RU2414755C1 - Method for simulating hyperhomocystein induced endothelial dysfunction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: hyperhomocytein induced endothelial dysfunction is simulated by intragastric introduction of amino acid methionine 3 g/kg to male Wistar rats once a day for 7 days. After methionine introduction completed, on the 8th day from the beginning of the experiment, the animals are anaesthetised to catheterise a left carotid artery to record haemodynamic values; bolus dosing of pharmacological agents (acetylcholine in a dose 40 mcg/kg, sodium nitroprusside in a dose 30 mcg/kg) is executed in a right femoral vein. The haemodynamic values are measured by an invasive sensor and Biopac computer program. A degree of endothelial dysfunction development is evaluated by an endothelial dysfunction coefficient. A degree of hyperhomocysteinemia development is evaluated by the homocystein content in blood serum of the experimental animals.

EFFECT: method provides development of an easily reproduced and toxicologically safe model of hyperhomocystein induced endothelial dysfunction.

1 ex, 2 tbl

Description

The invention relates to medicine, in particular to experimental cardiopharmacology, and can be used to evaluate the endothelioprotective effect of a number of pharmacological drugs in modeling hyperhomocysteinemia.

Closest to the claimed solution is a method for simulating hyperhomocysteinemia, which consists of introducing methionine in doses of 12 to 44 g / kg in food for experimental animals or adding methionine to drinkers for experimental animals (Sanjana Dayal, Murine Models of Hyperhomocysteinemia and Their Vascular Phenotypes, Arteriosclerosis, Thrombosis, and Vascular Biology. 2008; 28: 1596-1605).

The main disadvantages of the method are: insufficiently accurate dosing of methionine when modeling pathology; the lack of an assessment of the development of hyperhomocysteinemia and endothelial dysfunction at different periods of methionine administration in the indicated dosages; toxic effect of methionine on physiological vital signs of experimental animals.

The objective of the invention is to develop an easily reproducible and toxicologically safe model of hyperhomocysteine-induced endothelial dysfunction based on intragastric administration of methionine.

The technical result is achieved by intragastric administration of 3 g / kg amino acids methionine to male Wistar rats once a day for 7 days, by evaluating the development of endothelial dysfunction using the coefficient of endothelial dysfunction and confirming the development of hyperhomocysteinemia by determining the concentration of homocysteine in blood serum.

Intragastric administration of methionine at a dose of 3 g / kg leads to the development of hyperhomocysteinemia in Wistar rats. Homocysteine is synthesized in the body from the essential amino acid methionine and serves as a precursor of cystathionine and cysteine. An increase in homocysteine levels correlates with a decrease in the severity of endothelium-dependent vasodilation. From these data, it can be concluded about the ability of the amino acid methionine to cause endothelial dysfunction.

The method is as follows.

The experiments were performed on sexually mature male Wistar rats weighing 180-220 g. The solution for intragastric administration of methionine was prepared ex tempore using a TWIN 80 solubilizer and 1% starch solution. In order to study the possibility of modeling endothelial dysfunction, methionine (LLC Polysynthesis, Belgorod) was administered according to the following schemes: 1) daily, 2 times a day, intragastric administration of methionine at a dose of 5 g / kg for 7 days (n = 10 animals ); 2) daily, 1 time per day, intragastric administration of methionine at a dose of 5 g / kg for 14 days (n = 10 animals); 3) daily, 1 time per day, intragastric administration of methionine at a dose of 3 g / kg for 7 days (n = 10 animals); 4) daily, 1 time per day, for 7 days, intragastric administration of 10% Tween 80 solution in a dose of 1 ml / kg (control, n = 10 animals).

At the end of the methionine administration, on the 15th and 8th days, from the beginning of the experiment, the surviving animals were anesthetized (chloral hydrate 300 mg / kg), a catheter was inserted into the left carotid artery to record blood pressure (BP), bolus administration of pharmacological agents was carried out in the right femoral vein. Hemodynamic parameters: systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) are measured continuously using the sensor and the Bioshell computer program. Functional tests: endothelium-dependent vasodilation (ESV) - intravenous administration of acetylcholine (AH) at a dose of 40 μg / kg, endothelium-independent vasodilation (ENZV) - intravenous administration of sodium nitroprusside (NP) at a dose of 30 μg / kg.

The degree of development of endothelial dysfunction is estimated using the coefficient of endothelial dysfunction, which is the ratio of the area of the triangle over the trend of the reaction of blood pressure recovery in response to the introduction of NP to the area of the triangle over the trend of the reaction of blood pressure recovery in response to the introduction of AH.

The degree of development of hyperhomocysteinemia is estimated by the content of homocysteine in the blood serum of experimental animals. Homocysteine concentration is determined by immunoturbodimetry using a Pliva-Lachema Diagnostika s.r.o. kit.

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

An example of a specific implementation.

The results of the experiments showed that daily, for 7 days, intragastric administration of methionine twice a day at a dose of 5 g / kg causes the death of 60% of experimental animals. Starting from the second day of methionine administration, the animals became lethargic, inactive, refused food and water. Starting from the third day, the death of animals with a clinical picture characteristic of acute cerebrovascular accident was observed. At the autopsy of dead animals, a morphological picture of ischemic stroke was found, which is consistent with published data on increased thrombosis and hemorheological disorders under the influence of hyperhomocysteinemia.

