RU2349320C2 - Method of structural vessel alteration correction in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns experimental medicine, particularly cardiology, and can be applied in experimental biology and medicine for correction of structural vessel wall alteration in post-perfusion period. Correction of structural alteration of main blood vessels in experiment involves intravenous meksidol introduction after rehabilitation in fractions each 20 minutes in amount of 25 mg/kg.

EFFECT: extended range of agents for correction and prevention of re-perfusion complications in main blood vessels, reduced volume of binding tissue elements in vessel walls.

2 tbl, 7 dwg

Description

The invention relates to medicines and medicines for therapeutic purposes and can be used in experimental biology and medicine for the correction of structural changes in the vascular wall in the post-reperfusion period.

The process of successive compensatory-adaptive + structural remodeling of tissues is defined by the term "remodeling". Remodeling of the vascular wall includes transformation of the structure with migration, proliferation and transformation of GMCs, macrophages, fibroblasts, and proliferation of elements of the connective tissue. The result of the changes is a thickening of the wall, an increase in stiffness, as well as the initiation or significant acceleration of the mechanisms of atherosclerosis.

The trigger moment initiating the chain of adaptive reactions in the vascular wall under various influences is damage to the endothelium. The endothelium is an active barrier that is very actively involved in maintaining the tone and structure of blood vessels, local homeostasis and proliferation of vascular wall cells (Dzau V., Bernstein K., Celermaier D. et al. The relevance of tissue angiotensin-converting enzyme: manifestations in mechanistic and endpoint data // Am J Cardiol. - 2001. - No. 88. - P.1-20. Endothelial cells are constantly exposed to blood flow, platelet factors, neurohormones.

In recent years, reperfusion changes that develop in the vascular pool after total ischemia have been identified and actively studied (Petrishchev N.N., Vlasov T.D.The functional state of the endothelium with ischemia-reperfusion (literature review) // I.M. Russian Physiological Journal Sechenov. - 2000. - No. 2. - S.148-163).

This determines the relevance of the problem and its significance, since it has now been proven that reperfusion after hypoxia or ischemia often only exacerbates primary hypoxia (Dolgikh V.T., Rusakov V.V., Korpacheva O.V., Mordyk A.V. Leading pathogenetic factors of postresuscitative cardiodepression // Theoretical and clinical problems of modern resuscitation. Proceedings of the international symposium dedicated to the 90th anniversary of Academician V.A.Negovsky. - M., 1999. - P. 56).

Damage to the vascular wall during ischemia and subsequent reperfusion, probably leads to an imbalance in the intracellular regulatory systems, the launch of the remodeling stages. The mutual enhancement of the action of the ischemic / reperfusion factor with the development factors of atherosclerosis or mechanical damage to the vascular wall can lead to a multiple acceleration of the development of remodeling (Fitileva EB, Serov R.A., Yurpolskaya L.A. Role of reperfusion syndrome in the mechanism of development of restenosis // Angiology and vascular surgery. - 1998. - No. 3-4. - P.18-22), an increase in mechanical "rigidity" and a pronounced dysfunction of the walls of the great arteries (London CM, Guerin AP, Marchais SJ et al. Cardiac and arterial interactions in end-stage renal disease // Kidney Int. - 1996 - Vol.50. - P.600-608).

One of the most important aspects of preventing reperfusion complications is the development of measures related to the correction of post reperfusion rearrangements in the vascular wall.

The current arsenal of pharmacological agents used to correct or prevent the development of vascular remodeling is dominated by drugs aimed at eliminating endothelial dysfunction. Endothelial dysfunction is understood as an imbalance between the mediators secreted by the endothelium, which normally ensure the optimal course of all endothelium-dependent processes. Violation of the production and action of endothelial vasoactive factors is observed simultaneously with abnormal vascular reactivity, changes in the structure and growth of blood vessels associated with vascular diseases.

Medicines that are potentially capable of affecting the function of the endothelium can be divided into 4 main categories:

- replacing natural protective endothelial substances (stable PGI2 analogues, nitrovasodilators, r-tPA);

- inhibitors or antagonists of endothelial constrictor factors (angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, TxA2 synthetase inhibitors and TxF2 receptor antagonists);

- cytoprotective substances: free radical scavengers of superoxide dismutase and probucol, a lazaroid inhibitor of free radical production;

- lipid-lowering drugs.

