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Dibucaine in ionic-gradient liposomes

Epilepsy is a neurological disorder treated with antiepileptic drugs (AEDs). Since AEDs are administered in women in childbearing age, it is critical to study if drugs are capable of inducing developmental toxicity. In the present study, we evaluated the teratogenic and anticonvulsant effects of six different AEDs: carbamazepine (CBZ), lamotrigine (LTG), levetiracetam (LEV), phenobarbital (PB), phenytoin (PHT) and valproic acid (VPA).

Zebrafish was the selected animal model because of its small size, rapid external development and similar neurophysiology to mammals. In addition, the neuro physiology of zebrafish is similar to those of mammals Zebrafish larvae exposed to pentylenetetrazol display increased locomotor activity, seizure-like behavior, and epileptiform activity. Zebrafish embryo and larvae were exposed to AEDs. Embryo development was monitored by their hatching and morphology. In larvae, locomotor activity was measured as a parameter of neurotoxicity. Finally, anticonvulsant effect was determined after exposure to AEDs in zebrafish larvae treated with the proconvulsant drug pentylenetetrazole.

Locomotor activity and development of a model of epileptiform behavior. After 1 h of AEDs-treatment, the spontaneous movement of 121 hpf larvae was studied in a multichannel ADC system (WMicrotracker, Designplus SRL). Briefly, zebrafish were placed in 96-well microplates and subjected to illumination with infrared beams, which signal is interrupted by larvae movements. Swimming activity was recorded during 15 min, and variations>3% (empirically determined threshold) were considered as activity events. Zebrafish larvae were driven to an epileptiform behavior with the proconvulsant drug pentylenetetrazol (PTZ). Briefly, after 1 h of AEDs-treatment, 121 hpf larvae were exposed to 2.5mM PTZ for 10 min to develop an epileptiform behavior. Finally, spontaneous movement was measured as described above. PTZ controls were exposed to the same concentration of PTZ without previous antiepileptic treatment. The assays were considered valid only if spontaneous movements of PTZ controls were significantly different from non-treated larvae. Locomotor activity of zebrafish larvae and anticonvulsant effect of AEDs. The spontaneous movement of zebrafish larvae of 120 hpf was studied as a parameter of neurotoxicity. For this purpose, 120 hpf larvae were exposed to different AEDs and their locomotor activity was quantified at 121 hpf. No variations of spontaneous movement compared to controls were observed for any AEDs, except for PB at a 100 μM concentration.  In order to evaluate the anticonvulsant effect of AEDs, 120 hpf larvae treated for 1 h with AEDs were then exposed to the pro convulsant drug pentylenetetrazol (PTZ), and their spontaneous movement was quantified. PHT and LTG significantly reduced the epileptiform behavior of larvae at 50 and 100 µM, while VPA exhibited an anticonvulsant effect only at 100 µM. For CBZ, LEV and PB no reversion of epileptiform behavior was observed in larvae (Fig. 7). Our results suggest that lamotrigine and phenytoin could be suitable non-teratogenic and efficient anticonvulsant options for epilepsy treatment.

Fig. 7. Anticonvulsant effect studied as changes in spontaneous movement of 5-dpf zebrafish model of epileptiform behavior. Zebrafish larvae of 120 hpf were exposed to the antiepileptic drugs for one hour, followed by an incubation with the proconvulsant pentylenetetrazole (PTZ) during 10min. Locomotor activity was recorded for 15min and expressed as a percentage of spontaneous movement respect to non-treated control larvae. Results are shown as mean ± SEM. Significant differences respect to PTZ control were analyzed by ONE-WAY ANOVA test followed by Dunnett’s multiple comparisons post-test (*p < 0.05, ***p < 0.001, ****p < 0.0001).

Neurotoxicology and Teratology, 03 Feb 2018, 66:17-24 DOI: 10.1016/j.ntt.2018.01.008.

C.S. Martinez, D.A. Feas, M. Siri, D.E. Igartúa, N.S. Chiaramoni, S. del V. Alonso, M.J. Prieto