Method for simulating sepsis

Abstract

FIELD: gynecology.SUBSTANCE: C57BI / 6 mice were administered 3.1% sodium thioglycolate intraperitoneally at a dose of 2 ml. After two days, alpha-galactosylceramide is administered intraperitoneally at a dose of 1.5 μg / mouse. And a day later, E. coli lipopolysaccharide is administered intraperitoneally at a dose of 2 to 32 μg / mouse.EFFECT: increasing the lifespan of mice when modeling sepsis.1 cl, 2 dwg, 1 tbl, 2 ex

Description

[0001] The invention relates to the field of pharmacology and medicine, namely to a method for experimental modeling of sepsis in laboratory animals.

[0002] Sepsis is a pathological process based on the body's response in the form of generalized inflammation to an infection of various origins, leading to organ dysfunction. Septic shock is the most severe variant of sepsis, characterized by disorders that cause an increased risk of death. Sepsis (in adults). Clinical guidelines of the Ministry of Health of the Russian Federation. ID: 898_1. 2024. The duration of sepsis in humans can vary from 1-3 days (fulminant sepsis), up to 4 weeks (acute sepsis) and up to 3-4 months (subacute sepsis). According to research in Russia, the proportion of patients with infection in intensive care units was 34.1% of all hospitalized; septic shock among them developed in 20.2% of cases, and the mortality rate among people with various forms of infection was 30.4%. Thus, sepsis is an unsolved medical problem, and there is a need to create new effective technologies for the treatment of sepsis.

[0003] Modeling human diseases in laboratory animals is a necessary step in the development of medical technologies preceding clinical trials in humans. Since sepsis is of infectious origin, the main approach to modeling sepsis is the administration of infectious agents to animals, for example, Staphylococcus aureus (RU2507601C1), or a bacterial inoculum consisting of a known number of bacteria mixed with adjuvant or feces. Weinstein WM et al. Infection and Immunity. 1974. 10(6):1250-1255. Volk HW et al. European Surgical Research. 990. 22(6):347-355. Onderdonk AB et al. Infect Immun. 1976. 13(1):22-6. Polymicrobial models of sepsis include the intraperitoneal fecal pellet model, the cecal puncture and ligation model, and the ascending colon peritonitis model. Ayala A, Chaudry IH Shock. 1996:27-38. Song M et al. Crit. Care Med. 2005. 33:463-465. Neumann B et al. Int. Immunol. 1999.11:217-227. Maier S et al. Shock. 2000. 14:187-192. Swathi M et al. Immunology Letters. 2019. 10:1016.

[0004] Experimental models of "sterile" sepsis are known that exclude the use of infectious agents, which allows research to be conducted in laboratories where it is impossible to use infectious agents. These models are based on the introduction of bacterial fragments, in particular E. coli lipopolysaccharide (LPS), which is capable of inducing generalized inflammation in the absence of an infectious agent. Remick DG, Ward PA. Shock 2005, 24(Suppl 1):7-11. The use of LPS as the sole inducer of inflammation is insufficient for modeling sepsis, since, unlike sepsis observed in humans, the introduction of LPS alone is characterized by rapid resolution of inflammation and low mortality, although it is accompanied by a short-term increase in proinflammatory cytokines. Rittirsch D et al. J Leukoc Biol 2007, 81:137-143.

[0005] A non-infectious sepsis model in C57Bl / 6 mice is known, caused by intravenous administration of alpha-galactosylceramide (αGalCer), an agonist of invariant T-killer receptors, followed by intravenous administration of E. coli lipopolysaccharide (LPS), where the role of αGalCer is to sensitize the immune system to the subsequent administration of LPS, an agonist of Toll-like receptors 4 (TLR4). Ito H et al. Lethal endotoxic shock using alpha-galactosylceramide sensitization as a new experimental model of septic shock. Ito H, Lab Invest. 2006, 86(3):254-61. This model is characterized by severe sepsis (septic shock) with high mortality, reaching 100% within the first 12 hours after intravenous administration of LPS at a dose of ≥1 μg / mouse.A critical drawback of this model is the discrepancy between the duration of sepsis in this model and that observed in humans. Specifically, the period to 100% mortality (12 hours) is too short, which is shorter than that observed in fulminant sepsis (1-3 days). Therefore, there is a need to develop non-infectious sepsis models with a duration longer than 12 hours, at least approaching the level of fulminant sepsis (1-3 days) with a high mortality rate, for testing candidate sepsis treatments. The sepsis model of Ito et al. was used as a prototype for the sepsis modeling method of the present invention.We have found that modification of the above-mentioned prototype model by replacing the intravenous administration of alpha-galactosylceramide and lipopolysaccharide with intraperitoneal administration, as well as the use of an additional agent - sodium thioglycolate, allows for a significant increase in the lifespan of animals, with mortality parameters observed in sepsis in humans.

[0006] The present invention relates to a method for modeling sepsis in C57Bl / 6 mice, comprising the following steps: (a) intraperitoneal administration of 2 ml of 3.1% sodium thioglycolate; (b) subsequent two days later intraperitoneal administration of 1.5 μg / mouse alpha-galactosylceramide; and (c) subsequent 24 hours later intraperitoneal administration of E. coli lipopolysaccharide at a dose of 2 to 32 μg / mouse.

