Method of modelling paroxysmal convulsive disorder

FIELD: medicine.

SUBSTANCE: invention can be used both for studying mechanisms of development of paroxysmal states with progredient course (epilepsy) and for screening (selection) of novel medications, possessing potential anti-epileptic activity. For this purpose, rats are given fractional injections of convulsant 1.5-pentamethylentetrazole (pentylenetetrazole) with 15 minute interval in dose 10 mg/kg in volume 0.1 ml solution per 100 g of body weight until fist generalised convulsive seizure develops. After that calculated is threshold dose of convulsant reflecting base level of CNS excitability for each animal. When sessions are repeated, dynamics of change of initial dose of pentylenetetrazole is taken into account, interval between sessions of pentylenetetrazole titration can constitute from 24 and more than 168 hours.

EFFECT: method ensures high accuracy of evaluation of change of seizure development threshold due to the fact that expression of modelled progredient state can be changed at any stage of carrying out chronic research without application of special methods of examination.

1 tbl, 2 dwg

 

The invention relates to medicine, namely to methods for modeling of convulsive States, and can be used for studies of the mechanisms of development of paroxysmal States with progredient course over (epilepsy), and screening (selection) of new drugs with potential antiepileptic activity.

The development of modern antiepileptic drugs in the preclinical stage requires the study of their specific activity on adequate models of epileptic disease. Despite the significant achievements of epilepsy, search models of this disease still remains an issue. For example, the classical model for the selection of anticonvulsants are not always able to identify therapeutic activity of new anti-epileptics.

The most promising may be the model of epilepsy, taking into account possible, on the one hand, the spontaneity of formation of epileptic seizures, and on the other, having a progressive nature of current pathology. Such combinations of pathological signs are hardly compatible with precision measurements of the severity of the pathological signs.

Currently, the preferred models of the disease, based on genetic disorders, resulting in changes in the functioning of neurochemical the fir and neurophysiological activity of individual neurons and brain regions (formation of an epileptic focus). Such models can be attributed photosensitive and audiogone convulsions in animals. Electrophysiological correlates of such seizures are formed in the respective specific areas of the cortex and in the case of long/powerful impact of pathological factor epileptiform discharges generalized, leading to the development of behavioral convulsive seizure.

In these models, there are both pathological factor, but the severity of progression is difficult to predict, because the mechanisms of formation of these pathological changes is important sensitivity of peripheral receptors to the perception of audio and futurestate, as well as mechanisms for abnormal discharges in the Central nervous system. In this regard, the threshold stimulus for the formation of seizures can have individual animals vary considerably.

Other models of epilepsy have a higher predictive value for studies of the pathogenesis and the identification of new drugs belong to the class of models of kindling/swing" (kindling) [Shandra A.A., godlewsky PS, Brusentsov A.I. Kindling and epileptic activity.- Odessa: Extra Print, 1999. - 274 S.]. The basis for the formation of paroxysmal States defines the physical or chemical factor dosed mode subthreshold stimulation. Continue the compulsory presentation of such stimuli causes that over time the sensitivity to the damaging factors increase and decrease the thresholds of occurrence sugoroku the end, after some period of time seizures begin to develop themselves already in the absence of the damaging factor. Although this model of epileptic disease also carries the above-mentioned disadvantages, but has the advantage that the variability seizures may be significantly less pronounced than in the above models, because the pathological stimulation clearly measurable (electrical current, the dose of anticonvulsant).

The ideal can be genetic model, where the basis of initiation of seizures are the mechanisms of the failure of the neurochemical processes in the Central nervous system, caused by damage of the genetic apparatus. However, registration of paroxysmal activity in these models is quite complicated and requires some tricks (EEG monitoring), because the probability of occurrence of seizures during the recorded period is unpredictable.

Other models of epilepsy maximum electroshock, chemical generalized convulsions, local focal chemical convulsions currently have less prognostic importance for the evaluation of antiepileptic activity of drugs, because time-consuming and/or require the presence of the Oia special equipment.

The closest in technical essence and the achieved result is a simulation method paroxysmal disorders by conducting fractional injections pentylenetetrazole 1,5-pentamethylenetetrazol-corsola) [Shandra A.A., godlewsky PS, Gratkowski CENTURIES of Systemic mechanisms of pharmacological resistance convulsive activity // Archive wedge. and experts. medicine. - 2002. - T, No. 1. - S-114.].

