Neuroprotective effects of deep hypothermia in refractory status epilepticus

Abstract

Objective: Pharmacoresistance develops quickly during repetitive seizures, and refractory status epilepticus (RSE) remains a therapeutic challenge. The outcome of RSE is poor, with high mortality and morbidity. New treatments are needed. Deep hypothermia (20°C) is used clinically during reconstructive cardiac surgery and neurosurgery, and has proved safe and effective in those indications. We tested the hypothesis that deep hypothermia reduces RSE and its long-term consequences.

Methods: We used a model of SE induced by lithium and pilocarpine and refractory to midazolam. Several EEG measures were recorded in both hypothermic (n = 17) and normothermic (n = 20) animals. Neuronal injury (by Fluoro-Jade B), cell-mediated inflammation, and breakdown of the blood-brain barrier (BBB) (by immunohistochemistry) were studied 48 h following SE onset.

Results: Normothermic rats in RSE seized for 4.1 ± 1.1 h, and at 48 h they displayed extensive neuronal injury in many brain regions, including hippocampus, dentate gyrus, amygdala, entorhinal and pyriform cortices, thalamus, caudate/putamen, and the frontoparietal neocortex. Deep hypothermia (20°C) of 30 min duration terminated RSE within 12 min of initiation of hypothermia, reduced EEG power and seizure activity upon rewarming, and eliminated SE-induced neuronal injury in most animals. Normothermic rats showed widespread breakdown of the BBB, and extensive macrophage infiltration in areas of neuronal injury, which were completely absent in animals treated with hypothermia.

Interpretation: These results suggest that deep hypothermia may open a new therapeutic avenue for the treatment of RSE and for the prevention of its long-term consequences.

Figure 1

Experimental flow. Status epilepticus (SE) was induced by lithium administration followed by pilocarpine injection. Midazolam (3 mg/kg) was injected 12 min after the second “stage 3” seizure. Then the animals were placed in ice packs and cooled to 20°C for 30 min. In early cooling, hypothermia was initiated after midazolam injection. In delayed cooling, hypothermia was initiated 15 min after midazolam only in animals that were refractory to midazolam. When midazolam alone stopped SE, the animals were discarded.

Figure 2

Delayed hypothermia transiently reduces relative EEG power. (A) The upper panel shows the compressed 4‐hr EEG of a normothermic (red) and a hypothermic rat (blue, 20°C for 30 min) with rectal temperature. Both rats received midazolam 3 mg/kg ip (arrow) and cooling was initiated 15 min later. The lower panel shows the magnified 4‐sec EEG traces marked by vertical lines (a‐d) (vertical bar = 0.5 mV; horizontal bar = 1 sec). (B) The left y‐axis of this graph shows the ratio of EEG power at each time points to initial EEG power at baseline, before pilocarpine injection. Note the logarithmic scale. Time points between 15 and 135 min following the initiation of cooling showed a significant difference between normothermic and hypothermic rats (***P < 0.001; **P < 0.01; *P < 0.05). The right y‐axis shows the rectal temperature scale.

Figure 3

Reduction of neuronal injury in animals treated with deep hypothermia. (A and B) These images show fluoro‐jade B staining in CA1 area 48 h following status epilepticus (SE) in a normothermic (A) and hypothermic (B) rat. Note that background of hypothermic hippocampus was enhanced to show anatomy. (C) This graph shows the number of fluoro‐jade B‐ positive CA1 cells counted by an unbiased stereological method. Deep (delayed) hypothermia significantly reduced CA1 injury. (D–I) These images show fluoro‐jade B staining in the hilus of dentate gyrus (D–E), the frontoparietal neocortex (F and G), thalamus (H) and the piriform cortex (I) 48 h following SE in normothermic (D, F, H, and I) and hypothermic (E and G) animals. Bars = 100 microns.

Figure 4

Reduction in blood–brain barrier breakdown and of macrophages in animals treated with deep hypothermia. (A and B) These images show IgG‐like immunoreactivity in the frontoparietal cortex 48 h following status epilepticus in a normothermic (A) and hypothermic (B) animal. Bars = 100 microns. (C–F) These images show staining with a macrophage marker in frontoparietal cortex (C and D), hippocampus (E) and piriform cortex (F) of normothermic (C, E, and F) and hypothermic animals (D). Bars = 100 microns.