RU2539630C1 - Method for prevention of ischemia of retina experimentally - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: simulating this pathology is accompanied by pharmacological preconditioning. That is ensured by using Nicorandil in a single dose of 0.86 mg/kg of body weight which is introduced intragastrically through a probe in the form of a starch solution into a laboratory animal. Ischemia of the retina is simulated 30 minutes later by delivering mechanical pressure of 110 mm Hg on an anterior eye chamber for 30 minutes.

EFFECT: method provides the pronounced correction of ischemic ocular injuries.

1 ex, 1 tbl

Description

The invention relates to medicine, in particular to experimental pharmacology and ophthalmology.

According to well-known literature, retinal ischemia is the leading link in the pathogenesis of a number of ocular fundus vascular diseases and can lead to disability (Neroev V.V. Retinal ischemia and nitric oxide // Bulletin of the Russian Academy of Medical Sciences. - 2003. No. 5. - P. 37. -40). Prevention and correction of retinal ischemic diseases is an urgent problem that can be solved with the help of pharmacological preconditioning, the essence of which is the activation of endogenous defense mechanisms that reduce the degree of damage in the subsequent long-term ischemic episode (Kolesnik I.M. Pharmacological preconditioning with erythropoietin - new possibilities for optimizing the survival of ischemic tissues // Kursk scientific and practical bulletin “Man and his health.” - 2010. No. 3. - P.32-36).

There is a method of dibornol correction of acute ischemic retinopathy in an experiment (Tsoi Yu.R. Influence of dibornol on the morphofunctional structure of rat retina when modeling total transient cerebral ischemia // All-Russian 69th final scientific student conference dedicated to the 200th anniversary of N.I. Pirogova (Tomsk, May 11–13, 2010): collection of articles / edited by VV Novitsky, L. M. Ogorodova. - Tomsk: Siberian State Medical University, 2010. - P.376-378), which is characterized by the fact that the experiments were carried out on 30 mature white rats, Wistar female line weighing 230-250 g Animals were divided into 3 groups. Rats of the first group (n = 10) were reproduced total transient cerebral ischemia (IHM) according to the method W.A. Pulsineli. 1 day before the simulation of total transient IGM in anesthetized rats (ethaminal sodium 60 mg / kg), both vertebral arteries were thermocoagulated at the level of the first cervical vertebra. IHM was reproduced under ether anesthesia by clamping the common carotid arteries for 30 minutes. The consistency of the model was evaluated by blanching of the visible part of the choroid, expansion of the pupils, and development of hyperventilation. Reperfusion was performed by removing the occluders, after which the wound was sutured. Rats of the second group (n = 10) were dibornol at a dose of 100 mg / kg in 1 ml of starch mucus once a day for 7 days. The control group (n = 10) - intact animals kept in vivariums identical to experimental conditions. The study material was the retina of rats seized after their killing.

The main disadvantage of this method is that the model of total transient cerebral ischemia is not specific for the study of the correction of acute ischemic retinopathy, since in addition to the retina, the brain and soft tissues of the head are exposed to ischemia, which leads to the development of massive post-reperfusion damage to tissues and organs with the possibility of crash development Syndrome. In addition, the use of this model does not exclude the presence of collateral circulation between the pool of the common carotid and subclavian arteries.

There is a method of endothelio- and cardioprotection by pharmacological preconditioning of nicorandil at a dose of 4 mg / kg when modeling nitric oxide deficiency, according to the description of experimental studies in the thesis for the degree of candidate of medical sciences on the topic: “Using pharmacological preconditioning of nicorandil for correction of endothelial dysfunction on the L- model NAME-induced nitric oxide deficiency (experimental study) "Staroseltseva OA The experiments were carried out on white Wistar male rats weighing 250-300 g. N-nitro-L-arginine methyl ether (L-NAME) was administered daily once a day at a dose of 25 mg / kg for 7 days. When modeling -NAME-induced NO deficiency, the endothelial protective effect of the pharmacological preconditioning of nicorandil was studied. Nicorandil was administered at a dose of 4 mg / kg as a solution intragastrically through a probe 2 times a day for 7 days immediately before administration of L-NAME.

The main disadvantage of this method is that only male animals are included in the model of endothelial dysfunction due to the presence of cyclic hormonal background in females. Due to the endothelioprotective properties of estrogens, the purity of the experiment is impaired when evaluating the endotheliotropic properties of drugs, in particular, nicorandil.

In addition, the study revealed only the endothelio and cardioprotective properties of pharmacological preconditioning with nicorandil.

Closest to the claimed solution is a method for the prevention of retinal ischemia in an experiment described in the work of T. DEMIR et al. Trimetazidine for prevention of induced ischemia and reperfusion of guinea pig retina. Clin http: //Ophthalmol.-2010.-Feb. No. 2 (4), 21-26, ref., Including modeling of retinal ischemia with subsequent reperfusion, using trimetazidine to protect against retinal ischemia.

The disadvantage of this method is the complexity of the experiment.

The objective of the invention is to provide an effective method for the prevention of retinal ischemia in an experiment, including the preconditioning agent nicorandil.

This object is achieved by the fact that the proposed method for the prevention of retinal ischemia in the experiment, including pharmacological preconditioning, using nicorandil at a dose of 0.86 mg / kg body weight, which is administered to the laboratory animal once in the form of a starch solution intragastrically through a probe, and after 30 minutes, retinal ischemia is simulated by applying a mechanical pressure of 110 mm Hg. on the anterior chamber of the eye for 30 minutes.

