Protective effect of PGSF on myocardial ischemia injury induced by ISO in neonatal rats and its possible mechanism / 中国医师杂志

Objective:To study the protective effect of polygalasaponin F (PGSF) on isoproterenol (ISO)-induced myocardial ischemia (MI) injury in neonatal rats and its possible mechanism.Methods:Fifty newborn Sprague Dawley (SD) rats were randomly divided into control group, model group, low, medium and high dose PGSF groups (5, 10, 50 mg/kg), with 10 rats in each group. The rats in the control group were treated with normal saline; Myocardial ischemia (MI) model was established in model group by subcutaneous injection of isoproterenol (ISO, 85 mg/kg, once a day); The MI model was established in rats of low, medium and high dose PGSF group after intraperitoneal injection of 5, 10 and 50 mg/kg PGSF for 7 days. The cardiac function of rats in each group was evaluated by echocardiography; pathological changes of myocardial tissue of rats in each group were observed by hematoxylin and eosin (HE) staining; The serum activities of troponin I (cTnI), creatine kinase isoenzyme (CK-MB), myoglobin (Mb) and lactate dehydrogenase (LDH) of rats in each group were detected by enzyme linked immunosorbent assay (ELISA); the content of malondialdehyde (MDA) and active oxygen species (ROS) in myocardial tissue were detected ; the expression of nuclear proliferation antigen (Ki67) and caspase-3 protein in myocardial tissue was detected by immunohistochemical staining; The expression of protein kinase B (AKT) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein in myocardium was detected by Western blot.Results:In the model group, the myocardial structure was disordered, the cells were congested and swollen, and there were a lot of inflammatory cells infiltrating and large necrotic foci. The left ventricular wall thickness (LVWT), left ventricular ejection fraction (LVEF), fractional shortening (FS), heart rate (HR) and expression of Ki67 positive protein in the model group were lower than those in the control group (all P<0.05), while the left ventricular end systolic volume (LVESV), activities of cTnI, CK-MB, Mb, LDH in serum, content of ROS and MDA in myocardial tissue and caspase-3 positive protein in the model group were higher than those in the control group (all P<0.05). Compared with the model group, the degree of myocardial pathological changes in neonatal rats of the low, medium and high dose PGSF groups gradually decreased. Compared with model group, the LVWT, LVEF, FS, HR and expression of Ki67 positive protein increased in low, medium and high dose PGSF groups (all P<0.05), while the LVESV, activities of cTnI, CK-MB, Mb and LDH in serum, content of ROS and MDA in myocardial tissue and the expression of caspase-3 positive protein decreased (all P<0.05); Western blot results showed that the relative expression of phosphorylated(p)-AKT/AKT, p-Nrf2/Nrf2 protein in myocardium of model group was lower than that of control group (all P<0.05); The relative expression of p-AKT/AKT, p-Nrf2/Nrf2 protein in myocardium of low, medium and high dose PGSF groups were higher than that in the model group (all P<0.05). Conclusions:PGSF has protective effect on MI injury in neonatal rats, and its mechanism may be related to anti-apoptosis and anti-oxidative stress.

Relationship between intestinal fatty acid-binding protein expression and necrotizing enterocolitis in neonatal rat model / 中华围产医学杂志

Objective To analyze the role of intestinal fatty acid-binding protein (I-FABP) expression in a neonatal rat model of necrotizing enterocolitis (NEC).Methods A total of 24 newborn rats were randomly divided into two groups:control group (n=6) and NEC group (n=18).Rats in the NEC group were fed with formula and experienced hypoxia,reoxygenation,cold stress and sequentially Lipopolysaccharide (10 mg/kg) lavage for three consecutive days to establish NEC model,after which were respectively sacrificed on day 1,2 and 3 (six for each day).Those in the control group were all sacrificed on day 3.Ileocecal tissues were collected for morphological and histological analysis.I-FABP expression was detected using Western blot and immunohistochemistry (IHC).One-way analysis of variance,LSD-t test,Kruskal-Wallis H test,Mann-Whitney U test and Pearson's correlation analysis were used for statistical analysis.Results The NEC model (intestinal pathological score ≥ 2) was established successfully without causing death.Compared with the control group,the NEC group showed less body weight gain [M (P25-P75):1.00 (0.48-1.35) vs 1.74 (1.62-1.86),1.25 (0.75-1.40) vs 2.61 (2.53-2.99),1.35 (0.88-1.48) vs 3.60 (3.48-3.73);Z=-2.898,-2.903,-2.892;all P＜0.05] and higher intestinal pathological scores [(2 (2-3),3 (2-3),4 (3-4) vs 0 (0-1);all P＜0.05] on day 1,2 and 3.The intestinal pathological score on day 3 was significantly higher than that on day 2 and day 1 (both P＜0.05).Expression of I-FABP and the number of I-FABP positive enterocytes in the NEC model group were increased compared with those in the control group [Western blot:0.179 (0.179-0.186),0.231 (0.211-0.245),0.202 (0.192-0.225) vs 0.091 (0.086-0.093);IHC:59 (55-60),80 (83-86),80 (84-88) vs 44 (39-47);all P＜0.05].Moreover,the expression of I-FABP protein and the number of I-FABP positive enterocytes on day 2 and day 3 were significantly higher than those on day 1 (all P＜0.05).I-FABP expression was positively associated with intestinal pathological score (Western blot:r=0.932,95％CI:0.872-0.969;IHC:r=0.709,95％CI:0.484-0.872).Conclusions I-FABP is an efficient marker for NEC and correlates with the severity of intestinal injury.

Influences of different microbial environment on gut microbiota colonization in early life of mice / 中华围产医学杂志

