RU2785847C1 - Method for modeling chronic toxic coagulopathy in rats in experiment - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: invention relates to experimental medicine, namely to toxicology and ecology, and can be used to model chronic toxic coagulopathy in experimental animals. To do this, a solution of mercury chloride at a dose of 0.5 mg/kg in terms of metal is injected through a probe into the stomach of a rat every day for 60 days. At the same time, a unit of solution equal to 0.3 ml contains 0.05 mg of mercury.

EFFECT: method ensures the development of toxic coagulopathy in an animal due to the optimal dose and duration of exposure to the metal, which can be used in the search for means for the prevention and treatment of toxic coagulopathy.

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Description

The invention relates to experimental medicine, namely to toxicology and ecology, and can be used to model chronic toxic coagulopathy in experimental animals.

Mercury is one of the most common heavy metals (Raj D., Maiti S. Sources, Toxicity, and Remediation of Mercury: An Essence Review. Environ. Monit. Assess. 2019; 191 doi: 10.1007/s10661-019-7743-2. ), which have high biological activity in relation to living organisms. The effect of mercury compounds on cells and the human body is currently being actively studied (Avtsin A.P. et al. 1991). It has been shown that long-term intake of mercury compounds into the body can lead to its accumulation in human organs and tissues and have a genotoxic, cytotoxic and organotoxic effect, contributing to multiple organ structural and functional damage.

An analysis of modern literature shows that the processes of intravascular blood coagulation can be a component of many pathological processes in the body: pathologies of the cardiovascular system, hypertension, cerebrovascular accidents, pathologies of the liver and kidneys (Byshevskiĭ A.Sh., Galian S.L. 2006; Momot A. P. et al. 2011; Melnik A. A. 2016). The study of the processes of the hemostasis system and their role in the development of somatic pathology in chronic intoxication is relevant (Vlasov A.P., Grigoryeva T.I., Leshchankina N.Yu., 2009).

Current treatment regimens for chronic intoxication include anticoagulant therapy and long-term, sometimes lifelong, oral anticoagulants (Shafi S.T., et al. 2014; Oliveira M., et al. 2017; Monahan R.C., et al. 2017). However, recent studies have found that this can lead to the development of anticoagulant-associated nephropathy in both patients with chronic kidney disease and patients without kidney disease (Kalaitzidis R.G., DuniA., Liapis G., Balafa O., et al. Anticoagulant -related nephropathy: a case report and review of the literature of an increasingly recognized entity. Int Urol Nephrol. 2017; 49: 1401-7.). At the same time, the mechanisms of development of renal pathology under the influence of anticoagulants are not fully known.

Thus, the problem of correcting the hemostasis system in chronic intoxication remains open. Relevant is the development of methods for modeling toxic coagulopathy, for the subsequent study of methods for the prevention of this pathology.

There are only a few works describing multidirectional and contradictory changes in the hemostasis system under the influence of various mercury compounds. A statistically significant increase in clotting time with increased thrombin formation has been described in workers occupationally exposed to mercury vapor (Nowakowska, B et al. parameters of hemostasis] Medycyna pracy vol. occupationally exposed to mercuric vapors J Trace Elem Exp Med 2002 15 21-29 https://doi.org/10.1002/jtra 1055 Song YG Effects of chronic mercury poisoning on blood coagulation and fibrinolysis systems.// Zhonghua laodong weisheng zhiyebing zazhi (Chinese journal of industrial hygiene and occupational diseases) vol. 2005, 23.6: 405-7.).

