The Specific Features of Red Blood Cell Membranes in Premature Neonates Due to Multiple Pregnancy

Objective: to substantiate the efficacy of bipolar impulses of different shapes and duration. Method. The investigation used the dynamic model II of the mammalian ventricular cardiomyocyte membrane in the guinea-pig, proposed by Luo and Rudy (1994—2000). The cardiomyocyte membrane was acted by substituting the current density of the impulse under study. The threshold impulse energy ratio considered as an integral index of the threshold action (a measure of efficiency) of an impulse measured in μA 2 ms/cm 4 was then calculated. A comparison was made between a classical quasi<sinusoidal impulse, a stepwise quasi-sinusoidal, rectilinear, classical trapezoidal, trapezoidally modulated (the presence of high-frequency, high-amplitude oscillations of current) impulse and its unmodulated equivalent, and a trapezoidal low<angle front tail impulse of the duration equal to that of the classical quasi-sinusoidal one. The shape of the impulses corresponded to the 100-ohm resistance of the chest. Results. The most effective impulses  proved  to  be  a  quasi-sinusoidal  stepwise  impulse  (229.6  μA 2 ms/cm 4 ),  next  a  classical  quasi-sinusoidal  impulse (249 μA 2 ms/cm 4 , +9%) and a trapezoidal low<angle front tail one (253.0 μA 2 ms/cm 4 , +10%). The trapezoidally modulated impulse (397 μA 2 ms/cm 4 , +73%) turned out to be lowest effective (in the threshold impulse energy ratio). The other  impulses  were  intermediate  between  the  above  impulses  in  the  following  order:  a  modulated  trapezoidal impulse equivalent (272.0 μA 2 ms/cm 4 ), next a rectilinear impulse (273.5 μA 2 ms/cm 4 ), and a classical trapezoidal one (307.0 μA 2 ms/cm 4 ). Conclusion. In terms of the excitation threshold of the Luo-Rudy model of the guinea-pig cardiomyocyte membrane, the most effective impulses are quasi<sinusoidal stepwise, next quasi<sinusoidal and trapezoidal low-angle front tail ones.

www.reanimatology.com,www.niiorramn.ruЛитература ined using an atomic force microscope (AFM).The objects to be examined were residual umbilical cord blood (RUCB), central venous blood 7 hours after birth and venous blood had been taken from the premature neonates on day 7 of life.The placentas were also examined.The data were compared with gestational age, birth health status, and clinical symp toms.Results.In babies at birth, planocytes are a major morphological type of red blood cells; at the same time, there are also stomatocytes and other abnormal cells that are intermediate reversible forms.The first order surface is the least stable and prone to pathological changes on the red blood cell membrane and the spectrin matrix and protein clus ters are unchanged.The partial pressure of carbon dioxide and hemoglobin oxygen saturation affected the heights h 1 and h 2 of the red blood cell membrane in residual UCB.The internal, cytoplasmic surface is resistant to long term hypoxia exposure, as the heights h 1 and h 2 of the red blood cell membrane at birth corresponded to the similar heights of healthy neonates and remained unchanged throughout the study.The first postnatal life hours are characterized by the active transformation of red blood cells from one form to another.The fact that diskocytes are lacking in premature neonates during the early neonatal period suggests that the resistance of red blood cell membranes to unfavorable intrauterine factors is diminished.The consequences of prenatal exposure of red blood cell membranes are retained for a certain time and are beyond the early neonatal period.A baby's birth stops a cascade of pathological reactions, but there is no normalization in the indicators in question by postnatal day 7.The high membrane flickering is conserved; the morphological composition of red blood cells does not persist.Key words: multiple pregnancy, membrane nanos tructure, red blood cells, premature neonates, placenta.

Molecular and cellular mechanisms for the development of critical conditions
Alterations of erythrocytes may contribute to mech anisms of critical illness development [1][2][3][4][5][6][7].A great atten tion has currently been given to the issues of perinatology because the rate of premature and sick children birth is still high.Special attention has been given to multiple pregnan cies in which there is a high risk of antenatal and intranatal deaths of fetuses and birth of pre term infants including the ones with low and extremely low birth weight [8,9].