Objective To investigate the influences of exposure to different environmental microbes on early-life gut microbiota colonization in mice.Methods Male (n=8) and female (n=16) adult specific pathogen free (SPF) BALB/c mice were caged together at a ratio of 2:l.After conception,the mice were divided into four groups according to the environments where the offsprings were reared at three different periods (fetal period,breastfeeding period and childhood).Group A:Offsprings were kept in a SPF environment throughout the study;group B:SPF environment during fetal and breastfeeding periods,and then ordinary environment during childhood;group C:SPF environment during fetal period,and then ordinary environment during breastfeeding period and childhood;group D:ordinary environment all the time.Fecal samples were collected at the end of week 3 and 5.Total bacterial DNA was extracted from each sample and analyzed by high throughput analysis.Kruskal-Wallis and Dunn-Bonferroni test were applied for statistical anaysis.Results 1.At the end of three weeks:(1) Diversity:① Phylum level:There were significant differences in the abundance of Firmicutes,Verrucomicrobia,Proteobacteria and Actinobacteria among the four group (all P＜0.01).Compared with group C and D,group A and B showed significantly decreased abundance of Firmicutes [30.876(23.448-41.218)× 10-2,3.317(1.116-4.641) 10-2 vs 71.936(53.587-86.713)× 10-2,79.105(56.305-82.736)× 10-2],but increased abundance of Verrucomicrobia and Proteobacteria [Verrucomicrobia:17.249(9.748-35.106)× 10-2,58.883(0.017-6.047)× 10-2 vs 0.152(0.066-1.890)× 10-2,0.003(0.000-0.016)× 10-2;Proteobacteria:12.640(0.336-15.070)× 10-2,3.653(3.362-4.5955)× 10-2 vs 0.219(0.134-0.325)× 10-2,0.124(0.116-0.165) × 10-2,all P＜0.05 or 0.01].② Genus level:There were significant differences in the abundance of Lactobacillus,Akkermansia and Bacteroides among the four groups (all P＜0.01).Compared with group C and D,group A and B showed significantly decreased abundance of Lactobacillus [19.283(8.618-31.541)× 10-2,0.339(0.264-22.278) × 10-2 vs 58.414(34.874-71.942)× 10-2,66.007(55.141-76.940)× 10-2],but increased abundance of Akkermansia,Bacteroides and Klebsiella [Akkermansia:17.247(9.748-35.106)× 10-2,58.883(0.017-60.475)× 10-2 vs 0.152(0.066-1.890)× 10-2,0.003(0.000-0.017)× 10-2;Bacteroides:3.978(0.683-25.171)× 10-2,8.216(6.023-9.946)× 10-2 vs 0.141(0.061-0.281)× 10-2,0.568(0.149-1.455)× 10-2;Klebsiella:0.209(0.050-8.888)× 10-2,1.402(0.865-1.692)× 10-2 vs 0.003(0.000-0.039) 10-2,0.000(0.000 0.001)× 10-2,all P＜0.05 or 0.01].(2) Alpha diversity:Significant differences were found in operational taxonomic unit (OTU) and Chaol index (P＜0.05),but not in Shannon index among the four groups (P＞0.05).The OTUs of group A and B were significantly lower than that of group D [246(221-348),257(209-280) vs 387(324-478),P=0.045 and 0.008,respectively].2.At the end of five weeks:(1) Diversity:① Phylum level:There were significant differences in the abundance of Firmicutes,Verrucomicrobia and Proteobacteria among the four groups (P＜0.05 or 0.01).The abundance of Firmicutes in gut microbiota in group A was lower than that in group B,C and D [13.765(64.181-24.238)× 10-2 vs 48.912(37.280-59.466)× 10-2,86.065(50.149-89.856) × 10-2,53.847(31.946-72.936) × 10-2],while that of Verrucomicrobia was higher [58.089(22.459-61.285)× 10-2 vs 0.001(0.000-0.005)× 10-2,0.000(0.000-0.001)× 10-2,0.003(0.000-0.006)× 10-2],all P＜0.05 or 0.01.② Genus level:There were significant differences in the abundance of Lactobacillus and Akkermansia among the four groups (P＜0.01).The abundance of Lactobacillus in gut microbiota in group A was lower than that in group B,C and D[1.755(0.805-8.833)× 10-2 vs 26.391(17.550-37.265)× 10-2,70.688(45.713-77.953) × 10-2,28.675 (15.660-57.224) × 10-2],while that of Akkermansia was higher [58.089(22.460-61.285)× 10-2 vs 0.000(0.000-0.006)× 10-2,0.000(0.000-0.001)× 10-2,0.003(0.000-0.006)× 10-2,all P＜0.05 or 0.01].(2) Alpha diversity:There were significant differences in OTU,Chaol and Shannon index among the four groups (P＜0.05 or 0.01).The OTU of group A was lower than that of group B,C and D [268(241-410) vs 438(380-516),562(533-588),546(473-599)],and the OTU,Chaol and Shannon index of group B were all lower than those of group C and D [OTU:438(380-516) vs 562(533-588),546(473-599);Chaol index:1 033(883-1 181) vs 1 285(1 220-1 338),1 328(1 155-1 516);Shannon index:3.85(3.25-4.50) vs 4.28(3.30-5.11),4.17(3.62-4.38),all P＜0.05 or 0.01].Conclusions Early-life exposure to different environments has an obvious impact on the diversity and composition of intestinal microbiota in mice.The less clean the living environment is,the more diverse the gut microflora will be.Furthermore,the window of opportunity for gut microbiota colonization seems to be related to breastfeeding period.

Effects of gestational exposure to mobile phone radiation on parietal cortical astrocytes in rat offspring / 中华围产医学杂志

Objective To evaluate the effects of gestational exposure to mobile phone radiation on astrocytes in parietal cortex of rat offspring.Methods Nine male Sprague Dawley rats were mated with nine female rats at the age of 12 weeks.Pregnant rats were randomly divided into three groups including control group,short-term gestational exposure group (STE group) and long-term gestational exposure group (LTE group) with three in each group.From day 2 to day 18 of pregnancy,rats in STE and LTE groups were exposed to a mobile phone in talking mode for 6 h and 24 h per day,respectively.Meanwhile,the mobile phone used in control group was kept in standby mode.Morphology and ultrastructure of cells in parietal cortex of 1 month-old offspring rat were studied by Nissle staining and electron microscope,respectively.Cells positive for glial fibrillary acidic protein (GFAP) and expression of GFAP in parietal cortex were detected by immunohistochemistry and Western bloting.One-way ANOVA,followed by SNK post hoc tests,were used for statistical analysis.Results (1) No significant difference in the morphology of cells in parietal cortex of rat offspring was observed among the three groups by Nissle staining.These cells were regularly arranged with intact cell membrane,clear nuclear membrane,evenly distributed chromatin and distinct nucleoli.(2) Neurons with normal morphology and intact synapse were observed in rat offspring in both control and STE groups.But condensation and migration of nuclear chromatin in neurons and blurred and widened synaptic clefts were observed in LTE group.(3) In offspring of control and STE groups,there were few GFAP-positive cells with small cell body and short processes in parietal cortex.But in LTE group,more GFAP-positive cells with large body and long processes were found.Moreover,long-term exposure to mobile phone during pregnancy resulted in enhanced GFAP expression in parietal cortex (0.79±0.04) as compared with control group (0.37±0.03) and STE group (0.41 ±0.04) (F=147.059,P＜0.001).Conclusions Long-term gestational exposure to mobile phone radiation might lead to activation of astrocytes,increased expression of GFAP and changes in the ultrastructure of neurons in parietal cortex of rat offspring.

Effects of maternal high-protein diet during pregnancy and lactation on growth of male rat offspring / 中华围产医学杂志

