RU2547692C1 - Method for stimulating neoangiogenesis in ischemic skeletal muscle - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: shin muscle ischemia is simulated in laboratory animals on the second experimental day by surgical removal of a portion of great vessels, including femoral, popliteal, anterior and posterior tibial arteries. The ischemia is corrected by the intragastric administration of dihydroquercetin in a daily dose of 5.5 mg/kg every 46 hours of the first 7 experimental days.

EFFECT: effective treatment of the skeletal muscle ischemia by stimulating neoangiogenesis that is proved by laser Doppler flowmetry and morphological analysis.

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Description

The invention relates to medicine, in particular to experimental surgery and experimental pharmacology, and can be used to correct ischemia.

A known method for the correction of skeletal muscle ischemia by distant ischemic preconditioning RU 2438192 C1. Its disadvantage is that the method is not without the potential danger of ischemic episodes for pathologically altered tissues and is applicable mainly to a healthy body.

Closest to the claimed solution is the "Method of pharmacological correction of limb ischemia, including with l-name induced nitric oxide deficiency" (RU 2426174 C1). Correction of ischemia is carried out by recombinant erythropoietin. The advantage of this method is that the activation of the preconditioning process is carried out by a pharmacological preparation. That is, there is no episode of ischemia, which can have a damaging effect on the tissue. However, the disadvantage of this method is that erythropoietin is chosen as the initiator of the preconditioning process. Numerous researchers tend to consider him the humoral agent of preconditioning. However, this is a hormonal protein preparation and there are a number of contraindications for its use, namely arterial hypertension, cancer. In addition, methods are known to activate the preconditioning process by direct stimulation of the main effector link of ATP-dependent potassium channels. Undoubtedly relevant is the study of drugs that can activate various stages of the preconditioning process.

The technical result of the invention is the development of an effective method of stimulating neoangiogenesis in ischemic skeletal muscle, including the use of a pharmacological drug, non-toxic, with a minimum number of contraindications.

The technical result is achieved by the fact that skeletal muscle ischemia is simulated on a laboratory animal on the second day of the experiment, neoangiogenesis is stimulated by intragastric administration of dihydroquercetin in a daily dose of 5.5 mg / kg every 46 hours for the first 7 days of the experiment.

The main advantage of the proposed method is the absence of side effects (except for individual intolerance) of the pharmacological agent, since it belongs to the class of flavonoids. It is known that with preliminary oral administration, the drug under conditions of severe hypobaric hypoxia exerted both an antioxidant and antihypoxic effect on the body. Such properties of the drug are probably due to the fact that dihydroquercetin, due to the hydrophobic nature of the structure, is able to penetrate the hydrophobic region of the membranes and thereby significantly reduce lipid mobility. Thus, this drug stabilizes membranes under conditions of oxidative stress and acts as a structural antioxidant. In addition, it is known that flavonoids are capable of shunting the work of NADH-dehydrogenase, taking electrons from NADH and transferring them to the next - the third mitochondrial chain complex, normalizing the processes of oxidative phosphorylation (L. Lukyanova, 2011). Based on the results obtained, it can be assumed that dihydroquercetin is able to activate the preconditioning process, one of the effects of which is neoangiogenesis. The drug is administered every 46 hours, given that by this time the preconditioning effect is weakening, the first 7 days of the experiment, since it is at this stage that the activation of neoangiogenesis in ischemic muscle is most effective.

The method is carried out as follows.

The experiments are carried out on white rats of the Wistar line weighing 220-250 g.

Ischemia is modeled on the second day on the muscles of the right lower leg of the rat by surgical removal of a section of the great vessels, including the femoral, popliteal, anterior and posterior tibial arteries.

When assessing the state of the microvasculature in the ischemic muscle, the level of microcirculation in it and the data of the morphological study are taken into account.

The microcirculation level in the calf muscles is determined using Biopac systems equipment: MP 100 polygraph with LDF100C laser Doppler flowmeter module and TSD144 invasive needle probe. Registration and processing of laser Doppler flowmetry (LDF) results is performed using the AcqKnowledge version 4.2 software. Microcirculation values are expressed in perfusion units (PE). The microcirculation level is recorded at five points (the middle of the muscle length, points 3-5 mm above and below, lateral and medial of the first).

The level of microcirculation is recorded in the group of intact animals and in the groups of false-operated animals, with modeling of ischemia of the calf muscles and in the group of animals that were injected with dihydroquercetin on days 21 and 28 after the operation.

Morphological research is carried out on the material of standard muscle areas taken after removing animals from the experiment. Material processing is carried out according to a standard method with formalin fixation, paraffin embedding, hematoxylin and eosin staining. General morphological and morphometric studies are performed using the MiraxDesk image scanning and archiving system.

Dihydroquercetin is administered intragastrically at a daily dose of 5.5 mg / kg every 46 hours for the first 7 days of the experiment.

