Rapamycin action upon kindled seizure activity in rats

ABSTRACT

The mTOR signal system's activity plays a prominent role in chronic epileptic activity development, which is modeled via repeated subconvulsive dosage of pentylenetetrazol (PTZ) administration. The investigation aimed to verify the effects of Rapamycin – blocker of mTOR upon kindled seizures induced with PTZ in a dosage of 35,0 mg/kg, i.p. Rapamycin was administered in dosages 0,3; 1,0 and 3,0 mg/kg, i.p. during ten days after fully developed kindled seizures. Sodium valproate (50,0; 100,0 and 250,0 mg/kg, i.p.) was used as a reference drug. Methocel solution was administered to control kindled animals under identical conditions. The testing dosage of PTZ (35,0 mg/kg, i.p.) was used on the 11th day from the moment of treatment beginning, and behavioral seizures scored. It was established that Rapamycin in a dosage of 3,0 mg/kg prevented generalized seizures in all kindled rats, and the pronouncement of seizures was in correspondence with the effectiveness of valproic acid administered in a dosage of 250,0 mg/kg. The conclusion of the pronounced antiseizure activity of Rapamycin was made. Such an activity might be explained by Rapamycin ability to prevent neoangiogenesis, which is very important for kindling- induced chronic epileptic activity establishment.

Key words: Rapamycin, pentylenetetrazol, chronic epileptic activity, antiepileptic drugs

Introduction. Inhibitors of the mammalian target of rapamycin (mTOR) pathway are recognized as perspective antiepileptic compounds (19,21). Such pharmacons are different from classical antiepileptic drugs and cause a broad spectrum of effects, including modification of cell growth, proliferation, autophagy, and epigenetic effects. The last one underlay changes in protein synthesis, which, in turn, impacts synaptic plasticity (5, 7, 20). Vice versa, activation of the mTOR pathway induced brain epileptization intensified in genetic and acquired rodent models of epilepsy (16, 21).

Such data favors the mTOR inhibitors as a new and promising approach to epilepsy treatment (4, 8, 9, 11, 15, 18). Antiseizure action of rapamycin – well-known mTOR inhibitor has been shown on kainic acid induced continuous seizures (21), epileptic status induced with pilocarpine (4, 11) as well as on status epilepticus induced with brain structures electrical stimulations (18). But there are no data on effects caused by rapamycin on kindling – induced seizures.

That is why this work's main aim was to investigate the effects of rapamycin upon pentylenetetrazol (PTZ)- induced kindled convulsions. Considering that PTZ-kindled seizures are highly sensitive to valproic acid's antiseizure action [10], the comparison of rapamycin and valproic acid effects was the additional aim of the investigation.

Materials and Methods. Experiments were performed on male Wistar rats with initial bodyweight 200-250 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).

Kindled convulsions were induced, as described previously [6]. PTZ ("Sigma Aldrich") was given intraperitoneally (i.p.) daily in a dose of 30.0 mg/kg for 21 days. The severity of convulsions was evaluated according to the following criteria:

• 0, absence of symptoms of seizures;
• 1, facial tremor and separate myoclonic jerks;
• 2, whole-body clonic convulsions;
• 3, clonic convulsions of the whole body with rearings;
• 4, generalized clonic-tonic convulsions with rearings and falling;
• 5, repeated convulsions as at stage 4 or lethal outcome as a result of a seizure fit.

Those rats, which failed into generalized fits as a response to both 20-th and 21-th PTZ injections, were taken for further observations and screening effects of compounds.

Rapamycin ("Pfizer", USA) was administered in doses of 0.3 – 3.0 mg/kg, i.p., and sodium valproate ("Sigma Aldrich", USA) was injected in doses of 50.0 – 250.0 mg/kg, i.p. Both compounds were dissolved in 5.0% methylcellulose (Methocel, "Sigma Aldrich"). Rapamycin and valproic acid were administered daily for ten days. The last injection was performed in 60 min before testing PTZ administration. Control animals were treated with Methocel only.

Data were analyzed by nonparametric analysis of variance ANOVA (Kruskall-Wallis test), followed by a Mann-Whitney test when appropriate; p<0.05 was accepted as a defining statistical difference.

Results. Behavioral characteristics of the convulsions in kindled rats

Repeated i.p. administration of PTZ (35.0 mg/kg) resulted in the progressive development of seizure manifestations, starting from the third to sixth injection. At the moment of completion of kindling – after the 21-t PTZ administration, a prevalent number of rats (two thirds) demonstrated generalized clonic – tonic fits. Those rats with generalized seizures were used for further investigations.

Effects of rapamycin and valproic acid

Administration of rapamycin in doses of 0.3; 1.0 and 3.0 mg/kg, i.p. resulted in a dose-dependent decrease in the severity of kindled convulsions. Statistical significance was achieved at a dosage of 1.0 mg/kg (Table 1), at which dose 6 of 9 animals were protected against stage 4-5 seizures (p<0.05) as compared with control. When administered in a 3.0 mg/kg dose, rapamycin completely prevented the development of stage 4-5 convulsions.

Administration of valproic acid to kindled animals (50.0; 100.0 and 250.0 mg/kg, i.p.) resulted in a dosedependent seizure-protecting effect that was statistically significant for two doses, 100.0 and 250.0 mg/kg (Table 1) as compared with controls (P<0.001). Both doses prevented stage 5 seizures in all animals, while the higher dosage (250.0 mg/kg) completely prevented generalized clonic-tonic seizures (stage 4 and 5) (Table 1).

Table 1 - Effect of rapamycin and sodium valproate upon 35.0 mg/kg i.p. PTZ-induced kindled seizures

Statistics performed by Kruscall-Wallis and Mann-Whitney U tests.

Discussion. Hence, gained data revealed that rapamycin caused anticonvulsive action on the PTZ - kindled seizures in rats, which was pronounced to prevent generalized clonic-tonic fits. Such effect corresponds with other author's data on antiepileptic potency of rapamycin and blocking the mTOR signaling pathway (19).

Considering mechanisms of antiepileptic effects, it is worth noting that rapamycin is able to inhibit tyrosine kinase B [20], which is of importance for vascular endothelial growth factor (VEGF) synthesis and neoangiogenesis promotion (3, 17, 19). Newly created vessels are highly porous, and the situation with the broking down the brainblood barrier (BBB) is induced finally (2, 13, 14). The long-term process of stimulated neoangiogenesis might be considered as specific pathogenesis of kindled – induced chronic seizures (12). Such an assumption corresponds with earlier shown antiseizure effectiveness of axitinib – another pharmacon, which caused blocking effects upon tyrosine kinase B activity (6).

Also, the comparatively high effectiveness of rapamycin, which is equal to the highest dosages of sodium valproate, is of great interest. It might be supposed that the ability to inhibit tyrosine kinase B, which is essential for the maintenance of kindled seizures (1), is of charge for such effectiveness of rapamycin.

Conclusion. Obtained data and analysis of mechanisms of rapamycin effects favor of a possible role played by VEGF and remodeling of vessels in PTZ – kindled seizures development.

• he pronouncement of action of rapamycin delivered in the highest dosage (3.0 mg/kg, i.p.) corresponds to the effects of sodium valproate administered in a dosage of 250.0 mg/kg, i.p.

Financial support: Ministry of HealthCare of Ukraine.

Conflict of interests: there are any conflicts.

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