Objective To investigate the influence of maternal high-protein diet during pregnancy and lactation on the growth of male rat offspring.Methods Female Wistar rats were mated with male rats and the pregnant ones were randomly assigned into three groups (n=10):Group Ⅰ,Group Ⅱ and Group Ⅲ.Three isocaloric purified diets,which were Diet Ⅰ (protein,14.0％;carbohydrate,69.3％;fat,16.7％),Diet Ⅱ (protein,24.0％;carbohydrate,59.3％;fat,16.7％) and Diet Ⅲ (protein,34.0％;carbohydrate,49.3％;fat,16.7％),were offered ad libitum to the rats in the three groups,respectively.On the 3rd day after birth,only three male rat offspring of each litter were kept.All male rat offspring at the age of 21 days were weaned onto the same normal diets until 77-day-old.Food intake (FI),body weight (BW),body length (BL) and visceral fat mass (VFM) of them were recorded.Blood lipids levels on the 21st,49th and 77th days after birth were detected.Weight gain (WG),food efficiency (FE) and percentage of VFM (VFM ％) were calculated to assess the growth of male rat offspring.One-way ANOVA,LSD or Tamhane T2 test was used for statistical analysis.Results (1) On the 3rd day after birth,male rat offspring in Group Ⅱ and Group Ⅲ had higher fast BW than those in Group Ⅰ [(9.77±0.81),(9.58±0.54) and (9.17±0.68) g;F=3.222,P=0.026].On the 7th,14th and 21st days after birth,male rat offspring in Group Ⅱ [(17.59± 1.24),(39.51 ±2.68) and (67.77±4.22) g] had higher BW than those in Group Ⅰ [(15.96±1.17),(35.35±4.11) and (63.43±3.98) g] and Group Ⅲ [(16.52±1.05),(37.06±3.27) and (64.43±3.81) g] (F=23.684,17.070 and 84.195,all P＜0.001).Male rat offspring's BL of Group Ⅱ were longer than those of Group Ⅰ on the 7th and 14th days after birth[(7.53±0.29) vs (7.53± 0.29) cm,(10.38 ±0.24) vs (9.99± 0.30) em,both P＜0.05].BL of Group Ⅲ was longer than that of Group Ⅰ,but shorter than that of Group Ⅲ on the 14th day after birth[(10.22 ± 0.25) vs (9.99± 0.30) cm,(10.22 ± 0.25) vs (10.38±0.24) cm,both P＜0.05].Male rat offspring's average WG from the 3rd to the 7th and the 7th to the 14th day after birth were higher in Group Ⅱ [(7.70±0.41) and (22.08±1.20) g] and Group Ⅲ [(7.00±0.40) and (20.75± 1.72) g] than in Group Ⅰ [(6.73±0.55) and (19.68± 1.73) g] (F=86.925 and 38.876,both P＜0.001),and the differences between Group Ⅱ and Group Ⅲ were statistically significant (both P＜0.05).(2) No significant differences in WG,FI and FE was observed among the three groups (all P＞0.05).Male rat offspring's BL on the 49th day after birth was longer in Group Ⅱ than in Group Ⅰ and Group Ⅲ [(22.03±0.26),(21.57±0.43) and (21.77±0.33) cm,F=3.222,P=0.026).VFM ％ of Group Ⅱ (3.87±0.32 and 5.13±0.32) and Group Ⅲ (3.90±0.27 and 5.15±0.33) on the 49th and 77th days after birth were higher than those of Group Ⅰ (3.50±0.34 and 4.68±0.38) (F=3.631 and 3.611,both P＜0.05).(3) Triglyceride (TG) level was higher in Group Ⅰ than in Group Ⅱ and Group Ⅲ on the 21st day after birth [(1.12±0.13),(0.89±0.10) and (0.97±0.12) mmol/L,F=7.283,P=0.004].However,Group Ⅱ and Group Ⅲ had a higher level of TG than Group Ⅰ on the 77th day after birth[(2.64±0.37),(2.43±0.32) and (1.90±0.21) mmol/L,F=12.321,P＜0.001].Conclusion Maternal high-protein diet can increase the birth weight of male rat offspring to a certain extent,which is influenced by carbohydrate content.Moreover,male rat offspring of dams fed with high-protein diet during pregnancy and lactation will have increased visceral fat accumulation and serum TG level during adulthood.

Effects of discoidin domain receptor1 mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage in neonatal rats / 中华围产医学杂志

Objective To study the effects of discoidin domain receptor 1 (DDR1) mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage (HIBD) in neonatal rats and its possible mechanism.Methods Sixty-four seven-day-old male specific-pathogen-free Wistar rats were randomly divided into four groups with sixteen in each: Sham, HIBD, HIBD with normal saline (HIBD+NS) and HIBD with DDR1 inhibitor (HIBD+DI) groups. A rat model of HIBD was established by subjecting the rats to left common carotid artery ligation, followed by exposing them to hypoxia for two hours. In HIBD+DI group, the inhibitor of DDR1 was immediately injected into lateral cerebroventricles of the rats following modeling. Forty-eight hours after injection, tissues of left cerebral cortex were collected from each rat to evaluate histopathological changes with HE staining. Western-blotting was used to assess the phosphorylation levels of DDR1 and protein Tau. Enzyme-linked immunosorbent assay was performed to detect the concentrations of acetylcholine. Analysis of variance ort test were used for statistical analysis.Results (1) Damages in cerebral cortex: Percentages of abnormal neurons in the rats of HIBD group were higher than those in Sham group [(80.28±4.51)% vs (10.40±2.17)%,t=39.491,P<0.01]. Pyknotic or necrotic neurons in the rats of HIBD+DI group were less than those in HIBD+NS group [(31.91±3.05)% vs (82.01±7.20)%,t=18.123,P<0.01]. (2) Phosphorylation of DDR1 and protein Tau: Levels of phosphorylated DDR1 in the cerebral cortexes of rats in HIBD group were higher than those in Sham group (0.922±0.199 vs 0.095±0.023,t=10.379,P<0.01), and those levels in HIBD+NS group were higher than those in HIBD+DI group (1.200±0.171 vs 0.255±0.111,t=11.901, P<0.01). The phosphorylation of protein Tau was similar to that of DDR1 (0.919±0.228 vs 0.194±0.224 in HIBD and Sham groups,t=7.347; 1.100±0.167 vs 0.291±0.210 in HIBD+NS and HIBD+DI groups,t=9.447;bothP<0.01). (3) Levels of acetylcholine: Levels of acetylcholine in cerebral cortexes of rats in HIBD group were lower than those in Sham group [(3.685±0.472) vs (7.429±0.861) ng/g protein,t=10.781,P<0.01], and that levels in HIBD+DI group were higher than those in HIBD+NS group [(7.058±0.915) vs (2.521±0.723) ng/g protein,t=10.989,P<0.01].Conclusions Activation of DDR1 plays a key role in enhancing the phosphorylation of protein Tau and in reducing the secretion of acetylcholine in cerebral cortexes of rats with HIBD. Inhibitor of DDR1 could protect neonatal rats from HIBD through the decreasing of protein Tau phosphorylation and increasing of acetylcholine release by inhibiting the activation of DDR1.

Sodium pyruvate protect occipital cortex of rats with repetitive and severe neonatal hypoglycemia detected by high resolution 1H nuclear magnetic resonance spectroscopy / 中华围产医学杂志

Objectives To investigate the occipital cortex metabolite alterations in repetitive and severe neonatal hypoglycemia rats treated with sodium pyruvate and to reveal the protective role of sodium pyruvate using high resolution 1H nuclear magnetic resonance spectroscopy.Methods Thirty-six 2-dayold Sprague-Dawley rats were randomly divided into hypoglycemia group and pyruvate group with 18 rats in each group.Rats in both groups received intraperitoneal injections of insulin (40 U/kg body weight) at 2,4 and 6 days of age to induce severe hypoglycemia (blood glucose value ≤ 1.4 mmol/L).In the hypoglycemia group,2.5 hours after insulin injection,intraperitoneal injection of 50％ glucose (2 ml/kg) was administered to terminate hypoglycemia,while in the pyruvate group,50％ glucose (2 ml/kg) and sodium pyruvate solution 2.5 ml/kg (500 mg/kg) were injected.Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay was used to observe the status of injured neurons in six neonatal rats,and metabolite changes in occipital cortex of the other 12 rats were detected by 1H nuclear magnetic resonance spectroscopy.The difference between the two groups was compared by independent-samples t test.Results Neonatal rats of both groups reached severe hypoglycemia level 2.5 hours after insulin injection.Compared with hypoglycemia group,pyruvate group had fewer injured neurons (45±5 vs 113 ± 12,t=0.782,P=0.013) and lower injured index in the occipital cortex (0.15 ± 0.03 vs 0.36 ± 0.06,t=l.143,P=0.020).Pyruvate group showed significant decreases in the concentration of taurine [(13.31 ± 2.06) vs (18.44 ± 3.86) mol/kg,t=8.231],glutamine[(1.50 ± 0.24) vs (2.02 ± 0.40) mol/kg,t=3.137],glutamate[(7.04 ± 0.95) vs (9.40 ± 1.73) mol/kg,t=6.449],aspartate[(1.51 ± 0.28) vs (2.15 ± 0.58) mol/kg,t=2.561] and creatine [(6.37±0.99) vs (8.46± 1.77) mol/kg,t =4.226] in the occipital cortex (all P'＜0.017).Conclusions Simultaneous use of glucose and sodium pyruvate to terminate hypoglycemia in repetitive and severe neonatal hypoglycemia rats can effectively alleviate severe hypoglycemia-induced occipital lobe damage via regulating excitatory amino acid neurotransmitters,energy metabolism and other metabolic pathways.

