OBJECTIVE: β-amyloid precursor protein (β-APP) is thought to be a sensitive marker for brain white matter damage (WMD) and participates in the mechanisms of hypoxic-ischemic brain damage. This paper aims to study the influence of ischemia and IGF-1 treatment on the expression of β-APP in white matter of near-term fetal sheep. METHODS: Romney-Suffolk fetal sheep were instrumented at 117 to 124 days of gestation (term=147 days). Reversible cerebral ischemia was induced by occlusion of bilateral carotid arteries for 30 mins. After damage the sheep were randomly divided into two groups: the Ischemic group (n=8) and the IGF-1 treatment group (Treatment group, n=9). The sham-operation group (n=5) was used as Control group. In the Treatment group, 3 μg (1 ml) recombinant human IGF-1 (rhIGF-1) was infused into the left lateral ventricle, 90 mins after reperfusion. The Control group received infusion of 1 ml artificial cerebrospinal fluid into the left ventricle. Ninety-six hrs after ischemia, the sheep were sacrificed and the brains were fixed. Immunohistochemical staining was performed to assess glial fibrillary acidic protein (GFAP), β-APP positive cells and the myelin basic protein (MBP) density in the white matter. Fluorescent staining was performed for double labeling. RESULTS: The MBP density of the Ischemic group ( 4.7± 7.1) was significantly reduced as compared with the Control group ( 27.8± 4.8, P< 0.001). β-APP positive cells were not observed in the Control group. β-APP positive cells of the Ischemic group increased significantly after ischemia ( 49.6± 23.7, P< 0.001). IGF treatment significantly reduced the β-APP positive cells ( 17.9± 16.5, P< 0.01). Fluorescent double labeling showed that the β-APP positive cells were co-localized with GFAP positive cells. CONCLUSIONS: Ischemia increases the expression of β-APP by astrocytes in near-term fetal sheep white matter, which may underlie the mechanisms of ischemic WMD. IGF-1can reduce the expression of β-APP, which may be mechanism of its protective effect against WMD.

Objective Asthma is recognized as a chronic airway inflammatory disease. Reactive oxygen species can induce airway inflammation. The aim of this study was to explore the effect of melatonin (MT) on the content of reactive oxygen species and airway inflammation in rats with bronchial asthma. Methods Twenty-four Sprague-Dawley (SD) rats were randomly assigned into 3 experimental groups (8 in each): 1) Asthma group: the rats were immunized on day 1 by intraperitoneal injection of 100 mg ovalbumin (OVA) in 1 ml saline with 100 mg of aluminum hydroxide. After 14 days, the rats were challenged with aerosolized 1% OVA for 20 mins per day for 7 consecutive days; 2) MT group: OVA-sensitized rats were given intraperitoneal injection of 10 mg/kg MT 30 mins before each OVA challenge; and 3) Control group: OVA was replaced with normal saline. Airway responsiveness to aerosolized acetylcholine was detected 6 hrs after the last challenge. Then the rats were lavaged and total and differentiated leukocytes counts in bronchoalveolar lavage fluid (BALF) were performed after Wright-Giemsa staining. At the same time, the content of reactive oxygen species (ROS) in the lung tissues was assessed with chemical colorimetry. Results After OVA challenge, there was a significant decrease in airway responsiveness and the number of lymphocytes and eosinophils in the BALF of the MT group compared with the Asthma group (P< 0.05). The MT group also showed a significantly lower ROS level in the lung tissues compared with Asthma group (P< 0.05). Conclusions MT can decrease airway inflammation and the content of ROS in asthmatic rats, which may be the underlying protective mechanisms of MT against asthma.

