RU2500041C1 - Method of simulating atherosclerosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for the purpose of diagnosing, preventing and treating the same disease, a cholesterol powder in the amount of 1%, margarin 10%, mercazolilum 10 mg/kg and vitamin D 2.5 IU per kg of body weight is added to the laboratory rats' feed. That is combined with performing an operation of ligating a left renal pedicle with a non-absorbable suture and underrunning a right upper pole with 2/3 of the organ being preserved.

EFFECT: method is easy to implement, causes no animal death; it represents an adequate model of an endothelial damage and atherosclerotic process development.

12 dwg, 4 tbl, 1 ex

Description

The invention relates to experimental medicine, pathophysiology, can be used for the diagnosis, prevention and treatment of the atherosclerotic process.

Atherosclerosis and its complications continue to lead in the structure of morbidity and mortality in Western countries and Russia. Mortality in cardiovascular pathologies in the world is twice as much as from oncological diseases, and 10 times higher than from accidents [Vorobeva E.N., Schumacher G.I., Osipova I.V. et al. // Cardiovascular therapy and prevention. - 2006, No. 5 (6). - S.129-136; Lupach N.M., Khludeeva E.A., Lukyanov P.A. et al. // Russian Medical Journal. - 2010, No. 4. S.71-74; Titov V.N. // Clinical laboratory diagnostics. 2006, No. 4. S.310].

One of the main risk factors (RF) for the development of atherosclerosis is a violation of lipid metabolism in the body. Dyslipidemia, which is a decrease in high-density α-lipoproteins (HDL), with an increase in β-lipoproteins, or low-density lipoproteins (LDL), pre-β lipoproteins, or very low-density liproproteins (VLDL), contributes to the development of atherosclerosis. Moreover, modified, subjected, most often, to peroxidation, oxidized (oxy-LDL) possess atherogenic properties. They contribute to an increase in the synthesis of caveolin-1, which leads to a decrease in the formation of NO by the endothelium [Vorobeva E.N., Schumacher G.I., Osipova I.V. et al. // Cardiovascular therapy and prevention. - 2006, No. 5 (6). - S.129-136; Zotova I.V., Zateyshchikov D.A., Sidorenko B.A. // Cardiology. - 2002, No. 4. - S. 57-67; Titov V.N. // Clinical laboratory diagnostics. 2006, No. 4. S.310]. Oxidized lipoproteins are active stimuli for monocytes that penetrate into the subendothelial space, turning into macrophages, and then, as modified LDL are accumulated in them, into foam cells. Activated macrophages and foam cells release biologically active substances - growth factors, anti-inflammatory cytokines, cell adhesion molecules, which promote platelet aggregation, vasoconstriction and leukocyte adhesion, and consequently the development of the inflammatory process in the arterial wall and the progression of atherosclerosis. Also, oxy-LDL induce proliferation of smooth muscle cells (MMC) of blood vessels, HDL on the contrary carry out the reverse transport of cholesterol (cholesterol) from the vascular wall and macrophages to the liver [Titov V.N. // Clinical laboratory diagnostics. 2006, No. 4. S.310].

Arterial hypertension (AH) is the second important risk factor for the development of atherosclerosis. It was proved that drug pressure control in hypertensive patients reduces the risk of strokes by 40%, myocardial infarction by 8%, and the overall mortality from heart disease by 10% [Chicherina EN, Milyutina OV // Clinical medicine. 2009. - No. 2. - S.18-21]. With isolated hypertension in men aged 47.5 ± 8.4 g, the lipid profile is shifted toward an increase in total cholesterol (GC), triglycerides (TG), LDL cholesterol, lower HDL cholesterol, and an increase in atherogenic coefficient (CA) [Ovchinnikova L .K., Yagudina R.I., Ovchinnikova E.A. // Russian pharmacies. - 2007. - No. 14. - S. 26-31]. Hypertension helps to increase the permeability of the endothelium and the accumulation of lipoproteins in the intima [Shlyakhto EV, Gavrisheva NA, Ovchinnikova OA et al. The effect of induced inflammation on collagen metabolism in atherosclerotic plaques in mice // Medical Immunology. 2008, No.6. S. 507-512]. It is proved that the reason for the activation of peroxidation (PO) of proteins and lipids in rats with spontaneous hypertension is to increase the production of oxygen radicals and the inefficiency of endogenous systems of their inactivation. It is also known that the development of spontaneous hypertension in rats accompanies the systemic inflammatory response syndrome: its initial stage is the activation (priming) of polymorphonuclear leukocytes (neutrophils), increased production and secretion of the active forms of O ₂ - and H ₂ O _2, and intensification of PO proteins and fatty acids (FAs) at the same time. The reaction of O ₂ - with nitric oxide (NO) s forms ONOO - and deprives NO of its biological effect as a relaxation factor. A decrease in NO leads to an increase in blood pressure by the type of development of the vicious circle [Zotova I.V., Zateishchikov D.A., Sidorenko B.A. // Cardiology. - 2002, No. 4. - S. 57-67].

