Effects of high-dose caffeine on the cerebrum of the immature ovine brain

Preterm birth is a worldwide clinical problem and despite advances in obstetric care, infants born preterm have higher rates of morbidity including cerebral palsy and neurodisability. Infants born very preterm (<34 weeks) are at an increased risk of developing apnea of prematurity (AOP) a condition of irregular/intermittent breathing which can contribute to unfavourable neurodevelopmental outcomes. Caffeine administration is one of the few treatments available for these infants, with proven efficacy. The current treatment involves a loading dose of 20mg/kg caffeine (citrate) followed by a daily maintenance dose of 5-10mg/kg and this dose of caffeine is associated with improved neurodevelopmental oucomes. Infants that do not respond to the current clinical dosing of caffeine may require higher doses. However, the effects of higher doses of caffeine on the developing brain have not been subject to rigorous neuropathological evaluation in a suitable animal model. Thus there is an urgent need to evaluate the safety of a high-dose caffeine treatment on the development of the immature brain. The global aim of this thesis has therefore been to investigate the effects of high-dose caffeine administration on the development of the immature brain. To do this, I have used an ovine model, where caffeine was administered to fetal sheep at a stage of brain development similar to that of a very preterm infant. In Chapter 3, the fetal physiological response to high-dose caffeine was assessed to determine if caffeine has a direct effect on the brain or an indirect effect through alterations in fetal physiology. As caffeine was administered in utero, assessment of maternal physiological response was necessary in order to determine if any changes in fetal physiology was a result of altered maternal status. The data presented in Chapter 3 demonstrated that the dosing regimen of caffeine base used (25 mg/kg loading; 20 mg/kg maintenance) produced high fetal plasma caffeine levels, which were comparable to plasma caffeine concentrations measured in preterm infants administered higher than standard doses of caffeine. Exposure to high-dose caffeine did not adversely affect fetal blood chemistry over the 15-day administration period, nor did it affect fetal growth. High-dose caffeine did lead to transient alterations in fetal cardiovascular physiology in the first 3 days of caffeine exposure. As we did not measure cardiovascular variables after this period it is unknown whether these transient alterations continued. We conclude that any observed neuropathological effects of caffeine are likely to be a direct, rather than an indirect action on the brain. Chapter 4 assessed the impact of high-dose caffeine on the developing white matter, a region of high vulnerability in the preterm infant. The results from this chapter suggested that daily administration of high-dose caffeine did not cause any structural alterations to the developing white matter, or alterations to associated glial cells, in the very immature ovine brain. We conclude that high-dose caffeine does not overtly injure the developing ovine white matter. The potential effect of caffeine on the grey matter was assessed in Chapter 5. Here high-dose caffeine was associated with increased Ctip2-positive subcerebral projection neurons, GFAP-positive astrocytes and Olig2-positive oligodendrocytes in the cortical grey matter; there was no effect on somatostatin-positive GABAergic interneurons. It is possible that high-dose caffeine may increase proliferation of these cells, however this requires further investigation. The findings from this chapter suggest that high-dose caffeine may have adverse consequences for the ovine cortical grey matter. Since subcerebral projection neurons are excitatory glutamatergic neurons in the cortex, an increase in this cell type with no alterations in inhibitory GABAergic neurons may lead to cerebral dysconnectivity and thus cognitive and/or motor deficits. Thus further investigation is required to determine if these changes persist in the long-term. In Chapter 6, the long-term effects of high-dose caffeine were investigated. The data from this chapter showed that although caffeine exposure did not compromise growth trajectory at 2 months after birth, it did decrease kidney weight by 14%. As nephrogenesis coincided with the period of caffeine administered, we conclude that early caffeine exposure may alter long-term kidney growth by increasing the functional demands of the kidney and subsequently disrupting developing nephrons. Thus the long-term renal effects of high-dose caffeine need to be assessed further. Prenatal high-dose caffeine exposure did not adversely affect white matter structure, detected using diffusion MRI at 2 months of postnatal age. Grey matter structure was not assessed in the present study, however, given the increase in neuronal and glial cell density described in Chapter 5, this region requires further investigation. In conclusion, the studies presented in this thesis increase our understanding of the effects of high-dose caffeine on neurodevelopment and provide substantial evidence that high-dose caffeine can affect fetal development. This thesis also provides a model that may help us better understand the effects of high-dose caffeine on the developing brain. As caffeine has been, and will remain, the treatment of choice for AOP, and infants who do not response to standard caffeine doses will continue to receive higher doses, we need to further investigate the neuropathological outcomes associated with caffeine use. The results from this thesis add to our understanding of the safety of high-dose caffeine. Studies must now focus on determining the maximal dose of caffeine that is not only effective for AOP, but also safe for developing and highly vulnerable preterm infants.