RU2703709C1 - Method for simulating an experimental soft tissue wound in rats for developing a therapeutic approach - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to experimental surgery, and can be used to simulate an experimental wound of soft tissues in rats for developing a therapeutic approach. Wound cavity is created by insertion of the implant into soft tissues for period of 6–7 days. Soft tissue layer-by-layer incision is performed at the required depth: skin, subcutaneous fat, superficial fascia and muscular tissue. Hydrophilic polymer implant of spherical shape is introduced to create model of experimental aseptic wound. To create a purulent wound, a porous polymer implant is also introduced in a spherical shape, pre-saturated with a suspension of a bacterial agent in concentration of 10⁵–10¹² microbial cells of the investigated agent per 1 ml of the suspension. Wound is closed in layers to form the appropriate wound cavity model. After that, implant is surgically removed, surface area (S) and volume (V) of which are calculated by formulas: S=4πr², V=4/3πr³, where r is implant radius, π – 3.14.

EFFECT: method provides the soft tissues wound simulation in the animal experiment with the possibility of creating the specified parameters of the wound as a result of introduction of the polymer material into the experimental wound to the required depth, which allows varying the diameter of the implant depending on the exposure variation in the given solution.

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Description

The alleged invention relates to medicine, in particular to experimental surgery, to methods for modeling aseptic and purulent wounds of soft tissues in experimental conditions on laboratory animals.

The proposed "Method for modeling an experimental soft tissue wound" can be used in experimental medicine, as well as in veterinary medicine for modeling soft tissue wounds in laboratory animals at the stages of preclinical studies of various medical devices, drugs, treatment tactics.

Models of wounds in experimental animals are used to study the healing process, its features under the influence of various systemic, local factors, medications and physical effects.

The relevance of developing new methods for experimental modeling of this pathology is due to the lack of an optimal method for modeling soft tissue wounds that meets all the necessary criteria, as well as the need to find the most effective methods of treating wounds and caring for them in accordance with the phases of the wound process [1].

Known methods for modeling aseptic wounds by applying a linear wound followed by implantation of suture material into the wound cavity, or simultaneous suturing of the wound I.A. Sychennikova, 1974 [2] and Yu.A. Furmanova, 1985 [3].

The disadvantages of these methods are the ability to simulate only small aseptic linear wounds, not complicated by a purulent-inflammatory process; uncontrolled area of the wound - due to the lack of fixation of its edges, which indicates the lack of universality of the methods, the impossibility of creating a model that is close to the real conditions of the course of the wound process.

Known methods for modeling infected and aseptic wounds by mechanical action on them. So in his research V.P. Petrov 1989 [4] inflicted smashed skin-muscle wounds on a laboratory animal, followed by infection with Staphylococcus aureus (Staphylococcus aureus) and sutured the wound. A.V. Volenko, 1998, [5] on the contrary, after injury by a hemostatic clamp, a piece of silk ligature and a section of crushed fiber were introduced into the wound.

In the above methods of modeling surgical wounds in the experiment, there are certain drawbacks that affect the accuracy of the assessment of the results, especially with qualitative and quantitative control of the microbial load on the wounds. In addition, reproducing one of the most important components of a surgical wound - mechanical trauma, it is practically impossible to determine in each case the force of the traumatic effect created during the experiment by the researcher’s hands on the tissue by crushing them or injuring with hemostatic clamps.

Known invention S.A. Lepekhova, which consists in the following: an experimental animal is excised with a skin flap, the edges of the wound are sutured, after 3 days the scab and necrotic tissues are excised, sanitized, after which a semipermeable membrane is installed for membrane-sorbing drainage complexes, a bacterial suspension containing 0.5 ml of E. coli 10 ⁹ and 0.5 ml of Ps. aeraginosa 10 ⁹ [6].

In all of the above models of purulent wounds, we are talking about the formation of a model of only superficial wounds, in the model of S.A. Lepekhova does not exclude the development of maceration under the membrane, anaerobic conditions are created under the dressing, the method is laborious and rather traumatic for the animal.

