| Objectives:Brain injury in premature infants, particularly infants with very low birth weight (VLBW), is of enormous public health importance because of the large number of infants that survive with serious neurodevelopment disabilities, including major cognitive and behavioral defects. Periventricular white matter damage (PWMD), is the dominant neurological lesion in preterm infants who survive brain injury. And the enormous burden of neurological disability is expected to continue. Prevention of the brain injury underlying PWMD is urgently needed. However, no specific therapy for PWMD is presently available.Recombinant human erythropoietin (rh-EPO) has been used to treat anemia in premature infants for almost 20 years. Recently, it has been reported that erythropoietin (EPO) has many unexpected non-hematopoietic functions, including neuroprotection and etc. Studies observed the effect of rh-EPO on protecting of brain injured animal model following peripheral administration. These results implied that rh-EPO can penetrate the blood brain barrier to protect injured brain. At present, studies on the neuroprotective effect of rh-EPO are underway. However, the precise mechanism of this association remains to be determined. Especially, the relationship to PWMD is unknown.Therefore, the aim of this study was to make a neonatal rat model of PWMD and administrate rh-EPO on the rat to observe its protection and explore its mechanism on PWMD.Methods:All of postnatal day 3 rats were randomized into three groups:the sham group (without hypoxia-ischemia [HI]), the HI group (HI with saline administration) and the EPO group (HI with rh-EPO administration). Rat pups underwent permanent ligation of the right common carotid artery followed by 6% O2 for 4 h or sham operation and normoxic exposure. Immediately after the HI, rats received a single intraperitoneal injection of rh-EPO (5U/g) or saline.First, the development of rats was observd. Rats were sacrificed when they were 14 days old and adopt HE staining to observe the pathologic change of the brain. Functional outcomes were tested at postnatal day 30 (hanging test, open field test, rejection reaction test, cylinder rearing test) and postnatal day 90 (Y-maze test). Each subgroup had at least 6 rat pups.Second, rats were sacrificed when they were 5,7,10 and 14 days old and brains were obtained. Eythropoietin receptor and neuroglobin were tested by immunohistochemistry, western blotting or real-time fluorescence quantitative PCR (qRT-PCR). Each subgroup had at least 6 rat pups.Third, rats were sacrificed when they were 5,7,10 and 14 days old and brains were obtained. Angiogenesis-related cells (CD34+ cells), anglogenic factors (VEGF and Ang-1) and arteriovenous related genes (ephrinB2 and EphB4) were examined by immunohistochemistry, western blotting or real-time fluorescence quantitative PCR (qRT-PCR). Each subgroup had at least 6 rat pups.Results:1. During the experiment, two rats from the sham group died (2.5%), fifteen rats from the HI group died (16.12%), and seven rats from the EPO treated group died (8.23%). The mortality rates among the three groups are significantly different (P< 0.05). There is no difference of rats’weight among three groups (F=0.448, P= 0.647) before operation For HI rats, the weight gain was less than sham rats after damage. For rh-EPO treated rats, the weight gain was also less than sham rats, but more than HI rats. 2. In the right brain, we find that the white matter under the cortex and corpus callosum exhibit serious leukoaraiosis, the ventricles appeare to expand, and disordered arrangement of neurons or apoptosis are present in the gray matter. For rh-EPO treated rats, they show lighten damage.3. Functional outcomes appeare significant difference among three groups (hanging test, open field test, rejection reaction test and Y-maze test). The scores of HI rats are less than sham rats and rh-EPO treated rats are more than HI rats (P< 0.05). Sham rats show symmetrical use of both forepaws. HI rats show a marked preference for the use of the non-impaired forepaw, however, treatment with rh-EPO reduce this asymmetry (cylinder rearing test).4. EPOR presents a low level in the sham rats group. For the HI rats, the level of EPOR initially increas to a greater level than that of the sham rats (P5), and maintains at a higher level (P7, P10 and P14). EPOR in the rh-EPO administrated rats increas greater than that of HI rats at P5 (P< 0.05), however decrease sharply (compared with sham rats, P< 0.05; compared with HI rats, P> 0.05)5. Ngb slowly increases with age in the sham rats group. For the HI rats, Ngb increase greater than that of the sham rats from P5 to P14 (P< 0.05). Ngb in the rh-EPO administrated rats increase greater than that of HI rats from P5 to P10. However, it decrease at P14 (compare with HI rats, P> 0.05; compare with sham rats, P< 0.05).6. CD34+ cells gradually increase with age in the sham rats group. For the HI rats, the level of CD34+ cells initially increase to a greater number than that of the sham rats (P5 and P7), and then decrease to fewer than that of the sham rats (P10 and P14). CD34+ cells in the rh-EPO administrated rat follow the same pattern, but are greater than that of the HI rats. The level of CD34+ cells among the three groups are significantly diffierent (P<0.05).7. Both VEGF and Ang-1 mRNA levels increase in a time-dependent manner from P5 and peaked at around P10, then decrease slightly by P14. The VEGF mRNA was elevated in the HI rats compared to the sham rats at P5, P7 and P10. There is a significant increase in VEGF mRNA levels in the rh-EPO treated rats compare with the HI rats at P7, P10 and P14 (P< 0.05). HI causes an increase in Ang-1 mRNA levels at P7 and P10, compare with sham controls. Ang-1 mRNA levels are increased at all time points (P5, P7, P10 and P14) in rh-EPO treated rats compare with HI rats (P< 0.05).8. The mRNA of both EphrinB2 and EphB4 gradually increase with age and the ratio of EphrinB2/EphB4 is approximately 0.5 in the sham rats group. For HI rats, the mRNA of both EphrinB2 and EphB4 don’t increase greater than sham rats at P5 (P> 0.05). However, they increase rapidly greater than sham rats at P7, and the mRNA level of EphrinB2 is more than EphB4. After that, they decrease lower than Sham rats (P10 and P14) and the ratio of EphrinB2/EphB4 is more than 0.5. The mRNA of both EphrinB2 and EphB4 in the rh-EPO treated rats increase greater than that of HI rats (P< 0.05) and the ratio of EphrinB2/EphB4 is 0.49 (P5). And they peak at P7. After that, they decrease, however they still higher than HI rats. And the mRNA level of EphrinB2 is more than EphB4.Conclusions:1. Although cerebral hypoxia-ischemia causes white matter damage, a regenerative response is also triggered in the early stage around the ischemic core area. EPOR increases in a high level, which offers more receptors for EPO. Ngb augments to provide more oxygen. Furthermore, there is angiogenesis around damage areas, such as EPCs aggregates, angiogenic factors (VEGF and Ang-1) and microvessel increase.2. There is the powerful therapeutic potential of rh-EPO treatment of PWMD. Rh-EPO can lighten the damage of the brain and improve the outcomes of rats. It can increase the level of EPOR in order to provide more receptors of EPO to protect the injured brain.3. When EPO combin to it’s receptor, it can increase the level of Ngb which could provide more oxygen to the brain, and it can augument the angiogenesis response which could provide more blood flow to brain tissue. |
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