Abstracts

Rationale: Acquired epilepsy (AE) results from a previous neurological insult such as stroke, traumatic brain injury (TBI), or SE, accounting for ~ 40% of all epilepsy cases. We have shown that SE causes prolonged alterations in intracellular calcium levels ([Ca²⁺]_i) that underlie the development of AE. Interventions preventing SE induced Ca²⁺ plateau could prove to be beneficial in lowering AE. Hypothermia is used clinically to prevent neurological complications associated with TBI, cardiac arrest, and stroke. Here we investigated whether hypothermia prevented the development of Ca²⁺ plateau in various models of SE. Methods: SE was induced in hippocampal neuronal cultures (HNC) by exposing them to no added MgCl₂ solution for 3-h. To terminate SE, low Mg²⁺ solution was washed with 31°C (hypothermia) or 37°C physiological solution. [Ca²⁺]_i were measured using Fura-2 ratiometry. For in vivo SE, rats (male Sprague-Dawley, 200-250 g) were treated with scopolamine (1 mg/kg, ip) 30-min prior to pilocarpine (PILO, 375 mg/kg, ip). Following 1-h SE (grade 4-5, Racine scale), diazepam (5 mg/kg, ip) at 1, 3 and 5-h terminated SE. For organophosphate induced SE, rats were injected with paraoxon (POX, 4 mg/kg, sc) followed by atropine sulfate (2 mg/kg, ip) and 2-PAM (25 mg/kg, im) 1-min later. SE was terminated by diazepam and 2-PAM injections given at 1, 3 and 5-h post SE. Hypothermia was induced at 1-h post-SE onset by spraying rats with chilled ethanol (17°C) and placing them in a cold room (5-8°C) for 8-10 min. [Ca²⁺]_i was estimated in isolated hippocampal neurons 24-h after SE using established procedures. Results: [Ca²⁺]_i was significantly elevated after in vitro SE (0.49± 0.03-SE vs. 0.26± 0.02-control). After 20-min of hypothermia, [Ca²⁺]_i in SE HNC had returned to baseline ratio of 0.25±0.01 which was not significantly different from control. In contrast, [Ca²⁺]_i remained significantly elevated (0.36±0.02) in 37°C HNC's (n=186 neurons, p<0.05, t-test). Hippocampal neurons isolated from rats 24-h after PILO and POX SE exhibited significant elevations in [Ca²⁺]_i (0.88±0.07 and 0.83±0.04 respectively vs 0.49±0.04- control). Neurons isolated from hypothermia-treated PILO or POX SE rats exhibited [Ca²⁺]_i ratios of 0.65±0.07 and 0.58±0.02 respectively, which were similar to control neurons and significantly lower than ratios obtained from PILO or POX SE (n=6 rats, p<0.05, t-test). Conclusions: These results demonstrate that hypothermia is effective at blocking the development of the Ca²⁺ plateau in both in vitro and in vivo models of SE. Formation of the SE induced Ca²⁺ plateau is dependent upon NMDA receptor activation while the maintenance is mediated via ryanodine receptors. Blocking the development of Ca²⁺ plateau has been shown to prevent the development of AE, and we have recently demonstrated that hypothermia reduces Ca²⁺ entry via NMDA and ryanodine receptors in HNC. The ability of hypothermia to reduce elevated Ca²⁺ following SE by targeting these two receptor systems could interfere with epileptogenesis and prove to be an effective therapeutic intervention for reducing SE-induced AE.