Introduction. It was previously found that rapamycin, an mTOR blocker, has an anticonvulsant effect in a rat model of pharmacological kindling [1]. Suppression of type B tyrosine kinase activity by administration of axitinib also provides an anticonvulsant effect in a model of chronic epileptic activity [2]. However, until recently, the nature of the focal epileptic activity was not investigated in the conditions of the use of rapamycin and axitinib.
Therefore, the purpose of this study was to determine the features of the dynamics of the activity of epileptic foci created in rats in the frontal parts of the cerebral cortex under conditions of separate and combined use of rapamycin and axitinib.
Material and methods.The observations were carried out under the conditions of an acute experiment on male Wistar rats weighing 180-250 g. Animals were kept in standard conditions (constant temperature 23o C and relative humidity 60%, 12 hrs dark/light cycles, standard diet, and tap water were given ad libitum). They were acclimatized to laboratory conditions for at least seven days before experimentation. At the stage of experimental work planning, principles outlined ARRIVE guidelines and the Basel declaration (http://www.basel- declaration.org), including the 3R concept, have been considered. All experiments were approved by Danylo Halytsky Lviv National Medical University Bioethics Committee (UBC).
The frontal parts of the cerebral cortex of both hemispheres, where the active electrode was located, were exposed under ketamine (100.0 mg/kg, i.p.) anesthesia and in conditions of fixation in a SEZh-5 stereotaxic apparatus. An indifferent electrode was fixed in the nasal bones of the skull. After 30 min, the animals were injected with d-tubocurarine (Orion, Finland, 0.15 mg/kg, i.v.) and transferred to artificial respiration. The sites of dissection of the head tissues and points of compression were infiltrated with a solution of novocaine (0.25%). The electrical activity of the brain structures was recorded 1.5 hours after the operation using the DX-5000 computer system (Kharkiv, Ukraine).
Foci of epileptic activity (EpA) were created by applying to the surface of the cerebral cortex pieces of filter paper soaked in a freshly prepared solution of benzylpenicillin sodium salt (30,000 IU / ml). Rapamycin (Pfizer, USA) was used at a dose of 1.0 mg/kg, i.p. Axitinib (Sigma-Aldrich, USA) was used at doses of 0.5 and 2.0 mg/kg, i/p. DMSO was administered to animals of the control group under similar conditions. The preparations were injected against the background of generation of focal spike activity, stable in frequency and amplitude. The severity of focal EpA was assessed by the frequency-amplitude characteristics of spike potentials and the total duration of the existence of foci.
Statistical processing of research results (latency and power of bioelectrical activity) was carried out using the one-way ANOVA method and Newman-Keuls criterion. The severity of seizures was assessed using the Kruskal-Wallis test.
Results and discussion. The creation of foci by applying a penicillin solution to the cerebral cortex in rats of the control group was accompanied by the formation of interictal potentials, the latent period of development of which ranged from 2.5 to 5.5 minutes. Within 5-10 minutes from the moment of onset, an increase in the frequency and amplitude of the discharges was noted, respectively, up to 25-45 per minute and 1.5-2.0 mV. EpA, stable in frequency and amplitude, in the foci, was recorded for 15–25 min, after which a gradual decrease in the magnitude and frequency of spikes occurred within 30–50 min. The duration of the existence of foci in the control group ranged from 57 to 89 minutes (on average, 71.6+10.5 minutes).
In 30 min from the moment of intravenous injection of rapamycin (1.0 mg/kg, i.p.), the frequency of generation of discharges in the foci was 18.7+2.4 per minute and was by 19.3% less than that in the control group (P <0.05). Significant differences between the groups persisted during the next 30 min of observation. During the next 50 min after the application of rapamycin, the amplitude of the discharges was also lower than in control by 18.4% and was equal to 1.42+0.30 mV (P <0.05). The duration of the existence of foci created under the conditions of the use of rapamycin ranged from 45 to 80 min (on average 65.3+8.4 minutes) and did not differ from the indicator in the control group (P>0.05).
A significant decrease in the frequency of generation of spike discharges in the foci - by 27.8% in comparison with the control group on the 30th min after penicillin solution application, under the influence of axitinib (2.0 mg/kg) (P <0.05). Further observation for 30 min revealed the persistence of significant differences between the groups, which at 60 min was 37.9% (P <0.05). Under the influence of axitinib, applied at a dose of 0.5 mg/kg, at the 40th min, there was a decrease in the amplitude of the discharges compared to that in the control group by 21.5% (P<0.05). Significant differences between the groups remained until the end of the observation, and at the 60th minute, the differences were 24.8% (P <0.05). At the same time, the duration of the existence of penicillin- induced foci did not change, despite it was less than the corresponding index in the control group by 7.5% and by 12.1% after treatment with the axitinib at doses of 0.5 and 2.0 mg/kg (P> 0.05).
Administration of rapamycin and axitinib in independently ineffective doses (0.5 mg/kg) caused the decrease of the frequency of spike potential generation by 22.8% in comparison with the corresponding data in the control group (P <0.05). A significant decrease in the amplitude of discharges - by 26.0% was noted at the 30th minute (P <0.05). At the same time, significant differences between the groups remained until the end of the observation, and the duration of the foci life span was shortened up to 47.9+5.1 min (with fluctuations from 27.5 to 62.0 min) (P<0.05).
Thus, the presented results showed that the penicillin-induced model of epileptogenesis proved to be sensitive to the antiepleptic action of rapamycin and axitinib. Both drugs used in higher dosages were able to suppress focal epileptic discharges, but they failed to affect their lifespan. Under conditions of combined administration of rapamycin (0.5 mg/kg, i.p.) and axitinib (0.5 mg/kg, i.p.), both the suppression of frequency, amplitude of spike discharges, and shortage of lifespan of foci were observed. Such a result favors the synergy of rapamycin and axitinib with regard to their antiepileptic activity. Considering that the epileptogenic activity of penicillin is confined to GABAergic inhibition breaking down, the mechanism of the synergy of investigated drugs might be explained by their ability to restore GABAergic inhibition in the cortical neuronal network.
Conclusions.1.Rapamycin and axitinib demonstrate synergy in suppressing focal epileptic activity induced in the rat cortex with the application of sodium salt of benzylpenicillin. 2. The combination of blocking mTOR with the suppression of tyrosine kinase B is promising with searching for new and more effective approaches to epilepsy treatment.
References
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