RU2431890C1 - Method of modelling infected skin wound - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: skin is incised, and wound edges are anchored to subjacent tissues. Each wound angle is underrun additionally with a separate diagonal suture. A formed scab and necrotising tissues are excised within healthy tissues. Then the wound is treated with an antiseptic and washed with physiologic saline. Thereafter, the treated wound surface is coated with a semipermeable membrane under the angular diagonal sutures to introduce a bacterial suspension containing 0.5 ml E.coli 109 and 0.5 ml Ps.aeraginosa 109. ^ EFFECT: method provides a model of the infected skin wound with preset bacterial population with its characteristics as much as possible approached to an actual clinical course of the wound process. ^ 3 tbl, 7 dwg

Description

The invention relates to medicine, namely to experimental surgery and pathophysiology.

There are various methods for modeling purulent wounds. So known is a method of modeling an infected soft tissue wound. For this purpose, a rabbit (3-4 kg in weight) is laid on the back with a plate inside which a hole with a diameter of 2.0 cm is made. Under general anesthesia, a skin in the form of a cone 0.9-1.1 cm high is pulled out and excised. The focus of necrosis is formed by applying a gauze swab dipped in 70% acetic acid solution to the wound for 3-5 seconds. After 3-5 days, the necrotic scab is removed. Then, the wound is irrigated with St.aureus culture at a concentration of 5 × 10 ⁵ CFU (Method for modeling an infected soft tissue wound / Sukhovey Yu.G., Tsiryatyeva S.B., Minin A.S., Samusev R.S., Sych A.S. ., Kostolomova EG // RF patent №2321898 from 04/10/2008, publ. Bull. No. 1).

The disadvantages of this method include obtaining a model of a wound that has a mixed nature of damage, i.e. chemical burn with bacterial contamination. In addition to the disadvantages of this method should also include early closure of the wound surface, due to the lack of fixation of the edges of the wound. These disadvantages do not allow to standardize the course and healing process of the wound.

The closest in technical essence to the proposed one is a method for modeling an infected musculoskeletal wound, including excision of the skin, scab removal 3 days after applying the skin defect and the introduction of bacterial suspension into the wound.

The known method is as follows. A 2 × 2 cm skin area is excised on the back of a laboratory animal (rat). To form granulation tissue, the skin defect is left without a bandage - “under the crust”. After 3 days, the crust is removed, the bottom of the wound is incised with a blade, after which 1 billion microbial bodies of the daily culture of St.aureus are introduced into the wound. An occlusive dressing is applied to the wound with an infect (Immunohistochemical study of purulent wounds in rats after application of crab collagenase / I.Yu. Sakharov, B.M.Shekhonin, S.P. Gligos, F.E. Litvin // Bulletin of experimental biology and medicine. - 1993, - No. 4, - p.267).

The disadvantages of this method include:

- the impossibility of obtaining a "standard" wound necessary to study the course and healing processes of the wound under the influence of antibacterial drugs;

- the impossibility of dynamic control over the quantitative content of strains of infect introduced into the wound;

- joining the infection, since before the introduction of the infect, the wound is infected with both its own microflora and the microflora of the environment;

- uncontrolled depth of the lesion, due to the implementation of notches of the bottom of the wound with a blade;

- uncontrolled area of the wound due to the lack of fixation of the edges of the wound.

Therefore, the known method does not allow to obtain a model of an infected skin wound with a controlled microbial landscape and a controlled area and depth of the wound.

The task of the invention is to develop a method for modeling an infected skin wound with a given microbial landscape, as well as with a controlled depth and area of damage.

The technical result of the proposed method is the possibility of obtaining an infected skin wound with a given bacterial contamination of the wound, as well as providing a controlled depth and area of the wound.

The technical result is achieved by the fact that the method of modeling an infected skin wound includes excision of the skin, removal of the scab and the introduction of infect into the wound.

Distinctive techniques of the proposed method are:

- hemming the edges of the wound to the underlying tissues after excision of the skin,

- each corner of the wound is additionally stitched with a separate diagonal suture,

- excision of the scab and necrotic tissue within healthy tissue,

- treatment of a wound with an antiseptic followed by washing it with physiological saline,

- closing the treated surface of the wound with a semipermeable membrane, the ends of which are inserted under the corner diagonal seams,

- the introduction under a semi-permeable membrane of bacterial suspension containing 0.5 ml of E. coli 10 ⁹ and 0.5 ml of Ps.aeraginosa 10 ⁹ .

A comparative analysis with the prototype showed that the proposed method differs from the known above methods and, therefore, meets the criteria of the invention of "novelty."

Comparison of the claimed technical solution not only with the prototype, but also with other technical solutions in experimental medicine did not allow us to identify signs that distinguish the claimed solution from the prototype.

Using the above methods of the proposed method allows to obtain a model of an infected skin wound with a given microbial landscape, as well as with a controlled depth and area of damage.

Thus, fixing the edges of the wound by suturing them to the underlying tissues prevents premature contraction of the edges of the wound, which makes it possible to "standardize" the area of the wound, regardless of the individual characteristics of the healing process in the experimental animal, and to evaluate the effect of the studied drug.