From modern positions, a key link in the pathogenesis of atherosclerosis is endothelial dysfunction (ED), which is an imbalance between the main functions of the endothelium: vasodilation and vasoconstriction, inhibition and promotion of proliferation, antithrombotic and prothrombotic, antioxidant and oxidative [Lupach N.M. ., Lukyanov P.A. et al. // Russian Medical Journal. - 2010, No. 4. S.71-74; Allison B. Reiss, Amy D. // Journal of investigative medicine. 2006. Vol. 54, N. 3. P.123-131; Huber S. A., Sakkinen P., David C. // Circulation. 2001. - N. 103. - P. 2610-2616]. Nitric oxide is an important regulator in the cardiovascular system, a messenger that mediates auto and paracrine effects. In the body, the NO synthesis reaction is catalyzed by the NO synthase (NOS) family. NOS uses L-arginine as a substrate and NADPH-diaphorase as a cofactor. NADPH-diaphorase is involved in transporting electrons to the prosthetic enzyme group. The determination of NADPH-diaphorase is based on the formation of diformazan in the presence of endogenous β-NADPH and tetrazolium salts [Zotova IV, Zateishchikov DA., Sidorenko B.A. // Cardiology. 2002, No. 4. S.57-67; Shumatova T.A., Prikhodchenko N.G., Grigoryan L.A. et al. // Pacific Medical Journal. 2010, No. 3. S.59-61; Allison B. Reiss, Amy D. Glass // Journal of investigative medicine. 2006. Vol. 54, N. 3. P.123-131].

Clinical and epidemiological studies have proven the pathogenetic effect of hypertension and hyperlipidemia on the vascular wall, however, the period of ED formation under the combined action of these factors in the experiment is not clearly established [Ovchinnikova LK, Yagudina RI, Ovchinnikova EA // Russian pharmacies. - 2007. - No. 14. - S. 26-31; Vorobyova E.N., Schumacher G.I., Osipova I.V., Khoreva M.A. et al. // Cardiovascular therapy and prevention. - 2006. - No. 5 (6). - 129-136; Nagornev V.A., Voiskants A.N. // Vestn. RAMS, 2006. - No. 9-10. S.66-74; Davignon J. Ganz P. // Circulation. - 2004; 109: 27-32].

Experimental animal models play an important role in the study of diseases, including atherosclerosis. Rats are often used in modeling hyperlipidemia as a risk factor for atherosclerosis [Meshcherskaya K.A., Borodina G.P., Koroleva N.P. On the methodology for selecting funds that affect cholesterol metabolism. // Eleutherococcus and other adaptogens from Far Eastern plants. / Ed. K.A. Meshcherskaya. - Vladivostok, 1966. - S.289-294; Sannikova A.A., N.N. Chuchkova, Gaysina E.Sh. Immunomodulatory effect of glucosaminylmuramyl dipeptide with altered lipid metabolism and atherosclerosis. // Bulletin of the Ural medical economic science. - 2008. - No. 1. - S.64-66. 10; Yudina T.P., Charevach E.I., Tsybulko E.I., Maslennikova E.V., Plaksen N.V. The lipid-lowering effect of a complex emulsifier containing algae laminal and an aqueous extract from the roots of the soap dish Saponaria officinalis L., in an experiment on rats. // Nutrition issues. - 2008. - T. 77, No. 2. - S.76-79]. Their acquisition and maintenance are relatively inexpensive, animals are easy to handle, breed well in captivity. Of all the experimental animals in rats, metabolism is the best studied [Kulikov V.A., Chirkin A.A. Features of lipoprotein metabolism in rats // Pathological physiology and experimental therapy. - 2004. - No. 1. - S.26-27].

