An Infected Wound Simulation in Rats with Long-Term Diabetes Mellitus

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Despite the achievements of medicine and endocrinology in recent decades, the issues of treatment and rehabilitation of patients with diabetic foot syndrome still remain one of the most urgent problems of purulent surgery. The number of patients suffering from diabetes is steadily increasing. Diabetic foot syndrome is one of the saddest complications of long-term diabetes mellitus, as in most cases it ends in amputation and disability, causing significant socio-economic damage [1-5]. Diabetic foot syndrome is detected in 80% of patients with diabetes mellitus with a disease duration of more than 20 years and there is no tendency to reduce the number of these patients. In addition, patients with DM, complicated by SDS in 25% of cases require inpatient treatment and in terms of the frequency of occurrence occupy a leading position among other complications of diabetes mellitus[2, 6, 7, 8]. The statistics of the Russian registry indicate that the frequency of high amputations in patients with DM complicated by SDS is from 0.76 to 18.2 per 1 thousand patients, and the national average is 6.4 per 1 thousand patients. The share of large amputations, according to the statistics of the Russian Register of patients with diabetes, among this category of patients is 48.9–60.0%.
Today, many tools, methods and techniques for the treatment of purulent-necrotic lesions of the feet in diabetes mellitus are being introduced into clinical practice, but despite the many treatment methods used, the number of high amputations and deaths in patients with this pathology remains significant, and the problem is relevant and not completely solved [4, 9, 2, 10].
The study and evaluation of the effectiveness of new methods of treatment of purulent-necrotic complications in patients with SDS is extremely necessary.
Recently, methods of local local ozone therapy have been increasingly recognized in the world medical practice. Ozonated solutions are used for the treatment of wounds and body cavities, parenteral methods of using ozonated solutions and various methods of using ozonated allocrovi are widely used. Modern scientific studies confirm the safety of this method of treatment and its effectiveness [16]. It is considered proven that the therapeutic effect of ozone therapy occurs due to the physiological activation of free radical reactions. According to the feedback principle, ozone triggers and, if necessary, activates its own antioxidant system [10, 14, 15, 16].
Over the past 10-20 years, ultrasound radiation has been widely used in various fields of science, technology, and medicine [11, 12, 13]. Ultrasonic waves have a high mechanical energy and trigger a number of biological, physical and chemical processes, which explains the interest in studying the mechanism of action of ultrasound on biological objects and acquires a high interest in studying its effectiveness in long-running purulent processes. But to date, there is no reliable data on the effectiveness of the combined use of local ozone therapy and ultrasound on a purulent wound.
The aim of the study: to simulate a long-term current DM and an infected wound on the background of a long-term current diabetes mellitus.
Materials and methods. The experimental study included 120 Wistar rat populations weighing from 180.0 to 200.0 g and aged 4 months. The experiment was carried out in accordance with the requirements set out in the directives of the European Community and the Helsinki Declaration and approved by the Bioethical Commission for work with animals and the Local Ethics Committee of the Krasnoyarsk State Medical University named after prof. V. F. Voino-Yasenetsky of the Ministry of Health of the Russian Federation dated June 28, 2018, minutes of the meeting No. 2.
Rats of 40 animals in each group were distributed in three series of experiments: the first group – control, healthy animals; the second group – rats, which on an empty stomach, after 24 hours of fasting, formed alloxan-remaxol – induced diabetes mellitus, which was caused by intraperitoneal administration of 5% alloxan at a dose of 200 mg/kg (40 mg), after 15 minutes intraperitonially administered hepatoprotector-remaxol 13 ml/kg (2.5 ml), detoxification and membrane-protective effects; the second group – rats in which, after 24 hours of fasting, alloxan-induced diabetes mellitus was induced, which was modeled by intraperitoneal administration of 5% alloxan at a dose of 200 mg / kg (40 mg).
The model of alloxan diabetes mellitus is one of the most common and studied. Alloxan is a structural analog of glucose, which allows it to accumulate in the B cells of the pancreas and leads to the death of B cells with the subsequent development of diabetes. At the same time, degenerative changes in the renal parenchyma and hepatocytes contribute to the defeat of B cells. In addition, when simulating DM with alloxan, oxidative stress develops in animals. These changes are irreversible and they are likely to lead to high mortality of laboratory animals in the first day after the introduction of alloxan. Therefore, to simulate long-term diabetes, we used the drug Remaxol, which has a hepatoprotective and antioxidant effect. Remaxol (LLC "NTFF "POLISAN", St. Petersburg) is one of the most effective drugs in this field. Remaxol converts anaerobic processes into aerobic ones, normalizes the energy demand of hepatocytes, accelerates the synthesis of macroergs, prevents the destruction of liver cell membranes by lipid peroxidation products, and restores the activity of antioxidant defense enzymes. Remaxol reduces cytolysis in the liver and reduces the activity of aminotransferases and the content of bilirubin, improves the excretion of direct bilirubin in the bile. The drug reduces the toxic effect of cholestasis on the body.
Results and discussion. The animals after the DM simulation were kept in the same living environment and received the same nutritious diet. The animals were kept for 93 days without any intervention. In the first group of experimental animals without diabetes, 38 rats survived, in the second group 30 rats survived, and in the third group 23 rats survived. In the rats of the first group, no statistically significant weight loss was observed during the entire duration of the experiment, in the animals of the second group, the average body weight did not significantly decrease, while in the rats of the third group, the average weight by the end of the third month of observation decreased by an average of 40% from the initial one and was significantly lower than in the animals of the first and second groups. In the second group of rats, the clinical signs of toxic manifestations were completely stopped after three months. This effect is explained by the antioxidant and hepatoprotective effect of remaxol. Meanwhile, as in the experimental animals of the third group, a clinic of toxic manifestations was observed throughout the entire duration of the experiment, which, apparently, caused the high death rate of animals in this group in the future.
Ascension - Bayer. In the morning, on an empty stomach, venous blood was taken from the tail vessel from the rats of the second and third groups. The blood glucose level of rats of the second group for 3 months was 7.3±0.4 mmol/l, and in animals of the third group -7.9±0.48 mmol/l. Blood sugar in the animals of the second and third groups was significantly higher during the entire observation period compared to the animals of the control group (p0. 05). A consistently high level of sugars throughout the experiment proves the presence of DM in the animals of the second and third groups (Table 1). However, it should be noted that in animals of the second group against the background of alloxan-remoxol diabetes mellitus, the sugars were consistently high, but during the entire observation period did not have statically significant increases in contrast to animals with alloxan-modeled diabetes mellitus.

