RU2536556C2 - Method for simulating generalised amyloidosis in animals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to experimental nephrology and can be used for simulating generalised amyloidosis in animals for studying pathogenesis, prevention and treatment of amyloidosis. The method involves administering native sheep plasma into golden hamsters subcutaneously at 0.025 mg/g of body weight. Administering is performed for 30 experimental days.

EFFECT: method is easily reproducible, provides reducing the time of the simulation of the above pathology by greater antigenic strength of the selected protein preparation for the above animal series; the method enables studying the functional state of the renal system, mineral exchange and other body characteristics successfully.

1 ex, 2 tbl, 4 dwg

Description

This invention relates to medicine, experimental pathology and can be used to model experimental amyloidosis in animals, study the pathogenesis, prevention and treatment of amyloidosis, especially in the study of the functional state of the kidneys.

Amyloidosis (amyloid dystrophy) is a violation of protein metabolism, accompanied by the formation and deposition in tissues of a specific protein-polysaccharide complex - amyloid.

The development of amyloidosis is associated with a distortion of the protein-synthetic function of the reticulo-endothelial system, the accumulation in the blood plasma of abnormal proteins that serve as autoantigens. As a result of the interaction of the antigen with the antibody, the coarsely dispersed proteins involved in the formation of amyloid precipitate. Being deposited in tissues (for example, in the walls of blood vessels, glandular, etc.), the amyloid displaces the functionally specialized elements of the organ.

Amyloid is a complex glycoprotein in which fibrillar and globular proteins are closely related to polysaccharides. The amyloid consists of polypeptide chains, which determines the birefringence when staining Congo red, characteristic of amyloid. In addition to the fibrillar protein, the composition of the amyloid includes another protein - the so-called P-component, which is the same for all forms of amyloidosis.

It has been established that amyloid contains albumin, globulin, fibrinogen, fibrin; γ-globulin predominates among plasma proteins (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.

However, the mechanisms of the formation of amyloid deposits and organ dysfunctions have been studied very poorly.

There is a classical method for modeling experimental amyloidosis (A. Gritsman. Some issues of experimental therapy of amyloidosis and amyloid resorption // Abstract of dissertation for the degree of Candidate of Medical Sciences. M., 1974), which consists in the introduction of white mice every other day, 0.5 ml of 5% sodium caseinate subcutaneously for 60 days. Its disadvantage 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 related to the influence of sodium and its determination.

Also known is a method of modeling experimental amyloidosis in animals (RF patent for the invention No. 2269825, Zaalishvili T.V., Kozyrev K.M., registered 02.20.2009, application No. 2004127649/14 dated 09.15.2004, IPC 'G09B 23/28), which consists in administering to white mice every other day 1 ml of native egg albumin subcutaneously for 30 days of the experiment.

The disadvantage of this method 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 due to the small physiological volumes and anatomical dimensions, as well as the complexity and duration of the reproduction of the amyloidosis model.

The prototype is a method for modeling experimental amyloidosis in animals (RF patent for the invention No. 2347279, I. Pukhova, K. M. Kozyrev, B. B. Brin, registered on 02.20.2009 application No. 2007135901/14 dated 09.27.2007 g., IPC 'G09B 23/28), which consists in administering to the Syrian hamsters native pork plasma every other day subcutaneously at the rate of 0.025 ml / g body weight for 60 days of the experiment.

The disadvantage of the prototype is that the experiment uses pork plasma, which has less antigenicity and is introduced within 60 days.

The invention is aimed at solving the problem of developing a method for modeling generalized amyloidosis in animals.

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

The proposed method differs from the prototype in that native sheep plasma (with high antigenic properties) is used to model generalized amyloidosis at the rate of 0.025 ml / g body weight for 30 days of the experiment in order to obtain and the possibility of studying the pathogenesis, prevention and treatment of amyloidosis, especially when study of the functional state of the kidneys. The introduction of native sheep plasma leads to the development of systemic secondary amyloidosis with predominant damage to the kidneys, spleen, liver and myocardium within 30 days of the experiment.

The problem is achieved in that the method of modeling generalized amyloidosis in animals involves administering to the Syrian hamsters a protein preparation subcutaneously every other day at the rate of 0.025 ml / g body weight and differs in that native sheep plasma is administered as a protein preparation to the Syrian hamsters during 30 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."

Experimental animals (Syrian male hamsters weighing 100-120 g) as a protein preparation are injected with native sheep plasma (whose proteins have the highest antigenicity) every other day at the rate of 0.025 ml / g of body weight for 30 days of the experiment, which provides expanded modeling capabilities generalized amyloidosis in animals, due to the faster development of pathology, especially when studying the functional state of the kidneys, circulatory system, mineral metabolism and other experimental indicators animals, which allows us to conclude that the criterion of "inventive step".

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 quickly reproducible method for modeling generalized amyloidosis in animals, which allows us to conclude that the invention meets the criterion of "industrial applicability".

This method is as follows.

To obtain generalized amyloidosis, sexually mature Syrian male hamsters weighing 100-120 g were selected as experimental animals.

