MORPHOLOGICAL CHANGES IN CONTAMINATED WOUNDS

1,2Blinova N.P., 2Valiakhmedova K.V., 1Alekseev A.M., 1Bondarev O.I.

 1Novokuznetsk State Institute of Postgraduate Medicine, the branch of Russian Medical Academy of Continuous Professional Education,
2Novokuznetsk City Clinical Hospital No.1, Novokuznetsk, Russia

 MORPHOLOGICAL CHANGES IN CONTAMINATED WOUNDS

The problem of surgical site infections (SSI) in urgent abdominal surgery has the high social and economic significance. Currently, the rate of infections of postsurgical wounds varies from 3 to 65 % and has no tendency to decrease [1, 2]. Moreover, the relative risk of lethal outcome in surgical patients increases 2.2 times on average in development of SSI [3]. Also the surgical site infections result in significant material losses, with increasing costs for treatment by 54 % [2].
Currently, the traditional technique for preventing the postsurgical infection is antibiotic prophylaxis [1, 2, 4]. But despite of antibiotic prophylaxis in 100 % of cases, the surgical site infection still develops. So, the research of cytokine prevention remains the actual scientific and practical task [2, 6].
The investigation of the role of cytokines is important, considering the fact that the wound process is the complex biological mechanism, which involves the cellular elements of connective tissue and multiple factors of the immune system, including cytokines. From the perspective of the wound process, cytokines influence on the cells providing the inflammation phase (granulocytes, macrophages, T-lymphocytes) and the regeneration phase (mononuclear cells, fibroblasts, endothelial cells) and on the cells responsible for the immune response. As the main regulators of inflammation, cytokines can influence on the course and the outcome of a disease, controlling the degree of the immune response and intensity of regeneration processes [5]. Introduction of rIL-2 provides the adequate and targeted medicated correction of immune dysfunctions with replacement of deficiency of endogenous regulatory molecules and with full reproduction of their effect [7].
Currently, there is well clinical experience with systemic administration of rIL-2 for prevention and treatment of SSI. But despite the fact that the influence of systemic administration of rIL-2 on the treatment of purulent and infectious postsurgical wound complications has been studied sufficiently, the problem of efficiency of local administration of rIL-2 for prevention of SSI has not been studied enough [5].
Objective – to show the effectiveness of the preparations of recombinant interleukin-2 for contaminated wounds in the experiment.

MATERIALS AND METHODS

The experimental study of 200 white rats of Wistar line (body mass of 200-300 g, male and female) without external signs of the disease was conducted on the basis of the vivarium of Novokuznetsk State Institute of Postgraduate Medicine, the branch of Russian Medical Academy of Continuous Professional Education in the period from October 2016 to June 2017. The study protocol was approved by the ethical committee of Novokuznetsk State Institute of Postgraduate Medicine (the protocol No.75, the clause 2, October 24, 2016). Animal management and care were standard and corresponded to the requirements: Sanitary rules for construction, equipment and management of vivaria, April 6, 1973, No.1045-73, and No.1179 of USSR Health Ministry, October 10, 1983, No.267 of Health Ministry of the Russian Federation, June 19, 2003, the Rules for performance of work with use of experimental studies, the Rules for management, analgesia and euthanasia of experimental animals confirmed by Health Ministry of USSR (1977) and Health Ministry of RSFSR (1977), the principles of European convention (Strasbourg, 1986) and WHA Declaration of Helsinki of Humane Treatment of Animals (1996). The rats were kept in the conditions of the vivarium of Novokuznetsk State Institute of Postgraduate Medicine. They received 12 hours of light, with room temperature of 20 ± 2 °C, humidity – 50-70 %. The nutrition corresponded to the diet with use of ProKorm feed concentrates for rats and mice (BioPro, the factory article P-22; GOST P50258-92, RF). The nutrition was arrested 18 hours before the surgery. Water was available.

The animals were divided into three groups:
1. The control group No.1 – the rats with skin incision without formation of SSI and without introduction of rIL-2 (50 rats).
2. The control group No.2 – the rats with skin incision with formation of superficial SSI, but without introduction of rIL-2 (75 rats).
3. The main group – the rats with skin incision with formation of superficial SSI and introduction of rIL-2 (75 rats).

The operations were conducted under general analgesia with halothane in sterile conditions. The rats of all three groups received the longitudinal skin incision (the length of 2.0 cm) in the region of the withers (Fig. 1) with subsequent wound suturing. Formation of SSI included the introduction (subcutaneously, subfascially) of 10 % fecal mass into the wound (Fig. 2). After skin incision and SSI formation, the main group received the injective introduction of human recombinant interleukin-2 (NPK BIOTEKH, Russia) at the dosage of 2,500 IU subcutaneously (Fig. 3).

