RU2600926C2 - Method for modeling tuberculosis of female genital organs - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to phthisiology, and can be used for modeling of tuberculosis of the female genital organs. To do this a suspension of Mycobacterium tuberculosis Erdman culture is introduced to rabbits at a dose of 10⁷ CFU/0.2 ml under a serous membrane of the fallopian tube at a distance of 5 cm from the uterine horn. At the same time before the operation the estrogenization of animal with intramuscular introduction of 0.1 % solution of synestrol is carried out on 0.5 ml every second day in a course of 4 treatments. From 2 to 6 days after surgery estrogenization is continued in the same mode and prevention of infectious complications using broad-spectrum antibiotics, having no anti-tuberculosis activity is carried out.

EFFECT: method allows to obtain a model of local tuberculosis in the female genital organs, available for reproduction and as close as possible to the development of specific process to natural, and also to carry out in vivo monitoring of the development and course of specific inflammatory process and to choose the tactics of treatment.

1 cl, 1 ex, 5 dwg

Description

The invention relates to medicine, namely to TB, and can be used to model and develop methods for the treatment of tuberculosis of the female genital organs.

Tuberculosis of female genitalia is one of the forms of extragenital tuberculosis, most often develops in women of reproductive age from 20 to 45 years [1, 2, 3, 4, 5]. Tuberculosis of female genitalia accounts for about 5% of all inflammatory diseases of the female genital area [6] with outcome in tubal-peritoneal infertility, the frequency of which reaches 97% of clinical observations [7]. After a comprehensive anti-TB treatment, fertility recovery was noted in only 7.2% of women [8]. In this regard, the search for reliable methods of modeling tuberculosis of the female genital organs for the subsequent development of new methods of treating pathology is extremely relevant.

A known method of modeling tuberculosis of the female genital organs in experiments on rabbits (A.S. 912147. A method of modeling tuberculosis of the female genital organs). With it, local infection of animals with a culture of mycobacterium tuberculosis is made, which is introduced under the mucous membrane of the ampulla of the fallopian tube.

The disadvantages of the prototype:

1. The ability to repeat the implementation of the method is called into question, since the biological properties (species, virulence, sensitivity to anti-TB drugs) of the culture of mycobacteria used to infect animals are not indicated. The calculation of the infecting dose of mycobacteria is incorrectly presented, namely: the number of CFU (colony forming units) in 1 mg of the culture of mycobacterium tuberculosis is not indicated.

2. It is controversial to provide standard conditions for the technical and pathophysiological stages of the model proposed by the authors, since the introduction of a calculated infectious dose of mycobacterium tuberculosis directly under the mucous membrane of the fallopian tube under visual control is problematically and inevitably accompanied by a mechanical injury to the ampullar department with the subsequent development of a non-specific inflammatory reaction.

3. The target modeling of tuberculosis, set by the authors as an appendix to the genitals, dictates the need to take into account the anatomical features of blood circulation in the uterine artery basin. In the ampullar section of the fallopian tube, recommended by the authors as a zone of local infection with the culture of mycobacterium tuberculosis, the blood flow is minimal and, accordingly, the least favorable conditions for the life of aerobic mycobacterium tuberculosis.

4. It does not allow to observe the dynamics of the development of the pathological process, since monitoring the progress of the development of tuberculosis infection is carried out only when animals are withdrawn from experience. The latter, in turn, does not allow the choice of treatment tactics and leads to unjustified overstatement of both the number of animals used in the study and the financial costs of their acquisition and maintenance.

5. It is necessary to use narcotic analgesics (Promedol 1 ml of 1% solution per 1 kg) as an anesthetic aid.

The objective of the proposed method is to develop a method for modeling tuberculosis of the female genital organs with the ability to monitor the development of a specific inflammatory process and the development of treatment methods.

The problem is solved due to the fact that rabbits are injected with a suspension of Mycobacterium tuberculosis Erdman culture at a dose of 10 ⁷ CFU / 0.2 ml with an injection needle under the serous membrane of the left fallopian tube (without entering its lumen) outside at a distance of 5 cm from the left uterine horn.

The proposed method for modeling tuberculosis of the female genital organs allows:

1. To obtain a model of the local tuberculosis process in the female genital organs, accessible for reproduction by introducing an intraoperatively standardized culture of M. tuberculosis Erdman with a given infectious dose and providing favorable conditions for the life of aerobic tuberculosis mycobacteria by choosing a zone with optimal blood supply to the fallopian tube (at a distance 5 cm from the uterine horn), as well as improving vascularization and microcirculation of the fallopian tubes in estrogenized rabbits.

2. To bring the experimental model of development of the specific process to the natural one as close as possible, as well as to carry out intravital monitoring of the development and course of the specific inflammatory process from the moment of infection to euthanasia of each animal included in the study, according to the results of a skin test - Diaskintest (recombinant tuberculous allergen, DST), complex laboratory (hematological and biochemical parameters of peripheral blood) and radiation (hysterosalpingography and laparoscopy) studies.