Table  1. The blood glucose content in the blood serum of Wistar rats after modeling diabetes mellitus.

Сутки	Контрольная группа (ср. глюкозы ммоль\л)	1 группа (ср. глюкозы ммоль\л)	2 группа (ср. глюкозы ммоль\л)
0	4,1±0,4	5,1± 0,8	3,9±1,1*
3	4,1±0,4	7,25 ±1,	8,06±1,2*
6	5,1±0,4	7,6±0,9	6,9±1,8
9	4,7±0,3	7,7±0,9	8,06±1,2*
12	4,9±0,5	7,2±0,9	9,45±1,2*
15	4,6±0,4	7,4±0,7	9,3±1,2*
30	4,68±0,6	6,9±0,4	8,06±1,04*
50	4,3±0,4	6,6±0,6	7,56±1,3
60	5,1±0,5	7,4±0,6	8,03±1,3*
70	4,8±0,5	6,3±0,7	7,2±0,9*
80	4,7±0,7	7,7±0,9	7,5±1,2
90	4,5±0,4	7,8±0,9	7,95±1,6
100	4,5±0,4	7,4±0,7	8,3±1,8*
110	4,6±0,2	7,6±0,7*	8,73±0,9*
120	4,8±0,4	7,9±0,8*	8,6±1,2*

Note: * - the significance of differences compared to the second group of animals
Note: * - the significance of differences compared to the second group of animals
Thus, the experimental animals suffered from DM for 4 months, which corresponds to 6-8 years of human life. The blood sugar level was maintained by adding carbohydrate-containing foods to the animals ' food.
After 4 months from the formation of diabetes mellitus, 30 rats with alloxan-remaxol-induced diabetes mellitus and 5 healthy rats were simulated with an infected wound.
Under aseptic conditions, under anesthesia (using the drug zoletil 100 - 15 mg/kg/m), the animals were fixed motionless, in the interscapular region, the skin was excised to the surface fascia in the form of a circle with a diameter of about 35-40 mm. The superficial fascia was dissected by transverse and longitudinal mutually perpendicular incisions in the area of the wound bottom. At the edges of the wound, a pouch suture was applied to disrupt microcirculation, and then the skin around the wound was tightened, reducing the wound to 15-20 mm in diameter. For the formation of an infected wound, 3 billion rubles were used. Staphylococcus aureus, strain 209P, which is characterized by lecithinase, hemolytic activity and gives a positive dermatonecrotic test.

About the authors

Anna Borisovna Kulikova

V. F. Voino-Yasenetsky Moscow State Medical University of the Ministry of Health of the Russian Federation

Email: nyura.84@mail.ru
ORCID iD: 0000-0003-3238-0346

Associate Professor of the Department of General Surgery named after Professor M. I. Gulman

Russian Federation, г.Красноярск , ул. Партизана -Железняка 1 , 660022

Lyudmila Viktorovna Kochetova

V. F. Voino-Yasenetsky Moscow State Medical University of the Ministry of Health of the Russian Federation

Email: dissovetKrasGMU@bk.ru
ORCID iD: 0000-0001-5784-7067

  PhD,Professor Department of General Surgery named after Professor M. I. Gulman

Russian Federation, Partizana Zheleznyaka str., Krasnoyarsk, 660022, Russian Federation

Elena Alekseevna Khapilina

V. F. Voino-Yasenetsky Moscow State Medical University of the Ministry of Health of the Russian Federation

Author for correspondence.
Email: hapilina_elena@mail.ru

PhD, Associate Professor  Departments of Human Anatomy

Réunion, 1 Partizana Zheleznyaka str., Krasnoyarsk, 660022, Russian Federation

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