Protective Effects of Activated Protein C on Neurovascular Unit in a Rat Model of Intrauterine Infection-Induced Neonatal White Matter Injury

Summary： Activated protein C （APC）, a natural anticoagulant, has been reported to exert direct vascu- loprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. In- traperitoneal injection of 300 ~tg/kg lipopolysaccharide （LPS） was administered consecutively to preg- nant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infec- tion-induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats imme- diately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eo- sin （H＆E）. Immunohistochemistry was used to evaluate myelin basic protein （MBP） expression in the periventricular white matter region. Blood-brain barrier （BBB） permeability and brain water content ~were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 （PAR1）. Typical pathological changes of white matter injury were ob- served in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely acti- vated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein ex- pression levels were both down-regulated. Our results suggested that APC exerted neuroprotective ef- fects on multiple components of the neurovascular unit in neonatal rats with intrauterine infec- tion-induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

A Study on the Traditional Chinese Medicine Jinyebaidu for Prevention and Treatment of Intrauterine Infection with Guinea Pigs Cytomegalovirus

The purpose is to study the prophylactic and therapeutic effect of the traditional Chinese Medicine （TCM）-Jinyebaidu （JYBD） to guinea pig cytomegalovirus （GPCMV） intrauterine infection. The virus-free female and male guinea pigs were screened with nest-polymerase chain reaction （N-PCR）. After inbred, pregnant guinea pigs were selected and divided into 3 groups randomly： 5 guniea pigs of the blank control group were not given either GPCMV or JYBD. 31 guniea pigs of the positive control group were inoculated 1 mL （107 TCID50 ） suspension of GPCMV intraperitoneal. 10 gunlea pigs of the experimental group were inoculated GPCMV firstly and then perfused stomach with JYBD for 14 days （Dosage in accordance with the modulus of the weight ratio of human to guniea pig）. The effects of JYBD on the intrauterine infection of GPCMV were observed. The results showed that JYBD could decrease the maternal infection rate from 100 % （31/31） to 50 % （5/10） （P〈0. 001）, the intrauterine infection rate from 100% （72/72） to 75 % （21/28） （P〈 0. 001）, and the rate of abnormal outcome of pregnancy from 64.4 % （29/45） to 25.0 % （7/28） （P〈0. 001）, the infective symptoms being relieved. It can be concluded that traditional Chinese medicine- JYBD can prevent and treat GPCMV intrauterine infection, and can be expected a prophylactic drug for HCMV intrauterine infection.

Relationship between HBV viremia level of pregnant women and intrauterine infection:neated PCR for detection of HBV DNA

IM To determine the incidence of hepatitis B virus (HBV) intrauterine infection and to explore the relationship between HBV viremia level of pregnant women and HBV intrauterine infection.METHODS Sixtynine pregnant women were divided into three groups. Group A, 41 HBsAg positive patients, 14 of them were HBeAg positive (group A1), and 27 HBeAg negative (group A2); Group B, 12 HBsAg negative patients, but positive for antiHBs and/or antiHBe and/or antiHBc; and Group C, 16 patients negative for all HBV markers. Blood samples of mothers were taken at delivery, samples of their infants were collected within 24 hours after birth (before injection of HBIG and HBV vaccine). All the serum samples were stored at -20℃. HBV serum markers were tested by radioimmunoassay and HBV NDA were detected by nested polymerase chain reaction.RESULTS In group C, all of 16 newborns were negative for HBsAg and HBV DNA. In group A, 7 infants were HBsAg positive (171%), and 17 (415%) were HBV DNA positive (P<005). The incidence of intrauterine HBV infection was much higher in group A1 than that in group A2 (HBsAg 429% vs 37%, HBV DNA 929% vs 148%, P<005). The incidence of HBV intrauterine infection was significantly different between high and low HBV viremia of mothers (933% vs 429%, P<005).CONCLUSION The incidence of HBV intrauterine infection is high when HBV DNA in newborns detected with nested PCR is used as a marker of HBV infection. It is related to HBV viremia level of mothers.