Objective Matrix metalloproteinase-9 (MMP-9), a metalloproteinase, is capable of degrading type IV, V collagens, as well as gelatins. Increased levels of MMP-9 have been detected in aortic aneurysms in adult human, suggesting its role in arterial wall destruction and aneurysm formation. This study was designed to investigate the potential role of MMP-9 in the pathogenesis of coronary artery lesions in Kawasaki disease (KD) patients. Methods Twenty-seven children with KD [17 with coronary artery lesions (CALs) and 10 without] and age-matched 10 febrile and 10 healthy controls were enrolled in this study. Gelatin zymography and ELISA were used to detect the activity and levels of serum MMP-9. MMP-9 mRNA expression in the leucocytes was detected using reverse transcription-polymerase chain reaction (RT-PCR). Results 1) The activity ( 10.2± 2.2) and levels of MMP-9 (1 116 ±691 ng/ml) in KD patients with CALs in the acute phase were significantly higher than those without CALs ( 6.2± 2.1, 457±133 ng/ml, respectively; P< 0.05). Both of them in either the KD patients with CALs or without were higher than those of the healthy controls ( 0.1± 0.0, 72±24 ng/ml, respectively; P< 0.01) and the febrile controls ( 3.1± 1.4, 221±154 ng/ml, respectively; P< 0.05). 2) There was a significantly positive correlation between the serum MMP-9 protein levels and the circulating leucocytes counts in KD patients in the acute phase (r= 0.480, P< 0.05). 3) The MMP-9 mRNA expression in the leucocytes of KD patients in the acute phase were significantly elevated, as compared with the febrile and healthy controls (P< 0.01). There were no significant differences in the MMP-9 mRNA expressions between the two KD groups. 4) The activity, protein levels and mRNA expression of MMP-9 in the KD patients decreased obviously from the subacute through the convalescent phases, as compared with the acute phase (P< 0.01). Conclusions The MMP-9 expression in KD patients in the acute phase was significantly elevated, especially in those with CALs. MMP-9 may be involved in the development of coronary artery lesions in KD.

Objective Leptin resistance is thought to be a main mechanism of human obesity. Although some studies suggested that leptin resistance could be relevant to the level of leptin receptor and its downstream signaling pathway, there has been little research on leptin receptor regulation. This paper studied the effect of leptin on its receptors. Methods The human hepatocellur carcinoma cell line HepG 2 was incubated in serum-free medium containing 0, 10 -9, 10 -8, 10 -7, 10 -6 M of human leptin respectively for 24 hrs. Then semi-quantitative RT-PCR was used to measure the changes of long (OB-Rb) and short (OB-Ra: OB-R219.1, OB-R219.3) leptin receptors mRNA expressions in HepG2 cells. Results Both OB-Ra and OB-Rb mRNA were expressed in HepG2 cells, which provided a useful model for studies of leptin receptors regulation. Leptin (10 -7-10 -6 M) induced a significant decrease in the OB-Rb mRNA expression, with the maximum effect at 10 -6 M( 0.43± 0.14 vs 1.01± 0.22), when compared with the control (incubation in the absence of leptin). Similarly, the expressions of OB-R219.1 and OB-R219.3, two isoforms of OB-Ra, were also markedly reduced in cells treated with 10 -8-10 -6 M leptin, with the maximum inhibition for OB-R219.1 at 10 -7 M (44% of the control) and for OB-R219.3 at 10 -6 M (49% of the control). Conclusions Leptin can inhibit the expressions of both OB-Ra and OB-Rb mRNA in HepG2 cells, which may be associated with leptin resistance in vivo.

Objective To investigate the protective effects of hyperbaric oxygen (HBO) against hypoxic ischemic brain damage (HIBD) of neonatal rats. Methods Seven-day-old Sprague-Dawley (SD) rat pups were randomly divided into 4 groups (n=10 each): Control group, HIBD group, Hyperbaric air (HBA) group, and Hyperbaric oxygen (HBO) group. The HIBD model was produced by permanent occlusion of left common carotid artery and followed by exposure to a mixture of 8% oxygen and 92% nitrogen for 2 hrs (at 37℃). HBO and HBA treatments [2 atmosphere absolute (ATA) for 1 hr] were administered once daily to rats in the HBO and HBA group respectively after hypoxia for 7 days. Radial arm maze and sensorimotor functional tests were administered from 30 to 35 postnatal days. At the end of the behavior trials, the rats were sacrificed and cerebral histology was analyzed. The CA 1 subfield neurons numbers were counted to evaluate the brain damage. Results In the behavior test, the HIBD group showed different degrees of neurological damage. HBO treatment resulted in significant protection against hypoxia-ischemia induced behavior impairments (all P< 0.01). However, the HBA group did not show any significant improvement. There was a significant reduction of CA 1 neuron density in the left hemisphere of HIBD and HBA groups, compared with that of the Control group and the HBO group (all P< 0.01). Conclusion HBO therapy can attenuate brain damage and improve learning, memory and sensorimotor functions in neonatal rats after HIBD.

