RU2525704C1 - Method of experimental modelling of migration of bacteria into brain at background of burn trauma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: process modelling is carried out on rabbits with the weight of 2000-2500 grams, which are after the quarantine term kept for 8 hours at a temperature of surrounding air of 34-36°C. After that, the air temperature is reduced to 24-26°C and after 24 hours a thermal burn of an area of 10-20% of the body surface is applied under narcosis. Within an hour after the burn additional conditions for respiration with the application of a gas mixture, containing 80% of oxygen and 20% of nitrogen, are created for the rabbits. After 24 hours 1 ml of each non-culturable bacteria Pseudomonas aeruginosa and Staphylococcus aureusare are introduced to the animals subcutaneously in concentrations of 104-105 microbial cells in 1 ml. After 12 hours the temperature of surrounding air is increased again to 34-36°C within 8 hours. After that signs of the brain affection and lethality of the animals are determined for 5 days.

EFFECT: method makes it possible to create conditions of the maximal approximation of the modelled process to the natural conditions of development and course of the burn disease.

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Description

The invention relates to the field of experimental medicine and can be used to develop effective measures aimed at preventing the death of an infectious process against a background of burn injury, as well as for treating a burn disease using effective pathogenetic and etiotropic treatment methods.

According to the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), mortality from thermal injuries in children in the world takes third place among all traumatic factors after road accidents and drowning. More than 50,000 people die from burns every year [L. Budkevich. Modern methods of surgical treatment of children with severe thermal injury: Abstract. dis doct. medical sciences. - M., 1998. - 33 p .; World Report on Child Injury Prevention. - World Health Organization and UNICEF. - Geneva, 2008. - 39 p.]. In the works of S.P.Sakharov et al. [Sakharov S.P., Ivanov V.V., Shen N.P., Suchkov D.V. Lethal outcomes of a burn disease in children: 18 years of experience // Russian Scientific and Practical Journal "Ambulance". - 2011. - No. 3. - S.52-57.] Found that the frequency of deaths from burns with an area of more than 40% is 56.6%.

The main cause of mortality is associated with the infectious process in the body of burn patients. In the periodic literature there are reports of brain damage during a burn disease. S.E. Khrulev found the presence of brain damage in 33.7% of disabled people after suffering a burn injury [Khrulev S.E. Burn injury with cerebral complications in adults and children (clinic, developmental mechanisms, prevention). Abstract. dis doct. medical sciences. - Nizhny Novgorod, 2009. - 30 p.]. According to the results of the study of neuron-specific enolase, 2 peaks of possible cerebral complications were revealed (1-5 and 16-18 days). In many cases, infection of a burn wound in patients determines the severity of the course of the burn disease. Therefore, the study of the mechanism of development of the infectious and pathological process in case of a fatal burn disease is relevant in order to develop effective pathophysiological treatment methods and preventive measures aimed at preventing burn wound infection.

In the periodic and patent literature does not describe methods for experimental modeling of the infectious process with a fatal outcome in burn injury. V.A. Shatrov et al. [Patent RU No. 2286154 of 10.27.2006] proposed a method for the treatment of inflammatory diseases of the brain with autoleukocytes obtained by plasma leukocytapheresis using helium-neon laser irradiation.

A method for predicting purulent meningitis in burned patients is described [Patent RU No. 2321861 of 04/10/2008] by increasing the concentration of glucose in the blood in the first 3 days of illness and increasing the level of ESR. An increase in glucose concentration in the first 3 days of illness may be due to the multiplication of bacteria that form capsules or biofilms. With the enzymatic destruction of the surface polysaccharides of microbial cells (capsules and biofilms), it is obvious that the authors observed an increase in the concentration of glucose in the blood associated with the multiplication of bacteria. After 3 days, as a rule, bacteria cannot be detected in the blood as a result of the bactericidal action of blood serums.