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

EXAMPLE OF SPECIFIC PERFORMANCE

Experimental animals were divided into the following groups:

1) intact animals (n = 20);

2) false-operated animals (n = 20);

3) modeling of ischemia of the leg muscles (n = 20);

4) modeling of ischemia of the leg muscles + dihydroquercetin (n = 20);

Ischemia of the muscles of the right lower leg was simulated on the second day under anesthesia (chloral hydrate intraperitoneally 300 mg / kg of weight) by operative removal of the section of the great vessels, including the femoral, popliteal, anterior and posterior tibial arteries. Neoangiogenesis was stimulated in animals of group 4 by intragastric administration of dihydroquercetin in a daily dose of 5.5 mg / kg every 46 hours for the first 7 days of the experiment.

When assessing the state of the microvasculature in the ischemic muscle, the level of microcirculation in it and the data of the morphological study were taken into account.

The microcirculation level in the calf muscles was determined using Biopac systems equipment: MP 100 polygraph with laser Doppler flow meter LDF100C and invasive needle probe TSD144. The registration and processing of laser Doppler flowmetry (LDF) results was performed using the AcqKnowledge version 4.2 software. Microcirculation values were expressed in perfusion units (PE). The microcirculation level curve was recorded at five points (the middle of the muscle length, points 3-5 mm above and below, lateral and medial of the first) for 30 seconds at each point. From the five values obtained, the average was deduced, which was entered into the protocol and was taken as the level of microcirculation in the calf muscles in this animal. From the 10 obtained values, the average was calculated, which was taken as the level of microcirculation in this group of animals at this time of the study. The level of microcirculation was recorded in the group of intact animals and in the groups of falsely operated animals, with modeling of ischemia of the calf muscles and in the group of animals that were injected with dihydroquercetin on 21 (n = 10) and 28 (n = 10) days after surgery.

A morphological study was performed on the material of standard sections of the leg muscles taken after the animals were removed from the experiment. The material was processed according to a standard method with formalin fixation, paraffin embedding, hematoxylin and eosin staining. General morphological and morphometric studies were performed using the MiraxDesk image scanning and archiving system.

The average microcirculation level in the intact muscle of the tibia of rats is 526 ± 34 PE. During histological examination, myocytes form dense bundles, in the thickness of which pass full-blooded venules and arterioles containing single red blood cells. The lumen of the vessels is wide, endothelial cells without pathological changes.

The average value of the microcirculation level in the muscles of the lower leg of false-operated rats is 527 ± 10 PE on day 21 - 532 ± 34 PE. The results of laser Doppler flowmetry do not have significant differences from those obtained in the group of intact animals (p = 0.19; p = 0.43, respectively), and histological examination did not reveal any differences from the structure of intact muscles.

After modeling ischemia of the leg muscles, the level of microcirculation in them sharply decreases, its recovery begins only after 14 days, but up to 90 days it remains significantly lower than the value recorded in the group of intact animals.

Considering the results obtained in the analysis of the possibility of compensatory restoration of regional blood circulation in the model used by us, as well as literature data, the 28th day (361 ± 9 PE) was regarded as the most informative, the 21st (323 ± 7 PE) were considered intermediate.

On the 21st day, ischemic muscle looked macroscopically swollen, it contained large areas of grayish-brown color. Histologically, these sites were large foci of organized necrosis with resorption of necrotic fibers and the growth of granulation tissue. On the 28th day, the color of the muscle returned to normal, but it looked somewhat hypotrophic. During histological examination, the areas of resorbable necrosis were much smaller, and in the microvasculature, plethora and neoplasm of single capillaries were noted. Bundles of atrophied muscle fibers were found near necrosis sites.

The introduction of dihydroquercetin significantly increased the level of regional blood flow in the ischemic muscle of the tibia of rats. LDF indices in these groups were significantly higher than those in the control group at the corresponding time - 21st day - 453 ± 8 PE (p <0.05), 28th day - 700 ± 21 PE (p <0.05).

That is, we can say that the compensatory restoration of regional blood circulation in the ischemic muscles of animals of the experimental groups occurred before the 28th day of the experiment. In the control series, the restoration of the initial value is observed much later (up to 90 days).

Upon macroscopic evaluation of the muscles of the ischemic tibia of rats after the administration of dihydroquercetin on the 21st and 28th day, ischemia did not differ in color or appearance from intact ones. During histological examination, necrotic changes in them were not detected. However, in some areas there were small foci of atrophying myocytes with proliferation of perimizium cells.

With a large increase in the ischemic muscles of the lower leg, there was an enhanced neoplasm of the capillaries, more pronounced on the 28th day of ischemia.

Thus, the results obtained suggest the possibility of stimulation of neoangiogenesis in ischemic skeletal muscle with dihydroquercetin.

Claims (1)

A method of stimulating neoangiogenesis in ischemic skeletal muscle, characterized in that the laboratory animal is modeled for ischemia of the calf muscles on the second day of the experiment, neoangiogenesis is stimulated by intragastric administration of dihydroquercetin in a daily dose of 5.5 mg / kg every 46 hours for the first 7 days of the experiment.