RU2455703C1 - Method of surgical simulation of oxidative stress in laboratory animals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: simulation procedure is enabled by creating a acute and chronic body injury. After a surgical site is processed with an alcohol solution, an acute process - soft tissue abscess is invoked under regional anaesthesia with 0.5% Novocaine 20-200 ml. For this purpose skin is incised with subcutaneous fat, superficial and thoracolumbar fascias, a fascia of erector muscle of spine are dissected. A sterile gauze sponge impregnated in a daily culture of a pathogenic bacterial strain of the concentration of 10³/ml-10⁸/ml or impregnated in the liquid 1 ml prepared by dissolving animal's excrements 1 gram in 0.9% sodium chloride 1-10 ml. Then the wound is closed by a purse-string suture applied on skin edges. Thereafter within 72-120 hours after the implantation of the infected foreign body, the process is transformed into the chronic form by creating a septic wound. That is ensured by removing cutaneous sutures, extracting the foreign body and performing abscess cavity sanitation.

EFFECT: method provides the severity and length-controlled, two-phase experimental model of the oxidative stress enabling the high-efficiency induction of a required imbalance of the pro-oxidant and antioxidant systems, and having a recovery progression.

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Description

The invention relates to experimental medicine, namely to modeling the pathophysiological state in experimental animals, and can be used to create oxidative stress in order to study the patterns of its course and evaluate the effectiveness of antioxidant therapy.

One of the key mechanisms that shorten the duration of an active human life is considered to be an imbalance in the systems of peroxidation of tissue substrates and antioxidant protection towards increasing prooxidant potential [Skulachev V.P. Phenoptosis: programmed death of an organism // Biochemistry. - 1999. - T.64, No. 12. - S.1679-1688, Dubinina E.E. The role of reactive oxygen species as signaling molecules in tissue metabolism under conditions of oxidative stress // Questions of medical chemistry. - 2001. - T. 47, No. 6. - S. 561-581, Vladimirov Yu.A. Active forms of oxygen and nitrogen: importance for the diagnosis of prevention and therapy // Biochemistry. - 2004 .-- T.69, issue 1. - S.5-7]. Free radicals can occur in the body in excess due to radioactive and ultraviolet radiation, smoking, excessive consumption of fats and carbohydrates, in case of inactivity with its low level of biological enzymatic oxidation, as well as under the influence of an electromagnetic field, various external chemicals, air pollution, hyperoxia and other factors. An excessive amount of free radicals is observed with a decrease in the intake of antioxidants, such as tocopherol, ascorbic acid, flavonoids, and a decrease in the activity of antioxidant enzymes, which is found in congenital enzymes of antioxidant systems. The timely and dosed use of effective antioxidant agents (vitamins, lipoic acid, glutathione, combined antioxidant complexes) is important in the prevention of phenomena associated with impaired functioning of the antioxidant systems of the body [Balabolkin MI, Kreminskaya VM, Klebanova E. M. The role of oxidative stress in the pathogenesis of diabetic neuropathy and the possibility of its correction with α-lipoic acid preparations. // Problems of endocrinology. 2005. - T. 51, No. 3. - P.22-33, Ivashkin V.T., Drapkina O.M., Shulpekova Yu.O. Diagnosis and treatment of non-alcoholic fatty liver disease. // Russian medical news. - 2009. - T.XIV, No. 3. - S.1-12].

Studies over the past two decades have convincingly proven that a shift in balance in the antioxidant-prooxidant balance system accompanies many physiological and most pathological processes. In particular, a number of authors have proved that the wound healing process is inevitably associated with the activation of the prooxidant system [Zakharov V.V., Mamedov L.A., Nikolaev A.V., Gudz T.I., Kudryashov Yu.B., Goncharenko E. N., Ragimov Ch.R., Gorodovikova E.N., Kovsh I.V. The state of anti- and prooxidant systems in the healing of aseptic and infected wounds in the experiment // Bulletin of experimental biology and medicine. - 1988. - No. 6. -C.686-689; Grief R., Akca 0., Horn E.P., Kurz A., Sessler D.I. Supplemental perioperative oxygen to reduce the incidence of surgical-wound infection // New England Journal of Medicine. - 2000. - No. 342. - P.161-167; Kivisaari J., Vihersaari T., Renvall S., Niinikoski J. Energy metabolism of experimental wounds at various oxygen environments // Annals of Surgery. - 1975. - No. 181. - P.823-828, Gordillo G.M., Sen C.K. Revisiting the essential role of oxygen in wound healing // Am. J. Surg. - 2003. - No. 186. - P.259-263].