Figure 1. Skin incision in the withers region

Figure 2. Introduction of 10 % fecal mass into the wound

Figure 3. Injection of recombinant interleukin-2

To estimate the course of the wound process in conditions of SSI and local introduction of rIL-2, the experiment was completed by means of ether anaesthesia overdosing on the days 3, 7, 14 and 28 after the surgery. These time intervals were selected with consideration of duration of the wound process.
The first stage included the macroscopic estimation of the wound in the vivid rats on the day of theexperiment completion (Fig. 4, 5).

Figure 4. Macroscopic appearance of the wound in the control group No.1 and in the main group

Figure 5. Macroscopic appearance of the wound in the control group No.2

Each criterion (hyperemia, edema,infiltration, wound discharge, presence of fibrin and limited accumulations of pus) was estimated with degree intensity. After completion of the experiment, the second stage was dissection of the soft tissues in the region of the withers (3 × 3 cm) for histological examinations, which were conducted in the pathologic anatomy research laboratory of Novokuznetsk State Institute of Postgraduate Medicine. The samples were being fixed in 10 % solution of universal paraformaldehyde within 24 hours at the temperature of 36 °C in the thermostat TS-80M-2. Then the materials were washed and unwatered in ethanol and xylol. The received materials were covered with wax. The materials were cut on the sledge microtome. The slices of 5-7 µm were made and stained with hematoxylin-eosin and according to van Gieson. The microscopic picture was studied with the microscope Nikon Eclipse E200 with the digital camera Nikon digital sight-Fi 1, BioVision 4.0. During the morphological study, the section was estimated according to inflammation intensity (weak, moderate, high degree), necrosis zone, granulation and fibrosis (Fig. 6-8).

Figure 6. Microscopic view – necrosis region

Figure 7. Microscopic view – inflammatory infiltration

Figure 8. Microscopic view – granulation tissue with signs of fibrosis; full-blooded vessels with remodulation

Mann-Whitney non-parametric test and Kruskal-Wallis test were used for comparison of the studied groups. The level of significance was p < 0.05.

RESULTS

The control group 1 and the main group showed some insignificant local signs of inflammation, without discharge, on the day 3 after the surgery. The wound revision did not show any fibrin or limited fluid foci. The local signs of inflammation were absent on the days 7, 14 and 28.
The microscopic examination of the control group 1 showed weak inflammatory infiltration and granulation zone on the day 3. Necrosis zone was of middle intensity, and without signs of fibrosis. However the granulation tissue zone was increasing and some initial signs of fibrosis appeared on the seventh day. Inflammatory infiltration decreased on the days 14 and 28. The previous intensity of granulation tissue and of fibrosis zone was persistent. During the microscopic examination in the main group on the third day, the inflammatory infiltration and necrosis zone were unexpressed; granulation tissue was as previously, with evident development of granulation tissue and moderate fibrosis. On the days 14 and 28, the inflammatory infiltration was insignificant, without signs of necrosis, with evident development of granulation and fibrous tissue.
On the third day, the control group 2 showed the following features: moderate hyperemia and edema, evident infiltration of the wound borders, purulent discharge. During revision, the walls and the bottom were covered with high amount of fibrin, with limited purulent foci. On the seventh day, the macroscopic picture was without changes. The wounds had the small amount of purulent discharge. The purulent foci were on the walls and the bottom of the wound. On the 14th day, hyperemia, edema and infiltration of the wounds were insignificant, without discharge. But after separating the wound boundaries, the walls and the bottom were covered with fibrin (insignificantly) and contained some abscesses. There were not any local signs of inflammation on the 28th day. The wound walls included rare purulent foci. The microscopic examination showed intense inflammatory infiltration with extensive necrosis on the 3rd day. On the 7th day, inflammatory infiltration and necrosis zone were without changes. The zone of granulation tissue increased. The signs of fibrosis were absent. Inflammatory infiltration was without changes on the days 14 and 28. The amount of necrotic tissues decreased slightly. The previous intensity of granulation tissue persisted. Some initial sings of fibrosis appeared. 

DISCUSSION

The comparison of the studied groups identified the evident differences in the macroscopic and microscopic picture of the surgical wound on the days 3, 7, 14 and 28 (p = 0.003). The further analysis showed less severe macro- and microscopic signs of inflammation in the group of rIL-2 as compared to the results in the group without SSI (p = 0.7). Also the signs of regeneration were more intense in the group of rIL-2 as compared to the groups without it. 

CONCLSUION

1. The local introduction of rIL-2 promotes the favorable course of the wound process, decreases the intensity of inflammatory changes in the wound and stimulates the regeneration process.
2. It is possible to use rIL-2 agents for prevention of infections in surgical intervention site.

REFERENCES:

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