Dynamic changes in adenosine monophosphate-activated protein kinase in neonatal rats with hypoxic ischemic encephalopathy / 中华围产医学杂志

Objective To determine the dynamic changes in adenosine monophosphate-activated protein kinase (AMPK) in neurons of neonatal rats suffering from hypoxic ischemic brain injury.Methods Twenty-four-hour old and seven-day old neonatal rats were used in this study.A classic primary cortical neuron oxygen glucose deprivation (OGD) model and neonatal rat hypoxic ischemic encephalopathy (HIE) model were employed.Lactic dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were used to evaluate neuron viability and damage.The expression of phosphorylated adenosine monophosphate-activated protein kinase (P-AMPK),phosphorylated activated protein kinase (P-Akt) and Cleaved Caspase-3 in neurons and brain tissue was measured by Western blot at different time points after OGD or HIE.The Student-t test was used for statistical analysis.Results (1) Compared with the control group,LDH levels at 2,4,8 and 24 h after OGD were higher (all P＜0.05) and optical absorption levels of MTT were lower (all P＜0.05).(2) Levels of P-AMPK in the OGD group were higher than those in the control group,and showed a time-dependent increase at 30 min and 2,4,8 and 24 h (all P＜0.05).The expression levels of P-AMPK in the HIE group were higher than those in the control group (0.345 ± 0.038,0.387 ± 0.112 and 0.618 ± 0.075 at 1,3 and 7 days after HIE,and 0.132±0.032 in the control group,all P＜0.05).(3) The levels of P-Akt increased above the control levels at 30 min (0.991 ±0.134 vs 0.304±0.050),reached a maximum level at 2 h (1.183± 0.107),and then gradually declined,whereas the levels of Cleaved Caspase-3 started to increase at 30 min,and remained elevated at 24 h (all P＜0.05).Conclusion Following hypoxic ischemic brain damage,the expression of P-AMPK is significantly increased in both in vivo and in vitro studies in a time-dependent manner.

Application of modified retinal flatmount combined with immunofluorescence staining in oxygen-induced retinopathy model / 中华实验眼科杂志

Background Retinal flatmount of oxygen-induced retinopathy (OIR) animal models is a useful tool in the study of ischemic retinopathy.The retinas of OIR of rat or mouse pups were small and thick and difficult in operating of conventional preparation and quantitative analysis of retinal flatmounts.Objective This study was to explore an easy and stable operating method of retinal flatmount combined with immunofluorescence staining in rodent.Methods Forty ＜6-hour-old SD rat pups were randomly assigned to OIR model group and normal control group.The pups were raised with nursing mothers in hyperoxia environment (80％) and normal oxygen environment (21％) alternately at a 24-hour interval for 14 days in the OIR model group,and the pups were raised in the room air for 14 days in the normal control group.The eyeballs of the rats were extracted to isolate the retinas intactly.The retinas were stained with glutamine synthetase (GS)-isolectin B4 firstly and then expanded into flatmounted and cut into 4 petals radially.Adobe Photoshop CS3 imaging analysis system was used to match the pictures into entire retinal vascular images and analyzed under the fluorescence microscope.The pixel values of retinal avascular areas and the entire retina were quantified by this system.The percentage of avascular areas to the entire retina was calculated to analyze the severity of non-perfusion areas.The use and care of the animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results An intact and smooth retinal flatmount could be obtained by firstly staining method.Ora serrata structure was seen surrounding the whole retina.Strong green fluorescence was exhibited in retinal vessel net with clearly visible vascular branches;while the background fluorescence was weaker.Fully developed blood vessels were displayed in the retinas of the normal control group.Non-capillary areas around the central optic disk and large peripheral avascular areas could be seen in the retinal flatmounts of OIR models.Conclusions The preparation of retinal flatmount is easy and feasible by first immunofluorescence staining for retinal vessels followed by radially cutting of retina.This method of retinal flatmount can ensure the integrity of retinal vascular system and it is available for the observation and evaluation of retinal vascular structure in OIR models.

Exposure of high concentration oxygen inhibits Sox17 expression in vascular endothelial cells of neonatal mice lungs / 中华围产医学杂志

Objective To study the effects of high concentration oxygen exposure on the Sox17 expression of vascular endothelial cells of neonatal mice lungs,and to explore the pathogenesis of blocked lung vascular development.Methods Thirty two C57B1/6J newborn mice within six hours after birth were randomly divided to hyperoxia group (n=16) and room air group (n=16).Mice of hyperoxia group were exposed to 85％ oxygen.Eight mice of either group were sacrificed at 7 and 14 days after birth respectively to observe the lung morphology and calculate radial alveolar counts (RAC),which is the number of alveoli on the straight line from the center of respiratory bronchioles to the nearest fibrous septa or the pleura.Sox 17 expression in the pulmonary vessels was detected by immunohistochemical staining.Sox17 mRNA was measured by reverse transcription polymerase chain reaction.Sox17 protein level was measured by Western blot.Two independent samples t-test was used for statistical analysis.Results Compared with day 7,the lung structures matured with more uniformed alveoli and the septas became thinner on day t4 in room air group.However,the lungs developed slowly with simplified and non-uniformed alveoli on day 14 in hyperoxia group.The Sox17 protein was positive on endothelial cells of pulmonary arteries,veins and alveolar capillarys,as well as the alveolar epithelial cells.The RAC on day 7 and day 14 in hyperoxia group were both lower than that in room air group (3.7±0.7 vs 5.0±0.8,5.3±0.6 vs 8.3±0.9,respectively,t=3.057 and 8.148,both P ＜ 0.01).Sox17 mRNA on day 7 and day 14 in hyperoxia group were both lower than that in room air group (0.62±0.10 vs 0.88±0.11,0.44±0.06vs 0.90±0.15,t=3.607 and 6.926,both P ＜ 0.01).Sox17 protein level on day 7 and day 14 in hyperoxia group were both lowered than that in room air group (0.32±0.04 vs 0.76±0.04,0.36±0.07 vs 0.96±0.06,t=3.102 and 8.421,both P ＜ 0.01).Conclusions Exposure of high concentration of oxygen may cause impairment of lung vascular development by inhibiting Sox17 expression in lungs of neonatal mice.