The disadvantages of this method is its lack of accuracy, due to the fact that the intensity of the simulated paroxysmal disorders cannot be accurately measured at any stage of chronic studies without the use of special methods of examination (EEG monitoring).

The technical result, which directed the present invention is to improve the accuracy of the simulation method paroxysmal convulsive disorders, due to the fact that the intensity of the simulated having a progressive condition can be accurately measured at any stage of chronic studies without the use of special methods of examination (EEG monitoring).

This technical result is achieved in that in the method of modeling paroxysmal convulsive disorders by conducting fractional injections pentylenetetrazole, according of the bretania, rats with an interval of 15 min spend subcutaneous injection of pentylenetetrazole at the rate of 10 mg/kg in a volume of 0.1 ml per 100 g of body weight to development of generalized convulsive seizure, and then calculate the threshold dose convulsant, reflecting a basic level of excitability of the Central nervous system for each animal during the repeated sessions take into account the dynamics of the original dose pentylenetetrazole, the interval between sessions titration of pentylenetetrazole can be between 24 and more than 168 hours, and the first session of the titration is carried out before the injection.

In the proposed simulation method having a progressive paroxysmal convulsive disorders take into account the dose of pentylenetetrazole necessary for the formation of the first/one generalized convulsive seizure. On the dynamics of progression of chronic disorder is judged to change the doses of pentylenetetrazole data regarding its original dimensions. The solution pentylenetetrazole administered to rats subcutaneously so that the dose injected per injection convulsant was 10 mg/kg in a volume of 0.1 ml per 100 g of body weight of the animal. Repeat injections after 15-minute interval during a single experimental day (session) prior to the occurrence of generalized convulsions, what is the criterion to stop further introductions convulsants this day. Further, the specified mode experiment is dictated by the purpose of the study, depending on which, or the session is repeated on the next day, or a day or a few days (weeks)that allows you to achieve different speeds of progression. Every experienced the day ends with a count of the total (cumulative) dose convulsant necessary for the development of a single generalized seizures for each animal. The value of convulsive dose of pentylenetetrazole registered on the first day, is a control for each animal (case-control) in a chronic experiment that allows you to individualize the assessment of the measurement of paroxysmal activity in each case (for each animal).

The method is as follows:

In our studies was used 70 white mongrel of male rats, weighing 200-300 g, which in accordance with the objectives have been divided into groups in accordance with different strategy-setting experiment (scheme - a, B, C).

Conditions of the introduction of pentylenetetrazole (corsola) were standardized in all experimental schemes. Pentylenetetrazol was administered subcutaneously in a volume of 0.1 ml per 100 g of body weight at a dose of 10 mg/kg rats, again, with an interval of 15 min before the advent of generalized seizures. Each series of experiences Sataniv the Lee, calculating for each animal the minimum dose of pentylenetetrazole, causing convulsions, in the future, were used to determine average convulsion dose of the drug in each experimental group of rats.

Scheme And (case-control)

All test animals were divided into groups:

1.) sessions titration 1,5-pentamethylenetetrazol" (to convulsions) was performed every day for 6 days with an interval between individual experiments 24 hours;

2.) same, but the spacing between the individual experiments was 72 hours;

3.) same as in 1, but the titration 1,5-pentamethylenetetrazol" was performed 1 time per week during the month (interval 168 hours).

The results of determination of the threshold for the development of seizures in these groups of rats reflected in figure 1.

