RU2559937C1 - Method for pharmacological stimulation of neoangiogenesis in treating acute experimental limb ischemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: acute limb ischemia is simulated in Wistar male rats by excising a segment of great vessels containing a femoral artery, a popliteal artery and initial segments of shin arteries. The operation is performed under chloral hydrate anaesthesia in a dose of 250-300 mg/kg. Through 5 days immediately following the surgery, Actovegin is introduced intraperitoneally in a dose of 50 mcg/kg daily once a day.

EFFECT: method provides stimulating neoangiogenesis and activation of developing collateral blood flow in the ischemic limb and thereby improvement of the arterial blood inflow from the proximal portions of the limb into distal ones.

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Description

The invention relates to medicine, namely to experimental pharmacology and vascular surgery, and can be used in the treatment of critical limb ischemia.

Closest to the claimed solution is a method of pharmacological correction of skeletal muscle ischemia with resveratrol (patent RU 2518965 C1 of 06/10/2014). In this experimental study, shin muscle ischemia was modeled by the operative removal of a section of the great vessels, including the femoral, popliteal, anterior and posterior tibial arteries on the second day of the experiment; Resveratrol was administered intragastrically to a laboratory animal in a daily dose of 2.0 mg / kg every 46 hours for the first 7 days of the experiment; muscle microcirculation was evaluated on day 28 of ischemia using laser Doppler flowmetry. However, in this study, shin muscle ischemia is modeled on the second day of the experiment operatively after applying the ischemic preconditioning effect, which, as you know, consists of two periods: early (lasts up to 2 hours and protects cells from damage) and late ("second protective window" develops 24 hours after the onset of the causative factor and lasts about 48 hours). In addition, the disadvantage of this method is that the authors of the drug resveratrol, which has a proven cytoprotective effect, is used just in the phase of the late period of ischemic preconditioning, namely every 46 hours, which contributes to the fact that the level of microcirculation becomes higher than even in intact animals. This suggests that pharmacological correction of skeletal muscle ischemia is achieved not only due to neoangiogenesis, but also due to the phenomenon of intermittent ischemia or metabolic adaptation.

The main advantage of the proposed method is that, against the background of the use of the Actovegin preparation, microcirculation recovery is achieved only by stimulating neoangiogenesis and activating the development of collateral blood flow in the ischemic limb and, thus, improving arterial blood flow from the proximal extremities. The drug is administered intraperitoneally at a dose of 50 μg / kg daily 1 time per day for 5 days immediately after surgery in the absence of the effect of ischemic preconditioning, which in real life conditions in patients suffering from acute thromboembolism of lower limb arteries does not work.

The technical result of the invention is the development of a method for the treatment of acute experimental limb ischemia using the drug "Actovegin".

The technical result is achieved by the fact that against the background of modeling acute experimental limb ischemia in male Wistar rats by excision of the main vessel, including the femoral artery, popliteal artery, and the initial sections of the lower leg arteries under anesthesia with chloral hydrate at a dose of 250-300 mg / kg, the correction of the pathology of intraperitoneal the introduction of the drug "Actovegin", obtained from the blood of healthy dairy calves, at a dose of 50 μg / kg daily 1 time per day for 5 days immediately after surgery. This leads to a pronounced correction of microcirculation disorders in the ischemic limb, as confirmed by laser Doppler flowmetry.

The method is as follows.

The experiments were carried out on 160 male Wistar rats weighing 300-350 g. Male rats were divided into four series: intact, control, experimental and comparisons. In each of the series, 40 male rats were included, which were divided into four groups depending on the breeding period; 10 male rats were included in each group.

In male rats of all series, the dynamics of the level of microcirculation in the ischemic limb was studied without and against the background of treatment of acute experimental ischemia with the Actovegin drug at 5, 10, 21, and 28 days. Acute hind limb ischemia was modeled according to the method of M. Sugano et al. (2004), S. Takeshita et al. (1998) by excision of the main vessel, including the femoral artery, popliteal artery and the initial sections of the arteries of the leg.

Assessment of the level of microcirculation in the muscles of the tibia of male rats was performed by laser Doppler flowmetry (LDF). The study was carried out using an apparatus - a laser-Doppler flowmeter "Biopac-systems MP-100" and a sensor "TSD-144". Data recording and processing was performed using the AcqKnowledge 38 program. The study was performed under anesthesia with chloral hydrate at a dose of 250-300 mg / kg, which was administered intraperitoneally in the form of an aqueous solution. After the onset of narcotic sleep, a skin site was excised on the anterolateral surface of the right hind leg of the hind limb. The male rat was laid on the opposite side. The sensor was closely attached to the muscle and performed flowmetry. The microcirculation level was recorded at five points: 1) the middle of the muscle length; points 3-4 mm 2) higher, 3) lower, 4) lateral and 5) medial from the first (patent No. 2353977 dated 04/27/2009). The microcirculation level curve was recorded for 30 seconds at each point. From the five values obtained, the average was derived, which was entered into the protocol and was taken as the level of microcirculation in the calf muscles in this male rat. From the 10 obtained values, the average was deduced, which was taken as the microcirculation level in this group of male rats at this study period.

