RU2735838C1 - Method for pharmacological correction of experimental critical skeletal muscle ischemia with a combination of simvastatin and mononuclear fraction of autologous bone marrow - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to experimental pharmacology and surgery, and can be used to model critical skeletal muscle ischemia. Method for pharmacological correction of experimental critical skeletal muscle ischemia with a combination of simvastatin and mononuclear fraction of autologous bone marrow, consisting in experimental modeling of critical skeletal muscle ischemia in rats and performing its correction by intragastric introduction of simvastatin in dose of 0.86 mg/kg once a day daily for 14 days and single parenteral introduction of mononuclear fraction of autologous bone marrow on 7th day after simulating critical ischemia by 50 mcl to 4 points: paravasally above the inguinal ligament at a point of retraction from a lateral artery bypassing the internal iliac artery surrounding the femur, under the inguinal ligament in the area of the peripheral artery circumscribing the iliac, in area of a branch of a muscular branch of a femoral artery r. muscularis, attachment point for the pectineal muscle and long adducting hip muscle to the upper third of the gastrocnemius muscle.

EFFECT: method described above enables correcting ischemia of skeletal muscle with simvastatin and mononuclear fraction of autologous bone marrow in conditions of combined therapy.

1 cl, 1 tbl, 1 ex

Description

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

The closest to the claimed solution is a method for correcting limb ischemia using trental (pentoxifylline), which is a drug that improves microcirculation, described by B.C. Saveliev, V.M. Koshkin. "Critical ischemia of the lower extremities". Chapter 7. Treatment of patients with chronic obliterating diseases of the arteries of the lower extremities. Moscow, 1997 Publishing house "Medicine", which consists in intra-arterial, intravenous, intramuscular and oral administration of these drugs in patients for the correction of peripheral circulatory disorders.

The main disadvantage of this method is the limited pathogenetic orientation associated with the absence of an endothelium-dependent vasodilating effect while taking trental. The mechanism of action of trental is based on a decrease in blood viscosity and an increase in the elasticity of erythrocytes, an improvement in microcirculation and an increase in the concentration of oxygen in tissues. The increase in the elasticity of erythrocytes, apparently, is due to inhibition of phosphodiesterase and an increase as a result of this content of cAMP in erythrocytes. A decrease in blood viscosity may be due to a decrease in the concentration of fibrinogen in plasma and suppression of the aggregation of erythrocytes and platelets.

While statins act on several links of pathogenesis at once, at any initial level of cholesterol statins lead to its decrease, it is important to note the fact that they are characterized not only by hypolipidemic, but also non-lipid (pleiotropic) effects. Thus, they improve the function of the endothelium, have an antithrombotic effect, reduce the activity of inflammatory processes, inhibit fibrosis processes, enhance perfusion and angiogenesis. Thus, the use of simvastatin in critical limb ischemia is quite reasonable.

For the purpose of regenerative cell therapy, bone marrow stem cells are currently used due to their pluripotency. Stem cells (hematopoietic and mesenchymal) are contained in the mononuclear fraction of bone marrow cells. Possessing multipotent properties, these cells can promote replacement of tissue defects and neoangiogenesis. In addition, the lymphoid cells of the fraction, due to their morphogenetic abilities, can take part in the processes of restorative regeneration of damaged organs and tissues.

Combined therapy with simvastatin and the mononuclear fraction of autologous bone marrow can reduce not only the daily dose of simvastatin to 0.86 mg / kg, but also the course dose.

Thus, the combined use of simvastatin and the mononuclear fraction of autologous bone marrow in limb ischemia, which act on different links of pathogenesis, is quite reasonable. This non-competitive interaction allows to achieve the maximum physiological response, to provide a good rheological effect in the ischemic focus and to reduce the dose of simvastatin in the combination we proposed, which in turn increases the safety of this combination in terms of side effects, and is also pharmacoeconomically more beneficial.

The technical result of the invention is the development of a method for correcting skeletal muscle ischemia with simvastatin and a mononuclear fraction of autologous bone marrow in a combination therapy.

The technical result is achieved by the fact that against the background of modeling ischemia of skeletal muscle, its correction is carried out by intragastric administration of simvastatin at a dose of 0.86 mg / kg 1 time per day daily for 14 days and a single parenteral administration of a mononuclear fraction of autologous bone marrow on the 7th day after modeling critical ischemia, 50 μl at 4 points (paravasal above the inguinal ligament to the place of origin from the internal iliac artery of the lateral artery, bending around the femur; under the inguinal ligament in the area of the superficial artery, bending around the ilium; in the area where the muscular branch of the femoral artery r. muscularis, the place of attachment of the comb and long adductor muscles of the thigh; in the upper third of the gastrocnemius muscle).

THE METHOD IS CARRIED OUT AS FOLLOWS

Experiments are carried out on white Wistar rats weighing 220-250 g.

