Агонист α2-адренорецепторов дексмедетомидин в практике современной седации

The problem of sedation in intensive care units has obvious scientific-and-practical relevance. Many current studies deal with the practical introduction of novel medications for sedation, with the specific features of their pharmacodynamic effects in different clinical situations, with the advantages and disadvantages of their use. Recent years are marked by an increase in the number of publications on the o^-adrenoceptor agonist dexmedetomidine used for sedation. The paper reviews current publications on o^-adrenoceptor agonists used in anesthesiology and resuscitation, considers their physiology and the mechanism of sedative action of dexmedetomidine, its pharmacokinetics and pharmacodynamics at different plasma concentrations of the drug. The experience in using dexmedetomidine sedation in clinical practice is discussed in detail, by analyzing the data of current multicenter randomized trials in which the drug has been compared with other sedative medications (propofol, midazolam, lorazepam). Some aspects of the clinical pharmacology of dexmedetomidine, such as its effect on the brain and its blood flow, nociception, sympathoadrenal system, hemodynamics, respiratory system, thermoregulation, immunity, and neuroendocrine system, are analyzed. The clinical value of different receptor-dependent effects of the drug and the specific features of its application in different situations are also analyzed. Key words: dexmedetomidine; sedation in intensive care patients; sedative drugs; o^-adrenoceptor agonists; delirium in intensive care unit patients.


Ключевые слова: мембраны эритроцитов; нелинейные деформации; атомно-силовая спектроскопия
Modifiers of membranes cause local defects on the cell surface.Measurement of the rigidity at the sites of local defects can provide further information about the structure of defects and mechanical properties of altered membranes.
The purpose of the study: a step-by-step study of the process of a nonlinear deformation of red blood cells membranes under the effect of modifiers of different physico-chemical nature.
Materials and methods.The membrane deformation of a viscoelastic composite erythrocyte construction inside a cell was studied by the atomic force spectroscopy.Nonlinear deformations formed under the effect of hemin, Zn 2+ ions, and verapamil were studied.
Results.The process of elastic deformation of the membrane with the indentation of a probe at the sites of local defects caused by modifiers was demonstrated.The probe was inserted during the same step of the piezo scanner z displacement; the probe indentation occured at the different discrete values of h, which are the functions of the membrane structure.At the sites of domains, under the effect of the hemin, tension areas and plasticity areas appeared.A mathematical model of probe indentation at the site of membrane defects is presented.

Introduction
The ability of red blood cells (RBC) to deform determines the possibility of their passage through capillary network and rheology of a blood [1].This problem is especially urgent in practical reanimatology and rehabilitology since in critical states due to a blood loss and multitrauma the intrinsic parameters of erythrocyte membranes vary in a wide range [2][3][4][5].
The atomic force spectroscopy (AFS) makes it possible to obtain images of the nanosurface of membranes and to study their ability to deform under the influence of the indenter [6,7].A conclusion about the firmness of the material and its mechanical properties [8,9] is made by the deformation of the biological structure.The deformability and rigidity of the erythrocyte membranes varies depends on the disease studied and inclludes diabetes mellitus, myocardial ischemia, hypertension [10,11], and tumors [12,13].
According to the force curves measured in the superficial layer, the Young's modulus is calculated, which is attributed to the entire volume of the material [14][15][16].In this context, the measurement of the hardness of the cell membrane can be carried out on membranes fixed on the solid substrate [17].But in this case, it is not possible to obtain data about the deformability of individual membrane components.One can only estimate the Young's modulus of the surface layer.The task of studying the mechanical properties of erythrocyte membranes with the help of AFS should be set differently.Namely, a study of the ability of the viscoelastic composite membrane to deform inside a cell on an integral erythrocyte construction [18,19].
Modifiers of membranes may produce local defects on the cell surface.The measurement of the hardness at sites of local defects can give additional information about the structure of defects and the mechanical properties of membranes, in general.Therefore, to describe the biomechanics of membranes, it is necessary to investigate not only the Young's modulus of the superficial layer, but also changes in the rigidity of membranes in their various zones and at different depths of deflection.For this purpose, the effect of modifiers of different nature on the cells was studied: a toxic agent hemin, ions of heavy metals (Zn 2+ ), and a pharmaceutical verapamil.