Along with the complex examination of the newborns the study of red blood cells (RBCs) and other cells in resid ual umbilical cord blood (RUCB) remains an important source of information, especially in multiple pregnancy and preterm birth, because the detection of abnormal cells and cell elements could be due to the process of adaptation to the labor or a certain disease.
At the same time, hypoxia is an important factor affecting the RBCs condition.Acute hypoxia was found to increase the number of RDCs and hemoglobin concentra tion.Intrauterine hypoxia increases the number of nor moblasts in cord blood, which indicates the stimulation of erythropoiesis [10].
Active hemopoiesis in extremely pre mature infants with extremely low body is a necessary condition for the adaptation in the early neonatal period.The lack of bone mar row hemopoiesis, low hemoglobin content, low number of RBCs, low hematocrit, change of RBCs morphometric char acteristics -poykilocytosis -are negative prognostic symp toms.The normalization of erythropoiesis is observed only at the end of the 1-2 nd month of life in pre mature infants with favorable process of adaptation [11].The morphological structure of RBCs, the structure of their membranes, the detection of reversible and irreversible changes have diagnos tic significance along with their quantitative characteristics.The oxygen transport function of RBC depends on the func tional state of its membrane [12].Studing oRBCs morpholo gy using light immersion microscopy showed that echinocytes, оvalocytes, spherocytes, shistocytes were the most often common forms of RBCs in newborns with multi ple organ failure by the 7 th day of life, which may be caused by the influence of various factors, including effects of oxidative stress on RBCs membrane [13,14].
Studying the RBC ultrastructural characteristics using light and electron microscopy shows that the indi vidual ultrastructure of diskocytes and planocytes is not always homogeneous.The thickening of the outer zone of cell membrane lipid bilayer is typical for diskocytes.The ultrastructure of stomatocytes is more heterogeneous if compared with diskocytes and planocytes.
When forming, less optical density is typical for stoma tocytes and increases during further cell transformations.Double cell membranes are formed on the surface of stomato cytes.In some cases the transformation of diskocytes into stomatocytes causes additional exit of hemoglobin and parts of the inner content from the cell through small areas of cell mem brane lipid bilayer.The ultrastructure of stomatocytes is char acterized by less homogeneous membrane the larger number of its local damages and less even intracellular distribution of hemoglobin if compared with diskocytes;.The results obtained show that the reversible transformation «diskocyte stoma tocyte» and back cannot be exactly the same because the trans formation of stomatocyte into diskocyte will require so called «healing» of local destructive changes in cells [15].Atomic force microscopy allows to study the microstructure of RBC membranes in various diseases and critical conditions [16,17].
Objective: to study the morphology of RBCs and the nanostructures of their membranes in premature children born from a multiple pregnancy.

Materials and methods
A study of RBCs membrane nanostructure of 28 newborns including 14 premature newborns born from a multiple pregnancy, which formed the study group, was performed.The basic indica tors of pre mature infants are presented in table 1.The mean ges tational age was 34.1±1,7 week, birth weight 2065,4±304,8.4 spontaneous twins (3 bichorionic biamniotic and 1 monochorionic biamniotic) and 2 trichorionic triamniotic triplets, received by the method of in vitro fertilization (IVF) were included in the study.4 (28,6%) neonates with respiratory distress syndrome (RDS) needed artificial lung ventilation (ALV).The duration of artificial lung ventilation was 52±23 hours.
In most cases the amniotic fluid was transparent, in two cases there was blood due to the placental abruption.The male gender was prevaliant.
Exclusion criteria: newborn isoimmunisation by the ABO and rhesus factor.
The data on the newborn mother's health condition, the course of pregnancy, the way of delivery are very important.The medical anamnesis of the mothers is presented in table 2.
