Effect of Perfluorane on the Regulation of Skin Blood Flow in Acute Blood Loss : Experimental Study

Материалы и методы. Эксперименты проведены на 31 беспородных крысах-самцах массой 300—400 г под наркозом (нембутал 45 мг/кг внутрибрюшинно). С целью измерения АД, забора, реинфузии крови и введения инфузионных растворов катетеризировали хвостовую артерию. Кровоток в микрососудах внутренней поверхности правого уха регистрировали методом лазерной доплеровской флоуметрии (ЛДФ) (аппарат ЛАКК-02, НПП «ЛАЗМА», Россия). Использовали модель острой, фиксированной по объему кровопотери. Целевой объем забора крови составил 30% ОЦК. На 10-й минуте после забора крови животным вводили раствор 0,9% NaCl (ФР, n=15) или перфторан (ПФ, n=16) в дозе 3 мл/кг массы тела. На 60-й минуте после забора крови проводили аутогемотрансфузию, после чего следовал реперфузионный период (60 мин). При анализе ЛДФграммы определяли следующие параметры: показатель микроциркуляции (ПМ); максимальные амплитуды колебаний кровотока в эндотелиальном (Аэ), нейрогенном (Ан) и миогенном (Ам) диапазонах частот методом вейвлет-анализа. Статистическую обработку данных проводили с использованием программы Statistica 7.0.


Introduction
The goal of the experimental study of acute blood loss was to elucidate new aspects of its pathogenesis and the effect of perftoran as a therapeutic intervention helpful at a certain stage of the pathological process.
The centralization of circulation, along with the mobilization of deposited blood and autohemodilution, are the main compensatory reactions to hemorrhagic hypovolemia [1].The centralization of circulation is a redistribution of cardiac output in favor of the vital organs (heart and brain), by vasoconstriction in regions that are less sensitive to ischemia (skin, muscle, gastrointestinal tract).At the same time, alterations of microcirculation and blood rheology, hypoxic cell damage are appearing and progressing in the ischemic tissues.Those, in turn, are leading factors in the pathogenesis of hemorrhagic shock and multiple organ failure [2].Reperfusion complications, coagulopathy and systemic inflammatory response to injury have an important pathophysiological significance as well [3].
The cutaneous blood flow is expected to become sharply decreased following blood loss due to the centralization of circulation.Non-invasive methods allow assessing the dynamics of body's compensatory reactions and links of microcirculatory alterations to central hemodynamics, as well as the impact of therapeutic interventions on these processes [8,9].One of methods of investigation includes the laser Doppler flowmetry (LDF) that allows not only to investigate the microcirculatory blood flow, but also to assess the regulatory mechanisms of microcirculation and dynamics of their activity when assisted by mathematical analysis of oscillations (fluxmotions) [10].
The goal of current study was to characterize effects of PF administration on the patterns of cutaneous microcirculatory blood flow in rats during a fixed-volume blood loss and post-resuscitation with the aid of LDF and wavelet analysis.

Materials and Methods
Experimental studies were started after the approval of the Ethical Committee of the V. A. Negovsky Institute of General Reanimatology.Experiments were carried out in 31 male outbred rats weighing 300g to 500g during spontaneous breathing and room temperature of 20-22°C.The animals were anesthetized by intraperitoneal injection of pentobarbital (45 mg/kg).Anesthesia was maintained by additional intraperitoneal injections of anesthetic (pentobarbital 15 mg/kg at intervals of 40 to 50 min or as required).Polyethylene catheter was advanced through the tail artery for invasive measurement of blood pressure, blood withdrawal/ reinfusion and drugs infusion.The catheter was flushed intermittently with saline solution (0,1 ml) containing 50 IU/ml of unfractionated heparin.
The cutaneous blood flow in the rat's ear was recorded by LDF capable to non-invasive optical sensing of tissue reactions by monochromatic laser and analyzing the light reflected from moving red blood cells.Backscattered light from moving red blood cells has a Doppler shift relative to the probe beam.This variable component of the reflected signal is proportional to the number of red blood cells in the probed area and to their velocity.Then the computer calculates the index of perfusion (IP) that reflects the tissue perfusion in the test volume (about 1 mm 3 ) per unit time and is measured in arbitrary perfusion units (PU).
The probe of the LDF device LAKK-02 (SPE «LAZMA», Russia; wavelength 0.63 microns) was set over the inner surface of the right ear with a minimal clearance.Care was taken to place the probe at a skin area with minimal vascularization.The LDF-gram registration was performing for 8 min at each stage of the experiment.When there were significant «noise factors» (due to the movements of the rat, external noise etc.) LDF-gram fragments that lasted at least for 4 minutes (without «noise») were allocated.In the present study we investigated active flux motion components.The following parameters were analyzed: the mean value of IP in the time interval of registration; the maximum oscillation amplitudes of the local cerebral blood flow in the respective frequency bands (Ae, An, Am) obtained by the wavelet analysis.
The stages of experiment: 1.A baseline.
2. Blood loss.We employed an acute fixed-volume hemorrhage model that allow evaluating the natural course of the pathological process and the dynamics of compensatory reactions in posthemorrhagic period [14].The total blood volume (TBV) was calculated as 6.5% of rat's body weight [15,16].The target blood loss volume was 30% of TBV.Blood was withdrawn by a syringe containing 0.5 ml of heparinized saline, in three equal portions (10% of the TBV) during 20 min (1 st , 10 th and 20 th minute).
Registration of systemic blood pressure (BP) and the LDF parameters were observed at: a) a baseline (after 20 min of animal stabilization); (b) 1-10 minutes after the third step of blood loss (before drug administration); (c) Этапы эксперимента.
Введение исследуемых препаратов на 10 минуте гиповолемии в последующие 15 минут при- Statistical processing of the data was performed using Statistica 7.0.To assess the significance of differences between groups we used Mann-Whitney U test and a paired t-test for the dynamics of indexes within the groups.Differences at P<0.05 were considered as significant.The analyzed values were reported as median and 25% and 75% quartile ranges: Me (25%, 75%).Body weight and the amount of blood loss were reported as a mean (M) and a standard error of the mean (SE).

