Experimental Research Of Effect Of Oxygen Therapy For Intrauterine Growth Restriction In Fetal Rats And Impact Of Blood System Of Pregnant Rats

BackgroundFetal growth restriction(FGR) was named intrauterine growth retardation(IUGR) in the past. It means the fetal birth weight less than 2500 gram after 37 weeks of pregnancy or less than two standard deviations of the same gestational age average weights or lower than 10 percentile of the same gestational age of normal weights. The incidence of FGR in developed countries is about 2%-3%, and the average incidence in our country is 6.39%, it is one of the main complications in the perinatal period. Perinatals fatality rate of FGR is 4-6 times of normal newborns, in death is accounted for about 30% of perinatals and 50% of intrapartum intrauterine hypoxia perinatals children are FGR fetus. FGR is not only affect fetal development, also influence childhood and adolescence physical ability and intelligent development, its pathogenes are various and complicated, including pregnant women factors, fetus factors and placenta factors,40% of patients have unknown causes. FGR can lead to increases of fetal malformation, premature and full-term low birthweight, perinatals morbidity and mortality increased, and cause long-term complications such as development of nervous system disorders, mental retardation,coronary heart disease, high blood pressure and diabetes incidence increased after adult time. Therefore, effective and timely treatment is the key to improve the prognosis of FGR, with the development of perinatology, people put more and more emphasis on prevention and treatment of FGR. In addition to routine therapy, hyperbaric oxygen therapy has been reported that has obvious effect for FGR, but HBO therapy pressure is higher than normal pressure so as prone to oxygen toxicity, the time is long, long duration malpractice, compression, decompression is likely to cause pregnant or fetus adverse reactions. Therefore, HBO therapy when pregnancy in clinical is not widely application, then the normobaric hyperoxia and low flow oxygen whether benefit for FGR and influence of oxygen therapy for pregnant rat,are all needs further research.In high pressure(more than normal pressure) under the environment of high concentration of oxygen, breathing on treatment of certain diseases method is hyperbaric oxygen therapy, HBO therapy is a special kind of oxygen cure method, it has effect which in atmospheric environment generally oxygen treatment cannot achieve. Clinical has been found, HBO is benefit to miscarriage,hypertension of pregnancy and FGR.Atmospheric high concentration oxygen therapy is called normobaric hyperoxia(NBH) for short,and is also called high flow oxygen outside cabin, means high flow pulsed oxygen,in atmospheric environment oxygen mask or high flow hoods breathing high concentration oxygen, high oxygen flow reaches 8 L-10 L/min, concentration to 80%-99%, reported that NBH achieves good effects in fetal distress, fetus arrhythmia, placenta functions declined, fetal tricuspid regurgitation therapy.Experiments have shown hyperbaric oxygen will damage late pregnancy fetus and neonatal retinal, cause premature and neonatal retinopathy, says eye type oxygen toxicity and blocked HBO therapy promotion in the maternity and pediatric. So does NBH can use to healing FGR? If it has remedial effect, how is the effect, whether it can replace HBO therapy? And security, dose, duration and frequency problem of NBH therapy is still pending further study, theoretical mechanism of NBH for fetal intrauterine treatment is unclear, there is no evidence-based medicine evidences of clinical application. Therefore, this topic research plans to determine whether NBH therapy has certain treatment effect to FGR and its mechanism through influence of three oxygen therapy ways to pregnant rat and fetal rats born situation.ObjectiveEstablish animal models of fetal growth restriction, give pregnant rats of FGR different oxygen therapys, observe fetal rats birth weight, placenta structure and weight changes, calculate mortality and incidence of FGR in fetal rats, pregnancy growth status of pregnant rats, detect blood viscosity relevant index of pregnant rats before and after oxygen therapy,explore feasibility and curative effect of NBH therapy and ordinary low flow oxygen therapy to