| Hypoxic-ischemic encephalopathy (HIE) caused by perinatal asphyxia is one of the common diseases which do harm to neonates, lead to neonatal death or later neurologic developmental disorder. Activin A (ACT A) has been confirmed to have neuroprotective effect in vitro. Our previous study indicated that the exogenous ACT A can protect the brain tissue of neonatal rats with hypoxic-ischemic brain damage (HIBD) by inhibiting the cerebral edema and neurocytal apoptosis. However, the correlativity between endogenous ACT A and HIBD has not been reported yet. In this study, both animal experiment and clinical research are performed in order to investigate the expression and its significance of endogenous ACT A in both brain tissue of neonatal rats with HIBD and blood of neonates with asphyxia. The result of this study is expected to provide an experimental evidence for further study of HIE in its pathogenesy, prevention and therapy. Part I: Experimental Study1. Materials and Methods1.1 Animals and materialsTotal 100 seven-day-old Wistar neonatal rats, with average weight of(15.6±3.2) g, regardless of female or male (Experimental Animal Centerof Shandong University) ; Activin A monoclonal antibody(R&D SystemCompany, United States), SP immunohistochemical test kit (Lab VisionCorporation, United Kingdom)1. 2 MethodsAll animals are divided randomly into 3 groups: normal control group, fake surgery group and HIBD model group. For modeling of HIBD, According to the means of Rice, unilateral carotid ligation and hypoxia was performed on rat pups. These pups were sacrificed at 8 different time points (10n forn each) of 1h, 3h, 7h, 10h, 12h, 24h, 48h, 72h.Brain tissues were fixed in 4% paraformaldehyde at certain time point for a certain period, then anhydrated in gradient ethanol, lucidified in xylene, embed in paraffin. Coronaryserial sections with H&E staining were observed under light microscope. Immunohistochemistry was performed to detect ACT A.1.3 Statistical treatmentAll data was represented by (x±s). T-test and rank sum test were usedto compare two groups. One way ANOVA and Newman-Keuls(q-test) were used to compare many groups. P-value is significant at the standard of 0.05. All statistical analysis was completed by the package of SAS6. 0. 2. ResultPups of normal contral group and fake surgery group were sacrificed at the time point of 10h. Seldom ACT A positive cells were seen in both sides of the sections under light microscope. By ststistics, there was no significant difference between both sides of the same group and the same side of different groups (P>0. 05) . Seldom ACT A positive cells were seen in right side of model group at different points. There was no significant difference compared with control groups (P>0. 05) . At the time point of 1h, there were some ACT A positive cells in left side of model group. There were significant differences to compare with right side of model group and left side of control groups (2. 50±1. 51 vs 1. 13±0. 64, P<0. 05; 2.50±1.51 vs 1.00±0.76, P <0. 05) . At the later time points, ACT A positive cells increased and their immunostaining enhanced gradually. At the time point of lOh, the expression of ACT A positive cells reached its high-peak. There were extensive ACT A positive cells in left side. There were significant differences to compare with right side of model group and left side of control groups (19. 75±2. 19 vs 1. 25±0. 71, P<0.01; 19.75±2. 19 vs 1.00±0. 76, P<0.01) . At the time point of 72h, there were still ACT A weakly positive cells in some area of left side. There was significant difference to compare with right side (12. 75±3. 62 vs 1.38±0.74, P<0. 05). Comparing the expression of ACT A positive cells among different time points in model group, there were significant differences in left side (F=28. 61, P=0. 0001<0. 01) and no significant difference in right side. Positive reaction localized mainly in cytoplasm , axon and dendron, nonstaining in nucleus.Part II: Clinical Study1. Objects and Methods 1. 1 objects1.1.1 Asphyxia groupTotal 58 full-term newborn infants with asphyxia by spontaneous delivery in Obstetrics of Jinan Central hospital from 2004 to 2006 were selected in this group. They were divided into 3 groups according to the standard of symptoms and laboratory findings: All 23 cases in mild group were cured; For 26 cases in moderate group, 25 cases were cured or clinical cured and 1 died; For 9 cases in severe group, 7 cases were cured or clinical cured and 2 died.1.1.2 Normal control groupTotal 30 full-term normal newborn infants by spontaneous delivery in Obstetrics of Jinan Central hospital from 2004 to 2006 were selected in this group.There was no significant difference in birthweight, gestational age, days after birth and gender between two groups.1.2 MethodsFor both groups, 3ml cord blood and 3ml femoral venous blood was collected instantly after birth and in postnatal day of 7～10 respectively. ACT A, fibroblast growth factor (FGF) and tumor necrosis factor (TNF)-αin blood serum was detected by enzyme linked immunosorbent assay (ELISA) .1.3 Statistical treatmentAll data was represented by (x±s). T-test and rank sum test were used to compare two groups. One way ANOVA and Newman-Keuls(q-test) were used to compare many groups. P-value is significant at the standard of 0. 05. All statistical analysis was completed by the package of SAS6. 0. 2. ResultThe concentration of serum ACT A,FGF and TNF-αincreased in cord blood of neonates with asphyxia. There was significant difference to compared with those of normal controls. (3.64±0.69 ng/ml vs 2.95±0.34 ng/ml, P<0. 001; 77. 55±23. 18 pg/ml vs 36.43±9.51 pg/ml, P<0. 001; 10.72±1. 50 ng/ml vs 9. 15±0. 99 ng/ml, P<0. 001) . In convalescence stage, the concentration of serum ACT A went on increasing; FGF decreased a little but remained a higher level. Both serum ACT A and FGF were obviouse higher than those of normal controls(5. 59±1. 33 ng/ml vs 2.93±0.25 ng/ml, P <0. 001; 64. 21±18. 70 pg/ml vs 41.69±9. 08 pg/ml, P<0. 001); Serum TNF-αdecreaed in convalescence stage and no significant difference to compared with normal controls(9. 41±0. 87 ng/ml vs 9.03±1.06 ng/ml, P >0.05).Part III: ConclusionThe resits of this study indicated that HIBD in neonates with asphyxia can induce increased expression of endogenous ACT A both in brain tissue and blood. Furthermore, There' s a positive correlation between the level of endogenous ACT A and severity of HIBD. It' s presumed that HIBD stimulate the secrete of endogenous ACT A which can protect the brain tissue from damage. This study illuminated that endogenous ACT A may play an important role in the pathogenesis of HIE in neonate with asphyxia. Its expression product could be developed as a sensitive target to evaluate cellular metabolism disorder and effect of medication after HIBD. |
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