However, the above researchers evaluated only the lipid composition of the blood over a short observation period (from 16 days to 3 months), the models do not contain data on morphological and functional changes in the vessel wall, and the inclusion of long-term compensatory mechanisms that impede the formation of vascular lesions is not taken into account.

Known methods for modeling atherosclerosis (cl. RU No. 2033646; cl. G09B 23/28, 1995; cl. RU No. 2223228, cl. G09B 23/28, 2008, bull. No. 17; cl. RU No. 2127113, cl. A61K 31 / 70, A61K 31/505, 1999).

However, the above methods include the introduction of medications (obzidana - 1 mg per 100 g of weight, a suspension of hydrocortisone acetate - 1.5 mg per 100 g of animal weight, uridine at a dose of 50 mg per 1 kg of body weight once a day for 6-8 days) against the background of a diet rich in fats, artificially change the metabolism of the animal and inadequately reflect the formation of natural pathogenetic mechanisms that play a key role in the development of atherosclerosis.

For the prototype adopted modeling of hyperlipidemia in rats for a long time [A. Kropotov Influence of the Daurian Tsimifuga and the Forest Kaluga on some indicators of lipid metabolism and the reproductive system (experimental study). Abstract. thesis for the degree of candidate honey. Sciences, Vladivostok - 1975, p. 5]. The known method gives the diet pronounced atherogenic properties. Rats are on a diet with excess fat for 7 months. Cholesterol powder in an amount of 1%, margarine 10%, mercazolil 10 mg / kg and vitamin D in an amount of 2.5 ME per kg of rat body weight are added to animal feed.

However, in the prototype, the change in the functional and morphological properties of the vascular endothelium was not evaluated, the researchers observed only changes in the lipid spectrum in the blood and in biopsy samples of rat liver.

Given the characteristics of the metabolic processes of rats, contributing to the formation of their resistance to fat load, the inventors used a combination of hyperlipidemia with arterial hypertension for the most pronounced endothelial damage. The method enhances the violation of the processes of cholesterol metabolism, the formation of persistent signs of atherosclerotic vascular damage, taking into account the inclusion of urgent and long-term compensatory mechanisms.

The task of the invention is to develop an experimental model of endothelial dysfunction based on the study of the combined effect of hyperlipidemia and hypertension on the morphological structure of rat vessels.

The objective of the proposed method is achieved by a combination of feeding laboratory animals with an atherogenic diet, consisting of adding cholesterol powder in an amount of 1%, 10% margarine, 10 mg / kg mercazolilum, and vitamin D - 2.5 ME per kg of rat body weight, and an operation involving ligature on the kidney of the left kidney with non-absorbable suture material and suturing of the upper pole of the right kidney, while leaving 2/3 of the organ, which contributes to the development of persistent renovascular arterial hypertension. During the experiment, the following steps were performed:

- The state of serum lipid metabolism was monitored with isolated experimental hyperlipidemia (EG) and with the combined effects of an atherogenic diet and arterial hypertension (D + AH).

- Monitoring the level of blood pressure in the models of EG and D + AH.

- Determination of the activity of NADPH-diaphorase in the endothelium of the aorta, femoral arteries and microvessels of the anterior abdominal wall (PBS) in two experimental models.

- Assessment of the state of the vascular lumen in experimental animals by the method of computed magnetic resonance imaging (MRI).

The technical result of the proposed method is to obtain persistent structural disorders of the vascular wall, compared with an isolated atherogenic diet, in order to create a model of atherosclerosis in laboratory animals for the diagnosis, prevention and treatment of atherosclerosis.

The essence of the claimed invention consists in the combination of hyperlipidemia and renovascular hypertension in laboratory rats.

Hyperlipidemia was achieved by adding cholesterol powder to the feed in the amount of 1%, 10% margarine, 10 mg / kg of mercazolyl and vitamin D - 2.5 ME per kg of rat body weight.

Renovascular hypertension was performed by ligating the renal leg of the left kidney with non-absorbable suture material and suturing the upper pole of the right kidney (leaving 2/3 of the organ).