Materials and Methods
The method of deformations measuring, the object, as well as the procedure of plotting and analyzing of force curves in the atomic force spectroscopy has been described in details in Part 1 of this article.
Blood was collected from donors on a voluntary basis during preventive examinations, and all further studies were performed in vitro.Blood from 6 male donors aged from 23 to 34 years old was used in the study.All studies were carried out in accordance with the requirements of the Declaration of Helsinki by the World Medical Association «Ethical Principles for Medical Research Involving Human Subjects», as amended in 2000, as well as with the requirements of the Ethics Committee of the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology.
Studies of local hardness of erythrocyte membranes after in vitro exposure of blood to modifiers: hemin, heavy metal ions (Zn 2+ ), verapamil.These modifiers had different nature and different mechanisms of action on the nanostructure of the membranes.It is important that these modifiers generated specific topological local defects on the erythrocyte membranes, which are characteristic only to this modifier.The choice of modifiers is substantiated by the need to obtain a wide range of effects of their influence on the nanostructure and mechanical properties of membranes.
Hemin is a product of hemoglobin oxidation.Malaria, sickle cells, ischemia, blood loss, bleeding from gastric erosions can lead to the formation of hemin [2,18,20].Zn 2+ ions cause clustering of protein structures and generate local membrane defects [21,22].Verapamil is one of the most commonly prescribed calcium channel blockers, used in the treatment of hypertension, angina pectoris, tachyarrhythmia and cardiomyopathy [23].
Dry hemin was used in the study (Sigma, USA).50 mg of dry hemin was dissolved in a 0.1 M solution of NaOH in distilled water.The final in vitro concentration of hemin in the blood was 1.5 mM.A ZnSO 4 solution (Sigma, USA) was added to the whole blood.The in vitro concentration of Zn 2+ ions in the blood was 2 mM.The in vitro concentration of verapamil hydrochloride (Nycomed, Austria) in blood was 0.6 mM.
The statistical processing of data was carried out using the standard Origin 9 software, (Origin Lab Corporation, USA).The histograms were constructed, the mean value, the standard deviation value were determined and an interval estimate, an estimate of the reliability of the obtained results, and an estimate of the reliability of the differences were obtained.The reliability of the differences was assessed using single-factor analysis of variance (One-way, ANOVA).

Process of probe indentation into RBC membrane under the effect of hemin
Hemin forms domains on the membrane surface [24].An example of the nanosurface of the red blood cell membrane after the action of hemin is shown in fig. 1, a. Zones of large domains (350-1000 nm), «Ld», zones of small domains (200-300) nm, «Sd», and zones of flat membranes surface, «Fl», are shown.The characteristic size of granular structures in domains is 100-120 nm, which is close to the characteristic cell size of the spectrin matrix.The sizes of domains are determined by the number of grains in it [25].

Dependences
h/ z (h) (fig. 1, b) and μ (h) (Fig. 1c) are presented for the three selected cell zones, shown in Figure 1a.The process of probe introduction into the zones of flat surface (fig. 1, Fl) was similar to the same process on the planocyte (part 1, fig. 4, Pl).The function h/ z (h) fell from 0.8 (h=6 nm) to 0 at the depth 32 nm.The process of probes indentation into the Sd and Ld domain zones differ from that for a flat surface of the same cell (fig. 1, b).In the domain zones, characteristic Н Sd and Н Ld tension zones and the zones of plasticity of the membrane composite material Т Sd and Т Ld for small and large domains, respectively, were registered.In zones H, the local membranes hardness coefficient μ increased with increase of h; and in zones T the coefficient μ almost did not change.These zones were more expressed for the large domain.In the zone Н Ld (26-36 nm), h/ z fell from 0.94 to 0.41.Then a zone of plasticity Т Ld (36-90 nm) appeared.In this zone, h/ z practically did not decrease (0.41-0.38).At a depth of h=94 nm, the probe stopped its indentation into the membrane.In the zone of a small domain, the indentation process was similar.The tension zone Н Sd was wider (28-58 nm), and the drop of h/ z was from 0.83 to 0.3.The plasticity zone T Sd was from 58 to 64 nm, and the maximum value of the probe indentation was h=74 nm.The function μ (h) (fig. 1, c) for a flat zone of the membrane Fl was an increasing one, similar to this function for the planocyte (part 1, fig. 4, c).For a large domain Ld, the function μ (h) had a characteristic flat zone Т Ld within which the value of the local hardness coefficient μ of the membrane remained practically unchanged.This zone is highlighted in fig. 1, c as a colored rectangle.At local deformation of the membrane to the depth h=90 nm, the coefficient μ sharply increased resulting in the stop of the probe indentation.