A known method of modeling oxidative stress [A.A. Hovsepyan, N.I. Venediktova, M.V. Zakharchenko, R.E.Kazakov, M.N. Kondrashova, E.G. Litvinova, I.R. Sahakyan, T. V. Sirota, I.G. Stavrovskaya, P.M. Schwarzburg. Antioxidant and immunoprotective effect of extract of wax moth larvae under oxidative stress in rats caused by consumption of feed enriched with iron // Bulletin of new medical technologies. - 2010. - No. 1 (electronic edition)], based on food overload by iron. Long-term consumption of animal feed fortified with iron in doses close to those accepted in veterinary medicine leads to the development of oxidative stress, as well as impaired immune and mitochondrial functions. In particular, to carry out the rat model, the latter, along with the main diet, is orally given an iron preparation (1 ml / alive / day).

The specified method has the following disadvantages:

a) modeling of oxidative stress takes a long time (10-12 weeks), which requires additional material costs for the maintenance of animals and the implementation of research work;

b) the model being formed is chronic sluggish due to the slow development of oxidative stress, which does not allow us to study processes in pathological conditions with an acute course and to test the effectiveness of drugs with antioxidant properties used in emergency therapy;

c) it is difficult to regulate the severity of oxidative stress due to the slow accumulation and excretion of a provoking agent (iron), which does not allow predicting the degree of disturbance of oxidative metabolism in the experimental group at the planning stage of the experiment;

d) wide variability of the results is possible due to the dosage of the provocative drug due to the fact that the latter is administered together with part of the diet, which can lead to more false results in the experimental group.

The method is not effective enough due to these disadvantages.

The method [N.V. Dovzhenko, A.V. Kurilenko, N.N. Belcheva, V.P. Chelomin was adopted as the closest analogue. Oxidative stress induced by cadmium in the tissues of the bivalve mollusk Modiolus Modiolus // Marine Biology. - 2005. - t.31 (No. 5) - p. 358-62], which consists in the fact that adult individuals of the bivalve mollusk Modiolus modiolus, selected by size (7-10 cm), are used, they are kept in aquariums for at least at least 7 days Then, a solution of CdCl _{2 is} added to aquariums with an experimental group of animals to a concentration of 100 μg / L. Water in aquariums is changed after 24 hours. The control group of mollusks was kept under the same conditions as the experimental one, but without the addition of a metal salt. The authors of the method found that in this model, oxidative stress is induced through the disorganization of the antioxidant system. A decrease in the ability of the antioxidant system to inactivate free radicals can be considered as a probable cause of the formation of oxidative stress and the accumulation of lipid peroxidation products.

The specified method has the following disadvantages:

a) the model being formed is chronic sluggish due to the slow development of oxidative stress associated with preliminary chronic inhibition of the activity of antioxidant enzymes, which can lead to a heterogeneous experimental group in terms of the intensity of free radical oxidation, due to different degrees of inhibition of the activity of antiradical defense enzymes;

b) a species of animal is used that does not breed in captivity and must be caught in the natural environment for experiments, which complicates the cyclic laboratory experiments;

c) the time of the experiment is delayed due to the fact that quarantine measures are required, leading to an increase in the cost of performing research work;

d) evaluating the effectiveness of treatment methods in this model is difficult due to the only method of drug delivery - by dissolving them in the mollusk's habitat, this approach to assessing antioxidant activity depends more on the solubility and bioavailability of the drug (indirect indicators) than on its direct antioxidant activity.

The method is characterized by high resource consumption and low efficiency due to these disadvantages.

The task is to develop a method for the surgical modeling of oxidative stress in laboratory animals, which makes it possible to cause a highly effective controlled imbalance between the prooxidant and antioxidant systems, which has the dynamics of the course of model pathology aimed at recovery, and is easily reproducible and uncomplicated in technical implementation.