In ex vivo experiments, an increase in the prothrombotic activity of erythrocytes was shown due to an increase in the expression of phosphatidylserine on the outer surface of erythrocyte membranes (Maseko R.V, van Rooy M., Taute H. Whole blood ultrastructural alterations by mercury, nickel and manganese alone and in combination: An ex vivo investigation Toxicology and industrial health, 2021, 37(2), 98-111 Notariale, R., Infantino, R., Palazzo, E., & Manna , C. (2021) Erythrocytes as a Model for Heavy Metal-Related Vascular Dysfunction: The Protective Effect of Dietary Components International journal of molecular sciences, 22(12), 6604. https://doi.org/10.3390/ijms22126604 ). The disadvantage is the difficulty of extrapolating the data of ex vivo experiments to the processes occurring in a living organism.

It should be noted that a number of modern authors argue that exposure to high doses of mercury does not affect the blood coagulation system (Nielsen, V.G. Lethal concentrations of mercury or lead do not affect coagulation kinetics in human plasma // J Thromb Thrombolysis 2019, 48, 697- 698. doi.org/10.1007/s11239-019-01921-x.). So in the experiment, no significant difference was noted in the values of the kinetic parameters of coagulation compared with the values obtained for plasma without the addition of mercury.

Such inconsistency and fragmentation of information determined the purpose of the present invention - the creation of a model of chronic toxic coagulopathy.

A known method (Arbi S, Oberholzer HM, Van Rooy MJ, Venter C, Bester MJ. Effects of chronic exposure to mercury and cadmium alone and in combination on the coagulation system of Sprague-Dawley rats // Ultrastruct Pathol. 2017 Jun 15:1 -9. doi: 10.1080/01913123.2017.1327909), which includes the introduction of cadmium and mercury salts dissolved in drinking water into the body of Sprague-Dawley rats at a dose exceeding the MPC by 1000 times for 28 days. It was found that cadmium and mercury salts are able to activate the procoagulant properties of platelets.

The disadvantage of this method is the difficulty in determining the amount of mercury entering the body with drinking water per unit body weight of the animal and the lack of information about the state of the plasma link of the hemostasis system when exposed to heavy metal on the body of an experimental animal.

Closest to the claimed is a method for modeling coagulopathy in rats with the introduction of an organic compound of mercury (Kostka B, Michalska M, Krajewska U, WierzbickiR. Blood coagulation changes in rats poisoned with methylmercuric chloride (MeHg) // Pol J Pharmacol Pharm. 1989 Mar-Apr ; 41 (2): 183-9., PMID: 2594581). Administration of a single dose of methylmercury (17.9 mg Hg/kg) and a repeated dose (5 x 8 mg Hg/kg/day) resulted in hypercoagulability. There was a decrease in clotting time, an increase in the level of fibrinogen in plasma, and changes in thromboelastographic parameters characteristic of hypercoagulation. At the same time, there were signs of impaired platelet activity: a decrease in the rate of aggregation and retraction of the clot, as well as an increase in bleeding time.

The disadvantage of this method is the choice of mercury compounds, dose, and duration of administration. It is known that different mercury compounds have different physicochemical properties, and, accordingly, different toxicity, and different target organs of damaging effects (Eto K, Takizawa Y, Akagi H, et al. Differential diagnosis between organic and inorganic mercury poisoning in human cases-the pathologic point of view Toxicol Pathol 1999;27(6):664-671 doi:10.1177/019262339902700608). Thus, the toxicity of organic compounds of mercury (including methyl mercury), as well as metallic mercury vapor, is due to their high lipophilicity and, consequently, bioavailability and distribution throughout all organs and systems, with a predominant lesion of the nervous system. Inorganic mercury compounds predominantly have a nephrotoxic effect, they can accumulate in the epithelium of the renal tubules and cause damage. Previously, some mercury compounds were used in the clinic as antiparasitic agents, as well as mercury diuretics - promeran, merkusal and novurite. Currently, mercury-containing preparations are prohibited for use due to high toxicity.

The disadvantage of this method is also that the effect of mercury on the body for a short time (5 days) and high doses, close to semi-lethal, does not allow to obtain a method for modeling chronic coagulopathy. The experiment, carried out for two months with the introduction of low dosages, is closer to natural conditions, simulates the intake of mercury into the body with food and water from the environment under conditions of anthropogenic pollution. More informative at different times (two weeks, one and two months) in dynamics, the study of the mechanisms of development of coagulopathy.