In 83.3% of cases the obstetric history was complicated: the pregnancies followed previous medical abortions, spontaneous abortions, which gave the background for the threat of interrup tion and pre term delivery from the earliest days.The physiologic cause of pregnancy was observed in one case.
The primary and secondary infertility, multiple pregnancy, obtained by the method of IVF are the risk factors for pathological cause of pregnancy.In our study 2 pregnancies, obtained with the help of high reproductive technologies, were under the threat of interruption from the earliest days.One of them was complicated by isthmic cervical insufficiency, the other one was complicated by gestational diabetes, preeclampsia of the second half of a pregnan cy, which was the cause of pre term delivery.In one case the third child was born without any signs of live birth, the intranatal fetal death was confirmed.
The causes of preterm birth were premature pouring out of amniotic fluid, the detachment of low lying placenta, rapid deliv ery, preeclampsia of the second half of pregnancy, isthmic cervical insufficiency.The majority (5-83,3%) of pregnant women held an emergency caesarean cessation by Gusakov's method.
All the pregnant women included in the study received treat ment aimed at the prolongation of pregnancy and improvement of maternal placental blood flow.
The RUCB of 14 full term newborns with favorable course of pregnancy and urgent delivery was used to obtain normal RBCs in the field of AMS.
The mean gestational age of the infants was 39,4±0.5 weeks, the birth weight -3131,7±588,8 grams, one minute Apgar score of 8 ±of 0.4 points.78.5% of the newborns were born by vaginal deliv ery, 21,4% -by emergency caesarean cessation.At birth all the infants had no signs of acute hypoxia.Pathological changes of the placenta were not detected during the morphological examination.
The following methods were used: 1.
The study of RBCs of the newborns in the field of the atomic force microscope (AFM).
The objects to examine were: RUCB of premature neonates, central venous blood after 7 hours of birth and venous blood of pre mature infants taken on day 7 of life.The samples of RUCB were taken immediately after crossing the umbilical cord.They were taken in special 0,25 ml tubes containing EDTA as preservative, the blood was mixed with the preservative, settled within 30 min utes, then a monolayer of erythrocytes for further processing in the field of the atomic force microscope was prepared: 20 μl of blood were put on a slide glass and a monolayer of erythrocytes with the aid of centrifuges Diff Spin 2 (USA) was prepared.The picture of erythrocytes was received using the atomic force microscope «NTEGRA prima» (Russia) in semi contact mode.Cantilevers NSG01 A were used as probes.The Number of scan dots was 512 and 1024, the area of scan was 100 100 microns, 10 10 microns.The analysis of RBC membrane nanostructures was performed using a spatial transformation with the use of the Fourier decom position of RBC membrane surfaces into three orders according to spectral windows of 1000-600 nm., 80-300 nm., 20-60 nm. for the first, second and third orders, correspondingly.
The first order correlates with the phenomenon of flickering surface «waves» on the erythrocyte membrane and reflecting macro structural properties of the membrane; the second and the third orders correlate with the configuration of spectrin matrix and reflect the condition of protein connection nodes.The method of analysis of nanostructure has been described in details in previ ously published papers [18,19].
2. Hystological examination of the placenta.The morpho logical data was compared with gestational age, the condition of the children at birth, clinical symptoms and the morphology of RBCs. 3.
The statistical processing of data was carried out using the standard program of Origin 6.1.,whichis included in Microsoft Office (USA) set.The significance of the differences was estimat ed by the factor analysis of variance (One way ANOVA).The dif ference was considered to be significant at (p<0.05).

Results and discussion
RUCB is the most important object to be examined in newborns.The results indicate the antenatal and intranatal condition of the fetus.The study showed that in case of normal pregnancy and urgent delivery in RUCB of newborns there were 85% of flat shape RBCs -planocytes and 15% of transformed RBCs, 3% of which were present ed by ekchinocytes and stomatocytes, 9% -by other abnormal cells representing intermediate forms of erythro cytes associated with the severity of intrauterine poykilo cytosis (Fig. 1).