The penetration of bacteria into the brain most often occurs with an increase in the permeability of the blood-brain barrier (BBB). An increase in the BBB permeability was registered in animals with hypothermia [Patent RU No. 2012219 of 05.15.1994]. I.A. Medyak et al. [Patent RU No. 2391107 of 06/10/2010] proposed the use of ozone to increase the permeability of the BBB. The penetration of bacteria into the nervous system was noted against the background of immunosuppression [http://www.mielin.ru/ru/ healing_ru / diseases / neuroinfection /]. It was also established that the encapsulated bacteria have components on their surface that allow them to pass through the BBB [http://medbiol.ru/medbiol/ infect_har / 00122bd3.htm]. Bacterial capsules consist of polysaccharides, during the enzymatic destruction of which glucose is formed, which is used by brain cells as a nutrient substrate. The penetration of glucose into the brain as a result of facilitated diffusion is known.

The closest technical solution is the material published in the patent of A.G. Novartis [Patent RU No. 2478385 of 04/10/2013] and we have chosen as the closest analogue of the invention (ie, "prototype"). A method for the treatment of cystic fibrosis is proposed. Therefore, the proposed method is not a means of the same purpose of the present invention, namely, a method of experimental modeling of the migration of bacteria into the brain against a burn injury. In the closest analogue of the invention, the causative agent of the disease is also Pseudomonas aeruginosa. In the United States, the incidence of cystic fibrosis is 1 per 1000 population. In Victoria, Australia has an incidence of 1 in 3,600 in Australia. In northern Italy, the incidence of cystic fibrosis is 1 in 4,300. The most common disease is found among Europeans and Ashkenazi Jews. Patent author David Reid proposed drugs to interrupt biofilm formation with P. aeruginosa bacteria and, as a result, microbes did not enter the brain. Prevention of biofilm formation by P. aeruginosa bacteria allowed the author of the patent to carry out etiotropic treatment of this infectious disease. Thus, it was found that P. aeruginosa bacteria, often detected on the surface of burn wounds with the possibility of biofilm formation, can penetrate through the BBB into the brain and, as a result, a lethal outcome of the disease is possible.

The objective of the present invention is to develop a method for experimental modeling of the migration of bacteria into the brain against a burn injury in order to develop effective measures aimed at preventing intracerebral lesions in burn injuries.

The technical result of the proposed method is based on the maximum approximation of experimental modeling of the migration of bacteria into the brain to the natural conditions of the development and course of burn disease. As a laboratory model for the implementation of the proposed method, rabbits were selected, in the body of which there is a sufficient amount of an enzyme that destroys insoluble polysaccharides, the main surface chemical compounds of capsules and biofilms of bacteria.

To reproduce the experimental simulation of the penetration of bacteria into the brain, the influence of additional factors affecting the penetration of bacteria into the brain was taken into account: a decrease in the body's immune status as a result of a burn disease, an increase in BBB permeability for bacteria with polysaccharide structures on its surface, and the ability of sugars to penetrate through BBB as a result of facilitated diffusion.

In the human body, there are enzymes that destroy polysaccharides (surface structures of microbial cells), as a result of this, microbial cells freed from biofilms can penetrate the brain. Microbial polysaccharides are a source of glucose and therefore, in the brain, obviously, against the background of a burn injury, the anaerobic method of generating energy (anaerobic glycolysis) predominates. Hypoxia occurs in the brain, which is the main cause of brain damage in burn injuries. With hypoxia, death of neurons is noted [S. Wolf et all. Die Blut-Hirn-Schranke: ′ Eine Besonderheit des cerebralen Mikrozirkulationssystems // Naturwissenschaften. - 1996.- No. 83.- S.302-311; S. Ohtsuki. New Aspects of the Blood-Brain Barrier Transporters; Its Physiological Roles in the Central Nervous System // Biological Pharmaceutical Bulletin. - 2004. - No. 27 (10). - p. 1489-1496].

In burn patients, hemorrhagic and ischemic stroke, meningitis and encephalopathy occur [Khrulev S.E. Burn injury with cerebral complications in adults and children (clinic, developmental mechanisms, prevention). Abstract. dis. Doct. honey. sciences. - Nizhny Novgorod, 2009. - 30 p.]. An increased concentration of glucose in the brain causes swelling of the brain tissue.

As additional factors affecting the increase in BBB permeability, an increase in the ambient temperature, usually recorded in the summer season, was used, and they also provided an increased concentration of oxygen in the air inhaled by animals.

