[Effect of Berberine on the Insulin Resistance and TLR4/IKKbeta/NF-kappaB Signaling Pathways in Skeletal Muscle of Obese Rats with Insulin Resistance].

OBJECTIVE: To explore the possible mechanism of berberine improve insulin resistance in skeletal muscle of obese rats. METHODS: Forty Wistar rats were divided into normal control group (NC group, n =10) and high fat food group (HF group, n=30). After the obese model rats established successfully, the rats of NC group (n=10) and HF group (n=10) were sacrificed. The level of fasting plasma endotoxin (ET) was detected. The expression of Toll-like receptor 4 (TLR4) mRNA in skeletal muscle were detected by real time quantitative PCR. The expression of TLR4, IkappaB kinase beta (IKKbeta), phospho-IKKbeta(Ser181) (p-IKKbeta(Ser181)), nuclear factor-kappaB (NF-kappaB), tumor necrosis factor-alpha (TNF-alpha), insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phospho-IRS-1(Ser307) (p-IRS-1(ser307)) and the tyrosine phosphorylation of IR and IRS-1 (p-IR(Tyr) and p-IRS-1(Tyr)) protein in skeletal muscle were detected by Western blot. The twenty HF group rats were randomly divided into two group: the fat model control group (FC group, n=10) were fed with high fat diet and distilled water. The fat berberine group (FB group, n=10) were fed with high fat diet and berberine. All rats were sacrificed after effective interventions of eight weeks. The same indexes as the first part of experiment were detected. Results The results showed that the level of ET was increased. The TLR4/IKKneta/NF-kappaB signaling pathway is activated and the expression of TNF-alpha was increased of the skeletal muscle in obese rats. However, the insulin signaling pathways of the skeletal muscle in obese rats was inhibited. Berberine could reduce the level of ET of obese rats, down-regulate the TLR4/IKKbeta/NF- kappaB inflammation signaling pathway and improve insulin resistance of skeletal muscle in obese rats. CONCLUSION: Our study revealed that berberine could reduce the level of ET of obese rats, down-regulate the TLR4/IKKbeta/NF-kappaB inflammation signaling pathway in skeletal muscle and berberine can improve insulin resistance of skeletal muscle through inhibiting the active of the TLR4/IKKbeta/NF-kappaB signaling pathway.

[Umbilical Cord-derived Mesenchymal Stem Cells Grafts for Neonatal Rats Model of HIBD: the Mechanism of PI3K/Akt Signaling Pathway].

OBJECTIVE: To investigate the role of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway for umbilical cord-derived mesenchymal stem cells (UC-MSCs) treating in neonatal rats with hypoxia-ischemia brain damage (HIBD). METHODS: P10 SD rats were divided into sham group, MSCs group, inhibitor (LY 294002) group (LY group) and HIBD group randomly. To establish a neonatal rat model of HIBD, UC-MSCs labeled with Brd U were transplanted into the lateral ventricle of rats. At 24 and 48 h after transplantation, the number of apoptotic cells was detected by TUNEL, while the expression of caspase3 protein and phosphorylation of Akt (P-Akt) were quantified by Western blot. RESULTS: At 24 and 48 h after transplantation, both the apoptotic cells and caspase3 protein expression in MSCs group were less than those in LY group and HIBD group (P<0. 05), while the expression of P-Akt was higher than those in LY group and HIBD group (P<0.05), and with the passage of time after transplantation, the expression of each index showed a downward trend. CONCLUSION: The apoptotic cells of brain and the expression of caspase3 protein decrease, while the expression of P-Akt increase. PI3K/Akt signaling pathway may be an important mechanism for UC-MSCs transplantation in HIBD rats.

[The risk factors of ventilator-associated pneumonia in newborn and the changes of isolated pathogens].