4. Choose a treatment strategy.

5. To exclude the use of narcotic analgesics.

6. Reduce the number of experimental animals.

The method is illustrated by drawings, where in: FIG. 1 - Erythema 72 hours after intradermal administration of Diaskintest. A - intact, B - infected rabbit; FIG. 2 - Infected left fallopian tube after 60 days. A - intact, B - infected rabbit; FIG. 3 - Hydrosalpinx of the infected left fallopian tube after 120 days. A - intact, B - infected rabbit; FIG. 4 - Hysterosalpingography. A - intact rabbit, B - infected rabbit; FIG. 5 - Histological picture of the mucous membrane of the infected uterine rabbit horn. Hematoxylin and eosin stain × 400.

The method is as follows:

Stage 1 - estrogenization of animals with synestrol - is administered 0.1% sinestrol solution of 0.5 ml intramuscularly every other day with a course of 4 procedures in the pre- and postoperative periods.

2nd stage - preoperative preparation and implementation of surgical access. An anesthesiological aid is performed: intramuscular injection of the drug for anesthesia of zoletil (zolozepam + tiletamine Virbach CA, France) at a dose of 25 mg / kg and 2% solution of muscle relaxant rometar (Xylazinum, Biovet, Czech Republic) in a volume of 1.0 ml. The position of the rabbit is “on the back”, the limbs are fixed on a special machine in the most extended position. The hair on the anterior abdominal wall is trimmed, the skin is treated with a 5% iodine solution twice. The surgical field is covered with sterile wipes. 5 cm skin incision is made along the midline. The anterior abdominal wall is opened in layers. After opening the peritoneum, the fallopian tubes and uterine horns are discharged into the surgical wound and fenced off from the abdominal cavity with sterile material.

Stage 3 - preparation of mycobacterial suspension and inoculation of infect. A 2-week (second generation) standard laboratory virulent test strain of M. tuberculosis tuberculosis M. tuberculosis Erdman is used, which is sensitive to anti-TB drugs (from the collection of the Federal State Budget Scientific Institution of the State Scientific and Research Institute for Standardization and Control of Medical Biological Preparations named after L. Tarasevich of the FMBA of Russia). Mycobacterial suspension is prepared extemporaneously in sterile physiological saline in a predetermined concentration of 10 CFU / 0.2 ml. To inoculate a suspension of culture of mycobacterium tuberculosis at a dose of 10 ⁷ CFU / 0.2 ml, an injection needle is inserted under the serous membrane of the left fallopian tube outside (without entering its lumen) at a point 5 cm away from the uterine horn. At the injection site, a papule forms 0.3 cm

Stage 4 - suturing the peritoneum with a continuous vikrilovy seam, the imposition of individual vikrilovy seams on the aponeurosis, muscles and skin of the anterior abdominal wall. Postoperative suture treatment with 5% iodine solution.

Stage 5 - postoperative period. In the postoperative period, from 2 to 6 days a day, the course of estrogenization of rabbits continues with a 0.1% sinestrol solution (intramuscularly, 0.5 ml), as well as the prevention of infectious complications using broad-spectrum antibiotics that do not have anti-tuberculosis activity (penicillins, cephalosporins 1 or 2 generations, 5 days course).

Assessment of the development and activity of the tuberculosis process in the female genital organs is confirmed by:

- staging a skin test with the drug "DIASKINTEST®" (recombinant tuberculosis allergen) with intradermal administration of the drug at a concentration of 2 μg / ml in an amount of 0.1 ml per skin test;

- computed tomography of the lungs to exclude generalization of a specific process;

- performing diagnostic laparoscopy;

- studies of blood taken from the marginal ear vein: counting the leukocyte formula, determination of ESR, acute phase proteins;

- conducting hysterosalpingography;

- bacteriological examination of biopsies or homogenates of the serous membrane of the uterine horn and fallopian tube (seeding on a dense nutrient medium of Levenshtein-Jensen);

- histological examination of preparations of the walls of the uterine horn and fallopian tube.

A positive result of the DST skin test and an increase in the value of C-reactive protein in the peripheral blood (from 3.86 ± 2.58 mg / L to 6.057 ± 2.83 mg / L, p <0.05) on the 30th day from the moment of infection, and the absence of pathological changes in the lungs is evidence of the development of an isolated form of tuberculous inflammation in the female genital organs of rabbits.

Example.

Rabbit No. 9 of chinchilla breed (gender - female, initial weight - 4.2 kg, temperature + 39.0 ° C) was preliminarily administered intramuscularly by estrogenization of 0.5 ml of 0.1% sinestrol solution from the seventh day before infection every other day. On the eve of infection, a biochemical and clinical blood test was performed.

