Effects of COX and 5-LOX Pathways on Different Seizure Models

Purpose This study aimed to examine the relationship between epilepsy and COX/5-LOX inammation pathways in the penicillin and pentylenetetrazole (PTZ) induced epilepsy models. Methods For this purpose, forty-two 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. behavioral parameters. valuable


Introduction
Epilepsy is characterized by repeated spontaneous bursts of neuronal hyperactivity and high synchronization in the brain (Trinka et al., 2015). In general, epileptic seizures predicate a disequilibrium between inhibitory (GABAergic) and excitatory (glutamatergic) neurotransmission. However, the mechanism of epileptogenesis is still unclear . In many cases, antiepileptic drugs (AEDs) can partially control seizures and help patients to maintain their everyday lives. Although advanced in antiepileptic drugs and research, seizures occur in approximately 30% of patients with epilepsy (Rahim et al., 2021). In this manner, many factors have essential roles in the pathophysiology of epilepsy, and we must consider these, including in ammation, in the treatment of epilepsy.
In ammation 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 (Paudel et al., 2018). Initially, metabolized arachidonic acid through cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways play a critical role in epilepsy and in ammation. Cyclooxygenase is the critical enzyme that converts arachidonic acid to prostaglandins (PGs) (Dhir et al., 2006). Arachidonic acid causes some alterations and in ammatory mediators' production on microglia, astrocytes, and brain capillary endothelial cells. The activation of in ammatory mediators such as COX-2 and nuclear factor kappa B (NF-κB) and the over-production of down-stream in ammatory factors including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2) contribute to the pathophysiology of seizures (Wang et al., 2018). Emerging evidence suggests that in ammatory pathways play a crucial role in the pathophysiology of epilepsy .
Although there are various reports regarding the increase of cyclooxygenase enzyme following seizure activity (Takemiya et al., 2003), some studies have con icting results. Selective and non-selective COX inhibitors can cause a delay in the progress of seizure (Tanaka et al., 2009), increase the seizure threshold (Akula et al., 2008), or worsen the seizure activity (Taskiran et al., 2017). 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 in ammation 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; thereby, 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 (Kim et al., 2008). Similarly, Acetyl-11-Keto-β-Boswellic Acid (AKBA), a 5-LOX inhibitor, did not affect the seizure activity in the kainic acid-induced epilepsy. Whereas, AKBA+indomethacine increased the seizure latency (Bishnoi et al., 2007).
A number of animal studies showed that both COX and 5-LOX pathways have contradictory results (Akula et al., 2008;Tanaka et al., 2009;Taskiran et al., 2017). In addition, there can be different effects on seizures in terms of acute or chronic phase in ammation. 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, forty-two 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 ± 3% humidity level, and 22 ± 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). After the stabilization of ECoG activity in 30 minutes, licofelone and esculetin were administered. In the PTZ-induced 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 arti cial cerebrospinal uid. Doses of the drugs were selected based on the studies in the literature. The animals were anesthetized with urethane (1.25 g/kg, i.p.) and xed in a rat stereotaxic frame. A midline incision was made through the scalp. Two stainless-steel screw electrodes were implanted on the left somatocortex skull bone ( rst 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 701N.
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 16/SP (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 (Fischer and Kittner, 1998) ( Table 1).
After having reached at least ve seizures of stage 3-5, fully kindled animals were anesthetized with ketamine/xylazine and xed in a rat stereotaxic frame. The surgical process and the implantation of the electrodes were performed as described in 2.4.1. All animals were allowed to recovery for seven days.
After a seven-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, rst 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 ten 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: In the PTZ-induced epilepsy model, ECoG activity was recorded for 30 min and total spike count were evaluated for 30 min. Behaviors of the rats were monitored to evaluate the seizure score, the number of needed injections for kindling, and the rst myoclonic jerk (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 ± S.E.M. The Shapiro-Wilk test was applied to determine the normality of data. In the penicillin groups, data were analyzed using two-way 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 signi cant at con dence limits of p < 0.05.  Figure 4. The mean spike frequency of each group is given in Table 2. Data are presented as mean ± SEM. Licofelone group was statistically signi cant between 10 and 60 minutes compared to the penicillin+ saline group.