Objective The insulin-like growth factor-1 (IGF-1) can protect damaged neurological system, but it is unknown whether exogenous IGF-1 can inhibit the productions of endogenous IGF-1 and IGF-1 receptor. This study aims to observe the changes of the expressions of IGF-1 and IGF-1 receptor mRNA in neonatal rats with hypoxic-ischemic brain damage (HIBD) and to explore the effects of IGF-1 treatment on endogenous IGF-1 and IGF-1 receptor. Methods Seven-day-old rats were subjected to unilateral carotid artery ligation and hypoxia exposure to establish a HIBD model. In situ hybridization was used to observe the changes of the expressions of IGF-1 and IGF-1 receptor mRNA in the injured regions of neonatal rats at differenft time points following HIBD. The expressions of endogenous IGF-1 and IGF-1 receptor mRNA between the HIBD group and the IGF-1-treated group at 12 hrs and 72 hrs following HIBD were compared by semi-quantitative analysis. Results The expressions of IGF-1 and IGF-1 receptor mRNA in the hippocampus began increasing at 48 hrs following HIBD, and reached a peak at 72 hrs. At 120 hrs post-damage, the expression of IGF-1 mRNA decreased to normal, while the expression of IGF-1 receptor mRNA maintained at a high level. In the cortex, the expressions of IGF-1 and IGF-1 receptor mRNA began increasing at 24 hrs post-damage, and decreased to normal at 96 hrs but their increased extent was less than that in the hippocampus. The expression of endogenous IGF-1 mRNA was not changed after IGF-1 treatment compared with the un-treated HIBD group. The expression of endogenous IGF-1 receptor mRNA remained unchanged 12 hrs after IGF-1 treatment, but significantly increased at 72 hrs. Conclusions IGF-1 and IGF-1 receptor mRNA in the cortex and the hippocampus increase after HIBD. Exogenous IGF-1 has no effect on the expression of endogenous IGF-1, but can up-regulate the expression of IGF-1 receptor.

Objective The pathogenic mechanism of asthma has not yet been explained. This paper aims to study the relationships between IL-4, IL-10 and IFN-γ and asthma so as to explore the immunologic pathogenesis of asthma. Methods The Wistar rats were randomly assigned into 2 groups: a Normal group (n=11) and an Asthma group (n=11). Asthma was induced by intraperitoneal injection of OVA and fog inhalation of OVA. Five days after asthma induction, the rats were sacrificed and their spleens were removed. Then the mononuclear cell suspension was prepared, cultured, and stimulated with OVA. The levels of IL-4, IL-10 and IFN-γ in the supernatant were measured by enzyme linked immunosorbent assay (ELISA). Results The IL-4 level in the mononuclear cells of the Asthma group ( 64.56± 5.83 pg/ml) was significantly higher than that of the Normal group ( 24.66± 3.68 pg/ml) (P< 0.05), while the levels of IL-10 ( 34.13± 0.85 pg/ml) and IFN-γ ( 3.87± 0.48 pg/ml) were significantly lower than those of the Normal group [( 85.12± 6.13) and ( 14.51± 1.32) pg/ml, respectively] (P< 0.05). Conclusion IL-4, IL-10 and IFN-γ might be involved in the pathogenesis of asthma.