To simulate generalized amyloidosis, experimental animals undergo subcutaneous injections of native sheep plasma every other day at the rate of 0.025 ml / g body weight for 30 days. Native sheep plasma is obtained from the blood of sheep slaughtered in a slaughterhouse. Blood from the carotid artery is collected in a sterile vessel with the preliminary addition of heparin at a rate of 5000 units / liter of blood. The vessel is covered with a sterile plastic cover. Stabilized blood is poured into centrifuge tubes and centrifuged at a speed of 2500 rpm for 15 minutes. Next, the resulting plasma is collected using a sterile syringe with a capacity of 20 ml and poured into plastic tubes and frozen at a temperature of -10 ° C. Before injections, the plasma is thawed at a temperature of 30 ° C.

In control and experimental animals, a 6-hour spontaneous diuresis is examined. Glomerular filtration is determined by the clearance of endogenous creatinine, and tubular water absorption is calculated. The sodium and potassium content in the urine is determined by flame photometry, the calcium content is determined spectrophotometrically. The main processes of urination and excretion of electrolytes are determined after 15 and 30 days of the experiment.

After the experiment (1 month), the animals are killed under anesthesia. Morphological examination for the detection of amyloidosis (stained with hematoxylin and eosin, Congo red) undergoes internal organs. 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 study of the slices is carried out in transmitted light using a Mikmed-1 microscope under magnification x 80, x 200, x 400. The essence of the proposed method is confirmed functionally and morphologically.

Studies have shown that in conditions of spontaneous diuresis against the background of subcutaneous administration of native sheep plasma, an increase in diuresis after 1 month is noted (Table 1). The increase in diuresis is due to a decrease in tubular reabsorption of water.

Table 1 Changes in the volume of spontaneous diuresis and the main processes of urination during the formation of amyloid nephropathy Experience Conditions Statistical indicator Urination processes Mg / ml protein content Diuresis ml / hour / 100 SKF m / h / 100 Reabsorption
% Background M ± m 0.15 ± 0.007 21.89 ± 1.065 99.32 ± 0.026 0.89 ± 0.096 1 month the experiment M ± m 0.21 ± 0.007 22.0 ± 0.905 99.03 ± 0.031 1.79 ± 0.046

The excretion of potassium and calcium under the influence of amyloidogen under conditions of spontaneous 6-hour diuresis increases during the experiment by more than 1.5 times (Table 2).

table 2 Changes in urinary electrolyte excretion during the formation of amyloid nephropathy Experience Conditions Statistical indicator Electrolyte excretory function Excretion Na Excretion K Excretion Sa Background M ± m 11.18 ± 0.389 6.76 ± 0.394 0.55 ± 0.043 1 month experiment M ± m 10.82 ± 0.805 11.67 ± 0.492 0.99 ± 0.093

Under the influence of the introduction of native sheep plasma, there was a progressive increase in proteinuria by a factor of 2 on the 30th day of the experiment (Table 1).

Morphologically noted:

Figure 1. Plasma impregnation of the walls of the central arteries of the lymphoid follicles, perivascular edema (1), passing to the intercellular spaces with the accumulation of congophilic substances (2).

Figure 2. Congophilia of the vascular glomerulus (1), basal membranes of the tubules (2) and stromal-vascular structures of the kidney (3). Coloring Congo red.

Figure 3. Swelling of cardiomyocytes, focal disappearance of transverse striation (1) and focal congophilia of certain groups of cardiomyocytes (2).

Figure 4. Congophilia of the vascular wall (1), small-drop fatty degeneration and necrosis of hepatocytes (2), perivascular lymphohistiocytic infiltrate (3).

Example

A series of experiments from 2 groups were carried out:

1st group (10 Syrian male hamsters) - control,

Group 2 (10 Syrian male hamsters) - amyloid - the animals were given subcutaneous injections of native sheep plasma every other day at the rate of 0.025 ml / g of body weight of Syrian male hamsters for 30 days.

Morphological examination for the detection of amyloidosis (Congo-red color) subjected to internal organs, blood vessels.

As a result of the studies, morphological evidence was obtained of the effectiveness of the use of native sheep plasma for modeling experimental amyloidosis in Syrian male hamsters.

Histologically, in the 2nd group of animals in the kidneys (Fig. 2), when Congo-red sections were stained, focal congophilia of the microvasculature, vessels and stromal structures of the renal cortical and brain layers was noted, hyperemia, plasma impregnation and perivascular edema, most pronounced in the cortical layer. Hypertrophy of individual glomeruli with compression by the swollen capillaries of the malpighian body of the Shumlyansky-Bowman capsule. Thus, the initial manifestations of a model of systemic amyloidosis obtained by subcutaneous administration of native sheep plasma for 30 days are morphologically documented.

Based on the results of the study, the use of native sheep plasma for modeling amyloidosis in Syrian hamsters is justified.

Summing up, it should be noted that subcutaneous administration of native sheep plasma to Syrian hamsters is an effective way to model experimental amyloidosis.

Claims (1)

A method for modeling generalized amyloidosis in animals, comprising administering to the Syrian hamsters a protein preparation subcutaneously every other day at the rate of 0.025 ml / g body weight, characterized in that native sheep plasma is administered as a protein preparation to the Syrian hamsters for 30 days of the experiment.

Images