The main advantages of the proposed method is that:

- the introduction of nicorandil at a dose of 0.86 mg / kg of animal body weight once leads to a pronounced correction of retinal ischemia, since nicorandil opens ATP-dependent K + channels and further induces metabolic adaptation mechanisms, due to which the preconditioning effect is realized;

- reproduce the model of retinal ischemia 30 minutes after the administration of nicorandil by applying mechanical pressure (110 mm Hg) to the anterior chamber of the eye for 30 minutes on the 1st day of the experiment, which is specific for the study of the correction of ischemic retinopathy, since ischemia only the tissues of the eye are exposed without involving the soft tissues of the head and brain.

In addition, the anti-ischemic effect of pharmacological preconditioning with nicorandil in modeling retinal ischemia was detected in animals of both sexes.

The level of microcirculation in the retina was recorded after 72 hours of reperfusion after modeling of retinal ischemia.

The method is carried out as follows.

The experiments were performed on 40 white laboratory rats of both sexes weighing 180-220 g. Each group included 10 rats. The first group is a group of intact animals, the second group is with retinal ischemia (control), the third group is with distant ischemic preconditioning (DIP); the fourth group - with pharmacological preconditioning with nicorandil.

Retinal ischemia was simulated by applying mechanical pressure (110 mmHg) to the anterior chamber of the eye for 30 minutes on the 1st day of the experiment. Confirmation of the formation of ischemia was the absence of ocular blood flow (whitening of the eyes of a laboratory animal).

In the third group of animals, DIP was performed on day 1 of the experiment with 10 minutes of compression of the femoral artery 40 minutes before modeling of retinal ischemia, followed by a 30-minute episode of reperfusion. DIP was performed by applying a tourniquet to the proximal third of the thigh. The control of the correct application of the tourniquet was the absence of a pulse on the leg arteries.

In the fourth group of animals, nicorandil was introduced at a dose of 0.86 mg / kg of body weight of the animal 30 minutes before the simulation of the pathology (the therapeutic dose was obtained by transferring doses from a person to a rat using the interspecific transfer formula and the dose conversion factor for different animal species depending on by body weight according to IP Ulanova) once intragastrically (through a probe) in the form of a starch solution. 15 minutes prior to the modeling of ischemia, anesthesia was performed by intraperitoneal injection of a solution of chloral hydrate at a dose of 300 mg / kg of animal body weight.

The severity of the anti-ischemic effect was judged by the level of microcirculation in the rat retina using a Biopac-systems MP-150 laser-Doppler flowmeter and a TSD-144 needle-type sensor (USA) for 72 hours of reperfusion after modeling the pathology. For this, under anesthesia, the animal was fixed and the level of microcirculation in the retina was recorded at ten points around the circumference of the sclera.

When statistical data processing is calculated, the average value, the standard deviation. Differences are considered significant at p <0.05.

EXAMPLE OF SPECIFIC PERFORMANCE

Microcirculation was evaluated using a Biopac-systems MP-150 laser-Doppler flowmeter and TSD-144 sensor (USA). For this, under anesthesia (chloral hydrate 300 mg / kg of the animal’s body weight, intraperitoneally), the animal was fixed and the level of microcirculation in the retina was recorded at ten points around the sclera circumference with a step of 1 mm. The microcirculation level curve was recorded for 20 seconds at each point. From the obtained ten values, the average was deduced, which was entered into the protocol and was taken as the level of microcirculation in the retina in this animal. From 10 obtained values, the average was deduced, which was taken as the level of microcirculation in the retina in this group of animals.

The results of assessing the level of microcirculation in the rat retina at 72 hours of reperfusion after modeling retinal ischemia and its correction with nicorandil at a dose of 0.86 mg / kg of animal body weight are presented in the table.

Table The results of assessing the level of microcirculation in the rat retina at 72 hours of reperfusion (M ± m), p.u. (perfusion units) Intact (n = 10) 743.9 ± 5.0 Retinal Ischemia (n = 10) 353.3 ± 11.7 ∗ DIP correction (n = 10) 638.5 ± 15.8 ∗∗ Correction with nicorandil, 0.86 mg / kg (n = 10) 705.2 ± 15.5 ∗∗ Note: ∗ - p <0.05 in comparison with a group of intact animals; ∗∗ - p <0.05 compared with a group of animals with retinal ischemia

The level of microcirculation in the retina of intact rats was 743.9 ± 5.0 p.u. (perfusion units). The microcirculation level after modeling of ischemia in the control group amounted to 353.3 ± 11.7 p.u. at 72 hours of reperfusion, which was significantly lower than the value in the group of intact animals (p <0.001). Against the background of correction of the pathology of DIP, the level of microcirculation in the retina at 72 hours of reperfusion significantly increases to 638.5 ± 15.8 p.u. (p <0.001) compared with the control group. With the correction of retinal ischemia with nicorandil, the level of microcirculation in the group rises to 705.2 ± 15.5 p.u. and significantly different from the values in the control group (p <0.001).

Thus, the proposed method is effective for the prevention of retinal ischemia. The obtained results indicate a pronounced correction of ischemic damage to the eyes under the conditions of a model of retinal ischemia with systemic administration of nicorandil to bisexual white laboratory rats. The anti-ischemic effects of pharmacological preconditioning with nicorandil were revealed in modeling retinal ischemia due to the discovery of ATP-dependent K + channels and the further activation of metabolic adaptation mechanisms.

Claims (1)

A method for the prevention of retinal ischemia in an experiment, including pharmacological preconditioning, characterized in that they use nicorandil at a dose of 0.86 mg / kg body weight, which is administered to the laboratory animal once as a starch solution intragastrically through a probe, and after 30 minutes, ischemia is simulated retina by exerting a mechanical pressure of 110 mm Hg on the anterior chamber of the eye for 30 minutes.