The data presented indicate that the pronounced decrease in the dose of 1.5-pentamethylenetetrazol leading to convulsions were observed only in the first group of rats. Apparently, the repeated series "titrations", repeated at intervals of 24 hours, to form the state similar to the chemical buildup (kindling) [Shandra A.A., godlewsky PS, Brusentsov A.I. Kindling and epileptic activity. - Odessa: Extra Print, 1999. - 274 S.]. It is also necessary to emphasize that in this experiment we over time has also registered the transformation of the behavioral response of animals in the CTE is the introduction of repeated doses convulsant. So, from the third day of injections of 1.5-pentamethylenetetrazol, the animals began to react already at a dose of 20-30 mg/kg, although convulsive dose of pentylenetetrazole at this time even increased, in comparison with the results of the first injection. Behavioral sensitivity was shown by the twitching of individual body parts, generalized wince rats, sometimes celebrated elements of stereotypical behaviour (animals at this time did not react to external audio signals). Described behavioral phenomena were absent in the first two days of the fractional introduction convulsant. By the end of the experiment all experimental animals of the first group, the dose of 1.5-pentamethylenetetrazol, causing the development of generalized clinico-tonic convulsions, decreased by 10 mg no Such dependence we observed in groups 2 and 3, in which the titration is conducted after 72 and 168 hours, respectively.

Scheme B (a comparative study with low providentially to identify prostoronnih effects):

1.) sessions titration 1,5-pentamethylenetetrazol" (to convulsions) was performed once a 7 days with an interval between experiments 168 hours, the animals received standard laboratory food;

2.) sessions titration 1,5-pentamethylenetetrazol" (to convulsions) was performed once a 7 days with an interval between experiments 168 hours, but within a month the experimental animals were on a diet, enriched with exciting amino acids (HAC: glutamate and aspartate).

Table 1 presents the results, showing the change of excitability of the Central nervous system in rats that two weeks was on the food enriched HAC.

The results of these experiments showed that a daily Supplement in food HAC increases the sensitivity of rats to the convulsant effect of 1,5-pentamethylenetetrazol.

Because the control group in this experiment we observed a dose reduction convulsant within three weeks, whereas rats from 7 days consumption HAC minimal convulsive dose of pentylenetetrazole was significantly lower. Alignment doses of pentylenetetrazole in the experimental and control groups after four weeks due to the fact that, beginning with the 15th day of the test animals again went on a diet of standard laboratory food. The increase in the functional activity of the HAC system for the development of epilepsy is well known.

The scheme In (a comparative study with high providentially to study the anticonvulsant effects):

1.) sessions fractional introduction of pentylenetetrazole (prior to the development of seizures) was performed every day for 6 days (the interval between experiments 24 hours), the animals through the mouth received distilled water;

2.) sessions fractional introduction of pentylenetetrazole(prior to the development of seizures) was performed every day for 6 days (the interval between experiments 24 hours), animals received through the mouth verapamil at a dose of 25 mg/kg;

3.) sessions fractional introduction of pentylenetetrazole (prior to the development of seizures) was performed every day for 6 days (the interval between experiments 24 hours), the animals through the mouth received diltiazem at a dose of 20 mg/kg;

4.) sessions fractional introduction of pentylenetetrazole (prior to the development of seizures) was performed every day for 6 days (the interval between experiments 24 hours), the animals through the mouth received diazepam at a dose of 0.5 mg/kg

The results, showing the change convulsive dose of pentylenetetrazole with repeated injections suggests that over time increases the sensitivity of animals to convulsive action convulsant. A significant reduction of the minimum convulsive dose of the agent to 80,57 mg/kg was noted already from the second day of the experiment. The initial dose of pentylenetetrazole in the control group of rats was 67,332,48 mg/kg, and by the end of the experiment was 58,641,53 mg/kg (P<0,01). Thus, the re-introduction of pentylenetetrazole caused increased sensitivity of the CNS to the convulsive action.

The effect of BCA on the convulsive effect of pentylenetetrazole in our experiment are presented in figure 2.

All drugs with repeated injections increased the threshold dose convulsant necessary for the development of seizures: verapamil (25 mg/kg) to 105.8%, diltiazem (20 mg/kg) - 102%, diazepam (0.5 mg/kg) - 113%in the control group (animals received distilled water through the mouth) this value corresponded to 87.1%. Thus, all subjects in the calcium channel blockers, as well as diazepam, have lowered the sensitivity of rats to the convulsant action of pentylenetetrazol. The medicines were prescribed by mouth once a day immediately after the session definition convulsive dose of 1.5-pentamethylenetetrazol. Diazepam (0.5 mg/kg) was administered according to the same scheme. This method of setting the experiment made it possible to exclude acute effects of drugs on the development of 1,5-pentamethylenetetrazol cramps and dynamically evaluate the change of the threshold convulsions within five days.

