Method of experimental pseudorheumatism modelling

FIELD: medicine.

SUBSTANCE: invention concerns experimental medicine, namely to experimental rheumatology and can be used for pseudorheumatism modelling. For this purpose 4 mg of an ovalbumin into 1 ml of full Freund's adjuvant (FFA) in the knee joint area are entered subcutaneously. After ovalbumin introduction in FFA, intravenously a papain in a dose of mass of a body of 8-10 mg/kg is entered. Then for 12 days from the beginning of formation of model 5 mg of an ovalbumin is entered into a cavity of a knee joint, thus enter an ovalbumin in FFA unitary.

EFFECT: reduction of quantity of stages and terms of modelling of pseudorheumatism, formation of more expressed system inflammatory reactions in an organism that allows to spend approbation of complex ways of treatment with use not only locally - but also is system operating preparations.

1 ex

 

The invention relates to experimental medicine and can be used for modeling of rheumatoid arthritis in animals when testing different methods and means of treatment.

There is a method of modeling of rheumatoid arthritis in rats by injection of 0.07 to 0.08 ml complete adjuvant's adjuvant (PAF), after which the animal is exposed to immobilization stress for 1 hour daily in the period from 14-th to 28-th day (patent RF №2305328, CL G 09 23/28. Publ. 27.08.2007, bull. No. 23).

The disadvantages of the method are the use of long-term (two-week) daily hour immobilization stress, and intradermal PAF, which creates certain difficulties of a technical nature. In addition, the way a long time (28 days).

There is also known a method of simulating rheumatoid arthritis in rats by injecting PAF and bovine serum albumin followed by electrical stimulation through electrodes implanted in the region of the base of the tail and ankle (copyright certificate №1748174, CL G 09 23/28. Publ. 18.06.1990, bull. No. 26).

The disadvantages of this method are considerable complexity and the complexity of modeling arthritis. For complete method requires surgery in rats, for which you commit to operating with the OLE, intraperitoneal anesthesia with thiopental, subcutaneous implantation of electrodes in the region of the base of the tail and ankle, suturing of the skin in the area of surgical intervention. In addition, carry out the introduction of the PAF in the base of the tail and bovine serum albumin pad hind legs.

This method allows you to play only severe form of arthritis that is designed to explore advanced stage of the disease with a strong inflammatory destruction of the joints, when therapeutic interventions, including medication, are ineffective. At the same time, the need to study thin pathogenetic mechanisms of the disease and improving diagnosis of metabolic disorders, as well as testing new patterns of drug therapy perspective it is in the initial stage, when the minimum degree of activity of the inflammatory process.

Closest to the proposed invention in its technical essence is the following method modeling of rheumatoid arthritis in rabbits: injected subcutaneously with 4 mg of ovalbumin in 1 ml of PAF in the region experienced knee joint, after 14 days of repeated immunization, 5 days after which experienced knee enter the resolving dose of ovalbumin (5 mg; 2-3 days to register the formation of the tion arthritis (Pettipher E.R., Henderson C., Moncada S. et al. Leucocyte infiltration and cartilage proteoglycan loss in immune arthritis in the rabbit // Br. J. Pharmacol. 1988. V.95. P.169-176).

The disadvantages of this method are a lot of stages and the duration of the formation model (21-22 days), as well as the lack of significant systemic manifestations of inflammatory reactions in the body.

The task, which is aimed by the invention is the reduction of the number of stages and timing simulation of rheumatoid arthritis, the formation of a more pronounced systemic inflammatory reactions in the body, which allows for the testing of complex methods of treatment using not only locally but also systemically acting drugs.

The problem is solved in that in the method of modeling the experimental rheumatoid arthritis by subcutaneous injection of 4 mg of ovalbumin in 1 ml complete adjuvant's adjuvant (PAF) in the region of the knee joint with subsequent intra-articular introduction 5 mg of ovalbumin, optionally, after subcutaneous injection of ovalbumin in PAF, intravenous papain at a dose of 8-10 mg/kg of body weight, and on the 12th day since the beginning of the formation of the model - ovalbumin into the cavity of the knee joint, while ovalbumin in PAF enter once.

The inventors found that an additional intravenous injection of papain, a proteolytic enzyme of plant p is ashodaya, simultaneously with subcutaneous injection of 4 mg of ovalbumin in 1 ml of PAF in the region experienced knee joint leads to rapid degradation of the protein and carbohydrate components of the matrix of connective tissue and joint structures, increasing their inflammatory destruction that leads to more pronounced and systemic inflammatory reactions in the body.