Results and Discussion
The animals of compared groups did not differ in body weight and blood loss volume.Body weight was 379±10 g and 358±15 g, and the blood loss volume was 6.5±0.2 mL and 6.4±0.2 mL for the groups with S or PF administration, respectively.
At a baseline the groups did not differ in all investigated parameters of local cutaneous blood flow as well as in the level of blood pressure (BP).After the blood had been withdrawn (30% of TBV), BP decreased in both groups on average by 58.3% (S) and 62.2% (PF) compared to the baseline (P<0.01).Against this background, in both groups there was a significant decrease in IP and increase in An (Table 1).
10 minutes after S or PF administration (the 15-25 minutes of hypovolemia), BP and IP increased in both groups compared to the previous stage of the experiment (before drugs administration).In PF group this parameters was higher than in the rats of comparing group (Table 1).At the same stage, Ae increased in both groups compared to the baseline and An continued to remain at an elevated levels.Along with these changes in the PF group, in contrast to the S group, An decreased compared to the 1-10 minutes of hypovolemia (before S and PF administration).Perhaps this change was due to the higher elevation of BP in response to the PF administration.This assumption is based on the results of current study as well as our earlier studies [9,17,18], which showed an increase in An during BP decreasing.
К 30-40 минутам гиповолемии у животных с введением ПФ произошло снижение АД до «active» frequency band have been considered as a compensatory response of microvessels, aimed at maintaining both tissue perfusion and transcapillary exchange [19][20][21].PF is known to have a positive effect on microcirculatory alterations [22,23].Therefore, we can assume that one of the mechanisms of PF positive influence on the microcirculation is associated with stimulating effect of the drug on the functional activity of the endothelium.
BP dynamics observed in the present study in a group of animals with PF administration is consistent with data obtained in another study [24] devoted to investigation of PF effects in different doses on BP dynamics, the bioactivity of NO and its derivatives (S-nitrosothiols) in experimental settings on anesthetized rats.It was shown that intravenous PF administration in a dose of 1 g/kg (about 5 ml/kg) was accompanied by a two-phase change in BP: its initial increase then was followed by decreasing this index to means lower than the baseline value.At the same time, PF administration at a dose of 0.14 g / kg (0.9 ml/kg) resulted in a moderate BP decrease from the first minutes with no initial increase.A transient increase in BP was explained by NO sorption on drug's micelles, leading to vasoconstriction.When PF was used in relatively small doses or after decreasing its blood levels, NO oxidation in the micelles of the drug was enhanced associated with S-nitrosothiols production at high concentrations.S-nitrosothiols are known to induce vasodilation that explains the development of hypotension.
However, in the current study we investigated the effect of PF on the microcirculation during hypovolemia, i.e. when skin vessels were vasoconstricted.Also, an injection of PF led not only to a rise in BP, but it doubled the blood flow values (IP) that hardly could be explained by an increased vasoconstriction.BP increase in the study [24] was explained by vasoconstriction.Our data is likely indicate the participation of other mechanisms of PF effect on BP during hypovolemia.On the other hand, BP decrease during the reperfusion without blood flow reduction is consistent with the authors' conceptions on the mechanisms of delayed BP lowering after PF administration in relatively high doses.

Conclusion
PF administration (3 ml/kg of body weight) in rats during hemorrhagic hypovolemia (30% of the TBV), in the first 15 minutes leads to a more pronounced increase in BP and IP compared to the animals with S administration at the same dose.Both S and PF administration leads to an increase of Ae compared to baseline values of this parameter.However, if in S group an increase of Ae was limited to fifteen minutes after the injection, PF administration led to increased values of Ae maintained throughout the period of hypovolemia.These results demonstrate that PF stimulates endothelium-dependent mechanisms of fluxmotions in the skin of rat ear ло 0,9 мл/кг) с первых минут приводило к умеренному снижению АД без фазы его повышения.Преходящее повышение АД авторы объясняют сорбцией молекул NO мицеллами препарата, что приводит к вазоконстрикции.При введении ПФ в относительно малых дозах или снижении его концентрации в крови, усиливаются процессы окисления NO в мицеллах препарата с образованием повышенных концентраций S-нитрозотиолов, обладающих вазодилатирующим действием, чем и объясняется развитие артериальной гипотензии.
during hypovolemia.After blood reinfusion all investigated microcirculatory parameters did not differ from baseline values in both groups.w w w .r e a n i m a t o l o g y .c o m DOI:10.15360/1813-9779-2015-6-19-27