Serial MRI after Experimental Febrile Seizures: Altered T2 Signal without Neuronal Death

Animals and Induction of Experimental Febrile Seizures

Sprague-Dawley–derived rats were born and maintained in quiet facilities under controlled temperatures and light schedule. Experimental procedures were approved by Institutional Animal Care Committees and conformed to NIH guidelines. Prolonged experimental FSs were elicited as described.^8–10 In brief, on postnatal day (P) 11, pups were placed in a glass container, and their core temperature was raised using a regulated stream of heated air to approximately 41°C (simulating high fever). Core temperatures were measured at baseline, seizure onset, and every 2 minutes during the seizures and maintained in a narrow range. Hyperthermia was induced for 30 minutes resulting in 20-to 22-minute seizures. The behavioral seizures in this paradigm are stereotyped, consisting of arrest of heat-induced hyperkinesis and facial automatisms, often followed by body flexion. They correlate with electrographic hippocampal seizures.^9–11

Magnetic Resonance Imaging Procedure

MRIs were performed on a 4T scanner Picker console (Marconi/Philips Medical, Cleveland, OH). Baseline scans were performed on P10, in five control rats and eight in which seizure were evoked on P11. All animals then were imaged on P12 and 7 to 8 days later. Age-matched littermate controls served to account for potential maturational changes in the MRI signal.

Rats were anesthetized (pentobarbital, 40mg/kg, IP) 30 minutes before acquisitions. For each scan, a pair of rats was positioned prone inside the radiofrequency coil on a handmade support, along with several phantoms (distilled water and mixed water-gadolinium). Sagittal and coronal localizing acquisitions were obtained using a fast spin-echo sequence (repetition time [TR]: 3,000 milliseconds; echo time [TE], 104 milliseconds; eight echoes; field of view [FOV], 120mm; acquisition matrix size, 128 × 256). Brains then were scanned coronally: posterior cuts corresponded to A −0.4 to −0.1mm (referring to bregma¹²) for entorhinal cortex visualization; most anterior cuts corresponded to A −5.5 to −5.6 mm, at the septal level, and five to six contiguous 1.1mm slices were imaged. T2-weighted anatomical images were acquired using fast spin-echo: TR, 5,500 milliseconds; TE, 105 milliseconds; eight echoes; FOV, 65mm; matrix size, 128 × 256; eight excitations.

Magnetic Resonance Imaging Analysis

Images were analyzed without knowledge of group (“blindly”): regions of interest, delineated manually from anatomical images,¹² included dorsal and ventral hippocampus, amygdala, piriform and entorhinal cortices, and medial ventroposterior thalamic nucleus. T2-weighted signal intensity was determined using standards calibrated using “phantoms” of water and water-gadolinium mixtures falling within, as well as outside of, the range of observed values. The ratio of intensity of the structure of interest and a reference structure (corpus callosum) then was calculated, permitting normalization among sections and animals.

Determination of Neuronal Injury

A total of 39 animals (not those undergoing MRI) were analyzed for the presence of neuronal death. Because the time course of injury visualizable by Fluoro-Jade¹³ in the immature rat typically commences at 24 hours, peaks at 3 to 4 days, and dissipates after a week,^14,15 eight animals were perfused at 24 hours, five at 48 hours, six at 4 days, and eight at 7 days and compared with three controls per time point. Animals were perfused transcardially with saline followed by buffered 4% paraformaldehyde under pentobarbital anesthesia.¹⁰ Brains were cryoprotected and sectioned coronally (30μm), and sections were processed for Fluoro-Jade visualization of neuronal death.^13,15

Statistical Considerations

Seizure-experiencing animals were compared with age-matched controls. Effects of the seizures and of age (maturation) were evaluated using analysis of variance (ANOVA), with post hoc tests as indicated. Significance was set at p value of 0.05, and values are reported as means ± SEM.

Prolonged Experimental Febrile Seizures Induce Acute T2 Signal Enhancement in Limbic Regions of Immature Rat Brain

Prolonged experimental FSs led to increased T2 signal in 75% of the rats 24 hours later, compared with age-matched controls. In general, signal changes were more pronounced at 8 days, distinguishing 87.5% of the seizure group (seven of eight animals) from their age-matched controls (Figs 1 and 2). These signal changes involved dorsal hippocampus, amygdala, and piriform cortex (see Fig 1) and were quantitatively analyzed (see Fig 2). In dorsal hippocampus, seizures increased T2 signal in a single animal at 24 hours and caused significant increase in T2 signal of the aggregate seizure group at 8 days (p < 0.01, Bonferroni's post hoc test; see Fig 2A). At that time point, the ratio of T2-weighted signal intensity in hippocampus over that of corpus callosum exceeded “1” only in rats experiencing seizures (see Fig 2F).

An effect of the seizures, with significant increases of signal intensity, was found also for other limbic regions including piriform cortex (see Fig 2B) and the medial ventroposterior thalamic nucleus (see Fig 2C). In amygdala, variability among seizure-experiencing animals was notable. Striking enhancement of T2 signals occurred in two seizure animals at both 24 hours and 8 days (see Fig 1). In these animals at 8 days, normalized signal was 1.34 ± 0.03 versus 1.16 ± 0.05 in the controls (p = 0.08; see Fig 2D). Limbic regions without seizure-evoked T2 signal changes included entorhinal cortex (see Fig 2E) and ventral hippocampus (not shown).

T2 Signal Intensity Changes in Limbic Regions Do Not Result from Neuronal Death

Enhancement of T2 signal on MRI within hours or days of a neurological insult is generally considered to indicate increased tissue water content (edema), associated with neuronal injury that may progress to death. Such progression from enhanced T2 signal to subsequent tissue atrophy and cell loss has been documented in animal models of status epilepticus.^16–18 Therefore, we studied whether cell injury and death occurred in immature rats after experimental prolonged FSs, specifically in regions demonstrating altered T2 MRI signal. We used the Fluoro-Jade method, shown by several groups to sensitively detect irreversible injury of individual neurons^13–15 and validated its use by concurrently analyzing sections from adult rats where excitotoxic death was provoked by kainic acid–induced status epilepticus.

Prolonged, kainic acid-provoked limbic seizures in adult rats resulted in the typical pattern of limbic cell death,^19,20 which was well visualized using Fluoro-Jade in hippocampal cornu ammonis 3 (CA3) region pyramidal cells, piriform cortex, and basolateral amygdala (Fig 3A–C). In contrast, prolonged experimental FSs in immature rats did not lead to observable neuronal injury either at 24 hours (see Fig 3D–F) or at other time points (not shown). These findings suggest that profound neuronal injury, such as detected by Fluoro-Jade and similar methods^8,14 is not a prerequisite for the occurrence of T2 signal intensity changes. They also affirm the relative resistance of limbic structures of infant rats to seizure-provoked cell injury.^8,15,21