Effects of intrauterine infection in different periods on the placenta and endometrial blood vessel formation of pregnant mice and the growth and development of fetal rats

Objective:To investigate the effects of intrauterine infection in different periods on the placenta and endometrial blood vessel formation of pregnant rats and the growth and development of fetal rats.Methods:According to the random number table method,32 pregnant rats were divided into the early infection group,the mid-term infection group,the late infection group and the control group,with 8 rats in each group.On the 3rd,9th and 15th day of pregnancy,lipopolysaccharide was injected intraperitoneally to construct intrauterine infection models.The pregnant rats in the control group were intraperitoneally injected with the same dose of 0.9%sodium chloride solution.On the 18th day of pregnancy,the inflammatory factors[interleukin-6(IL-6),tumor necrosis factor-(TNF-)],the blood vessel density of placenta and endometrium in the placental tissues of pregnant rats,dead fetus+absorbed fetus,the inflammatory factors IL-6,TNF-and oxidation reaction indicators[malondialdehyde(MDA)and myeloperoxidase(MPO)]in the fetal rat lung and brain tissues were detected.Results:The changing trend of IL-6 and TNF-levels in the placental tissues of pregnant rats with intrauterine infection in different periods was:the control group<the late infection group<the mid-term infection group<the early infection group,the differences were statistically significant(p<.05).The changing trend of fetal rat weight,placental weight and placental coefficient in the intrauterine infection groups in different periods was:the control group>the late infection group>the mid-term infection group>the early infection group,the differences were statistically significant(p<.05).The blood vessel density of placenta and endometrium,the mean number of fetuses,brain coefficient and lung coefficient in the late infection group were significantly increased in comparison with the early infection group and the mid-term infection group.The total number and the ratio of dead fetus+absorbed fetus,the levels of IL-6,TNF-,MDA and MPO in brain and lung tissues were significantly reduced,and the differences were statistically significant(p<.05).The blood vessel density of placenta and endometrium,brain coefficient and lung coefficient of pregnant rats in the mid-term infection group were significantly increased in comparison with the early infection group,and the differences were statistically significant(p<.05).There was no statistically significant difference in the other indicators between the two groups(p>.05).Conclusions:Intrauterine infection in different periods can inhibit placental and endometrial angiogenesis,and affect the survival rate of fetal rats and the growth and development of brain and lung.The reason may be related to the aggravation of fetal inflammatory responses and oxidative stress.The earlier the intrauterine infection occurs,the severer the adverse effects on the fetal rats will be.

Long-term Impact of Intrauterine MCMV Infection on Development of Offspring Nervous System

This study examined the impacts of intrauterine murine cytomegalovirus（MCMV） infection on the long-term learning and memory of offspring.Sexually matured male and female BALB/C mice without MCMV infection were identified by ELISA and then mated.Seventy pregnant mice were randomly divided into the virus group（n=40） and the control group（n=30）,in which the pregnant mice were subjected to placenta inoculation of MCMV suspension（1 μL,1×106 PFU） or the same amount of cell culture medium,respectively,at gestational age of 12.5 days.Some pregnant mice [virus group（n=20）,control group（n=15）] were sacrificed by cervical dislocation at gestational age of 18.5 days,and the head circumference and brain weight of the mouse fetuses were measured,and the MCMV infection in their brain tissues was detected by PCR.The other pregnant mice [virus group（n=20）,control group（n=15）] delivered naturally,and the learning and memory capability of the offspring at 70-day-old was analyzed by Morris water maze test.The results showed that 28.57% mouse fetuses in the virus group developed viral infection in the brain.Their head circumference and brain weight were significantly reduced as compared with those in the control group（P0.01）.The Morris water maze test revealed that the mouse offspring in the control group found the platform with straight-line trajectories after training.In contrast,the counterparts in the virus group intended to enter the central area,but looked for the platform with a circular trajectory.And the infected mice exhibited prolonged swimming distance and swimming latency（P0.01）.It was concluded that：（1） placenta inoculation of MCMV can cause fetal brain infection and intrauterine development retardation;（2） the offspring of MCMV placenta inoculation mice showed a long-term decline in learning and memory capability.

Diagnostic value of human cytomegalovirus late mRNA detection in active intrauterine infection

To study the diagnostic value of human cytomegalovirus (HCMV) late mRNA detection in active intrauterine infection Methods The HCMV late mRNA in peripheral blood of 42 HCMV IgM positive pregnant women and their fetal attachments (such as chorionic villi, amniotic fluid, umbilical blood and placenta) were detected by reverse transcription polymerase chain reaction (RT PCR) Results Late mRNA was detected in 23 of 42 HCMV IgM positive cases, a rate of 54 3% Fetal appendages in 13 cases of late mRNA positive mothers were also tested, of which 7 were positive, with a vertical transmission rate of 53 8% In 12 late mRNA negative mothers, only 1 case of fetal appendages tested was positive, with a vertical transmission rate of 8 3% There was significant difference between the transmission rates of these two groups Conclusions Positive results of HCMV IgM cannot accurately reflect the activity of HCMV at the time of testing However, the activity of HCMV is closely related to the mother fetus vertical transmission rate As an indicator of active HCMV infection, late mRNA can not only reflect the mother fetus transmission rate during active HCMV infection, but also provide some information about HCMV activity in fetal tissue