Excision of the scab and necrotic tissue within healthy tissue provides a controlled depth and area of damage.

Treatment of a wound with an antiseptic, after removal of necrotic tissue, eliminates the development of both the animal’s own microflora and the environment’s microflora in the wound. This technique creates optimal conditions for the development of microflora of the introduced infection - hospital strains of E. coli and Ps.aeraginosa.

Subsequent washing with physiological saline eliminates the negative effect of the antiseptic remaining in the wound on the introduced microflora.

The introduction and placement of a semipermeable membrane in the wound (for membrane-absorbing drainage complexes) prevents the bacterial suspension from flowing out of the wound, eliminates wound contamination in the early postoperative period and creates optimal conditions for the development of introduced microflora.

The introduction of a bacterial suspension containing microbial cultures of E. coli 10 ⁹ and Ps.aeraginosa 10 ⁹ of 0.5 ml each, under a semi-permeable membrane creates optimal conditions for the development of introduced infection in the early postoperative period. In addition, a semi-permeable membrane prevents environmental microflora from entering the wound. The use of E.coli and Ps.aeraginosa cultures is due to their prevalence in purulent wounds.

When analyzing known methods for modeling purulent wounds, they revealed a lack of information about the influence of the distinguishing features of the proposed method on the achievement of a technical result. The above allows you to establish compliance with the criterion of "inventive step".

The method comprising the claimed invention is intended for use in experimental medicine. The possibility of its implementation is confirmed by the methods and means described in the application. From the foregoing, it follows that the claimed invention meets the condition of patentability "industrial applicability".

The inventive method is as follows. Experimental animal rat under general anesthesia spend excision of the skin flap 2 × 2 cm (figure 1). On the square of the defect, the edges of the wound are sutured with a continuous suture to the underlying tissues (atraumatic 4/0) (figure 2). Moreover, each corner of the wound is additionally stitched with a separate diagonal suture.

After 3 days under general anesthesia, the scab and necrotic tissues are excised within healthy tissues (Fig. 3). Wound sanitation is carried out with an antiseptic solution (0.02% Anavidin), washed with physiological saline, after which a semipermeable membrane for membrane-sorbing drainage complexes is installed on the treated surface of the wound with placement under angular diagonal seams. A bacterial suspension containing 0.5 ml of E. coli 10 ⁹ and 0.5 ml of Ps.aeraginosa is introduced under a semipermeable membrane

10 ⁹ (Fig. 4).

The proposed method is illustrated by an example of a specific implementation.

The study was conducted on white Vistar male rats of 6 months of age with a body weight of 200-250 g. The animals were kept in vivarium with free access to water and food, which meets the standards of GOST "The content of experimental animals in the nurseries of the research institute" (vivarium I category, vet certificate 238 No. 0015220 dated March 25, 2009, the Veterinary Service of the Irkutsk Region). Animal experiments were carried out in accordance with the rules for the humane treatment of animals, regulated by the "Rules for the use of experimental animals", approved by Order of the Ministry of Health of the USSR No. 742 of 11/13/84, "On the approval of the rules for working with experimental animals" and No. 48 from 01/23/85, "On the control of work using experimental animals." All surgical interventions were performed under sterile conditions under general anesthesia.

Under aseptic conditions and under general anesthesia, the animal was fixed on the table of A.I.Sechenov in the position on the stomach. After preparation of the surgical field in the back, excision of the skin and subcutaneous fat was performed with an area of 4 cm ² . The edges of the wound were sutured to the underlying tissues with an atraumatic needle with absorbable ligature 4/0, in increments of 5 mm. Additionally, each corner of the wound was stitched with a separate diagonal suture.

In each animal of the main group consisting of 40 animals, after 3 days, the scab and necrotic tissues were excised. Necrotic tissues were excised within healthy tissues to a depth of 0.3 cm. Each animal of this group was treated with the Anavidin antiseptic and then washed with saline. Then, a semipermeable membrane was installed in the wound (for drainage compositions). The fixation of the semipermeable membrane on the wound was achieved by placing its corners under the corner diagonal seams. Under a semipermeable membrane, i.e. 1 ml of daily bacterial suspension containing 0.5 ml of E. coli 10 ⁹ CFU / ml 0.5 ml of P. aeruginosa 10 ⁹ CFU / ml was introduced into the wound.

The control group consisted of animals that were modeling wounds by the prototype method. The number of animals in this group was 10.

The starting point of the day experiment in all groups was the time of wound infection.

Wound microflora, wound surface area, morphological changes in the wound in the dynamics of the experiment on days 1, 3, 7, 9, 11, 19 were studied in all animals of the main and control groups.

The clinical picture of wounds in both groups of animals on the 1st day was the same. The wounds were characterized by sagging, scar-fibrinous edges, sluggish and dull granulations, foci of necrotic changes.

The dynamics of microbial contamination of the infected wounds of animals of the main group are presented in table 1 (Lg CFU / cm ² , median and quartile).