This technique allows you to get persistent structural disorders of the vascular wall, compared with isolated experimental hyperlipidemia.

The essence of the proposed method is illustrated by drawings, where Fig.1 a-1 shows the increase in experimental rats of the width of the common carotid artery, brachiocephalic trunk and thoracic aorta, respectively, at 2 months of study, figure 2 shows the definition of D + AG definition uneven contrasting of the arteries, which suggests local atherogenic changes in the wall of the arteries, figure 3 - in the aorta of experimental rats when stained with hematoxylin and eosin shows changes in the architectonics of elastic fibers, the displacement of the nuclei of myocytes to the periphery, their compact ies, cell wall infiltration, thickening of the endothelium, Magnification × 400 (A × Cam MRc camera, Germany), hematoxylin and eosin staining, figure 4 visualizes perinuclear optically empty formations, magnification × 400 (A × Cam MRc camera, Germany), hematoxylin and eosin staining; figure 5 - staining with hematoxylin and eosin of the aorta (control), magnification × 100 (camera A × Cam MRc, Germany), staining with hematoxylin and eosin; Fig.6 in the femoral arteries visualized perinuclear optically empty formations magnification × 400, Coloring hematoxylin and eosin; Fig.7 - staining with hematoxylin and eosin femoral artery (control), magnification × 400 (camera A × Cam MRc, Germany) staining with hematoxylin and eosin; on Fig - in the group of rats with D + AG when staining the aorta with Sudan 4 (according to the Okamoto method), shows the infiltration of the vessel with fatty inclusions, the color of the vessels according to the Okamoto method, magnification × 100; figure 9 in the group of rats with D + AH when staining the femoral artery with Sudan 4 (according to the Okamoto method) shows infiltration of the vessel with fatty inclusions, magnification × 400; figure 10 shows a graph of the thickened walls and intima of the aorta and femoral arteries of rats in the model of hyperlipidemia (group I) and in the complex model: hyperlipidemia and arterial hypertension (group II).

Concrete example

The material for experimental studies were Wistar rats - 45 males weighing 200-250 g. Animals were divided into 2 groups:

Group 1 - 15 male rats were on a cholesterol diet for 6 months (prototype). The diet consisted of adding 1%, 10% margarine, 10 mg / kg of mercazolilum and vitamin D - 2.5 ME per kg of rat body weight to the feed.

2 group of 15 male rats 15 days before feeding with the same atherogenic diet (adding 1%, 10% margarine, 10 mg / kg of mercazolilum to the feed and vitamin D 2.5 ME per kg of rat body weight) an operation was performed - applying a ligature to the renal leg of the left kidney with non-absorbable suture material and suturing the upper pole of the right kidney, leaving 2/3 of the organ (the claimed method). This operation develops persistent renovascular arterial hypertension by 8-10 weeks of the experiment.

Group III - control - 15 healthy male rats fed a normal diet. After 6 months, studies of animals of each group were withdrawn from the experiment under ether anesthesia by decapitation. Blood serum, aortic fragments, femoral arteries and PBS were collected. The experiment was carried out with strict observance of the requirements of the European Convention (Strasbourg, 1986) on the maintenance, feeding and care of experimental animals, as well as their withdrawal from the experiment and subsequent disposal. The experiments were guided by the requirements of the World Society for the Protection of Animals (WSPA) and the European Convention for the Protection of Experimental Animals. The study was approved by the interdisciplinary ethics committee (minutes No. 4, case No. 21 dated January 24, 2011).

Determination of OH content; TG; LDL cholesterol, HDL was performed using the standard colorimetric method using Olveks Diagnostic reagents (Russia).

Blood pressure was measured in the caudal artery using an MLU / 4C 501 analyzer (MedLab PRC). During the experiment, the animals were under anesthesia, which saved them from worries and the associated pressure surges.

The method of magnetic resonance imaging is as follows.

