RU2361283C1 - Method of simulation and examination of hemorrhagic stroke consequences - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine, particularly to experimental neurology and can be used to estimate protective action of heparin in hemorrhagic stroke in experiment. The invention is ensured by stroke simulation by autoblood action on cerebral neurons. Herewith convoluted section of cerebral neurons are prepared with registering the initial parametres of caused bioelectric activity of cells, followed with autoblood action and registering the same parametres after the sections are washed. They are compared to the same parametres derived in heparin model wherein the sections are pre-kept within 30 minutes in the incubatory heparin medium 0.5 ml containing 5000 Units/ml. Thereafter the sections are inserted in autoblood for 300 minutes and washed. When the specified parametres are compared to the model with and without heparin, protective action of heparin for integrity of neuron activity in hemorrhagic stroke is estimated.

EFFECT: maintained protection of the cerebral neurons from destructive effect of autoblood in developed simulated hemorrhagic stroke.

2 ex, 2 tbl

Description

The invention relates to physiology, in particular to a method for determining the effect of a biologically active substance in an in vitro experiment in studying the occurrence of hemorrhagic stroke and the development of its consequences.

There is a method of modeling and studying the consequences of hemorrhagic stroke [1], adopted as a prototype, including the preparation of surviving sections of nerve cells of the brain, exposure of autologous blood to sections of the brain, washing of sections, registration of initial and subsequent parameters, after exposure to sections of autologous blood, comparison of the parameters of initial and after exposure.

The goal is to protect brain nerve cells from the damaging effects of autologous blood while modeling a hemorrhagic stroke in an experiment.

This problem is solved as follows.

The essence of the proposed method for modeling and studying the consequences of hemorrhagic stroke, including the preparation of surviving brain slices, recording the initial and subsequent parameters after incubation of the slices in autologous blood and determining the effect by comparing the initial and subsequent parameters, is that the sections are preliminarily placed after preparation heparin (0.5 ml, 5000 U / ml) dissolved in a 1 ml incubation medium and kept there for 30 minutes. Then the sections are placed in autologous blood with a volume of 2 ml (which simulates a hemorrhagic stroke) for 300 minutes (this time corresponds to the “therapeutic window”, after which irreversible damage occurs in the nervous tissue). Following this, the sections are washed with saline from autologous blood and the evoked bioelectric activity of the cells is recorded. The bioelectric activity determines the effect of heparin on the preservation of nerve cells in a simulated hemorrhagic stroke compared to a model without exposure to heparin.

The method is illustrated by an example of determining the protective effect of heparin on brain tissue when modeling hemorrhagic stroke.

Example 1

Determination of the effect of heparin on brain tissue by bioelectric activity parameters - individual components of focal potentials (AF). Sections were kept in an incubation medium with heparin for 30 minutes; this time was established empirically. Then the sections were kept in autologous blood for 300 minutes, the indicated time corresponds to the “therapeutic window”, after which irreversible disturbances in the functioning of nerve cells occur.

As can be seen from the data presented in the table, the preliminary exposure to heparin prior to the action of autologous blood protects the functioning of individual mechanisms of electrogenesis of nerve cell sections. So, the activity of presynaptic processes, estimated by the value of the LOT PD, decreases by 9%. The postsynaptic ones (AMPA and NMDA EPSP) are reduced by 8% and 14%, respectively. The activity of the braking mechanisms, determined by the amplitude of the TPPSm, is reduced by 13%. Statistically, these differences from the initial values are unreliable: n = 10, control; n = 16, heparin; U = 56 - for the amplitude of the PD LOT; U = 54 - for the amplitude of AMPA EPSP, U = 51 - for the amplitude of NMDA of EPSP, U = 49 - for the amplitude of TPPSm, p≥0.05. Exposure to brain sections of autologous blood inhibits the functioning of excitatory mechanisms (PD LOT, AMPA, and NMDA EPSP) and blocks inhibitory activity (TPSPm). Therefore, heparin has a protective effect on nerve cells from the damaging effects of blood.

As a comparison drug, aspirin (acetylsalicylic acid) was used, which is widely used in the clinic as an antiplatelet agent in the treatment of ischemic and hemorrhagic strokes.

Example 2

Determination of the effect of aspirin at a concentration of 0.02 mg / ml on brain sections by the parameters of bioelectric activity - individual components of AF.

As can be seen from the data presented in table 2, the preliminary exposure of aspirin to the action of autologous blood on the nervous tissue does not protect the functioning of individual mechanisms of electrogenesis of nerve cell sections, as evidenced by a sharp decrease in the amplitudes of individual AF components after the action of blood. So, the activity of presynaptic processes, determined by the magnitude of the amplitude of the PD LOT, decreases by 71%. Postsynaptic (AMPA and NMDA of EPSP) are reduced by 72% and 95%, respectively. The activity of the braking mechanisms, determined by the amplitude of the TPPSm, is completely blocked. Statistically, these differences from the initial values are significant: n = 11, control; n = 12, aspirin; U = 50, 45, 49, 52, respectively, for PD LOT, AMPA, NMDA EPSP and TPPSm; p≤0.05. Therefore, aspirin does not protect nerve cells from the negative effects of blood.

The data presented prove that preliminary exposure to heparin on nerve cells of brain sections protects their functioning from the destructive effect of autoblood. Therefore, heparin acts as a neuroprotective substance in the development of the negative effects of hemorrhagic stroke in the experiment.

Thus, a method is proposed that provides protection of nerve cells of the brain, in combination of distinctive features leading to a new technical problem, meets the criteria of the invention: "novelty", "inventive step".

The invention recommends heparin as a drug that protects against the effects of hemorrhagic stroke during clinical trials.

Literature

1. Mokrushin A.A., Hama-Murad A.X., Pavlinova L.I., Smirnova G.P. A method for modeling and studying the effects of hemorrhagic stroke. Patent for invention RU No. 2307396, IPC G09B 23/28, 2007. Prototype.

Claims (1)

A method for determining the protective effect of heparin in hemorrhagic stroke in an experiment, including modeling a stroke by exposing autologous blood to brain nerve cells, preparing surviving sections of nerve cells in the brain, registering the initial parameters of the induced bioelectric activity of cells, influencing autologous blood, recording the same parameters after washing the slices and compared with the same parameters obtained in the model with heparin, in which the sections were pre-incubated for 30 in in 0.5 ml of the incubation medium with heparin containing 5000 U / ml, more sections are placed in autologous blood for 300 minutes, then washed slices; when comparing the specified parameters of the model with and without heparin, the protective effect of heparin on the preservation of the activity of nerve cells during hemorrhagic stroke is determined.