The Effects Of In Utero Hypoxia On Acetylcholine-mediated Vasodilatation In Fetal Sheep Middle Cerebral Artery And Its Underlying Mechanisms

Background:Cerebrovascular diseases are important causes of human death.Hypoxia could lead to adult middle cerebral artery dysfunction and increase risks of cerebrovascular diseases.Hypoxia in utero is common during pregnancy and labor,which can induce fetal growth restriction,abnormal neurogenesis,and even fetal death.It is largely unknown whether intrauterine hypoxia affects fetal middle cerebral artery vasodilatation.This study investigated effects and mechanisms of intrauterine hypoxia on fetal middle cerebral artery vasodilatation.Methods:Near-term pregnant sheep(122±3 days,term:approximately 145 days)were randomly selected.The operation was performed under aseptic conditions.After anesthesia,the fetal was placed with an inflatable occluder around the umbilical cord.At the same time,the catheters were inserted into fetal femoral arteries at both sides.After 4-5 days of postoperative recovery,the compression of umbilical cord was performed to reduce the umbilical blood flow until fetal arterial oxygen pressure decreased about 50%of the baseline.The compression was maintained for 50 min,and then the occluder was deflated,allowing recovery of umbilical blood flow to baseline.This hypoxia treatment was repeated every day for 5 days continuously.The fetus exposed to intrauterine hypoxia was defined as in utero hypoxia group.The fetus without the intrauterine hypoxia served as the control.After 5 days of intermittent intrauterine hypoxia,the sheep were euthanized for collections of the middle cerebral artery.Human umbilical vein endothelial cells(HUVEC)were exposed to hypoxia in cellular experiments.Vascular tone measurement,molecular analysis,dihydroethidium assay(DHE),transmission electron microscope(TEM),enzyme linked immunosorbent assay(ELISA),and molecular analysis were utilized to determine vascular functions,tissue anatomy,and molecular pathways in fetal sheep middle cerebral artery.Results:In fetal sheep middle cerebral artery,both sodium nitroprusside(SNP)and acetylcholine(ACh)induced reliable relaxation.ACh-induced relaxation was obviously attenuated by intrauterine hypoxia,but SNP-mediated dilation was not significantly changed.Atropine(the inhibitor of muscarinic receptors),P-F-HHSiD(inhibitor of CHRM3),L-NAME(inhibitor of NOS3),and u0126(ERK1/2 inhibitor)blocked most of ACh-mediated dilation,while AF-DX 116(inhibitor of CHRM2)and Tropicamide(inhibitor of CHRM4)partially inhibited the dilation.Indomethacin(inhibitor of COX)and SB203580(inhibitor of MAPK14)did not significantly change ACh-mediated dilation.Tempol(SOD analogue)and PS-341(ERK1/2 agonist)could restore the attenuated ACh-mediated vasodilatation following intrauterine hypoxia.The NO production,mRNA expressions of CHRM2,CHRM3,and protein levels of CHRM3,p-NOS3,SOD2,MAPK14,p-MAPK14,ERK1/2 and p-ERKl/2 were significantly reduced by intrauterine hypoxia.Dihydroethidium assay showed that production of ROS was increased under intrauterine hypoxia.TEM analysis revealed damaged endothelial cells by intrauterine hypoxia.In the fetal sheep circulation,ELISA showed that intrauterine hypoxia decreased Total SOD,Mn-SOD,CAT,increased ROS and MDA level.In human umbilical vein endothelial cells(HUVEC),hypoxia increased ROS formation and decreased expressions of CHRM3,p-NOS3,SOD1,SOD2,SOD3,p-MAPK14,ERK1/2 and p-ERK1/2,while tempol and PS-341 potentiated p-NOS3 protein expression.Conclusions:In utero hypoxia reduced the ACh-mediated vasodilatation in fetal sheep middle cerebral artery predominantly via decreased CHRM3 and p-NOS3,and the decreased NOS3 bioactivities might be attributed to ROS and ERK1/2 pathways.