The essence of the invention is a method for surgical modeling of oxidative stress in laboratory animals, comprising introducing an animal initiating oxidative stress pathogenic factor, characterized in that after processing the surgical field with an alcohol solution, under local anesthesia with a solution of novocaine 0.5% - 20-200 ml cause an acute process - soft tissue abscess - make an incision, dissect the subcutaneous tissue, superficial and lumbar-spinal fascia, the own fascia of the muscle of the rectifier of the back, enter the wound into the wound a gauze ball impregnated with a daily culture of a pathogenic strain with a concentration of 10 ³ / ml-10 ⁸ / ml or 1 ml of liquid obtained by dissolving 1 gram of animal feces in 1-10 ml of a 0.9% sodium chloride solution, then the wound is closed by suturing the edges of the skin a purse-string suture, after 72-120 hours from the moment of implantation of the infected foreign body, the process is transformed into a chronic one - a purulent wound is formed - skin sutures are removed, a foreign body is removed: and the abscess cavity is reorganized.

The technical result of the invention is:

1) providing modeling of oxidative stress with a natural focus on recovery, including the acute and chronic phases of thanatogenesis;

2) the ability to regulate the severity of developing oxidative stress from mild to extremely severe (up to potentially lethal) using strains that differ in virulence and the introduction of various amounts of microbial infection;

3) a high degree of reproducibility of oxidative stress - the ability to simultaneously obtain a model in many laboratory animals, using many strains of bacteria;

4) technically simple manipulations with laboratory animals to create a model of oxidative stress (does not require general anesthesia, resuscitation devices, with an elementary knowledge of the basics of surgical procedures).

The method is as follows

On the eve of a laboratory animal, the hair is cut and shaved on the middle and lower third of the back. The next day, after twice processing the surgical field (shaved area) with an alcohol solution under local anesthesia with a solution of novocaine 0.5% - 20-200 ml, an incision 1-5 cm long is made depending on the size of the animal. Subcutaneous tissue, superficial and lumbar-spinal fascia, intrinsic fascia of the muscle of the rectifier of the back are dissected. A sterile gauze ball with a diameter of about 10 mm, impregnated with 1 ml of liquid with a daily culture of a pathogenic strain, for example, Staphylococcus aureus, concentration of 10 ³ / ml-10 ⁸ / ml, or 1 ml of liquid obtained by dissolving 1 gram of animal feces in the wound is injected. 1-10 ml of 0.9% sodium chloride solution. Then the wound is closed by stitching the edges of the skin over the ball with a purse string suture, that is, an acute purulent disease of soft tissues - an abscess (acute phase of oxidative stress) is generated using a foreign body. It is possible to perform several wounds on one animal. 72-120 hours after the implantation of the infected foreign body, the skin sutures are removed, the foreign body is removed and the abscess cavity is reorganized. As a result, the abscess transforms into a chronic purulent process - a purulent wound (chronic phase of oxidative stress), which can be treated by daily dressings using aseptic material and ointment preparations.

The technical result is an experimental model of oxidative stress that is adjustable in severity and duration and allows for high efficiency to cause the necessary imbalance between the prooxidant and antioxidant systems and has a flow-oriented recovery.

The technology of the method for surgical modeling of oxidative stress consists in creating a soft tissue abscess (acute phase of oxidative stress) in a laboratory animal, which, after removal of sutures and removal of a foreign body, is transferred to a purulent wound (chronic phase of oxidative stress).

Justification of the results.

The model of oxidative stress was tested on laboratory rabbits (24 pieces), the control group consisted of laboratory rabbits comparable in age and weight, receiving a normal diet and not undergoing surgery (11 pieces). Blood was drawn from the ear vein on days 1, 3, 5, 7, and 10 from the start of the experiment. The severity of oxidative stress was determined by changing the intensity of chemiluminescence and the ability to oxidize under the influence of direct electric current.

Antioxidant activity of blood plasma measured by the amperometric method [Basov A.A., Fedosov S.R., Kanus I.S., Eremina T.V., Pshidatok D.V., Malyshko V.V. Modern methods of standardization of antioxidant drugs and biologically active additives // Modern problems of science and education. - 2006. - No. 4. - Appendix No. 1, p.149], from 1 to 5 days it decreased by 38%, from 5 to 7 days it stabilized, after 7 days it increased by 7%.

