The role of recombinant human erythropoietin (rhEPO) and its analogues as agents that prevent cell injury from various stresses and also promote repair after injury has not been fully defined. In particular, the mechanisms to protect the developing brain from hypoxic-ischaemic injury need further definition, especially around 24-28 weeks gestation when white cell precursors are at particular risk. The central hypotheses of this project were that: (1) in a rat model representing the 24-28 week old neonatal human brain, apoptosis and autophagy will play critical roles in determining brain damage after hypoxic injury; (2) the delivery of supraphysiological doses of rhEPO or its non-erythropoietic analogue carbamylated EPO (CEPO) will protect the brain by minimising the effects of apoptosis and autophagy; (3) mechanisms involved in the cytoprotective nature of rhEPO and CEPO are activated signalling pathways that will improve tissue remodelling of healthy oligodendrocytes and astrocytes, and maintenance of a healthy neurons and vasculature; and (4) cytoprotection by rhEPO of the fetal or neonatal brain from hypoxia-ischaemia-induced injury will be further improved by rhEPO pre-treatment strategies to mothers prior to delivery.
The first original research Chapter 3 investigated the benefits of rhEPO in pre-term neonates when their brains are at a particularly vulnerable stage of development. To do this, an in vivo model of hypoxia-injured neonatal brain was used. Quantification of apoptosis was used to measure the level of brain injury, with and without rhEPO. Assessment of white matter injury and expression of glial cell markers (for oligodendrocytes and astrocytes) and neuronal markers was conducted. In this study, mild hypoxia caused a significant increase in apoptosis in the neonatal brain, which then was ameliorated by rhEPO delivered prior to the hypoxic insult. There was also stimulation of glial cells with rhEPO delivery as well as the stimulation of a glial cell developmental protein, nestin, in white matter. The signaling pathways for possible rhEPO protective mechanisms were also investigated. This work demonstrated the neuroprotective benefit of treatment with rhEPO in hypoxia-injured neonatal brain.
In Chapter 4, the development of autophagy in the hypoxia-injured neonatal brain was investigated, using the model developed in Chapter 3. One of the autophagy-related-genes, LC3-II, was used as an autophagy cell marker. Immunohistochemistry and histochemistry were used to assess the extent of the autophagy in this model and to see whether rhEPO would affect autophagy or autophagic cell death in the neonatal brain after hypoxic insult. The results indicate that autophagy is increased by hypoxic injury and rhEPO minimises this effect.
In Chapter 5, one of the EPO analogues, CEPO, was investigated to see whether it had a similar cytoprotective effect as rhEPO in the hypoxia-injured neonatal brain, especially by preventing apoptosis. Using the same model as used in Chapter 3, CEPO did not decrease apoptosis at 4 h of hypoxic treatment. CEPO also did not promote the expression of the glial cell markers. This may be due to the short time frame of 4 h which might not be long enough for CEPO to be taken up into cells. In addition, from other research papers, the dose of CEPO used in this investigation (25μg/kg) may be too low. Further definition of the model and analysis are needed in the future.
In Chapter 6, the cytoprotective ability of rhEPO delivered to mothers in utero for fetuses with hypoxic-ischaemic challenge was investigated. To do this, bilateral uterine artery ligation of the pregnant rodents was carried out 2 days prior to term delivery, with and without daily delivery of rhEPO to mothers. The fetuses were taken by Caesarean section at term, and some were again exposed to hypoxia with and without rhEPO, thereby mimicking the further hypoxic stress to newborns. Quantification of apoptosis was conducted to measure the damage to the immature brain. EPO signalling pathways and expression of markers for glial and neuronal cells were analysed to see any changes throughout the experiment. Delivery of rhEPO to pregnant rats when fetuses were under hypoxic-ischaemic stress decreased apoptosis in the developing brain, but not to a significant amount. When the neonates, that already had hypoxic-ischaemic stress in utero, had to face the additional problem of oxygen restriction after birth, apoptosis was further increased. rhEPO delivery post-birth did not have any great positive effects on the injury, but neonatal response variation, perhaps induced by the previous hypoxia-ischaemia in utero, certainly affected the outcome. This study needs refining and repeating in the future.
In summary (Chapter 7), this project established that rhEPO, but not CEPO, may be useful in protecting key structural, neuronal and vascular support cells in the hypoxia-injured neonatal brain. The project also provided novel insight into molecular mechanisms of cytoprotection by rhEPO. The results of these experiments may pave the way for human trials of rhEPO to improve outcome in disease resulting from the hypoxic or ischaemic stressors in the brain.