RU2426174C1 - Method of pharmacological correction of extremity ischemia including in l-name induced nitrogen oxide deficiency - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: extremity ischemia is modelled by removing a portion of a great vessel, including with simultaneous additional modelling of nitrogen oxide (NO) deficiency by intraperitoneal introduction of a NO synthesis blocker N-nitro-L-arginine of methyl ester (L-NAME) 25 mg/kg daily for 7 days. In both cases, the ischemia is corrected by subcutaneous introduction of recombinant erythropoein 50 IU/kg for the first, third and fifth days of experiment. ^ EFFECT: effective ischemia treatment in experiment ensured by improved extremity microcirculation. ^ 1 ex, 1 tbl

Description

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

Closest to the claimed solution is a method for correcting limb ischemia using a single daily administration of L-arginine at a dose of 30 to 200 mg / kg, proposed by M.V. Pokrovsky et al. Patent for invention RU 2302239 C1.

However, this decision does not evaluate the effect of recombinant erythropoietin on limb ischemia, including with L-NAME-induced nitric oxide deficiency.

The objective of the invention is to develop a method for the correction of limb ischemia, including with L-NAME-induced deficiency of nitric oxide, including the use of recombinant erythropoietin.

The task is achieved in that the correction of limb ischemia, including when simulating a deficiency of nitric oxide (NO) by intraperitoneal administration to a laboratory animal for 7 days, a NO synthesis blocker of N-nitro-L-arginine methyl ester (L-NAME) at a dose of 25 mg / kg daily, carried out by subcutaneous administration of recombinant erythropoietin at a dose of 50 IU / kg on the first, third and fifth days of the experiment.

The method is carried out as follows.

The experiments were carried out on white Wistar female rats weighing 200-220 g. Nitric oxide deficiency was simulated by intraperitoneal administration of an N-nitro-L-arginine methyl ether (L-NAME) blocker for the synthesis of NO at a dose of 25 mg / day to the laboratory animal. kg daily.

Limb ischemia modeling is carried out on the second day of the experiment under anesthesia (chloral hydrate 50 mg / kg ip) by removing a section of the main vessel, including the femoral artery, popliteal artery, and the initial parts of the leg artery.

Microcirculation in the muscles of the tibia of an animal is assessed using a Biopac-systems MP-100 laser-Doppler flowmeter and TSD-144 sensor (USA). To do this, under anesthesia (chloral hydrate 50 mg / kg of weight intraperitoneally), a section of skin is excised in the anterolateral region of the leg region. 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).

Microcirculation level is recorded in groups with modeling of limb ischemia, with modeling of limb ischemia in L-NAME-induced nitric oxide deficiency and in animal groups that corrected limb ischemia and limb ischemia in L-NAME-induced nitric oxide deficiency with recombinant erythropoietin.

Recombinant erythropoietin ("Epocrine" epoetin alpha; Federal State Unitary Enterprise "State Research Institute for Highly Clean Preparations" of the Federal Medical and Biological Agency St. Petersburg, RUSSIA) is administered subcutaneously at a dose of 50 IU / kg on the first, third and fifth days of the experiment in morning hours.

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

EXAMPLE OF SPECIFIC PERFORMANCE

The level of microcirculation in the right and left muscles of the leg of intact animals did not differ from each other and amounted to 533.2 ± 9.7 p.u. (perfusion units). Experimental animals were divided into the following groups:

1. modeling of limb ischemia (n = 20);

2. modeling of limb ischemia + recombinant erythropoietin (n = 20);

3. modeling of limb ischemia in L-NAME-induced nitric oxide deficiency (n = 20);

4. modeling of limb ischemia in L-NAME-induced nitric oxide deficiency + recombinant erythropoietin (n = 20).

All animals underwent modeling of limb ischemia on the second day of the experiment under anesthesia (chloral hydrate 50 mg / kg ip) by removing a section of the main vessel, including the femoral artery, popliteal artery, and the initial parts of the leg arteries. Nitric oxide (NO) deficiency was simulated in animals of groups 3 and 4 by intraperitoneal injection of N-nitro-L-arginine methyl ester (L-NAME) at a dose of 25 mg / kg daily for the first 7 days. Correction of limb ischemia was performed in animals of the 2nd and 4th groups by subcutaneous administration of recombinant erythropoietin at a dose of 50 IU / kg on the first, third and fifth days of the experiment.

Microcirculation in the muscles of the calf of the animal was assessed using a Biopac-systems MP-100 laser-Doppler flowmeter and TSD-144 sensor (USA) on days 21 and 28. To do this, under anesthesia (chloral hydrate 50 mg / kg ip), a section of skin was excised in the anterolateral region of the leg. 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 microcirculation level curve was recorded 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 10 obtained values, the average was deduced, which was taken as the level of microcirculation in this group of animals at a given time of the study.

The results of assessing the level of microcirculation in animals with modeling of hind limb ischemia, with modeling of hind limb ischemia with L-NAME-induced nitric oxide deficiency and the groups treated were presented in the table (Table 1).

Table 1 The results of assessing the level of microcirculation in the muscles of the leg (M ± m) 21 days (n = 10) 28 days (n = 10) Limb ischemia 322.43 ± 13.9 361.454 ± 11.955 Limb Ischemia + Erythropoietin 435.33 ± 27.3 * 768.83 ± 14.575 * Limb Ischemia + L-NAME 350,757 ± 35,111 427,253 ± 12,902 Limb Ischemia + L-NAME + Erythropoietin 359.93 ± 18.078 682.2393 ± 42.794 ** * - p <0.05 in comparison with a group of animals with limb ischemia; ** - p <0.05 compared with a group of animals with limb ischemia treated with L-NAME.

The table shows that the level of microcirculation after surgery to model ischemia of the hind limb at 21 days 322.43 ± 13.9 p.u., at 28 days 361.454 ± 11.955 p.u., which is significantly lower than the value in the group of intact animals (p < 0.001).

Against the background of treatment with recombinant erythropoietin for 21 days, the level of microcirculation significantly increases to 435.33 ± 27.3 p.u. (p <0.05), and by 28 to 768.83 ± 14.575 p.u. (p <0.0000000087).

When modeling limb ischemia against the background of L-NAME-induced nitric oxide deficiency, the level of microcirculation on day 21 in the treated group did not significantly differ from the group of animals that did not receive treatment (359.93 ± 18.078 bp versus 350.757 ± 35.111 bp ) A statistically significant difference was noted in these groups on day 28 682.2393 ± 42.794 p.u. versus 427.253 ± 12.902 (p <0.0002).

Thus, the results obtained make it possible to ascertain the pharmacological correction of limb ischemia, including limb ischemia, with L-NAME-induced nitric oxide deficiency by recombinant erythropoietin.

Claims (1)

A method for the pharmacological correction of limb ischemia, including with L-NAME induced nitric oxide deficiency, characterized in that the correction of limb ischemia, including with modeling nitric oxide (NO) deficiency, by an intraperitoneal injection of a blocker of NO N- synthesis synthesis into a laboratory animal for 7 days nitro-E-arginine methyl ester (L-NAME) at a dose of 25 mg / kg daily, is carried out by subcutaneous administration of recombinant erythropoietin at a dose of 50 IU / kg on the first, third and fifth days of the experiment.