For the closest analogue, a method for modeling a purulent wound according to the well-known method of P.I. Tolstoy, 1976 [7], which is carried out by full-layer excision of the skin area, the edges and the bottom of the wounds are crushed with a Kocher clamp for tissue devitalization, after which 1 ml of suspension of Staphylococcus aureus is introduced into the wound. To do this, skin with a subcutaneous tissue 1 × 1 cm in size is excised on the back in a shaved area and treated with an antiseptic, then a gauze swab containing 10 ¹⁰ microbial bodies of the daily culture of Staphylococcus aureus 592 is introduced into the wound. To standardize the treatment conditions, prevent wound deformation, as well as to prevent drying, contamination of the wound surface and bites by other animals, a gauze dressing with a polyethylene pad is placed on the wound to create a "greenhouse" effect. On the 3rd day, the wound is a surface covered with blood clots, fibrin, pus, tissue edema is observed in the wound and around its perimeter.

However, with this method, a surface wound is formed with possible maceration under a plastic film. Another disadvantage is the ability to simulate the conditions for the development of only anaerobic infections under gas and waterproof linings, therefore, the proposed method is not universal and is not suitable for assessing the clinical picture of aerobic wound infection. The method is time-consuming, difficult to standardize under the specific conditions of the experiment. The volume of the gauze swab may vary depending on the method of its placement in the wound, since its shape is unstable and has virtually no rigid boundaries. Therefore, this model cannot be used to study the possibilities of prevention and treatment of such lesions as soft tissue wounds of various origins.

Thus, the analysis of existing models of purulent wounds indicated the following disadvantages:

- the impossibility of obtaining a “standardized" wound with predetermined geometric parameters in all experimental animals;

- the possibility of changing the microbial landscape of the wound during the experiment due to the prevalence of anaerobic microflora due to the creation of anaerobic conditions under the film is not ruled out;

- 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;

- requires additional equipment, and the implementation of laborious manipulations;

- uneven distribution of the introduced suspension with microbial load under a semipermeable membrane along the bottom of the wound;

- early necrectomy, the amount of execution, which largely depends on the preferences of the experimenter, disrupts the natural course of the phases of the wound process, which reduces the objectification of the results.

Tasks: Development of a method for modeling soft tissue wounds in an animal experiment with the ability to create specified parameters of this wound and control them by depth, area of damage, degree of bacterial load, severity of the inflammatory reaction; reducing the complexity of the wound modeling process; decrease in the level of intraoperative aggression (trauma) when creating a wound model.

The essence of the invention is a method for modeling an experimental soft tissue wound in rats for the development of treatment tactics, including creating a wound cavity by introducing an implant into the soft tissues for a period of 6-7 days, characterized in that the soft tissue is layered at the required depth: skin, subcutaneously - fatty tissue, superficial fascia and muscle tissue, and to create a model of an experimental aseptic wound, a hydrophilic polymer implant of spherical shape is introduced, and pori are injected to create a purulent wound The quick polymer implant is also spherical in shape, pre-saturated with a suspension of the bacterial pathogen at a concentration of 10 ⁵ -10 ¹² microbial cells of the studied pathogen per 1 ml of suspension. Then the wound is sutured in layers to form the corresponding cavity (model). After the indicated period, the implant is surgically removed, the surface area (S) and volume (V) of which are calculated by the formulas: S = 4πr ² , V = 4 / 3πr ³ , r is the implant radius, π is 3.14.

The novelty is: the introduction into the experimental wound in laboratory animals to the required depth: skin, subcutaneous fat, surface fascia and muscle tissue of the polymer material, which allows you to control the depth, area of damage and volume of the wound cavity due to the possibility, depending on changes in the exposure of the polymer material in a given solution, vary the diameter of the implant.

The method is as follows:

a) form an implant of the required size by exposure of a hydrophilic polymer material in an antiseptic solution (chlorhexidine bigluconate 0.05%). The polymer is capable of sorbing low molecular weight organic and inorganic substances, this ability of the polymer was used and found application in the proposed invention. A quantitative relationship was established between the exposure of the implant in the solution and the geometric parameters of the material used due to sorption of substances from the pharmacological solution in which it was previously placed (Appendix Fig. 1. Dependence of the volume of the hydrophilic implant on the exposure in the antiseptic solution (in% of the initial volume)) .

