Mitochondrial oxidative stress is the leading cause of age-related degenerative diseases and may contribute profoundly to seizure instigation (Beal, 2005; Waldbaum and Patel, 2010). Oxidative mitochondrial damage accelerates neuronal excitability via affecting mitochondrial roles crucial for normal brain functioning (Rowley and Patel, 2013). The deleterious role of mitochondrial dysfunction in epileptogenesis stems from the correlation between epilepsy and the frequent incidence of inherited mitochondrial disorders such as those occurring with childhood encephalopathies (Waldbaum and Patel, 2010). Parallel to the impediment of sufficient adenosine triphosphate (ATP) production by neurons, the resultant mitochondrial damage affects antioxidant defenses, fatty acid oxidation, neurotransmitter biosynthesis, regulation of cytosolic Ca²⁺, and synaptic glutamate homeostasis (Waldbaum and Patel, 2010). Mitochondrial dysfunction is, hence, implicated in excessive neuronal excitability and epileptogenesis. It accelerates apoptotic neuronal death following glutamate excitotoxicity, which independently participates in seizure-induced neuronal loss especially in the hippocampus (Nicholls and Ward, 2000; Noh et al., 2006). The inner mitochondrial membrane protein, uncoupling protein 2 (UCP2), could largely preserve mitochondrial membrane potential and hamper mitochondrial oxidative stress through controlling mitochondrial-derived reactive oxygen species (Pierelli et al., 2017).

Glutamate transporters are responsible for preserving synaptic glutamate in non-neurotoxic levels (Todd and Hardingham, 2020). Though they are highly expressed on both neurons and neuroglia, it has been supposed that astroglial transporters, namely, GLT-1 and GLAST, are responsible for the uptake of the majority of glutamate (Danbolt, 2001). Their dysfunction may accordingly contribute to an increase in glutamate beyond the normal levels, promoting seizure development. This was further verified when the use of threo-β-benzyloxyaspartate, a glutamate transport inhibitor, extended epileptiform activity (Shimamoto et al., 1998). GLT-1 knockout mice develop spontaneous seizures (Tanaka et al., 1997), while those lacking GLAST show high susceptibility to seizures (Ueda et al., 2002). GLT-1 and GLAST hypofunction or hypo-expression has been observed with aging and is indeed implicated in several neurodegenerative disorders, including epilepsy (Todd and Hardingham, 2020). The age-related decline in astroglial glutamate transporters in mitochondrial superoxide dismutase knockout mice coincided with epileptic seizure susceptibility (Liang and Patel, 2004). Glutamate transporters are highly vulnerable to oxidative damage resulting in the impairment of glutamate uptake machinery (Trotti et al., 1998). These observations imply that mitochondrial oxidative stress and secondary dysfunction may be sufficient to increase seizure vulnerability through modifications of astroglial glutamate transporters.

Trimetazidine [1-(2,3,4-trimethoxybenzyl) piperazine dihydrochloride] (TMZ) is mostly used as an anti-ischemic drug. It optimizes oxygen demands via shifting fatty acid oxidation to glucose oxidation (Peng et al., 2014). Potentiation of glucose oxidation in an ischemic cell preserves cellular ATP, as energy obtained during glucose oxidation requires less oxygen than fatty acid oxidation (Sandhiya, 2015). TMZ was reported to prevent cerebral ischemia-reperfusion injury in experimental animals (Dhote and Balaraman, 2008). It improved brain activity by increasing brain glucose uptake (Nowak et al., 2006), conserving brain mitochondrial membrane, and protecting neuronal cells against intracellular acidosis (Jain et al., 2010). It also augmented the central ATP level (Al-Kuraishy and Al-Gareeb, 2017) and hindered hippocampal oxidative damage via the upregulation of antioxidant enzymes and inhibition of lipid peroxidation in an animal model of Alzheimer’s disease (Hassanzadeh et al., 2015). TMZ profoundly elevated seizure threshold in the increasing-current electroshock seizure test (Jain et al., 2010) and prevented pentylenetetrazol (PTZ)-induced kindling in mice (Jain et al., 2011). The exact mechanistic rationale behind its anti-seizure potential, however, has not been fully elucidated yet.

The current study was, therefore, conducted to investigate whether TMZ could modulate mitochondrial redox status and glutamate homeostasis in the hippocampi of rats following lithium/pilocarpine (Li/PIL) injection. It also explored the impact of such effects on the incidence and intensity of induced seizures.

The current investigation emphasizes the efficacy of TMZ in reducing the incidence and severity of seizures in rats subjected to Li/PIL. This anti-seizure potential was associated with a marked reduction in the hippocampal glutamate content along with a noticeable upregulation of the gene expression of astroglial transporters: GLT-1 and GLAST. These effects were correlated with the ability to preserve the integrity of neurons in the different hippocampal regions.

