RU2317543C1 - Method for blood sampling in laboratory mice - Google Patents

Abstract

FIELD: physiology.

SUBSTANCE: the present innovation deals with blood sampling while studying the influence of protein preparations upon the values of natural resistance. Murine blood should be sampled with the help of decapitation of murine heads pre-placed into a thermostat for about 2-5 min at 40-42°C. The innovation enables to simplify blood sampling in white mice and increase quantitative values of blood and serum under collection.

EFFECT: higher efficiency.

2 ex, 4 tbl

Description

The invention relates to physiology, namely to taking blood with subsequent separation of serum in the study of the effect of protein preparations on indicators of natural resistance.

A known method of obtaining blood of laboratory animals using decapitation, described in SU No. 1296117 A1 "Device for blood sampling in small laboratory animals", published 03/15/1987.

The disadvantages of this method are the small amounts of collected blood, insufficient to study the physiological parameters of blood when protein preparations affect the animal’s body.

A simplification of the method of taking blood from white mice and an increase in the quantitative and qualitative indicators of the collected blood and serum is achieved by taking blood from white mice: control and experimental (immunized with a bone marrow preparation, protein nature). For this, healthy animals are selected that are in the same weight and age categories and in one physiological state. Groups of control and experimental animals are formed. Animals are placed in the same conditions after immunization of the experimental and monitor their physiological condition until the first immune response to the introduced antigen. A preparation for insertion into the hind pit of a mouse is obtained from the bone marrow of the tubular bones of wild and farm animals by precipitation of proteins from bone marrow cells using a 10% trichloroacetic acid solution and subsequent dialysis of the protein solution.

Blood sampling in mice of both groups is carried out by decapitation of the animal's head. The animals are preliminarily kept in a thermostat for 2-5 minutes at a temperature of 40-42 ° C (there is an increase in metabolism and blood thinning of mice). Then the animal with tweezers is grabbed by the fold of skin on the nape, fixed and cut off the head with sterile scissors. Blood is collected using a funnel into thin Ulengut tubes until dying convulsive convulsions of the animal’s body occur and the blood is ejected in portions into the funnel.

After taking 25-30 minutes, the blood is kept in an thermostat (35 ... 37 ° C), then the clot is surrounded by a thin metal stick (separation of the clot from the walls of the vessel) and put in the refrigerator for 16-18 hours. The serum is pipetted with a pear, transferred to a sterile vessel, closed with a lid and stored in the refrigerator. Thus, you can get up to 1.5 ... 2 ml of blood and ≈0.5 ml of serum.

Example 1

Outbred white mice, males in the same age category (4-6 months) weighing 18.8 ± 0.3 g are immunized with a bone marrow preparation of foxes containing 50.3 g / l of total protein in their composition (table 1), in a small pad hind paw at a dose of 0.025 ml per mouse. After 7-8 days from the animals of the experimental and control groups, blood is collected for the study of serum for total protein and its fractions. Decapitate the heads of animals without first placing them in a thermostat. Blood is collected and serum is separated as described above. Thus, up to 0.5 ± 0.04 ml of blood is obtained.

Table 1
Electrophoresis of bone marrow of foxes in agarose gel according to the method of Chekishev V.M. (1977) Try Total protein (g / l) Albumin (g / l) Globulins (g / l) γ ₁ + γ ₂ % ∑γ ₁ + γ ₂ α ₁ α ₂ β γ ₁ γ ₂ one 50.3 6.18 7.06 10.59 11.47 11.47 3.53 15.00 29.82 2 50.3 6.29 7.19 10.78 10.78 10.78 4.48 15.26 30.33 3 50.3 7.06 8.83 11.47 8.83 10.59 3.52 14.11 28.05 four 50.3 6.40 9.15 10.97 9.15 11.89 2.74 14.63 29.09 Average data 50.3 6.48 ± 0.2 8.06 ± 0.7 10.95 ± 0.4 10.23 ± 0.7 11.18 ± 0.4 3.57 ± 0.3 14.75 ± 0.3 28.32 ± 0.5

Example 2

Immunize laboratory mice with a protein preparation from the bone marrow of foxes according to example 1, but before blood sampling by decapitation, animals are placed in a thermostat for 2-5 minutes at a temperature of 40-42 ° C. Thus, 1.7 ± 0.07 ml of blood and 0.48 ± 0.01 ml of serum are obtained. Tables 2 and 3 show the blood test data of mice obtained by the claimed method, divided into two groups: with normal body temperature (control) and heated in a thermostat, and in table 4 - the difference between the average electrophoresis values of the selected blood serum of mice.

table 2
Blood leukogram of mice before and after heating Groups Band neutrophy. Segmented neutrophy. Monocytes Eosinophils Lymphocytes The control M ± m 8.3 ± 0.51 47.9 ± 1.0 2.1 ± 0.04 3.2 ± 0.30 38.2 ± 0.38 % 100.0 100.0 100.0 100.0 100.0 Heated animals M ± m 8.3 ± 0.11 48.1 ± 0.16 2.1 ± 0.17 3.3 ± 0.18 38.2 ± 0.20 % 100.0 100,4 100.0 103.1 100.0

With an increase in the body temperature of animals, the number and species composition of leukocytes did not change significantly in comparison with the control group of animals.

Table 3
The dynamics of the indicators of the immune defense of mice subjected to heat Indicators The body temperature of mice, ° C 37.5 38.3 one 2 3

Continuation of table 3 White blood cells 6.09 ± 0.070 6.15 ± 0.07 Total protein 83.7 ± 0.45 85.4 ± 1.32 Phagocytic activity 25.8 ± 1.57 29.6 ± 1.11

According to table 3 shows that the indicators of cellular and humoral protection of animals with a body temperature of 38.3 ° C changed slightly upward. FA indicators - by 14.7%, total protein - by 2.03% in comparison with the control.

Table 4
Evaluation of the difference in average values of two samples of mouse serum electrophoresis (control experiment) n = 10 Indicators of electrophoresis g / l Laboratory mice Difference of indicators R the control experience Total protein 49.5 ± 0.91 85.4 ± 1.32 +25.90 <0.001 Albumin 12.0 ± 0.64 25.0 ± 1.41 + 12.93 <0.001 Globulins α ₁ 7.0 ± 0.40 11.4 ± 0.43 + 4.42 <0.001 α ₂ 7.3 ± 0.28 11.7 ± 0.85 +4.44 <0.001 β 9.1 ± 0.33 10.3 ± 0.72 +1.21 > 0.05 γ ₁ 10.3 ± 0.26 20.3 ± 0.73 + 10.05 <0.001 γ ₂ 3.2 ± 0.13 6.4 ± 0.42 +3.28 <0.001 γ ₁ + γ ₂ 13.6 ± 0.18 26.8 ± 0.41 +13.2 <0.001 % ∑ γ ₁ + γ ₂ 27.6 ± 0.43 31.4 ± 0.59 +4.4 <0.001

A distinctive advantage of the proposed method of taking blood from mice is the availability of the proposed method, which allows to increase the yield of collected blood and serum without changing its quality indicators, and to study the effect of immunostimulating drugs on the animal's body. So, for example, in experimental animals immunized with a bone marrow preparation, the total protein content increased by 1.7 times and the γ ₁ + γ ₂ globulins in blood serum increased by 1.9 times compared to the control.

Claims (1)

A method of taking blood from laboratory mice to determine the concentration of total protein, including blood sampling by decapitation of animals, the samples of which are placed in microtubes suitable for centrifugation, characterized in that the blood is collected from animals previously placed in a thermostat for 2-5 minutes at a temperature 40-42 ° C.