RU2587039C1 - Method for simulating osteoarthrosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine, namely to rheumatology, and can be used for simulating osteoarthrosis (SO), approbation agents and methods of treating it. Method for simulating osteoarthrosis in Wistar rats by daily subcutaneous injections of 1 % 0.1 ml mezaton solution throughout 2 weeks. After each injection provide animals with swimming for 20 minutes. Further, animal is placed on 2 months in close cells with its provision of caloric supply. Diet includes not less than 55 % of carbohydrates, not less than 30 % of fats and not less than 9 % proteins from diet.

EFFECT: method provides for development of osteoarthrosis articular cartilage with 2-3 degree 2 stages, being non-injuring, maximum allow for etiopathogenesis of disease, does not require special equipment and high time expenses.

1 cl, 3 tbl, 20 dwg

Description

The invention relates to the field of experimental medicine, namely to rheumatology, and can be used both for biological modeling of osteoarthritis (OA) with lesions of articular cartilage (CX) of the 2-3 degree 2 stage, and for testing means and methods of its treatment.

OA (in the foreign literature “osteoarthritis”) is the most common age-associated group of diseases of the musculoskeletal system with a similar pathogenesis, clinical picture, and damage to all elements of the joint elements, primarily cartilage and subchondral bone. Total prevalence, physical suffering, disability of potentially able-bodied individuals and significant financial losses act as a generator of the search for new approaches to the study of OA. In recent scientific papers [9, 10], the term “early” OA is used, which has clear histological criteria (1-3 degrees of SC lesion according to the OARSI classification [11]). Early OA is an object of research of scientists of recent years and represents an urgent problem of rheumatology [9]. But a non-traumatic intravital study of cartilage in humans is difficult, as well as there is no possibility of a dynamic assessment of his condition during treatment, which dictates the need for OA modeling in the experiment.

For experimental reproduction of OA, various methods are used that can be conditionally divided into several groups: the use of animals with genetically determined traits, surgical (operative) manipulations, intra-articular administration of chemical compounds and traumatic agents, mathematical modeling, methods of indirect effect on the joint (by changing hormonal status, violation of biomechanics, the intake of toxic substances, etc.).

In a group of methods involving the use of animals with a modification of the genetic apparatus, a model of spontaneous OA is known that develops in STR / OPT mice [12]. All animals aged 32-35 weeks develop premature degeneration of CX and other tissues of the knee joint, most pronounced in the medial plateau of the tibia. This model is extremely difficult to reproduce, since the STR / 1N mice are required to obtain the STR / OPT line, the closely related crossing of which under certain conditions (after the non-inbreeding period) ensures the birth of animals with the necessary genetic and phenotypic characters. Also important disadvantages of this model are: a long period of OA formation (on average 6 months), individual variability of damage to joint tissues, synovitis is not typical for the model.

Among the group of surgical methods for producing OA, a violation of regional blood circulation of the joint by femoral artery intersection is known [3]. Under the operating conditions in dogs, the femoral artery is isolated on two hind limbs at the level of the middle third of the femur, ligated in two places and intersected between the ligatures, then the surgical wound is sutured in layers and the limbs are immobilized by the Ilizarov apparatus for 28 days. Excretion of animals from the experiment is carried out 28 days after x-ray confirmation of the development of OA of the knee joints. Disadvantages of the model: the development of gross destructive changes in the joints that do not involve the study of the early stages of the disease, a complex technique of execution (requires a surgical team, sterile operating room, equipment, anesthetic, Ilizarov apparatus, possession of surgical skills), tissue trauma, the risk of animal death, the development of postoperative complications .

Among the methods for modeling OA by intraarticular injection of chemicals, there is a method involving the use of trypsin [8]. Outbred white mice of both sexes were injected with 0.1 ml of a 0.1% trypsin solution into the knee joint under intraperitoneal anesthesia with ketamine and xalazine; animals were withdrawn from the experiment on days 14 and 28.

The disadvantages of the model are: trauma to joint-forming structures, acute local destruction of cartilage tissue under the influence of the drug (mismatch of the resulting pathology to a human disease), limited possibility of testing drugs for treating OA, technical difficulties in implementing the method (conditions for manipulations, drugs for anesthesia), risk of complications after infestations, the development of monoarthrosis (in humans, OA usually affects several joints).

Among the methods of non-invasive effects on the joint, a method for producing systemic steroid OA in rats is known [2]. To reproduce it, the authors performed three intramuscular injections of dexamethasone phosphate at a dose of 7 mg / kg with an interval of one week to male Wistar rats weighing 250-300 g. The key disadvantage of the model is the mismatch between the etiopathogenesis of corticosteroid arthropathy and the experiment with OA in humans: chronic overdose dexamethasone provides the development of secondary, drug-induced OA in rats. The reproduced pathology is accompanied by systemic side effects, this prevents the correct study of the condition of the joints, including when testing drugs. Thus, the existing models suggest specific triggers for the development of OA that differ from the real causes of its development in humans, and an optimal biological model of early OA has not yet been proposed.