OBJECTIVE: To study the risk factors of ventilator-associated pneumonia (VAP) in newborn and the profiles of isolated pathogens. METHODS: The clinical data of 179 neonates in the previous group (from December 2005 to December 2008) and 331 neonates in the present group (from January 2009 to January 2012) admitted into the neonatal intensive care unit (NICU) and received mechanical ventilation for equal or longer than 48 hours were respectively reviewed and analyzed,and their isolated pathogen profile and drug sensitivity were also compared in two groups. RESULTS: The incidence of VAP in the previous group and the present group were 22.35% and 20.24% respectively, no significant difference between two groups (P > 0.05) was observed. Gram-negative bacteria was the main pathogens (93.40%), the first 5 common pathogens in the previous group were Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter aerogenes. However, the first 5 of isolated gram-negative pathogens in the present group were somewhat different from the previous group, which were Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli and Enterobacter aerogenes. The drug sensitivity test showed that these pathogens had a general decline in sensitivities to commonly used antibiotics, of which the carbapenem antibiotic drug sensitivity decreased significantly, there was statistically significant difference between the previous group and the present group (P < 0.05). The risk factors of VAP include the duration of mechanical ventilation (OR = 5.680, 95% CI: 2.867-11.253), the frequency of tracheal intubation (OR = 2.219, 95% CI: 1.037- 4.748), birth weight (OR = 2. 127, 95% CI: 1.067-4.238) and neonatal asphyxia (OR = 2.025, 95% CI: 1.079-3.799) as well as the duration of hospital stay (OR = 2.012, 95% CI: 1.215-3.967). CONCLUSIONS: According to the data in our hospital, the main pathogenic bacteria of VAP were gram-negative pathogens, which showed a general decline in sensitivities to commonly used antibiotics. The incidence of VAP was closely related to the duration of mechanical ventilation, the frequency of intubation, birth weight and the hospitalization as well as the neonatal asphyxia.

Incidence of brain injuries in premature infants with gestational age ≤ 34 weeks in ten urban hospitals in China.

BACKGROUND: There is a large number (1.5 million per year) of premature births in China. It is necessary to obtain the authentic incidences of intraventricular hemorrhage (IVH) and periventricular leukomalacia (PVL), the common brain injuries, in Chinese premature infants. The present multicenter study aimed to investigate the incidence of brain injuries in premature infants in ten urban hospitals in China. METHODS: The research proposal was designed by the Subspecialty Group of Neonatology of Pediatric Society of the Chinese Medical Association. Ten large-scale urban hospitals voluntarily joined the multicenter investigation. All premature infants with a gestational age ≤ 34 weeks in the ten hospitals were subjected to routine cranial ultrasound within three days after birth, and then to repeated ultrasound every 3-7 days till their discharge from the hospital from January 2005 to August 2006. A uniform data collection sheet was designed to record cases of brain injuries. RESULTS: The incidences of overall IVH and severe IVH were 19.7% (305/1551) and 4.6% (72/1551), respectively with 18.4% (56/305) for grade 1, 58.0% (177/305) for grade 2, 17.7% (54/305) for grade 3 and 5.9% (18/305) for grade 4 in nine hospitals. The incidences of overall PVL and cystic PVL were 5.0% (89/1792) and 0.8% (14/1792) respectively, with 84.3% (75/89) for grade 1, 13.5% (12/89) for grade 2, and 2.2% (2/89) for grade 3 in the ten hospitals. The statistically significant risk factors that might aggravate the severity of IVH were vaginal delivery (OR=1.883, 95% CI: 1.099-3.228, P=0.020) and mechanical ventilation (OR=4.150, 95% CI: 2.384-7.223, P=0.000). The risk factors that might result in the development of cystic PVL was vaginal delivery (OR=21.094, 95% CI: 2.650-167.895, P=0.000). CONCLUSIONS: The investigative report can basically reflect the incidence of brain injuries in premature infants in major big cities of China. Since more than 60% of the Chinese population live in the rural areas of China, it is expected to undertake a further multicenter investigation covering the rural areas in the future.

Epidemiological survey on newborns born at the obstetric departments in hospitals in mid-southern region of China in 2005.

OBJECTIVE: To investigate the situations at birth of newborns in the mid-southern region of China by performing a survey on the newborns born at urban hospitals. METHODS: A total of 23 hospitals in the mid-southern region of China were selected to participate in this survey. The data of 15,582 newborns who were born at the obstetric departments from January 1, 2005 to December 31, 2005 were retrospectively investigated. RESULTS: The male to female ratio among newborns was 1.16:1. The incidence of preterm birth was 8.11%, while very low birth weight (VLBW) infants accounted for 0.73%. The rates of spontaneous vaginal delivery and cesarean section ware 57.52% and 40.82%, respectively, while the other delivery modes accounted for 1.66%. The incidence of neonatal asphyxia was 3.78% (0.75% for heavy asphyxia). The mortality of newborns was 0.55% (5.56% for preterm infants). CONCLUSIONS: The incidences of preterm birth and neonatal asphyxia are relatively high in the mid-southern region of China. The rate of cesarean section is abnormally high, which is due mainly to social factors.