It should also be noted that in the complex treatment of burn patients, the leading place is taken by the method of oxygen therapy.

It is known that oxygen consumption by the central nervous system is 20% of the total amount of oxygen consumed by the body. Compared to other organs, the brain has the smallest supply of nutrients and therefore nerve cells cannot provide their energy needs through anaerobic glycolysis alone. There is an aerobic and anaerobic mechanism for generating energy in the brain [http://ru.wikipedia.org/wiki/]. In the aerobic type of respiration, CO ₂ (carbon dioxide) is released as a result of redox reactions. The periodic literature describes a method for increasing the BBB permeability in laboratory animals by inhaling carbon dioxide [http://nt.ru/nj/nz/1986/1101.htm]. It should be noted that bacteria of the genus Pseudomonas do not have the phosphofructokinase enzyme and therefore, enzymatic cleavage of glucose (tricarboxylic acid cycle) produces CO ₂ (carbon dioxide) [Medical Microbiology, Virology and Immunology: Textbook / Ed. A.A. Vorobyov. - M .: Medical informative agency, 2004. - 691 p., Pp. 57-58.]. Therefore, in our method of experimental modeling of the migration of bacteria into the brain, carbon dioxide served as an additional factor reducing the BBB permeability.

The proposed experimental modeling method reflects the main mechanisms of the penetration of microbial populations into the brain and traces the main pathophysiological changes in the body when bacteria enter the brain, which will allow developing the main measures aimed at reducing pathological processes in the brain in burn patients.

The technical result is achieved by the fact that according to the invention, healthy rabbits weighing 2000-2500 grams after quarantine are kept for 8 hours at an air temperature of 34-36 ° C, then the air temperature is reduced to 24-26 ° C and after 24 hours a thermal anesthesia is applied the burn, with an area of 10-20% of the body surface area, creates additional breathing conditions for rabbits within an hour after the burn, using a gas mixture containing 80% oxygen and 20% nitrogen, then after 24 hours 1 ml of non-cultured Pseudomonas bacteria is injected subcutaneously into the animals aer uginosa and Staphylococcus aureus at concentrations of 10 ⁴ -10 ⁵ microbial cells in 1 ml and after 12 hours they again increase the air temperature to 34-36 ° C for 8 hours, and then signs of brain damage are determined within 5 days and the death periods are recorded animals.

The essence of the method is achieved by the fact that for the development of pathophysiological processes in the brain, a certain sequence of exposure to various factors affecting brain damage in laboratory animals is used.

The use of oxygen in the process of breathing rabbits with the formation of the final product of CO ₂ had an effect on the decrease in BBB permeability. The formation of carbon dioxide also occurred during the reproduction of P. aeruginosa bacteria. An increase in the ambient temperature also contributed to an increase in BBB permeability, and a burn injury influenced a decrease in the immunobiological reactivity of the animal organism.

As a laboratory model, a laboratory animal was chosen, in the body of which there is a sufficient amount of an enzyme that destroys capsules and biofilms of bacteria. For infection of animals, a culture of P. aeruginosa was selected, which has the ability to form a biofilm and, when bacteriological examination of patients, was often plated from the surface of burn wounds, and the penetration into the brain of bacteria having polysaccharide chemical structures on their surface occurred by the type of nutrients entering the brain.

Bacterial biofilms consist of insoluble polysaccharides that can pass through the BBB as a result of facilitated diffusion by special transport systems. Thus, the penetration of glucose into the brain is provided by the GLUT-1 transporter [Agus D.B. et all. Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters // J. Clin Invest. - 1997. - No. 100. - p. 2842-2848.]. In the brain, bacteria, freeing themselves from biofilms, cause an infectious process. In the closest analogue of the invention (ie, “prototype”), the above described mechanism of penetration of P. aeruginosa into the brain is noted, however, the method described in the “prototype” is intended to prevent the possibility of penetration of P. aeruginosa into the brain in order to ensure effective treatment of patients with cystic fibrosis. Our proposed method for experimental modeling of bacterial migration into the brain is intended to identify the mechanisms of penetration of P. aeruginosa into the brain and to develop effective methods of treating burn disease.

