M. Mantskava, N. Momtselidze,
L. Davlianidze.

I.Beritashvili Center of Experimental Biomedicine, 14 Gotua St., Tbilisi, Georgia, 0160.

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The aim of our work was a red blood cell deformation (rheological rheological ) at rheological degrees of severity of experimental hemorrhagic shock. The study of erythrocyte deformability violation and a compulsory monitoring of this physical value deserve more attention of fundamental and applied sciences. The modeling of hemorrhagic shock of different stages was performed in the group of anesthetized animals by means of standard blood discharge from the femoral artery (2,5 ml – first stage of shock; 5 ml – second stage of shock; 5ml – third stage of shock, n=12).[/su_animate]

Data show, that index deformability decreases at increase in number of hemorrhagic shock’ stage (12% – one subgroup, 13% – second subgroup and 15% – third subgroup). While studying the deformability of erythrocytes, we monitor the rheological parameters that will promote the development of the algorithm of intensive therapy and a diagnosis of massive blood loss, as well as of hemorrhagic shock.

           Hemorrhagic shock appears to be the sequels of a variety of diseases, especially in abdominal surgery and in gynecological departments. In spite of this the mechanism of development of microcirculational disorders as one of the basic links of shock pathogenesis has not been appropriately studied yet. In not so far past shock was viewed only from the aspect of alteration of general hemodynamics. In the present day scientific literature studies are often related only to the intravascular hemocoagulation. From the point of view of blood rheological properties the crucial factors in the microcirculation system are: local hematocrit, erythrocyte aggregability, erythrocyte deformability and plasma viscosity. As regards the erythrocyte deformation, this rheological parameter must cause far graver results in the organs  the diameter of the capillary lumen of which is equal to or less than is the erythrocyte size.

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Hemorheological shock appears to be a complex pathological process with the mechanism unexplored up to the end. A particular link, which is involved in various stages of hemorheological shock, is blood rheology.biz [5, 8]. The aim of our work was a red blood cell deformation (rheological marker) at different degrees of severity of experimental hemorrhagic shock. The study of erythrocyte deformability violation and a compulsory monitoring of this physical value deserve more attention of fundamental and applied sciences. Erythrocyte deformability depends on their elasticity and rigidity, therefore the determination of erythrocyte deformation index is particularly important for the treatment and prognosis of hemorrhagic shock.

Materials and Methods. Experiments were carried out adult rats of both sexes weighting 250-300 g., anesthetized with 4% solution chloral hydrate (0.15 ml/100 g,) administered intramuscularly. (n=50). The modeling of hemorrhagic shock of different stages was performed in the group of anesthetized animals by means of standard blood discharge from the femoral artery (2,5 ml – first stage of shock; 5 ml – second stage of shock; 5ml – third stage of shock, n=12) [1,4]. Arterial pressure was measurement in tail vein at manometer MPX5050D (Motorola). Experimental animals was without pharmacology drugs. We reproduced useful model. The index of RBC deformability was determined with nucleopore membrane filter method [6] during hemorrhagic shock and control group. Evaluation of erythrocytes deformability was performed with an aid of the nucleopore membrane filter method, which is based on assessing velocity of the erythrocytes passage through the very small pores (5 µm, which is a diameter of the smallest capillary) of the filter, at constant pressure (10 cm of water column) and temperature (37°C). Obtaining the pure erythrocytes was performed by centrifuging the blood sample at 3000 rpm, for 15 min. Resulting plasma was aspirated with micropipette and the remaining blood cells were added with bovine serum albumin (0.2 mg per 5 ml) dissolved in the phosphate buffer. Then the blood was centrifuged second time at 1000 rpm for 5 min. The precipitated erythrocytes, as well as thin layer of leukocytes and thrombocytes, were separated from the phosphate buffer. This procedure was repeated three times. Purified erythrocyte mass was diluted in the phosphate buffer, with hematocrit of 10%. Evaluation of the deformability index implied measuring a velocity of the erythrocyte passage through the filter (mm/min) was recorded. The high quality polycarbonate filters (with 5 µm diameter pores) were used in measuring procedures.