[A survey of neonatal births in maternity departments in urban China in 2005].

OBJECTIVE: To study the epidemiology of births in urban China. METHODS: A retrospective study was conducted on neonates born in 2005 in the maternity departments of 72 urban hospitals from 22 provinces in China. RESULTS: A total of 45722 infants born between January 1, 2005 and December 31, 2005 were enrolled. The male to female sex ratio was 1.13:1. Preterm births accounted for 8.1%. The incidence of very low birth weight infants was 0.7%. A total of 99.7% of mothers delivering at term had conceived naturally and 0.3% had experienced assisted reproduction. A total of 98.4% of mothers who delivered preterm had conceived naturally and 1.6% had experienced assisted reproduction. The proportion of vaginal deliveries was 50.8% compared to 49.2% delivered by cesarean sections. Many cesarean sections (38.1%) were due to social factors. Infants with an Apgar score≤7 at 1 minute accounted for 4.8%, and 1.6% of infants had an Apgar score≤7 at 5 minutes. Of all the infants included in the study, 7.14% were admitted to neonatal units for treatment. The death rate of all included infants was 0.74%. CONCLUSIONS: The proportion of preterm births was higher in 2005 than in 2002-2003. The proportion of cesarean section deliveries was much higher in urban China than in most other Asian countries and America.

Epidemiologic survey on hospitalized neonates in China.

OBJECTIVE: To carry out a nationwide epidemiologic survey on the neonates in urban hospitals with an attempt to understand the disease spectrum and treatment outcomes of hospitalized neonates in China. METHODS: The clinical data of 43,289 hospitalized neonates from 86 hospitals in 47 Chinese cities (22 provinces) between January 1, 2005 and December 31, 2005 were retrospectively analyzed. RESULTS: The male:female ratio was 1.73:1. Premature infants accounted for 26.2% of the hospitalized neonates, which was higher than that reported in 2002 (19.7%). The top three diseases during the neonatal period were jaundice, pneumonia, and hypoxic-ischemic encephalopathy. The incidences of pneumonia, meconium aspiration syndrome, and bilirubin encephalopathy in term infants were higher than those in premature infants, while the incidences of asphyxia, respiratory distress syndrome, and pulmonary hemorrhage in term infants were lower than those in premature infants. The incidences of asphyxia, small for gestational age infant, and wet lung were higher in neonates whose mother had pregnancy induced hypertension. The outcomes of these hospitalized neonates included: recovered, 63.9%; improved, 27.3%; discharged due to the family's own decisions, 7.6%, and died, 1.2%. Nearly half (46.4%) of the neonatal death occurred within 24 hrs after admission. CONCLUSION: The incidence of premature birth shows an increasing trend among hospitalized neonates. Since the neonatal deaths mainly occur within 24 hrs after admission, monitoring during this period should be enhanced.

[An epidemiologic investigation of newborns from obstetric departments in the central south region of China].

OBJECTIVE: To investigate the birth information of newborn infants from obstetric departments in the Central South Region of China. METHODS: A retrospective investigation was carried out in 15582 newborns from obstetric departments of 23 hospitals in the Central South Region of China between January 1 and December 31 of 2005. RESULTS: The sex ratio (male/female) of neonates was 1.16∶1. The proportion of preterm infants was 8.11%. The very low birth weight infants accounted for 0.73%. The neonates born by spontaneous labor accounted for 57.52%. Cesarean sections accounted for 40.82% (social factor of cesarean section: 29.91%). The incidence of neonatal asphyxia was 3.78%, in which 0.75% of the cases were severe asphyxia. The mortality of newborn infants was 0.55%, in which the mortality of preterm infants was 5.56%. CONCLUSIONS: The proportion of preterm infants and the incidence of neonatal asphyxia is high in the Central South Region of China. The proportion of births delivered by cesarean section is high, and social factors are probably responsible for the high rate.

[Multicenter investigation for incidence of periventricular leukomalacia in premature infants in China].

