RU2306617C1 - Method for modeling experimental amyloidosis in animals - Google Patents

Abstract

FIELD: medicine, experimental biology.

SUBSTANCE: the method suggested deals with injecting a protein preparation subcutaneously. As such protein preparation one should inject native bovine plasma for golden hamsters every other day at 0.025 ml/g body weight for 60 d during experimental period.

EFFECT: higher efficiency.

5 dwg, 1 ex

Description

The invention relates to medicine, experimental biology and can be used to model experimental amyloidosis in animals with the aim of studying the pathogenesis, prevention and treatment of amyloidosis, especially when studying the functional state of the circulatory system, kidneys, mineral metabolism and other indicators.

Amyloidosis is a stromal-vascular dysproteinosis characterized by the deposition of abnormal fibrillar proteins in tissues and organs, the precursors of amyloid - a complex glycoprotein in which fibrillar and globular proteins are closely associated with glycosaminoglycans. Possessing chemical inertness and immunological tolerance, amyloid uncontrollably accumulates in organs and tissues in large quantities, becoming a morphological substrate of many diseases and pathological processes. Severe amyloidosis leads to atrophy and sclerosis of organs, which is accompanied by the development of their functional insufficiency (Dammacco F. Amyloidosis: clinical picture, immunological and biomolecular features, treatment prospects. // Ann. Ital. Med. Int. - 1991. - Jan-Mar- 6 (1 Pt 2). P. - 107-116).

In this regard, there was an urgent need to create and develop methods for modeling amyloidosis in order to study the pathogenesis, prevention and treatment of amyloidosis.

Native bovine plasma attracted attention due to its availability, natural sterility, isotonicity, constant concentration, stable pH, and the absence of side impurities.

There is a classical method for modeling experimental amyloidosis (A. Gritsman. Some questions of experimental treatment of amyloidosis and amyloid resorption. // Abstract of dissertation for the degree of candidate of medical science, M., 1974), which consists in the introduction of white mice every other day, 0.5 ml of 5% sodium caseinate subcutaneously for 60 days, which was taken by us as an analogue.

The disadvantage of the analogue is that the preparation of a caseinate solution is associated with certain difficulties, such as the search for pure casein, the need to dissolve its exact weight in a boiling 0.25% sodium hydroxide solution, filtering, sterilization, storage. The finished solution of sodium caseinate has an alkaline reaction, a high sodium content, which sharply limits the use of this model in the study of the functional state of the kidneys, circulatory system, mineral metabolism and other indicators associated with the influence of sodium and its determination.

The closest is a method for modeling experimental amyloidosis in animals (RF patent for the invention No. 2269825, Zaalishvili T.V., Kozyrev K.M., registered 02.10.2006, application No. 2004127649/14, from 09.15.04. IPC G09B 23/28 ), which consists in introducing to white mice every other day 1 ml of native egg albumin subcutaneously for 30 days, which we took as a prototype.

To obtain experimental systemic amyloidosis, subcutaneous injections of native bovine plasma are given every other day at the rate of 0.025 ml / g of body weight of Syrian male hamsters aged 5-6 months. within 60 days.

The disadvantage of the prototype is that white mice are small animals, which greatly complicates the study of the functional state of various systems of experimental animals. Given that in white mice the volume of blood obtained after decapitation does not exceed 1.0 ml, it is difficult to use it for the needs of biochemical and pathophysiological studies. It is also difficult to study the functional state of the kidneys and circulatory system due to small physiological volumes and anatomical dimensions.

The invention is aimed at solving the problem of developing a method for modeling experimental amyloidosis in animals, including the administration of a protein preparation subcutaneously.

The solution to this problem provides the expansion of modeling capabilities of experimental amyloidosis in animals, especially when studying the functional state of the circulatory system, kidneys, mineral metabolism, and other indicators of experimental animals.

To achieve this technical result, the claimed invention of a method for modeling experimental amyloidosis in animals has the following essential features: the animals are given subcutaneous injections of native bovine plasma every other day at the rate of 0.025 ml / g of body weight of Syrian male hamsters aged 5-6 months. within 60 days.

The proposed method differs from the prototype in that native bovine plasma is used to model experimental amyloidosis in animals with the aim of studying the pathogenesis, prevention and treatment of amyloidosis, especially in the study of the circulatory system, functional state of the kidneys, mineral metabolism and other parameters of experimental animals. The introduction of native bovine plasma leads to the development of systemic secondary amyloidosis with a primary lesion of the spleen, liver, and kidneys within 60 days of the experiment.

The claimed method is effective, cost-effective and easily reproducible.

According to the information available to the author, the set of essential features characterizing the essence of the claimed invention is not known, which allows us to conclude that the invention meets the criterion of "novelty."

