RU2539629C1 - Method for prevention of ischemia of retina with erythropoetin experimentally - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves simulating retinal pathology and preventing it by administering recombinant erythropoietin as a preconditioning agent into a laboratory animal. Erythropoietin is administered daily once on the first experimental day intragastrically in a dose of 50 IU/kg 30 minutes before simulating the pathology. The method promotes the pronounced correction of ischemia of the retina ensured by a mechanism of opening of ATP-dependent K+-canals followed by inducing mechanisms of metabolic adaptation in implementing the preconditioning effect.

EFFECT: preventing the ischemic conditions of the retina.

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 (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 ischemia is theoretically possible using pharmacological preconditioning, the essence of which is the activation of endogenous defense mechanisms that reduce the degree of damage during a subsequent prolonged ischemic episode (Kolesnik I.M. Pharmacological preconditioning with erythropoietin - new possibilities for optimizing the survival of ischemic tissues // Kursk Scientific -practical bulletin "Man and his health." - 2010. No. 3. - P.32-36). The most promising for studying the preconditioning action on the retina is erythropoietin. Recombinant erythropoietin in a sub-erythrostimulating dose has a pronounced cytoprotective effect on the model of an isolated skin flap on a feeding leg (Kolesnik IM The effect of distant preconditioning and recombinant erythropoietin on the survival of ischemic tissues and neovasculogenesis (experimental study) [text]. Sciences: 14.03.06, 01/14/17 / I.M. Kolesnik; Kursk, State Medical University - Kursk, 2010 .-- P.19).

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) were intact animals kept in the vivarium identical to the 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.

Closest to the claimed solution is a Method for the prevention of retinal ischemia in an experiment by prior administration of erythropoietin as a retinal protective agent (MOWAT, FM Endogenous erythropoietin protects neuroretinal function in ischemic retinopathy / FM Mowat, F. Gonzalez, UF Luhmann et al. // Am J Pathol. - 2012 / - Apr. No. 180 (4). - P / 1726-1739).

The main disadvantage of this method is that the introduction of recombinant erythropoietin is carried out intraperitoneally, starting from the 14th day of postnatal development and ending on the 25th day with a dose of 5000 U / kg, which leads to a change in hematocrit and angiogenesis.

The objective of the invention is a method for the prevention of retinal ischemia with erythropoietin in an experiment, comprising recombinant erythropoietin as a preconditioning agent.

The objective is achieved in that in a method comprising modeling retinal pathology by applying mechanical pressure (110 mm Hg) to the anterior chamber of the eye for 30 minutes and preventing it from being administered to a laboratory animal by recombinant erythropoietin, the recombinant erythropoietin being administered as a preconditioning agent once on the 1st day of the experiment, intraperitoneally at a dose of 50 IU / kg 30 minutes before modeling the pathology.

The main advantage of the proposed method is that:

- as a preconditioning agent, recombinant erythropoietin is administered intraperitoneally once in the 1st day of the experiment in a suberythrostimulating dose (50 IU / kg animal body weight), due to this, ATP-dependent K + channels open after 30 minutes (first protective window) after administration which subsequently leads to an increase in the resistance of retinal tissues, 12-48 hours after the introduction of recombinant erythropoietin, 2 protective windows open in which the preconditioning effect is realized mainly due to the action of nitric oxide (NO) and lneyshego inclusion metabolic adaptation mechanisms. In addition, erythropoietin in a sub-erythrostimulating dose (50 IU / kg animal body weight) is administered once, as a result of which the microcirculation in the ischemic retina is improved not due to the formation of new capillaries, but a preconditioning action.

The method leads to a pronounced correction of retinal ischemia, since recombinant erythropoietin at this dose opens ATP-dependent K + channels and further induces metabolic adaptation mechanisms, due to which the preconditioning effect is realized.

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 recombinant erythropoietin at a dose of 50 IU / kg of animal body weight.

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, recombinant erythropoietin (Epocrine epoetin alpha; FSUE State Research Institute for Highly Clean Drugs, Federal Medical and Biological Agency of St. Petersburg, Russia) was administered intraperitoneally at a dose of 50 IU 30 minutes prior to modeling the pathology. kg once. 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 was calculated, the average value, the standard deviation. Differences are considered significant at p <0.05.

EXAMPLE OF SPECIFIC PERFORMANCE

Microcirculation was assessed using a Biopac-systems MP-150 laser-Doppler flowmeter and a 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 recombinant erythropoietin at a dose of 50 IU / kg are presented in the table.

Table The results of assessing the level of microcirculation in the retina of rats 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 ** Erythropoietin Correction, 50 IU / kg (n = 10) 724.0 ± 4.1 ** 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 level of microcirculation after modeling of ischemia in the control group for 72 h of reperfusion was 353.3 ± 11.7 bp, which is 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 for 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 erythropoietin, the level of microcirculation in the group rises to 724.0 ± 4.1 p.u. and significantly different from the values in the control group (p <0.001).

Thus, the obtained results indicate a pronounced correction of ischemic damage to the eyes in the rat retina ischemia model with recombinant erythropoietin at a dose of 50 IU / kg of animal body weight with a single systemic administration.

Claims (1)

A method for preventing retinal ischemia with erythropoietin in an experiment, including modeling retinal pathology and preventing its introduction to a laboratory animal with recombinant erythropoietin, characterized in that the recombinant erythropoietin is administered as a preconditioning agent once per day on the experiment at a dose of 50 IU / kg, for 30 minutes before modeling pathology.