Strain differences in the extent of brain injury in mice after tetramethylenedisulfotetramine-induced status epilepticus

Abstract

Acute intoxication with tetramethylenedisulfotetramine (TETS) can trigger status epilepticus (SE) in humans. Survivors often exhibit long-term neurological effects, including electrographic abnormalities and cognitive deficits, but the pathogenic mechanisms linking the acute toxic effects of TETS to chronic outcomes are not known. Here, we use advanced in vivo imaging techniques to longitudinally monitor the neuropathological consequences of TETS-induced SE in two different mouse strains. Adult male NIH Swiss and C57BL/6J mice were injected with riluzole (10 mg/kg, i.p.), followed 10 min later by an acute dose of TETS (0.2 mg/kg in NIH Swiss; 0.3 mg/kg, i.p. in C57BL/6J) or an equal volume of vehicle (10% DMSO in 0.9% sterile saline). Different TETS doses were administered to trigger comparable seizure behavior between strains. Seizure behavior began within minutes of TETS exposure and rapidly progressed to SE that was terminated after 40 min by administration of midazolam (1.8 mg/kg, i.m.). The brains of vehicle and TETS-exposed mice were imaged using in vivo magnetic resonance (MR) and translocator protein (TSPO) positron emission tomography (PET) at 1, 3, 7, and 14 days post-exposure to monitor brain injury and neuroinflammation, respectively. When the brain scans of TETS mice were compared to those of vehicle controls, subtle and transient neuropathology was observed in both mouse strains, but more extensive and persistent TETS-induced neuropathology was observed in C57BL/6J mice. In addition, one NIH Swiss TETS mouse that did not respond to the midazolam therapy, but remained in SE for more than 2 h, displayed robust neuropathology as determined by in vivo imaging and confirmed by FluoroJade C staining and IBA-1 immunohistochemistry as readouts of neurodegeneration and neuroinflammation, respectively. These findings demonstrate that the extent of injury observed in the mouse brain after TETS-induced SE varied according to strain, dose of TETS and/or the duration of SE. These observations suggest that TETS-intoxicated humans who do not respond to antiseizure medication are at increased risk for brain injury.

Keywords: MRI; Neurodegeneration; Neuroinflammation; PET; Tetramine.

Fig. 1.. Dosing paradigm and imaging schedule.

Adult mice were pretreated with riluzole 10 min prior to the administration of vehicle or tetramethylenedisulfotetramine (TETS). NIH Swiss mice were injected with TETS (0.2 mg/kg) or VEH, and C57BL/6J were injected with TETS (0.3 mg/kg) or VEH. 40 min later, a rescue dose of midazolam (MDZ) was administered to stop seizure behavior. Brains of TETS-intoxicated and vehicle control mice were imaged using MRI and PET at 1, 3, 7, and 14 days post exposure. Sample sizes for each strain and exposure group are shown in the table to indicate repeated imaging time points within subjects for mice in this study.

Fig. 2.. Acute TETS intoxication-induced neuropathology, as detected by diffusion-weighted imaging (DWI).

Representative anatomical (T2W, left column) and parametric maps (DWI, right column) of vehicle (VEH) and TETS-intoxicated C57BL/6J and NIH Swiss mice. Geometric mean ratio (dot) of the standard deviation (SD) of the apparent diffusion coefficient (ADC SD) average for TETS vs. VEH mice with 95% confidence intervals (bars). Confidence intervals that do not include 1 (gray line) indicate a significant difference between TETS and VEH (those identified in turquoise survived the FDR correction). All VEH groups had n = 3 for each time point. Sample sizes for TETS time points: Day 1: n = 8–9; Day 3: n = 8–9; Day 7: n = 7–8; Day 14: n = 3. Individual data points used to generate this figure can be found in the supplemental material (Tables S1 and S2).

Fig. 3.. Acute TETS intoxication-induced neuroinflammation, as detected by TSPO PET imaging.

Representative anatomical (T2W, left column) and parametric maps (TSPO PET, right column) of vehicle (VEH) and TETS-intoxicated C57BL/6J and NIH Swiss mice. Geometric mean ratio (dot) of the TSPO standard uptake value (SUV) for TETS vs. VEH mice with 95% confidence intervals (bars). No statistically significant differences were detected between brain regions for NIH Swiss mice, so these data were collapsed. Confidence intervals that do not include 1 (gray line) indicate a significant difference between TETS and VEH (those identified in turquoise survived the FDR correction). All VEH groups had n = 3 for each time point. Sample sizes for TETS time points: Day 1: n = 8–9; Day 3: n = 8–9; Day 7: n = 7–8; Day 14: n = 3. Individual data points used to generate this figure can be found in the supplemental material (Table S3).

Fig. 4.. Seizure duration correlates with severity of neuropathology at 7 days post-exposure in NIH Swiss mice acutely intoxicated with TETS.

(A) Representative anatomical T2W MRI (first column), diffusion-weighted MRI (second column), TSPO PET (third column), and histology (last columns) images from VEH mouse (top row), TETS-intoxicated mouse that experienced SE for 40 min (middle row), and TETS-intoxicated mouse that exhibited SE > 120 min (bottom row). The middle row shows a TETS animal that responded normally to rescue by midazolam at 40 min after seizure initiation (40 min seizure), whereas the bottom row is of a TETS animal that continued to seize after midazolam treatment yet survived (>120 min seizure). Arrows point to regions of hypointensity (T2W & DWI) and high radiotracer uptake (TSPO PET), indicating neurodegeneration and neuroinflammation, respectively. IBA-1 (red) identifies microglia; FJC (green), degenerating neurons; DAPI (blue), nuclei. (B) Quantitative data table reporting regional ADC SD, average PET SUV, % area IBA1-positive, and FJC-positive cells/mm² from the vehicle (n = 1), TETS SE (40 min; n = 1), and prolonged TETS SE (>120 min; n = 1) mice.