RU2776130C1 - Method for simulating kidney tuberculosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely phthisiology and urology, and can be used to simulate kidney tuberculosis in an experiment. The method involves the introduction of a culture of tuberculosis mycobacterium into the cortical layer of the kidney. At the same time, a fine needle puncture of the cortical layer of the parenchyma of the lower pole of the kidney is performed under ultrasound control to the rabbit, after which a suspension of a standardized strain of M. tuberculosis H₃₇Rv is injected through a puncture needle in a dose of 10⁶ microbial cells in 0.2 ml of saline solution.

EFFECT: invention provides for the creation of an experimental model close to the clinical course of the disease, using minimally invasive surgical intervention and reducing the risk of postoperative complications.

1 cl, 6 dwg

Description

The invention relates to medicine, namely to phthisiology and urology, and can be used to simulate kidney tuberculosis in an experiment, to study the mechanisms of formation and development of nephrotuberculosis and to find the optimal tactics for its treatment.

The proportion of tuberculous lesions of the genitourinary system occupies one of the leading positions in the structure of extrapulmonary forms of this disease. Infection of the kidneys with mycobacterium promotes the formation of granulomas in the glomeruli and their calcification, which leads to terminal chronic renal failure in 5.7% of patients [1]. The pathological process that develops in the renal tissue further spreads to the urothelium, leading in 54.5-84.7% of cases to the formation of ureteral strictures and then to the formation of hydronephrotic transformation of the kidneys [2, 3, 4], which subsequently requires reconstructive plastic surgery on the upper urinary tract, often with the use of different parts of the gastrointestinal tract [5, 6]. Despite the high efficiency of anti-tuberculosis drugs, the development of fibrotic changes in the urinary tract in some cases leads to loss of organ function [7, 8]. The cure in 60-80% of cases is associated with surgical treatment in the amount of nephrectomy [8, 9, 10].

The search for new methods of combined therapy of nephrotuberculosis, aimed at maintaining a functioning renal parenchyma, continues tirelessly, and therefore it is relevant to create a model of tuberculous kidney damage in laboratory animals.

A scientific study is known, the purpose of which is to model tuberculous lesions of the kidney in rabbits with intravenous injection of a culture of bovine mycobacteria, where an indispensable condition for the reproducibility of the model is the additional formation of a violation of the outflow of urine from the kidneys by applying a ligature to the ureter [11]. The main disadvantage of this method is the high lethality of laboratory animals due to the development of an acute generalized specific process.

There is a known method for modeling destructive tuberculosis of the kidney, which makes it possible to approach the model to the clinical course by creating a tuberculous cavity in an experimental animal (dog), previously sensitized with horse serum [12]. Under anesthesia, the upper pole of the kidney is compressed, cooled to -2 -6 ° C and infected with a culture of M. tuberculosis H37Rv (at a dose of 50 mg in 0.5 ml of horse serum). However, the proposed model does not reproduce the actual mechanism of development of tuberculous lesions of the kidney, since the formation of destructive forms (cavities) in the kidney occurs by artificially creating an area of ischemia as a result of experimental exposure. The disadvantages of this method should also include the choice of dogs as model animals, which significantly limits the breadth of use of the proposed method due to the high material costs for the acquisition and maintenance of animals, as well as for the performance of surgical and medical procedures.

E.N. Bellendir and co-authors [13] conducted a study to create a model of limited destructive kidney tuberculosis in rabbits by performing lumbotomy to inoculate a culture of mycobacteria (prototype). However, this method is traumatic and difficult to implement: the very manipulation of the formation of a channel in the cortical layer of the kidney (about 1 mm in diameter and 8-10 mm in length) is accompanied by blood loss, mechanical trauma, followed by the development of a nonspecific inflammatory reaction. The disadvantages of the method should also include the uncertainty of the qualitative (phenotypic and genotypic) characteristics of the clinical strain used for infection, as well as the lack of clear data on the infecting dose (the number of mycobacterial cells per 1 mg of dry culture). In addition, the method does not allow for in vivo monitoring of the development of the tuberculous process, which makes it impossible to obtain comparable results in the clinical course of the disease.

The objective of the invention is to create an experimental high-performance model of kidney tuberculosis, most accurately reflecting the clinical course of the disease, using minimally invasive surgery and reducing the risk of postoperative complications.

The problem is solved by the fact that, under ultrasound control, laboratory animals (rabbits) produce a fine-needle puncture of the cortical layer of the parenchyma of the lower pole of the kidney, after which a suspension of a standardized strain of M. tuberculosis H ₃₇ Rv is injected through the puncture needle at a dose of 10 ⁶ microbial cells in 0.2 ml physiological solution.

The proposed method for modeling tuberculous lesions of the kidney is simple to implement and is characterized by low trauma. The choice to create a model of rabbits, sufficiently large laboratory animals, opens up the possibility of reliable monitoring of pathophysiological changes from the very beginning of the infectious process until its completion, without resorting to euthanasia.