Before the start of the planned studies, an increase in air temperature to 34-36 ° C for 8 hours was used to identify the effect of premorbid background in rabbits on the experimental results. With an increase in the body temperature of rabbits above physiological norms, rabbits were not taken into the experiment.

The proposed method is as follows:

Healthy rabbits weighing 2000-2500 grams after quarantine are placed in small experimental cages (Application No. 2013119320 of 01/18/13 “Device for conducting experiments on rabbits), equipped with a pipe for supplying a mixture of oxygen and nitrogen. The duration of adaptation of animals to new cells was 3 days, then for 8 hours the cells kept the ambient temperature at 34-36 ° C to detect premorbid background, then the air temperature was reduced to 24-26 ° C and after 24 hours a thermal burn was applied under anesthesia an area of 10-20% of the body surface area.

Anesthesia was carried out according to the following method: premedication was carried out with a 2% solution of xylazine hydrochloride (rometar) at a dose of 5.0 mg / kg, and against its background, a 5% solution of zoletin at a dose of 7.5 mg / kg of rabbit body weight was injected intramuscularly respectively. Hair was removed from the back and side of the rabbit's body. Then the surface cleaned of wool was immersed in a water bath for 10 s at a temperature of 90 ° C. A sterile aseptic gauze dressing with levomekol ointment was applied to the burn surface.

Within an hour after the burn, the rabbits were provided with additional breathing conditions using a gas mixture containing 80% oxygen and 20% nitrogen. An oxygen mixture was supplied from the PRANA KISLOROD K161 oxygen canister to the tube of the experimental cell; then, after 24 hours, animals were injected subcutaneously with 1 ml of non-cultured bacteria P. aeruginosa and S. aureus at concentrations of 10 ⁴ -10 ⁵ microbial cells in 1 ml and after 12 hours again increased ambient temperature to 34-36 ° C for 8 hours, and then signs of brain damage and the timing of death of animals were determined within 5 days.

Examples of using the proposed experimental model

Example 1

The rabbit was kept in a cage in accordance with the requirements of sanitary rules (Approved by the Chief State Sanitary Doctor No. 1045-73). A healthy chinchilla rabbit weighing 2000 grams after quarantine was placed in an experimental cage (Application for Utility Model No. 2013119320 of January 18, 2013, “A device for conducting experiments on rabbits”) equipped with a pipe for supplying a gas mixture containing oxygen and nitrogen to the cage.

Within 3 days, the animal adapted to the experimental cell. Then, air temperature was maintained at 34-36 ° С for 8 hours in order to detect premorbid background in the animal. Animals with a body temperature above or below physiological norms were rejected. Before thermal injury, the animals were kept at an air temperature of 24-26 ° C for 24 hours, then anesthesia was performed according to the following procedure: premedication was carried out with a 2% solution of xylazine hydrochloride (rometar) at a dose of 5.0 mg / kg, and against its background a 5% solution of zoletin was administered intramuscularly at a dose of 7.5 mg / kg rabbit body weight, respectively.

After anesthesia, a thermal burn was applied. Hair was removed from the back and side of the rabbit's body. The surface of the rabbit, cleaned of wool, was immersed in a water bath for 10 seconds at a temperature of 90 ° C. The deep burn area amounted to 14.5% of the body surface area. A sterile aseptic gauze dressing with levomekol ointment was applied to the burn surface.

Within an hour after the burn, the rabbit was provided with additional breathing conditions using a gas mixture containing 80% oxygen and 20% nitrogen. A gas mixture was supplied from the PRANA KISLOROD K161 oxygen canister to the tube of the experimental cell, then after 24 hours 1 ml of non-culturable P. aeruginosa and S. aureus bacteria was injected subcutaneously to the animal at a concentration of 10 ⁵ microbial cells in 1 ml and after 12 hours the temperature was raised again cell to 34-36 ° C for 8 hours. On the second day after the introduction of microbial suspension, the animal died in the presence of clinical signs (lethargy, moan, cry, convulsions). At autopsy, cerebral edema was noted, and P. aeruginosa culture was isolated during bacteriological examination. The animal died as a result of cerebral edema and penetration of P. aeruginosa into the brain tissue through the BBB.