Evaluation of seizure scale, number of injections, and FMJ in the PTZ-induced epileptiform activity
In the PTZ group, the mean number of needed injections for kindling was 4.22±0.66 (p=0.04) ( Figure 5). According to the Fischer and Kittner seizure scale, the seizure severity stage was 4.01±0.23 in the PTZ group (p<0.0001) ( Figure 5). The seizure severity was found signi cantly lower in the esculetin group (3.32±0.16, p=0.12) and the licofelone group (2.32±0.11, p=0.003) compared to the PTZ group. 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 signi cantly increased (18.20±1.82) compared to the PTZ and esculetin groups (p=0.002). In the esculetin group, the number of needed injections was 8.6±1.72 compared to the PTZ group (p=0.186). In the PTZ group, the onset of FMJ was 143.2±7.08 sec (p<0.0001). In the licofelone group, FMJ was signi cantly increased at 787.8±70.04 (p<0.001). In the esculetin group, FMJ was 183.9±17.08 sec (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±145.9 spike/30 min (p=0.0087). Both licofelone and esculetin affected the seizure activity and decreased the total spike at 809±42.55 and 1302±221.45 spike/30 min, respectively (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. Both licofelone and esculetin decreased the seizure activity in penicillin-induced epilepsy, while licofelone was more effective in preventing PTZinduced seizure activity.
Previous studies revealed that metabolized arachidonic acid has an important role in epilepsy and the in ammation process. Arachidonic acid causes some alterations resulting production of in ammatory mediators on microglia, astrocytes, and brain capillary endothelial cells. In ammation 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 con icting results.
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 ( There are not enough studies conducted with acute models and in ammation. Related studies showed that these con icting results might be due to the ligand type, dosage, used epilepsy model, activated in ammatory regulators, the complexity of the in ammatory 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 the results section, 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 (Payandemehr et al., 2015). Another study demonstrated that 10 mg/kg licofelone had similar activity in the Lithium-pilocarpine model (Eslami et al., 2016). 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 (Akula et al., 2008). Another study showed that 2 mg/kg celecoxib has protective effects against PTZ induced seizures (Oliveira et al., 2008). 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 electroshock-induced seizures (Dhir et al., 2006). Administration of PTZ may affect the brain-blood barrier (BBB), leading to the structure's disruption (Cacheaux et al., 2009). With this breakdown, especially in the PTZ-induced model, TGF-β, an in ammatory regulator, may have a key role in epileptogenesis.
Con icting 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 10-69% (Kunz and Oliw, 2001). In the temporal lobe epilepsy of pilocarpine, parecoxib (10 mg/kg), a COX-2 inhibitor, has been found to cause neuronal damage in both the hippocampus and the piriform cortex (Polascheck et al., 2010). The opposite results can be seen by changing the injection time in the same study. Kunz and Olive examined pre-treatment and post-treatment 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 to the pre-treatment of nimesulide (Kunz and Oliw, 2001).
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 signi cant. However, esculetin exhibited anticonvulsant 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 acid-induced 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 (Kim et al., 2008). In another study, Acetyl-11-Keto-β-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 (Bishnoi et al., 2007). Although esculetin was administered alone in our study, we think that the main effect of co-administered COX and 5-LOX inhibitors shows a nity to the COX pathway.

Conclusion
Epilepsy is the most common and important neurological disorder in the world. In ammation is an important physiological process that is very critical in the body. The possible adverse effects of antiin ammatory drugs should be considered, especially in patients with epilepsy. In the present study, both acute and chronic application of inhibitors revealed that in ammation pathways might have a vital role in the pathophysiology of epilepsy. Licofelone is more effective than esculetin. Licofelone will be bene cial in epilepsy in further studies where quantities and changes of pro-in ammatory cytokines will be determined.       First myoclonic jerks (FMJ) and total spike count on PTZ-induced epilepsy. A) Time for rst myoclonic jerks (FMJ). In the PTZ and esculetin groups, rst myoclonic jerks were seen approximately 200 sec. after PTZ injection. Licofelone increased the time for the rst myoclonic jerks at about 800 sec (*, p<0.05). B) Total spike count for 30 min. Esculetin and Licofelone decreased the total spike count for 30 min compared to the PTZ group (*, p<0.05). The results are expressed as mean ± standard error (SEM).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. PTZRawData.pzfx PenicillinRawData.xlsx