Prototype

As a prototype, a method was used consisting in exposing Wistar rats to a two-week course of daily subcutaneous injections of 1% 0.1 ml of a solution of mesatone, followed by swimming after each injection for 20 minutes in a tank of water [4]. This drug - an adrenergic agonist - activates hypertensive mechanisms and, in combination with intense physical activity, is used to produce chronic heart failure (CHF) according to V.I. Incineration [6, 7].

When assessing the changes in the joints of animals, we can say that they correspond to the 1st degree of the 1st stage of cartilage damage according to the OARSI classification. However, the method does not allow to reach and investigate the further stages of cartilage damage (up to the 3rd degree according to the OARSI classification), this has become a key disadvantage of the method.

The objective of the invention is the creation of the most physiological, non-traumatic, taking into account the pathogenetic and pathomorphological aspects of the disease of the biological model of OA with damage to the articular cartilage of the 2nd to 2nd degree stage 2 for its further study and testing of drugs and treatment methods.

The essence of the invention lies in the fact that Wistar rats are subjected to a two-week course of daily subcutaneous injections of 1% 0.1 ml of a solution of mesatone, followed by swimming for 20 minutes after each injection in a tank of water, after which the animals are placed in tight cages for 2 months and provided with a high-calorie diet, including at least 55% carbohydrates, at least 30% fat and at least 9% protein from the diet.

The implementation of the method

The proposed method was reproduced in an experimental study on 20 sexually mature Wistar male rats weighing 304-498 g. All animals were randomly divided into 3 groups. The rats of the first - control - group (5 animals) were on the standard diet of the vivarium, then they were withdrawn from the experiment on its last day by an overdose of ether anesthesia. The remaining rats (15 animals) were subjected to a daily 20-minute swim after subcutaneous injection of 0.1 ml of a 1% solution of mesatone for 14 days. Of 15 animals, 5 rats (the second group) were withdrawn from the experiment using ether anesthesia. The remaining 10 animals made up the third group, were placed in tight cages, and for 2 months were on a high-calorie multicomponent diet, including millet (21%), muffin (30%), potato fried in vegetable oil (20%), raw pork lard ( 12%), chocolate (4%), hazelnuts (4%), cookies (5%), sunflower halva (4%), 20% free fructose solution was used instead of water (total proteins - 9.61%, fats - 31.87%, carbohydrates - 58.52%). Animals were weighed on the 1st, 30th, and 60th days, and the total and specific weight gain were determined. In each group, a clinical assessment of rats was performed. After 2 months, the animals were also withdrawn from the experiment by an overdose of ether anesthesia.

All rats were decapitated, for morphological studies they took knee joints and internal organs (heart, liver, kidneys, lungs) to confirm the development of vascular and metabolic disorders. After standard histological posting, the preparation of serial sections with a thickness of 5-6 μm was carried out on a rotational microtome MPS-2. For panoramic microscopy, paraffin sections were stained with Mayer hematoxylin and eosin [5]. The identification of siderophages in the lungs of rats with heart failure was carried out by Perls reaction [5]. Glycosaminoglycans were determined with 0.5% toluidine blue at different pH values (7.4; 4.7) with control (treatment with hyaluronidase). On paraffin sections on Super frost plus glasses, collagen type II was identified using a set of monoclonal antibody reagents for immunohistochemistry (BioCenex, USA), expression of caspase-3, matrix metalloproteinase MMP-9 was detected. Coloring was carried out according to the protocol of manufacturers. The degree of expression of type 2 collagen and MMP-9 was assessed visually by a point system from 1 to 4, the number of caspase-3 expressing cells was calculated and expressed as a percentage. Using the MOV-1-1.5 * eyepiece micrometer, the thickness of the CX layers was determined in 4.2 using an ocular insert network, and the relative bulk density of the vessels of the microvasculature of the synovial membrane (SM) was calculated [1]. Statistical data processing was performed using the software package "MS Excel 2007" and "Statistica 6.6 for Windows". The arithmetic mean values of the absolute and relative values, the errors of the mean values and standard deviations were calculated. The significance of differences in the compared parameters was determined by Student t-test, differences in average values were considered significant at a significance level of p <0.05%.

Research results

A clinical assessment of the condition of the animals of the control group did not reveal pathology. The second group of animals showed signs of heart failure: cyanosis, rapid breathing, rats rested their forelimbs on the side of the cell to engage additional respiratory muscles, the rear ones were swollen and cyanotic. The duration of normalization of the respiratory rate was 10-15 minutes, acrocyanosis (cyanosis of the skin of the ears, paws, tail) remained at rest. A clinical assessment of the condition of rats of the third group, in addition to the symptoms of heart failure, revealed a change in the behavior of rats: an increase in the number and frequency of meals and a solution of fructose, slow reactions, inactivity, and weighing of animals revealed an increase in their body weight by 30-36% from the original (Table 1).