Before scanning, animals were euthanized with solutions of rometar (Xylazinum, SPORA, PRAHA) at a concentration of 1 mg / ml and Relanium at a concentration of 2 mg / ml, intraperitoneally. MRI diagnostics were performed on a PharmaScan US 70/16 experimental tomography scanner (Bruker, Germany) with a magnetic field strength of 7.0 Tesla, a frequency of 300 MHz and a BGA 09P type coil. For angiography, the Head_Angio protocol was used with the following parameters: TR / TE = 50.0 / 5.6; tilt angle 25.0; image field 3.0 / 3.0 / 3.0; effective cut thickness 30 mm; overlap 30.0 mm; matrix of 256/256/64 elements; one signal averaging, scan time 14 min.

Histological preparations were fixed in 10% neutral formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, Van Gieson, Mallory and Sudan-4 (Okamoto Method). The micropreparations were described using an Olympus BX 41 microscope. Images were taken with an Olympus DP 12 electronic camera at a constant magnification of 100 and 400. Morphometry was carried out using an MOB eyepiece-micrometer 1-16 ×.

The experiment used the histochemical method for NADPH-diaphorase according to the standard recipe Hope, Vincent (1989): fragments of animal vessels were isolated using a blade and lowered into a cooled, prepared on 0.1 M phosphate buffer (pH 7.4) 4% paraformaldehyde, which of the entire diaphoresis class, only NADPH-diaphorase remains active. The material was fixed for 2 hours at 4 ° C, the day was washed at the same temperature in a 15% sucrose solution, changing the solution 7-8 times. Slices 10 μm thick were made from tissue samples frozen in a cryostat, mounted on glass slides and placed in an incubation medium. The composition and final concentration of the medium were as follows: 50 mM Tris buffer (pH 8.0), 1 mM NADPH (Sigma), 0.5 mM nitro blue tetrazolium (Sigma), and 0.2% Triton X-100 ( "Serva"). Incubation was carried out for 60 minutes in a thermostat at a temperature of 37 ° C. Then the sections were rinsed in distilled water, dehydrated and enclosed in a balm according to the generally accepted methodology in histology.

The enzyme activity was measured in the endothelium and smooth aortic myocytes, femoral arteries, and microvessels of rat PBS.

The enzyme activity was determined using the program "ImageJ1.37 v" and expressed in units of optical density. There is evidence of a direct relationship between the concentration of the studied enzyme and the optical density of the precipitate resulting from the histochemical reaction [Djuricic B.M., Rodac L.S., Spatz M., Mrsulja B.B. Drain microvessels Enzymic Activities. Adv In Neurology 1978; 20: 197-205].

For mathematical processing of the obtained data, the SPSS v program was used. 16. Comparison of mean values in the samples was carried out using the nonparametric Wilcoxon-Mann-Whitney U-test.

Blood pressure monitoring showed that in the II experimental group (D + AH), blood pressure was higher than in the I group and in the group of healthy rats throughout the experiment (2, 4, 6 months), which confirms the formation of the renovascular and renoprotein mechanisms of arterial hypertension (table 1).

Table 1 Rat blood pressure in experimental atherosclerosis models Rat groups 2 months experiment 4 month experiment 6 months experiment Systolic. HELL (mmHg) Diastolic. HELL (mmHg) Systolic. HELL (mmHg) Diastolic. HELL (mmHg) Systolic. HELL (mmHg) Diastolic. HELL (mmHg) Group I (IG) 113.8 ± 3.6 68.8 ± 1.22 122.06 ± 1.05 66.18 ± 7.08 141.70 ± 4.41 • 90.89 ± 1.83 II group (D + AG) 131.3 ± 1.5 •; * 83.4 ± 3.2 •; * 140.12 ± 3.25 •; * 90.24 ± 4.44 •; * 161.70 ± 1.66 •; * 99.33 ± 3.41 •; * Group III (control) 115.1 ± 0.7 73.4 ± 0.53 116.25 ± 0.84 70.20 ± 2.18 116.01 ± 3.05 71.44 ± 1.70 * - significance of differences between groups I and II (pu <0.05); • - reliability between the experimental groups and the control group (p _u <0.05).