Protective Effects of Biifdobacterium on intestinal tissue of newborn rats with necrotizing enterocolitis and its regulation / 中华围产医学杂志

Objective To discuss the possible molecular mechanisms involved in the protective effects of Biifdobacterium on intestinal tissue of necrotizing enterocolitis (NEC) newborn rats. Methods Seventy-five newborn Sprague-Dawley rats (born within 2 h) were randomly divided into five groups, each group with 15 rats. Group A was the NEC model group, and the rats were fed lipopolysaccharide (LPS) and formula. Group B was the Biifdobacterium treatment group, and the rats were fed LPS and formula and Biifdobacterium micro-capsule. Group C was the artificial feeding control group, and the rats were fed formula. Group D was the Biifdobacterium control group, and the rats were fed formula and Biifdobacterium micro-capsule. Group E was the breastfeeding control group, and the rats were fed rat breast milk by mothers. LPS 30 mg/kg was administered by gavage once per day for 3 days. Bifidobacterium micro-capsules were given as 1×1010 colony forming units/ml by gavage with formula once per day. After fed for 72 h and fasted for 12 h, the five groups of rats were killed by decapitation. Morphological changes in the terminal ileum tissue were observed under a light microscope and intestinal injury was scored. The expression of Toll-like receptor (TLR) 2, TLR4, and nuclear transcription factor (NF)-κB p65 was detected by immunohistochemical methods. Kruskal-Wallis test, analysis of variance, corrected Chi-square test and Fisher's exact test were used for statistics. Results The morbidity of NEC in group A to E was 11/15, 4/15, 3/15, 2/15 and 0/15, respectively;the intestinal injury score in group A to E was 3.37±0.27, 1.53±0.44, 1.75±0.37, 0.92±0.39 and 0.30±0.18, respectively; the expression level of TLR2 in group A to E was 0.35±0.05, 0.30±0.03, 0.32±0.04, 0.30±0.02 and 0.29±0.03, respectively;the expression level of TLR4 in group A to E was 0.48±0.05, 0.34±0.03, 0.36±0.03, 0.37±0.04 and 0.35±0.02, respectively;the expression level of NF-κB p65 in group A to E was 0.43±0.03, 0.29±0.03, 0.35±0.02, 0.32±0.02 and 0.30±0.02, respectively. The differences in NEC morbidity, intestinal injury score, and the expression levels of TLR4, TLR2 and NF-κB p65 among the five groups were all statistically significant (χ2, H or F=23.863, 70.290, 8.803, 38.599 and 75.076, respectively, all P 0.05). The intestinal injury score in the Bifidobacterium treatment group was significantly higher than that in the Bifidobacterium control group and the breastfeeding control group (both P 0.05). The expression levels of TLR4 and NF-κB p65 in the Biifdobacterium treatment group were significantly lower than those in the artificial feeding control group and the Biifdobacterium control group (all P 0.05). The expression level of TLR2 in the Biifdobacterium treatment group compared with the three control groups was not significantly different (all P > 0.05). Conclusions Biifdobacterium may inhibit pathogenic bacteria or regulate the negative feedback of TLR2 to reduce the expression of TLR2 and TLR4 in intestinal mucosa cells, inhibit the NF-κB pathway, attenuate the inflammatory reaction, and play a role in the prevention and control of NEC.

Influence of intrauterine hypoxia on lung blood vessel development in rats and expression of vascular endothelial growth factor in the lungs / 中华围产医学杂志

Objective To observe the impact of intrauterine hypoxia on the development of rat lungs and expression of vascular endothelial growth factor (VEGF) in the lungs as the time of hypoxia was extended.Methods To create a model of intrauterine hypoxia,12 pregnant rats were divided into four groups as follows:air-control group,hypoxic 2-day group,hypoxic 6-day group,and hypoxic 10-day group.At birth,we performed pulmonary vascular morphometry in newborn rats with Nis software,and measured pulmonary arterial diameter,wall thickness and wall thickness/pulmonary arterial diameter.We detected expression of VEGF protein by immunohistochemistry and mRNA by real-time polymerase chain reaction.Changes in pulmonary capillary endothelium under electron microscope were observed.One-way analysis of variance and the Student Newman Keuls q (SNK-q) test were applied for statistical analysis.Results As the hypoxic time was extended,wall thickness and wall thickness/pulmonary arterial diameter increased.Compared with the air-control group,pulmonary vascular wall thickness in the hypoxic 10-day group increased [(16.4 ± 5.9) vs (10.8±2.8) μm; q=-8.04,P＜0.05].Wall thickncss/pulmonary artcrial diameter in the hypoxic 10-day group increased compared with that in the air control group,hypoxic 2-day group and hypoxic 6-day group [(31.3±5.1) ％,(22.2±4.9) ％and (23.6±3.9) ％vs (24.1±3.9) ％;q=-7.08,-4.92 and-5.0,all P＜0.05].Expression of VEGF protein in the lungs increased in the hypoxic 6-day group compared with the air-control group [(13.7±3.9) ％ vs (9.3±3.5) ％; q=-6.83,P＜0.05],while the expression was higher in hypoxic 10-day group than in the air-control group and hypoxic 2-day group [(15.2±4.7) ％,(9.3±3.5) ％ vs (11.8 ± 3.3) ％] (q=-9.16 and-5.19,all P＜0.05).Expression of VEGF mRNA in the lungs increased in the hypoxic 6-day group compared with the air-control group [(1.6±0.2)vs (0.8 ±0.2); q=-5.07,P＜0.05],while the expression was higher in the hypoxic 10 day group than in the air-control group and hypoxic 2-day group [(2.2±0.3),(0.8±0.2) vs (1.3±0.2)] (q=-9.54 and-6.42,all P＜0.05).Electron microscopy showed puhnonary capillary endothelial cell swelling as the hypoxic time was extended.In the air-control group,there was no capillary endothelial cell hyperplasia and swelling; in hypoxic 2-day group,there was mild swelling of the capillary endothelial cells and a small amount of hyperplasia; in hypoxic 6-day group,there was moderate swelling of the capillary endothelial cells; and in hypoxic 10-day group:there was significant swelling of the capillary endothelial cells,and pyknosis.Conclusions Intrauterine hypoxia resulted in higher expression of VEGF protcin and mRNA.VEGF in the lungs of newborn rats was involved in the vascular development process.

Role of hypoxia-inducible factor-1α in hypoxia-induced pulmonary hypertension of neonatal rats and its association with pulmonary vascular remodeling / 中华围产医学杂志