OBJECTIVE: Sponsored by the Subspecialty Group of Neonatology of Pediatric Society, China Medical Association, more than 10 large-scale hospitals participated in the near two-year multicenter investigation for brain injuries in premature infants in China. This study presented the investigation result for the incidence of periventricular leukomalacia (PVL) in premature infants from 10 Third Class A Level hospitals. METHODS: The premature infants with a gestation age<37 weeks in the 10 hospitals were given routine cranial ultrasound scanning within seven days after birth, and then repeated every 3-7 days until discharge from January 2005 to August 2006. The severity of PVL was graded based on de Vries classification. RESULTS: A total of 4 933 premature infants were enrolled. The total incidence of PVL and the incidence of cystic PVL were 2.3% (112/4 933) and 0.3% (16/4 933), respectively. Of the 112 PVL cases, 96 (85.7%) were with grade I, 14 (12.5%) with grade II, and 2 (1.8%) with grade III. The incidence of PVL in 4 maternal and child health care hospitals were significantly lower than that in 6 general or children's hospitals (1.4% vs 2.8%) (X2=10.284, P<0.01). Vaginal delivery and mechanical ventilation were possible high-risk factors for the development of cystic PVL. CONCLUSIONS: The data of the multicenter investigation can basically reflect the situation about the occurrence of PVL in premature infants in major big cities of China. It is important to improve the ability to recognize the sonogram of non-cystic periventricular white matter injury.

[Protective effect of calpain inhibitor-3 on hypoxic-ischemic brain damage of neonatal rats].

OBJECTIVE: The mechanisms of hypoxic-ischemic brain damage (HIBD) are still largely unknown. Elevation of intracellular calcium concentration and subsequent calcium-dependent proteases activation such as calpains seem to play an important role in the process of neuronal death. Calpain inhibitors showed neuroprotective effects in adult rat cerebral ischemia models. This study aimed to investigate the protective effect and associated mechanisms of calpain inhibitor-3 (MDL28170) on HIBD of neonatal rats. METHODS: Seven-day old Sprague-Dawley rats were randomly divided into three groups: the control group (n = 18), HIBD group (n = 48) and calpain inhibitor-3 treated group (MDL group, n = 48). The mice in the latter two groups were subjected to hypoxia-ischemia (HI) insult. The puppies in MDL group were intraperitoneally injected with MDL28170 (25 mg/kg) at 0, 2 and 4 h after HI, while those in the other two groups were intraperitoneally injected with normal saline instead. All the pupies were sacrificed at 6 h, 24 h and 72 h after HI. Quantitative real-time fluorescent polymerase chain reaction was employed to detect micro-calpain gene expression, immunoblotting technique was used to measure mu-calpain and caspase-3 protein activation, apoptosis of ipsilateral cortex was detected by terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling staining (TUNEL). CA1 neuronal loss was counted 24 h after HI by light microscopy. RESULTS: After HI mu-calpain mRNA began to increase at 6 h and reached peak at 24 h compared to the control (1.805 and 4.83 vs. 1, P < 0.05); mu-calpain was activated through autolysis, the ratio of its activated fragment (76 000) vs. whole fragment (80 000) was significantly higher at 6 h (0.547 +/- 0.095) compared to the control (0.095 +/- 0.016, P < 0.05), it reached peak at 24 h (0.921 +/- 0.058, P < 0.01) and was still at a high level at 72 h (0.708 +/- 0.025, P < 0.05). Expression of activated caspase-3 protein reached peak at 24 h (3.78 +/- 0.30, P < 0.01), decreased to the same level as the control (1.56 +/- 0.07) at 72 h (1.82 +/- 0.11, P > 0.05). Apoptotic cells in the cortex ipsilateral to HI insult increased after HIBD, reached peak at 24 h (135.46 +/- 17.52/visual field) and was still markedly higher at 72 h (79.32 +/- 17.79/visual field) compared with the control (5.33 +/- 1.53/visual field, P < 0.01). At 24 h after HI CA1 neuronal loss (30.0 +/- 6.2/oil immersion lens field) in the HIBD group was significantly higher than that of the control (2.4 +/- 0.3/oil immersion lens field, P < 0.01). However, in the MDL group the expressions of mu-calpain and caspase-3 proteins were diminished, TUNEL positive cells at 6 h and 24 h were decreased and CA1 neuronal loss (18.2 +/- 2.4/oil immersion lens field, P < 0.05) was alleviated. The amount of micro-calpain mRNA was decreased in the MDL group, but there was no significant difference compared with the HIBD group. CONCLUSION: mu-calpain gene and protein expressions increased after HI, which may contribute to the pathogenensis of HIBD. Calpain inhibitor-3 may intervene neural necrosis and apoptosis by diminishing expressions of mu-calpain and caspase-3 to play a protective role after HI insult of neonatal brain.