1. Replacing natural protective endothelial substances.

Currently, three organic nitrates are vital for cardiac patients: nitroglycerin; isosorbide dinitrate; isosorbide 5-mononitrate.

Inside the vascular smooth muscle cell, including the coronary artery, nitrates entering the body interact with SH groups (nitrate receptors) to form nitric oxide (NO), which structurally corresponds to the physiological endothelial relaxing (relaxing) factor. In patients with coronary artery disease, the production of endothelial relaxing factor (NO) by the coronary and other arteries is significantly reduced, nitrates make up for this deficiency.

Thus, nitrates eliminate coronary spasm, expand the coronary arteries, and improve coronary collateral blood flow.

However, the use of these drugs has a number of side effects:

- mild: mild headache (due to vasodilation and difficulty in the outflow of venous blood from the brain), a feeling of fullness in the head, tinnitus, facial flushing, tachycardia, burning under the tongue;

- Significant: severe headache, dizziness, significant arterial hypotension.

2. Inhibitors of angiotensin-converting enzyme (ACE).

Among the known ACE inhibitors, quinaprilat (the active metabolite of quinapril) has the highest affinity for tissue renin-angiotensin of the aldosterone system (RAAS), which is 2 times higher than perindoprilat in terms of tissue affinity, ramiprilat is 3 times higher and enalaprilat is 15 times higher. The positive effect of quinapril on endothelial dysfunction is associated not only with its modulating effect on bradykinin metabolism and improved B2 receptor function, but also with the ability of this drug to restore the normal activity of muscarinic (M) endothelial receptors, which leads to mediated artery dilatation due to a receptor-dependent increase in synthesis endothelial relaxation factor (EGF-NO). Currently, there is evidence that quinapril has a direct modulating effect on the synthesis of EGF-NO (Belousov Yu.V., Grigoriev V.Yu., Miroshenkov PV, Karpov OI Clinical and economic evaluation of the use of quinapril // Qualitative Clinical Practice. - 2004. - No. 1. - S.46-56).

Thus, the use of ACE inhibitors eliminates the vasoconstrictor effects, prevents or slows down remodeling of the walls of blood vessels, heart.

However, in addition to the positive effect, significant disadvantages of the use of ACE inhibitors were identified:

- lack of parallelism between concentration and severity of the clinical effect;

- increased concentration of aldosterone during long-term therapy;

- a negative effect on the central nervous system and an increase in the activity of the natriuretic factor (Sidorenko B.A., Preobrazhensky D.V. New in the treatment of cardiovascular diseases and thrombosis. Cardiology. 1999. No. 4. P.86-96).

3. Hypolipidemic drugs.

Currently, the most popular theory is that atherosclerosis is considered as a reaction to damage to the vascular wall (primarily, the endothelium). The most important damaging factor is hypercholesterolemia.

So statins, having a hypolipidemic effect, have a direct anti-sclerotic effect: it was found that fluvastatin and simvastatin reduced myocyte proliferation, and simvastatin, in addition, also smooth muscle cell migration into the vascular intima, and this action was independent of the magnitude of the lipid-lowering effect. Statins also affect the immune mechanisms of atherogenesis (in particular, the metabolism of macrophages).

The disadvantage of these drugs is that they are used after the occurring changes in the vascular wall.

4. Antioxidants.

One of the best known antioxidants is vitamin E - alpha-tocopherol. A number of studies have been performed that demonstrate that vitamin E at a dose of 400-800-1000 ME per day (100 ME corresponds to 100 mg of tocopherol) reduces LDL sensitivity to oxidation and protects against the development of endothelial dysfunction and the progression of atherosclerosis - IHD (Nunes GL, Robinson K, Kalynych A, King SB III, Sgoutas DS, Berk BC Vitamins C and E inhibit Ch - production in the pig coronary artery // Circulation. - 1997. - Vol. 96. - P.3593-3601).

Of particular note are the antioxidant properties of probucol. In the blood, it binds to lipoproteins, including LDL, protecting them from peroxide modification, which reduces the risk of developing atherosclerosis (Sasahara M, Raines EW, Chait A, et al. Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol. I. Is the extent of atherosclerosis related to resistance of LDL to oxidation? // J Clin Invest. - 1994. - Vol. 94. - P.155-164).