Example 2

The rabbit was kept in a cage in accordance with the requirements of sanitary rules (Approved by the Chief State Sanitary Doctor No. 1045-73). A healthy chinchilla rabbit weighing 2400 grams after quarantine was placed in an experimental cage (Application for utility model No. 2013119320 of January 18, 2013, “A device for conducting experiments on rabbits”) equipped with a pipe for supplying a gas mixture to the cage.

Within 3 days, the animal adapted to the experimental cell. Then, air temperature was maintained at 34-36 ° С for 8 hours in order to detect premorbid background in the animal. Animals with a body temperature above or below physiological norms were rejected. Then the air temperature was lowered to 24-26 ° C and after 24 hours a thermal burn was applied under anesthesia.

Anesthesia was carried out according to the following method: premedication was carried out with a 2% solution of xylazine hydrochloride (rometar) at a dose of 5.0 mg / kg, and against its background, a 5% solution of zoletin at a dose of 7.5 mg / kg of rabbit body weight was injected intramuscularly respectively.

After anesthesia, the hairline was removed from the back and lateral surface of the rabbit's body. The surface of the rabbit, cleaned of wool, was immersed in a water bath for 10 s at a temperature of 90 ° C. The deep burn area amounted to 19.9% of the body surface area. A sterile aseptic gauze dressing with levomekol ointment was applied to the burn surface.

Within an hour after the burn, the rabbit was given breathing conditions with a gas mixture containing 80% oxygen and 20% nitrogen. A mixture of gases from an oxygen canister PRANA KISLOROD K 161 was fed into the pipe of the experimental cell; then, after 24 hours, 1 ml of non-culturable P. aeruginosa and S. aureus bacteria was injected subcutaneously at a concentration of 10 ⁴ microbial cells in 1 ml and the temperature was raised again after 12 hours ambient air to 34-36 ° C for 8 hours. 23 hours after the introduction of microbial suspension, the animal died in the presence of clinical signs (excitement, moan, scream, cramps). At autopsy, cerebral edema was noted, and P. aeruginosa culture was isolated during bacteriological examination. The animal died as a result of cerebral edema and penetration of P. aeruginosa into the brain tissue through the BBB.

Example 3

The rabbit was kept in a cage in accordance with the requirements of sanitary rules (Approved by the Chief State Sanitary Doctor No. 1045-73). A healthy chinchilla rabbit weighing 2300 grams after quarantine was placed in an experimental cage (Application for utility model No. 2013119320 of January 18, 2013, “A device for conducting experiments on rabbits”), equipped with a pipe for supplying a gas mixture to the cage.

Within 3 days, the animal adapted to the experimental cell. Then, air temperature was maintained at 34-36 ° С for 8 hours in order to detect premorbid background in the animal. Animals with a body temperature above or below physiological norms were rejected. Then the air temperature was reduced to 24-26 ° C and after 24 hours a thermal burn was applied under anesthesia.

Anesthesia was carried out according to the following method: premedication was carried out with a 2% solution of xylazine hydrochloride (rometar) at a dose of 5.0 mg / kg, and against its background, a 5% solution of zoletin was administered intramuscularly at a dose of 7.5 mg / kg of rabbit body weight, respectively.

After anesthesia, the hairline was removed from the back and lateral surface of the rabbit's body. The surface of the rabbit, cleaned of wool, was immersed in a water bath for 10 s at a temperature of 90 ° C. The deep burn area amounted to 13.8% of the body surface area. A sterile aseptic gauze dressing with levomekol ointment was applied to the burn surface.

Within an hour after applying the burn, the rabbit was provided with additional breathing conditions using a gas mixture containing 80% oxygen and 20% nitrogen. KISLOROD K161 IRANA oxygen mixture was injected into the test tube nozzle, then after 24 hours 1 ml of non-culturable P. aeruginosa and S. aureus bacteria was injected subcutaneously to the rabbit at a concentration of 10 ⁴ microbial cells in 1 ml and after 12 hours the cell temperature was raised again to 34-36 ° C for 8 hours. On the third day after the introduction of the microbial mixture, the animal died in the presence of clinical signs (lethargy, moan, scream, cramps). At autopsy, cerebral edema was noted, and P. aeruginosa culture was isolated during bacteriological examination. The animal died as a result of cerebral edema and penetration of P. aeruginosa into the brain tissue through the BBB.