[The influence of oxygen and glucose deprivation on the expression of neurite growth inhibitor Nogo-A in oligodendrocyte precursor cell of rat].

OBJECTIVE: To investigate the Nogo-A expression and its regeneration inhibition role in oligodendrocyte precursor cells (OLPs) after oxygen & glucose deprivation (OGD). METHODS: The OLPs were separated by the improved procedure of separation and purification through agitation and then cultured in chemically defined medium. The OGD model of OLPs was set up by using Na2S2O4 and Earle's fluid in the medium in vitro. Immunofluorescence assay was applied to identify the OLPs with its specific antibodies such as A2B5, O4 and O1. Western blot and immunofluorescence assay were used to analyze the expression of Nogo-A in OLPs at 10 min, 30 min and 60 min after OGD. The livability rate of cells in each group was detected by MTT. RESULTS: The expression of Nogo-A was increased significantly compared with control at 10 min, 30 min and 60 min after OGD (P < 0.05). MTT result showed that the livability rate of OLPs with OGD was significantly lower than that of control (P < 0.05). CONCLUSION: The expression of Nogo-A is increased while the OLPs livability is dropping down after OGD, which suggests that Nogo-A may play a role in OLPs damage and in the pathogenesis of restraining OLPs' regeneration.

[Expression of chemokine receptor CCR2 in cerebral tissue of newborn rat with experimental hypoxic-ischemic brain damage].

OBJECTIVE: To observe the expressions of mRNA and protein for the CCR2 in cerebral tissue with experimental hypoxic-ischemic brain damage (HIBD) to newborn rat. METHODS: The HIBD model of SD rat was set up by ligating the right carotid artery and exposing the animals to 8% oxygen for 2.5 h. The dynamic change of CCR2 mRNA level was studied by using quantitative, real-time, fluorescence PCR assay (TaqMan), and the change of CCR2 protein was detected by SDS-PAGE assay. RESULTS: The highest expression of CCR2 mRNA was showing at 24 h after HIBD, of which the level was significantly higher in hypoxic-ischemic (HI) cerebral hemisphere than in control (P < 0.05). The CCR2 mRNA level of 72 h after HIBD was still higher than that of control (P > 0.05) but had no difference on 7 d after HIBD when compared with control (P > 0.05). The increased expression of CCR2 protein was detected at 12 h after HIBD and peaked at 24 h after HIBD. The significant increase of CCR2 protein persisted in HI hemisphere up to 3 d after HIBD. CONCLUSION: The expressions of CCR2 mRNA and protein increase significantly in cerebral tissue of newborn rat with HIBD. The dynamic change of CCR2 expression is consistent with the process of HIBD, which suggests that CCR2 expression after HIBD may play an important role in the mechanism of brain damage.

[NgR expression in oligodendrocyte precursor cells and its changes after oxygen & glucose deprivation in neonatal rats].

OBJECTIVE: This study examined the NgR expression in oligodendrocyte precursor cells (OLPs) and its changes after oxygen & glucose deprivation (OGD) in order to explore the role of NgR expression in the regeneration of OLPs after OGD in neonatal rats. METHODS: The OLPs from 2-day-old neonatal rats were separated by improved separation and purification through agitation and then cultured in chemically defined medium. OLPs OGD model was prepared using the medium consisting of Na2S2O4 and Earle's fluid in vitro. Immunofluorescence assay was applied to identify the OLPs with its specific antibodies such as A2B5, O4 and O1. Western blot was used to detect the NgR expression in OLPs 10 and 30 minutes after OGD. The livability rate of cells was detected by MTT. RESULTS: NgR expression was found in both the cell body and the prominence of purified OLPs. NgR expression in OLPs increased significantly 10 and 30 minutes after OGD compared with that in OLPs without OGD (controls, P < 0.05). MTT showed that the livability rate of OLPs at 30 minutes following OGD was significantly lower than that of controls (65.97+/-3.69% vs 97.17+/-6.88%, P < 0.05). CONCLUSIONS: NgR is expressed in both the cell body and the prominence of OLPs. NgR expression increases while cell livability decreases following OGD, suggesting that NgR may play a role in the inhibition of regeneration of OLPs.

