Effect Of Erythropoietin On The Ability Of Learning And Memory In2-day-old Immature Rats With Cerebral White Matter Damage

Objective：To establish a neonatal rat model of premature infant brain white matter damage andclarify erythropoietin on the protective effect of cerebral white matter damage inpremature infants.Methods：1.experimental animalsNew2nd age (counted as0days old on the day of birth) in SD rats with clean108, maleand female halves, provided by the laboratory animal Centre in Anhui MedicalUniversity.2.The model preparation and grouping2-day-old immature neonatal rats with anhydrous ether inhalation anesthesia, ligation ofbilateral common carotid arteries (Bilateral carotid artery occlusion, BCAO). Wererandomly divided into BCAO model group (BCAO, n=36) and EPO treatment group(EPO, n=36). In another2-day-old newborn SD rats as normal control group, shamoperation group (sham, n=36), anesthetized and separation of the bilateral commoncarotid artery ligation. EPO in common carotid artery ligation treatment groupimmediately after intraperitoneal injection of EPO (diluted with saline concentration500u/ml)5000u/Kg, surgical group, BCAO group, injection of the same amount of saline solution (0.1ml/10G). Postoperative recovery1h and returned to the mothersquirrel cage kept on37°C incubator. Three groups of rats at postoperative24h,48h,72h and7d,14d,35d executed each time point (n=6).3.brain tissue pathological detectionPreparation of brain after weight measuring, immediately to the brain in4%paraformaldehyde fixed48h, at room temperature, the conventional dehydration,embedded in paraffin, select the first joint level, lateral ventricle planar continuouscoronary slice, slice thickness4um, every interval of10and1, for the same levelsections were compared analysis. HE staining was observed under the light microscope,pathological change; microscope connected to the multimedia color pathological imageanalysis system, through the anterior commissure and the lateral ventricle on the planeof the lateral ventricle area measurement.4. immunohistochemical staining methodUsing immunohistochemical staining method for the measurement of brain tissue MBPimmunohistochemical staining integral optical density: using image analysis system, theanterior fontanelle planar brain slice (x10) from the corpus callosum middle part ofboth sides of the cingulate and internal capsule, a total of five perspective view, it takethe average.5.determination of body weight and brain weightThree groups of rats were executed in corresponding time before measurement weight.Then ether anesthesia, local disinfection after open abdominal skin, followed by openabdominal, thoracic, diaphragm, exposure of the heart, the left ventricle with4%paraformaldehyde+0.1ml/LPBS perfusion to the outflow of liquid crystal.Determinations and brain, measuring brain weight. 6. brain water content determinationEach group with72h after operation (4each), after ether anesthesia cardiac blood,craniotomy and brain, resection of olfactory bulb and hindbrain, with longitudinalcerebral fissure and optic chiasma midpoint is a mark, left and right brain left, electronicbalance weighing wet weight, the70degree C drying box full dehydration of72h, thenget dry weight, the calculated cerebral water content, cerebral water content of formula:=(wet weight--dry weight/wet weight) of X100%.7.Determination of behavior35d after hypoxic-ischemia, groups of experimental animals, respectively, platform tests,Morris water maze test and closed dark test.8.statistical analysisAll data by mean±SD (x±s) said. Using SPSS17.0for Windows software was used forstatistical analysis, among the two groups were compared with t test, multiple groupwere compared with single factor analysis of variance, group two two compared with Qtest, P <0.05was considered statistically significant.Results：model group bad rat body weight and brain weight growth, for each time point modelrat body weight and control groups and Sham operation, so there are significantdifferences (P<0.05); for each time point sham operation, so weight and control groupsof rats were no significant differences (P>0.05).②model of water content in braintissue than normal control groups and significant increase in the surgery Group (p<0.05).③The HE dyeing model group big mouse appears around the ventricles of the brain thewhite matter damage, under the light microscope displays for the ventricles of the brain around the white matter cell swelling, pouch necrosis, the organization is loose; Thenormal control group and the sham-operation group brain organization has not seenexceptionally.④After immunity group dyeing technique24h,48h,72h three group ofbig mouse average integral light density difference non-significance significance(P>0.05); After the technique7d the model group big mouse MBP integral light densityand the control group and the treatment group quite is even reduces obviously, thedifference has the significance significance (P<0.05); The control group and thesham-operation group compares the difference non-significance significance mutually(P>0.05); After technique when14d,35d the model group and the control group andbetween the treatment group’s difference is more remarkable.⑤35days of age, modelgroups reduced locomotor activity in rats, slow to react flexibility reduces, Morris watermaze test and closed dark test, platform test results were worse than the control groupand surgical group, differences of significance (P<0.05).Conclusion：Successful establishment of animal model of cerebral white matter damage in prematureinfants, EPO in immature brain injury in neonatal rats can promote the expression ofMBP, also improves cognition, learning ability, for clinical application of EPO treatmentof brain injury in preterm infants provide a experimental basis.