After anesthesia of the animal (laboratory rats in this case), preparation of the surgical field, the surgical procedure is performed as follows: under aseptic conditions, a linear incision is made with a scalpel along the paravertebral line with a length of 1.0-1.5 cm by layer-by-layer dissection of tissues to the required depth (depending from research tasks, the implant can be placed in the intramuscular space, under the superficial fascia of muscles or above it, in subcutaneous fat). Then the implant is placed in the created wound defect, then the wound is sutured in layers.

In the postoperative period, the animal is monitored for six to seven days, during which the formation of a connective tissue capsule around the implant takes place for the subsequent creation of an experimental aseptic wound. On the seventh day, in the prescribed manner, in compliance with the requirements for the humane treatment of animals under general anesthesia (by administering 0.1 ml of “Telazole” IM) operatively by linear section of the underlying tissues 1.0-1.5 cm long, the implant is removed. An experimental wound is formed.

The study was carried out on 50 white laboratory non-linear male rats of five months of age with a body weight of 300-350 g (Appendix Fig. 2. Preparation of the surgical field). The animals were kept in vivarium with free access to water and food, which corresponds to GOST 33044-2014 “Principles of Good Laboratory Practice” (approved by Order of the Federal Agency for Technical Regulation and Metrology No. 1700-st of November 20, 2014), entered into force 1 August 2015

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.

Rats were divided into two conditional groups, 35 animals each. The first group of animals inside the wound was placed a hydrophilic implant in the form of a ball weighing 0.65-0.7 g and a diameter of 5-6 mm, the exposure of which took place for 4 hours in an aqueous solution of “Chlorhexidine bigluconate 0.05% - YuzhFarm” to form models of aseptic wounds.

b) The second group was injected with a polymer porous implant of the same volume with a bacterial suspension containing 0.4 ml of P. aeruginosa monoculture in 10 ⁹ CFU / ml.

When testing the method, it was found that the optimal range of the concentration of bacterial culture for the formation of a model of purulent wounds is the range of 10 ⁵ - 10 ¹² CFU / ml. This was established when the following pathogens were introduced into the experimental wound: E. coli from 10 ⁵ to 10 ¹² CFU / ml, P. aeruginosa from 10 ⁵ to 10 ¹² CFU / ml, S. aureus from 10 ⁵ to 10 ¹² CFU / ml, S. pyogenes 10 ⁵ to 10 ¹² CFU / ml and K. pneumoniae 10 ⁵ to 10 ¹² CFU / ml. In particular, it was experimentally determined that the optimal concentration for creating the desired model of a purulent wound in rats weighing 300-350 g. When using P. aeruginosa was the concentration of 10 ⁹ CFU / ml, which was used in the experiment.

Under aseptic conditions and under general anesthesia (by administering 0.1 ml of Telazole IM), the animal was fixed on the table of A.I. Sechenov in the position on the stomach. After preparation of the surgical field (shaving of the skin in the scapular region 6.0 × 4.0 cm in size and its triple treatment with iodopyron 5%, once with a solution of chlorhexidine bigluconate 3%), a linear skin wound 1.0 was applied to the rats with a scalpel using a scalpel -1.5 cm by layerwise dissection to the required depth: skin, subcutaneous fat, superficial fascia of muscles and muscle tissue, an implant was placed in a given layer.

An experimental soft tissue wound with an implant immersed in it was sutured in layers by a nodal suture (two in each layer) (atraumatic “Prolene” 3/0 - skin with subcutaneous fatty tissue, atraumatic “Prolene” 4/0 - muscle layer with underlying superficial fascia).

On the 7th day, animals from each group underwent repeated surgical intervention under aseptic conditions, the implant was removed under general anesthesia (Appendix Fig. 3. Removing a hydrophilic implant), the degree of formation of a standardized cavity was visually assessed (Appendix Fig. 4. Formed aseptic cavity) after which the formed capsule with surrounding tissues was excised for histological examination.