FGR, explore its whether can improve pregnancy rat blood viscosity and therapeutic to FGR through that, do animal experiment research for late clinical application of oxygen therapy to FGR.Materials And Methods1. Choose SPF level female never mated SD rats,2-3 mouths old, weight 240-280 g. Vaginal smear were obtained everyday at 18:00 pm to assess ovulatory status. Estrous cycle were observed under microscope rats, select female rats with proestrusstage or estrus stage,put the female and male in one cage according to 2:1, the next morning vaginal smear found sperm is judged as already mating, remember for day 1 of pregnancy.2.Eastablish FGR animal mode experiment:grope for the best time and way of ligation of bilateral uterine vessels in pregnant rat:32 maternal rats with pregnancy were divided into four groups,each group has 8 maternal rats. Group A, group B and group C were accepted partial ligation of both uterine arteries and veins respectively on day 12,14 or 16 of gestation.Did nothing to the control group. Celiac anesthesia with 0.3% pentobarbital, give medicine according to lml/100g, since the belly line open abdominal cavity, exposure bilateral uterine, group A with 2-0 # gutstring for pad, group B and C groups with 3-0 # gutstring for pad, all ligated uterine blood vessels with 3-0 # silksuture, pull out gutstring, put uterine back to abdominal cavity, take out the other side of uterine, do the same operation just before, close the abdominal incisions using continuous suture with 4 # silksuture,close the abdominal cavity. On day 21 of gestation, the fetuses and placentas in all groups were surgically taken weighted, and the incidence of FGR and mortality in the fetus were observed.3.Experimental research of effect of oxygen therapy for intrauterine growth restriction in fetal rats and impact of blood system of pregnant rats:96 female SD rats were randomly divided into six groups:①lank control group:without any drug treatment(n=16);②sham surgical control group(n=16);③FGR modle group(n=16);④HBO therapy group(n=16);⑤NBH therapy group(n=16);⑥atmospheric low concentration oxygen therapy group(n=16).③④⑤⑥groups are all use one method to make FGR modle,④group accept HBO therapy after surgical,⑤group accept NBH therapy after surgical,⑥group accept atmospheric low concentration oxygen therapy after surgical. Pregnant rats weigh one to two times a week,six groups of pregnant ratswere executed eight each group respectively on day 12 and 21 of pregnancy,take blood from heart before put to death and then test hemorrheology index,weigh fetal rats and placentas.Put the placenta in 10% formalin overnight,conventional paraffin embedding,section and Hematoxylin-Yihong(HE) dyeing.Results1. Establishment of a fetal growth restriction model in pregnant rats.All rats accept cesarean section on day 21 of gestation to take births, anesthesia with 0.3% pentobarbital and then open abdominal cavity, exposing the uterus, cut open myometrium, punctured fetal membrane, cut umbilical cord, dry new born rats body blood and amniotic fluid,then fetal rats and placenta weight.On day 21 of pregnancy, the control group pregnant rats are heaviest, weight of four groups have difference (P < 0.01).Four groups compared with each other, fetal rats and placental average weight all have difference (P< 0.01). The control group fetal rats average birth weight and placenta weight are more than the other three groups (P< 0.01).In experimental groups, the fetal rats birth weight and placenta of experimental group A is the minimum weight, lower than the other two groups, the fetal rats birth weight and placenta weight of experimental group B is haeviest, all have difference (P< 0.01). Incidence of FGR in the experimental group A (83.1%) is the highest,control group is the lowest,there were significant differences between the four groups (P< 0.01). Mortality of fetal rats in four groups from high to low for experimental group A 11.27%, experimental group B 8.70%, experimental group C 4.11%, the control group1.33%, there was no statistically difference between four group (P= 0.06).2.Influence of oxygen therapy to pregnant rats weight and blood viscosity.