The experimental animals were divided into the following groups:

1) intact (n = 30);

2) sham-operated animals (skin incision along the thigh along the inner surface with the release of the neurovascular bundle of the thigh and wound closure with a continuous suture) (n = 30);

3) modeling of critical ischemia of the leg muscles (removal of the femoral, popliteal, anterior and posterior tibial arteries and veins, as well as the sciatic nerve) (n = 30);

4) modeling of critical ischemia of the leg muscles and its correction with simvastatin and mononuclear fraction of autologous bone marrow (n = 30).

Modeling of critical ischemia of the left leg muscles was carried out under anesthesia (chloral hydrate 300 mg / kg of weight intraperitoneally), surgical removal of a portion of the great vessels, including the femoral, popliteal, anterior and posterior tibial arteries and veins, as well as the sciatic nerve.

Correction of critical limb muscle ischemia was carried out with simvastatin at a dose of 0.86 mg / kg, intragastrically once a day, daily for 14 days and a single parenteral administration of a mononuclear fraction of autologous bone marrow (from the tibia by puncture of the bone marrow cavity and isolation according to the Boyum method) on 7th day after modeling critical ischemia, 50 μl at 4 points (paravasal above the inguinal ligament to the place of origin from the internal iliac artery of the lateral artery, bending around the femur; under the inguinal ligament in the area of the superficial artery, bending around the iliac bone; in the area of origin of the muscular branch femoral artery r. muscularis, the place of attachment of the comb and long adductor muscles of the thigh; in the upper third of the gastrocnemius muscle).

The level of microcirculation in the leg muscles was determined using equipment manufactured by Biopac systems: an MP100 polygraph with an LDF100C laser Doppler flowmetry module and an invasive TSD144 needle sensor on days 10, 21 and 28. The registration and processing of the results of laser Doppler flowmetry (LDF) was performed using the AcqKnowledge program version 3.8.1., The microcirculation values were expressed in perfusion units (PU). 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 to the first) for 30 seconds at each point. From the obtained five values, the mean was deduced, which was entered into the protocol and taken as the level of microcirculation in the leg muscles in this animal. From 10 obtained values, the mean was calculated, which was taken as the level of microcirculation in this group of animals at this time of the study. Registration of the level of microcirculation is carried out in all experimental groups.

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

EXAMPLE OF A SPECIFIC PERFORMANCE

The results of assessing the level of microcirculation in animals of the experimental groups are presented in Table 1.

Table 1

Results of assessing the level of microcirculation in the leg muscles of the experimental groups (M ± m) in perfusion units 10 days (n = 10) 21 days (n = 10) 28 days (n = 10) Intact 528.6 ± 11.04 531.6 ± 12.08 533.4 ± 11 Sham-operated animals 521.4 ± 11.46 523 ± 13.79 524.1 ± 15.1 Modeling critical ischemia of the leg muscles 160.5 ± 3.28 * 249.1 ± 7.31 * 302.9 ± 6.46 * Modeling of critical ischemia of the leg muscles and its correction with simvastatin and mononuclear fraction of autologous bone marrow 283.8 ± 17.52 439.6 ± 9.14 540.1 ± 15.26 Note: * - p <0.05 in comparison with the intact group of animals; ** - p <0.05 in comparison with the group of animals with critical ischemia of the leg muscles

The average value of the level of microcirculation in the intact muscle of the leg of rats was 531 ± 12 PU (perfusion units).

In the group of sham-operated animals, the average value of the level of microcirculation in the muscles of the left leg at all periods did not differ significantly from those in the group of intact animals (523 ± 13 PU). In the group modeling the critical ischemia of the leg muscles, the level of microcirculation at all periods led to a significant decrease in the level of regional blood flow in the ischemic leg muscle of rats (day 10, p <0.05, day 21, p <0.05; day 28, p <0.05).

Correction with simvastatin and a mononuclear fraction of autologous bone marrow contributed to a significant increase in the level of regional blood flow in the ischemic muscle of the leg of rats compared with the parameters of the group of animals with critical ischemia of the leg muscles at the corresponding period (day 10, p <0.05, day 21, p < 0.05; 28th day p <0.05). The level of microcirculation in this group on the 28th day approaches that in the group of intact animals.

Thus, the results obtained allow us to state the correction of skeletal muscle ischemia by a combination of simvastatin and a mononuclear fraction of autologous bone marrow by stimulating neoangiogenesis.

Claims (1)

A method of pharmacological correction of experimental critical ischemia of skeletal muscle by a combination of simvastatin and a mononuclear fraction of autologous bone marrow, including experimental modeling of ischemia of the leg muscles, characterized in that critical ischemia of skeletal muscle is modeled in rats and corrected by intragastric administration of simvastatin at a dose of 0.86 mg / kg 1 time per day every day for 14 days and a single parenteral administration of a mononuclear fraction of autologous bone marrow on the 7th day after modeling critical ischemia, 50 μl at 4 points: paravasally above the inguinal ligament at the site of origin from the internal iliac artery of the lateral artery, bending around the femur, under the inguinal ligament, into the area of the superficial artery that bends around the ilium, into the area of origin of the muscular branch of the femoral artery r. muscularis, the place of attachment of the comb and long adductor muscles of the thigh, in the upper third of the gastrocnemius muscle.