Disrupting the complex neuroglial circuitry has been shown to robustly contribute to neurological disorders including epilepsy (Diaz Verdugo et al., 2019). Any change in the efficiency of glutamate clearance, especially by astrocytic phenotypes, largely affects synaptic function and accordingly seizures susceptibility (Wallraff et al., 2006). The depletion of astrocytic transporters in various neurodegenerative diseases supports the strong association between the accumulation of neurotoxic levels of glutamate and reactive astrogliosis (Rothstein et al., 2005). Striking morphological and function changes are detected in reactive astrocytes altering neurotransmitter homeostasis to provoke neuronal hyperexcitability (Seifert and Steinhäuser, 2013). Preictal astrocyte activation that shortly begins after a brain insult is likely to be an adaptive response to remove damaged tissues and restore normal function (Sanz and Garcia-Gimeno, 2020). Consistently, initial astrocytes activity is directed to hamper neural activity outbreaks (Diaz Verdugo et al., 2019). During this state, the highly synchronous glial cells activity ensures their homeostatic function. Glial cells networks can efficiently reallocate (Danbolt, 2001) and absorb excessive cations and glutamate by various astrocytes transporters (Pines et al., 1992). Nevertheless, excessive astrogliosis exhausts this homeostatic function and exacerbates inflammatory response, triggering hyperexcitability of neurons and generalized seizures. The mounting efflux of K⁺ around astrocytes accompanied with glutamate uptake reverses the working way of transporters and abruptly alters the harmonized glia–neuron interaction (Danbolt, 2001).

In the current study, GFAP was used as a tool to assess astrogliosis during seizures. Rats injected with Li/PIL showed a massive expression of GFAP in the CA1, CA3, and hilar regions of the hippocampi, which were shown to be the same sites of injury by histopathological examination. Our findings harmonize with previous studies that revealed a rapid amplified GFAP expression in various animal models of epilepsy, including PTZ (Torre et al., 1993; Sedky et al., 2017), electrical kindling (Stringer, 1996; Miyazaki et al., 2003), and kainic acid (Bendotti et al., 2000; Choi et al., 2003; Glushakova et al., 2012; Lee et al., 2012). Similarly, Alese and Mabandla (2019) recorded a marked increase in GFAP hippocampal expression 30 min following PIL administration in rats with or without a history of prolonged febrile seizure. Genetically induced extensive astrogliosis in mice disrupted glutamate uptake and triggered spontaneous seizures (Robel et al., 2015). In the same context, a previous study synchronizes with our finding, where the anticonvulsant activity of TMZ in PTZ-treated mice was associated with a marked decrease in hippocampal GFAP expression (Sedky et al., 2017).

In the current study, enhanced GFAP expression was also accompanied with an increase in the hippocampal glutamate level as well as a marked reduction in mRNA expression of both GLT-1 and GLAST. In agreement with these results, astrocytes activation was coupled with decreased GLT-1 and extracellular glutamate flooding in models of peripheral nerve injury (Cavaliere et al., 2007; Cirillo et al., 2011). Furthermore, the expression of GLAST and GLT-1 has been diminished in the cortices as well as in the hippocampi of rats with genetic absence seizures (Dutuit et al., 2002) and in a mouse model of focal epilepsy (Ingram et al., 2001).

Interestingly, GFAP and p-ERK1/2 were found to be co-expressed in the hippocampi of mice subjected to PIL-induced status epilepticus after 6 h. However, GFAP, but not p-ERK1/2, was still expressed after 3 days (Li et al., 2009). Similarly, Choi et al. (2003) reported a rapid and transient p-ERK1/2 increase in hippocampal neurons and astrocytes following kainic acid–induced seizures. These results go in line with the present study and suggest the possible contribution of p-ERK1/2 in the protective effect of astrogliosis in the early stages of epilepsy. In the same context, p-ERK1/2 was previously claimed to mediate GLT1 expression in astrocytes (Frizzo et al., 2007); however, its elevated level attained after Li/PIL injection in the present study was probably not enough to increase the gene expression of glutamate transporters. In addition, during astrogliosis, the activated protease, calpain-I, was previously accused of astrocyte transporters degradation (Cavaliere et al., 2007). Coherent with these results, Berkeley et al. (2002) demonstrated that PIL caused initial ERK activation, while its inhibition had not prevented PIL-induced seizures, yet increased its severity. The further increase in p-ERK1/2 observed after TMZ may be a partial mechanism through which it could increase mRNA expression of astroglial transporters and, consequently, glutamate uptake.

Simultaneously, mitochondrial dysfunction associated with the obvious alterations in mitochondrial oxidative status exerts an important role in modulating glia–neuron interplay (Jain et al., 1991; Wallace et al., 1992; Lin et al., 2020). Li/PIL injection in the current study provoked a state of mitochondrial oxidative burst as manifested by diminished TAC and GSH along with enhanced lipid peroxidation. In the same context, Waldbaum et al. (2010) reported a persistent perturbation of mitochondrial redox status during acute and chronic phases of Li/PIL-induced epilepsy. Under the condition of ATP deficiency coupled with mitochondrial oxidative damage as demonstrated herein, the expression and function of astroglial transporters are disrupted (Frizzo et al., 2007). Though extracellular ATP release from damaged neurons succeeded in inciting GLT1 expression via ERK1/2 activation (Neary et al., 1999, Neary et al., 2003), the reduced ionic gradient following energy failure may refute the driving force essential for glutamate uptake (Kauppinen and Swanson, 2007). Accordingly, synaptic glutamate increase was ascribed to glutamate uptake impairment (Colangelo et al., 2014) paralleled with glutamate efflux by the reversed action of transporters (Seki et al., 1999; Phillis et al., 2000). Additionally, the enhancement of hippocampal large-conductance Ca²⁺-activated K⁺ channels in response to redox status change may pose a plausible mechanism toward increasing hippocampal neuronal excitability after exposure to extracellular glutamate (Waldbaum and Patel, 2010).