The dynamics of free radical oxidation was determined using a laboratory system [Pavlyuchenko II, Basov A.A., Fedosov S.R. Laboratory system for the diagnosis of oxidative stress. Utility Model Patent No. 54787. - Declared. 01/19/2006; publ. 07.27.2006 - B.21] with special software [Pavlyuchenko II, Fedosov SR, Basov A.A. Chemiluminotester LT-1 Signal Registration Program. Certificate of official registration of the computer program No. 20066611562. - Declared. No. 20066610783 dated 03.16.2006] for the maximum and area of luminol-dependent H ₂ O ₂ -induced chemiluminescence. In the course of the experiments, it was found that the indicators of the maximum fast outbreak of blood plasma chemiluminescence were characterized by relative constancy: the highest values were noted before the abscess cavity was rehabilitated (an increase of 162%), their constant decrease was after the reorganization of the abscess cavity (by 3-8% per day).

Based on the totality of the indicators evaluated, the proposed model of oxidative stress can be evaluated as functionally suitable with the following main indicators: two-phase, adjustable severity of the course, the ability to quickly create the necessary working model.

Example No. 1. Before the introduction of rabbit No. 3 into the experiment, the maximum outbreak for plasma chemiluminescence was 0.481, the area was 9.830, and the antioxidant activity was 2907.

Surgical modeling of the acute phase of oxidative stress was performed, the first stage of which is shown in Figure 1, which shows how a sterile gauze ball with a diameter of about 10 mm, impregnated with 1 ml of fluid with a daily culture of a pathogenic strain, is introduced into the wound formed during the operation (0 day of the start of the experiment) .

After the formation of a soft tissue abscess (acute phase of oxidative stress) on day 5, the maximum outbreak of plasma chemiluminescence was 1.175 (+ 124% to the initial values), the area index was 55.02 (+ 460% to the initial values), and the antioxidant activity was 1591 (- 55% of the initial indicators).

Figure 2 shows the full opening of the soft tissue abscess in the stage of resolution in the acute phase of oxidative stress (5th day of the experiment).

By the time of purification of the purulent process in the wound on day 12, the maximum outbreak of plasma chemiluminescence was 0.815 (+ 69% to the initial values), the area index was 40.82 (+ 315%), the antioxidant activity was -1783 (-39%).

Figure 3 shows the chronic phase of oxidative stress at the resolution stage - a purulent wound cleans (12 days of the experiment).

This invention allows:

- simulate oxidative stress in laboratory animals of sufficient size, of any kind and in the necessary (large) quantities;

- reduce the cost of introducing the animal into the experiment by reducing the time for the formation of the pathological process with oxidative stress by an average of 35%;

- to study the features of the course of oxidative stress by the phases of its development: in the acute phase (before opening the abscess) and the chronic phase (after opening the abscess and the formation of a purulent wound), which determines the two-phase nature of the experimental model on the same laboratory animal, reducing the possibility of obtaining false results when comparing indicators in the process of further analysis and statistical processing;

- determine the effectiveness of drug treatment of oxidative stress depending on the phase of its development, which will allow us to differentiate the need for separate drugs with antioxidant properties in the acute or chronic phase of the development of oxidative stress, and therefore make treatment regimens more rational for practical use.

The practical result of the proposal is the ability to study the phases of the course of oxidative stress and the effectiveness of its drug treatment.

Claims (1)

A method of surgical modeling of oxidative stress in laboratory animals, including the introduction of an pathogenic factor initiating oxidative stress to an animal, characterized in that after treating the surgical field with an alcohol solution under local anesthesia with a 0.5% novocaine solution of 20-200 ml, an acute process is caused - soft tissue abscess , for this, an incision is made, subcutaneous tissue, superficial and lumbar-spinal fascia are cut, the fascia of the muscle of the rectifier of the back is its own fascia, a sterile gauze ball is introduced into the wound impregnated with a daily culture of a pathogenic strain with a concentration of 10 ³ / ml - 10 ⁸ / ml or 1 ml of liquid obtained by dissolving 1 g of animal feces in 1-10 ml of 0.9% sodium chloride solution, then the wound is closed by suturing the edges of the skin with purse string suture, after 72-120 hours from the moment of implantation of the infected foreign body, the process is transformed into a chronic one - a purulent wound is formed, for which skin sutures are removed, the foreign body is removed and sanitation of the abscess cavity is performed.

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