Pilocarpine-Induced Status Epilepticus

Male Sprague Dawley rats (Charles River, Wilmington, MA) were housed in a controlled environment with food and water ad libitum. Animal experiments were performed in accordance with Institutional Animal Care and Use Committee regulations and protocols approved by the University of Colorado Anschutz Medical Campus. Status epilepticus was induced at 8–9 weeks of age according to a previously reported protocol (Brooks-Kayal et al., 1998; Shumate et al., 1998). To reduce the peripheral effects of pilocarpine, an intraperitoneal (i.p.) injection of scopolamine methyl nitrate (1 mg/kg, Sigma, St. Louis, MO) was applied 30 min before administration of pilocarpine hydrochloride (385 mg/kg i.p, Sigma, St. Louis, MO). If rats did not exhibit convulsive seizures 1 h after pilocarpine injection, a second or third dose of pilocarpine (192.5 mg/kg) was administered in order to achieve seizure equivalence between animals. To slow down seizure progression and decrease mortality, diazepam (6 mg/kg, i.p.; Hospira, Lake Forest, IL) was administered 1 h after SE onset and additional doses (3 mg/kg, i.p.) were administered every 2 h if seizures persisted. Control rats were handled similarly but received a subconvulsive dose of pilocarpine (38.5 mg/kg, i.p.) and 1/10 of the dose of diazepam (0.6 mg/kg, i.p.). As criteria for inclusion, all rats used had confirmed stage 5 behavioral seizures.

MDL-28170 is cell permeable peptide that inhibits both calpain-1 and calpain-2 (Markgraf et al., 1998; Thompson et al., 2010). To evaluate if MDL-28170 (50 mg/Kg, i.p., Bachem, Torrance, CA) prevented cellular alterations linked to epileptogenesis, two treatment paradigms were used: a low-dose treatment consisting of two acute injections applied at 1 and 5 h after SE onset with a final dose the following morning; and, a high-dose treatment that consisted of four acute doses at 1, 3, 5 and 9 h after SE onset with a final dose the following morning. A third group, SE plus vehicle (Veh), was a composed of rats that received vehicle injections (DMSO) with the same frequency of the low- and high-dose. The vehicle group was predicted to undergo all pathophysiological events promoting SRS and was used as reference to evaluate possible disease-modifying effects of MDL-28170. To determine if MDL-28170 altered seizure burden, in addition to the acute doses, daily doses of the drug were administered at 1, 2 and 3 days post-SE. Animals were randomly assigned to each of these groups. The concentration and administration frequency were chosen based in studies describing MDL-28170 delivery to the brain (Li et al., 1998; Markgraf et al., 1998; Araujo et al., 2008). MDL-28170 has a plasma half-life of 1 to 2 hours and is capable of penetrating the blood-brain barrier and cell membranes. In naïve rats, a single intravenous bolus administration of MDL-28170 (30 mg/kg) resulted in protease inhibition within the brain in 30 min and declined over a period of 4 hours with a half-life of approximately 2 hours and no apparent toxicity (Markgraf et al., 1998).