RU2669024C2 - Method of modeling experimental liver cirrhosis - Google Patents

Abstract

FIELD: experimental medicine.SUBSTANCE: invention relates to experimental medicine, namely to experimental toxicology and hepatology, and concerns the modeling of liver cirrhosis. To do this, intragastric administration to laboratory animals for 3 weeks, every other day, 40 % ethanol at a dose of 3 g/kg is performed. At the same time, intraperitoneal administration of a 1 % solution of N-nitrosodimethylamine at a dose of 5 mg/kg for 4 consecutive days of each week is performed.EFFECT: method makes it possible to increase the reproducibility, shorten the period of modeling cirrhosis of the liver and bring the model closer to the clinical course of this pathology in humans.1 cl, 3 tbl

Description

The invention relates to medicine, is aimed at modeling chronic toxic liver damage in the form of fibrosis and cirrhosis and can be used to study toxic liver damage in a preclinical study of hepatoprotective drugs and in the development of new methods of treating liver cirrhosis. Currently, in economically developed countries, the incidence of liver cirrhosis is about 20-40 patients per 100 thousand people, and this indicator is growing steadily, about 350 thousand people per year die from this disease. More often, cirrhosis develops with prolonged intoxication with alcohol (according to various sources, from 40 to 80% of cases). Existing means and regimens for cirrhosis are not always effective and are characterized by a large number of contraindications and side effects. One of the possible reasons for this state of affairs is the lack of adequate experimental models of liver cirrhosis. The role of alcohol as an etiological factor in the development of cirrhosis is generally recognized.

A known method of initiating toxic liver damage by oral administration of 40% ethyl alcohol at a dose of 14 ml / kg for 60 days [Dorkina EG Hepatoprotective properties of flavonoids: author. dis. ... Dr. Biol. sciences / E.G. Dorkin. - Volgograd, 2010. - 45 p.].

The disadvantage of this method is the duration of the introduction of 40% ethanol - 60 days or more, and also the fact that as a result of such exposure usually only toxic hepatitis and / or fatty hepatosis develops, but not fibrosis and cirrhosis. In this regard, when modeling alcohol-induced experimental liver cirrhosis, according to the literature, there is a need to enhance the hepatotoxic damaging effect of ethanol with other hepatotoxicants and vice versa [Popper N. Histogenesis of alcoholic fibrosis and cirrhosis in the baboon / H. Popper, C.S. Lieber // Am. J. Pathol. - 1980. - Vol. 98, No. 3. - P. 695-716].

There is a method for creating a model of toxic hepatitis and cirrhosis of mammalian liver, according to which, as a hepatotoxicant, a 50% solution of Sovtol-1 in olive oil is administered intragastrically at the rate of 0.25 ml per 100 g of body weight and the animals are given daily for water for 1 month for drinking 10% ethanol solution (Patent for invention RU No. 2197018, 2003]. The use of ethanol in this model allows the development of chronic toxic hepatitis, and Sovtol is intended for rapid toxic damage to the liver. With a combination of two components their toxic effects are summarized, which ultimately leads to the development of both toxic hepatitis and cirrhosis 4-6 weeks after the start of the introduction of toxicants.

The disadvantage of this method of modeling liver cirrhosis is that the replacement of the drinking water regimen with alcohol is not physiological, and therefore, according to the literature, about 30% of animals refuse to drink alcohol [Patent for the invention RU No. 2373927, 2007; Filatova E.V. The influence of social conditions on the formation of ethanol preferences in rats / E.V. Filatova [et al.] // Reports of the Academy of Sciences. - 2010. - T. 430, No. 4. - S. 35-37].

There is a method in which an aqueous solution of dimethylnitrosamine is administered intragastrally at a rate of 50-100 mg / kg of animal weight 1 time per week for 2-3 months, while the liver is usually examined 2 weeks after the last injection. Another option of the same method is the introduction of an aqueous solution of dimethylnitrosamine intraperitoneally for 2-3 months every week for three days in a row, followed by a 4-day break [I. Harutyunyan. Modeling of liver cirrhosis in laboratory animals / I.V. Harutyunyan [et al.] // Clinical and experimental morphology. - 2012. - No. 2. - S. 45-50].

The disadvantage of this method is the duration of the experiment.