Objective To investigate the serum concentration and the lung tissue expression level of hypoxia-inducible factor-1α (HIF-1α) in neonatal rats with hypoxia-induced pulmonary hypertension (HPH),and the role of HIF-1α in pathogenesis of HPH and pulmonary vascular remodeling.Methods Eighty neonatal Wistar rats were randomly assigned into normoxia and hypoxic group,forty in each group.HPH models were established on rats in hypoxic group.On the 3rd,7th,14th and 21st day after modeling,ten rats in each group were taken to measure the mean pulmonary arterial pressure (mPAP).Meanwhile,serum HIF-1α concentration were determined by enzyme-linked immunosorbent assay,while expression of HIF-1α in lung tissue were detected by immunohistochemistry and real time polymerase chain reaction.After hematoxylineosin and Van Gieson staining,morphological changes of pulmonary arterioles of rats in both groups were observed by optical microscope.Intima-media thickness/external diameter ratio (MT) and medial wall cross sectional area/vessel total cross-sectional area ratio (MA) were determined.Wilcoxon rank sum test,t-test,Ridit analysis,Pearson correlation and Spearman rank correlation test were applied for statistical analysis.Results mPAP in both groups kept increasing as the time went on.mPAP in hypoxic group were higher than that innormoxiagroup[(8.59±1.57) mmHgvs (6.14±1.02) mmHg,(11.63±2.56) mmHgvs (8.33±0.76) mmHg,(15.29±2.88) mmHgvs (10.92±2.74) mmHgand (18.04±2.69) mmHgvs (12.17±1.64) mmHg on the 3rd,7th,14th and 21st day after hypoxia modeling (t=4.14,3.91,3.48 and 5.89,all P＜0.05).Serum concentration,expressions of mRNA and immune response intensity of HIF-1α in hypoxic group were higher than those in normoxia group on the 3rd,7th and 14th day after hypoxia [serum concentration:(805.67±19.52) μg/molvs (204.34±83.21) μg/mol,(906.29±24.24) μg/molvs (548.93±11.78) μg/mol and (939.25±28.57) μg/mol vs (674.44± 17.41) μg/mol,t=6.43,4.11 and 2.34,respectively,all P＜0.05; relative expressions of HIF-1α mRNA:0.98±0.35 vs 0.23±0.16,1.30±0.49 vs 0.69±0.13 and 1.45±0.76 vs 0.64±0.21,respectively,t=4.56,2.72 and 4.63,all P＜0.05; immune response intensity of HIF-1α (average Ridit value):0.318 vs 0.183,0.303 vs 0.198 and 0.374 vs 0.176,respectively,u=3.26,2.73 and 3.91,all P＜0.05].MT and MA in hypoxic group were higher than those in normoxia group [MT:52.71％ (45.91％-63.23％) vs 47.42％ (41.14％ 56.17％),61.28％ (52.25％-68.63％) vs 50.22％(43.79％ 58.40％) and 65.24％ (56.02％-71.28％) vs 53.78％ (48.02％-64.58％) at 7,14 and 21st day respectively,u=2.08,4.54 and 4.37; MA:60.89％ (54.04％ 66.21％) vs 55.19％ (50.32％-63.59％),66.09％ (58.52％-72.58％) vs 59.77％ (53.55％-67.09％) and 68.25％(62.02％-74.51％)vs 61.40％(50.74％-70.55％),respectively,u=2.16,2.27 and 3.18,all P＜0.05].There were positive relations between HIF-1α and mPAP (r=0.294),as well as MT and MA (r=0.548 and 0.265,all P＜0.05).Conclusions HIF-1α may play a role in the pathogenesis of HPH.Pulmonary vascular remodeling occurred after seven days of hypoxia induction and may be exacerbated as the hypoxic time is prolonged,which might be prevented with early interventions.

Effects of hydrogen sulfide on hyperoxia-induced lung injury in neonatal rats / 中华围产医学杂志

Objective To explore the effects of hydrogen sulfide (H2S) on hyperoxia-induced lung injury in neonatal rats.Methods Eighty full-term Sprague-Dawley rats within 12 hours after birth were randomly divided into control group,hyperoxia group,sodium hydrosulfide (NaHS) + hyperoxia group (NaHS 90 μmol/kg injected intraperitoneally) and propargylglycine (PPG) +hyperoxia group (PPG 50 mg/kg injected intraperitoneally).Except for the control group,the other three groups were exposed to 95％ O2 for seven days.Pulmonary histopathology was observed after HE staining,numeration of leukocyte and albumin in bronchoalveolar lavage fluid (BALF) were processed by optical microscope and Lowry methods.The plasma H2S concentration,activity of cystathionine-γ-lyase (CSE) and contents of malondialdehyde (MDA) in lung tissues were also detected.Analysis of variance and LSD-t test were used for statistics.Results (1) Compared with the control group,alveolar hemorrhage,interstitial edema,inflammatory cell infiltration were observed in the hyperoxia group.The number of white blood cells,neutrophils and albumin content in BALF increased in the hyperoxia group [(130.2± 15.3) × 107/L vs (15.1 ±2.5) × 107/L; (64.6± 12.4) × 107/L vs (2.1 ±0.5) × 107/L; (934.6± 106.4) mg/L vs (254.3±50.7) mg/L,respectively.LSD-t=-14.65,5.78 and 2.97,all P＜0.01],but the plasma H2S concentration and the activity of CSE in lung tissue decreased [(112.6± 20.4) μmol/Lvs (182.3±15.7) μ mol/L,LSD-t=-9.90; (3.4±0.4) μmol/ (min·g) vs (6.8± 1.4) μ mol/ (min · g),LSD-t=-4.59; both P＜0.01].However,the contents of MDA increased [(1.7± 0.3) nmol/ml vs (0.9±0.1) nmol/ml,LSD-t=3.03,P＜0.01].(2) Compared with the hyperoxia group,inflammatory exudation and structural disorder of lung tissue were alleviated in the NaHS+hyperoxia group.White blood cells [(56.3± 11.6) × 107/L],neutrophils [(34.8±7.8) × 107/L] and albumin content [(753.8± 89.6) mg/L] in BALF decreased significantly (LSD-t=-9.66,-11.81 and-5.78,P＜0.01).The plasma H2S concentration [(235.7±32.7) μ mol/L] and the activity ofCSE [(5.8± 1.1) μ mol/(min · g)] increased significantly (LSD-t=11.34 and 5.98,P＜0.01) in the NaHS+hyperoxia group.(3) Compared with the hyperoxia group,inflammatory exudation and structural disorder of lung tissue were more severe in PPG+ hyperoxia group.White blood cells,neutrophils and albumin content in BALF increased significantly (LSD-t=5.52,6.37 and 8.23,P＜0.01),the plasma H2S concentration and the activity of CSE decreased (LSD-t=-4.29 and-3.97,P＜0.01),the contents of MDA increased (LSD-t=3.02,P＜0.01).Conclusions H2S is involved in the pathophysiological process of hyperoxia-induced lung injury in neonatal rats.Exogenous H2S can alleviate the pulmonary injury by inhibiting inflammatory reaction and oxidative stress.

Relationships of cardiac compromise after hypoxic injury and expression of β1 and β2 adrenoceptors in a neonatal swine model of asphyxia / 中华围产医学杂志

