An involvement of COX and 5‐LOX pathways in the penicillin‐ and pentylenetetrazole (PTZ)‐induced epilepsy models

This study aimed to examine the relationship between epilepsy and COX/5‐LOX inflammation pathways in the penicillin and pentylenetetrazole (PTZ)‐induced epilepsy models. For this purpose, 42 albino male Wistar rats were used in this study. In the penicillin and PTZ‐induced epilepsy models, epileptiform activity was induced by injection of penicillin (500 IU, i.c.) and PTZ (35 mg/kg, i.p., three times a week), respectively. Licofelone (20 mg/kg, i.p.), a dual inhibitor of COX/5‐LOX, and esculetin (20 mg/kg, i.p.), a 5‐LOX inhibitor, were given. In the penicillin‐induced epilepsy model, ECoG activity was recorded for 180 min. In the PTZ‐induced epilepsy model, both ECoG activity was recorded, and behavioral parameters were performed. In the penicillin groups, both licofelone and esculetin decreased the mean spike frequency and amplitude during the experiments. In the PTZ groups, licofelone (20 mg/kg, i.p.) was more effective than esculetin (20 mg/kg, i.p.). Licofelone showed its protective effects both in ECoG activity and in behavioral parameters. Esculetin was less effective when compared to licofelone. The electrophysiological and behavioral data from the present study indicated that inflammation pathways might have a crucial role in controlling epileptiform activity in rats. Licofelone might be a valuable candidate in advanced studies.


| INTRODUCTION
According to ILAE, epilepsy be considered to be a disease of the brain defined by any of the following conditions: (1) at least two unprovoked (or reflex) seizures occurring >24 h apart; (2) one unprovoked (or reflex) seizure and a probability of further seizures similar to the general recurrence risk (at least 60%) after two unprovoked seizures, occurring over the next 10 years; and (3) diagnosis of an epilepsy syndrome [1]. In general, epileptic seizures predicate a disequilibrium between inhibitory (GABAergic) and excitatory (glutamatergic) neurotransmission. However, the pathophysiology of epilepsy is still unclear [2]. In many cases, antiseizure medications (ASMs) can partially control seizures and help patients to maintain their everyday lives. Although advanced in ASMs and research, seizures occur in approximately 30% of patients with epilepsy [3]. In this manner, many factors have essential roles in the pathophysiology of epilepsy, and we must consider these, including inflammation, in the treatment of epilepsy.
Inflammation is a complex process, and in general, it is the defense state against stimuli such as injury, trauma, microbial activity, and toxin in the body [4]. Initially, metabolized arachidonic acid through cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways play a critical role in epilepsy and inflammation. COX is the critical enzyme that converts arachidonic acid to prostaglandins (PGs) [5]. Arachidonic acid causes some alterations and inflammatory mediators' production on microglia, astrocytes, and brain capillary endothelial cells. The activation of inflammatory mediators such as COX-2 and nuclear factor kappa B (NF-κB) and the overproduction of downstream inflammatory factors including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2) contribute to the pathophysiology of seizures [6]. Emerging evidence suggests that inflammatory pathways play a crucial role in the pathophysiology of epilepsy [7]. In general, increased neuroinflammatory activity appears detrimental since anti-inflammatory treatments are successful in reducing brain pathology in animal models of CNS injury [8]. Similarly, PG analogs could decrease the number of epileptic bursts in generalized penicillininduced epilepsy [9].
Although there are various reports regarding the increase of COX enzyme following seizure activity [10], some studies have conflicting results. Selective and nonselective COX inhibitors can cause a delay in the progress of seizure [11], increase the seizure threshold [12], or worsen the seizure activity [13]. These effects can vary according to ligand type, seizure type, the dose of agents, injection time, etc. In this manner, the acute or chronic phase of inflammation might have a vital role in the pathophysiology of epilepsy.
The effects of the 5-LOX pathway on epilepsy are not as effective as the COX pathway; therefore, the 5-LOX pathway has received less attention. Kim et al showed that 5-LOX inhibitors do not have protective effects alone, but when aspirin, a COX-2 inhibitor, and esculetin, a 5-LOX inhibitor, were combined, the protective effects against neurotoxicity appeared in the PTZ-induced seizure [14]. Similarly, acetyl-11-keto-β-boswellic acid (AKBA), a 5-LOX inhibitor, did not affect the seizure activity in the kainic acid-induced epilepsy. However, AKBA + indomethacin increased the seizure latency [15].
A number of animal studies showed that both COX and 5-LOX pathways have contradictory results [11][12][13]. In addition, there can be different effects on seizures in terms of acute or chronic phase inflammation. With this background, the present study was aimed to explain the effects of COX and 5-LOX in the penicillin and PTZ-induced epilepsy models.