Thus, these drugs are used after the occurring changes in the vascular wall and their further correction.

In addition, the above pharmacological agents have the following disadvantages:

- lack of parallelism between concentration and severity of the clinical effect;

- increased concentration of aldosterone during long-term therapy;

- a negative effect on the central nervous system and an increase in the activity of the natriuretic factor;

- mild: mild headache (due to vasodilation and difficulty in the outflow of venous blood from the brain), a feeling of fullness in the head, tinnitus, facial flushing, tachycardia, burning under the tongue;

- Significant: severe headache, dizziness, significant arterial hypotension.

The objective of the invention is to expand the arsenal of existing means for correcting and preventing reperfusion complications of the great vessels and reducing the volume of connective tissue elements in the vascular walls.

The problem is solved in that in the known method for correcting structural changes in blood vessels in an experiment involving the administration of drugs, Mexidol is used as a means for correcting structural changes in the great vessels, which is administered intravenously during the first hour after a resuscitation fractionally every 20 minutes at a dose of 25 mg / kg

The present invention meets the criteria of the invention of "novelty" and "inventive step", since the conducted patent information research did not reveal the sources of scientific, technical and patent information that would discredit the novelty of the proposed method. The study showed the absence of technical solutions with significant features of the proposed method.

Currently, vascular remodeling is the morphological equivalent of the pathogenesis of various diseases and pathological conditions - hypertension, atherosclerosis, thrombosis, restenosis. That is why the problem of correcting morphological rearrangements in the vascular wall is relevant and remains one of the most important in medicine.

At the moment, with prolonged hypertension, remodeling correction is aimed at eliminating endothelial dysfunction, since it was found that endothelial damage due to an increase in shear stress is the trigger mechanism for remodeling.

Mexidol (succinate-2-ethyl-3-methyl-3-hydroxypyridine) is a crystalline powder, white or white with a creamy tint. Easily soluble in water. According to the chemical structure, Mexidol is the salt of succinic acid (succinate) corresponding to emoxipin (Mikhailov IB, Clinical Pharmacology. St. Petersburg: Foliant, 2000. - 525 p.).

Mexidol combines the antioxidant properties of a base (3-hydroxypyridine derivative) with the antihypoxic activity of succinate (Bergeron M., Ferriero DM, Vreman HJ Hypoxia-ischemia, but not hypoxia alone, induces the expression of heme oxygenase-1 (HSP32) in new born rat brain // Cerebral blood flow and metabolism. - 1997. - Vol.17. - No. 36. - P.647-658). The use of organic derivatives of succinate contributes to its better penetration through biological membranes. In this case, after the substance enters the cell, it dissociates or cleaves the succinate molecule, which is used directly by the respiratory chain as an energy substrate. At the same time, the succinate oxidase pathway is enhanced (Golovacheva T.V., Dudaeva N.G., Pavlyuk A.V., Dolgova M.A. The use of mexidol in combination therapy with traditional antihypertensive drugs in patients with arterial hypertension with signs of chronic cerebrovascular insufficiency) / Pat. Fiziolol. - 2003. - No. 4. - S.11-13).

Mexidol is prescribed intramuscularly or intravenously. Oxymethylethylpyridine succinate is rapidly distributed to organs and tissues. It is determined in blood plasma for 4-9 hours. It is metabolized in the liver by glucuronidation with the formation of phosphate-3-hydroxypyridine, glucuron conjugates. It is rapidly excreted in urine, mainly in the form of metabolites and in insignificant amounts unchanged (Pashina I.V. Study of the effect of nitroglycerin on the ischemic myocardium when combined with mexidol: Author's abstract. Diss .... Candidate of medical science. Kursk, 1997 . - 45 p.).

The drug Mexidol is effectively used in various fields of clinical medicine: neurology, psychiatry, cardiology, ophthalmology, surgery, dentistry, endocrinology, etc. The drug can be used both to quickly achieve a therapeutic effect, and to reduce the frequency of adverse reactions and shorten the time hospital stay (VV Abramchenko. Antioxidants and antihypoxants in obstetrics (oxidative stress in obstetrics and its treatment with antioxidants and antihypoxants). - St. Petersburg., 2001. - 400 p.).