The process of probe indentation into membranes under the effect of Zn 2+ ions and verapamil.
Red blood cells with membrane defects, caused by the effect of zinc ions and the calcium channel blocker verapamil are shown in fig. 2. Ions of heavy metals and, in particular, Zn 2+ , at high concentrations (2 mM and more) always cause local membrane defects (fig.2, a) [26,27].Verapamil in clinical concentrations does not cause destruction of membranes.This study used a tenfold dose of the drug in vitro, which caused a toxic effect, and as a result, specific local membrane defects (fig.2, b).
The function h/ z (h) for membranes damaged by zinc ions had the character of a tension zone, it decreased almost linearly.The probe stopped its indentation at the depth of h=18 nm, thus indicating a 3.6-fold increase in hardness as compared to the normal limits.The same function for verapamil practically repeated the normal curve, and the probe was indentated at 65 nm, as under normal conditions (64 nm) (fig.2, c).The hardness coefficient μ (h) for verapamil did not change practically during the entire process of the probe indentation and remained at the normal level (fig.2, d).
If there is a task to assess the average hardness of erythrocyte membranes over the ensemble of cells, then the approach to its solution should be different [28].The parameter h max is taken as the average hardness in the indentation zone.In this case, in different zones of the discocyte torus we should measure the value h max m times for n cells and average it over a certain ensemble.For illustration, the histograms of distributions of the average hardness of erythrocyte membranes are shown in fig. 3 under the effect of the modifiers of membranes on blood: ions of heavy metals (Zn 2+ ) and a calcium channel blocker verapamil.
In this example, the values hmax were measured at 6 points of the tours of 34 red blood cells for the reference group and the same amount for the groups of action of each modifier.The averaging and plotting of the histograms were carried out using an ensemble of 204 points for each group.Zinc ions (2 mM in vitro) caused local defects on the membrane [26,27] and increased its average hardness by 3.6-fold.
Verapamil in a concentration that is 8-10 times as high as the clinical dose caused damage of the membrane nanostructure [2], but in this case it did not change their average hardness, that remained similar to that in the reference group (P<0.05).
Зоны натяжений Н i -это участки h i , в которых коэффициент локальной жесткости μ возрас-trin fiber is a tetramer consisting of spiral α and β monomers connected by actin microfilaments.The length of the fiber can vary over a wide range: from 50 to less than 260 nm [29,30].Binding to the inner side of the membrane by means of band 4.1 and band 3 proteins, ankyrin, actin and others, spectrin stabilizes the phospholipid layer and limits lateral diffusion of integral proteins.
Due to the numerous and quite strong links between the phospholipid bilayer and the spectrin matrix under the action of the indenter on the cell, these structures behave as a single heterogeneous (in the sense of its elastic properties) composite cell membrane.
The process of the probe indentation into the erythrocyte membrane after the action of hemin (fig. 1) had other tendencies compared to kinetics of indentation in the normal red blood cell, presented in fig. 4 part 1.
Hemin (hydrochloric acid hematin) disrupts the conformation of spectrin, band 4.1 protein and weakens the bond between them [31].It modifies the conformation of the «spectrin-protein band 4.1, band 3» binding complex and induces the formation of local defects on the membrane surface [2,5,32].The effect of modifying the matrix is manifested in the immersion of the probe into the cell membrane, which was affected by the hemin.