A known method of modeling liver damage in the experiment by a single administration to rats of N-nitrosodimethylamine intraperitoneally at doses of 7 and 15 mg / kg [Tomilin N.V. Experimental evaluation of the genotoxic effect of N-nitrosodimethylamine with a single administration in toxic doses to white rats / N.V. Tomilin [et al.] // Actual problems of diagnosis, prevention and treatment of occupationally caused diseases: Proceedings of the All-Russian Scientific and Practical Conference, Sochi, October 14-15, 2013. - Sochi, 2013. - S. 423-425].

However, under the action of such doses of N-nitrosodimethylamine with a single injection, liver fibrosis and cirrhosis do not develop, and the effect of damage to this organ is recorded only by cytogenetic indicators.

With the introduction of dimethylnitrosamine in doses of 5.0-7.5 mg / kg for 2-3 weeks, all rodents develop chronic toxic hepatitis, and a significant proportion (50-70%) also have fibrosis, but liver cirrhosis does not occur [ Constandinou C. Modeling liver fibrosis in rodents / C. Constandinou, N. Henderson, JP Iredale // Methods Mol. Med. - 2005. - Vol. 117. - P. 237-250].

A known method of modeling liver cirrhosis, which is the introduction into the stomach of a laboratory animal twice a week for a month, a 50% solution of Sovtol-1 in olive oil at the rate of 0.25 ml per 100 g of body weight and 10% ethanol solution instead of drinking water [Patent for the invention RU No. 2197018, IPC G09B 23/28, 01/20/2003].

The disadvantage of this method is the low degree of accuracy of reproduction of liver damage in the experiment.

The technical result of the developed invention is to increase the reproducibility and approximation of the model of experimental cirrhosis to the clinical course of this human liver pathology.

The technical result is achieved by the fact that in order to obtain a model of liver cirrhosis in an experiment, dosed ethanol is administered to rats, moreover, ethanol is administered intragastrically for 3 weeks every other day at a dose of 3 g / kg and at the same time intraperitoneal administration of a 1% solution of dimethylnitrosamine (DMNA) is carried out in dose of 5 mg / kg.

Table 1 presents the dynamics of the weight of the animals during the experiment.

Table 2 presents the morphological parameters in animals when modeling experimental toxic liver cirrhosis.

Table 3 presents the biochemical parameters in animals when modeling experimental toxic liver cirrhosis.

The method is as follows.

To obtain a model of cirrhosis of the liver, ethanol was administered intragastrically to experimental animals for 3 weeks every other day at a dose of 3 g / kg and intraperitoneal administration of 1% dimethylnitrosamine (DMNA) at a dose of 5 mg / kg was simultaneously administered.

To prove the conformity of the claimed technical solution to the criteria of the invention, an experiment was conducted on 80 white outbred male rats weighing 180-220 g. Animals were divided into four groups:

- 1st group (n = 20) - 40% ethanol was administered intragastrically at a dose of 3 g / kg for 3 weeks after 1 day;

- 2nd group (n = 20) - 1% solution of N-nitrosodimethylamine (DMNA) was administered intraperitoneally at a dose of 5 mg / kg for 4 consecutive days of each week for 3 weeks;

- 3rd group (n = 20) - 40% ethanol was administered intragastrically at a dose of 3 g / kg for 3 weeks after 1 day and a 1% solution of N-nitrosodimethylamine (DMNA) was administered intraperitoneally at a dose of 5 mg / kg for 4 consecutive days of each week;

- 4th group (n = 20) - control group; animals were injected intragastrically with 0.5 ml of physiological saline after 1 day and intraperitoneally with 0.5 ml of physiological saline for 4 consecutive days of each week for 3 weeks.

Observation of laboratory animals was carried out for 2 weeks before the experiment (quarantine), during the 3-week intragastric and intraperitoneal administration of hepatotoxicants, as well as within 3 weeks after the end of their administration. Assessment of clinical, laboratory and morphological parameters in laboratory animals of the experimental and control groups was carried out before the experiment, 3 and 6 weeks after the start of hepatotoxicant administration (the second study was performed to clarify the possibility of resuming spontaneous fibrosis after the hepatotoxicants were withdrawn).