The claimed invention is aimed at solving the problem, which consists in developing a method for modeling chronic toxic coagulopathy in rats in the experiment.

The solution of this problem makes it possible to more fully study the dynamics of the pathophysiological mechanisms of the development of coagulopathy in chronic mercury intoxication, to create a method for modeling chronic toxic coagulopathy, which increases reproducibility, is convenient for experimenting on animals and is cost-effective.

To achieve this technical result, the claimed method for modeling chronic toxic coagulopathy in rats in an experiment, including the administration of a mercury compound to experimental animals, differs in that white Wistar rats are injected every day for 60 days with a solution of mercury chloride through an atraumatic probe into the stomach at a dose of 0, 5 mg/kg of animal weight, where per solution unit equal to 0.3 ml, there are 0.05 mg of mercury for metal.

This method differs from the prototype using mercury chloride (an inorganic compound of mercury) as a toxic substance, the duration and dosage of the introduction of the metal.

Between the distinctive features of the claimed invention and the technical result there is the following causal relationship: long-term administration of mercury chloride at a dose of 0.5 mg/kg leads to the development of chronic toxic coagulopathy in experimental animals. This is a convenient and close to natural model.

According to the information available to the authors, the set of essential features that characterize the essence of the claimed invention is not known, which allows us to conclude that the invention meets the criterion of "novelty".

According to the authors, the essence of the claimed invention does not follow for specialists explicitly from the known level of medicine, since it does not reveal the above possibility of obtaining a method for modeling chronic toxic coagulopathy in rats in the experiment. In the scientific and medical literature, we have not identified a description of the use of mercury chloride for modeling chronic toxic coagulopathy in experimental animals, which allows us to conclude that the invention complies with the patentability condition "inventive step".

The set of essential features that characterize the essence of the invention, in principle, can be reused in medicine to obtain the result, which consists in a more accurate and easily reproducible method for the development of chronic toxic coagulopathy, which allows us to conclude that the invention meets the criterion of "industrial applicability".

This method is carried out as follows. To obtain a toxic substance, mercury chloride is dissolved in distilled water in such a way that a solution unit of 0.3 ml contains 0.05 mg of mercury (in terms of metal). For every 100 g of the rat's weight, 0.3 ml of a toxic solution is injected, which is not an excessive water load on the organism of the experimental animal.

A solution of mercury chloride is administered through an atraumatic probe into the stomach at a dose of 0.5 mg/kg, daily 1 time per day for 60 days to a group of animals of 10 rats. The material for the study is whole blood, as well as platelet-rich and platelet-poor blood plasma. Blood sampling, stabilization and obtaining plasma samples are carried out taking into account international standards for clinical laboratory diagnostics for studies in the field of hemostasis (Momot A.P. Pathology of hemostasis. Principles and algorithms of clinical and laboratory diagnostics, St. Petersburg: Format, 2006, 208 p.) .

In blood plasma samples, the following indicators are determined:

• platelet aggregation activity (inducer ADP - 10.0 μg/ml) (Barkagan, Z.S., Momot A.P. Diagnosis and controlled therapy of hemostasis disorders, M.: Nyudiamed-AO, 2008, 292 p.).

• APTT-activated partial thromboplastin time according to Caen et al. (1968);

• PT - prothrombin time according to Quick (1935);

• VPFM - relative polymerization time of fibrin monomers,

• F-gene - the content of fibrinogen in plasma according to Clauss (1961);

• Protein C activity;

• AT(III) - activity of antithrombin III in blood plasma according to V.A. Makarov et al. (2002);

• SEL - time of spontaneous euglobulin lysis;

• RFMK - the amount of soluble fibrin-monomer complexes (Elykomov V.A., Momot A.P. Method for determining the amount of soluble fibrin-monomer complex in blood plasma / Author's certificate 1371219, 1987, USSR);

Coagulological studies were performed using reagent kits from NPO Renam and OOO Tekhnologiya-Standard, Russia, using an AR-2110 turbidimetric aggregometer, a CGL-2110 coagulometer, a PV-1251C spectrophotometer, Solar (Belarus).