Different forms of RBCs were detected studying the morphology of RBCs of premature infants born from multi ple pregnancies.Diskocytes as the basic physiological form of RBCs were not typical for the infants of this group.They were present in the blood 3 newborns, only in one of which (the second child from bichorionic biamniotic twins) the content of diskocytes reached 43% of the total cells num ber; in other infants only isolated cells were visualized.
Planocytes were visualized in 12 (85,7%) infants, but the frequency of their occurrence was different: in 9 children the content of planocytes was from 41 to 89.4%, so they were the prevailing form of RBCs; in 3 infants the frequen cy of their occurrence was 5-29% and in 2 infantsplanocytes were not detected.
Stomatocytes were found in 12 (85,7%) infants, but their number varied from 3 to 95%.Significant stomacyto sis was diagnosed in the first child of bichorionic bianmi otic twins with the collision of twins in childbirth.
Echinocytes were almost never detected in the cord blood.35% of echynicytes were found in one child from bichorionic biamniotic twins; in four infants isolated echynocytes were visualized.They were not found in new borns of monochorionic biamniotic twins and triplets.
In twins the morphological features of RBCs didn't not depend on the placenta chorionality.There were three types of cells in the RUCD: planocytes, stomatocytes and other abnormal cells associated with the severity of fetal poykilocytosis.This phenomenon was typical for all the Molecular and cellular mechanisms for the development of critical conditions

Fig. 1. Abnormal forms of red blood cells due to intrauterine poykilocytosis (the image was received by the method of AFM)
infants included in the study.The frequency of their occur rence was 30,4±22,3% of the total number of RBCs.
In triplets one half of RBCs was presented by planocytes, the second half -by stomatocytes and other abnormal cells.When intrauterine death of the third fetus of the triplet occurred, 55% planocytes and 45% of other abnormal cells were detected in his RUCB, which indicated his recent death.
4 newborns needed mechanical lung ventilation.2 of them were newborns from bichorionic biamniotic twins and the third and the fourth ones were the second and the third infants of trichorionic triamniotic triplet, respective ly.The twins were born with rapid delivery at 32 weeks of gestation, which resulted in acute hypoxia and intrauterine respiratory distress syndrome.The morphological struc ture of RBCs in RUCB of the first infant was as follows: 89,4% of planocytes and 10,6% of stomatocytes; of the sec ond newborn -41,3% of planocytes, 22,6% of stomatocytes and 35,5% other abnormal cells.The same morphological structure of RBCs was observed in two newborns of a triplet, who needed artificial lung ventilation.
Thus, planocytes were found to be the main morpho logical form of RBCs, typical for preterm newborns.
The presence of stomatocytes and other abnormal cells indicated prenatal poykilocytosis.Planocytes are transient physiological form of erythrocytes of newborn regardless of gestational age and the number of fetuses.
Analyzing the nanostructure of RBCs membranes, the height of the first order (h 1 ) was found to exceed the indicator of the infants in the group of comparison (p<0.05) in 8 (57.1%) newborns, that indicated in utero violation if RBCs membrane (membrane «flickering»).
It was tyipcal for the second child of the twins and for the second and third child of a triplet regardless the pla centa chorionality; the value of this indicator significantly (p<0.05)increased from the first newborn to the third one.For example, h1 for the first child was 3 nm,for the second one -5 nm, for the third one -7.5 nm; that might be due to hypoxia, which is often experienced by infants in multi ple births.The collision of twins was associated with mem brane flickering in both infants, but the height of nanos tructure h 1 was 12 times greater in the first newborn and it was associated with almost absolute stomacytosis (Fig. 2).
The heights h 2 and h 3 did not differ significantly from the corresponding indicators of children in the group of comparison, i. e. the spectrin matrix and the structural state of RBCs proteins were stable.