Thus, the presented results indicate that the BPC, although to a lesser extent than diazepam, but also reduce the level of excitability of the Central nervous system.

Calcium channel blockers are considered by researchers as potential antiepileptic drugs [Kryzhanovsky GN, Karpov M.N., Punks O.Y // Influence of organic calcium antagonists and magnesium on the development corpolongo of kindling. - Bull. Exp. Biol. and the Honey. - 1990. No. 1. - P.53-54], which was confirmed in our study using the proposed model.

Thus, the proposed method can accurately and unlimited time to register the change of the threshold of the development of the Penta is interatomic seizures. In the study of anticonvulsant/antiepileptic activity of new drugs. The first session "titration" is recommended prior to the introduction of the test drug (experimental group) or its solvent (control animals). This allows you to identify the initial level of excitability of the Central nervous system, which will help in future to establish the dynamics of changes convulsive reaction in each case.

This experiment is necessary especially when the objectives of the study is required to assess the effect of test drug on animals with a low threshold for the development of seizures. Such conditions, in our opinion, the most adequately reflect the pathogenesis of epilepsy, in which over time the threshold of excitability of the Central nervous system gradually decreases. It should be emphasized that in the day "titration" of pentylenetetrazole to enter the study drug needed after the experiment in order to exclude acute anticonvulsant effect of the test substance.

The proposed method of modeling paroxysmal convulsive disorders by conducting fractional injections pentylenetetrazole allows a high degree of accuracy to assess over time the change in the threshold of excitability of the Central nervous system (change convulsive dose of pentylenetetrazole) when testing new anticonvulsant/protivopul the practical tools. Presents the particular scheme of the production experiment, which reflects the main stages of preclinical studies of potential antiepileptic drugs. For example, in some cases may be justified multiple changing intervals between the sessions titration 1,5-pentamethylenetetrazol" in the course of a single experiment or the introduction of the test of anticonvulsant with its subsequent cancellation during one experiment. This will allow to estimate the effect of the investigational product, as in natural conditions and under conditions of increased excitability of the Central nervous system.

The proposed method of modeling paroxysmal convulsive disorders by conducting fractional injections pentylenetetrazole gives the ability to accurately quantify the level of excitability of the Central nervous system when the variation of the research depending on the conditions and objectives of the experiment, allowing to vary the sensitivity of animals to the stimulating effect of 1,5-pentamethylenetetrazol. However, in contrast to the known method, "chemical drive", the essence of which is that the introduction of 1,5-pentamethylenetetrazol [Shandra A.A., godlewsky PS, Brusentsov A.I. Kindling and epileptic activity. - Odessa: Extra Print, 1999. - 274 S.] is repeated at regular intervals of time, the method in our modification provides the register dynamics convulsive reaction, allowing assessment of changes in the sensitivity (irritability), Central nervous system and at the request of the researcher, changing the intensity of the "shakedown" by variation in timing of repeated sessions of fractional introduction of pentylenetetrazole.

Thus, the proposed method of modeling paroxysmal convulsive disorders by conducting fractional injections pentylenetetrazole suitable for studying the activity of the new anticonvulsant/antiepileptic medicines, because it allows high accuracy rate of change threshold for the development of seizures.

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Table 1
The effect of diets enriched HAC, to change the minimum convulsive dose of corazol
Groupday 7day 1421 days28 day
Control58,14,259,03,261,55,452,14,1
Experimental52,53,251,02,1**54,31,8*51,23,2
Note - the figures show the changes in the minimum convulsive dose of pentylenetetrazole
** P<0,01; * P<0,05

The simulation method paroxysmal convulsive disorders by conducting fractional injections pentylenetetrazole, characterized in that the rats with an interval of 15 min spend subcutaneous injection of 1.5-pentamethylenetetrazol at the rate of 10 mg/kg in a volume of 0.1 ml per 100 g of body weight to development of the first generalized convulsive seizure, and then calculate the threshold dose convulsant, reflecting a basic level of excitability of the Central nervous system for each animal during the repeated sessions take into account the dynamics of the original dose pentylenetetrazole, the interval between sessions titration of pentylenetetrazole can be between 24 and more than 168 hours



 

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