Mexidol provides much more powerful antioxidant and antihypoxic properties among other drugs. Combining all the positive effects of the effect on metabolic processes and blood supply, Mexidol deserves attention also due to its good tolerability and the minimum of contraindications and side effects (Uteshev DB, Mexidol as an antioxidant // Experimental and Clinical Pharmacology. - 1997. - № 2. - S.83-88).

At the same time, there is no data in the literature on the use of pharmacological agents for the prevention or correction of remodeling of major vessels under conditions associated with the short-term exposure to pathological factors such as ischemia and reperfusion.

To confirm the positive effect, experiments were carried out on 60 sexually mature (5 months) white non-linear rats - males weighing 200-250 g, contained in a standard diet of vivarium.

The experiments used the model of total ischemia by compressing the cardiovascular bundle for 10 min according to V.G. Korpachev. with subsequent traditional resuscitation and restoration of cardiac activity, blood circulation and respiration (Korpachev V.G., Lysenkov S.P., Tel L.Z. Modeling of clinical death and postresuscitation disease in rats // Patol. physiology and experimental therapy. - 1982. - No. 3. - S.78-80).

After resuscitation, Mexidol was administered intravenously during the first hour fractionally (every 20 minutes) at a dose of 25 mg / kg.

All animals were divided into 3 groups:

Group 1 - intact

Group 2 - post-reperfusion period of 60 days (control),

Group 3 - the introduction of Mexidol after resuscitation, a post-reperfusion period of 60 days.

Biochemical, histological and electron microscopic analysis of the tissue of the great vessels and determination of blood biochemiluminescence was performed on the 60th day after total ischemia.

For morphological studies, sections of blood vessels were taken according to the accepted scheme. Vascular fragments of not more than 1 mm were fixed in neutral formalin and glutaraldehyde and dehydrated in alcohols. Sections prepared on a microtome and ultratome were stained with hematoxylin and eosin, according to Van Gieson, fuchselin according to Weigert (Microscopic technique / Edited by D.S. Sarkisov, Yu.L. Petrova. - M .: Medicine, 19967. - 544 p. ) and uranyl acetate and lead citrate according to the Reynolds method (Reynolds ES / The use of lead citrate at high pH as an electronopaque stain in electron microscopy / J. Cell. BioL - 1963. - Vol.l7. - P.208-212). Histological preparations were examined using a Leica DMLS light microscope; ultrathin sections were examined with a FEI Morgagni 268D electron microscope.

To determine the intensity of free radical oxidation (SRO) and antioxidant activity (AOA) of blood plasma, we used the measurement of chemiluminescence (CL) on a BHL-06 biochemiluminometer (Vladimirov Yu.A., Sherstnev M.P. Chemiluminescence of animal cells // Biophysics. - 1989.- T.24. - P.176).

The analysis of the content of malondialdehyde in blood vessels was carried out according to the reaction with thiobarbituric acid (TAC) (Lankin V.Z., Gerasimova EN, Kasatkin LB Lipid peroxides and atherosclerosis. Enzymatic detoxification of lipid peroxides in the blood of patients with coronary heart disease due to atherosclerosis of the coronary arteries. // Cardiology. - 1979. - No. 6. - P.71-75).

Statistical processing of the obtained data was carried out on a PC using the Statistica 5.5 application packages for Windows 98. Paired intra-group and inter-group comparisons of means were determined according to Wilcoxon and Mann-Whitney criteria. Differences were considered significant at a significance level of p <0.05.

Studies of the maximum intensity of blood plasma glow (according to biochemiluminescence) in the long-term post-reperfusion period (60 days) revealed a significant 2.14-fold increase in plasma free radical activity in the control relative to the level of intact animals and a decrease in the total antioxidant activity (AOA) of the plasma in terms of the light sum of bio-chemiluminescence 2.04 times (Table 1).

With the introduction of Mexidol, a significant increase in total plasma AOA by 1.74 times was revealed in the long-term post-reperfusion period. Comparison with intact animals showed that Mexidol brought the activity values of the pro- and antioxidant systems of the plasma closer to normal.