The equation of regression was received the following correlation links were detected by statistical correlation analysis in RUCB: • a feedback link of medium strength between hemo globin saturation with oxygen (%SO 2 c) and the height h 1 (r=a 0.7; p=0.03); y=7,18181672 -0,0527131533*x; • a feedback link of medium strength between the partial pressure of carbon dioxide (pCO 2 ) and the height h 2 (r= 0,6; p=0.04); y=3,31985946 -0,0370085327*x; • a direct link of medium strength between the fifth minute Apgar score and the height h 1 7 hours after the birth (r=0,6, p=0.02); y = 15,0731707 + 2,97926829*x.
Other indicators of RUCB affected RBCs mem branes, for example pCO 2 , the content of which was physi ological and reached 47,5±10,4 mm hg., that favorably affected the spectrin matrix of RBCs.
Low intranatal hemoglobin saturation with oxygen affected the value of membrane flickering.Low Apgar score indicated acute intranatal hypoxia, which was not observed in the majority of the newborns in our study.
The placenta it was found to match the term of gesta tion; the combination of chronic subcompensated and acute placental insufficiency, moderate disturbances of water salt metabolism (swelling of the decidual tissue, fiber's stroma Vartanov's jelly) were registered.Other dystrophic changes were also registered: fibrin, fibrinoid, calcifica tions; some fibers were immured in fibrinoid.The vascular ization was poor.
Insufficient vascularization of the fibers and the obliteration of vessels, moderate dystrophic changes of the vessels walls were also were observed.The following signs of acute placental insufficiency were seen: vascular paresis, hemorrhages.In case of low placentation and premature detachment of the low lying placenta the vascularization of fibers was absent.
Individual morphological changes of placenta and abnormality of umbilical cord was typical for triplets.In one case the placenta corresponded to the age of gesta tion of the first and the second fetuses, there were not violations of the fiber maturation.Moderate dystrophic changes were identified: fibrin, calcifications, pseudoin farctions, cell dystrophy.There were small hemorrhages in intrafiber space and blood circulation disorders.Diffusive leukocyte infiltration was registered in decid ual layer.Paracentral attaching of the umbilical cord of first fetus and regional attaching of umbilical cord of the second fetus were also found.The umbilical cord of both fetuses was short.
The third fetus (intranatal death) revealed decom pensated placental insufficiency: significant dystrophic changes, calcified sinticial nodes, violation of maturation and branching of fibers, sclerosis of fiber stroma, violation of vascularisation, acute placental insufficiency (vascular paresis, focal hemorrhage, regional attaching and short umbilical cord) were registered.
In other case there was violation of maturation and branching of fibers and their chaotic arrangement.In other case there was prevalence of small fibers and dystrophic changes (fibrin, calcifications, pseudoinfarc tions; cell degeneration), focal hemorrhage in intrafiber space, blood circulation disorders, partial obliteration of fiber supporting vessels.
The changes in placenta were accompanied by distur bances of blood circulation, and promote the development of hypoxia, adversely affected the prenatal state of RBCs, causing the damage of membrane nanostructure.
At birth the cord blood parameters of pulmonary gas exchange reflected the intranatal state of the neonates: the partial pressure of oxygen (pO 2 ) -39,3±16 mm Hg, the sat uration of hemoglobin by oxygen (SO 2 c) -58,6±23.7%.The clinical condition of 10 (71,4%) neonates was stable, there was not respiratory failure, oxygen dependence was moder ate, infusion therapy, oxygen therapy through the front mask with oxygen fraction = 30-40% were carried out.The fol lowing early period of adaptation was uneventful.
7 hours after the birth morphological structure of RBCs was almost unchanged.Three types of RBCs were determined in newborns: stomatocytes, planocytes and other abnormal cells.A significant change in the number of these cells, in comparison to RUBC, was not revealed (p>0.05).
Single diskocytes were visualized in three children regardless the way of delivery, placenta chorionality and other factors.The same was for ekchynocytes.Thus, in the early hours of postnatal life the morphological composition of erythrocytes was stable.
The dynamics of h 1 и h 2 of RBCs membranes in the following stages of the study is presented on the graph (fig ure 3).7 hours after the birth there was an increase in h 1 , but the changes were not statistically significant (p> 0,01).
