Effect of eugenol on lithium-pilocarpine model of epilepsy: behavioral, histological, and molecular changes

Abstract

Objectives: Epilepsy establishment gives rise to biochemical and morphological changes in the hippocampus. Oxidative stress, morphological changes, and mossy fiber sprouting (MFS) in the hippocampus underpin the epilepsy establishment. Eugenol is the main component of the essential oil extracted from cloves with the potential to modulate neuronal excitability. Therefore, we investigated the effect of eugenol on convulsive behavior, oxidative stress, and histological changes of the hippocampus in lithium-pilocarpine model of epilepsy.

Materials and methods: Male Wistar rats weighing 220-250 g were divided into 4 groups; Control, Pilocarpine, Eugenol-Pilocarpine, and Eugenol. Oxidative stress markers were assayed by a biochemical method. Nissl and Timm staining were used to show neuronal survival and MFS, respectively. Behavioral convulsions were evaluated using the modified Racine scale.

Results: Eugenol decreased seizure stage and duration as well as mortality. Neuronal numbers were preserved by eugenol treatment in epileptic animals, while eugenol alone reduced the number by itself in all hippocampal sub-regions including DG, CA3, and CA1. Furthermore, eugenol alone increased MDA, GPx and SOD markers, while it increased MDA not only in combined treatment with pilocarpine but also in pilocarpine-treated animals. In contrast to MFS enhancement in naïve animals, eugenol partially reversed the MFS enhancement induced by pilocarpine.

Conclusion: Eugenol could prevent behavioral convulsions and show neuroprotective effects through increasing neuronal survival probably by decreasing MFS and increasing the GPx antioxidant marker.

Keywords: Epilepsy; Eugenolm Hippocampus; Mossy fiber sprouting; Oxidative stress.

Figure 1

Effect of eugenol on behavioral convulsions. Eugenol increased latency to the clonic convulsions compared to pilocarpine (A). Seizure stages (B) and GC duration (C) decreased due to eugenol. Pilocarpine increased mortality, and eugenol partially reversed the effect (D). † P<0.05, PL; Pilocarpine, EUG; Eugenol, GC; Generalized Clonic Convulsions

Figure 2

Eugenol activated the oxidative processes in the hippocampus. MDA increase was due to pilocarpine epilepsy and/or eugenol (A), while GPx increase was seen only in eugenol treated animals (B). SOD increase was shown due to eugenol in naïve animals (C). * P<0.05, ** P<0.01, *** P<0.001, * compared to Ctrl, Ctrl; Control, PL; Pilocarpine, EUG; Eugenol

Figure 3

Eugenol prevented the hippocampal neuronal loss. While pilocarpine-induced neuronal loss in the total hippocampus, eugenol prevented that effect (A). Pilocarpine and eugenol increased and prevented, respectively, neuronal loss in DG (B), CA3 (C) and CA1 (D) Eugenol alone decreased neuronal survival in the total hippocampus, DG, and CA3 * P<0.05, ** P<0.01, *** P<0.001, †P<0.05, * compared to Ctrl and † compared to PL, Ctrl; Control, PL; Pilocarpine, EUG; Eugenol

Figure 4

Photomicrographs showing Nissl staining of the hippocampal dentate and CA regions of epileptic animals. Control sections showed normal stained DG, CA3, and CA1 neurons (well-arranged and distributed neurons). PL sections reveal reduced and dispersed neuronal numbers, while eugenol restored the number, but not the dispersion except in DG. eugenol itself decreased neuronal survival and increased dispersion in DG, CA3, and CA1. Ctrl; Control, PL; Pilocarpine, EUG; Eugenol

Figure 5

Eugenol prevented hippocampal mossy fiber sprouting in the dentate gyrus. The mean Timm index was increased due to epilepsy (PL) and was prevented after eugenol administration, while eugenol alone partially increased the index (A). Coloring time was reduced in epileptic animals while it took longer due to eugenol IP injections (B) ** P<0.01, *** P<0.001, †† P<0.01, ††† P<0.001, * compared to Ctrl and † compared to PL, Ctrl; Control, PL; Pilocarpine, EUG; Eugenol