RU2584402C1 - Method for simulating traumatic osteomyelitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine and can be used for simulating traumatic osteomyelitis of long bones. Method includes preliminary sensitisation of rats by triple intraperitoneal introduction of low virulent Staphylococcus aureus culture weakened by heating at 60 °C for 30 minutes. At that, every 3 days sensitisation is performed with gradual increase in dose from 1 to 3 million bacterial bodies. Further, three days after sensitisation in sterile conditions under anaesthesia soft tissues are incised. Through distal femoral metaphysis hole in bone-medullary canal is formed with large needle, into which active aureus in dose of 6 mln bacterial bodies in 0.1 g 30 % solution of gelatine is introduced. Thereafter, soft tissues are closed with catgut; and wound is treated.

EFFECT: method enables to obtain physiologically accurate model of various stages of postoperative traumatic osteomyelitis while reducing mortality of test animals.

1 cl, 3 dwg, 2 tbl

Description

The invention relates to medicine and can be used to simulate acute and chronic traumatic osteomyelitis.

In the modern concept of the pathogenesis of osteomyelitis, unresolved problems remain. The impossibility of solving them clinically actualizes the search for a model of osteomyelitis, which corresponds to the laws of occurrence and provides an opportunity to study the mechanism of its development.

A known method of modeling osteomyelitis, involving the use of rabbits [1, 2]. For the development of the disease, an initial sensitization of the body is carried out, followed by the deposition of microbes into the bone marrow using viscous substances (lanolin or gelatin) [3].

The indicated model is quite effective and acceptable for studying the pathogenesis of the disease, but it requires significant material costs and is not always advisable for conducting screening studies.

To test new drugs for the treatment of the disease, an effective and easily implemented model that includes various stages of osteomyelitis development is necessary. It seems appropriate to improve the indicated model of osteomyelitis using rats, which will significantly reduce the cost of the experiment and make the possibility of its use more accessible.

A known method of modeling the development of osteomyelitis in rats [4]. It is as follows. A hollow tube with a blind end is inserted into the bone marrow canal of the tibia. A transverse bone defect is produced. At the same time, a through hole is made in the tube wall. The infectious material is introduced fractionally through the free end of the tube outside the wound. The introduction of infectious material is carried out in place of a bone defect. However, this model has several disadvantages:

- the model does not involve preliminary sensitization of the body, in connection with this a large percentage of unhealthy animals is possible, which requires re-infection;

- the needle is constantly located in the bone marrow canal, additionally traumatizes animals and causes inconvenience in their livelihoods, which is not consistent with the “Laboratory Practice Rules in the Russian Federation (GLP) (approved by Order of the Ministry of Health of the Russian Federation of June 19, 2003 N 267);

- the diameter of the tibia of the beauty is quite small, which complicates the introduction of an injection needle into the bone marrow canal;

- the introduction of staphylococcus is carried out without the use of viscous substances, so in some cases there is a severe course of the disease with generalization of infection and death of animals.

A new technical result is to obtain a more physiologically accurate model of the various stages of traumatic osteomyelitis corresponding to the diagnosis, expanding the scope of the model and reducing the mortality of the tested animals.

In order to achieve a new technical result in a method for modeling traumatic osteomyelitis, including the formation of a defect in the bone tissue of the tibia in an experimental animal, followed by the introduction of an infectious material into the defect, the rats are preliminarily sensitized by triple intraperitoneal administration with a weakly virulent golden culture weakened at 60 ° C for 30 minutes staphylococcus, and every 3 days sensitization is carried out with a gradual increase in dose from 1 d 3 million bacterial bodies, then, three days after the end of sensitization, under sterile conditions, an incision of soft tissues is performed under anesthesia, a hole in the bone marrow canal is formed with a thick needle through the distal metaphysis of the femur, into which staphylococcus is injected at a dose of 6 million bacterial bodies in 0.1 g of a 30% solution of gelatin, after which the soft tissues are sutured with catgut, the wound is treated.