Daily, once a day, intragastric administration of methionine at a dose of 5 g / kg for 14 days caused the death of 40% of the animals by 7 days from the start of the experiment. The morphological and clinical picture of animal death corresponded to that in animals of the aforementioned experimental series.

In groups of animals treated with methionine at a dose of 3 g / kg and a 10% solution of TWEEN 80 at a dose of 1 ml / kg, the death of animals was not observed throughout the entire period of administration.

Surviving animals were removed from the experiment in accordance with the above study design.

In the table. 1 presents data on the effect of intragastric administration of methionine on the functional parameters of the cardiovascular system of experimental animals.

It was found that daily administration of methionine according to the indicated schemes does not cause significant changes in blood pressure and heart rate (Table 1).

Processing of the obtained experimental data during functional tests for endothelium-dependent (acetylcholine 40 μg / kg iv) and endothelium-independent (nitroprusside 30 mg / kg iv) vascular relaxation in experimental animals allowed us to establish that intragastric administration of 10% Tween 80 solution in a dose 1 ml / kg does not cause an increase in the coefficient of endothelial dysfunction. Intragastric administration of methionine at a dose of 5 g / kg 2 times a day for 7 days causes an increase in the coefficient of endothelial dysfunction to 3.7 ± 0.5 (Table 1).

The highest value of QED was obtained in the group of animals with intragastric administration of methionine at a dose of 5 g / kg once a day for 14 days. In this group, QED was equal to 3.9 ± 0.2. A significant increase in the coefficient of endothelial dysfunction was also obtained in the group of animals with intragastric administration of methionine at a dose of 3 g / kg for 7 days (QED is 3.3 ± 0.3) (Table 1).

Table 1 The effect of methionine on hemodynamic parameters and the coefficient of endothelial dysfunction in modeling homocysteine-induced endothelial dysfunction (M ± m) Groups of animals Functional test GARDEN, mmHg DBP, mmHg S of the vascular reaction during ESVD with AH and ENVD with NP, conventional units QED, conventional units 10% TWEEN 80 Source 129.2 ± 4.3 82.4 ± 5.9 1 ml / kg OH 74.1 ± 2.9 39.4 ± 3.1 1124.2 ± 63.7 0.9 ± 0.2 The control NP 67.2 ± 5.1 42.9 ± 5.4 1011.8 ± 94.6 Methionine 5 g / kg 2 times / day for 7 days Source 117.2 ± 4.1 78.4 ± 5.6 OH 77.6 ± 6.1 45.4 ± 4.1 794.3 ± 54 * 3.7 ± 0.5 * NP 65.4 ± 7.1 38.3 ± 3.4 2938.1 ± 184 * Methionine 5 g / kg 1 time / day for 14 days Source 121 ± 4,2 79.6 ± 2.4 3.9 ± 0.2 * OH 81.4 ± 4.8 42.4 ± 2.5 658.1 ± 71.2 * NP 56.1 ± 8.6 34.2 ± 8.3 2566.6 ± 181.4 ** Methionine 3 g / kg 1 time / day for 7 days Source 118.9 ± 10.1 76.6 ± 7.2 OH 80.1 ± 2.9 41.4 ± 2.3 854.6 ± 61.4 * 3.3 ± 0.3 * NP 72.3 ± 6.7 45.9 ± 4.3 2820.2 ± 210.4 * Note: * - p <0.05 - in comparison with a group of animals that received 10% TWEEN 80. GARDEN - systolic blood pressure, DBP - diastolic blood pressure, S - area above the curve for restoration of blood pressure during pharmacological tests, QED - coefficient endothelial dysfunction.

The results of determining the concentration of homocysteine in blood serum allowed us to establish a significant increase in homocysteine levels in all groups of experimental animals. The concentration of homocysteine significantly differed from those in the control group of animals (table 2).

The highest increase in homocysteine levels was found in the blood serum of animals treated with methionine at a dose of 5 g / kg for 14 days (Table 2).

table 2 Effect of methionine on homocysteine concentration in modeling homocysteine-induced endothelial dysfunction (M ± m) Groups of animals 10% TWEEN 80
1 ml / kg
The control Methionine 5 g / kg 2 times / day for 7 days Methionine 5 g / kg 1 time / day for 14 days Methionine 3 g / kg 1 time / day for 7 days Homocysteine Concentration (μmol / L) 8.6 ± 2.1 92.3 ± 11.3 * 104.7 ± 8.9 * 53.5 ± 3.2 * Note: * - p <0.05 - in comparison with a group of animals treated with 10% TWE 80.

Thus, modeling of hyperhomocysteine-induced endothelial dysfunction using intragastric administration of methionine at a dose of 3 g / kg once a day for 7 days should be considered an adequate experimental model of hyperhomocysteinemia, which can be used to study the endothelial and cardioprotective effects of various groups of pharmacological drugs this model of pathology

Claims (1)

A method for modeling hyperhomocysteine-induced endothelial dysfunction, characterized by intragastric administration of 3 g / kg amino acids methionine to male Wistar rats once a day for 7 days, evaluating the development of endothelial dysfunction using an endothelial dysfunction coefficient and confirming the development of hyperhomocysteinemia by determining the concentration of homocysteine in homocysteine blood serum.