The content of the secondary product of lipid peroxidation of MDA in the tissue of arterial vessels in the control was significantly higher than the level of intact animals by 4.4 times. With the introduction of Mexidol, the concentration of MDA decreased 2.17 times relative to the control series, which corresponded to the level of intact animals (Table 1).

Table 1
The effect of Mexidol on blood plasma biochemiluminescence and MDA content in the tissue of arterial vessels in the long-term post-reperfusion period (M ± m) Groups of animals Imax, mV S, mV MDA, nMol / g of tissue Intact 1.89 ± 0.18 19.48 ± 1.64 1.66 ± 0.212 The control 4.05 ± 0.25 * 39.75 ± 1.71 * 5.63 ± 0.14 * Mexidol 2.67 ± 0.24 ** 22.91 ± 1.85 ** 2.59 ± 0.32 ** Note: * - significance of differences with intact level, p <0.05 ** - significance of differences with control, p <0.05

Thus, on the 60th day of the post-reperfusion period, an increase in the level of free radical oxidation in the blood plasma was observed against the background of a reduced power of antioxidant systems according to the bio-chemiluminescence of blood plasma. The increased activity of lipid peroxidation recorded in the tissue of arterial vessels in this period, as evidenced by the high level of MDA, indicates impaired lipid metabolism even two months after short-term total ischemia and reperfusion.

Our studies showed that mexidol, administered immediately after resuscitation, significantly reduced the indicators of the maximum intensity of luminescence and light sum of BChL to almost the level of intact animals in the long-term period of 60 days. The results indicate that the concentrations of Mexidol used were effective and safe in preventing the imbalance of pro- and antioxidant systems in the long-term post-reperfusion period.

We found that the introduction of Mexidol after resuscitation prevents an increase in the long-term post-reperfusion period of the concentration of MDA in the tissue of rat arterial vessels. This confirms information about the ability of the drug to inhibit LPO processes and increase the resistance of cell membranes.

Thus, Mexidol significantly corrects the metabolism of tissue of arterial vessels and blood plasma of rats under reperfusion, which prevents an imbalance in the balance of pro- and antioxidant activity of the body, and in particular, the tissues of the great vessels, in the long-term post-reperfusion period. The positive effects of the drug are associated with the inhibition of free radical processes upon activation of the antioxidant enzymatic system.

Microscopic study of the effect of Mexidol on the structure of the aorta, carotid and femoral arteries in the long-term post-reperfusion period for 60 days revealed a number of morphological features (Fig.1b).

A decrease in the severity of connective tissue structures in the structure of the media and in the subendothelial layer was recorded. Unlike the control series, in the series with the introduction of Mexidol, the formation of a single network by the fibrous structures throughout the entire thickness of the vessel from intima to adventitia was not revealed. There was also no violation of a clear strictly layered structure of the arrangement of fibers in all main vessels, there were no additional connecting bundles of collagen fibers. This gave reason to assume that Mexidol has properties that prevent the development of the process of active vessel remodeling in the distant post-reperfusion period due to the growth of connective tissue in the thickness of the arteries.

1. The ultrastructure of the intima of the vascular wall.

Changes in the intima of the aorta, carotid and femoral arteries both in the control and in the series with the introduction of Mexidol showed similar results: the restoration of a significant part of the endothelial layer and some mosaic cell elements. It should be noted that the restoration of the structure of the endothelial layer of the femoral artery was slower due to, apparently, maximum damage during reperfusion.

2. Luminal surface and the state of intercellular contacts

Microvilli and microgrowths were noted on the entire surface of the endothelium in the usual amount. The intercellular contacts of the endothelium were fairly dense, in places moderately widened. On the inner surface of some EC protrusions of the cytoplasm into the lumen of the vessel are noted.

In the group with Mexidol, unlike the control, there were no elements of repeated deendothelization (Fig. 4b; 6d).

3. The structure of endothelial cells

Cytoplasm. Most of the EC in the control and with the introduction of Mexidol had a dense cytoplasm. Pinocytotic vesicles were present in large numbers in most of the cells on the luminal and basal sides. In contrast to the control, in the series with Mexidol there were no cells with vacuoles, which can be explained by a more complete restoration of these cells.

