Natalia M. Surina

Epilepsy is a heterogeneous disease, which determines the relevance of investigating the mechanisms of pathogenesis of its various types, including reflex epilepsy. Pharmacotherapy is common for the treatment of patients with epilepsy, however, despite the significant recent achievements, 20-30% of patients remain resistant to the ongoing treatment. The urgency of creating new antiepileptic drugs, in particular, immune ones is due not only to a significant proportion of pharmacoresistant cases, but also to the struggle for the quality of life of patients. The neuroinflammation system in animals with different genetically determined audiogenic epilepsy proneness was investigated. These genetic groups were Krushinsky–Molodkina rats (tonic seizures of maximum intensity in response to the action of sound) and “0” strain (control group, non-convulsive phenotype). The main proinflammatory cytokines levels in the dorsal striatum and brain stem in rats of these genetic groups were measured by multiplex immunofluorescence assay. Background levels of IL-1â, IL-6 and TNFá in the dorsal striatum of KM rats were significantly lower than in the control “0” strain rats, whereas in the brain stem in the “background” levels of these metabolites did not differ. Four hours after the sound exposure, the TNFá level in the dorsal striatum of KM rats was significantly lower than in “0” rats. In KM rats, after the sound exposure and subsequent tonic seizures, the levels of IL-1â and IL-6 in the dorsal striatum were significantly higher than in the background. The IL-2 content was not detected in the background in KM rats, whereas after audiogenic seizures its level was 14.01 pg/ml. In the brain stem of KM rats, the levels of IL-1â and TNFá after audiogenic seizures were significantly lower than in the background. In rats of the “0” strain, cytokine levels in the dorsal striatum after the sound exposure did not differ from those in the background, while IL-1ß levels in their brain stem were significantly lower than the background state. Particular modulating role of the studied proinflammatory cytokines in the pathogenesis of audiogenic epilepsy is assumed, as well as certain possibility of anti-inflammatory and immune drugs application in anticonvulsant and antiepileptic therapy.

The participation of the glutamatergic neurotransmitter system in the pathogenesis of audiogenic seizures (AS) and post-ictal catalepsy (PIC) in Krushinsky–Molodkina (KM) strain rats was analyzed. Effects of D-serine and disocilpine administration were investigated. In intact KM rats, the intensity of AS correlates with the duration of PIC. The administration of D-serine (acute administration, doses of 200, 400 and 600 mg/kg, as well as after chronic administration—5 days of 300 mg/kg, n = 34) had no significant effect on AS and PIC. Disocilpine (MK-801, a non-competitive NMDA antagonist) was administered in an acute experiment at doses of 0.1, 0.2 and 0.4 mg/kg (n = 41). MK-801 dose-dependently reduced the intensity of AS and caused a “two-wave pattern” of seizures in the most of animals, and removed PIC at a lower dose than AS seizures. PIC was completely eliminated already at a dose of 0.2 mg/kg, while the clonic component of AS still persisted. Thus, it was possible to show the “dissociation” between AS and PIC. It is assumed that although dopaminergic control is involved in the mechanisms of PIC development, glutamatergic neurotransmission is also taking part in the PIC manifestation.

Learning difficulties are a fairly common comorbid complication in patients diagnosed with epilepsy. The relationship between the predisposition to epilepsy and the ability to learn is an important problem. The aim of the study was to analyze how the learning capacity differs in rats with different predispositions to reflex audiogenic epilepsy (AE). The success of active avoidance (AAC) conditioning in the shuttle box in rats of 3 strains was evaluated. These were rats predisposed to audiogenic epilepsy—the Krushinsky–Molodkina strain (KM), strain “4” (selected from a population of F2 hybrids of the KM strain and AE non-prone Wistars) and rats of “0” strain, selected for the absence of AE from the same population (i.e. these strains differ radically in AE-proneness, but have a similar genetic background). The training was conducted continuously for 5 days (20 presentations per day). Experiments have shown significantly more successful acquisition of this skill in rats of the “0” strain: in total, 75% of animals reached the learning criteria for 5 days of AAC training (70% AAC per day). The “4” strain rats indices were intermediate—41.7%, and only 1 from the 12 rats of the KM strain (8.3%) acquired the task according to this protocol. Thus, it was possible to identify more successful learning in rats, selected for the absence of AE seizures (strain “0”) compared to rats of the KM and “4” strains (expressing tonic seizures of maximum intensity in response to sound exposure). The weakest acquisition was revealed in the KM strain, in which the selection process duration for AE proneness was significantly longer than in the rats of the “4” strain.