The essence of the invention is illustrated by drawings.

Fig. 1 - Ultrasound image of the kidney of rabbit No. 9 infected with M. tuberculosis H37Rv at a dose of 10 ⁶ CFU / 0.2 ml. The arrow indicates the site of infection - the cortical layer of the parenchyma of the lower pole of the left kidney.

Fig. 2 - hyperergic reaction (erythema) in response to intradermal administration of ATP 30 days after inoculation of M. tuberculosis H ₃₇ Rv into the cortical layer of the parenchyma of the lower pole of the left kidney at a dose of 10 ⁶ microbial cells/0.2 ml. A - intact, B - infected rabbit No. 9.

Fig. 3 - Macropreparations of rabbit kidneys No. 9. A - contralateral (right, uninfected) kidney. B - left kidney 5 months after inoculation of M. tuberculosis H37Rv into the cortical layer of the parenchyma of the lower pole of the kidney at a dose of 10 ⁶ microbial cells / 0.2 ml. Near the capsule there is a cavity 1×1 mm in size, filled with loose caseous masses, in the surrounding tissue there are individual caseous foci 0.1×0.1 mm

Fig. 4 - Micropreparation of the left kidney of rabbit No. 9 5 months after inoculation of M. tuberculosis H37Rv into the cortical layer of the parenchyma of the lower pole at a dose of 10 ⁶ microbial cells/0.2 ml. Necrotic masses surrounded by pseudoeosinophilic leukocytes, macrophages, and emerging multinucleated giant cells. Stained with hematoxylin and eosin, SW. ×600

Fig. 5 - Micropreparation of the left kidney of rabbit No. 9 5 months after the inoculation of M. tuberculosis H37Rv into the cortical layer of the parenchyma of the lower pole at a dose of 10 ⁶ microbial cells/0.2 ml. acid-resistant bacteria. Coloring according to Ziehl-Nelsen, uv. ×1000.

Fig. 6 - Micropreparation of the left kidney of rabbit No. 9 5 months after inoculation of M. tuberculosis H37Rv into the cortical layer of the parenchyma of the lower pole at a dose of 10 ⁶ microbial cells/0.2 ml. The emerging focus of necrosis, surrounded by lymphoid-leukocyte infiltrate and the emerging capsule. Stained with hematoxylin and eosin, SW. ×300

The proposed method is carried out as follows.

Male rabbits of the "Soviet Chinchilla" breed weighing 3.2-3.5 kg in an operating room under general anesthesia using Zoletil intramuscular anesthesia drugs [zolozepam + tiletamine (Virbak SA, France) at a dose of 25 mg/kg of body weight] and Xila [(Xylazinum, Interchemie Werken "de Adelaar" BV, Netherlands) in the form of a 2% solution in a volume of 1.0-1.5 ml] were fixed on a special machine in the position on the right side, hair was removed in the left hypochondrium in projection area of the left kidney, the surface was treated with 5% iodine solution. An hour before the operation, antibiotic prophylaxis was performed using cefazolin (at a dose of 50 thousand U/kg, 1.5 ml intramuscularly). Then, under ultrasound control, a fine-needle puncture of the cortical layer of the parenchyma of the lower pole of the left kidney was performed (Fig. 1). To improve the positioning accuracy of the puncture needle, a puncture adapter for an ultrasound scanner was used. Through a puncture needle into the cortical layer of the renal parenchyma, a mycobacterial suspension of a 3-week (second generation) standardized virulent test strain M. tuberculosis H37Rv (obtained from the Federal State Budgetary Institution "Scientific Center for Expertise of Medical Applications" of the Ministry of Health of the Russian Federation), prepared ex tempore on the day of infection of animals and containing 10 ⁶ cells of mycobacteria in 0.2 ml of saline. Within 5 days after infection, infectious complications were prevented using broad-spectrum antibiotics that do not have anti-tuberculosis activity (penicillins, cephalosporins of the 1st or 2nd generation) for 5 days at average therapeutic doses.

Monitoring the development and course of tuberculosis infection in laboratory animals:

- clinical research methods: daily assessment of the general condition of animals (satisfactory / unsatisfactory), examination of the area of infection inoculation (abscesses, hematomas - yes / no); animal body weight dynamics (kg) once every 10 days;

- a study of peripheral blood, including an assessment of the activity of inflammation by the concentration of reactants of the acute phase (albumin and ceruloplasmin by the Ravin method), the activity of enzymes of the purinergic system - total adenosine deaminase (ADA) and its isoenzymes (ecto-ADA-1 and ecto-ADA-2) by the G method Giusti [14]; concentration of the biomarker of acute kidney injury - creatinine. In addition, the level of enzymes of the destructive process was studied: metalloproteinases (MMPs), including collagenase MMP-1 (CSB-E09744Rb), stromelesin MMP-3 (Rb) and their inhibitors (tissue inhibitor TIMP-1 (CSB-E06935Rb) and α2 -macroglobulin (MG) and elastolytic activity (EL) by the method of L. Visser and E.R. Blout [15];