When studying the lipid spectrum in the experimental groups of rats after 2 months of the experiment, an increase in the level of OX, TG, LDL, HDL and CA was established compared with the control group (p _u <0.05) (table 2). Moreover, in the group of rats with arterial hypertension, the values of OX, LDL, HDL and CA were significantly higher (p _u <0.05), and the level of TG was slightly lower (p _u > 0.05) than in the group of rats with isolated hyperlipidemia (table 2). At the 4th month of the experiment, lipid profile disorders persisted in group I rats, LDL levels significantly increased (p _u <0.05). In group II, the values of HDL and LDL decreased and became lower than in group I animals, while there was an increase in the level of TG and CA. By the 6th month of the experiment, the level of OX and TG significantly increased in both experimental groups of animals. In rats with an atherogenic diet, by this period of the experiment, an increase in the content of high-density lipoproteins was observed compared with their level at the 4th month of the study, while the LDL and CA values did not increase (p _u <0.05), while in group II rats (D + AH) continued decline in LDL and HDL. At the same time, the level of HDL in rats of this group became lower than in healthy rats (p _u <0.05), an increase in CA occurred - by 2.5 times compared to group I and 4.8 times compared to the control group of rats (table 2). The revealed changes confirm more pronounced lipid impairment in rats of group II (D + AH). A decrease in the serum content of LDL and HDL in rats with arterial hypertension and hyperlipidemia probably indicates an increase in their reception by vascular endothelium.

When assessing vascular NADPH-diaphorase, it was found that the content of NADPH-diaphorase was lower in the femoral arteries of the experimental and control groups of animals than in the aorta, which can be explained by the anatomical features of the structure of the walls of these vessels (in the femoral arteries the muscular component is more pronounced) (p _u <0.05). In the femoral arteries of group II rats, the values of NADPH-diaphorase were slightly lower than in the aorta, however, the indices did not have a significant difference, which may indicate a more pronounced disturbance in the synthesis of this coenzyme in the aorta when modeling renovascular hypertension. When monitoring NADPH-diaphorase, a decrease in its level in the fragments of the aorta and femoral arteries of experimental groups I and II of rats was recorded with significant differences with control (p _u <0.05) (Table 3).

No significant differences were found in the content of vascular coenzyme depending on the time of the experiment (2, 4, 6 months) in all experimental groups. The greatest decrease in the level of NADPH-diaphorase was determined at 2 months of the study with a relative stabilization of coenzyme values at a low level with subsequent monitoring.

In rats with hyperlipidemia and arterial hypertension, the value of NADPH-diaphorase in the dynamics of the entire experiment was lower than in the prototype (p _u <0.05), which indicates a deeper violation of the functional properties of the endothelium. In rats of group II, the level of NADPH-diaphorase in the vessels of the microvasculature decreased by 2 months of the study, while in the group of rats of group I (EG), a significant decrease in its level occurred only by the 6th month of the experiment.

When monitoring the condition of the arterial bed using magnetic resonance imaging (MRI), it was found that at 2 months of the study in experimental rats, the width of the common carotid artery, brachiocephalic trunk and thoracic aorta increased (table 4, figure 1, figure 2). This vascular reaction is due to the inclusion of protective and adaptive mechanisms to maintain central hemodynamics.

However, by the 6th month of the experiment, there was a narrowing of the lumen of these vessels (Table 4), most pronounced in group II rats (significance of differences with group I (p _u <0.05). In rats of group II, a decrease in the lumen width of the iliac arteries was recorded, which indicates multifocal lesions of the arterial bed under the combined action of hyperlipidemia and arterial hypertension. Uneven contrast of arteries was determined in the D + AG model, which suggests local atherogenic changes in the arterial wall (figure 2).