In all animals of the main group, on day 1, a high microbial contamination of the wounds was revealed by the association of the introduced microbial flora with growth of more than 10 ⁷ CFU (E. Coli + P. Aeruginosa), with a predominance of P. aeruginosa.

Table 1 Study Day 3 5 7 9 eleven 19 Lg CFU 7.0 • •• 5.5 ^∗ 5.0 ^∗ 5.5 5.5 ^∗ 4.0 ^∗ 4.0 ^∗ • •• (7.0-7.0) (5.0-6.0) (4.0-5.0) (4.0-6.0) (4.0-6.0) (3.0-5.0) (4.0-4.0) Note ^∗ —significant differences according to the Wilcoxon criterion in comparison with the indicator in the group for 1 day (p _w ≤0.05); • - compared with the indicator in the group on the 3rd day (p _w ≤0.05), •• - compared with the indicator in the group on the 5th day (p _w ≤0.05).

After 3 days, positive clinical changes were noted, consisting in the rejection of part of the necrotic foci, in increased growth of islands of healthy granulation tissue. In the control microbiological study, the seeding rate remained high 10 ^5-6 CFU (E. Coli and P. aeruginosa), with the predominance of P. Aeruginosa, but was significantly lower compared with 1 day (P _w = 0.007).

The revealed level of microbial contamination remained unchanged up to 11 days. A significant decrease was recorded by the 19th day (P _w = 0,0002).

The assessment of the course of wound healing was carried out according to two criteria: by the start of epithelization (in days) and by half-healing time - a 2-fold reduction in the area of the wound (in days). The measurement of the area of the wound was carried out by weighing a paper template on a torsion balance. The results are shown in table 2.

table 2 Estimated Score The timing of the onset of epithelization in days 14.3 (14.0-15.0) Half-healing time (reduction of the wound area by 2 times) in days 13 (12-15)

A morphological study of the animals of the main group in the wound area on the 1st day revealed: superficially microbial colonies, necrotic tissue, white blood cells, deuteritis, fibrin (Figure 5, position 1). In the underlying tissue: edema, plasma soaking, diffusely distributed white blood cells in the underlying connective tissue (Figure 5, position 2).

By the 19th day in the wound area, a scab of cell deuteritis is superficially located (Fig.6, position 3). Under the scab, newly formed connective tissue with fibroblasts and fibrocytes (Fig.6, position 4). The wound is superficial with the morphology of wound healing by the 19th day under the scab.

The dynamics of the microbial landscape of the infected wounds of the skin of animals of the control group are presented in table 3 (Lg CFU / cm ² , median and quartile).

When evaluating the microflora of the wounds of control animals, in addition to the introduced microflora (St.aureus), the presence of additional strains of microorganisms was revealed.

Table 3 Type of pathogen CFU Proteus 10 ⁵ Staphylococcus epidermidis 5 × 10 ⁴ Staphylococcus aureus 5 × 10 ⁴ Candida 5 × 10 ⁵ Klebsiella oxytoca 5 × 10 ⁵ Enterococcus faecalis 5 × 10 ⁵ Escherichia coli 5 × 10 ⁶ Pseodomonas aerug. 5 × 10 ⁶

The addition of extraneous microflora complicates the dynamic and quantitative study of microbial contamination of the wound by the introduced strain.

When morphological examination of infected wounds of control animals on the 1st day superficially in the wound area revealed: microbial colonies, necrotic tissue, white blood cells, deuteritis, fibrin (Fig.7, position 1). Aponeurosis (Fig. 7, position 2) in the underlying tissue, between the aponeurosis and adjacent fibers of the skeletal muscle, edema, plasma impregnation and moderate leukocyte infiltration. The wound is stratified, the aponeurosis separates the inflammation of the superficial and deep parts of the tissues to the muscle layer. A morphological evaluation of the preparations of this group revealed microabscesses, both superficial and in the deep parts of the tissues to the muscles, in the area of the notches applied with a scalpel to the bottom of the wound. All data obtained confirm the impossibility of controlling the depth and area of the lesion.

An analysis of bacteriological and morphological studies confirmed the possibility of obtaining a model of an infected skin wound with specified parameters.

Thus, the proposed method allows to obtain a model of an infected skin wound, in its characteristics as close as possible to the real clinical course of the wound process. The reproducibility of the model is 100%.

The model can be used in a chronic experiment to obtain an infected skin wound with controlled parameters, including both bacterial contamination of the wound and the depth and area of damage.

Claims (1)

A method for modeling an infected skin wound, including excision of the skin, removal of the scab and the introduction of infect into the wound, characterized in that after excision of the skin, the edges of the wound are sutured to the underlying tissues, with each corner of the wound being additionally stitched with a separate diagonal suture, the scab and necrotic tissue formed excised within healthy tissues, then the wound is treated with an antiseptic and washed with saline, and then on the treated surface of the wound under angular diagonal seams rehydrating a semipermeable membrane which is introduced by a bacterial suspension containing 0.5 ml of E.coli September ¹⁰ and 0.5 ml Ps.aeraginosa 10 ^9.

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