The method is as follows. In the experimental animal, a defect in the bone tissue of the tibia is formed, followed by the introduction of infectious material into the defect site, while the rats are preliminarily sensitized by triple intraperitoneal administration by attenuated heating at 60 ° C for 30 min, a culture of weakly virulent Staphylococcus aureus, and every 3 days sensitization is carried out with a gradual increase in dose from 1 to 3 million bacterial bodies, then, three days after the end of sensitization, under sterile conditions, under the drug Then the soft tissue is cut, through the distal metaphysis of the femur with a thick needle, a hole is formed in the bone marrow canal, where active staphylococcus is injected at a dose of 6 million bacterial bodies in 0.1 g of a 30% gelatin solution, after which the soft tissues are sutured with catgut, the wound is treated and the appearance on the 10th day of such signs of changes in the leukocyte blood count, the presence of such clinical manifestations as pain during palpation of the knee joint, an increase in the knee joint by 2-3 times compared with a healthy limb, the presence of pus of a discharge from the wound, registration during scintigraphy of accelerated and increased drug intake in the arteries on the affected side, as well as determination of areas of indicator hyperfixation in the soft tissue and bone phases of the study, confirms the formation of a model of the acute stage of traumatic osteomyelitis, and when signs of a decrease in the total number appear by day 21 myelokaryocytes, megakaryocytes, monocytes and lymphocytes in the bone marrow, a change in the leukoerythroblastic ratio of bone marrow cells towards sperm cells of the myeloid series, elements confirming purulent bone marrow infiltration confirms the creation of a model of the chronic stage of traumatic osteomyelitis

The proposed method is based on the analysis of the results of experimental studies. The experiments were performed on 20 nonlinear white male rats weighing 200-250 g. The animals were kept under standard vivarium conditions with free access to water and food. When conducting the experiments, they were guided by the "Rules for conducting work using experimental animals" (Appendix to the order of the Ministry of Health of the USSR dated 08/12/1977, No. 755).

Rats were divided into two groups: intact (group-1) and animals with experimental osteomyelitis (group-2). Preliminarily, group 2 rats were sensitized by intraperitoneal administration of a weakly virulent Staphylococcus aureus (60 ° C, 30 min) every 3 days. Sensitization was carried out with a gradual increase in dose (1 million, 2 million and 3 million bacterial bodies). Microbial suspension was prepared according to the standard turbidity GNIISK them. L.A. Tarasevich.

Three days after the end of sensitization under sterile conditions, under anesthesia (Zoletil, 10 mg / kg i / m), a dose of active staphylococcus (6 million bacterial bodies in 0.1 g of a 30% gelatin solution) was injected into the distal metaphysis of the right femur ) For this, an incision was made in the soft tissues, a hole was formed with a thick needle into the medullary canal, and infectious material was injected using a syringe. Then the soft tissue was sutured with catgut. The wound was treated with a brilliant green solution.

The day after the operation, observation of clinical manifestations began. We recorded indicators of the general condition of animals (lethargy, inactivity, rectally determined body temperature) and local manifestations (edema of soft tissues of the limb, local hyperthermia, sparing position of the limb, the presence of fistulas, abscesses). Markers for the development of inflammation were blood test data, bone marrow examination, scintigraphy, and histological examination.

A blood test was performed on the 7th day after the operation on an automatic hematology analyzer for veterinary medicine PCE 90 Vet (USA, High Technology). On the 21st day of the experiment, after euthanasia with CO ₂ asphyxia, the left femur was taken to quantify bone marrow cells, the right femur was placed in formalin solution for subsequent histological examination. Bone marrow was taken from the sternum for counting myelograms, smears were fixed in methyl alcohol for 5 min and stained with azure II - eosin prepared according to Noht. To determine the total number of myelokaryocytes (OKM), 1 ml of a 5% solution of acetic acid was washed out of the bone marrow from the channel of the left femur and resuspended. Additional dilution was performed using leukocyte melanger (final dilution of 20,000 times). The calculation of OKM was carried out using a Goryaev camera [5].

Three-phase scintigraphy with ^99m Tc-technofor (LLC Diamed, Russia, 18.5 MBq) was performed on the 10th day of the experiment on a Philips BrightView single-photon emission computer tomograph (USA). The study included radionuclide angiography, a soft tissue phase (blood pool) and a bone phase study (osteoscintigraphy).

For histological examination, the bone was decalcified according to Gripp [6], dehydrated in ascending alcohols and embedded in paraffin. Deparaffined sections with a thickness of 7 μm were stained with hematoxylin and eosin.

The results of experimental studies were statistically processed in the Statistica 10 program.

By the 6-7th day after the operation, the body temperature of diseased animals increased to 38.5 ° C, but by the 20th day there was a decrease in this indicator to 36.9 ° C. On day 2, in rats of group 2, immobility was observed, sparing the position of the limb, which remained throughout the experiment. By day 10, all rats of group 2 had pain on palpation, an increase in the knee joint by 2-3 times compared with a healthy limb, purulent discharge from the wound. By 15 days, three animals of group 2 formed abscesses in the region of the right femur. By 21 days the animals became more mobile, pain on palpation and swelling of the knee joint decreased.