Objective To investigate the hemodynamic changes and their association with the expression of β 1 and β 2 adrenoceptors after hypoxia-reoxygenation injury in a neonatal swine model of asphyxia.Methods One to four day-old piglets were randomly assigned to control group (n=6),acute hypoxia group (n=8) and subacute hypoxia group (n=8).The piglets in the control group were observed for 50 h under normoxic mechanical ventilation; while the acute and subacute hypoxia groups were subject to two hours of hypoxic injury induced by ventilation with 0.10-0.15 oxygen followed by 4 or 48 h of observation under normoxic mechanical ventilation,respectively.Blood gases were analyzed and the mean arterial blood pressure,heart rate,and cardiac output were recorded at different time points during the experiment.Tissues from the left ventricle were also harvested to assay lactate,glutathione and β adrenoceptors at the end of the experiment.Analysis of variance,the Tukey test and Pearson correlation analysis were used for statistical analysis of the data.Results Two hours after hypoxia,pH,HCO3-and partial pressure of oxygens (PO2) in the acute hypoxia group and subacute hypoxia group were lower than in the control group,however,pH and HCO3-in animals in the subacute hypoxia group recovered to 7.38 ± 0.05 and (23.04± 2.40)mmol/L,respectively,after reoxygenation,which was similar to those in the control group,and higher than in the acute hypoxia group [7.25±0.07 and (16.88±2.40) mmol/L,respectively,q=6.76 and 7.81,both P＜0.01].Mean arterial pressure,cardiac output and stroke volume in the acute group and subacute group were lower than those in the control animals following two hours of hypoxic injury (all P＜0.01).After reoxygenation,the mean arterial pressure in the acute hypoxia group and subacute group recovered to (42.17±6.14) and (43.19± 5.55) mmHg (1 mmHg=0.133 kPa),cardiac output recovered to (150.04± 56.17) and (169.75 ± 37.85) dl/min,respectively,and there were no differences compared with the control group (all P＞0.05).Expressions of β 1 and β 2 adrenoceptors in the left ventricle in the subacute hypoxia group (1.51 ±0.51 and 2.14±0.66,respectively),were higher than those in the control group (0.56±0.24 and 0.38±0.21,q=7.02 and 10.97,both P＜0.01) and the acute hypoxia group (0.65±0.20 and 0.45±0.11,q=6.86 and 11.38,both P＜0.01).The lactate level in the acute hypoxia group and subacute hypoxia group was higher than that in the control group [(6.95±0.32) and (6.92±0.40) vs (5.03±0.19) μ mol/mg protein,respectively,q=15.43 and 15.19,both P＜0.01].The level of glutathione in the subacute hypoxia group was lower than the control group and acute hypoxia group [(352.00± 16.51) vs (438.35±33.66) and (464.66±52.65) nmol/mg protein,respectively,q=6.00 and 8.46,both P＜0.01).In the subacute hypoxia group,the expressions of β 1 and β 2 adrenoceptors were negatively correlated with the changes in cardiac output (r=-0.60 and-0.59,respectively,both P＜0.05).Conclusions Severe metabolic acidosis and cardiac dysfunction resulting from perinatal asphyxia may recover after reoxygenation,which may be associated with the enhanced expression of β adrenoceptors in the left ventricle during the subacute phase.

Effects of stromal derived factor-1 and CXC chemokine receptor-4 in rats with white matter damage treated with human umbilical cord mesenchymal stem cells / 中华围产医学杂志

Objective To investigate the effects and the migration mechanisms of stromal derived factor-1 (SDF-1) and CXC chemokine receptor-4 (CXCR-4) in rats with white matter damage treated with human umbilical cord mesenchymal stem cells (hUC-MSCs).Methods A total of 108 three-day old Sprague-Dawley rats were randomly divided into the experimental group,control group and sham group.The left common carotid artery was ligated and then exposed to hypoxia of 6％ O2 and 94％ N2 in rats in the experimental and control groups.Rats in sham group were neither ligation nor hypoxia.After 24 hours,rats in the experimental group were administered 0.5 ml hUC-MSCs (1 × 106/ml) intraperitoneally,and rats in control and sham groups were administered 0.5 ml saline by the same way.Immunohistochemistry and reverse transcription-polymerase chain reaction were used to determine the expression of SDF-1 and CXCR-4 protein and mRNA in 5-,7-and 14-day-old rats.Analysis of variance and the LSD test were used for statistical analysis of the data.Results HE staining showed that,in 14 day-old rats of the experimental group,bilateral cerebral ventricles were similar with no cellular edema or necrocytosis.In the sham group,bilateral cerebral ventricles were also normal.However,in the control group,ventriculomegaly,cellular degeneration and necrocytosis were observed on the left side.On the 5th,7th and 14th day,SDF-1 protein levels were 0.15±0.06,0.24±0.01 and 0.12±0.01,and CXCR-4 protein levels were 0.35±0.16,0.60±0.21 and 0.72±0.25,respectively,in the experimental group; SDF-1 protein levels were 0.13 ± 0.01,0.16± 0.01 and 0.08± 0.01,and CXCR-4 protein levels were 0.18 ± 0.04,0.17 ± 0.09 and 0.25 ± 0.06,respectively,in the control group,and all were higher than those in the sham group (SDF-1 protein levels were 0.03 ± 0.01,0.04± 0.01 and 0.02±0.01; and CXCR-4 protein levels were 0.04±0.02,0.05±0.03 and 0.05±0.03,respectively) (LSD test,all P＜0.05).SDF-1 protein increased to a peak on the 7th day and decreased on the 14th day in the experimental group,however,these values were both higher than those in the control group (LSD test,both P＜0.05).CXCR-4 protein increased on the 5th day and continued to increase up to the 14th day in the experimental group,and these values were higher than those in the control group at the three time points (LSD test,all P＜0.05).In 5-,7-and 14-day-old rats,SDF-1 mRNA levels were 3.52 ± 0.33,4.18± 0.28 and 2.60± 0.21,respectively,in the experimental group,which were higher than those in the control group (2.07± 0.34,3.73 ± 0.28 and 2.08± 0.15,respectively),and were even higher than those in the sham group (0.99±0.17,1.00±0.16 and 1.31 ±0.32,respectively) (LSD test,all P＜0.05).In the experimental group,SDF-1 mRNA levels reached a peak on the 7th day,and on the 14th day,it decreased to the level lower than that on the 5th day (LSD test,all P＜0.05).In the control group,SDF-1 mRNA levels also reached a peak in 7-day-old rats,but not in 14-day-old rats,which was similar to 5-day old rats (LSD test,9＞0.05).In 5-,7 and 14-day-old rats of the experimental group,CXCR-4 mRNA levels were 1.32±0.29,1.75±0.36 and 2.33±0.49,respectively,higher than those in the sham group (1.00±0.16,0.94±0.16 and 0.81±0.14,respectively) and the control group (0.97±0.14,0.97±0.15 and 1.07±0.25,respectively) (LSD test,all P＜0.05).In the experimental group,CXCR-4 mRNA levels were higher in 14-day-old rats than that in 5-and 7-day-old rats (LSD test,both P＜0.05).Conclusions SDF-1/CXCR-4 may play a vital role in the migration of hUC-MSCs homing to damaged brain.

Effect of chronic stress in newborn rats on the progression of ligature-induced-periodontitis in adulthood

PURPOSE: To investigate the effect chronic stress in newborn rats on the progression of ligature-induced-periodontitis in adulthood. METHODS: Ten couples of adult Wistar rats were selected for mating. After birth, the female and their respective offsprings were allocated into two groups. Ligature Group (LG): offsprings were only watched during breast-feeding; Stress-ligature Group (SLG): after 24h of live birth, the offsprings were moved away from their mothers every day for four hours during breast-feeding for 20 days; in both approaches, after reaching ± 250g, ten rats were included in the groups. Periodontal disease was induced by a silk suture placed around the maxillary right second molar. The left side was used as control. After 15 days, the animals were subjected to euthanasia, maxillary bones were removed and stored in 10% formaldehyde. After 48h, radiographs were taken and revealed and were used for bone destruction analysis. Examiner was blind and calibrated for measurements. RESULTS: Stress-ligature group presented higher bone loss values in relation to ligature group (p<0.05). CONCLUSION: Exposure to chronic stress imposed on offsprings produced a greater progression of bone loss induced during adulthood.