Objective Perinatal asphyxia is an important cause of brain injury, but an early prediction of outcome is still difficult. This paper aims to study the changes of lactate levels in plasma and cerebrospinal fluid (CSF) of neonates with hypoxic-ischemic encephalopathy (HIE) so as to explore the relationship between lactate levels and HIE as well as asphyxia. Methods Plasma lactate levels were measured in 26 neonates with HIE (8 mild, 10 moderate and 8 severe cases) and 8 healthy neonates at 0-24 hrs, 48-72 hrs and 7-10 days of their lives. CSF lactate levels were measured at 48-72 hrs of their lives and brain MRI examination was taken 7-10 days after birth in neonates with HIE. Results Plasma lactate levels in neonates with HIE and healthy neonates decreased daily after birth. The plasma lactate levels of the HIE neonates were significantly higher than those of the healthy ones at 0-24 hrs and 48-72 hrs of their lives. Plasma lactate levels in severe HIE neonates ( 9.11± 3.29 mmol/L) were higher than those of neonates with mild and moderate HIE ( 6.03± 2.66 and 6.56± 1.42 mmol/L) in the first day after birth (P< 0.05), but not in the following days. The CSF lactate levels in the severe HIE neonates ( 2.53± 0.27 mmol/L) were significantly higher than those in mild and moderate HIE ones ( 1.80± 0.20 and 1.91± 0.28 mmol/L) (P< 0.01). CSF lactate levels in the asphyxiated neonates whose 5 min Apgar score were less than 5 scores ( 2.43± 0.34 mmol/L) were higher than those whose Apgar scores were more than 5 ( 1.83± 0.25 mmol/L) (t= 5.22,P< 0.01). CSF lactate levels in HIE neonates with severe MRI abnormalities were higher than those with mild and moderate ( 2.36± 0.44 mmol/L vs 1.72± 0.24 mmol/L, 2.14± 0.26 mmol/L) (F= 7.15, P< 0.01). Conclusions The plasma lactate level may be associated with the degree of hypoxia only in the first 24 hrs of life. The CSF lactate level can reflect the severity of brain injury.

Objective This paper aims to study the effects of the brain development and brain injury on early brain responses and to explore the relationship between early brain responses and neurodevelopment in preterm infants. Methods The intensity of brain responses after sound stimulation was evaluated by detecting cerebral oxygenation using near infrared spectroscopy (NIRS) in preterm infants. The neonatal behavioral neurological assessment (NBNA) was performed in preterm infants on the 40th week of their corrected gestational age (GA). The infants had follow-up visits for the neurodevelopment within one year of their lives. Results All subjects had responses to sound stimulation in different degrees. The maximum response value after stimulation in infants with GA of more than 34 weeks' was 4.1%±1.4%. It was not different from that of term infants ( 4.2%± 1.4%). There was a significant difference in maximum response value between the infants with GA of less than 34 weeks and the term infants ( 3.1%± 1.4% vs 4.2%± 1.4%; P< 0.05). The maximum response value of the preterm infants with brain injury ( 2.6%± 1.8%) was lower than those without ( 4.4%± 1.3%) (P< 0.05). The preterm infants who had normal NBNA results had higher maximum response values than those who did not have on the 40th week of their corrected GA ( 3.9%± 1.4% vs 2.1%±1.6%; P< 0.05). The follow-up visits found a significant difference between the normal and abnormal neurodevelopment infants for the early brain response (P< 0.05). Conclusions Preterm infants have brain responses to sound stimulation. The intensity of brain response may be correlated with GA. The perinatal brain injury may affect the brain response. Early response may be related to subsequent neurodevelopment in preterm infants.

