumulation in the extracellular space and its resulting At the end of the experiment, the animals were euthanized excitotoxicity after cerebral ischemia [1,7]. Recently, by increasing the inspired halothane concentration to 5 lamotrigine was shown to improve neurobehavioral and and injecting an intravenous dose of potassium chloride. histologic outcome following global cerebral ischemia [20]. 2.3. Drug administration In the present study, we evaluated the use of an easily reproducible model of transient global cerebral ischemia in Lamotrigine Glaxo Wellcome Inc, Greenville, NC was the rabbit. This animal model requires minimal surgical administered intravenously at a dose of 50 mg kg diluted preparation and allows for single or multiple episodes of in 20 ml of deionized water in the lamotrigine group. This cerebral ischemia of any desired duration. Using this dose of lamotrigine has been shown to prevent any model, we investigated the effect of the preischemic increase of glutamate during 10 min of ischemia [1], and intravenous administration of lamotrigine on ischemia- improved neurobehavioral outcome after a 6.5 min is- induced cytotoxic brain edema through serial monitoring chemic episode [20]. The drug was infused over 20 min of the ADC. starting 90 min before the onset of ischemia. The animals in the control group received an identical volume of deionized water without lamotrigine. The infusion medium

2. Materials and methods was prepared by an investigator not involved in image

acquisition and data analysis. To avoid any biasing of the 2.1. Animals results, blinding of the investigators was maintained until data analysis was completed. This study was performed under a protocol approved by the Institutional Animal Care and Use Committee at the 2.4. Induction of ischemia University of Texas Medical Branch. Fifteen New Zealand white rabbits, aged 4 months and weighing 3.560.25 kg After the animals had been positioned in the bore of the mean6S.D., were randomly assigned to one of three magnet, baseline images were recorded. Seventy minutes groups: control n56, lamotrigine n56, or sham n53. after completion of the lamotrigine infusion, the MAP was The animals were fasted for 24 h before the start of the decreased to between 25 and 50 mmHg with an intraven- experiment and housed one per cage at the institutional ous bolus of trimethaphan 5 mg. The neck tourniquet Animal Resource Center where they received routine was inflated to a pressure of 700 mmHg within 0.5 s using veterinary care. a regulated source of compressed air. This protocol reliably results in profound cerebral ischemia as evidenced by the 2.2. Surgical procedure prompt ,30 s appearance of an isoelectric electroence- phalogram [1,20]. After 12 min and 50 s of ischemia, the The animals were anesthetized in a Plexiglas box with neck tourniquet was deflated and MAP was restored to 5 halothane in oxygen. After loss of the righting reflex, between 80 and 100 mmHg with a bolus of phenylephrine endotracheal intubation was performed and the lungs were 5–10 mg given intravenously. mechanically ventilated FiO 51.0. The inspired 2 halothane concentration was reduced to 1 as soon as 2.5. Brain temperature measurements mechanical ventilation was established. After infiltration with 0.25 bupivacaine, the groin was incised and PE-90 Brain temperature measurements were carried out in an catheters were inserted into the femoral artery and vein. additional two animals to monitor the actual change during Mean arterial pressure MAP was measured throughout cerebral ischemia while in the magnet. A burr hole, 2 mm the study. Serial arterial blood samples were intermittently in diameter, was drilled 4 mm posterior and 4 mm lateral obtained during the experiment to measure pH, PO , and to the bregma in order to place a temperature probe into 2 PCO 1306 pH Blood Gas Analyzer, Instrumentation the dorsal hippocampus. After securing the temperature 2 Laboratory, Lexington, MA. Mechanical ventilation was probe with dental acrylic the animals were treated accord- adjusted to maintain the PCO between 35 and 40 mmHg. ing to the protocol described above. 2 Hemoglobin concentration was determined with a CO- oximeter 482 CO-Oximeter, Instrumentation Laboratory, 2.6. Magnetic resonance imaging Lexington, MA. Body temperature was monitored with a rectal temperature probe and maintained at 388C with a Following surgical preparation, the animals were se- circulating water heating pad Gaymar Industries Inc., cured in the prone position on a Plexiglas cradle. The head Orchard Park, NY which was placed around the animal’s of the rabbit was fixed in a nonmagnetic head holder and body throughout the experiment. An inflatable neck tour- positioned in the bore of the magnet. Magnetic resonance niquet 6 cm wide was secured loosely around the rabbit’s data were obtained using a 4.7 Tesla magnet Oxford H . Koinig et al. Brain Research 887 2000 301 –308 303 Instruments Ltd., Oxford, UK and a custom-built surface intravenously during the sixth frame of each movie. A 1 coil with a 9-cm diameter tuned to the H resonance washout period of 30 min was used after each bolus frequency 200.056 MHz. The magnetic