Impact And Mechanisms Of Prenatal Hypoxia On Bone Growth Of Offspring Rats

Part1:Impact of prenatal hypoxia on fetal bone growth and its mechanism1.1Objectives:To determine the effect of prenatal hypoxia on fetal bone growth and its mechanism1.2Methods:Pregnant rats were randomly divided into control group and hypoxia group,20 rats in each group.Rats in hypoxia group were provide with 10.5% oxygen concerntration in hypoxia box from the gestation day 4 to day 20,the control group rats into normoxic box.Fetuses were removed from pregnant rats by cesarean section on gestation day 20,Fetal body length and weight were measured immediately,and IUGR rate was calculated according to the criteria.Fetal left femurs(one per litter)were isolated under a dissection microscope.Femurs were fixed in 10% paraformaldehyde solution for 24 hours.Then the femurs were dehydrated and embedded in paraffin for histological investigation.The fetal femur sections were stained with hematoxylin and eosin(HE)for femur length(FL)and the calcifying zone length(CL).Safranin O-fast staining was performed to measure proteoglycan levels according to the manufacturer's instructions.Immunohistochemistry was performed with a DAB staining kit for determining expression of IGF1,IGF2,IGF1 R,AKT1/2,IRS1,Aggrecan,Col2a1,and Col1a1 proteins in femur growth plates.All other femurs were harvested for subsequent experiments.RT-PCR and Western-blot method were used to detected the expression levels of related genes and protein.We cultured fetal growth plate chondrocytes in normal or hypoxia environment(1%)for 48 hours,and then tested expression of ECM gene and IGF1 signaling gene by q RT-PCR.1.3Results:Compared to control,1)the IUGR rate was increased,the fetal body weight and lenth significantly lower,Fetal FL and CL/FL ratio was statistically reduced.2)the growth plates in hypoxia group exhibited a deceased level in proteoglycan,as determined by measuring mean optical density(MOD).Both the m RNA and protein levels of cartilage ECM genes,including Aggrecan,Col2a1,and Col1a1 were significantly lower in the prenatal hypoxia treated fetuses compared to control pups.The expression of IGF1,IGF1 R,IRS1,AKT1/2,not IRS2,in growth plates,was significantly decreased in the hypoxia fetuses.3)In vivo,q RT-PCR analysis indicated that hypoxia markedly reduced the m RNA expressions of IGF1,IGF1 R,IRS1,and AKT1/2 in growth plate chondrocytes.1.4Conclusion: The date suggested that prenatal hypoxia caused delay of fetal skeletal growth,and inhibited the synthesis of critical ECM components in fetal growth plates,which may induced by down-expression of IGF1,IGF1 R,IRS1,AKT1/2in growth plates.Part 2:The effects of hypoxia expose during pregnancy on ovariectomized osteoporosis in offspring rats2.1Objectives:To determine the effect of prenatal hypoxia on ovariectomized osteoporosis in offspring rats and its mechanism2.2Methods:The female offspring of the prenatal hypoxia rat and control female ratsreceived a bilateral ovariectomy by a minimally invasive surgical technique at 9 months old under isoflurane anesthesia.All rats were sacrificed for harvesting tissue 1 month following the surgery.The left femurs were for bone microstructure analysis using micro-computed tomographic(micro CT)for osteoporotic changes.All other femurs were harvested for subsequent experiments.RT-PCR method were used to detected the expression levels of related genes.2.3Result:1)Compared with control OVX rats,OVX offspring in the prenatal hypoxic group showed a greater bone loss.Micro-CT revealed a decrease in bone volume per tissue volume(BV/TV,P <0.01),trabecular thickness(Tb.Th,P<0.001),trabecular number(Tb.N1,P <0.05),bone mineral density(BMD,P <0.01),and an increase in structure model index(SMI,P <0.001),degree of anisotropy(DA,P <0.05),and trabecular pattern factor(Tb.Pf),in prenatal hypoxic OVX.There were no differences in trabecular separation(Tb.Sp,P <0.01)between the control and hypoxic rats receiving bilateral ovariectomy.2)the m RNA levels of IGF1,IGF1 R,Col2a1,and Col1a1 were decreased in the prenatal hypoxic OVX rats,whereas no significant differences were observed in Aggrecan compared with the control OVX rats.Conclusion:The date suggested that prenatal hypoxiaincreased damage to OVX-induced osteoporosis in there adult offspring,which may have a link with IGF-1 signal and ECM genes.