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

The volume of the formed cavity V = 4 / 3πr ³ , the wound surface area S = 4πr ² , the microflora of the wounds, and the morphological changes in the wound on the 7th day were studied in all animals. During a histological examination by electron microscopy (Appendix Fig. 5. The site of the capsule delimiting the aseptic cavity), it was recorded that the wall of the capsule is represented by granulation tissue containing a large number of thin-walled full-blood vessels. Cellular infiltrate is formed predominantly by neutrophilic granulocytes with a small admixture of lympho-macrophage cell infiltrate, fibroblasts. Infiltration of surrounding tissues with fibrin was noted.

Example 1. Modeling of an experimental aseptic wound of soft tissues was performed on a laboratory non-linear rat, a five-month-old male with a body weight of 335 g (Experiment Log No. 17 dated 09.09.2017). An implant was formed by exposure of a hydrophilic polymer material in an antiseptic solution (chlorhexidine bigluconate 0.05% - YuzhFarm) for 4 hours. The formed implant had a mass of 0.68 g and a diameter of 6 mm.

Under aseptic conditions and under general anesthesia (by administering 0.1 ml of Telazole IM), the animal was fixed on the table of A.I. Sechenov in the position on the stomach. After preparation of the surgical field (shaving of the skin in the scapular region 6.0 × 4.0 cm in size and its triple treatment with iodopyron 5%, once with a solution of chlorhexidine bigluconate 3%), a linear skin wound 1.0 cm long was applied with a scalpel using a scalpel by layer-by-layer dissection of the skin, subcutaneous fat, superficial fascia of muscles and muscle tissue into which the implant was placed. The wound was sutured in layers with a nodal suture (two in each layer) (atraumatic suture "Prolene" 3/0 - skin with subcutaneous fat, atraumatic suture "Prolene) 4/0 - muscle layer with underlying superficial fascia ) During the six days of the postoperative period, the animal was monitored with free access to water and food.

On the seventh day, in accordance with the established procedure, in compliance with the requirements for the humane treatment of animals under general anesthesia (by administering 0.1 ml of Telazole IM) by surgery, a linear incision of the underlying tissues 1.2 cm long was removed. Using the standard formulas: S = 4πr ² , V = 4 / 3πr ³ , r is the implant radius, π - 3.14 calculated the surface area (S) and volume (V) of the formed wound, which were 113 mm ² and 113 mm, respectively ³ . The area of the capsule delimiting the cavity was excised with a scalpel and histological examination was performed. It was found that the wall of the capsule is represented by granulation tissue containing a large number of thin-walled full-blooded vessels. Cellular infiltrate is formed by a lympho-macrophage pool, fibroblasts. Mild infiltration of surrounding tissue with fibrin was noted. The wall thickness of the formed cavity was 0.8 mm.

Example 2. Modeling of an experimental aseptic wound of soft tissues was performed on a laboratory non-linear rat — a five-month-old male with a body weight of 340 g (Experiment Log No. 35 of 09/19/2017). An implant was formed by introducing a bacterial suspension (containing 0.4 ml Ps. Aeraginosa 10 ⁹ CFU / ml) into the polymer porous implant. The formed implant had a mass of 0.53 g and a diameter of 5.8 mm.

Under aseptic conditions and under general anesthesia (by administering 0.1 ml of Telazole IM), the animal was fixed on the table of A.I. Sechenov in the position on the stomach. After preparation of the surgical field (shaving of the skin area in the scapular region 6.0 × 4.0 cm in size and its triple treatment with iodopyron 5%, once with a solution of chlorhexidine bigluconate 3%), apply a linear skin wound with a length of 1.4 cm with a scalpel along the paravertebral line by layer-by-layer dissection of the skin, subcutaneous fat, superficial fascia of muscles and muscle tissue into which the implant was placed. The wound was sutured in layers with a nodal suture (two in each layer) (atraumatic suture "Prolene" 3/0 - skin with subcutaneous fat, atraumatic suture "Prolene) 4/0 - muscle layer with underlying superficial fascia ) During the six days of the postoperative period, the animal was monitored with free access to water and food.