Results. The index of RBC deformability in control group was 2,25±0,03%. Index of RBC deformability at first, second and third stages of hemorrhagical shock see table. Data show, that index deformability decreases at increase in number of hemorrhagical shock’ stage (12% – one subgroup, 13% – second subgroup and 15% – third subgroup). The analysis of the data was performed using statistical programs “Origin 4.1” (Microsoft. Software, Ivc) and Microsoft Excel. All received results was evaluated statistically and significance of differences between mean values were assessed by Student’s Criterion.

The conditions for conducting the work on animals corresponded to the European Convention on the protection of experimental animals, adopted in 1986 in Strasbourg [8] and for the conducting the experiment the consent of Ethics Committee was obtained.

Discussion. A crisis of macro- and microcirculation has an especially specific character at hemorrhagic shock [3]. Hemorrhagic shock and the stages of its development depend on an adequate blood circulation, which is provided by the regulation of blood hemorheology. The blood circulating in the vessels is inhomogeneous, its parabolic profile is distorted with decreasing vessels caliber [2]. The disturbance of various hemorheological functions, independent from hematological parameters takes place. Tissue hypoxia and hypoglycemia that attend the blood rheological disorders are undoubtedly especially hard for the organ tissue. It is s known that neural structures have no supply of carbohydrates and oxygen.  It is known that most of operating compensatory reactions involved in the pathogenesis of shock in general and of hemorrhagic shock in particular are directed toward centralization of circulating blood volume and maintenance of relatively steady blood circulation. So far, under conditions of hemorrhagic shock the changes developed in the blood circulation system must substantially differ in the peripheral organs (for instance, in the small intestinal mesentery studied by us earlier) and the central organs.

This complex biomedical problem develops step by step at blood loss and appears to be a consequence of shock situations. Analyzing the experimental data, we observed changes in erythrocyte deformation of the first, second and third stages of shock. The change in erythrocyte deformation occurs after the inclusion of compensatory reaction of the organism, and/or deformation or deterioration appears to be the basis for the inclusion of this mechanism. The change and disorder of the feature of erythrocyte deformability takes place permanently from stage to stage. The change in elasticity and rigidity of erythrocytes causes a change in blood flow, which leads to a reduction in flow velocity in the microvessels up to the complete development of blood stasis, resulting in an occlusion of all open capillaries and disappearance of the blind, sometimes on the background of constant perfusion pressure [7]. We investigated hemorheological parameter  – RBC deformability index in healthy control and hemorrhagic shock. Hemorheological feature  is modified and changed. We are being registered for the first time. This data is important for researchers in microcirculation and rheological areas. We have great experience in studies of rheological and microcirculation mechanisms. Our research group first studied changes in blood flow during the first, second, third  type hemorrhagic shock. In this article, we are studying different hypervolemia effect in blood erythrocyte deformation. 

Our previous research provides practical recommendations.   Therefore, it is especially important to monitor the deformation of erythrocytes at a shock of any etiology. In the medicine of emergency situations, the tactics for the treatment of hemorrhagic shock is well known and widely used. However, at various stages of the shock the laboratory-diagnostic methods of the research deserve a special attention and completion. A correct diagnosis of hemorheological parameters is particularly important for correcting the violations of separate links of the homeostasis, which is an unavoidable cause of hypervolemia. While studying the deformability of erythrocytes, we monitor the rheological parameters that will promote the development of the algorithm of intensive therapy and a diagnosis of massive blood loss, as well as of hemorrhagic shock.

Acknowledgment. Shota Rustaveli National Science Foundation (FR/420/7-270/12)Table.  RBC deformability in hemorrhagic shock and control

*………………………………………………p<0.01

References

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