[0007] The technical result of the present invention is to increase the lifespan of C57Bl / 6 mice when modeling sepsis using the method of the present invention compared to the above-mentioned prototype method.

[0008] Sodium thioglycolate has the formula C2H5NaO2S, CAS number 367-51-1.

[0009] Alpha-Galactosylceramide has the IUPAC name N-[(2S,3S,4R)-1-(α-D-Galactopyranosiloxy)-3,4-dihydroxyoctadecan-2-yl]hexacosanamide, molecular formula C 50 H 99 NO9, CAS number 158021-47-7.

[0010] E. coli lipopolysaccharide, also known as endotoxin, is a component of the outer membrane of the cell wall of the gram-negative bacterium E. coli.

[0011] The following examples demonstrate the invention. The examples illustrate the invention and are not intended to limit the scope of the invention in any way.

[0012] Example 1

[0013] The example illustrates the method of the present invention.

[0014] The method for modeling sepsis in C57Bl / 6 mice includes the following steps:

[0015] (a) Stage of intraperitoneal administration of 2 ml of 3.1% sodium thioglycolate. At this stage, male C57Bl / 6 mice (Stolbovaya Branch of the Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency of Russia, Moscow Region), weighing 20-22 g, were administered 2 ml of an aqueous solution of 3.1% sodium thioglycolate intraperitoneally on the first day.

[0016] (b) Stage of subsequent intraperitoneal administration of 1.5 μg / mouse of alpha-galactosylceramide two days later. At this stage, mice were intraperitoneally administered 1.5 μg / mouse of α-GalCer two days after the administration of sodium thioglycolate.

[0017] (c) Stage of subsequent intraperitoneal administration of lipopolysaccharide at a dose of 2 to 32 μg / mouse 24 hours later. At this stage, mice that had already been administered sodium thioglycolate and αGalCer in the previous stages were intraperitoneally administered E. coli lipopolysaccharide (LPS) at doses of 2 to 32 μg / mouse 24 hours after the administration of αGalCer. The survival of animals after the administration of LPS at doses of 0, 2, 8, 16, and 32 μg / mouse was observed in groups for 48 hours, with each group containing five animals.

[0018] The results are presented in Table 1 as animal survival rates in groups. Differences in survival between groups were assessed using the Mantel-Cox log-rank test.

[0019] Table 1.

[0020] LPS, μg / mouse Mortality after LPS administration 12 hours 24 hours 36 hours 48 hours Total for 48 hours % 0 (control) 0 0 0 0 0 / 5 0 2 0 0 0 1 1 / 5 20 8 0 3 1 0 4 / 5 80* 16 0 2 3 0 5 / 5 100** 32 0 3 2 0 5 / 5 100**

[0021] *Different from control (p<0.05; log-rank test)

[0022] Thus, the sepsis modeling method of the present invention makes it possible to model sepsis in C57Bl / 6 mice with a mortality rate of up to 100% over an observation period of 48 hours.

[0023] Preliminary intraperitoneal administration of sodium thioglycolate in the method of the present invention is necessary to increase the lethality of sepsis, as illustrated by the following experiment. Sepsis was modeled in male C57BL / 6 mice in two ways. In the first group (n=5), sepsis was modeled according to the method of the present invention, using sequential intraperitoneal administration of 2 ml of a 3.1% aqueous solution of sodium thioglycolate, two days later, intraperitoneal administration of 1.5 μg / mouse αGalCer, and one day later, intraperitoneal administration of LPS at a dose of 16 μg / mouse. In the second group, the animals received 1.5 μg / mouse αGalCer intraperitoneally and one day later, 16 μg / mouse LPS intraperitoneally, but did not receive sodium thioglycolate (-TG). Kaplan-Meier survival curves for both groups are shown in Figure 1.Comparison of survival curves using the Mantel-Cox log-rank test revealed the presence of a statistically significant difference between the groups (p=0.0031), with the median mortality of animals in which sepsis was modeled using the method of the present invention (+TG) being 36 hours, but was not achieved in the group of animals with sepsis obtained without the use of sodium thioglycolate (-TG) within 48 hours of observation.

[0024] Example 2

[0025] The example illustrates a comparison of the method of the present invention with the prototype method.

[0026] Sepsis was modeled in male C57BL / 6 mice using two methods. In the first group (n=5), sepsis was modeled according to the method of the present invention, as described in Example 1, using sequential intraperitoneal administration of sodium thioglycolate, followed by intraperitoneal administration of 1.5 μg / mouse αGalCer two days later, and intraperitoneal administration of 16 μg / mouse LPS the following day. In the second group (prototype method), the animals received 1.5 μg / mouse αGalCer intravenously and 16 μg / mouse LPS intravenously the following day. The Kaplan-Meier survival curves for both groups are shown in Figure 2. Comparison of the survival curves using the Mantel-Cox log-rank test revealed a statistically significant difference between the groups (p=0.0027), with the median survival of animals in which sepsis was modeled using the method of the present invention being 36 hours, versus 12 hours in the group of animals with sepsis obtained using the prototype method.

[0027] Thus, modeling sepsis using the method of the present invention makes it possible to achieve a technical result and provides a statistically significant increase in the lifespan of C57Bl / 6 mice compared to the prototype method.

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