Animals were kept under standard vivarium conditions on a normal diet, with free access to water. The body weight of animals of the 1-3rd group before the experiment was (147.9 ± 5.2) g, of the 4th (control) group - (148.0 ± 6.3) g. Initial clinical and laboratory parameters in animals experimental and control groups also did not differ.

The presence and severity of toxic liver damage was assessed by visual observation of the clinical picture of intoxication, the study of biochemical parameters of peripheral blood and morphological studies of liver preparations.

As biochemical indicators characterizing the basic functions of the liver, we studied: total protein, creatinine, urea, glucose, bilirubin, potassium, sodium, chlorine, calcium, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase and an indicator of cholestasis - alkaline phosphatase. Evaluation of biochemical parameters was carried out using a Roche Omni C analyzer.

To confirm the development of experimental cirrhosis of the liver 1, 2, and 3 weeks after the start of the experiment, animals were euthanized (5 animals from each group) and liver samples were taken, followed by histological examination using standard methods. Liver sections were evaluated using Polyvar and Leica DMRE research microscopes, a digital video surveillance system and the Video Test-4 image analysis program. A morphological assessment of the degree of liver damage was carried out according to the results of measuring the area occupied by connective tissue, within 5-7 hepatic lobules on 6 sections of the liver, stained according to van Gieson [Guide for conducting preclinical studies of drugs / ed. A.N. Mironova. - M .: Grif and K., 2012. - 944 p.]. Using computer processing of the scanned image of a liver section, the relative area occupied in the liver tissue by the formed collagen was calculated.

The significance of intergroup differences in the average values of parametric indicators was evaluated using Student's t-test. In the tables, the data are presented in the form of arithmetic mean and error of the mean (M ± m).

The experiments showed (Table 1) that by the end of the 3rd week, the combined effect of ethanol and DMNA was accompanied by a significant increase (more than 30 g compared to background values) of body weight in animals of the 3rd group.

At autopsy 3 weeks after the start of the experiment, 50% of animals of the 3rd group showed the presence of developed ascites with a large amount (about 6-8 ml) of serous fluid in the abdominal cavity (Table 2). The weight coefficient of the liver in rats of the 3rd group was statistically significantly lower than in rats of the 4th (control) group (p <0.05), and the humidity of the liver in animals of the 3rd group was reduced.

The increase in reproducibility in the formation of liver fibrosis and cirrhosis is achieved due to the potentiation of the hepatotoxic effect of ethanol by N-nitrosodimethylamine and vice versa. In addition, modeling of toxic fibrosis and cirrhosis of the liver is carried out in a shorter time, and the developing clinical, laboratory and morphological picture of the lesion is largely close to the clinical course of this liver pathology in humans.

Using our model, the formation of a clinical, laboratory and morphological picture of liver cirrhosis with the development of severe ascites (up to 6-8 ml of fluid in the abdominal cavity) in 50% of laboratory animals begins 3 weeks after the start of the combined administration of two hepatotoxicants - N-nitrosodimethylamine and ethanol . In the remaining 50% of laboratory animals, fibrosis and cirrhosis of the liver are also formed at the same time, but up to 1-2 ml of fluid is sweated in the abdominal cavity. Thus, the proposed model allows us to reduce the time required for modeling liver cirrhosis compared to the standard experiment duration by an average of two times (from 6 weeks to 3 weeks), and also to increase the accuracy of reproducing the clinical, laboratory and morphological picture of toxic fibrosis and liver cirrhosis.

The combined effect of hepatotoxicants promotes the formation of experimental liver cirrhosis in rats already 3 weeks after the start of injection, which is manifested in an increase in the amount of connective tissue in the liver, its cirrhotic degeneration and the development of ascites. At the same time, in the first week after the beginning of the combined effect of ethanol and N-nitrosodimethylamine, central hemorrhagic necrosis is observed in the liver tissue, then septa and fibrous connective tissue are formed (2nd week), and after 3 weeks fine-cirrhosis. Thus, the model we developed allows us to trace the consistent development of all morphological signs of damage up to the development of cirrhosis.

Claims (1)

A method for simulating experimental cirrhosis of the liver, including dosed ethanol administration to rats, characterized in that ethanol is administered intragastrically for 3 weeks every other day at a dose of 3 g / kg, at the same time intraperitoneal administration of 1% solution is carried out for 4 consecutive days of each week dimethylnitrosamine (DMNA) at a dose of 5 mg / kg.