4. Discussion
The major findings of our present work demonstrate that Fer-1 could alleviate seizures in the acute stage and improve cognitive deficit in a mouse model of PTE with good safety and tolerability. Suppression of ferroptosis process may involve in the therapeutic effect of Fer-1 against PTE.
PTE is a very common acquired epilepsy, which is usually caused by TBI. Injection of FeCl3 in different brain regions such as somatosensory cortex (Willmore et al., 1978b) and amygdalar nuclear complex (Ueda et al., 2003) of rodents is useful to study the behavior and neuropathological characteristics of PTE. Notably, as early as 1978, Willmore et al. for the first time reported that unilateral injection of FeCl3 into the somatosensory cortex triggered the onset of seizures and epileptogenesis (Willmore et al., 1978b). Generally, epileptic discharges from the sensorimotor cortex are induced 15 minutes after FeCl3 injection and can last for more than 10 months (Moriwaki et al., 1992). Posttraumatic seizures are classified into three categories depending on the time delay from the TBI to the occurrence of the first seizure as follows: (1) immediate (<24 h); (2) early (1-7 d) and (3) late seizures (>7 d after TBI) (Zimmermann et al., 2017). Until now, the mechanism underlying the seizure generation is not well characterized. Our previous investigation has for the first time illustrated that ferroptosis, a novel type of cell death which is distinct from apoptosis, necroptosis, autophagy and so on, occurs in FeCl3-induced PTE (Li et al., 2019). In the present work, we revealed that Fer-1, a potent and selective inhibitor of ferroptosis (Dixon et al., 2012), ameliorated early seizures as it was observed that epileptiform discharge was significantly suppressed from day 1 to day 3 after FeCl3injection followed by Fer-1 treatment (Figure 2G). Similarly, our prior work also illustrated the protective effects of Fer-1 against seizure in various rodent models of epilepsy induced by pilocarpine or pentylenetetrazole (PTZ) (Mao et al., 2019). In vivo seizure model induced by kainic acid (KA), there are controversial results which show that Fer-1 counteracts KA-induced seizures in our previous study (Jia et al., 2020) while spontaneous seizures are not attenuated by Ye et al (Ye et al., 2019). It indicates that Fer-1 has different effects on distinct seizure models induced by different chemical reagents. In addition, it was also noted in our present work that treatment with Fer-1 did not evidently affect late seizures, indicating that Fer-1 hardly affects epileptogenesis. This is due to the notion that patients with chronic epilepsy usually exhibit neuropathological traits such as astrogliosis, mossy fiber and hippocampal sclerosis (Boison, 2008; Deshpande et al., 2020; Thom et al., 2011). On this occasion, cell death may not be dominant at this chronic stage.
Cognitive deficits are always very prevalent in patients with PTE (Jensen, 2009). The hippocampus is usually more vulnerable than other brain regions to memory processes (Cho et al., 2015; Fortin et al., 2002; Voss et al., 2017). In the present study, hippocampus-dependent learning and memory function was evaluated using two techniques, namely, NOR and MWM. Our findings demonstrated that Fer-1 ameliorated cognitive dysfunction in a mouse model of PTE. However, no significant difference was found in the aspect of spontaneous seizure on the chronic stage after Fer-1 treatment. It indicates the improvement of cognitive deficits by Fer-1 in PTE is not attributable to affecting spontaneous seizure. In line with our results, previous study in vivo KA-induced temporal lobe epilepsy, treatment with Fer-1 was also reported to attenuate cognitive dysfunction without suppressing SRS (Ye et al., 2019). Given that no evident toxic effect of Fer-1 on the normal mice, it indicates that Fer-1 may serve as a good candidate for improving cognitive deficits in PTE.
Ferroptosis is a novel type of regulatory cell death featured by lethal lipid peroxidation (Dixon et al., 2012). In our present work, toxic lipid hydroperoxide by-product 4-HNE was analyzed. It was demonstrated that elevation of 4-HNE level was detected in PTE. However, treatment with Fer-1 significantly decreased this index, indicating that inhibition of ferroptosis process in PTE when treatment with Fer-1. It was surprising that the activity of GPx, key enzymes converting toxic lipid hydroperoxides into non-toxic lipid alcohols, was increased in PTE while Fer-1 diminished GPx activity. It is possible that following PTE, enhanced GPx activity in order to counteract overproduction of lethal lipid hydroperoxides. In fact, prior work also illustrated the increase of GPx in TBI (Alim et al., 2019). Further investigation is indispensable to clarify this speculation.
In summary, we report that Fer-1 ameliorates seizure behavior in the acute stage of PTE and abrogates cognitive impairment in the chronic stage of PTE. Together with the results that Fer-1 exhibits no significant side effects in our present work, it indicates that Fer-1 may serve as a promising drug for curing patients with PTE. Besides, ferroptotic process may be associated with the protective effect of Fer-1 against seizure-induced brain damage. It warrants further investigation to explore the molecular mechanism underlying therapeutic potential of Fer-1 against PTE.