TMZ was effective in restoring mitochondrial oxidative homeostasis as evident by the enhanced TAC and restored GSH. Ultimately, it cured lipid peroxidation, an effect that reflected on mitochondrial efficacy in ATP production and, consequentially, function and expression of astroglial glutamate transporters. The activity of TMZ on glial uptake of glutamate was previously documented in an in vitro study using rat retinal Müller cell line. It revered glial transporter inhibition and protected the retina from ischemia-induced excitotoxicity (Payet et al., 2004). In agreement with our results, Jain et al. (2011) attributed the anticonvulsant activity of TMZ following PTZ-induced kindling in mice mainly to its robust antioxidant activity. The well-recognized antioxidant properties of TMZ are thought to be mediated indirectly by boosting the anti-oxidant enzymes (Tikhaze et al., 2000). This effect may be linked to the ability of TMZ to increase p-AMPK, which largely participates in restoring neuronal energy balance (Ronnett et al., 2009). AMPK activators were reported to induce nuclear retention of antioxidant transcription factors as forkhead box O1 (Yun et al., 2014) and nuclear factor erythroid 2–related factor 2 (Joo et al., 2016). In the same context, TMZ increased the ATP/ADP ratio in the hippocampus of diabetic epileptic rats (Mohamed et al., 2020). In addition, it restored ATP synthesis after cerebral mitochondrial respiration inhibition by cyclosporin (Zini et al., 1996).

Intracellular Ca²⁺ accumulation following excitotoxic insults was reported not only to participate in further mitochondrial ROS production and ATP synthesis inhibition but also inevitability to the activation of membrane transition pores and Cyt c release, leading to apoptotic neuronal death (Sullivan et al., 2005), as demonstrated herein by the increase in caspase-3 activity. The increase in mitochondrial UCP2 observed herein and previously (Diano et al., 2003) is postulated to reduce ROS production and guard against neuronal death. Usually, enhanced UCP2 in response to various neuronal stress is one of the neuroprotective responses, by increasing the number of mitochondria and consequently ATP production. However, these beneficial effects were obviously established after 5 days in a Li/PIL animal model (Dutra et al., 2018). Furthermore, hippocampal neurons employ another survival response via boosting ERK expression (Berkeley et al., 2002) to act in a mutual intervention with ERK induced in astrocytes during initial astrogliosis. The implication of ERK1/2 in neuroprotection in response to excitotoxicity was previously attributed to its ability to upregulate the antiapoptotic Bcl2 (Ortuño-Sahagún et al., 2014). In addition, phosphorylation of Kv4.2, fast-inactivating A-type potassium channels present postsynaptically, by activated ERK1/2 enables their localization to areas of hyperactivity in the hippocampus. Consequently, they accelerate repolarization and limit firing of action potential to guard against lethal seizures and neuronal death (Berkeley et al., 2002).

In the present study, the success of TMZ in limiting seizure incidence was associated with a marked reduction in apoptotic neuronal death rate as manifested by the obvious decrease in hippocampal Cyt c and caspase-3 activity. The anti-seizure as well as the antiapoptotic effects of TMZ could be linked to the potentiating effect of ERK phosphorylation in both neurons and glial cells. In addition, the effect of TMZ on AMPK could participate in its antiapoptotic activity via endorsing high mitochondrial membrane potential to preserve Ca⁺² homeostasis (Zhang et al., 2017). In agreement with these results, Liu et al. (2016) reported that TMZ could protect cardiomyocytes against ischemic injury via activation of both AMPK and ERK. Intriguingly, purified rat brain mitochondria have been shown to express binding sites for TMZ (Morin et al., 2000). Former in vitro studies advocated that TMZ could inhibit mitochondrial permeability transition pores' opening (Rothstein et al., 1992; Argaud et al., 2005) and thus hinder apoptotic neuronal loss following hyperexcitability.

In conclusion, TMZ showed a promising anti-seizure potential that was evident on seizure score as well as on various biochemical indicators. The beneficial actions of TMZ could be attributed to its ability to decrease mitochondrial oxidative damage as well as glutamate accumulation, parallel to a positive modulation of p-ERK1/2/p-AMPK signaling. Also, it succeeded in reducing ATP-dependent energy disruptions as well as astrocytes activation. This was all reflected as a reduction in neuronal apoptosis and preserved cellular integrity.