4. The energy apparatus.

Mitochondria of both series did not differ much in size and structure from mitochondria of intact cells. The matrix in most mitochondria is dense, enlightenment is extremely rare.

5. The synthetic apparatus is active in most ECs of both series.

In some young cells, intensification of both synthetic processes and secretory function is noted - the plasma membrane has exocytosis elements with electron-dense contents. A slightly increased number of ribosomes and polysomes; EPS is moderately expanded.

6. Nuclear apparatus

The nuclei of most EC in both series of normal elongated shape. The area of the nuclei is ordinary, in some cell fragmentation and invagination (Fig.3b). The perinuclear space and nuclear pores are expanded in cells with a significant increase in synthetic activity.

Subendothelial layer

In the group with the introduction of Mexidol, the volume of the subendothelial layer due to fibers and an amorphous substance is less pronounced compared to the control (Fig.2b; 4a). The inner elastic membrane is more uniform, without thickenings and additional processes (Fig.5b). In the series with Mexidol, myofibroblasts and leukocyte cells that migrated from the bloodstream are absent in the subendothelium.

Ultrastructure of the media of the vascular wall.

In contrast to the control, in the group with the introduction of Mexidol on the 60th day of the post-reperfusion period in the media of the aorta, carotid and femoral arteries, there was no significant increase in the number of synthetic cells and the volume of fibrous structures of connective tissue, which was a key link in vascular remodeling in the control.

In both groups, the following cellular elements were identified in the media of blood vessels: synthetic adrenal gland micro-genes, spindle-shaped adrenal glands, single fibroblasts. Process GMCs were more often noted in the part of the media adjacent to the intima. The extension of the processes towards endotheliocytes went through the places of ruptures of the fragmented inner elastic membrane. Less common were the process cavities of a part of the cytoplasm of endotheliocytes in the direction of smooth muscle cells. Often, contractile MMCs had signs of smooth muscle cells — myofilaments, an oval nucleus, and signs of fibroblast — an adolescent form, diffuse vesicles in the cytoplasm. Such cells were identified as myofibroblasts and prevailed in the control series compared to the series using mexidol. Also, single proliferating cells identified as fibroblasts with reduction of myofilaments were noted. Secreting fibroblasts in the control were detected more often in the carotid artery than in the aorta. More often than in the aorta, myofibroblasts were identified. Cells with the presence of myelin figures in a series with the introduction of Mexidol, in contrast to the control, were extremely rare.

The structure of smooth myocytes

Cytoplasm. The structure of the MMC cytoplasm in the distant reperfusion period was dense, with no signs of hydration (Fig. 2d). The density of the cytoplasm in the process cells of the synthetic type was higher than in the contractile. Cytoplasmic vesicles were located differently in different types of cells. In contractile cells, a large number of vesicles were distributed along the inner surface of the cytoplasmic membrane and less in the cytoplasm. In synthetic MMCs, the vesicles were diffusely located in the cytoplasm and near EPS, with a minimum concentration near the cytoplasmic membrane. Also in the cytoplasm of different cells there was a different number of myofilaments. In contractile spindle-shaped cells, there was a maximum number of myofilaments, usual for cells of this type. In process synthetic cells, the number of these structures was smaller, and in single fibroblasts they were absent. In the group with the introduction of Mexidol, extremely rare isolated dark degenerative forms of MMC containing vacuoles with the presence of myelin figures were extremely rare.

Energy apparatus. The mitochondria of smooth muscle cells in both series were of different sizes, mainly with clear cristae. In the process MMCs, part of the mitochondria is hyperplastic, and in the individual, slightly swollen. The area of mitochondria and cristae is close to normal. The matrix is dense and structural (Fig. 5g).

Synthetic apparatus. The key difference between GMCs of different types - the synthetic function was expressed differently in the cells of the synthetic and contractile types. Synthesizing fibroblasts, sprouting GMCs and transitional cells — myofibroblasts had an increased number of free and attached ribosomes and polyribosomes with adjacent fiber chains compared with contractile MMCs. In contrast to the control series, the smooth and rough EPS was less expanded and increased. Fiber synthesis was observed on all floors, part of the elastic and collagen fibers in the intermediate stage of assembly was at the exit from the expanded EPS (Fig.2b, c, d; 4a, c, d; 6b). The most active synthesis of fibers in cells morphologically occupying a transition state between MMC and fibroblasts was noted in the control, but not in the group with the introduction of Mexidol.