field homogen- tracking movie to reduce contrast agent concentration. The eity throughout the sample volume was then maximized by sequence of DWIs, bolus tracking movies, cerebral is- shimming on the water free induction decay FID using a chemia, and the measurement of physiologic variables are Varian Unity Inova NMR console coupled to a Sun depicted in Fig. 1. Microsystems host computer Ultra Sparc-Station 10 running VNMR 6.1B software Varian Inc., Palo Alto, 2.7. Data analysis CA. Sagittal and coronal pilot scans were acquired for the selection of a set of five transverse imaging slices. DWIs Computation of quantitative ADC images was per- were obtained using a multi-slice spin-echo diffusion formed on a Sun Microsystems computer Ultra Sparc- sequence with a diffusion gradient applied along the Station 10. Regional evaluations of ADC mean values transverse horizontal ‘X’ axis. Imaging acquisition pa- were carried out in two regions of interest ROI’s, i.e., rameters were as follows: five consecutive slices centered bilateral hippocampus Fig. 2. Size and location of the on the slice of interest, 1.6-mm slice thickness, repetition ROI’s were selected with the reference to an atlas of the echo times of 3000 65 ms, 10310 cm field of view, rabbit brain [34] and high-resolution images acquired using using128 phase encoding steps, and one echo was averaged a conventional spin-echo sequence. per phase encoding step. For quantitative determination of Phase files were made for each frame of the bolus the ADC, DWIs with different-weighting factors b-val- tracking experiment. These were converted into individual 2 ues50, 293, 661 s mm were recorded before ischemia frames of the bolus tracking movie in a proprietary format and after 10, 30, 60, and 90 min of reperfusion. To using an in-house developed software package Transit. improve temporal resolution in the periischemic period, An artery pixel at the base of the brain and the sagittal 2 additional single DWIs b5661 s mm were acquired vein outlined were then selected to provide arterial A RT every 6 min and 25 s. These images and the unweighted and venous residue times V . From these data the total RT 2 spin echoes b50 s mm of the previously recorded signal observed V was determined for each vessel. AUC baseline measurements were used for calculation of the The parenchyma of the whole brain was then outlined and ADC during ischemia and early reperfusion. the average flow curve determined to give WB and AUC Bolus track imaging was used to assess cerebral blood parenchymal residue time WB . These data were then RT flow CBF during baseline and reperfusion. Two bolus used to calculate the cerebral blood flow CBF before and tracking movies were acquired using a flash sequence with after ischemia using the following method: the following parameters: 50 single slices in rapid succes- V ? WB sion, total acquisition time per frame of 520 ms, repetition AUC AUC 21 21 ]]]]] CBF ml ? 100 g ? min 5 21 echo time of 8 3 ms, and field of interest equal to 11311 WB T 2 A R RT cm. The movies were recorded 25 min before ischemia and at the end of the experimental protocol. A bolus of 0.5 ml 2.8. Statistical analysis  gadopentetate dimeglumine contrast agent Magnevist , Berlex Imaging Laboratories, Wayne, NJ was injected Data were analyzed using a commercially available Fig. 1. Time-course of the experimental protocol. The small arrows indicate times when temperature and mean arterial blood pressure MAP readings were recorded. Plus 1 marks indicate times when arterial blood gas samples were obtained. B, baseline images; I, ischemia images; Re, reperfusion images. 304 H Fig. 2. Regions of interest were drawn on the image by a single observer. The hippocampus was selected for each animal using the help of both an atlas of the rabbit brain [34] and a high-resolution image acquired with a conventional spin-echo sequence. computer program StatView 5.0; SAS Institute Inc., San groups during the entire experiment. Brain temperature Francisco, CA. Physiologic parameters pH, PO , PCO , measurements in two animals showed a 3.0560.358C 2 2 hemoglobin, MAP, and body temperature were compared decrease at the end of the 12 min and 50 s ischemic with repeated-measures analysis of variance ANOVA and episode. As per the protocol, MAP during ischemia was Scheffe’s test. ADC and CBF values were compared using lowered to 25 to 50 mmHg in the control and lamotrigine factorial ANOVA and Dunnett’s test. A paired t-test was groups. After deflating the neck tourniquet, MAP returned used to test for statistically significant changes between to baseline values. No significant differences in MAP were baseline and minimum ADC values as well as pre- and observed between groups. CBF ml 100 g min during postischemic CBF values in both groups. Differences were baseline was 54617 in the control group, 52622 in the considered statistically significant at P ,0.05; data are lamotrigine group, and 5363 in the sham group. During presented as mean6S.D. reperfusion CBF was 5464 in the control group, 49612 in the lamotrigine group, and 5765 in the sham group. There were no significant differences in CBF between groups or

3. Results over time.