Method for simulating experimental amyloid cardiopathy in rats

FIELD: medicine.

SUBSTANCE: method consists in a single administration of a mixture consisting of a rat's homogenised myocardial tissue - 25%, egg albumin - 25% and Freund's adjuvant - 50%, into old male rats. The method involves administering in an amount of 0.3 ml into 5 injection points: intraperitoneally, into inguinal and axillary regions from the left and right.

EFFECT: being easy to reproduce and cost efficient, the method is effective for creating a model aiming at a possibility of studying the pathogenesis, preventing and treating cardiopathy.

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The invention relates to medicine, experimental biology and can be used to simulate the experimental amyloid cardiomyopathy in animals with the goal of studying the pathogenesis, prevention and treatment of cardiomyopathy.

Cardiopathy, cardiopathia (from the Greek. cardia - heart and pathos - suffering, defeat), the term applied to such changes of heart that are prolonged or persistent pathological condition which is a consequence of previous degenerative or inflammatory processes.

Of particular interest is the problem of amyloidosis of the heart, which is diagnosed in 2.3% of deaths before the age of 50 years, in the age group of 50-70 years it is detected in 30%, in the group of 70-80 years is already at 41%, and in persons who died at the age of 90 years, amyloidosis was found in 71%-90% of cases. In this regard, the aim of our study is to attempt to simulate a more simple way cardiopatico type of amyloidosis to obtain opportunities for further study of the pathogenesis, prevention and treatment of amyloid cardiomyopathy.

Amyloidosis, amyloid degeneration, impaired protein metabolism, accompanied by the formation in the tissue-specific protein-polysaccharide complex - amyloid. The development of amiloidosis associated with the perversion of the protein-synthetic function of the reticulo-�endoteliales system, the accumulation in plasma of abnormal proteins that serve as autoantigens and induce the formation of autoantibodies. As a result of interaction of the antigen with the antibody occurs deposition of particulate proteins involved in the formation of amyloid. Putting off in the tissues, amyloid displaces functionally specialized elements of the body, which leads to the death of this body.

Based on the above homogenized myocardium of rats applied with the aim of launching specific autoimmune reactions of the body, which in combination with amyloidogenic promotes the formation and deposition of amyloid masses in the myocardium.

A method of modeling of experimental amyloidosis in rats, taken by the authors for the prototype (patent of the Russian Federation for the invention 2410761 C1, gabuyeva A. A., Breen V. B., K. M. Kozyrev, registered 09.10.2009, the application 2009137437/14, 09.10.2009 IPC' G09B 23/28), which consists in the introduction of experimental animals equal mixture of native egg albumin and complete adjuvant of frand rate of 0.2 ml in 5 points of injection.

The disadvantages of the prototype is the low efficiency for specific modeling of amyloidosis of the heart.

Also known a method of simulation of experimental amyloidosis in animals, taken for analogue (RF patent for the invention №2306617 C1, Gioia Z. V., Zaalishvili T. V., Kozyrev, K. M., Breen B. V., registered�recorded 18.04.2006, application 2006113109/13, 18.04.2006 IPC' G09B 23/28), which consists in the introduction of the protein preparation subcutaneously. As the protein preparation is administered to Syrian hamsters native bovine plasma through the day from the calculation of 0.025 ml/g of body weight within 60 days of the experiment.

The disadvantages of analog is the need for repeated administration of protein of the drug subcutaneously every other day for 60 days of the experiment, and the relatively small size of the animals, which hinders functional methods of research models. In addition, Syrian hamsters difficult and uneconomical.

There is a way of modeling of experimental amyloidosis in animals, taken by the authors for the analog (RF patent for the invention №2347279 C1, Pukhov, I. W., K. M. Kozyrev, Bryn B. V., registered 27.09.2007, the application 2007135901/14, 27.09.2007 IPC' G09B 23/28), including the introduction of native porcine plasma through the day from the calculation of 0.025 ml/g of body weight within 60 days of the experiment.

The disadvantages of analog is the need for repeated administration of protein of the drug within 60 days through the day, and small size animals, which greatly complicates the study of the functional state of the cardiovascular system.

There is also a method of simulation of experimental amyloidosis in animals, also taken by the authors for analogue (paten� of the Russian Federation for the invention №2446482 C1, Breen V. B., Belikov A. T., K. M. Kozyrev, registered 13.11.2010, the application 2010146365/14, 13.11.2010 IPC' G09B 23/28), which proposes a single administration to Syrian hamsters native human plasma, mixed with the adjuvant of freind in equal proportions. The injection is carried out in a symmetrical axillary and inguinal region, and intraperitoneally at the rate of 0.1 ml in each point.

The disadvantage of this analogue is the low specificity bestselection effects on the myocardium.

The claimed invention is directed to solving the problem, which consists in the development of a new method of modeling of experimental amyloid cardiomyopathy in animals, consisting in the conduct of a mixture consisting of homogenized myocardial tissue of rats (25%), egg albumin (25%) and adjuvant of franda (50%), 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously to the left and right.