When conducting experiments on animals, we were guided by the rules of laboratory practice in the Russian Federation (Order of the Ministry of Health of the Russian Federation dated April 1, 2016 No. 199).

Statistical processing of the obtained results was carried out using the STATISTICA 10.0 software package (StatSoft) and Microsoft Excel 2016.

Data are presented as median (Me) and interquartile range (Q25÷Q75). Significance of differences was assessed using the nonparametric Mann-Whitney U-test. Differences were considered significant at p<0.05.

Example. For one month (10 rats) and two months (10 rats), male Wistar rats were injected with a solution of mercury chloride at a dose of 0.5 mg/kg of animal weight through a tube into the stomach once a day for one month (10 rats) and two months (10 rats). Intact animals (10 rats) kept under standard vivarium conditions served as controls. The state of the hemostasis system was assessed after one and two months. The parameters characterizing the state of the vascular-platelet link of hemostasis (the number of platelets and their aggregation activity), the coagulation link (APTT, PT, VPFM, protein C activity, AT (III) activity, SEL time), the content of fibrinogen and the level of thrombinemia ( RFMK).

A study of the state of the hemostasis system in animals after one month of experiments revealed the development of procoagulant tendencies with simultaneous activation of the anticoagulant and fibrinolytic components of the hemostasis system (Table 1.). In rats, the degree of ADP-induced platelet aggregation and their number increased. The amount of fibrinogen increased. There was an activation of blood coagulation both along the external pathway and along the internal pathway (by shortening the APTT and PT). An increase in the activity of protein C and antithrombin III was detected, and the time of spontaneous euglobulin lysis was shortened. Two months later, an increase in the degree of ADP platelet aggregation was detected, but their number decreased compared to the data after one month and did not differ significantly from the control. The prothrombin time was prolonged. The polymerization time of fibrin monomers was shortened. The concentration of fibrinogen decreased significantly relative to the experience after one month, reaching control values. At the same time, there was a depression of anticoagulant and fibrinolytic mechanisms. The activity of antithrombin decreased, the time of spontaneous euglobulin lysis was prolonged. Thrombinemia developed, the content of soluble fibrin monomer complexes significantly increased (The results obtained are shown in table 1.).

The data of the experiments performed demonstrate the development of a hypercoagulable syndrome, characterized by an increased readiness of the circulating blood for clotting, with an increase in the content of hemostasis activation markers in the blood, suppression of anticoagulant and fibrinolytic activity (A.P. Momot, L.P. Tsyvkina, I.A. Taranenko Modern methods recognition of thrombotic readiness, Barnaul, Alt.un-ta Publishing House, 2011, p.138.), which can lead to disruption of microcirculation processes, an increase in blood viscosity, slowing down of venous blood flow - the development of transient and initial signs of organ dysfunction.

The proposed method for modeling chronic toxic coagulopathy in experimental animals is effective, allows you to study in detail the pathophysiological mechanisms of the formation of toxic coagulopathy during prolonged mercury intoxication, and can contribute to the development and search for tools for the treatment and prevention of blood coagulation disorders.

Claims (1)

A method for modeling chronic toxic coagulopathy in rats in an experiment, including intragastric administration of a mercury compound to experimental animals, characterized in that the animals are injected every day for 60 days with a solution of mercury chloride through a tube into the stomach at a dose of 0.5 mg/kg of animal weight, where unit solution, equal to 0.3 ml, accounts for 0.05 mg of mercury for metal.