| Animals
In the present study, 42 male Wistar Albino rats (180-250 g, 8 weeks old) were used. The animals were maintained under standard laboratory conditions with a 12/12-h light/dark cycle, 60 AE 3% humidity level, and 22 AE 4 C constant room temperature. They had ad libitum access to water and food. All experiments were conducted between 09:00 and 17:00 h. The animals were purchased from Erciyes University Experimental Research and Application Center. This study was approved by the Ethical Committee for Animal Experiments at Erciyes University (approval number: 17/143).

| Experimental design
The animals were divided into the following groups:

| Drugs
In the penicillin-induced epilepsy model, the seizure activity was triggered by penicillin G potassium (I.E., Ulagay Pharmacy) (500 units, i.c., infusion rate; 0.5 μl/ min). After the stabilization of ECoG activity in 30 min, licofelone and esculetin were administered. In the PTZinduced epilepsy model, licofelone and esculetin were given 30 min before the administration of PTZ. In this study, licofelone (20 mg/kg), a COX/5-LOX dual inhibitor, and esculetin (20 mg/kg), a 5-LOX inhibitor, were used as chemical agents. All chemicals were bought from Sigma-Aldrich and dissolved in artificial cerebrospinal fluid. The application doses of licofelone and esculetin were determined according to the effective doses stated in the studies in the literature [16][17][18][19].

| Penicillin-induced epileptiform activity
The animals were anesthetized with urethane (1.25 g/ kg, i.p.) and fixed in a rat stereotaxic frame. A midline incision was made through the scalp. Two stainlesssteel screw electrodes were implanted on the left somatocortex skull bone (first screw 3 mm lateral and 4 mm rostral to bregma, second screw 3 mm lateral and 4 mm caudal to bregma). A third hole was opened for penicillin injection (3 mm lateral and 3 mm rostral to bregma), and penicillin G potassium was injected using Hamilton microsyringe type 701 N. Epileptiform activity occurred approximately within 3-4 min. In about 25-30 min, spike frequency and amplitude of the epileptiform activity became stable and continued for about 4-5 h. Licofelone (20 mg/kg) and esculetin (20 mg/kg) were performed 30 min after penicillin injection. ECoG activity was recorded by PowerLab/16SP (AD Instruments, Australia) for 180 min after drug injections ( Figure 1). Electrophysiological records were analyzed with a software program (LabChart v8), and the data were transferred to MS Excel.

| PTZ-induced epileptiform activity
In the PTZ-induced epilepsy model, the ECoG recording process is slightly different from the penicillininduced epilepsy model ( Figure 1). Licofelone and esculetin were administered 30 min before PTZ injection. PTZ (35 mg/kg) was administered for the kindling process three times a week (Monday, Wednesday, and Friday), and animal behavior was monitored to evaluate the behavioral parameters. In the kindling process, the Fisher and Kittner seizure scales were used [20] ( Table 1). After having reached at least five seizures of Stages 3-5, fully kindled animals were anesthetized with ketamine/xylazine and fixed in a rat stereotaxic frame. The surgical process and the implantation of the electrodes were performed as described in Section 2.4.1. All animals were allowed to recovery for 7 days. After a 7-day post-surgery recovery period, rats were placed separately in glass cages, and ECoG activity was recorded for 30 min. In this model, the seizure score, the number of needed injections for kindling, first myoclonic jerk (FMJ), and total spikes/30 min were determined in awake rats.

| Evaluation of ECoG activity and behavioral parameters
In the penicillin-induced epilepsy model, ECoG activity was recorded for 180 min after licofelone and esculetin injections. Mean spike frequency and amplitude were determined every 10 min over a period of 180 min. For evaluation of ECoG recording, mean amplitude and spike frequency in the 10 min between the 20th and 30th minute before penicillin administration were considered 0th control value. In the calculation of percentage, the following formula was used: Mean spike frequency or amplitude after drug administration Control value, 0 th min x100 In the PTZ-induced epilepsy model, ECoG activity was recorded for 30 min, and total spike count was evaluated for 30 min. Behaviors of the rats were monitored to evaluate the seizure score, the number of needed injections for kindling, and the FMJ.