On the seventh day, in accordance with the established procedure, in compliance with the requirements of humane treatment of animals under general anesthesia (by administering 0.1 ml of Telazol IM) by surgery, a linear incision of the underlying tissues 1.5 cm long was removed, the implant was removed. According to standard formulas: S = 4πr ² , V = 4 / 3πr ³ , r is the implant radius, π - 3.14 calculated the surface area (S) and volume (V) of the formed wound, which were respectively 106 mm ² and 102 mm ³ . The area of the capsule delimiting the cavity was excised with a scalpel and histological examination was performed. It was found that the wall of the capsule is represented by granulation tissue containing a large number of thin-walled full-blooded vessels. Cellular infiltrate is formed predominantly by neutrophilic granulocytes with a small admixture of lympho-macrophage cell infiltrate, fibroblasts. Infiltration of the surrounding tissues with fibrin was noted, as well as the presence of pathogen cells previously introduced into the implant on the inner surface of the capsule. The wall thickness of the formed cavity was 1.2 mm.

The advantage of the proposed method is that the proposed modeling method does not require additional equipment, allows to obtain aseptic and purulent (in case of combination with bacterial contamination) skin-subcutaneous wounds and skin-subcutaneous-muscular wounds with controlled: depth, area of damage and the possibility of obtaining standardized cavity due to the formation of the implant with a variable diameter depending on the exposure in the antiseptic solution, in addition, the implant promotes uniform distribution edeleniyu by oral bacterial suspension retains the properties at strong changes in temperature or humidity, has bioinertness, chemical inertness, which allows to follow the dynamics of wound healing in conditions as close as possible to clinical, in an experimental model of soft tissue wounds. The proposed model has confirmed its effectiveness:

- in all experimental animals it was possible to form the same wound models, standardized both by geometric parameters and by the type of wound process depending on the bacterial load and exposure of the soft tissue implant;

- the technology for the formation of a wound model is easy to use, takes little time, is less traumatic for the animal itself.

Literature:

1. Wounds and wound infection: a Guide for doctors / ed. Kuzina M.I., Kostyuchenko B.M. - Moscow: Medicine, 1990 .-- 591 p.

2. Sychennikov I.A. // Modeling, study methods and experimental therapy of pathological processes. M., 1974. - S. 69-73.

3. Furmanov Yu.A., Gorshevikova E.V., Adamyan A.A., Vinokurova T.I., Tsetlin B.L., Vlasov A.V., Silkis E.M., Moshkovsky G.Yu. Development and testing of surgical suture materials. Clinical Surgery 1985, 3, p. 25-28.

4. Zhitnyuk I.D. Treatment of infected wounds with a powder mixture. Herald of Surgery 1967, 12, p. 69-74 /.

5. Volenko A.V. Prospects and opportunities for preventive washing of surgical wounds with pulsating jets of fluid under pressure. Surgery 1998, 4, p. 45-50.

6. Lepekhova S.A., Koval E.V., Grigoriev G.E., Goldberg O.A., Zaritskaya L.V. Patent RU 2431890, G09B 23/28 - in medicine - Method for modeling an infected skin wound.

7. Bulletin of new medical technologies. Treatment of purulent wounds using wound coverings "biatrauma" and "resorb" Lazarenko V.A., Bezhin A.I., Guseinov A.Z., Cherdakov A.V., Ivanov A.V., Zhukovsky V.A., Volume XVII No. 3 2010 - p. 200.

Claims (1)

A method for modeling an experimental soft tissue wound in rats to develop treatment tactics, including creating a wound cavity by introducing an implant into soft tissue for a period of 6-7 days, characterized in that the soft tissue is layered at the required depth: skin, subcutaneous fat, surface fascia and muscle tissue and to create a model of an experimental aseptic wound, a spherical hydrophilic polymer implant is introduced, and a porous polymer implant is also introduced to create a purulent wound ovidnoy form, previously saturated with a suspension of bacterial pathogen at a concentration of 10 ^{5 to} 10 ¹² cells of the test microbial pathogen per 1 ml of suspension, the wound is then sutured layer by layer to form a pattern corresponding wound site, the implant through said term surgically removed, the surface area (S) and the volume (V) which is calculated by the formulas: S = 4πr ² , V = 4 / 3πr ³ , where r is the radius of the implant, π is 3.14.

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