Table 4 The diameter of the lumen of the vessels of rats (mm), established by MRI. Vessels I (diet) II group (diet + operation) Control (size in mm) 2 month 6 months 2 month 6 months 2 month 6 months Total sleepy 1.57 (1.49-1.63)! 1.41 (1.38-1.54) 1.34; (1.26-1.47) 1.14; (1.10-1.19) • 1.27 (1.19-1.32) 1.23 (1.20-1.31) Inner carotid 0.79 (0.76-0.81) 0.72 (0.70-0.73) 0.78 (0.76-0.84) 0.44 (0.42-0.50) •! 0.8 (0.78-0.89) 0.77 (0.75-0.91) Shoulder head barrel 1.54 (1.51-1.58)! 1.38 (1.43-1.50) 1.47 (1.60-1.65)! 1.23 (1.21-1.25) • 1.31 (1.28-1.33) 1.30 (1.27-1.32) Cerebral arteries 0.49 (0.46-0.56) 0.40 (0.38-0.41) 0.49 (0.45-0.52) 0.44 (0.42-0.50) 0.40 (0.37-0.47) 0.41 (0.39-0.44) Gr. part of the aorta 2.13 (2.05-2.16)! 1.78 (1.76-1.79) × 2.32 (2.26-2.33)! 1.51; (1.47-1.53) •! × 1.95 (1.83-1.97) 1.86 (1.80-1.93) Br part of the aorta 1,61 1.41 1.66 1,64 1.62 (1.54-1.63) (1.59-1.63) (1.40-1.44) (1.60-1.68) 1.53 (1.43-1.56) (1.60-1.66) Common iliac arteries 1.1 (0.94-1.05) 0.82 (0.80-0.87) 0.94 (0.92-0.96) 0.74 (0.71-0.75)! × 0.98 (0.96-1.2) 0.93 (0.90-0.99) Note: data presented in the form of Median (NK-VK). ! - the reliability between the experimental groups and the control group (p _u <0,05). • - significance of differences between groups I and II (p _u <0.05); × - significance of differences between indicators at 2 and 6 months of the experiment.

Assessment of the histological structure of the arterial wall showed that the most pronounced changes in blood vessels are recorded by the 6th month of the experiment. In the aorta and femoral arteries of experimental rats, when hematoxylin and eosin stained, changes in the architectonics of elastic fibers are observed, perinuclear optically empty formations, myocyte nuclei shift to the periphery, their compaction, cell wall infiltration, thickening of the endothelium are visualized (FIG. 3, FIG. 4, FIG. 6,) compared with intact rats (FIG. 5, FIG. 7). In this case, the most pronounced changes in the morphology of the arteries are recorded in the second experimental group (D + AH) (Fig. 4, Fig. 6). When staining arteries with Sudan 4 according to the Okamoto method in experimental rats with D + AH, vessel infiltration with fatty inclusions was detected. In this case, fat deposition is filled with voids identified during hematoxylin and eosin staining (Fig. 8, Fig. 9).

In PBS in experimental rats, a decrease in the number of microvessels is observed (5-7 microvessels are detected in group I rats, 3-4 microvessels in the field of vision in group II, while 8-10 microvessels in control rats). The vessels of the microvasculature in rats of the experimental group II in the form of strokes with proliferation of endotheliocytes, while in control rats - oval or round. The thickness of the microvessels of the anterior abdominal wall in the experimental groups of rats increased. In this case, the maximum thickening of the microvessel wall was observed in the II experimental group (M = 4.62 (4.36-4.72) μm in the second group, M = 2.31 (2.12-2.36) μm in the I group, and 1.54 (1.50-1.62) μm in control rats). An increase in the thickness of the wall of the aorta and femoral arteries was recorded in experimental rats. In rats with arterial hypertension, an increase in the thickness of the wall and vascular intima was recorded, compared with the model of isolated experimental hyperlipidemia (Fig. 10).

A comparative analysis of the proposed solution with the prototype shows that in the claimed method, combining arterial hypertension and hyperlipidemia, by the 6th month of the experiment, changes in the lipid spectrum of blood serum were established (increase in OX, TG, decrease in HDL, increase in CA) compared with the prototype. The inventive method allows to establish a persistent increase in systolic and diastolic blood pressure from 2 to 6 months of the study. Compared with the prototype recorded a decrease in the activity of NADPH-diaphorase in the vascular endothelium by 6 months of the experiment. Vascular damage was observed: deformation of elastic fibers, an increase in wall and intima thickness, cell infiltration, deposition of fatty inclusions in the wall, narrowing of the lumen of blood vessels, and a decrease in the number of PBS microvessels.

Claims (1)

A method for modeling atherosclerosis, including feeding the studied animals with an atherogenic diet, consisting of adding cholesterol powder in an amount of 1%, margarine 10%, mercazolilum 10 mg / kg and vitamin D - 2.5 ME per kg rat body weight, characterized in that Along with feeding an atherogenic diet, the animals undergo an operation consisting of applying a ligature to the renal leg of the left kidney with non-absorbable suture material and suturing the upper pole of the right kidney, while leaving 2/3 of the organ.

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