Protective mechanism of 1,25-Dihydroxyvitamin D3 on hyperoxia-induced lung injury in neonatal rats / 中华围产医学杂志

Objective To investigate the effects and mechanisms of 1,25-Dihydroxyvitamin D3 [1,25(OH)2 D3] on hyperoxia-induced lung injury of neonatal rats.Methods Neonatal Sprague-Dawley rats were randomly divided into air group,hyperoxia group and 1,25(OH)2D3 group within 12 hours after birth,eight in each group.Rats in air group were exposed to air,while those in hyperoxia and 1,25 (OH) 2 D3 group were exposed to hyperoxia (≥85 ％ oxygen concentration).Rats in 1,25(OH)2D3 group were injected with 1,25(OH)2D3 0.5 μg/(kg · d) intraperitoneally once a dayfor seven days,meanwhile the rats in the other two groups received 0.9 ％ saline in the same way.All rats were sacrificed on day 7.Lung tissue sections were HE stained in order to assess lung histological changes and lung radical alveolar count (RAC).The levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6 mRNA were measured by real time-polymerase chain reaction.Analysis of variance and LSD test were applied for statistics.Results Compared with the air group,the weight of rats in the hyperoxia group was significantly lower on day 7 [(8.48±1.34) g vs (12.51±0.47) g,t=8.05,P＜0.05],while the weight of rats in 1,25(OH)2D3 group [(10.29±1.00) g] was higher than that in the hyperoxia group (t=3.61,P＜0.05).Lung tissue structure was normal in the air group.In the hyperoxia group,inflammatory exudation was observed in pulmonary interstitial,the alveolar size was uneven,and the RAC was lower than that in the air group (5.6±0.1 vs 6.8±0.2,t=21.45,P＜0.05).The RAC in 1,25(OH)2D3 group (6.2±0.1) was significantly increased compared with that in the hyperoxia group (t=11.76,P＜0.05),but still lower than that in the air group (t=9.69,P＜0.05).The expressions of TNF-α,IL-1β and IL-6 mRNA in hyperoxia group (0.0348±0.0006,0.0269±0.0003 and 0.0368 ± 0.0006) were higher than those in the air group (0.0111±0.0007,0.0040±0.0003 and 0.0162 ±0.0007,t=56.54,111.12 and 49.26,P＜0.05,respectively).The expressions of TNF-α,IL-1β and IL-6 mRNA in the 1,25 (OH)2D3 group (0.0203±0.0009,0.0141±0.0004 and 0.0251±0.0009) were lower than those in the hyperoxia group (t=34.44,61.93 and 27.99,P＜0.05,respectively),but higher than those in the air group (t=22.10,49.19 and 21.27,P＜0.05,respectively).Conclusions 1,25(OH)2D3 could attenuate hyperoxia-induced lung injury by inhibiting the expression of inflammatory cytokines.

Hepatic damage in newborns from female rats exposed to the pesticide derivative ethylenethiourea / Lesões hepáticas em recém-nascidos de ratas expostas ao derivado de agrotóxico etilenotioureia

PURPOSE: To evaluate hepatic morphological-histological abnormalities in newborns from female rats exposed to ethylenethiourea. METHODS: A randomized study was conducted on fifty-five newborn Wistar rats were studied: 34 in the experimental group, whose mothers had been exposed to 1% ethylenethiourea; and 21 in the control group, whose mothers had received 0.9% physiological solution. The solution was administered via gavage on the 11th day of gestation. Cesarean section was performed on the 20th day of gestation. The newborns' livers were examined and any morphological-histological abnormalities were registered. The presence of megakaryocytes was quantified in 50 microscope fields, as the total number of these cells per mm². RESULTS: The entire experimental group presented abnormalities of embryonic formation, with musculoskeletal anomalies, digestive system anomalies, hepatic congestion and friability, hydrops and delayed intrauterine growth. The histopathological analysis showed that morphological-histological hepatic destructuring had occurred in all entire experimental with removal of the hepatic trabeculae and severe hepatic megakaryocytosis. The mean megakaryocyte density ranged from 107.9 to 114.2 per mm², and it was eight times greater than in the control group, thus characterizing a situation of extramedullary hematopoiesis. CONCLUSION: The fetal exposure to ethylenethiourea caused hepatic damage characterized by severe extramedullary hematopoiesis.

OBJETIVO: Avaliar alterações hepáticas morfohistológicas em recém-nascidos de ratas prenhes expostas à etilenotioureia. MÉTODOS: Realizado ensaio randomizado em animais de experimentação, onde foram estudados 55 recém-nascidos de ratas Wistar, 34 do Grupo Experimento, expostas a etilenotioureia 1% e 21 do Grupo Controle, em que a rata prenhe recebeu solução fisiológica 0,9%, ambos por gavagem no 11º dia de gestação. Realizada no 20º dia de gestação cesariana, analisados os fígados dos recém-nascidos e registradas as alterações morfohistológicas. Realizou-se a quantificação dos megacariócitos em 50 campos microscópicos, avaliando a quantidade total destas células por mm². RESULTADOS: Todos os recém-nascidos do Grupo Experimento apresentaram alterações na formação embrionária, com anomalias musculoesqueléticas, anormalidades do sistema digestório, congestão e friabilidade hepática, hidropisia e crescimento intrauterino retardado. A análise histopatológica mostrou desestruturação hepática morfohistológica em todos os recém-nascidos expostos à etilenotioureia, com destrabeculação dos hepatócitos e intensa megacariocitose hepática, apresentando média da densidade de megacariócitos de 107,9 até 114,2 por mm² sendo cerca de oito vezes maior que no Grupo Controle, caracterizando hematopoese extramedular. CONCLUSÃO: A exposição fetal a etilenotioureia provocou danos hepáticos caracterizados pela intensa hematopoese extramedular.

Expression and significance of hypoxia-inducible factor-1α in retinal neovascularization induced by metabolic acidosis in newborn rats / 中华围产医学杂志

Objective To investigate the expression and significance of hypoxia-inducible factor-1α (HIF-1α) in the retinal neovascularization by metabolic acidosis in newborn rats. Methods One hundred and twenty newborn SD rats were randomly divided into acidosis (experiment) and normoxia (control) groups. A total of 60 newborn rats in experiment group underwent tubal feeding day for 6 days and followed by a period of recovery. The rats in the two groups were sacrificed at the 3rd, 5th, 8th, 10th, 13th and 20th day after birth, respectively. The morphologic changes of retinal vessels were estimated by observing the vascular pattern in adenosine diphosphatase stained retina flat mounts. The newborn vessels were quantified by HE staining. Immunohistochemical method was used to detect HIF-1α expression. Results In experiment group, numerous neovascularization and un-perfused area at the periphery of vessels occurred on the 10th day. The result of HE staining showed that in experiment group of 10-day old,the number of neovascular nuclei extending into the vireo was 28.78±7.53, and that of the control group was 1.22±1.48 (t=11.169,P<0.01). The results of immunohistochemistry revealed that the expression of HIF-1α protein were stronger in the experiment group than in the control group on the 8th, 10th and 13th day, and there were significant differences between the two groups (108.87±15.21, 183.68±26.58 and 129.42±9.85 vs 74.98±4.50, 76.38± 3.38 and 74.78±1.86, t=4.625, 9.023 and 9.672,P<0.05). Conclusions HIF-lα might play an important role in retinal neovascularization.