- staging a test with recombinant tuberculosis allergen Diaskintest®, which was administered at a concentration of 2 μg/ml in 0.1 ml of saline to rabbits intradermally on the back in the kidney projection area;

- bacteriological examination of the urine of rabbits after 30 and 60 days (inoculation on a dense medium of Levenshtein-Jensen by the method of serial dilutions), the titer of mycobacteria was expressed in decimal logarithms (lg) of the number of CFU (colony-forming units);

- Ultrasound examination 60 days after inoculation of M. tuberculosis H37Rv suspension;

- computed tomography (tomograph Toshiba Aquilion 32) of the lungs 60 days after MBT inoculation to exclude the generalization of a specific process;

- pathomorphological examination, including macroscopic examination of the internal organs of the chest and abdominal cavity, histological examination of micropreparations of the kidneys when stained with hematoxylin and eosin, as well as by the Ziehl-Nelsen method for the detection of acid-resistant mycobacteria.

Statistical data processing was carried out using the Statistica 7.0 software package (StatSoftInc, USA). In case of deviation from the normal distribution (Shapiro-Wilk test), the median (Me), the first and third quartiles (Q1-Q3) were calculated. The reliability of differences in metric values (Wilcoxon test), their correlation between each other (Spearman test) and with quantitative signs (Kruskal-Wallace test) were evaluated.

30 days after MBT inoculation, in response to the introduction of the allergen in infected rabbits, the appearance of erythema measuring 15.3 ± 2.8 mm was recorded, which indicates the presence of sensitization and confirms the development of the tuberculosis process (Fig. 2).

The development of the tuberculous process in the kidneys was accompanied by an increase in the inflammatory response in the peripheral blood. At all periods of the study, the concentration of creatinine was increased, there was an increase in the activity of total ADA due to its isoenzyme ADA-1, the most significant at the height of inflammation (2 months after infection) ADA - 4, ADA-1 - 3.8 times compared with indicators of intact rabbits. The presence of correlations between degradation markers (MMP-1, MMP-3, TIMP-1, elastase) with ROF and creatinine, as well as between ADA-1 activity and creatinine concentration (r=0.9; p=0.037) on height of inflammation.

60 days after infection, ultrasound examination showed progressive edema in the region of the lower pole of the left kidney with a significant thickening of the parenchyma, expansion of the PCS. In crops of urine at this period, a moderate growth of the MBT culture was revealed (4.19±0.06 lg CFU).

Radiographically, no specific pathological changes were found in the lungs of a rabbit.

Five months after infection, the animals were euthanized by injecting the anesthetic drug Zoletil into the lateral vein of the ear at a dose 5 times higher than the therapeutic one.

During macroscopic examination, near the capsule of the left kidney (infected), the following were visualized: a 1×1 mm cavity filled with loose caseous masses, in the surrounding tissue - individual caseous foci 0.1×0.1 mm with the transition of the pathological process to the ureter and its obliteration (Fig. 3).

Histological examination of micropreparations of the tissues of the left kidney confirmed the presence of specific inflammation with the formation of cavities with necrotic masses surrounded by pseudoeosinophilic leukocytes, macrophages and forming giant multinucleated Langhans cells (Fig. 4). In addition, an emerging focus of necrosis was identified, surrounded by a lymphoid-leukocyte infiltrate and a developing capsule (Fig. 5). Acid-resistant mycobacteria were detected when staining micropreparations of the tissue of the left kidney according to the Ziehl-Nelsen method (Fig. 6).

The method has been tested on 20 mature male rabbits.

Efficiency of the method: The observation time was 5 months. The results of the experiments indicate that inoculation under ultrasound control through a puncture needle into the cortical layer of the renal parenchyma of a suspension of a standard laboratory virulent test strain H37Rv at a dose of 10 ⁶ microbial cells/0.2 ml leads to the development of specific inflammatory changes in the kidney in experimental animals, at the same time, it is combined with minimal intraoperative damage and a small risk of process generalization.

Thus, the proposed method makes it possible to reproduce tuberculous kidney damage under experimental conditions on rabbits using a minimally invasive surgical approach, as well as to carry out in vivo 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. The proposed model is of scientific and applied value for phthisiology and urology, since it reveals the mechanism of structural and functional disorders at different stages of the development of the disease and makes it possible to adjust the tactics of treating nephrotuberculosis.

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Claims (1)

A method for modeling kidney tuberculosis by introducing a culture of mycobacterium tuberculosis into the cortical layer of the kidney, characterized in that, under ultrasound control, the rabbit is subjected to a fine-needle puncture of the cortical layer of the parenchyma of the lower pole of the kidney, after which a suspension of a standardized strain of M. tuberculosis H ₃₇ Rv is injected through the puncture needle at a dose of 10 ⁶ microbial cells in 0.2 ml saline.

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