| BackgroupWith the development of perinatal medicine and neonatal intensive care unit,thesurvival rate of the premature has been obviously improved,however, the prematurecerebral injury rate was also increased. With more and more attention on this problem,the pathogenesis of premature infant brain injury has become the hot topics in the field ofthe medicine Periventricular leukomalacia(PVL) is the most common type of prematurebrain injury,which can cause spastic cerebral palsy,cognitive and behavioral abnormalitiesand other sequelae.Erythropoietin(EPO) as a hematopoietic growth factor also haveneuroprotective effect on the premature brain injury,in addition to the treatment of anemiain premature. Therefore, the research aims to give EPO to intervene on the basic ofestablishing the model of PVL though the3-day-old SD rats with unilateral carotid arteryligation and hypoxia, from pathology、 glial fibrillary acidic protein (GFAP)immunohistochemistry and long-term neurobehavioral tests and Y-maze test to detect ratnerve motor function and learning and memory abilities in the case in order to explore theneuroprotective effect of EPO on the rats with PVL.ObjectiveThrough the establishing of the model of3-day-old SD rats with PVL to explore the neuroprotection effect of erythropoietin(EPO)on3day-old neonatal rats with periven-tricular leukomalacia, and provide evidence of early treatment on preterm infants.Methods150three day-old Sprague-Dawley(SD)rats were randomly divided into the controlgroup and experimental groups.The experimental groups were divided into experimentalgroup1(EPO treatment group) and experimental group2(PVL group). There were noligations of carotid artery (CA) and hypoxia but only isolation of CA in controlgroup.Ratswere subjected to left carotid ligation (LCL)first, followed by exposure to hypoxiaenvironment(6%oxygen and94%nitrogen)for2.5hours in the experimental groups.EPO5000IU/Kg was injected by intra-peritoneal injection immediately in experimental group1(EPO treatment group) after modeling. The same volume of physiological saline wasinjected in experimental group2(PVL group) at the same time.Weight of brain,percentage of brain damage quality changes of rats were recorded at3d、7d、14d and21dafter operation. The histological(HEstaining)and immunohistochemistry methods wereused to determine the pathological changes and the GFAP expression levels in the brain.At30days,the rest of rats were tested neurobehavioral tests (hanging test、open field test)and Y-maze test to detect rat nerve motor function and learning and memory abilitiesResults1. Behavior:The control group rats had no obvious behavior abnormal, both the PVLand EPO group rats were abnormal behavior change after ischemia and hypoxia. The EPOrats mainly were lethargy, irritable easily, some rats were crawling or could not stand toone side, the feeding and activity were slightly reduced with uncoordinated action, thePVL rats behaved not only lethargy, irritable extremely, most of the rats were crawling orcould not stand to one side,the feeding and activity were obviously reduced, but alsosome of their bodies were cyanosis visible, restless, twitching limbs, and even death.2.The weight gain:The weight gain of rats in the PVL group were obviously fallbehind the EPO group and control group at3d、7d and14d after operation,the differencewere significant [3d:(9.8±0.62)vs(11.7±0.80)、(13.8±0.41),7d:(12.1±0.46) vs(16.1±0.62)、(18.2±0.53),14d:(23.5±0.89)vs(28.4±0.62)、(33.8±0.71),P<0.05]; 3. The brain weight/the body weight:The brain weight/the body weight in the ratswith PVL and EPO group were larger than in the control group at1d after operation, thedifference were significant [(5.37±0.38),(3.83±0.22)vs(2.92±0.32),P<0.01], however, thebody weight/the brain weight in the rats with PVL and EPO group were smaller than inthe control group at21d after operation, the difference were significant[(2.61±0.11),(2.81±0.07) vs (3.70±0.05),P<0.01];4.The percentage of brain damage quality:The percentage of brain damagequality changes in the EPO and PVL group were increased with the day went, which hadsignificantly increase compared to the control group at1d,7d and21d after operation[1d:(27.74±7.25),(13.36±5.40)vs(0.29±0.47),7d:(37.65±8.12),(25.32±6.51)vs(0.37±0.17),21d:(40.95±11.34),(34.38±10.37)vs (0.18±0.08),P<0.05];5.HE staining:HE staining in the control group was normal, in EPO group thestructure of callosum was roughly normally at7d post-operation and lateral ventriclesbecame expanded mildly at21d post-operation．However，the structure of callosum wasloose and lateral ventricle dilation was obvious at the same sampling point in PVL group;6.Immunohistochemistry:The expression of GFAP in PVL group were increased at3d post-operation(P<0.05).The expression of GFAP in EPO group and PVL groupincreased obviously compared to control group after7d modeling,especially PVLgroup(P<0.05).The expression of GFAP in both EPO and PVL group slightly fall but stillmore than control group(P<0.05)，and PVL group was higher than EPO group(P<0.05)after14d modeling. At21d post-operation,the expression of GFAP in each group dropbasically the normal level basically;7.Neurobehavioral tests:In the hanging test of neurobehavioral tests the PVL grouprats were unresponsive, there was little physical activity and can easily fell from the glassrod. The hanging test scores of PVL group and EPO group rats were lower than thecontrol group rats, there was significant difference [(1.00±0.76),(2.38±0.91) vs (3.87±0.83),P<0.05], In the open-field test,the majority of rats in the PVL group showed belowmovement and lower number of through the grid, the scores were lower than the controlgroup and the EPO group and there was statistically significant[(5.00±1.20) vs (7.37±0.92),(10.12±1.24), P <0.05]; 8. Y-maze test:In the Y-maze test the required training times in the PVL group andEPO group were more compared to the control group，and there was significantdifference [(31.25±2.60),(25.1±1.89) vs (19.63±3.38), P <0.05],the memory retentionafter24hours in the PVL group were lower than the EPO group and control group, thedifference was statistically significant [(43.0±5.65) vs (56.4±9.18),(75.4±5.51), P<0.05], with the PVL group was minimum.Conclusion1. The eatablishment of PVL model by LCL-hypoxia in3day-old SD rats wassuccessful;2. GFAP positive cells were increased when happened PVL, they came to appearat3d post-operation and reach peak at7d post-operation,at14d post-operation they came todrop,the expression of GFAP droped basically the normal level at21d post-operation;3.The expression of GFAP increased after white matter damage.EPO treatment cansignificantly reduce the expression of GFAP positive cells after white matter damage，possibly play an neuroprotective role on promoting nerve repair．4. Voluntary movement dysfunction and abnormal emotional ability appears in ratswith PVL, the learning and memory function was also reduced in rats with PVL, EPO canimprove long-term neurobehavioral abnormalities. |
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