The solution of this problem provides empowering simulation experimental cardiomyopathy in animals.

The proposed method differs in that for modeling of amyloid cardiopathy injected a mixture consisting of homogenized myocardial tissue of rats (25%), egg albumin (25%) and adjuvant of franda (50%), 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously to the left and right.

The manufacturer.�th method is effective, cost-effective and easily reproducible.

According to the information set of essential features that characterize the essence of the claimed invention, is not known, which allows to make a conclusion about conformity of the invention the criterion of "novelty."

Experimental animals (old rats at the age of 18-24 months, weighing 350-400 g) once injected a mixture of homogenized myocardial tissue of rats (25%), egg albumin (25%) and adjuvant of franda (50%), 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously to the left and right, which allows extending the modeling capabilities of experimental cardiomyopathy in animals and allows to make a conclusion on compliance with the criterion of "inventive step".

The set of essential features that characterize the invention, in principle, can be repeatedly used in medicine with the result, consisting in an effective and easily reproducible method of simulation of experimental amyloid cardiomyopathy in animals, which allows to make a conclusion about conformity of the invention the criterion of "industrial applicability".

This method is as follows.

To obtain experimental cardiopathy in experimental animals selected Kry�s-males line "Wistar" weighing 350-400 g at the age of 18-24 months, so how exactly senile bradytrophic fabric allows you to raise the issue of modeling cardiopathy. For modeling cardiopathy in experimental animals once injected a mixture of homogenized myocardial tissue of rats (25%), egg albumin (25%) and adjuvant of franda (50%), 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously to the left and right.

In control and experimental animals under hypentelium anesthesia investigated hemodynamic and invasive way in terms 60 days of the experiment, after which the animals were killed. The tissue samples are fixed in 10% neutral formalin, followed by the preparation of paraffin sections with a thickness of 5-6 microns. Sections were stained with hematoxylin and eosin, Congo red. The study of slices is carried out in transmitted light with microscope Mikmod-1 under magnification× 80, × 200, × 600.

The essence of the claimed method is confirmed by functionally and morphologically.

In the study of the main indicators of hemodynamics was significantly decreased: cardiac index (SI) and shock index (si) and the increase of specific peripheral vascular resistance (UPSS) compared with the control (tab. 1). Tended to decrease mean arterial pressure (SBP) and heart rate (HR).

Table 1
Changes in key indicators of hemodynamics during experimental formation of amyloid cardiopathy
GroupIndicatorGARDENCardiac indexShock indexUPSSHR
IntactM±M109,9±1,644 65±0,730,0020,116±0,0021,954±0,0043385±5,42
ExperiencedM±M107,3±2,0734,944±1,1200915±0,0042,407±0,8To 384.1±9,72
P>0,1<0,001<0001<0,001>0,1

Example No. 1. Conducted a series of experiments consisting of 2 groups:

1st group (20 rats-males line "Wistar") control

2nd group (20 rats-males line "Wistar") - experienced� - old rats-males (age 18-24 months, body weight 350-380 g) once introduced a mixture consisting of homogenized myocardial tissue of rats (25%), egg albumin (25%) and adjuvant of franda (50%), 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously to the left and right.

The morphological study to identify cardiomyopathy subjected to the myocardium and blood vessels.

In the studies provided morphological evidence for the effectiveness of native egg albumin in combination with the complete adjuvant of freind for modeling experimental cardiomyopathy in old male rats.

Histologically in the 2nd group of animals in the myocardium (Fig. 1, 2) when stained with sections of hematoxylin-eosin and Congo red revealed areas of parenchymal dystrophy protein, sometimes passing to lobular collapse and fragmentation of cardiomyocytes with the disappearance of the transverse iscertainly (1). Marked perivascular limfogistiocitarnom infiltration (2), stromal edema (3), the plethora of the vessels of the microvasculature (4). In addition, marked sclerotic changes of stroma. Is the plasmatic impregnation of the walls of the microcirculatory vessels and perivascular areas of plasturgie (5).

When stained with Congo-red marked ex�proposal amorphous masses of amyloid in the walls of blood vessels and stroma of the myocardium (6).

Thus, the results of morphological studies of tissues of experimental animals fed with amyloidogenic equal mixture consisting of homogenized myocardial tissue of rats was 25%, egg albumin 25% and adjuvant of frand - 50%, 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously on the left and right show the development of experimental amyloidosis with primary lesion of the myocardium.

Summing up, it should be noted that a single administration of a mixture consisting of a homogenized myocardial tissue of rats (25%), egg albumin (25%) and adjuvant of franda (50%), 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously to the left and to the right is an effective way of modeling the experimental cardiopathy.

The method of experimental simulation of amyloid cardiopathy in rats, consisting in a single administration to old rats-males of the mixture consisting of pre-blended fabric of the myocardium of rats was 25%, egg albumin 25% and an adjuvant of frand - 50%, 0.3 ml in 5 points of injection: intraperitoneally, in the inguinal and axillary region subcutaneously to the left and right.



 

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