In intact male rats, the microcirculation and shin muscles of the right hind limb were assessed by LDF. The obtained average microcirculation level in this group was taken as the “norm” and amounted to 535.22 ± 37.1 perfusion units (bp). The error of this average value (37.1) does not exceed 10% of the absolute value (53.52), which indicates a sufficient number of observation units in the group.

Male rats of the control series of treatment did not receive. In male rats of the experimental series, treatment was started immediately after surgery by the intraperitoneal administration of the drug Actovegin, obtained from the blood of healthy dairy calves, at a dose of 50 μg / kg daily 1 time per day for 5 days. In the comparison group, male rats were treated with pentoxifylline at a dose of 60 mg / kg intraperitoneally daily 1 time per day for 5 days.

Male rats were removed from the experiment by an overdose of anesthesia on days 5, 10, 21 and 28. Before being removed from the experiment in male rats, the dynamics of clinical and flowmetric parameters were evaluated in modeling limb ischemia at different times. The data obtained were processed statistically: the average values of the shifts (M) were calculated, the average error of the arithmetic mean (± m) and the probability of a possible error (p). Differences were evaluated as significant at p <0.05.

During a clinical examination, the majority of male rats showed pallor and cooling of the skin of the lower leg and foot, and limited participation of the limb in movement.

The effect of the drug "Actovegin" on the level of microcirculation in male rats with acute experimental limb ischemia is presented in table 1.

As can be seen from table 1, in the control series of male rats after the operation of modeling ischemia, there is a sharp and rapid decrease in the level of microcirculation compared to intact: on day 5 - by 71.5%; on the 10th day - by 61.3%; by 21 days - by 42.9%; on the 28th day - by 33.4%. There is a slow recovery in the level of microcirculation, which indicates the inclusion of the existing compensatory mechanisms of collateral circulation. However, up to 28 days, inclusive, microcirculation indices remain significantly below the normal value recorded in the group of intact male rats.

In a series of male rats treated with pentoxifylline, an increase in the level of microcirculation as compared with the control on day 5 was detected - by 30%; on the 10th day - by 11%; on 21 days - by 11%; on the 28th day - by 5%. It follows that pentoxifylline has a positive effect on microcirculation, but by the 28th day statistically significantly the latter does not reach normal values.

Compared with the control series, the level of microcirculation in the experimental series treated by the claimed method was significantly higher by 5 days - by 38%; on the 10th day - by 19%; on the 21st day - by 28%; on the 28th day - by 45%; and compared with a series of male rats treated with pentoxifylline, on the 5th day - it increases by 5.6%; on the 10th day - by 7.4%; for 21 days - by 14.5%; on the 28th day - by 38%.

So, the microcirculation level in the experimental series of male rats treated with Actovegin, in contrast to the series treated with standard Pentoxifylline, statistically reliably reached the normal value recorded in the series of intact male rats by 28 days, which indicates sufficient the effectiveness of the proposed method.

EXAMPLE OF SPECIFIC PERFORMANCE

The male rats of the comparison series in the experiment were administered the standard preparation Pentoxifylline at a dose of 60 mg / kg intraperitoneally daily 1 time per day for 5 days immediately after modeling acute limb ischemia, which led to an increase in microcirculation in the ischemic limb by 5 days to 198, 33 ± 11.48 b.p., on the 10th day - up to 232.20 ± 13.57 b.p., on the 21st day - up to 347.96 ± 17.36 b.p. and on day 28, 388.98 ± 13.34 p.u., however, the level of microcirculation did not statistically significantly reach the normal value recorded in a series of intact male rats.

Immediately after modeling acute limb ischemia, the male rats of the experimental series were intraperitoneally injected with Actovegin, obtained from the blood of healthy dairy calves, at a dose of 50 μg / kg daily 1 time per day for 5 days. This led to a significant increase in the level of microcirculation in the ischemic limb on the 5th day to 209.44 ± 19.32 p.u., on the 10th day - to 249.46 ± 9.49 p.u., on the 21st day - to 398, 42 ± 26.37 p.u. and on day 28, up to 535.34 ± 18.26 p.u., which is higher compared with a series of male rats treated with pentoxifylline and reaches normal values by 28 days.

Thus, the results of an experimental study convincingly indicate that the proposed method is more effective and pathogenetically justified in the treatment of acute limb ischemia compared to the known one due to stimulation of neoangiogenesis and the development of collateral blood flow in the ischemic limb and, thus, improving arterial blood flow from the proximal departments of the limbs into the distal.

Claims (1)

A method of pharmacological stimulation of neoangiogenesis in the treatment of acute limb ischemia in an experiment, characterized in that against the background of modeling acute limb ischemia in male Wistar rats by excision of a section of the main vessels, including the femoral artery, popliteal artery and the initial parts of the leg arteries, under anesthesia with chloral hydrate in at a dose of 250-300 mg / kg, the Actovegin drug is administered intraperitoneally at a dose of 50 mcg / kg daily 1 time per day for 5 days immediately after surgery.