Nuclear apparatus. In synthetic MMCs there were active nuclei containing clear nucleoli, densified karyoplasm, and an increase in marginal chromatin. The shape of the nuclei is irregular; some nuclei are invaginated. The nuclear pores are open, the perinuclear space is moderately expanded. In the contractile cells of the nucleus, a rounded oblong shape with dense karyoplasm. Nuclear pores are less open (Fig. 3c; 7a). In the series with the introduction of Mexidol, in contrast to the control, cells with enlarged hyperchromic nuclei and expanded perinuclear space, chromatin margin, with vacuoles and myelin figures in the cytoplasm were not found (Fig.6b).

The structure of the intercellular substance

In control, the remodeling process was manifested in a sharp activation of the synthetic function of GMCs and fibroblasts and their synthesis of connective tissue elements with the development of vessel wall fibrosis.

In the aorta, thickening and uneven elastic membranes having fuzzy boundaries are noted. In their thickness there were fibroblasts densely surrounded by collagen fibers, as if immersed in connective tissue. The intercellular substance of the aorta was represented by chaotic collagen and elastic fibers, surpassing the main substance in quantity. In the thickness of the media, the fibers were distributed almost uniformly, with some prevalence of fibrous structures in the inner layers. Due to the large amount of connective tissue, myocytes seemed scattered (Figure 2).

In the state of the intercellular substance of the media of the carotid artery in the control series, a regular increase in the volume of the fibrous component was also noted (Figure 4).

In the femoral artery in the distant post-reperfusion period, taking into account the muscle type of the structure of this vessel and the minimum, compared with the aorta and carotid artery, the number of connective tissue elements in the wall structure, the increase in the number of connective tissue in the control series was most noticeable. Synthetic GMCs of the process form and contractile MMCs of the spindle-shaped form were recorded. Classical fibroblasts were not detected, but transitional forms were noted - cells of an adolescent form without myofilaments with an invaginated nucleus and a developed synthetic apparatus. Towards adventitia, the number of fibers decreased slightly.

In the group with the use of Mexidol in the structure of the main vessels, the number of fibers did not exceed the amount of the main substance.

Thus, in the series with mexidol in the aorta, carotid and femoral arteries on the 60th day of the post-reperfusion period, compared with the control series, the phenomena of vascular remodeling are less pronounced: the synthesis and, as a consequence, the volume of fibrous structures of connective tissue are reduced. Therefore, the positive effect of Mexidol is to prevent the formation of connective tissue elements in the vascular walls.

The proposed method is as follows.

The rat is anesthetized with intraperitoneal administration of Nembutal at a dose of 25 mg / kg in order to intubate the trachea with a polyethylene catheter. Total ischemia is performed by completely compressing the vascular bundle of the heart intrathoracically without opening the chest and without pneumothorax with a special L-shaped hook made from an injection needle. The hook is inserted into the mediastinum at the level of the third intercostal space on the right along the parasternal line after a preliminary puncture of the skin, intercostal muscles and parietal pleura with the sharp end of the pressing part of the hook. Then the holder is rotated to a position perpendicular to the axis of the spine. During this manipulation, the bend of the hook went into the pleural cavity. Next, the hook is lowered down to the spine to a light stop and the holder is rotated counterclockwise by 100-110 °, and the pressing part of the clamp is brought under the vascular bundle of the heart. At the next stage, the hook is raised perpendicularly upward, pressing the inferior vena cava and the nameless veins, pulmonary artery and aorta to the sternum. The sternum is displaced upward with a finger. At the same time, the trachea is not pinched, and blood circulation completely stops - this is the beginning of total ischemia. After 3 min 30 s - 4 min, the hook is removed from the chest in the reverse sequence, and the animal continues to be in this state until resuscitation begins for 10 minutes from the moment the stopwatch is turned on.

Resuscitation is carried out using external cardiac massage and mechanical ventilation. Before resuscitation begins, 0.1 ml of a 0.1% adrenaline solution is administered endotracheally. External heart massage is carried out by strokes of the index and middle fingers on the sternum at the level of the fourth intercostal space with a frequency of 200 beats per minute. Artificial ventilation of the lungs is carried out by connecting an open circuit with a frequency of 60 per minute to the endotracheal catheter of the artificial respiration apparatus. The effectiveness of resuscitation measures is monitored by monitoring the ECG in the II standard lead.