Neuroinflammation plays an important role in epileptogenesis, however, most studies are performed using pharmacological models of epilepsy, while there are only few data available for non-invasive, including genetic, models. The levels of a number of pro-inflammatory cytokines were examined in the Krushinsky–Molodkina (KM) rat strain with high audiogenic epilepsy (AE) proneness (intense tonic seizure fit in response to loud sound) and in the control strain “0” (not predisposed to AE) using multiplex immunofluorescence magnetic assay (MILLIPLEX map Kit). Cytokine levels were determined in the dorsal striatum tissue and in the brain stem. Background levels of IL-1β, IL-6, and TNF-α in the dorsal striatum of the KM rats were significantly lower than in the rats “0” (by 32.31, 27.84, and 38.87%, respectively, p < 0.05, 0.05, and 0.01), whereas no inter-strain differences in the levels of these metabolites were detected in the brain stem in the “background” state. Four hours after sound exposure, the TNF-α level in the dorsal striatum of the KM rats was significantly lower (by 38.34%, p < 0.01) than in the “0” rats. In the KM rats, the dorsal striatal levels of IL-1β and IL-6 were significantly higher after the sound exposure and subsequent seizure fit, compared to the background (35.29 and 50.21% increase, p < 0.05, 0.01, respectively). In the background state the IL-2 level in the KM rats was not detected, whereas after audiogenic seizures its level was 14.01 pg/ml (significant difference, p < 0.01). In the KM rats the brain stem levels of IL-1β and TNF-α after audiogenic seizures were significantly lower than in the background (13.23 and 23.44% decrease, respectively, p < 0.05). In the rats of the “0” strain, the levels of cytokines in the dorsal striatum after the action of sound (which did not induce AE seizures) were not different from those of the background, while in the brain stem of the “0” strain the levels of IL-1β were lower than in the background (40.28%, p < 0.01). Thus, the differences between the background levels of cytokines and those after the action of sound were different in the rats with different proneness to AE. These data suggest involvement of the analyzed cytokines in pathophysiology of the seizure state, namely in AE seizures.

Animal models of epilepsy are of great importance in epileptology. They are used to study the mechanisms of epileptogenesis, and search for new genes and regulatory pathways involved in the development of epilepsy as well as screening new antiepileptic drugs. Today, many methods of modeling epilepsy in animals are used, including electroconvulsive, pharmacological in intact animals, and genetic, with the predisposition for spontaneous or refractory epileptic seizures. Due to the simplicity of manipulation and universality, genetic models of audiogenic epilepsy in rodents stand out among this diversity. We tried to combine data on the genetics of audiogenic epilepsy in rodents, the relevance of various models of audiogenic epilepsy to certain epileptic syndromes in humans, and the advantages of using of rodent strains predisposed to audiogenic epilepsy in current epileptology.

Dexamethasone is a synthetic glucocorticosteroid with anti–inflammatory and immunosuppressive effects. In order to identify new targets for pharmacotherapy, as well as to study immune mechanisms in the pathogenesis of epilepsy, the influence of dexamethasone on audiogenic epilepsy and catalepsy in Krushinsky–Molodkina (KM) rats has been studied. The chronic (but not acute) infusion of dexamethasone decreased the audiogenic seizure fits intensity in male rats of Krushinsky–Molodkina (KM) strain which is highly susceptible to audiogenic epilepsy, but was accompanied by part of animal deaths. In a month after dexamethasone injections the audiogenic sensitivity of the rescued animals restored to the control levels. The “0” strain rats, bred from F2 KM x Wistar hybrids, no dexamethasone induced mortality was found. The acute and chronic dexamethasone action in rats of “0” strain induced the emergence of catalepsy after the sound exposure, although the audiogenic seizure was found only in one animal. The chronic and acute dexamethasone decreased the postictal catalepsy in KM rats. Thus, pro-inflammatory mechanisms are involved in the pathogenesis of audiogenic epilepsy. Dexamethasone had a distinct anticonvulsant effect in the chronic experiment.

The review presents data which provides evidence for the internal relationship between the stages of rodent audiogenic seizures and post-ictal catalepsy with the general pattern of animal reaction to the dangerous stimuli and/or situation. The wild run stage of audiogenic seizure fit could be regarded as an intense panic reaction, and this view found support in numerous experimental data. The phenomenon of audiogenic epilepsy probably attracted the attention of physiologists as rodents are extremely sensitive to dangerous sound stimuli. The seizure proneness in this group shares common physiological characteristics and depends on animal genotype. This concept could be the new platform for the study of epileptogenesis mechanisms.

We studied parameters of specific receptor binding of D2-dopamine receptor ligand [³H]-sulpiride and NMDA-receptor ligand [³H]-MK-801 on the membranes of striatum cells in Krushinsky–Molodkina rats (predisposed to audiogenic seizures) and strain “0” selected for the absence of audiogenic seizures. No interstrain differences were observed in affinity (K_d) of both D2- and NMDA-receptors to ligands. At the same time, significant interstrain differences in receptor density (B_max) were found for both D2-receptors and NMDA-receptors. The reduced number of dopamine and glutamate receptors in the striatum can be associated with neurological peculiarities of Krushinsky–Molodkina rat strain (audiogenic seizures and postictal catalepsy).