| Statistical analyses
All statistical analyses were performed by GraphPad Prism 8 software. Electrophysiological data were analyzed using the LabChart program (Version 8). Values are expressed as means AE SEM. The Shapiro-Wilk test was applied to determine the normality of data. In the penicillin groups, data were analyzed using twoway ANOVA followed by post hoc test for multiple comparisons. In the PTZ groups, behavioral and ECoG data were analyzed using one-way ANOVA. Results were considered significant at confidence limits of P < 0.05.  Figure 4. The mean spike frequency of each group is given in Table 2.

| Evaluation of seizure scale, number of injections, and FMJ in the PTZinduced epileptiform activity
In the PTZ group, the mean number of needed injections for kindling was 4.22 AE 0.66 (P = 0.04) ( Figure 5). According to the Fischer and Kittner seizure scale, the seizure severity stage was 4.01 AE 0.23 in the PTZ group (P < 0.0001) ( Figure 5). The seizure severity was found significantly lower in the esculetin group (3.32 AE 0.16, P = 0.12) and the licofelone group (2.32 AE 0.11, P = 0.003) compared with the PTZ group (F(2, 22) = 10.67, P < 0.0001). In terms of the number of injections, similar results were seen in the seizure severity stage. In the licofelone group, the mean number of injections for kindling was significantly increased (18.20 AE 1.82) compared with the PTZ and esculetin groups (F(2, 21) = 18.14, P = 0.002). In the esculetin group, the number of needed injections was 8.6 AE 1.72 compared with the PTZ group (P = 0.186). In the PTZ group, the onset of FMJ was 143.2 AE 7.08 sec (P < 0.0001). In the licofelone group, FMJ was significantly increased at 787.8 AE 70.04 (F(2, 42) = 47.45, P < 0.001). In the esculetin group, FMJ was 183.9 AE 17.08 s (P = 0.948). Licofelone was more effective than the esculetin group in terms of all parameters ( Figure 6).

| Evaluation of ECoG recordings in the PTZ-induced epileptiform activity
In the PTZ model, before the implantation of electrodes, seizure scores, the number of injections, and FMJ were examined from video recordings. Later, ECoG activities were recorded for 30 min. In the PTZ group, the total spike count was 2495 AE 145.9 spike/30 min (P = 0.0087). Both licofelone and esculetin affected the seizure activity and decreased the total spike at 809 AE 42.55 and 1302 AE 221.45 spike/30 min, respectively (F(2, 17) = 6.304, P = 0.007, P = 0.019). Representative traces of ECoG recordings of the animals are shown in Figure 7.

| DISCUSSION
In the present study, licofelone, a dual inhibitor of COX and 5-LOX receptors, and esculetin, a 5-LOX inhibitor, were studied in two experimental models of epilepsy.  Notes: Data are presented as mean AE SEM. Licofelone group was statistically significant between 10 and 60 min compared with the penicillin + saline group. Both licofelone and esculetin decreased the seizure activity in penicillin-induced epilepsy, while licofelone was more effective in preventing PTZ-induced seizure activity. Previous studies revealed that metabolized arachidonic acid has an important role in epilepsy and the inflammation process. Arachidonic acid causes some alterations resulting in production of inflammatory mediators on microglia, astrocytes, and brain capillary endothelial cells. Inflammation is an essential condition in epileptic mechanisms, both in terms of being the primary mechanism involved in forming epilepsy and further triggering current seizures. There are many studies in the literature, but these studies include conflicting results.