Objective This study examined the levels of the superoxide anion produced by neutrophils after different stimulations in neonates with isolated intrauterine growth restriction (IUGR) in order to clarify the effects of IUGR on activated oxygen metabolism of neutrophils in neonates. Methods Fifteen samples of cord venous blood from neonates with isolated IUGR (IUGR group) were stimulated with either phorbol myristate acetate (PMA) or bacteria (Staphylococcus aureus and Escherichia coli), and then stained with hydroethedine, an indicator of superoxide. The samples were analyzed on granulocyte gate by flow cytometry. The mean fluorescence for superoxide anion production was acquired, and was compared with 23 samples from normal full-term neonates (Control group). Results The superoxide anion level (demonstrated by mean fluorescence intensity, MFI) of the IUGR group was significantly higher than that of the Control group after PMA stimulation (448±131 vs 314±89; P< 0.01). Significant differences were also found in the superoxide anion level between the IUGR and Control groups after either Staphylococcus aureus or Escherichia coli stimulations (471±142 vs 362±79 and 502±133 vs 396±94; both P< 0.01). The ability of bacterial phagocytosis was the same for the two groups. Conclusions Neutrophils from neonates with isolated IUGR may be activated by some pathologic factors in uterus and yield an increased level of activated oxygen metabolism.

Objective To evaluate the effects and regimen-related toxicity (RRT) of CEM (carboplatin, etoposide, melphalan) chemotherapy as a conditioning regimen in autologous peripheral blood stem cell transplantation (APBSCT) in children with high-risk neuroblastoma. Methods Six patients in stage IV neuroblastoma, who had gotten a complete remission after receiving high-dose chemotherapy, radiation and surgery were enrolled in this study. All the patients received CEM chemotherapy (carboplatin 425 mg/m 2 per day for 4 days; etoposide 338 mg/m 2 per day for 4 days; melphalan 70 mg/m 2 per day for 3 days) as a conditioning regimen in APBSCT. Transplantation related complications were observed and RRT was graded according to Bearman's proposal. Results The RRT was principally manifested as stomatitis (grade I in 6 cases) and gastrointestional symptoms (grade I in 5 cases and grade II in 1), and only 1 case had RRT in liver (grade I). The RRT symptoms were improved about 16 days after transplantation. The amount of white blood cells was recovered to over 1×10 9/L after about 33 days of transplantation. The follow-up visit (ranging from 8 months to 48 months ) revealed that five patients had event free survival and 1 died due to brain metastasis after APBSCT. Conclusion CEM chemotherapy as a conditioning regimen in APBSCT might be effective and safe for children with high-risk neuroblastoma.

Objective The purpose of this study is to evaluate the therapeutic effects of combined immunosuppressive (CIS) therapy on severe aplastic anemia (SAA) and refractory aplastic anemia (RAA) in children and to study the relationship between the effectiveness of IS therapy and the immune mediated pathological mechanism. Methods Thirty-six children with SAA (n=33) and RAA (n=3) were given CIS therapy (CIS group, a combination of antithymocyte globulin, cyclosporin A and high-dose immunoglobulin). The therapeutic effects were evaluated and compared with those of 43 children with SAA (n=42) and RAA (n=1) who received the treatment of single IS agent (Control group). Peripheral blood lymphocyte subsets levels were measured with a flow cytometer. Serum interleukin and TNF levels were examined with radioimmunoassay in the two groups. Results The total response rate [ 80.6%(29/36)] in the CIS group was significantly higher than that in the Control group [ 55.8%(24/43)] (P< 0.05). Among the CIS group, 78.8% (26/33) of SAA patients had responses to CIS therapy, which was more than that of the Control group [ 57.1%(24/42)]. All of RAA patients (3/3) were responsive to CIS therapy. The results of immunological function testing showed that the CD4/CD8 ratio decreased and IL-2R, IL-8 and TNF levels increased in the two groups. In the Control group, the children whose disease duration was less than 6 months or the absolute reticulocyte counting was greater than 10×10 9/L demostrated a higher response rate to single IS therapy. The therapeutic effects in the CIS group were not associated with the disease duration and the absolute reticulocyte counts. No severe side effects and treat-related death were found in both groups. Conclusions CIS therapy is effective and safe for childhood SAA and RAA, and the therapeutic effect of CIS is better than single IS agent. Abnormal immune mediated pathological mechanism of AA might be the base of IS therapy. In order to improve the therapeutic effects, it is necessary to make a therapeutic effect prediction for choosing cases and the treatment protocol before treatment of AA with single IS agent.

