| Background With the gradual improvement of perinatal medicine, the successes of treatment of premature, low birth weight infants, and multiple fetuses have been significantly improved. However, in the long run, the problems of the neural development at different degrees do not fall obviously, especially preterm infants with brain damage caused by periventricular white matter softening (PVL), attracted many scholars’ attention home and abroad. Myelin phosphatide Protein(MBP) is a immunohistochemical marker of mature oligodendrocyte, constitute the main components of the Myelin sheath, detection MBP can evaluate neural information conveyance function.Erythropoietin (EPO) is mainly used for prevention and treatment of anemia in the early stage. But currently, a large number of experimental studies show that erythropoietin (EPO) has protective effects on the central nervous system (CNS) injury. However, its working mechanism, the appropriate time of utilization, and the precise dosage are ambiguous for doctors. Recombinant human erythropoietin (rhEPO) is a kind of gene by recombinant DNA technology synthesis with natural EPO with exactly the same amino acid sequence, and the biological activity of protein is similar, in1989, which was approved by the United States Food and Drug Administration (FDA) and was widely used in clinical, showed good curative effects on experimental animals, and displayed a promising application prospect in pediatrics clinics.Objective Through the intraperitoneal injection of different doses of rhEPO to neonatal rats with periventricular white matter softening, we want to get a knowledge of the neuronal protection mechanism of rhEPO on PVL, the optimal dosage, and precise time in application, which will provide the basis for the intervention and treatment of preterm infants with PVL in the early clinical stage.Method3day old neonatal SD rats were selected for180pieces, and were randomly divided into sham operation group and control group, rhEPOS group, rhEPOM group, rhEPOH group, each group has36rats. Sham operation group separation of right common carotid artery, but only the operation line through, from the common carotid artery ligation under not, will also not hypoxia treatment.by hypoxia ischemia(HI), animal model making similar premature infants with periventricular white matter softening damage. rhEPOS group, rhEPOM group, rhEPOH group, ischemia and hypoxia in the later were respectively given rhEPO1000U/kg,rhEPO3000U/kg,rhEPO5000U/kg intraperitoneal injection.sham operation group, control group at the same time point were given injections of normal saline. Animal were killed after modeling success, a part of the brain tissues were embedded in paraffin, the preservation of the other part of the brain tissue in liquid nitrogen. By immunohistochemistry and Real-time fluorescence quantitative PCR technology to detect the expression of EPO,EPOR, MBP in protein level and gene level at different time points, explore the brain protective effect of law. SPSS17.0for statistical analysis, P<0.05had significant difference.Results1. Behavioral changes:The experimental group of rats appeared cyanosis, poor response, frequenttic, incontinence, etc in the process of ischemia hypoxia, and part of the animals were died during the process of hypoxic ischemia(HI). The experimental group rats after HI showed less to suck breast, poor response, uncoordinated movement, etc. Sham-operation group of rats showed good activities for infants, powerful, spiritual good response, and movement coordination. With the increase of EPO dosage, the animal performance was close to the sham operation group.2.Weight changes:After HI, newborn rat lose weight, and weight gains slowly.With the increasing of rhEPO dosage, the growth rate of the animal’s weight is close to the normal growth rate, each using single factor analysis of variance was statistically significant (P<0.05).3.Brain tissue pathological changes:In the control group after making the model appeared that disordered arrangement of cells in the areas of brain white matter, a osteoporosis necroticareas, thinning of the corpus callosum, and ventricle enlarges significantly. In the sham operation group, cerebral white matter cells arranged in order and are distinct, the nuclei are round, light pollution, left and right lateral ventricle are in symmetry. In rhEPOS, rhEPOM, rhEPOH group with the increase of rhEPO content, brain tissues were more close to the pseudo operation group performance.4. Immunohistochemical results and Real-Time PCR results:mRNA and protein expression of EPO and EPOR in normal brain tissue is in low quantity. EPOmRNA and protein expression in the control group afterl2h rises obviously,36h declines significantly; after giving rhEPO, declining rate of EPOmRNA and protein is slowing. the greater the rhEPO dosage is, the slower decline rate is. EPORmRNA and protein increased obviously after hypoxic hypoxia, up to peak after12h, with the prolongation of time, it decreased slowly,10days after birth still presents high expression; rhEPO group compared with the control group, no significant changes in expression of EPORmRNA and protein by rhEPO injection. MBPmRNA and protein in control group of brain tissue14days after birth,28days after birth time point showed low expression level, low growth.with the increase of the amount of injected rhEPO, while MBPmRNA and protein increases gradually.Compared with each time point, it has statistical significance (P<0.05).5. Correlation Analysis:Through the analysis of Spearman rank correlation, the expression of both EPO mRNA and EPO protein showed that the two had positive correlations (r=0.881, P<0.05); through the analysis of correlation between EPOR mRNA and EPOR protein, the expression showed that two had positive correlations (r=0.854, P<0.05); through the analysis of correlation between MBP mRNA and EPOR, expression of protein also showed positive correlations (r=0.891,P<0.05).Conclusions1. Recombinant human erythropoietin(rhEPO) has neuroprotective effect on PVL and mechanism of action may be by adjusting the EPO/EPOR system.2. Through the comparison of MBP, Studies have confirmed rhEPO treatment of PVL rats,the neuroprotective effects of using rhEPO in large dose (5000IU/Kg) is better than in middle doses(3000IU/Kg) or small doses (1000IU/Kg). |
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