Prevention of intrauterine infection by hepatitis B virus with hepatitis B immune globulin:efficacy and mechanism

Objective To evaluate the efficacy of hepatitis B immune globulin (HBIG) in preventing intrauterine infection by hepatitis B virus (HBV) and to investigate its mechanism. Methods Forty eight pregnant women positive for hepatitis B surface antigen (HBsAg) were randomly divided into 2 groups. The 34 women in the study group were injected with HBIG during pregnancy; the other 14 women were controls. Maternal blood samples were taken before HBIG injection and at delivery. Neonatal blood samples were taken within 24 hours after birth before HBIG and hepatitis B vaccine were given. HBsAg and antibody to HBsAg (anti HBs) were tested by radioimmunoassay. Results None of the 35 newborns (including 2 twins) in the study group was positive for HBsAg, but 3 (21%) in the control group were positive (P=0.02). The HBsAg titers in the women in the study group decreased after HBIG injection. Of the 35 newborns in the study group, 32 (91%) were positive for anti HBs. Conclusion Systematic injections of HBIG during pregnancy may prevent intrauterine HBV infection, the mechanism of which may be reduction of maternal HBV viremia and production of fetal passive immunity.

Expression and its clinical significance of HLA-G in HCMV-infected placenta] villi at early pregnant stage

Objective：To study the expression and its clinical significance of HLA-G in HCMV intrauterine infected placental villi at early pregnant stage. Methods：PCR （polymerase chain reaction） was used to screen the peripheral blood for HCMV-DNA in 462 women who had willingly undergone induced abortion. Then immunohistochemistry was also used to detect expressions of mouse anti-HCMV early antigen （HCMV-EA） and mouse anti-HLA-G in HCMV-DNA positive cases＇ placental villi. The difference of HLA-G expressions between the intrauterine infection group（HCMV-EA positives）, the intrauterine infection-free group（HCMV-EA negatives） and the normal control group （50 cases of healthy early placental villi） was compared. Results： Of the 78 cases, which were detected HCMV-DNA positive, 11 （14.10%） were HCMV-EA positive. Compared with the other two groups, HLA-G expressions in the intrauterine infection group were both obviously decreased（both P〈0. 001）. HLA-G expression positions in all three groups were mainly located in the cytotrophoblast. Conclusion：Intrauterine HCMV infection at early pregnant stage is closely related to HLA-G expression at the maternal-fetal interface. The virogenetic products may affect the expression of HLA-G at the maternal-fetal interface and that of its immunological function, thus leading to different clinical outcomes.

Effects of Jinye Baidu Granule (金叶败毒颗粒) on Fetal Growth and Development with Maternal Active Human Cytomegalovirus Infection

To evaluate the effects of Jinye Baidu Granule （金叶败毒颗粒, JYBDG）, a traditional Chinese medicine compound prescription, on fetal growth and development with maternal active human cytomegalovirus infection. Methods： A prospective, randomized and controlled trial was adopted during January 1996 to June 2002. From the pregnant women with an abnormal pregnant history, 240 cases were screened to be infected by human cytomegalovirus （HCMV） by enzyme-linked immunoabsorbent assay （ELISA） and reverse transcription polymerase chain reaction （RT-PCR）. They were assigned according to the random number table to two groups. The 122 cases in the treatment group were administrated with JYBDG, one package each time, three times a day for two continuous weeks, while the other 118 in the control group did not receive any treatment. The negative conversion rate of both HCMV-IgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate between the two groups were compared, and fetal growth and development in partial fetuses were also observed. Results： The negative conversion rate of both HCMV-IgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate in the treatment group were 77. 05% （94/122）, 48.98% （48/98） and 21.74% （10/46） respectively, while those in the control group were 38. 14% （45/118）, 67.50% （54/80） and 52.63% （20/38） respectively, all showing significant difference between the two groups （P〈0.01）. Totally 35 normal infants and 11 abnormal infants were born in the treatment group, and the number in the control group was 20 and 18 respectively, and comparison between the two groups showed significant difference （P〈0.01）. Six months of child birth, the scores of both mental development index （MDI） and psychomotor development index （PDI） of infants were higher in the treatment group （20 cases） than those in the control group （20 cases）, but there was no significant difference between the two groups （P〉0.05）. Conclusion： JYBDG could decrease the intrauterine transmission of HCMV and is beneficial to fetal growth and development.