Mexidol is administered once intraperitoneally during the first hour (fractionally every 20 minutes) after resuscitation at a dose of 25 mg / kg.

A specific example is given in the form of an extract from the experiment.

We took a sexually mature (5 months) white non-linear rat, a male weighing 249 g, contained in the standard diet of vivarium.

Total ischemia was modeled by clamping a cardiovascular bundle for 10 min according to V.G. Korpachev. with subsequent traditional resuscitation and restoration of cardiac activity, blood circulation and respiration (Korpachev V.G., Lysenkov S.P., Tel L.Z. Modeling of clinical death and postresuscitation disease in rats // Patol. physiology and experimental therapy. - 1982. - No. 3. - S.78-80).

After resuscitation, Mexidol was administered intravenously during the first hour fractionally (every 20 minutes) at a dose of 25 mg / kg.

Biochemical, histological and electron microscopic analysis of the tissue of the great vessels and determination of blood biochemiluminescence was performed on the 60th day after total ischemia.

For morphological studies, sections of blood vessels were taken according to the accepted scheme. Vascular fragments no more than 1 mm. fixed in neutral formalin and glutaraldehyde and dehydrated in alcohols. Sections prepared using a microtome and ultratome were stained with hematoxylin and eosin according to Van Gieson, fuchselin according to Weigert and uranyl acetate and lead citrate according to the Reynolds method. Histological preparations were examined using a Leica DMLS light microscope; ultrathin sections were examined with a FEI Morgagni 268D electron microscope.

To determine the intensity of free radical oxidation (SRO) and antioxidant activity (AOA) of blood plasma, we used the measurement of chemiluminescence (CL) on a BHL-06 biochemiluminometer (Vladimirov Yu.A., Sherstnev MP, Chemiluminescence of animal cells // Biophysics. - 1989. - T.24. - S.176).

The analysis of the content of malondialdehyde in blood vessels was carried out according to the reaction with thiobarbituric acid (TAC) (Pankin V.Z., Gerasimova EN, Kasatkin LB Lipid peroxides and atherosclerosis. Enzymatic detoxification of lipid peroxides in the blood of patients with coronary heart disease due to atherosclerosis of the coronary arteries. // Cardiology. - 1979. - No. 6. - P.71-75).

Indicators of biochemiluminescence of blood plasma and the content of MDA in the tissue of arterial vessels are presented in table 2.

Table 2. Imax, mV S, mV MDA, nMol / g of tissue 2.46 22.23 2.15

Histological examination of the wall of the main vessels: the preservation of the endothelial layer, single aggregates consisting of blood cells, a decrease in the severity of connective tissue structures in the media and in the subendothelial layer are noted (Fig.1b).

Ultrastructure of the intima of the vascular wall of the great vessels.

The intercellular contacts of the endothelium were fairly dense, in places moderately widened. EC mainly had a dense cytoplasm. Most cell mitochondria were in a state of physiological norm. The matrix in most mitochondria is dense, enlightenment is extremely rare. EPS is moderately expanded. The core is rich in chromatin. Contours of the nucleus with shallow invaginations of karyolemma. The inner elastic membrane is dense, uniform, without thickenings.

Ultrastructure of the media of the vascular wall of the great vessels.

The structure of the MMC cytoplasm was dense, with no signs of hydration. Mitochondria of smooth muscle cells with clear cristae. The matrix is tight. EPS is moderately expanded. Active synthesis of connective fibers is not observed. The shape of the nuclei is irregular; some nuclei are invaginated. The nucleus is rich in chromatin, the perinuclear space is moderately expanded. In the intercellular substance, an increase in the volume of the fibrous component is not observed (Figs. 3, 5, 7).

Claims (1)

A method for correcting structural changes in blood vessels in the post-reperfusion period in an experiment, which includes administering drugs, characterized in that mexidol is used as a means for correcting structural changes in the great vessels, which is administered intravenously during the first hour after a resuscitation every 25 minutes at a dose of 25 mg / kg

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