T A B L E 2 Mean spike frequency (spikes/min) of the penicillin-induced epileptiform activity in study groups
Results of the present study showed that both licofelone and esculetin decreased the spike frequency and amplitude of the seizure activity in the penicillin model. Both of them showed anticonvulsant activity with similar results in the acute model of epilepsy. In our previous studies, aceclofenac (10 and 20 mg/kg) and aspirin (150 and 500 mg/kg), COX-2 inhibitors, have conflicting results in the penicillin-induced epilepsy model. While aspirin has protective effects at low and high doses, aceclofenac used for rheumatoid arthritis in the clinic has proconvulsant effects [13,21]. Previous studies revealed that proinflammatory cytokines such as IL-1β, IL-6, and TNF-α might affect seizure activity through glutamatergic, GABAergic, and NMDA receptors [22][23][24]. TNF-α is the most critical cytokine in the acute phase response to inflammation [25]. Moreover, TNF-α exhibits a dual role in the pathophysiology of seizures, exerting pro-convulsive effects through TNF receptor 1 (TNFR1) and anticonvulsive effects via TNF receptor 2 (TNFR2) [26]. This may explain the conflicting results between the early stage (acute effect) inflammation and epilepsy.
There are not enough studies conducted with acute models and inflammation. Related studies showed that these conflicting results might be due to the ligand type, dosage, used epilepsy model, activated inflammatory regulators, the complexity of the inflammatory process, and the time of occurrence. In the present study, promising results were gained with ECoG recordings and behavioral observations in the PTZ-induced epilepsy model. Especially, licofelone appears to have more effects that are protective in the chronic model in terms of all parameters. As mentioned in Section 3, licofelone decreased the seizure severity and total spike count while increasing the number of needed injections for kindling and time for FMJ. It is suggested that licofelone has anticonvulsant activity with all these parameters and is worth investigating with advanced studies. Compatible with our results, licofelone was examined in different experimental epilepsy models. In another study, licofelone has anticonvulsant activity at 10 mg/kg and above in mice in a PTZ-induced model [17]. Another study demonstrated that 10 mg/kg licofelone had similar activity in the lithium-pilocarpine model [16]. However, while the two studies were compatible with our study, it was observed that not all of them had an altogether terminating seizure activity. There are also compatible studies with other COX inhibitors in the literature. Akula et al reported that rofecoxib (2 and 4 mg/kg) increased the seizure activity threshold but not at 1 mg/kg in PTZ-induced epilepsy [12]. Another study showed that 2 mg/kg celecoxib has protective effects against PTZ induced seizures [27]. Dhir et al found that nimesulide (2.5 mg/kg) and rofecoxib (2 mg/kg) increased the mean onset time of convulsions, decreased the duration of clonus, and decreased the mortality rate in bicuculline and picrotoxin-induced convulsions in mice while not in 1 mg/kg nimesulide and rofecoxib. On the other hand, these inhibitors did not affect the seizure activity in maximal electroshockinduced seizures [5]. Administration of PTZ may affect the brain-blood barrier (BBB), leading to the structure's disruption [28]. With this breakdown, especially in the PTZ-induced model, TGF-β, an inflammatory regulator, may have a key role in the pathophysiology of epilepsy.
Conflicting results with COX-2 inhibitors that act as a proconvulsant are confusing in epilepsy. Pretreatment with nimesulide augmented seizures and increased the mortality rate from approximately 10% to 69% [29]. The opposite results can be seen by changing the injection time in the same study. Kunz and Olive examined pretreatment and posttreatment of nimesulide (10 mg/kg) in the kainic acid-induced seizure. They found that nimesulide after the kainic acid had better effects on seizures compared with the pretreatment of nimesulide [29].
The other group in the present study was the 5-LOX inhibitor esculetin. According to the obtained data, the effects of esculetin were seen in ECoG activity. In behavioral parameters, the results were not statistically significant. However, esculetin decreased seizure activity in this study. In the literature, studies with both esculetin and other 5-LOX inhibitors are very limited. In these studies, 5-LOX inhibitors were not effective in controlling seizures. However, 5-LOX inhibitors have protective effects when administered in combination with other COX inhibitors. Kim et al examined the combined effects of COX inhibitor (aspirin) and 5-LOX inhibitors (NS-398 and esculetin) in a kainic acidinduced seizure model. They found that aspirin given with 5-LOX inhibitors has protective effects against neurotoxicity after the injection of kainic acid, but not given alone [14]. In another study, acetyl-11-ketoβ-boswellic acid (AKBA), a 5-LOX inhibitor, were not effective in kainic acid-induced seizure. When AKBA combined with other COX inhibitors, it increased the seizure latency [15]. Although esculetin was administered alone in our study, we think that the main effect of co-administered COX and 5-LOX inhibitors shows affinity to the COX pathway.

| CONCLUSION
Epilepsy is the most common and important neurological disorder in the world. Inflammation is an important physiological process that is very critical in the body. The possible adverse effects of anti-inflammatory drugs should be considered, especially in patients with epilepsy. In the present study, both acute and chronic application of inhibitors revealed that inflammation pathways might have a vital role in the pathophysiology of epilepsy. Licofelone is more effective than esculetin. We believe that more precise and beneficial results will be obtained with molecular analyzes in which the effects of licofelone on proinflammatory cytokines will be determined.