The given examples illustrate the possibility of using the proposed method for experimental modeling of the migration of bacteria into the brain to develop effective methods of treatment and prevent the development of pathological processes in the brain in case of burn injury. The work was performed on the basis of the vivarium at the State Agrarian University of the Northern Trans-Urals. Under supervision were 7 rabbits of the breed "chinchilla." Fatal outcome was observed in 7 rabbits. The animals showed clinical signs of brain damage (groaning, crying, agitation, convulsions), swelling of the brain tissue and the isolation of P. aeruginosa bacteria from the brain tissue. The results of the studies are presented in table 1.

An analysis of the data suggests that the pathological process in the brain occurs due to hypoxia in the brain cells, anaerobic type of respiration, accompanied by a high concentration of glucose in the brain tissues, and as a result, edema occurs in various parts of the brain. Additional factors affecting the development of the pathological process in the body are: the infectious process caused by P. aeruginosa, and redox reactions in the brain tissue, accompanied by the release of carbon dioxide. Summarizing, it should be noted that the temperature factor, carbon dioxide emission and burn injury have a significant effect on the decrease in BBB permeability in these laboratory animals.

Distinctive features of the proposed method for experimental modeling of the migration of bacteria into the brain against a burn injury compared with the closest analogue of the invention (ie, the "prototype" are as follows:

1. The proposed method for experimental modeling of the migration of bacteria into the brain against a burn injury is intended to improve the methods of treatment and prevention of infectious complications of a burn disease, and the “prototype” is for the treatment of the chronic disease “Cystic fibrosis”.

2. In the proposed method for experimental modeling of the migration of bacteria into the brain against the background of a burn injury, the bacteria are transferred from an uncultured to a cultured state, followed by the penetration of bacteria into the brain, and a complex chemical compound is used in the “prototype”: 4- [3,5-bis (2-Hydroxyphenyl) - [1,2,4] triazol-1-yl] benzoic acid or a pharmaceutically acceptable salt thereof to prevent the formation of biofilms by culture bacteria.

3. Based on the results of experimental studies, the proposed method for experimental modeling of the migration of bacteria into the brain allows us to determine the main directions for realizing the goal: improving methods for treating a burn disease and using the revealed mechanisms of bacteria to enter the brain to prevent cerebral complications of burn injuries.

The method of experimental modeling of the migration of bacteria into the brain against a burn injury

Table 1 Clinical and morphological characteristics of brain damage in rabbits caused by the introduction of non-cultured strains of S. aureus and P. aeruginosa against a burn injury (n = 7). Rabbit number Rabbit body weight (in grams) Area of deep burn (in%) Clinical symptoms of brain damage Cerebral edema Isolation of P. aeruginosa from brain tissue one 2500 12.6 + + 2 2000 14.5 + + + + 3 2400 19.9 + + + + four 2300 17.8 + + + 5 2200 20,0 + + 6 2100 15,5 + + 7 2300 13.8 + + + + M ± m 2257.1 ± 69.83 16.3 ± 1.03 one hundred% one hundred% 57.14% 42.86% Note: observation of rabbits was carried out in the acute period of a burn disease during the first 5 days.

Claims (1)

A method of experimental modeling of the migration of bacteria into the brain against a burn injury, namely, that healthy rabbits weighing 2000-2500 g after the quarantine period are kept for 8 hours at an air temperature of 34-36 ° C, then the air temperature is reduced to 24-26 ° C and after 24 hours a thermal burn is applied under anesthesia with an area of 10-20% of the body surface area, additional breathing conditions are created for rabbits within an hour after the burn, using a gas mixture containing 80% oxygen and 20% nitrogen, then after 24 hours animals are given a horseshoe but 1 ml nekulturabelnyh bacteria Pseudomonas aeruginosa and Staphylococcus aureus at concentrations of 10 ⁴ -10 ⁵ microbial cells in 1 mL and after 12 hours, again to increase air temperature 34-36 ° C for 8 hours and then for 5 days determine signs brain damage and record the timing of the death of animals.