Additional intravenous papain allows you to enter ovalbumin in PAF once, reducing the number of stages by eliminating repeated immunization, and implement the introduction of ovalbumin into the cavity of the knee joint on the 12th day since the beginning of the formation of the model, which leads to the reduction of the time of model generation for up to 15 days.

Selected empirically single dose of papain 8-10 mg/kg of body weight the most appropriate and effective for the formation of a pronounced systemic inflammatory reactions in the body. Dose of less than 8 mg/kg does not provide a full model, and the dosage more than 10 mg/kg does not give an additional positive effect.

The technical result is to reduce the number of stages and the acceleration simulation of rheumatoid arthritis, as well as in the formation of more pronounced systemic inflammatory reactions in the body.

The method is implemented as follows.

Laboratory animal, in particular a rabbit, fixed on with the Anke for laboratory animals. Conduct training of the knee joint: shave the hair, after which the skin is treated with a 5%solution of iodine or alcohol 1%solution of brilliant green. In the experiment used the PAF containing 20 mg of killed by autoclaving mycobacteria in the form of dried BCG vaccine, 10 ml of vegetable oil and 2 ml of physiological solution. PAF prepared immediately before use, sterile, whisking vigorously. As the antigen used ovalbumin. Immunization of the animal is carried out by subcutaneous injection of 4 mg of ovalbumin in 1 ml of PAF in two points (lateral and medial) in the field of experimental knee joint.

Thereafter processed in a 5%solution of iodine marginal ear vein of a rabbit injected with papain at a dose of 8-10 mg/kg of body weight. After carrying out the manipulation of the injection sites stick with bactericidal plaster.

On the 12th day after immunization sterile experienced in the knee joint of an animal injected with 5 mg of ovalbumin in 1 ml of physiological solution. After 2-3 days there was a picture of pronounced arthritis.

The experiment was conducted on 43 rabbits breed "Chinchilla Russian", weighing 3.0 to 3.6 kg, which was in standard vivarium conditions with free access to food and water. Of these 15 individuals modeling arthritis was carried out according to the method Pettipher E.R. et al., (1988) and in 20 - and h is th way. 8 intact (healthy) rabbits constituted the control group. The reproducibility of the experimental model of rheumatoid arthritis was 100%.

The formation of the model was confirmed by the analysis of both local and systemic clinical and laboratory manifestations of arthritis. Clinical research methods include inspection, an overall assessment of animal behavior, activity, weight and appetite, palpation of the joints, measurement of a circle, volume of movements in the joints. Also included imaging data, hematological, biochemical and histomorphometrical methods. Studies were conducted in serum and content of the articular cavity.

A comparative analysis of how the proposed and known, selected as a prototype, methods develops the classic pattern of arthritis. However, the inventive method has allowed to build the model in a shorter time than known. This revealed a higher degree of overall activity of the inflammatory process with abrupt shift indicators gamesforum, as well as more pronounced metabolic disorders. The latter are determined by the degree of inflammatory and destructive processes in the connective tissue, the intensification of the processes of lipoperoxidation and failure of antioxidant defenses, leading to the development of p is racino-antioxidant imbalance.

EXAMPLE. Rabbit No. 6, weight 3.4 kg, formed the experimental rheumatoid arthritis. On the 15th day after the beginning of the experiment, conducted a comprehensive survey of the rabbit in order to confirm the formation of the model.

When inspecting the animal is lethargic, a little flexible as compared with intact animals, wool dull with bald spots. The body weight of the animal was 3.2 kg Circumference experienced joint, in which were entered the resolving dose of the antigen-ovalbumin increased by 6 mm and was 14 mm (before the start of the experiment (8 mm). Broken anchor function experienced limbs due to swelling and pain reactions. Palpation experienced knee painful (animal draws back the limb). Volume of active and passive movements are reduced.

When thermal imaging survey detected a pronounced temperature asymmetry in the region of the knee. The temperature difference between the test and control joint was 1.3°C.