[Expressions of micro-calpain mRNA and protein in cerebral tissues of neonatal rats after hypoxic-ischemic brain damage].

OBJECTIVE: To identify the expression of micro-calpain mRNA and protein in cerebral tissues of neonatal rats after hypoxic-ischemic brain damage (HIBD) and its role in the pathogenesis of HIBD. METHODS: Seven-day-old Sprague-Dawley rats were randomly divided into seven groups: control group and groups with 0, 6, 12, 24, 48, and 72 hours of exposure to HIDB. Quantitative real-time fluroscent polymerase chain reaction and Western blot technique were employed to detect the expression of micro-calpain mRNA and its protein respectively. RESULTS: The micro-calpain mRNA in the ipsilateral cerebral tissues began to increase 6 hours after the HIDB and reached peak at 24 hours, and then gradually decreased but remained in a higher level than that of the control group until at 72 hours (P < 0.05). The activated micro-calpain protein started to increase 6-12 hours after the HIDB (P < 0.05) and reached peak at 24 hours (P < 0.01). Higher levels of micro-calpain protein were maintained until 48 and 72 hours after the HIBD (P < 0.05). CONCLUSION: The expressions of micro-calpain mRNA and protein increase with the HIBD. The micro-calpain may play a role in the pathogenesis of the HIBD.

[Roles of glutamate receptor 2 and cellular free calcium in the pathogenesis of periventricular leukomalacia].

OBJECTIVE: To study the expression of the AMPA receptor subunit glutamate receptor 2 (GluR2) and the cellular free calcium concentration in the white matter of neonatal rats with periventricular leukomalacia (PVL) and their roles in the pathogenesis of PVL. METHODS: A PVL model was prepared by unilateral carotid artery ligation (UCL) followed by exposure to 6% oxygen for 4 hrs in 2-day-old rats. The neonatal rats performed a sham operation, without hypoxia-ischemia (HI), were used as the control group. At 12, 24, 48 and 72 hrs of HI, the expressions of GluR2 mRNA and protein in the white matter were detected using real time quantitative PCR and Western blot respectively. Spectrophotofluorimetry and Fura 2/AM were used to detect the cellular free calcium concentration. RESULTS: The expressions of GluR2 mRNA and protein in the white matter were significantly reduced in the PVL group at 24 hrs of HI, and remained at lower expressions until 72 hrs of HI compared with the control group (P < 0.05). The cellular free calcium concentrations increased significantly in the PVL group at 12 hrs of HI, and remained at higher levels until 72 hrs of HI compared with the control group (P < 0.05). CONCLUSIONS: The expressions of GluR2 mRNA and protein in the white matter decreased whereas the cellular free calcium concentration increased in neonatal rats with PVL. The decreased expression of GluR2 might lead to the overloading of cellular calcium in the white matter, which may cause neuronal damage and death.

[Expression of P75NTR protein and RhoA mRNA in the brain of neonatal rats with white matter damage].

OBJECTIVE: Recent studies have indicated that the signal pathway of NgR-P75NTR- RhoA plays a key role in nerve injury and remodeling, but its exact mechanism and the role of the downstream molecule RhoA regulated by P75NTR remain unclear in hypoxia-ischemia (HI) neonatal animals. The present study was designed to assess the expression of P75NTR protein and RhoA mRNA in neonatal white matter and to investigate their relationship in newborn rats with white matter damage (WMD). METHODS: The rat WMD model was established by the ligation of right common carotid artery, followed by 6% hypoxia exposure for 4 hrs. The control group was sham-operated, without HI treatment. The histological changes of brain tissue were observed under light and electron microscopes. Expression of P75NTR protein and RhoA mRNA in the brain white matter after 12, 24, 48 and 72 hrs and 7 days of HI were detected by RT-PCR and immunohistochemistry, respectively. RESULTS: Periventricular white matter damage was observed by 48 hrs of HI. Expression of P75NTR protein increased in the striatum and callosum zones at 12 hrs, peaked at 48 hrs, and remained at a higher level than control until 72 hrs of HI in the WMD group (P < 0.01). After 7 days of HI expression of P75NTR protein was no longer statistically different from controls. The RhoA mRNA was higher in the WMD group for the first 72 hrs and then declined to control values. CONCLUSIONS: Increased P75NTR protein might mediate apoptosis of nerve cells and inhibit the regeneration of neuron axons. The subsequent decline back to control value may be correlated with the aggregation of necrosis of nerve cells after HI. The patterns of RhoA mRNA expression were consistent with those of P75NTR protein, suggesting that the increased P75NTR level may promote RhoA mRNA expression.