To confirm the development of pathology (CHF, obesity), a morphological study of the internal organs of rats (heart, liver, kidneys, lungs) was performed. In the control group, myocardial preparations stained with Mayer hematoxylin - eosin, determined contractile cardiomyocytes with oval nuclei, clear contours, uniform color, transverse striation, between them were layers of intermuscular loose connective tissue (Fig. 1, magnification 300). In the liver, hepatocytes formed beams, between which there were sinusoidal capillaries directed to the central veins, the liver cells were separated by bile capillaries (Fig. 2, magnification 600). In the kidneys, brain and cortical matter with renal bodies and convoluted tubules was determined, the glomeruli were normal in size, the capsule lumen was free, the tubule epithelium was cubic in shape with clear contours, and the lumen was preserved. Respiratory alveolocytes were formed in the lung tissue, forming interalveolar septa with macrophages, and interstitial tissue was located peribronchially. Microscopy of the internal organs of animals in the control group revealed no pathology.

A morphological study of the internal organs stained with Mayer hematoxylin - eosin in rats of the second group in the myocardium (Fig. 3, magnification 300) revealed areas of muscle contraction, convergence of insertion discs, and an increase in the proportion of connective tissue. In the liver (Fig. 4, magnification 600), stagnation phenomena (dilated sinusoidal capillaries with sulphated blood cells, the phenomena of small-drop fatty degeneration in hepatocytes of centrolobular zones) were noted. The vessels in the lungs were dilated, siderophages were identified in the lumens of the alveoli, and hemosiderin was determined in the alveolar septa. In the kidneys, venous congestion and initial signs of hyaline droplet dystrophy of the tubule epithelial cells were noted. These data confirmed the fact of the development of heart failure in rats.

Microscopic examination of the internal organs of animals of the third group revealed the most pronounced signs of their dystrophy. In myocardial micropreparations stained with Mayer hematoxylin and eosin (Fig. 5, magnification 400), connective tissue predominated, in its interlayers and under the epicardium there were accumulations of adipocytes, in cardiomyocytes there were areas of re-contraction and convergence of insertion discs. In the liver (Fig. 6, magnification 400), large-droplet steatosis was observed in the centrolobular (damage to the ischemic genesis) and peripheral zones (mainly due to the nutritional factor), part of the hepatocytes underwent necrosis with exposure of sinusoidal capillaries (a symptom of a “muscular” liver in heart failure). Lymphocyte infiltration sites were determined. Sites of fibrosis were found in the lung tissue, brown induction of the lungs (the interalveolar septa became thicker, siderophages were identified by the Perls reaction), and degenerative calcification (atherocalcinosis) was detected in the walls of some vessels. In the kidneys there were signs of hyaline droplet and hydropic dystrophy, accumulation of adipocytes in the interstitial tissue, hyaline droplets in the tubules, the vessels were dilated and filled with sweetened red blood cells. The data obtained confirmed the development of obesity in the presence of heart failure in rats of the third group.

Microscopic examination of rat knee joints revealed their unequal morphofunctional state, comparative characteristics of SM, CX, and subchondral bone of animals are shown in Tables 2, 3. Analysis of the morphology of the joints of rats of the first group revealed that they are healthy tissues, the rest showed signs of their reorganization. In the second group, the initial changes in cytoarchitectonics and functional activity of cells of a degenerative nature were found, in rats of the third group, the most pronounced signs of disorganization of the components of the joints were noted.

Technical result

The first signs of degenerative joint disease in experimental animals were found on the 14th day of OA modeling. Morphologically confirmed OA with CX lesion corresponding to stage 2–3 grade 2 according to the OARSI classification [11] develops over the next 2 months.

Thus, in experimental animals, a polyetiological, as close as possible to real clinical conditions and technically uncomplicated biological OA model with a 2–3 degree 2 stage of CX lesion was obtained. It is characterized by 100% reproducibility, zero mortality, lack of exposure to toxic substances, corticosteroid drugs, suggests an indirect, non-traumatic effect on the joint. This method of obtaining OA is cost-effective, it does not require special equipment, animals with a modified genetic apparatus, significant time costs, it is feasible in vivarium conditions.

The proposed solution is not a modification or summation of known methods; it is based on a completely original idea and is of great practical importance, since it allows carrying out experiments that are not feasible in the clinic to study the early stages of OA development, which are in the focus of attention of modern researchers, as well as to test new means and methods of treating this disease in experimental animals.

Information sources

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Claims (1)

A method for modeling osteoarthritis with damage to articular cartilage of degree 2 and 2 stages in Wistar rats by daily subcutaneous injections of 1% 0.1 ml of mesatone solution for 2 weeks, followed by swimming after each injection for 20 minutes and subsequent placement of animals for 2 months in cramped cells providing them with a high-calorie diet, including at least 55% carbohydrates, at least 30% fat and at least 9% protein from the diet.

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