(1) weight of pregnant rats in sham surgical control group and control group are steady growing in pregnant time.Each group of pregnant rats weight have no differences before ligation on 12 day of pregnancy(P>0.05),on day 14 of pregnancy, hyperbaric group and atmospheric high-concentration oxygen group pregnant rats weight even slightly lower than the weight on day 12,average weight of four groups compared with control group and sham surgical control group have statistical difference(P< 0.01). After oxygen treatment,weight of pregnant rats in hyperbaric oxygen group growing faster than other groups,hyperbaric group average weight(303.38±9.23)g is higher than the model group(291.75±13.56)g(P< 0.05),NBH group(293.88±5.64)g (P> 0.05) and low concentrations oxygen group(290.75±11.91)g(P< 0.05).Average weight of pregnant rats in model group and low concentrations oxygen group have no differences(P>0.05),respectively is 1 (311.25±12.17)g and(314.38±12.08)g,obviously lower than the other four groups(P < 0.01). Average weight of pregnant rats in HBO group and NBH group have no differences(P>0.05),respectively is(349.38±4.96)g and(346.25±6.94)g,and also has no difference between them and control group(P>0.05).(2) There are no difference between blood viscosity,plasma viscosity,red blood cell aggregation index,number of red blood cell,hemoglobin number and hematokrit of rats in six groups(P> 0.05) on day 12 of pregnancy.There are no difference between high cut and middle cut blood viscosity,number of red blood cell,hemoglobin number of rats in six groups(P> 0.05) on day 21 of pregnancy. Hemorrheology and blood viscosity of pregnant rats in FGR group increase, relevant indicators of pregnant rats in HBO and NBH group reduce, are similar with sham surgical group and control group.Low cut blood viscosity,plasma viscosity,red blood cell aggregation index and hematokrit of rats in modle group and atmospheric low concentration oxygen therapy group are higher than other four groups(P<0.05). Low shear blood viscosity(15.57±6.26) mPa.S,plasma viscosity(1.06±0.30) mPa.S,red blood cell aggregation index(4.60±1.01) and hematokrit(30.23±3.05)% in HBO group are lower than model group and low concentration oxygen group (P< 0.05), compared with the control group and sham surgical group, there are no statistical difference(P> 0.05).Plasma viscosity(1.12±0.14) mPa.S and hematokrit(29.86±3.78)% in NBH group are lower than model group(P<0.05).3.Influence of oxygen therapy to fetal rats weight and the placental weight.Average birth weight and placenta weight of fetal rats in the control group respectively is (4.57±0.37)g and (0.54±0.07)g,and that in the sham surgical group is (4.46±0.36)g and (0.53±0.08)g,there are no statistical difference between two groups(P> 0.05),but significantly higher than model group and low concentration oxygen group(P< 0.01). Average birth weight and placenta weight of fetal rats in the modle group is (3.26±0.49)g and (0.40±0.05)g,significantly lower than other five groups(P< 0.01).After oxygen treatment,average birth weight and placenta weight of fetal rats in the HBO group is (4.11±0.37)g and (0.47±0.05)g,and that in the NBH group is (4.08±0.32)g and (0.46±0.05)g,significantly higher than model group and low concentration oxygen group(P< 0.01),a little lower than control group and the sham surgical group(P< 0.01).There are no statistical difference between HBO group and NBH group(P> 0.05).Average birth weight and placenta weight of fetal rats in the low concentration oxygen group respectively is (3.37±0.39)g and (0.41±0.05)g,higher than modle group but there are no difference(P>0.05),and much lower than control group,sham surgical group,HBO group and NBH group(P<0.01).4.1nfluence of oxygen therapy to FGR incidence and mortality of fetal rats.There were statistically significant differences between FGR incidence of fetal rats in six groups.FGR incidence of fetal rats in modle group is the highest(83.1%), second is the low concentration oxygen group(75.71%).Control group is the lowest(1.33%),it is similar with sham surgical group(2.94%),FGR incidence of fetal rats in HBO group and NBH group respectively are 13.63% and 13.89%,lower than modle group and low concentration oxygen group(P<0.01). There were statistically significant differences between mortality of fetal rats in six groups(P=0.038). Mortality of fetal rats in control group and sham surgical group respectively is 1.33% and 1.47%.And that in HBO group and NBH group respectively is 6.06% and 6.94%.5.Influence of oxygen therapy to structure and