[Expression of ngR mRNA and ngR protein in brain tissues of newborn rats with hypoxic ischemic brain damage].

UNLABELLED: OBJECTIVE To study the expression of NgR mRNA and NgR protein in the brain tissues of newborn rats with hypoxic ischemic brain damage (HIBD) its implications. METHODS: Seventy two newborn SD rats (7 days) were randomly divided into two groups, with or without surgical induced HIBD. RT-PCR was used to detect the expression of NgR mRNA. The immunohistochemistry method and the Western Blot were used to detect the distribution and expression of NgR protein in the brain tissues. RESULTS: Compared with the rats without HIBD, the expression of NgR mRNA and protein in the rats with HIBD increased significantly (P< 0. 05), although the expression started to decrease after 24 hours in HIBD condition. CONCLUSION: Increased NgR mRNA and NgR protein may be associated with the hypoxic-ischemic injury and may inhibit the neuron regeneration. The decreased expression of NgR mRNA and protein at a later stage may be associated with the loss of nerve cells.

[Expression of F-actin and RhoA in experimental hypoxic-ischemic white matter damage in premature SD rats].

OBJECTIVE: White matter damage (WMD) in preterm infants is a well-recognized serious complication of prematurity. The collapse of cell skeleton of growth cone after hypoxia-ischemia (HI) is considered as the basic neuropathologic change of the long-term residuals of premature white matter damage. F-actin is the major component of cell skeleton and maintains the normal form of cells, its function and potential mechanism of WMD have not been reported. In this study, changes of F-actin and its influencing factor RhoA were investigated. METHODS: Totally 184 Sprague-Dawley (SD) rats (age 2 days, body weight 6 to 8 grams) were randomly divided into 14 groups: 7 different time WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d, 14 d, 28 d) and 7 corresponding control groups. The 2 day-old SD rats were subjected to ligation of right carotid artery (ischemia), and then they were put into a box full with 6% oxygen and 94% nitrogen for 4 hours (hypoxia). The light microscopy was used to observe the brain pathological changes and the electron microscopy was used to detect the brain ultrastructural changes after hypoxia and ischemia. Eighty SD rats were used for flurescent-immunohistochemical method to detect the distribution of F-actin in cell membrane and cytoplasm of both WMD groups and the control groups at 12 h, 24 h, 48 h, 72 h, 7 d after HI respectively. The distribution of F-actin was reflected by the percentage of non-integrity cells. Another 80 SD rats were used for real time RT-PCR to detect the expression of RhoAmRNA in the white matter tissue of both WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d) and the control groups. RESULTS: (1) Necrosis of lateral ventricle tissue was observed by 72 h after HI. Dilatation of ventricle and formation of capsular space beneath white matter had been observed by 14 d after HI. (2) Disregulation, pyknosis, mitochondrion swelling and chromatin agglutination were observed in WMD groups. The maldevelopment of myelins in WMD groups was detected at 1 h after HI. (3) The fluorescent stains decreased on cellular membrane, but increased in cytoplasm with time. The percentage of non-integrity cells was significantly higher (P < 0.05) in HI groups (0.32 +/- 0.04, 0.43 +/- 0.04, 0.56 +/- 0.03, 0.65 +/- 0.04, 0.87 +/- 0.03) than the controls (0.02 +/- 0.01, 0.02 +/- 0.01, 0.01 +/- 0.01, 0.02 +/- 0.01, 0.02 +/- 0.01). (4) The expression of RhoA mRNA was significantly increased (P < 0.05) in HI groups (1.205, 2.415, 4.830, 1.500) in the white matter tissue compared with the controls (0.300, 0.375, 0.375, 0.530) at 12 h, 24 h, 48 h, 72 h after HI. The expression of RhoA mRNA reached the peak value at HI 48 h, and then gradually decreased. The expression of RhoA mRNA at HI 7 d in WMD group (0.500) was not significantly different from the control (P > 0.05). CONCLUSION: (1) The pathological and ultrastructural changes of white matter in WMD groups after HI suggest that the WMD model was successfully set up in premature 2 days SD rats. (2) F-actin is redistributed within cells after HI: expression in membrane is decreased and expression in plasma was increased. The redistribution possibly results in the collapse and retraction of cells. (3) The expression of RhoA mRNA is increased significantly after HI, which may lead to the redistribution of F-actin. (4) The increase of the expression of RhoA mRNA is not persistent, but the redistribution of F-actin is continued, which suggests that RhoA may not be the only factor affecting the redistribution of F-actin.

