The lithium-pilocarpine model is widely used to study temporal lobe epilepsy (TLE), but its utility is limited by high and unpredictable mortality during status epilepticus (SE). Reliable epileptogenesis requires 30–60 min of SE, yet this duration greatly increases animal loss, reducing reproducibility and raising ethical concerns.

This study evaluated the dose-dependent effect of diazepam administered 60 min after SE onset on mortality, with the aim of identifying an optimal dosing strategy that minimizes mortality without abolishing epileptogenesis.

Male Wistar rats (n = 100) were divided into four groups: control (no diazepam), diazepam 10 mg/kg, 20 mg/kg, and 30 mg/kg. 9 animals were excluded across groups: 7 due to death during SE before diazepam administration and 2 due to pilocarpine resistance (failure to develop SE). SE was induced with lithium chloride and pilocarpine. Diazepam was given intraperitoneally 60 min after SE onset. Mortality was monitored for 14 days and analyzed using chi-square and Fisher’s exact tests.

Mortality was 100% in controls (10/10), 62.1% at 10 mg (18/29), 34.6% at 20 mg (9/26), and 23.1% at 30 mg (6/26). Group differences were significant (χ2 = 9.23, df = 2, p = 0.0099). Post-hoc analysis showed lower mortality at 30 mg versus 10 mg (p = 0.006), while differences between 20 and 30 mg were not significant, indicating possible saturation.

Diazepam reduces mortality in the lithium-pilocarpine SE model in a clear dose-dependent manner. High doses (20–30 mg/kg) offer the greatest survival benefit and provide a methodological solution to improve reproducibility, reduce animal loss, and support ethical implementation of this widely used model.