In the zones of domains, the graphs h/ z (h) had specific features, typical for them alone.On these graphs, there are tension zones Н Sd and Н Ld , when the gradient of the function h/ z (h) is maximal, and the plasticity sites Т Sd and Т Ld , when this gradient is close to 0.
The tension zones Hi are the sites hi in which the local hardness coefficient μ increases, and the indenter immersion slows down sharply.In the zones of plasticity of T i , the coefficient of local hardness μ remains practically unchanged (Fig. 1c), and, consequently, h does not change in this region.The dimensions of the tension zones and the plasticity zones of Н i and T i depend on the size of the domains formed on the surface of the membranes as a result of the action of hemin.
Кроме рассмотренных структур мембраны эритроцита на величины h и h max может влиять внутреннее содержание клетки.Это гемоглобин и цитоплазма.Учитывая, что молекула гемоглобина creased sharply.The difference h/ z was 0.5 for the small domain Sd and 0.53 for the large domain Ld.Matrix began to be deformed elastically.Its fibers were stretched, creating almost the maximum spiral elasticity of the medium.When the probe is introduced into the membrane at a depth close to h max , the spectral fiber that has already lost its spiral elasticity (configuration) is stretched to the maximum possible length.After that, the thread became almost completely hard, and the probe stopped its movement ( h=0).
In the context of the considered problem, it would be perspective to arrange experiments to study the elastic properties of the matrix on the isolated spectrin network.Although such experiments are conducted [33], but the extrapolation of their results the whole blood cell is still a difficult problem.
In addition to the considered structures of the red blood cell membrane, the internal content of the cell may influence on the values h and h max .This is hemoglobin and cytoplasm.Considering that the molecule of hemoglobin has a size of about 5 nm and these molecules are freely immersed in the liquid medium of cytoplasm, this may be modeled by a drop of liquid in a soft coating [18,34].The influence of these medium on the measurement of μ membrane is insignificant [35].
Modeling of the process of erythrocyte membrane deformation.We will consider the «membraneprobe with cantilever» system as consistently connected springs with hardness coefficients μ and K, respectively.The hardness of the probe is an infinitely large value, so the coefficient K is entirely determined by the hardness of the cantilever.
The following assumptions are made in the model: 1.The cantilever hardness coefficient K is not changed during the measurement.
2. The membrane hardness coefficient μ depends on h nonlinearly.This is caused by the change in the mechanical properties of the membrane as it deforms, in particular by modification of the configuration of the spectrin matrix [36].
Let us introduce the equation: where a, b, c are the parameters, c=-a/b 2 .The coefficient a determines the steepness of the function μ (h), that is, how sharply μ varies as the probe enters the membrane.The coefficient b has dimension and a physical meaning of h max .
The μ(h) dependence curve is presented in fig. 4.This dependence agrees with the data on the change in the mechanical properties when the configuration of spectral fibers is changed [37].
The condition of equilibrium: the cantilever force is equal to the membrane force: . Using a number of mathematical transformations and taking into account ( 1), ( 2) and ( 3), we obtain the graphs of dependencies F (z) and μ (h) for the discocyte cavity (fig.4).
For h h max , the membrane hardness coefficient tends to infinity (μ ), which is the reason for stop of the indenter.In this case, the hardness coefficient of the entire system K syst (successively connected springs with coefficients K and μ) will tend to the hardness of the cantilever: K syst K.
In practice, even at μ 5K, the hardness coefficient of the entire system K syst grows enough to cause the stop of indenter.In the model, the value b=38 nm, and in the experiment for the discocyte cavity (part 1, fig. 4, b), the probe stopped at a depth of h max =35 nm.Therefore, in the experimental dependences of μ (h) near the point h max , the graphs do not become vertical (μ ), but go at the some angle γ<π/4 (part 1, fig. 4, c).This is facilitated by the frictional forces (not included in the model) between the surfaces of the indenter and the membrane.
As it is seen in the graphs in fig. 4 at a given coefficients a and b, the dependences F (z) and μ (h) obtained at the modeling adequately describe these dependences obtained in the experiment.