In order to anesthetize, intramuscularly administered a solution of zoletil 0.3 ml and a solution of Rometar 2% - 1.0 ml. The rabbit is fixed on the machine in a position on its back, limbs - in the most extended position. The hair on the anterior abdominal wall is trimmed, the skin is treated twice with 5% iodine solution. A skin incision was made 5 cm long along the midline, a layered opening of the anterior abdominal wall. The fallopian tubes and the uterine horns are brought into the surgical wound and fenced with sterile material from the abdominal cavity. Then, a suspension of Mycobacterium M. tuberculosis Erdman was administered at a dose of 10 ⁷ CFU / 0.2 ml under the serous membrane of the left fallopian tube by external puncture with an injection needle at a distance of 5 cm from the uterine horn. A papule of 0.3 cm is visualized at the injection site. The peritoneum is sutured with a continuous Vicryl suture, the aponeurosis, muscles and skin of the anterior abdominal wall with individual Vicryl sutures. Postoperative suture treated with 5% iodine solution.

In the early postoperative period, the animal was observed daily, examination, weighing, estrogenization with a 0.1% solution of 0.5 ml sinestrol from 2 to 6 days a day, as well as the prevention of infectious complications with cefazolin solution intramuscularly in a dose of 1.5 ml were performed (based on 50 thousand units / kg) for 5 days. Further clinical examination was carried out 1-3 times a week.

30 days after inoculation of the MBT, a skin test was performed by intradermal injection of Diaskintest at a concentration of 2 μg / ml in an amount of 0.1 ml per skin test. It was found that in response to the introduction of an allergen in the back of an infected rabbit, the appearance of erythema of 15.7 ± 3.5 mm was recorded, which indicates the presence of sensitization and confirms the development of the tuberculosis process (Fig. 1). An increase in the level of C-reactive protein in the peripheral blood was determined in the peripheral blood of an infected rabbit (from 3.86 ± 2.58 to 6.057 ± 2.83 mg / L, p <0.05).

60 days after infection, revision of the abdominal cavity using laparoscopic techniques showed progressive swelling, hyperemia, and expansion of the infected left uterine horn and left fallopian tube (Fig. 2). The growth of MBT was determined in the cultures of biopsy samples of the serous membrane of the infected uterine horn. When computed tomography of the lungs in a rabbit, specific pathological changes were not detected.

When visual inspection of the abdominal cavity after 120 days after infection revealed loose and planar adhesions, obliteration of the fallopian tubes with the formation of hydrosalpinx in the infected left fallopian tube (Fig. 3). When hysterosalpingography of rabbit No. 9, there was a complete delay in the release of contrast medium into the abdominal cavity, which indicates obstruction of both infected and collateral fallopian tubes (Fig. 4). Performed euthanasia of the animal. Macroscopic examination revealed deformation of the left fallopian tube, obliteration of the site at the site of inoculation of a suspension of MBT culture. In other organs of the thoracic and abdominal cavity, tuberculous changes were not detected. Histological examination in the central sections of the muscle layer of the infected uterine horn showed signs of specific inflammation in the form of single productive tuberculous epithelio-unicellular granulomas with giant multinucleated cells with weakly expressed lymphoplasmacyte infiltration (Fig. 5).

The method was applied on 25 (5 of them intact) sexually mature female rabbits. The effectiveness of the method. The observation time was 120 days. The results of the experiments indicate that inoculation under the serous membrane of the left fallopian tube with a suspension of a standard laboratory virulent test strain of M. tuberculosis Erdman at a dose of 10 ⁷ CFU / 0.2 ml with additional estrogenization leads to the development of specific inflammatory changes in experimental animals against the background of adhesions in the uterus and fallopian tubes with obliteration of the lumen of the fallopian tubes and / or the formation of hydrosalpinxes.

Thus, the proposed method allows to reproduce in experiments on rabbits tuberculous lesions of the uterus and fallopian tubes, as well as carry out intravital monitoring of the development and assessment of the severity of a specific inflammatory process from the moment of infection to euthanasia in a particular animal included in the study. This makes it possible to develop tactics for treating tuberculosis of the female genital organs at different stages of the development of the disease.

Bibliography

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Claims (1)

A method for modeling female genital tuberculosis by local infection of rabbits with a culture of tuberculosis mycobacteria, characterized in that the animal is injected with a suspension of Mycobacterium tuberculosis Erdman culture at a dose of 10 ⁷ CFU / 0.2 ml under the serous membrane of the fallopian tube at a distance of 5 cm from the uterine horn, while before surgery the animal is estrogenized by intramuscular injection of 0.1% Sinestrol solution of 0.5 ml every other day with a course of 4 procedures, and from 2 to 6 days after the operation, estrogenization is continued in the same mode and inf ktsionnyh complications using broad-spectrum antibiotics that do not have antituberculosis activity.

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