morphology of placenta.In this experiment, histological change of each placenta is observed through light microscopy, the results indicate that general observed uterine placenta of rats in FGR model group is blood stasis, edema shape, dishes, central part of placental color thiner than the control group, placenta matrix was surrounded by white face, close to the placenta exudate covers a edge white calcification point focal distribution, shows the degree differs with pale, fetal face amniotic membranes yellow dye, cloudy, burnish is darker, placenta whole shows chronic blood anoxic change.Placental blood microcirculation in HBO group and NBH group are improving, with the increasing area, clearance decreased, and the metamorphosis of villi cellulose membrane flake necrosis significantly reduced, free villi structure is clear, not seen capillaries filled axis congestion, wall relatively complete; bottom exuviate membrane layer structure is clear, blood vessels number more.Percentage of placenta villi surface area in control group is (58.98±1.16)%,no difference between sham surgical group(58.67±0.28)%(P>0.05).Percentage of placenta villi surface area in modle group(42.49±1.36)% is lower than control group(P<0.05).Percentage of placenta villi surface areain HBO group and NBH group are(57.95±.22)% and(58.22±1.02)%,no difference between them(P>0.05).Percentage of placenta villi surface area in atmospheric low-concentration oxygen group is (43.21±1.34)%,similar with modle group(P>0.05), lower than other groups(P<0.05).Conclusions1. This study found that more ahead of time to ligating uterine blood vessels of rats during pregnancy,fetal rats birth weight and placenta weight is more lower, FGR incidence and mortality of fetal rats are more higher. The birth weight and placental weight of pregnant rats in 12 days of ligation group is lower obviously than other groups. The incidence of FGR is 84.13%, except a few(11.11%) of embryonic ligation for dysplasia or chronic blood anoxic led to death absorption,the rest were all survive in the day of full-term birth. so: that the ideal FGR model is part ligation of uterine vessels on day 12 of pregnant rats. Can be used in the experimental research for later.2.After oxygen treatment, the birth weight and placental weight of FGR pregnant rats is obviously weighter than without of HBO or NBH.The incidence of FGR and mortality also lower than modle group, only a slightly higher than normal control group.It showes that hyperbaric oxygen and atmospheric high-concentration oxygen have therapeutic effect in pregnancy rats,and atmospheric low-concentration oxygen have no obvious therapeutic effect on FGR pregnancy rats.The growth of pregnancy rats was very slowly or have no obviously growth, the birth weight and placenta weight obviously lower than normal control group.As the similar of FGR modle group, the incidence of FGR and mortality of pregnancy rats is higher than control group. Because hyperbaric oxygen therapy pressure is higher than normal pressure prone to oxygen toxicity, compression, decompression is likely to cause adverse effect to pregnant or fetus as abortion, etc, therefore premature pregnancy hyperbaricoxygen therapy in clinical did not widely used, this study is a foundation of hyperbaric oxygen treat FGR in the clinical. But its existing or future offspring is still needs further study. 3.The blood viscosity related indexes of pregnancy rats in FGR modle group was higher than that in the control group. Hyperbaric oxygen and atmospheric high-concentration oxygen can reduce blood low shear viscosity value, plasma viscosity, red cell aggregation index and hematokrit of FGR pregnancy rats in some different degree.This shows that oxygen therapy may be improve placental blood anoxic condition of pregnancy rats by reducing blood viscosity and so as to the purpose for treating fetal growth restriction..4.Placenta in FGR model group shows chronic ischemia changes,present extravasated blood condition under light microscope, see more fibrinoid necrosis, fluffy area decreasing, and hyperbaric oxygen and atmospheric oxygen can promote placenta repair of FGR rats, improve microcirculation placental blood anoxic, so as to achieve the purpose of treatment, it molecular mechanism, whether have recent or long-term influence to offspring, are all still needs further research.