Expression And Significance Of FP1 In Rat Models And Cell Models With Hypoxic-ischemic Periventricular Leukomalacia

ObjectivesTo establish hypoxic-ischemic PVL animal model of the 4-day-old SD rat and evaluate it by methods of morphology, molecular biology and behaviouristics.Methods1. The 4-day-old SD rats were randomly divided into a control group and a PVL group according to a random number table. The control group was subjected to sham surgeries and was exposed normoxic air during the experimental period. The PVL group was subjected to bilateral carotid ligation and then they were put into a box filled with 8% oxygen and 92% nitrogen for 30 minutes.2. HE staining was used to observe the brain pathological changes. Immunofluorescence staining and Western blot were used to investigate the distributions and expressions of APP and MBP for 12 weeks after HI.3. Examination of weight was started before HI and was carried out daily until 7 weeks after HI. The sensorimotor functions were inspected by sensation reflex, hanging test, cliff escaping test and inclined place test for 12 weeks after HI. Morris Water Maze Test was used to oberve the spacial and memory cognition of rats.Results1. The survival rate of control group was 100%, while that of the PVL group was 89.2%. About 10% of pups in PVL group had eye unopened or cataract.2. HE staining showed edema, rarefaction, gliosis and malacia lesion in the periventricular white matter. There existed light injury in the cortex and hippocampus.3. The expression of MBP was significantly decreased while the APP was dramatically increased in the PVL group compared with the control group(P<0.01).4. The weight of PVL group was lighter than that of the control group from 1 day to 7 weeks after HI(P<0.01).5. The mean time of eye opening in the PVL group (21±2.0d ) was significantly delayed compared with the control group(13.33±0.58d) (P<0.01).6. The mean appearing time of auricle reflex in the PVL group (20.67±1.53d ) was longer than that of the control group(15.33±0.58d) (P<0.01).7. The motor tests in PVL group showed the shorter hanging time in hanging test, the weaker ability of cliff escaping and the longer turning-round in inclined place test than that in the control group at 4weeks, 8weeks and 12weeks after HI(P<0.05).8. The Morris water maze test showed that the rats of PVL group had spatial and memory cognition defect through the swimming curve, average latency to target, average swimming speed, target crossings and residence time in the third quadrant at 4weeks, 8weeks and 12weeks after HI(P<0.05).Conclusions1. The rats of PVL group showed periventricular white matter rarefaction and malacia formation. They also had axon damage and myelin dysfunction. There exited light injury in the cortex and hippocampus.2. The rats of PVL group had growth and development dysfunction. They also had sensorimotor dysfunction, spatial and memory cognition defect at 4weeks, 8weeks and 12weeks after HI.3. The pathological and neurological changes of rats in PVL group were accorded with the characteristics of the PVL in infant.4. We can successfully establish the PVL animal model in the 4-day-old SD neonatal rat by the bilateral carotid ligation plus hypoxia. Objectives1. To determine the distribution and content of iron in different brain regions (cortex, hippocampus, periventricular white matter) within 4weeks after PVL.2. To determine the expression of FP1mRNA and protein in different brain regions (cortex, hippocampus, periventricular white matter) within 4weeks after PVL.3. To infer the significance of FP1 in the pathogenesis of PVL.Methods1. The 4-day-old SD rats were randomly divided into a control group and a PVL group. The control group was subjected to sham surgeries and was exposed normoxic air during the experimental period. The PVL group was subjected to bilateral carotid ligation and then they were put into a box filled with 8% oxygen and 92% nitrogen for 30 minutes.2. The distribution of iron in the different brain regions was detected by Prussian blue iron staining within 4 weeks after HI. The iron concentrations of the cortex, hippocampus and periventricular white matter in rats were measured by graphite stove atomic absorption spectrometry.3. The expression of FP1mRNA and protein in different brain regions was investigated by immunofluorescence histochemical staining, Real-time PCR and Western-blot methods within 4 weeks after HI.Results1. There was no difference in the iron positive granules at 1day and 3days after HI between the PVL group and control group. There was few iron positive granules in the cortex and hippocampus at 1 weeks after HI in the PVL group, while there were much more iron positive granules in the periventricular white matter. 