Method of detecting lifetime paranecrotic change of tissues and skeletal system in animals

FIELD: medicine.

SUBSTANCE: to detect the lifetime paranecrotic changes of tissues and skeletal system in experimental animals, average weight molecules in blood plasma are evaluated by spectrophotometric analysis in the wave length range 250-300 nM. The derived data are estimated by the area of a figure formed by an absorption curve and a zero mark axis. If the average weight molecules are increased in relation to the initial level in the wave length range 250-270 nM, the lifetime paranecrotic changes are observed.

EFFECT: method allows detecting the paranecrotic changes in tissues and skeletal system of experimental animals without killing.

1 ex, 1 tbl

 

The invention relates to the field of experimental medicine and can be used for modeling pathological reactions, processes, and States to determine in vivo parametricheskikh changes in the tissues of the organs and bones of experimental animals.

Parametricheskii are the changes in the tissues, which may, depending on conditions go to the irreversible stage of necrosis or be restored to its original state (1). It is known that parametricheskie processes increase in the structures of the nervous tissue and other organs under different effects on the organism of experimental animals (2), as well as disorders of endocrine regulation (3), which are accompanied by increased sorption vital dyes.

Still in experimental medicine structural and functional changes in tissues and organs revealed after the killing of animals, studies of biopsies of suspected damaged organs (4). There are also chemical method for detection of in vivo changes in the tissues of the organs by examining the chemical composition of the blood (5) and histochemical evaluation method for in vivo parametricheskikh changes in the tissues (6). However, these methods have several disadvantages associated with the necessity of killing animals and traumatise the tion of the studied organs.

As a prototype for closest to the technical nature of our chosen the way histochemical determination parametricheskikh of life changes in the tissues of the organs, namely, that the number of adsorbed cloth bodies paint neutralist judge available parametricheskikh life changes (7), as this paint stains denaturation changes in the cell at a reversible stage (8).

The method is as follows. Experimental animal (in this case the rat) injected intraperitoneally 0.5% aqueous solution naturalrate in the amount of 0.25 ml per 100 g mass. Then after 30 min the animal is slaughtered and retrieve the bodies, which are placed in loireau environment (acidified with concentrated sulfuric acid solution 70 ethyl alcohol) in an amount of 5 ml per 1 body. After 24 hours the eluate photometrate to determine the adsorbed dye cloth bodies. To assess the tissue distribution of the ink bodies are placed in the incubator for 24 hours at 37. The result is appreciated in mg per gram dry tissue.

This method for determining parametricheskikh life changes has the disadvantage associated with the necessity of killing animals for subsequent extraction of the dye from the tissues of the organs, and, therefore, there is no opportunity to study in vivo the pair is eroticheskih changes in the tissues of the organs and bones of experimental animals in the dynamics.

The technical result of the invention is the possibility of studying in vivo parametricheskikh changes in the tissues of the organs and bones of the experimental animal in the dynamics, as well as the definition of lifetime parametricheskikh changes in the tissues of the animal without killing.

The technical result is achieved by the fact that in experimental animals to determine the average molecule mass (MSM) in the range of 250-300 nm in plasma and when they increase from baseline in the range of 250-270 nm wavelengths determine the in vivo changes in the tissues of the organs and bones.

The method is as follows:

Plasma (0.5 ml) experimental animal according to the method Myamalfoy (8) determine the MSM. Blood samples of 1 ml is carried out with decapitate or from the tail vein (in rats). The deposition of macromolecular proteins spend 15% solution of trichloroacetic acid (0.5 plasma 0.5 ml of 15% R-RA trichloroacetic acid). Clot denaturirovannykh proteins stir with a glass rod, and then centrifuged at 5000 revolutions per minute. Then 0.5 ml of centrifugate add 9.5 ml of distilled water and photometrate spectrometer at wavelengths from 250 to 300 nm. The obtained results are judged by the size of the square shape formed by Krivovichev absorption and the axis zero point. Express this value in arbitrary units. Then experiment, which consists in the simulation of pathological reactions, processes and States. In this case, used a model of traumatic effects on the animal body by mechanical fracture of the bones of the leg. 24 hours after the injury examined the blood contains MSM and when they increase from baseline in the range of 250-270 nm wavelengths determine the in vivo changes in the tissues of the organs and bones of the experimental animal.

Distinguishing the essential feature of the proposed method is the determination in experimental animals of molecules of average weight (MSM) in plasma and when they increase from baseline in the range of 250-270 nm wavelength determination of in vivo changes in the tissues of the organs and bones.

The causal link between distinguishing the essential feature of the proposed method and achieve the result:

As is known, the impact of many pathogenic factors may cause significant changes in neuroendocrine regulation with subsequent disruption of the internal environment and the presence of several humoral factors endogenization pathological processes (9). While involved in the pathological process and is not damaged body is (in this case not injured organs), exacerbating the course of the basic pathological process. Currently, among the factors of endogenous intoxication of paramount importance MSM (10), which are represented by the classes of compounds with a molecular weight of up to D. The range of 250-270 nm wavelengths, this method for determining the in vivo changes in the tissues of the organs and bones of the experimental animal, chosen by the authors experimentally.

We first established correlative relationship between increased from baseline by MSM, defined in the spectrum of wavelengths from 250 to 270 nm, and lifetime parametricheskii changes in the tissues of internal organs and bones of the experimental animal.