The clinical picture of the disease is confirmed by the results of laboratory studies. Laboratory data blood: ESR - 8,00 mm/h at the rate of 2.8±0.20 mm/h, hemoglobin - 118,20 g/l at norm 124,30±0,80 g/l; erythrocytes - 3,33·1012/l at the rate of 4.04±0,10·1012/l; leucocytes - 10,14·109/l at norm 7,60±0,11·109/l; stab - of 5.82% but when the IU 3,96± 0,18%; segmented - 51,37% at the rate 42,60±0,81%; lymphocytes - of 33.26% at the rate 47,80±0,84%; eosinophils - to 2.57% at the rate 1,49±0,10%; monocytes - to 4.62% at the rate 3,44±0,12%; molecules of average weight - 0,312 eop the norm 0,236±0,002 eop; ceruloplasmin 38,01 $ with the norm 27,77± $ 1,03; malonic dialdehyde - 5.2 mmol/l at norm 3,59±0.15 mmol/l; glycosaminoglycans, as defined by uronic acid and 3.4 g·10-2/l at norm 1,47±0.03 g·10-2/HP

Laboratory data content of the articular cavity: General lymphocytosis - 2,12·109/l at the rate of 0.15·109/l; neutrophils - 42,00% at the rate of 9.51±0,11%; lymphocytes - 41,03% at the rate 8,43±0,09; macrophages - 38,22% at the rate of 0%; regolitopodobnykh cells 30,51% at the rate of 0%.

When histomorphometrical study experienced tissues of the knee joint revealed thickening paraartikulyarnye tissues, razvlechenie bundles of collagen fibers and weak perivascular metachromasia, proving the existence mulenga swelling is a sign of disorganization of connective tissue. Was determined moderately expressed the proliferation of fibroblasts and weak lymphoid cell infiltration. The sum of the scores of symptoms - 7.

The synovial membrane was thickened and crumbly. Was determined focal proliferation of synoviocytes, revealed hyperplasia of the villi and proliferation of cereals is s young pyroninophilia forms of fibroblasts. There were inflammatory and exudative characteristics loosening of the surface layer. Was determined proliferative endovascular. From the signs of disorganization of connective tissue revealed perivascular metachromasia as a result of enhanced synthesis of glycosaminoglycans by fibroblasts and synoviocytes and decomposition of protein-carbohydrate complexes of the basic substance of connective tissue. Found sclerosis, often perivascular fibrosis and hardening of the stroma subintimal layer. The amount of points the signs of destruction - 11.

And menisci revealed proliferation of fibroblasts, some weakening of metachromasia deep zones and plasma cells. The amount of points the signs of destruction - 5.

Joint the cartilage had degenerative changes typical of rheumatoid arthritis: focal loss of the outer layers of the superficial zone of the cartilage, the focal razvlechenie surface and partially the main areas, diffuse arthropathy, indicating the release of glycosaminoglycans, loss of chondrocytes and degeneration of the remaining, which was accompanied by paroniria and increased activity of acid phosphatase is a marker enzyme to the lysosomes. The sum of the scores of symptoms is 4.

At the same time the structure of subchondral bone was broken. Revealed thinning of the subchondral bone plate and tra is ekul, indicating a loss of bone substance. The sum of the scores of symptoms - 2.

Analysis of the results histomorphometrical research has established that this rabbit in the tissues of experimental knee joint developed rheumatoid inflammation, morphological substrate which also, as in patients with rheumatoid arthritis are two interrelated processes: inflammatory infiltration with the proliferation of synoviocytes and disorganization of the matrix of connective tissue structures that form the elements of the joint.

Conclusion on histomorphometrical the study: this rabbit has the characteristics of apparent chronic nonspecific productive synovitis.

Thus, on the 15th day from the start of the experiment the animal are pronounced local and General clinical manifestations of arthritis with a high degree of General and local activity of the inflammatory process and material breach of metabolic homeostasis.

The proposed method reduces the number of steps and timing modeling, rheumatoid arthritis, and also generates a more pronounced systemic inflammatory response in the body.

Reliability and efficiency reproduce experimental models of rheumatoid arthritis, the comparability of clinical manifestations and hematological, bi the chemical and morphological changes in animals and patients with rheumatoid arthritis, the availability of the drugs used allow us to recommend the model to study various schemes medication and non-medication therapy.

The method of modelling of experimental rheumatoid arthritis by subcutaneous injection of 4 mg of ovalbumin in 1 ml complete adjuvant's adjuvant (PAF) in the region of the knee joint with subsequent intra-articular introduction 5 mg of ovalbumin, characterized in that, in addition, after injection of ovalbumin in PAF, intravenous papain at a dose of 8-10 mg/kg of body weight, and on the 12th day since the beginning of the formation of the model - ovalbumin into the cavity of the knee joint, while ovalbumin in PAF enter once.



 

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