2. The increased iron concentrations of cortex, hippocampus and periventricular white matter were 21.2%, 11.21% and 40.46% respectively in the PVL group at 1 week after HI compared with control group. The iron concentrations of cortex and periventricular white matter were still higher than that in control group at 4 weeks after HI. While the iron concentrations of hippocampus recovered to the level of control.3. Immunofluorescence staining showed that FP1 was expressed in the cell membranes, cytoplasms and dendrites of cortical pyramidal neurons. It also can be seen in the cytoplasms and processes of hippocampal neurons. There was many FP1 positive cell and fibers in the periventricular white matter.4. Compared with the control group, the expression of FP1 mRNA and protein decreased in all brain regions in the PVL group at 1 weeks after HI, especially in the periventricular white matter.Conclusions1. Brain iron metabolism changed in the rats of hypoxic-ischemic PVL. The iron concentration increased in the cortex, hippocampus and periventricular white matter, especially in the periventricular white matter.2. The expression of FP1 can be down regulated in the hypoxic-ischemic PVL. It decreased in the cortex, hippocampus and periventricular white matter.3. The downregulation of FP1 can be involved in the increase of iron concentration and deposition in the different 4-day-old SD rat brain regions after HI, which may be one of the pathogenesis of PVL. Objectives1. To obtain pure and primary cultured oligodendtocyte/type-2 astrocyte (O2A) progenitors from cortices of the 4-day-old neonatal SD rats.2. To determine the expression and function of FP1 on the primary cultured O2A progenitors.3. To establish the feasible hypoxic-ischemic PVL cell model in vitro.4. To ascertain the expression and significance of FP1 in the PVL cell model.Methods1. The O2A progenitors were isolated and primarily cultured from the cortices of the 4-day-old neonatal SD rats by twice shaking and using the conditioned medium. The morphologies of O2A were observed under inverted phase-contrast microscope. And the immunofluorescence cytochemical staining was used to identify and determine the purity of the cells.2. Double immunofluorescence cytochemical staining was used to investigate the expression of FP1 on the O2A progenitors and iron efflux test was used to explore the function of FP1.3. O2A progenitors were treated with Oxygen-glucose deprivation (OGD) for 3, 6, 12 and 24h to mimic hypoxic-ischemic PVL in vitro and their morphologies were observed under inverted phase-contrast microscope. The survival rate of O2A progenitors was investigated by CCK-8 method and lactate dehydrogenase (LDH) leakage test.4. The level of FP1 in the PVL cell model was tested by real time PCR and Western blot.Results1. O2A progenitors were round or oval with strong refraction under inverted phase-contrast microscope. They had monopolar, dipolar or tripolar processes, which were fine and straight without branches. O2A progenitors were A2B5 positive cells and their purity is up to 95%.2. Double immunofluorescence cytochemical staining showed that O2A progenitors can express FP1, which was located in the cell membrane, cytoplasm and processes.3. Iron efflux test showed that the immunofluorescence intensity increased with time-dependence in the routine cultured and OX42 antibody treated O2A progenitors(P>0.05).while the immunofluorescence intensity of FP1 antibody treated cells did not increase with time.4. After 3h, 6h, 12h and 24h incubation with OGD, the morphologies of O2A progenitors had hypoxic-ischemic changes, and the survival rate decreased and the LDH viability in the cultured medium increased with time-dependence. The survival rate was only 49.4% at 24h after OGD. 5. The level of FP1mRNA and protein decreased with time-dependence after 0h,3h, 6h and 12h incubation with OGD.Conclusions1. O2A progenitors with purity up to 95% can be obtained by twice shaking and using conditioned medium from the cortices of the 4-day-old neonatal SD rats.2. O2A progenitors can express FP1, which was located in the cell membrane, cytoplasm and processes.3. FP1 can induce the iron efflux from O2A progenitors and it is an important iron-regulated protein.4. The feasible hypoxic-ischemic PVL cell model in vitro can be successfully established by treating the O2A progenitors with 3h, 6h and12h incubation of OGD.5. The level of FP1 in the hypoxic-ischemic PVL cell model decreased with time-dependence, which may reduce the iron efflux from the cells and then aggravate the cell hypoxic-ischemic injury.