[Brain mitochondrial DNA damage of newborn piglets following hypoxia-ischemia].

OBJECTIVE: This study investigated the 8003 base pair (bp) fragmentation damage of brain mitochondrial DNA in newborn piglets at different times after hypoxic-ischemic brain damage (HIBD) so as to explore the biomolecular foundation of neonatal neuronal metabolic disorders. METHODS: Fifty 3-day-old piglets were randomly assigned into Control and HIBD groups. The HIBD group was subdivided into groups sacrificed at 0, 24, 48 and 72 hrs post-HIBD (n=10). HIBD was induced by left carotid ligation and exposure to 8% oxygen for 2 hours. The Control group was exposed to air and was sham-operated. The left hippocampal cortexes of all subjects were obtained to amplify the fragments of 200 bp and 8003 bp by the LX-PCR method. The PCR products were electrophoresed on agaros gels to obtain the integral optical density (IOD). RESULTS: The IOD of 8003 bp fragment was markedly reduced in the HIBD 0 hr group (22.616 +/- 2.276) when compared with that of the Control group (56.995 +/- 0.317) (P < 0.05). The IOD value remained lower at 24 hrs (27.719 +/- 0.309) and 48 hrs post-HIBD (49.491 +/- 3.233) (P < 0.05). Until 72 hrs post-HIBD, the IOD (55.972 +/- 2.236) restored to the control value. CONCLUSIONS: The brain mitochondrial DNA was fragmented in newborn piglets following brain hypoxia-ischemia. It did not recover to normal until 72 hrs post-HIBD. The fragmentation damage of mitochondrial DNA may be related to the depression of mitochondrial respiratory enzymes activity and neuron apoptosis.

[Alternations of ADF and G-actin in renal tubular epithelial cell of neonatal rats during kidney ischemic Injury].

OBJECTIVE: To study the changes of ADF and G-actin in renal tubular epithelial cell of neonatal rats during kindney ischemic injury. To explore the interactions between ADF and G-actin and their relationships with actin cytoskeleton. METHODS: Twenty-four neonatal rats were used to the experiments due to have the weights from 30 to 40 g respectively. Renal ischemia was induced by clamping the left renal pedicle for different periods of time that was in 10 min or 30 min. The kidneys of normal neonatal rats were functioned as the control group. Two kinds of primary antibodies, which one was rabbit anti-chick ADF antiserum and another was mouse monoclonal G-actin antibody, were used on one same section of kidney tissue. FITC-labeled and TRITC-labeled secondary antibodies were used to identify ADF and G-actin respectively. These samples were examined by using a fluorescene microscope. RESULTS: In control group, ADF and G-actin showed to be diffused in cytoplasm of renal tubular epithelial cell. In ischemic injury group, the distributions of ADF and G-actin were changed significantly to apical region of tubular epithelial cells and the renal tubula lumen. Regardless of the ADF expression to not change but G-actin was increased after kidney ischemia. CONCLUSION: Under the physiolgical condition, ADF and G-actin got a diffuse distribution in cytoplasm of renal tubular epitbelial cell. However, when the kidney got in ischemia, the distributions of ADF and G-actin were changed significantly to apica region of tubular epithelial cell or the renal tubular lumen.