The development of the acute inflammatory process was indicated by a significant increase in the blood of animals of group 2 of the number of leukocytes, neutrophilic granulocytes and monocytes on the 7th day of the disease (Table 1). The number of lymphocytes was significantly reduced compared to intact ones.

As a result of scintigraphy on day 10 in the group of healthy rats, the simultaneous entry of the indicator into the main vessels of the hind limbs, the absence of drug hyperfixation sites in the soft tissue and bone phases of the study were determined. In the second group, accelerated and increased drug entry into the arteries on the affected side was recorded, and areas of indicator hyperfixation in the soft tissue and bone phases of the study were also determined (the average difference in counts compared to the symmetrical section was 115 ± 5% and 121 ± 10%, respectively), which was estimated by us as a manifestation of the acute stage of osteomyelitis (Fig. 1).

OCM in the bone marrow of diseased rats was significantly reduced compared to intact rats. As a result of the development of the disease, a predominantly myeloid germ was impaired, as indicated by a decrease in the neutrophil maturation index (ISN) of the diseased in comparison with healthy animals (Table 2). At the same time, the erythrokaryocyte maturation index (ISE) of animals of both groups did not differ significantly. As a result, the leukoerythroblastic ratio (LEO) of the bone marrow cells of animals of group 2 shifted towards a significant decrease in the cells of the myeloid series in comparison with group 1. In addition, in the bone marrow of diseased animals, the number of megakaryocytes decreased by 73%, monocytes by 61% and lymphocytes by 51% compared with the control.

A morphological examination of the femoral diaphysis revealed purulent bone marrow infiltration. There was necrosis and autolysis of the bone beams, the cortical layer of the bone, the walls of the channels of osteons. Bone resorption was accompanied by the appearance of small defects filled with pus in it, which merged into larger foci containing sequestra (Fig. 2). Along with inflammatory and necrotic changes in the bone tissue, reparative processes took place. On the periphery of the osteomyelitis lesion, the formation of granulation tissue containing a large number of fibroblasts and osteoblasts was noted. Necrosis sites were replaced by young bone tissue (Fig. 3). The results obtained characterized the classical picture of chronic osteomyelitis.

Information sources

1. Strelkov N.S. Modeling purulent osteomyelitis in the experiment: Proceedings of Izhevsky honey. in-that. - 1995. - T. 33. Part 3 - S. 114-115.

2. Peresylsky P.F. Experimental and theoretical aspects of the pathogenesis of hematogenous osteomyelitis. - Irkutsk, 1993 .-- 145 p.

3. Maslikov V.M. Comparative evaluation of surgical treatment methods for acute hematogenous osteomyelitis in children: author. ... candidate of medical sciences, M., 1979.

4. Matuzov S. A., Busoedov A. V., Kotlyarov V. N., Sizonenko V. A. Патент Patent No. 2139735 dated 04/27/1999.

5. A guide to practical exercises in hematologists: a training manual / Novitsky VV, Urazova OI - Tomsk: Tomsk University Press, 2007 .-- 230 p.

6. Pierce E. Histochemistry. M .: publishing house of foreign literature, 1962, 690 p.

Table 1. The results of blood tests of experimental animals

Table 2. Bone marrow myelogram of experimental animals

FIG. 1. Scintigraphy of rats on the 20th day after modeling of osteomyelitis. The increased concentration of the radiopharmaceutical in the soft tissue phase (a) and the bone phase (b) of the study in the affected area of the right femur.

FIG. 2. The femur of a rat on day 21 after modeling osteomyelitis. Necrosis and bone resorption in the area of purulent inflammation. Hematoxylin and eosin stain. SW 100 x

FIG. 3. The femur of a rat on day 21 after modeling osteomyelitis. Granulation tissue with a large number of osteoblasts. Plots of osteogenesis. Hematoxylin and eosin stain. SW 100 x

Claims (1)

A method of modeling traumatic osteomyelitis, including the formation of a defect in the bone tissue of the tibia in an experimental animal, followed by the introduction of an infectious material into the defect site, characterized in that the rats are preliminarily sensitized by triple intraperitoneal administration by attenuated heating at 60 ° C for 30 minutes of a culture of weakly virulent Staphylococcus aureus, and every 3 days, sensitization is carried out with a gradual increase in dose from 1 to 3 million bacterial bodies, d Then, three days after the end of sensitization, under sterile conditions, under soft anesthesia, an incision is made in the soft tissues, through the distal metaphysis of the femur with a thick needle, a hole is formed in the bone marrow canal, where active staphylococcus is injected at a dose of 6 million bacterial bodies in 0.1 g 30% gelatin solution, after which the soft tissue is sutured with catgut, the wound is treated.

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