The following changes of the research indicators were observed: in 8 (57.1%) infants with low h 1 in RUBC this indicator increased up to 4,5-12 7 days after the birth, that meant that the violations or RBCs membrane nanostruc ture remained..In other cases h1 decreased to 1.5-3 nm.
The state of spectrin matrix and protein clusters was stable.Significant changes were not observed (p<0,01).
The early neonatal period was characterized by an increase of the stomatocyte number and the reduction of planocyte number (p<0.05), the lack of discocytes and ekhinocytes.
By the 7 th of life the indicators h 1 and h 2 of RBCs membranes didn't change (p>0.01).
The changes of the membrane flickering were differ ent: in 5 newborns (35,7%) the value of h 1 decreased.In 2 newborns with high h1 there was a wave like change of h 1 in RUBC: it was decreasing by the 7 th hour after the birth and then the indicator increased dramatically; in other cases, the height of h1 was practically the same.
By the 7 th day of life the condition of the newborns was stable, there were not signs of respiratory failure and oxygen dependence in the infants, the infusion therapy in the majority of the infants were cancelled on the 4-5 days after the birth.
In all the 4 newborns the course of the RDS was favorable, by the 5 th day of postnatal life the respiratory insufficiency was resolved, however all the RBCs of the sec ond newborn of the bichorionic biamniotic twins were transformed into stomatocytes by the 7 th day of life; the stomacytosis was 74,6% in the 1 st newborn; in the 3 rd new born of triplet the stomacytosis was 51%.
It was not possible to identify the relationship between stomacytosis and the duration of mechanical ven tilation due to small size of sample.
In case of multiple pregnancy the onset of preterm birth was the result of chronic and acute placental insufficiency, the abnormality of the umbilical cord, which was confirmed by morphological study and indicators of gas exchange at birth.That resulted in perinatal change of RDCs memranes of dif ferent severity.The main RBCs form at birth were planocytes, stomaticytes and other abnormal cells, which represented intermediary, revercible forms were also identified.Changing of RBCs shape may be due to changing the ratio of external and internal area of monolayers.It is a reversible phenomenon.
Prenatal injuries of RBCs outer membrane -flicker ing remained, spectrinal matrix and protein clusters were intact, the surface of the first order, reflecting the damage to the membrane at this level was the least stable indicator.
Other indicators (pCO 2 , % SO 2 c) of RUCB affected the height h 1 and h 2 in RBCs membrane.
A correlation between the indicators and the research heights of RBCs membranes was also found.
The inner cytoplasmic surface was resistant to prolonged hypoxia, because the height h 2 and h 3 of RBCs membranes were similar to the corresponding indicators of healthy newborns at birth and were not changed later.
The first hours of postnatal life were characterized by active transformation of RBCs from one form to another.The lack of diskocytes in premature infants in the early neonatal period indicateed a reduced sustainable stability of RBCs membranes to the influence of unfavorable intrauterine factors.
The effects of prenatal exposure to the RBCs mem brane remained for a certain period of time, and lasted longer than early neonatal period.Baby's birth of interrupted the cascade of pathologi cal reactions, but the normalizing of research indicators didn't occur by the seventh day of life.High membrane flickering remained, no stabilization of RBCs morphologi cal composition occured.

Conclusion
1. Using atomic force microscopy we confirmed that the damage of RBCs nanostructure occurs in pre term newborns during complicated multiple pregnancy.
2. The early period of adaptation was found to be characterized by the change of morphological forms ery throcytes: planocytosis were replaced by stomacytosis, other abnormal cells were also detected.They appeared due to the active process on the RBCs membranes.
3. Membrane flickering was mostly diagnosed in the second newborn of twins, the second and the third child of triplets; it did not depend on the chorionality of placenta, its value increased from the first infant to third one.
4. The collision between the twins was associated with membrane flickering and significant stomacytosis in both children.

Fig. 3 .
Fig. 3.The dynamics of the heights h 1 and h 2 of RBCs mem branes in different periods of the study.