Anti-Epileptic Effect of Crocin on Experimental Temporal Lobe Epilepsy in Mice

Abstract

Temporal lobe epilepsy (TLE) is a common kind of refractory epilepsy. More than 30% TLE patients were multi-drug resistant. Some patients may even develop into status epilepticus (SE) because of failing to control seizures. Thus, one of the avid goals for anti-epileptic drug development is to discover novel potential compounds to treat TLE or even SE. Crocin, an effective component of Crocus sativus L., has been applied in several epileptogenic models to test its anti-epileptic effect. However, it is still controversial and its effect on TLE remains unclear. Therefore, we investigated the effects of crocin on epileptogenesis, generalized seizures (GS) in hippocampal rapid electrical kindling model as well as SE and spotaneous recurrent seizure (SRS) in pilocarpine-induced TLE model in ICR mice in this study. The results showed that seizure stages and cumulative afterdischarge duration were significantly depressed by crocin (20 and 50 mg/kg) during hippocampal rapid kindling acquisition. And crocin (100 mg/kg) significantly reduced the incidence of GS and average seizure stages in fully kindled animals. In pilocarpine-induced TLE model, the latency of SE was significantly prolonged and the mortality of SE was significantly decreased by crocin (100 mg/kg), which can also significantly suppress the number of SRS. The underlying mechanism of crocin may be involved in the protection of neurons, the decrease of tumor necrosis factor-α in the hippocampus and the increase of brain derived neurotrophic factor in the cortex. In conclusion, crocin may be a potential and promising anti-epileptic compound for treatment of TLE.

Keywords: crocin; kindling; pilocarpine; spontaneous recurrent seizure; temporal lobe epilepsy.

FIGURE 1

Schematic timeline diagram of experiments in each animal model. (A) Timeline of the experiment in hippocampal rapid kindling model; (B) timeline of the experiment in fully kindled model; (C) timeline of the experiment in pilocarpine-induced SE model; (D) timeline of the experiment in pilocarpine-induced SRS model. ADT, after discharge threshold; GS, generalized seizure; SE, status epilepticus; SRS, spontaneous recurrent seizure; OFT, open field test; NORT, novel object recognization test; EEG, electroencephalograms; ELISA, enzyme linked immunosorbent assay.

FIGURE 2

Effects of crocin on hippocampal rapid kindling acquisition (n = 8 for each group, x̄±S.E.M.). (A) Average seizure stage; (B) cumulative ADD of six stimulations each day; (C) the number of stimulations to reach seizure stage 3, 4, 5; (D) the number of stimulations to stay in seizure stage 0–2 and 3–5 during kindling acquisition; (E) Number of stage 4–5 seizures in the 8th day. ADD, afterdischarge duration; NS, normal saline; VPA, sodium valproate. Values are from 3 independent experiments. *p < 0.05, **p < 0.01, compared with NS group. Two-way ANOVA was performed for (A,B). Comparisons of the number of stimulations for each seizure stage (C,D) were made with the nonparametric Mann–Whitney U test. One-way ANOVA for repeated measures was used followed by Tukey’s t-test for (E).

FIGURE 3

Effects of crocin on the incidence of GS and seizure severity in fully kindled mice (n = 8 for each group, x̄±S.E.M.). (A) Mean incidence of GS, (B) average seizure stage, (C) average GSD and (D) average ADD during 10 days. GS, generalized seizure, GSD, generalized seizure duration, ADD, afterdischarge duration; NS, normal saline; VPA, sodium valproate. Values are from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with NS group; ^## p < 0.01, ^### p < 0.001, compared with VPA group. One-way ANOVA was used for (B–D), followed by Tukey’s t-test. The χ2 test was used for (A).

FIGURE 4

Effects of crocin on pilocarpine-induced SE and SRS. (A) average latency of SE (n = 12 for each group, x̄±S.E.M.); (B) mortality rate of SE (n = 12 for each group); (C) number of SRS (n = 7 for each group, x̄±S.E.M.); (D) mean seizure stage of SRS (n = 7 for each group, x̄±S.E.M.); (E) mean duration of SRS (n = 7 for each group, x̄±S.E.M.). SE, status epilepticus; SRS, spontaneous recurrent seizures; NS, normal saline; VPA, sodium valproate. Values are from four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with NS group. One-way ANOVA for repeated measures was used followed by Tukey’s t-test for (A,C,D) and the χ2 test was used for (B).

FIGURE 5

Effects of crocin on the behaviors of mice in OFT and NORT (n = 7 for each group, x̄±S.E.M.). (A) representative tracks of mice in OFT; (B) total travelling distance of mice in OFT; (C) movement distance of mice in center zone in OFT; (D) duration of mice staying in center zone in OFT; (E) representative tracks of mice in NORT (blue boxes mark the location of old object and green boxes mark the location of novel object); (F) DR of mice in each group; (G) DI of mice in each group. OFT, the open field test; NORT, the novel object recognization test; DR, discrimination ratio; DI, discrimination index; SE, status epilepticus; SRS, spontaneous recurrent seizures; nSRS, non-SRS; NS, normal saline; VPA, sodium valproate. Values are from 3 independent experiments. *p < 0.05, compared with nSRS group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, compared with NS group (with SRS); ^$ p < 0.05, ^$$ p < 0.01, compared with VPA group. Two-way ANOVA was performed for (B,C,D,F,G).

FIGURE 6

Effects of crocin on the neural damage in the hippocampus region in each group of mice. (A–E) H-E staining of neurons in the left hippocampal CA3 region; (F–J) H-E staining of neurons in the right hippocampal CA3 region; (K–O) H-E staining of neurons in the left hippocampal CA1 region; (P–T) H-E staining of neurons in the right hippocampal CA1 region. Bar = 200 μm. (U–X) The quantification of survival neurons in left CA3, right CA3, left CA1 and right CA1 (n = 3 for each group, x̄±S.E.M.). SRS, spontaneous recurrent seizures; nSRS, non-SRS; NS, normal saline; VPA, sodium valproate. Values are from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with naive group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, compared with NS group (with SRS). One-way ANOVA was used for (U–X), followed by Tukey’s t-test.

FIGURE 7

Effects of crocin on intracerebral concentration of BDNF, IL-1β and TNF-α in each group of mice (n = 6 for each group, x̄±S.E.M.). (A) the concentration of BDNF in the hippocampus of mice; (B) the concentration of IL-1β in the hippocampus of mice; (C) the concentration of TNF-α in the hippocampus of mice; (D) the concentration of BDNF in the cortex of mice; (E) the concentration of IL-1β in the cortex of mice; (F) the concentration of TNF-α in the cortex of mice. SRS, spontaneous recurrent seizures; nSRS, non-SRS; NS, normal saline; BDNF, brain derived neurotrophic factor; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α. Values are from 3 independent experiments. **p < 0.01, compared with nSRS group; #p < 0.05, compared with NS group (with SRS). One-way ANOVA for repeated measures was used followed by Tukey’s t-test for (A–F).