Figure 1.
Genetic deletion of sEH delays seizure onset induced by GABA antagonists.
Deletion of the sEH gene in mice stabilizes EETs and delays onset of seizures induced by pentylenetetrazole (PTZ) and picrotoxin (PIC). (A) Subcutaneous PTZ led to a set of stereotypical convulsive behaviors including myoclonic (black bars) and tonic (gray bars) seizures in mice. In sEH−/− mice both clonic and tonic phase of seizures were delayed compared to conspecific C57/BL6 mice (n = 6 mice/group, Student's t-test, sEH−/− vs. wt, *p = 0.049 and **p = 0.001). Note that animals that did not display tonic hind limb extension reflex within 30 min were excluded from these graphs (see Table 1 for survival data). (B) Onset of tonic seizures induced by subcutaneous PIC were delayed in sEH−/− mice (n = 5 mice/group, Student's t-test, sEH−/− vs. wt, ***p = 0.003), while onset of PIC induced clonic seizures did not differ (p = 0.75). (C) Seizures induced by 4-AP led to tonic seizure without a clear clonic phase and onset in sEH−/− and wt mice were not significantly different (n = 7/group, p = 0.39). Data are expressed as mean ± s.e.m for all figures.
Figure 2.
Potent inhibitors of sEH dose dependently delay onset of seizures induced by GABA antagonist.
In parallel to observations with sEH−/− mice, small molecule inhibitors of sEH delay onset of tonic phase of seizure in wild type Swiss mice. In all three experiments sEHI were administered one hour prior to convulsants to allow absorption and distribution of the inhibitor. (A) TUPS was administered at a volume of 1 µL/g body weight by intraperitoneal route following completely dissolving it in PEG400. Subcutaneous PTZ induced tonic seizure is delayed by sEH inhibitor TUPS (n = 8–22/group, Kruskal-Wallis One Way ANOVA on Ranks followed by Dunn's multiple comparison, vehicle vs TUPS, *p = 0.028, # p = 0.008, § p = 0.03), though onset of clonic seizure was not affected by inhibition of sEH. (B) In parallel to results obtained by TUPS, a structurally different sEHI, TPPU delayed the onset of tonic phase of PTZ induced seizure compared to vehicle (n = 8–14/group, Kruskal-Wallis One Way ANOVA on Ranks followed by Dunn's multiple comparison, vehicle vs TPPU, **p≤0.05). Clonic phase of seizures was not monitored in this experiment. See Table S1 for structure and potency information of TUPS and TPPU. (C) Seizures induced by 4-AP had a shorter onset in Swiss mice however consistent with data in Figure 1, an efficacious dose of TUPS did not significantly alter onset of seizure (n = 8/group, Student's t-test, p = 0.49).
Table 1.
The sEHI treatment reduced the proportion of mice experiencing tonic seizures but did not provide significant protection against clonic seizures.
Figure 3.
Potent inhibitors of sEH elevate threshold of tonic seizure in the timed i.v. infusion test.
Consistent with the subcutaneous PTZ test both TUPS and TPPU, administered 1(vehicle n = 8, TPPU n = 6, TUPS n = 3, One Way ANOVA followed by Student Newman Keuls post hoc analysis *p = 0.026, # p = 0.005).
Table 2.
The effects of allopregnenolone was significantly enhanced by sEHI and partially antagonized by finasteride (One Way ANOVA followed by Student Newman Keuls post hoc analysis TUPS+Allo vs TUPS, *p = 0.016, TUPS+Allo vs Allo, **p = 0.007, TUPS vs TUPS+FIN, #p = 0.02).
Figure 4.
Brain levels of sEHI correlate with observed activity.
The sEHI TUPS was administered at a single i.p. dose of 1/kg and seizures were induced at varying times using s.c. PTZ procedure (n = 8/time point). Following tonic hind limb extension and lethality, brains were rapidly excised and TUPS was quantified by LC-MS/MS. Protected animals (3 of 8 in each group) were sacrificed 30 min following PTZ treatment and are included on the left panel. (A) TUPS was well absorbed and blood and brain inhibitor levels reached well over in vitro IC50 values within an hour and remained relatively stable over 8 h. (B) The efficacy of TUPS was significantly correlated with brain inhibitor levels (Spearman's Rho, 0.90, p = 0.037) All time points are significantly different from the vehicle treated animals (One Way ANOVA, p = 0.034).
Figure 5.
Brain oxylipins display decrease in response to seizure and are elevated by sEHI.
(A–C) Bar graph of ratios of brain epoxy to dihydroxy-FAs in mice receiving vehicle (n = 6, black bars), sEHI (n = 6, gray bars), vehicle+PTZ (n = 8, dark gray bars) and sEHI+PTZ (n = 8, light gray bars). Inhibitors were administered 1 h prior to sampling, PTZ treated animals were sampled up immediately following tonic phase of the seizure. PTZ treatment resulted in a significant decrease in the levels of bioactive regioisomers 14,15-EET and 13,14- EpDPE (Kruskal-Wallis One Way ANOVA on Ranks followed by Dunn's multiple comparison, control vs PTZ, # p≤0.05). Although in the absence of seizures sEHI did not lead to changes in the brain levels of 14,15- EET and 13,14- EpDPE, the decrease mediated by seizures was recovered by inhibition of sEH for the ARA metabolite 14,15-EET (*p = 0.001) but not for the DHA metabolite 13,14-EpDPE (p = 0.061). There were no significant changes in the levels of EPA derived epoxy fatty acids among groups.
Figure 6.
Direct administration of EETs and sEHI into the brain delay onset of PTZ induced seizures.
Bar graph of percent change in onset of clonic (black bars) and tonic (gray bars) seizures following intracerebroventricular administration of EpFAs, sEHI and diazepam (n = 6–14mice/group). Methyl ester EpFAs, sEHI and diazepam were dissolved in PEG400 and 1 µL was given by i.c.v. route to lightly anesthesized mice in the amounts indicated on x-axis. Thirty minutes after treatments PTZ assay was performed. Among the treatment groups, only diazepam was effective in delaying the onset of clonic seizures (Kruskal-Wallis One Way ANOVA on Ranks followed by Dunn's multiple comparison, *p≤0.05). Diazepam, EET-methyl esters and high dose of sEHI and were effective in delaying the onset of tonic seizures (Kruskal-Wallis One Way ANOVA on Ranks followed by Dunn's multiple comparison, **p≤0.05).