Objective Matraine, a main ingredient of sophora, can inhibit the proliferation of many tumor cells such as K562 and HL-60. This study was designed to investigate the effect of matrine on acute lymphoblastic leukemia (ALL) cells and its possible mechanisms. Methods The samples were obtained from 24 children with initial ALL. The ALL cells were co-cultured with matrine. The inhibition effect of the proliferation of ALL cells was detected by the MTT assay. A flow cytometer (FCM) was used to detect the expression of Bcl-2 in ALL cells. Results Compared with the controls without matrine treatment ( 0.84± 0.27), the OD values (reflecting the survival cell amount) were reduced significantly after 48 hrs of different concentrations of matrine incubation ( 0.73± 0.16 at 0.1 mg/ml, 0.58± 0.15 at 0.2 mg/ml, 0.32± 0.16 at 0.5 mg/ml, and 0.24± 0.14 at 1.0 mg/ml). The positive rate of Bcl-2 protein was down-regulated significantly from ( 25.0± 5.8)% in the untreated controls to ( 21.3± 6.1)% in the cells treated with 0.1 mg/ml matrine, and ( 18.2± 3.7)% for 0.2 mg/ml, ( 14.5± 4.0)% for 0.5 mg/ml and ( 10.0± 3.2)% for 1.0 mg/ml at 48 hrs of incubation. Conclusions Matraine can inhibit the proliferation of ALL cells, and the possible mechanism may be associated with the down-regulated Bcl-2 expression in ALL cells.

Objective To study the changes of serum and cerebrospinal fluid (CSF) levels of neuron-specific enolase (NSE), S-100β protein (S-100β) and myelin basic protein (MBP) in children with epilepsy (EP) after seizures, so as to evaluate their significance in early diagnosis of neuronal damage. Methods The serum and CSF levels of NSE, S-100β and MBP in children with EP (EP group, including 12 cases of frenquent seizures, 13 infrenquent seizures and 6 status epilepticus) were determined respectively by electrochemiluminescence and enzyme-linked immunosorbent assay within 24 hrs after seizures. Samples were obtained from thirty-eight children with upper respiratory infection and these were used as the Control group. Results The serum and CSF levels of NSE and S-100β in the EP group within 24 hrs after seizures were significantly higher than those in the Control group (P< 0.01). Of the EP group, the serum and CSF levels of NSE and S-100β in children with frenquent seizures and children with status epilepticus were significantly higher than in children with infrenquent seizures (P< 0.05 and P< 0.01, respectively), while there were no significant differences between the children with frenquent seizures and ones with status epilepticus. There were no statistically significant differences in serum and CSF MBP levels between the EP and Control groups. Conclusions NSE and S-100β in serum and CSF may be markers of neuronal damage following seizures, while MBP is not correlated with neuronal damage.

Objective The mechanism of the fibrosis in muscles with progressive muscular dystrophy is not clear. Transforming growth factor β 1 (TGF-β 1), which can induce the extracellular matrix aggrogation, has roles in fibrogenesis and tissue repair. This study aims to explore the possible correlation between the immunolocalization of TGF-β 1 in muscle and fibrosis, and to study the mechanism of muscle progressive damages in children with progressive muscular dystrophy. Methods Eighteen children with progressive muscular dystrophy, including 7 casese of Duchenne muscular dystrophy (DMD), 6 Fukuyama type and 3 non-Fukuyama type congenital muscular dystrophy (FCMD and nFCMD), 2 Becker muscular dystrophy (BMD), were involved in this study. Thirteen cases of non-muscular disease served as the controls. The level of TGF-β 1 expression from biopsied frozen muscle were assayed by the immunohistochemical method and Western blot analysis. Results TGF-β 1 of active and latent forms were strongly expressed in the muscle fibers and emdomysium of the muscle in CMD and DMD, and the levels of expression in CMD were significantly higher. The Western bloting results also showed a stronger TGF-β 1 expression in FCMD than in DMD. TGF-β 1 expression levels in both FCMD and DMD were significantly higher than in controls. Conclusions The fibrosis in muscles might be one of the important causes of progressive muscular lesions in children with progressive muscular dystrophy.

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