Time-Restricted Feeding Schedule and Pilocarpine-Induced Seizure Model

Ninety-five young adult male Wistar rats (8 weeks of age) weighing approximately 220 g obtained from Harlan laboratories (USA) were used and maintained under constant temperature conditions (25°C) and a 12 h light/12 h dark cycle. Animals were fed with a standard diet of Lab Diet Rodent Laboratory Diet 5001 pellets (PMI Nutrition International, Inc., Brentwood MO) and water ad libitum (AL, control and pilocarpine groups) or underwent the time-restricted feeding (TRF and TRF plus pilocarpine groups) schedule described by Rivera-Zavala et al. (2011). Briefly, TRF consisted of allowing rats to feed freely for only 2 h daily for 20 days, and after this period of time, we proceeded to perform the acute seizure model at day 21. For the acute seizure model, we chose the lithium-pilocarpine model because it is one of the most widely used models to induce SE and is an excellent model that resembles human temporal lobe epilepsy (Lemos and Cavalheiro, 1995). Animals were first injected with lithium-chloride (3 mEq/kg, i.p.) at day 20; 18 h later, animals received a scopolamine methyl nitrate injection (1 mg/kg, s.c.) 30 min prior to minimize the peripheral cholinergic effects of pilocarpine. Pilocarpine was administered (60 mg/kg, s.c.) to induce SE, and the latter was maintained for 90 min; immediately afterwards, animals received an injection of diazepam (Valium²¹²², 5 mg/kg i.m.) to stop the seizures. The same procedure was performed for control animals that did not receive a pilocarpine injection but rather received a saline (0.9%) injection (Figure ​(Figure1A).1A). It is important to mention that SE was induced after 6 h of fasting in AL-pilocarpine rats and ≈ 22 h fasting in TRF-pilocarpine rats to avoid any changes in metabolism; both AL- and TRF-pilocarpine animals received a saline solution injection to avoid dehydration. Twenty-four hours after the pilocarpine injection, experimental animals were sacrificed with an overdose of sodium pentobarbital (26 mg/kg) to perform biochemical analyses. All experiments from the present study were approved by the Ethical Committee of the School of Medicine at UNAM following all of their statements to minimize animal suffering.

Influence of the time-restricted feeding (TRF) model on body weight, food intake, glucose, and β-hydroxybutyrate in the fasted/refed state and the ratio among kilocalorie consumption and basal metabolic rate (BMR) in rats. (A) Schematic representation of the experimental procedure of the dietary schedule and status epilepticus (SE) induction. Body weight of TRF rats showed a significant decrease at all time points measured (B); moreover, there is a reduction in food intake and the ratio of caloric intake/metabolic rate compared with that of ad libitum (AL)-fed animals (D,F). Regarding biochemical parameters, the blood glucose concentration showed no significant difference in the fasted state; however, AL-fed rats showed an inability to metabolize glucose at 15 and 20 days, which does not occur in TRF animals (C). Interestingly, the blood β-hydroxybutyrate concentration was high during the TRF schedule and it was maintained even in the refed state, even though it is lower than during the fasted state (E). Data are expressed as the mean ± SD from each determination (n = 30, **p < 0.01; ***p < 0.001).