According to the author, the essence of the claimed invention should not be obvious for specialists from a known level of medicine, since the above mentioned possibility of obtaining a method for modeling experimental amyloidosis in animals, including the administration of a protein preparation subcutaneously, characterized in that Syrian is introduced as a protein preparation, is not revealed from it. native bovine plasma every other day at the rate of 0.025 ml / g body weight for 60 days of the experiment, which extends the modeling capabilities of ex erimentalnogo amyloidosis in animals, especially in the investigation of the functional state of the circulatory system, kidneys, mineral metabolism and other parameters of experimental animals, allowing to conclude that the criterion "Inventive Level".

The set of essential features characterizing the essence of the invention, in principle, can be repeatedly used in medicine to obtain a result consisting in an effective and easily reproducible method for modeling experimental amyloidosis in animals, which allows us to conclude that the invention meets the criterion of "industrial applicability".

This method is as follows.

To obtain experimental systemic amyloidosis, subcutaneous injections of native bovine plasma are given every other day at the rate of 0.025 ml / g of body weight of Syrian male hamsters aged 5-6 months. within 60 days.

Native bovine plasma is obtained from the blood of bulls slaughtered in a slaughterhouse. Blood from the carotid artery is collected in a sterile vessel with a capacity of 3 l with the addition of heparin at the rate of 5000 IU / l of blood. The container is closed with a sterile plastic cover. Stabilized blood is poured into centrifuge tubes and centrifuged for 20 minutes at 3000 rpm. The resulting plasma is collected using a sterile syringe with a capacity of 20 ml, poured into plastic tubes and frozen at a temperature of -10 ° C. Before injections, the plasma is thawed at a temperature of 30 ° C.

After the experiment (2 months), the animals are killed under anesthesia. Morphological examination for amyloidosis (stained with hematoxylin and eosin, Congo red) undergoes internal organs, blood vessels. Tissue samples are fixed in 10% neutral formalin, followed by preparation of paraffin sections with a thickness of 5-6 microns. Sections are stained with hematoxylin and eosin, Congo red. The sections were studied in transmitted light using a Mikmed-1 microscope under magnification × 80, × 200, × 400.

The essence of the proposed method is confirmed morphologically:

Figure 1. Focal inflammatory myocardial infiltrates, phenomena of endo- and perivasculitis, focal deposits of amyloid, parenchymal dystrophy and foci of hemorrhage.

Figure 2. Amyloid deposits in the walls of blood vessels, mainly in the intima and muscle layer. Thickening and loosening of the walls of blood vessels.

Figure 3. Amyloid deposition in the liver is predominantly in the centers of lobules around the vessels.

Figure 4. Amyloid deposition in the tubules of the kidneys. Severe parenchymal dystrophy of the tubules.

Figure 5. Amyloid deposition in the spleen is predominantly around lymphoid follicles.

Example 1. A series of experiments from 2 groups:

1st group - control (20 Syrian male hamsters).

Group 2 (20 Syrian male hamsters) - (amyloid) - the animals received subcutaneous injections of native bovine plasma every other day at the rate of 0.025 ml / g of body weight of Syrian male hamsters aged 5-6 months. within 60 days.

Morphological research on the subject of amyloidosis (Congo-red color) subjected the internal organs, blood vessels.

As a result of the studies, morphological evidence of the effectiveness of the use of native bovine plasma for modeling experimental amyloidosis in animals was obtained.

Histologically, in the 2nd group of animals in the myocardium (Fig. 1), focal inflammatory infiltrates, phenomena of endo- and perivasculitis, focal deposits of amyloid, parenchymal dystrophy and foci of hemorrhages were found. Amyloid deposits were observed in the vessel walls (Fig. 2), mainly in the intima and muscle layer, which led to a thickening and loosening of the vessel walls. In the liver (Fig. 3), the amyloid was deposited mainly in the centers of the lobules around the vessels. In the kidneys (figure 4), the amyloid was detected along the direct vessels and collecting tubes. In the intermediate zone and pyramids, the stroma was saturated with plasma proteins. Amyloid was deposited not only in the pyramids, but also in the glomeruli. In the spleen (Fig. 5), the amyloid was deposited perifollicularly, lymphoid hyperplasia was noted.

Based on the results of the study, the use of native bovine plasma for modeling amyloidosis is substantiated.

From the above it follows that the introduction of native bovine plasma in animals is an effective way to model experimental amyloidosis.

Claims (1)

A method for simulating experimental amyloidosis in animals, comprising administering a protein preparation subcutaneously, characterized in that native bovine plasma is administered to Syrian hamsters as a protein preparation every other day at the rate of 0.025 ml / g body weight for 60 days of the experiment.

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