Distinctive significant feature of the proposed method is new and allows to determine in vivo parametricheskie changes in the tissues of the animal without killing that makes it possible to study in vivo parametricheskie changes in the tissues of the organs and bones of the experimental animal in the dynamics.

Specific example:

(Rat No. 3A and 4A of Protocol No. 7). Experiment: a fracture of the right ankle under General anesthesia. Study after 18 days on the prototype method and the proposed method.

In rat No. 3A of hvos the OIC Vienna was taken blood and investigated by the present method for the determination of plasma MSM in the range of 250-300 nm, used spectrophotometer. It was found that in the range of 250-270 nm MSM totaled 33.7 USD, while the control rats, this indicator was equal to 10.8 cu, indicating in vivo parametricheskikh changes in the tissues of the organs and bones of the rat No. 3A.

In rat No. 4A from the tail vein was also taken blood and investigated by the present method for the determination of plasma MSM in the range of 250-300 nm. It was found that in the range of 250-270 nm MSM reached 8.9 cu, in control rats, this indicator was equal to 10.8 cu, indicating the absence of in vivo parametricheskikh changes in the tissues of the organs and bones of the rat # 4A.

The obtained data confirmed the results obtained in the study of in vivo parametricheskikh changes in the tissues of the organs and bones of the rat according to the method prototype:

BodiesThe content of paints mg per 1 gram of tissue
ControlRat 3ARat 4A
The brain0.00390.00690.0041
0.00410.00730.0043
Liver0.00210.00810.0018
Heart0.00680.01090.0059
Kidney0.01180.04280.0109
Thigh0.00150.00460.0016
Shin bones0.00170.00390.0020

The essence of the method is illustrated by results obtained in rats with fractures of tubular bones (table - Comparative table of the evaluation of life parametricheskikh changes in tissues and organs of the skeletal system in rats with experimental fracture of the Shin bone on the prototype method and by the present method).

The data presented in table 1, indicate that in vivo parametricheskie changes in the tissues of rats, determined by the method prototype, identified using the proposed method.

Thus, the inventive method can be used to detect in vivo parametricheskikh changes in the tissues of the organs and bones of experimental animals without killing them, which gives the opportunity to study in vivo parametricheskikh changes in the tissues of the organs and bones of experimental animals in the dynamics.

Table 1
Comparative table of the evaluation of life parametricheskikh changes in tissues and organs of the skeletal system in rats with experimental fracture of the Shin bone on the prototype method and the present method
The follow-up periodCONTROL (n=9)After 24 hours (n=8)After 18-21 days (n=7)
Organs:
The brain0.0043+0.00020.0081+0.0006*0.0039+0.0006
Spinal cord0.0039+0.00060.0061+0.0004*0.0047+0.0008
Liver0.0016+0.00010.0072+0.0008*0.0013+0.0001
Heart0.0075+0.00080.0210+0.0010*0.0082+0.0004
Kidney0.0130+0.00200.0520+0.0110*0.0160+0.0020
Thigh0.0017+0.00010.00.0019+0.0003
Shin bones0.0021+0.0002035+0.0001*0.0022+0.0005
(method-prototype)0.0036+0.0002*
Molecules average11.3+2.7 $ 25.2+3.4 $ *9.8+3.7 $
mass (cu - conditional units)(250-270 nm)(250-270 nm)(250-270 nm)
31.5+3.1 $ 17.8+ $ 5.429.6+3.2 $
(the inventive method) (280-300 nm)(280-300 nm)(280-300 nm)
Note: * - significant change (p<0.05)

Sources of information

1. Alexandrov VA, Kisluk IM the Response of cells to heat shock: physiological aspect // Cytology. - 1994. T.36, No. 1. - C.5-60.

2. Novels S.N. The biological effect of mechanical vibrations. - L., 1983. - 208 S.

3. Khegay PPM Pathophysiological basis for the development of complications in insulin-dependent diabetes mellitus. - Abstract. Diss. for obtaining the academic degree of doctor. the honey. Sciences. - SPb., 1998. - 39 S.

4. Fingers M.A., Anichkov NM Pathological anatomy. - M., 2000. Vol. 1. C.11-23.

5. Homolo PS Neurogenic atherosclerosis and lipidosis aorta. - M., 1972. S-247.

6. Gramenitskiy E.M. Intravital staining cells and tissues. - Leningrad, 1963. - 149 C.

7. A.S. Troshin the Distribution of substances between the cell and the environment. - L., 1985. - 192 S.

8. Medical laboratory technology. Handbook / edited Aeacides, Vol.2. - St. Petersburg, 2002. 400 S.

9. Kryzhanovsky GN. Introduction to General pathology. - M., 2000. - 71 S.

10. Kopytova T.V. molecules of average weight as the substrate of endogenous intoxication in severe dermatoses // Successes of modern natural science. 2006. No. 9. P.7-10.

The method of determining the lifetime parametricheskikh changes in tissues and organs of the skeletal system the experimental is the rest of the animals, characterized in that to determine the average molecule weight in blood plasma by spectrophotometry in the range of 250-300 nm wavelengths, with the results evaluated according to the magnitude of the square shape formed by the curve of the magnitude of the absorption axis and the zero mark, and with the increase of molecules of average weight with respect to the initial level in the range of 250-270 nm wavelength, determine the lifetime parametricheskie changes.



 

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