Kai Craenen Arenberg Midterm Report: The effects of ionizing radiation and folic acid on neural tube closure, eye development and cognitive functions in mice

Over the past decades, research on the effects of radiation on biological specimens (cells and animal models) has enhanced our knowledge on a number of industrial, clinical and research-oriented applications. Besides the clear advantageous socio-economical impact of ionizing radiation in the medical sector, it is important to note the related detrimental health effects as well. Next to its well-known role in cancer induction, a number of studies have shown non-cancer effects after irradiation, such as cardiovascular and cognitive detriment. For instance, irradiation of the embryo during early development (e.g. neurulation) can have a negative impact on proper development and thus lead to so-called birth-defects or congenital abnormalities, which are often associated with structural and functional aspects of the central nervous system (CNS)1–8. This PhD project will focus specifically on rostrally oriented neural tube defects (NTDs), eye defects (EDs) and abnormal behavior/vision in mice resulting from prenatal radiation exposure. After the Chernobyl accident in 1986, large quantities of the radioactive isotope Cesium-137 were deposited throughout Ukraine with higher amounts found in the northern half of the country. The years following the disaster, an increase in NTD and ED occurrence was observed in a number of contaminated regions. It was suggested that this phenomenon originated from extensive maternal Cesium-137 exposure5. However, it should be noted that no clear-cut NTD increase was observed in western Europe the years following the disaster9,10. So far, increased occurrence of malformations and sub-optimal cognitive functioning in descendants exposed in utero to radiation are often reported in the literature about the Hiroshima and Nagasaki A-bombings11. Thus, suggesting a causative role of ionizing radiation in the increased prevalence of NTDs, EDs and cognitive malfunctioning. Currently, an important issue is the exposure of women during the earliest stages of pregnancy to ionizing radiation in the context of diagnostic or interventional procedures. The possibility of unexpected pregnancy between radiotherapy sessions and subsequent exposure of the embryo to low doses of radiation is a matter of concern. It would be of interest to assess exactly how prenatal ionizing radiation exposure may induce these congenital abnormalities. The discovery of afflicted cellular mechanisms may furthermore lead to the use of new compounds for the prevention and treatment of these defects. Folic acid (FA) and folates have a known track-record in ameliorating birth defect prevalence: it is thus often advised for pregnant women to consume additional FA during gestation. Although FA food fortification is not common practice in any of the EU member states, the consumption of food supplements containing the compound by pregnant women has been advised by various EU governments in the past. Important to note is that a number of previous experimental findings have suggested a positive effect of FA on cognitive functioning if administered postnatally12–16. Furthermore, a link between abnormal folic acid metabolism and irradiation events has been reported in the past17–19. It is then imperative to determine the efficacy of postnatal FA administration in improving the standard of life after prenatal exposure to radiation. Aim 1.1.: Mechanisms of prenatal radiation-induced NTDs and EDs In the first aim, the development of NTDs and EDs is assessed after prenatal irradiation at embryonic day (E)7.5, using different doses of X-rays (up to 1.0 Gy). To this end, (immuno)histochemistry (IHC) and subsequent morphometric analyses are being performed at different developmental stages following radiation exposure. In addition, to unravel underlying mechanisms responsible for radiation-induced NTDs and EDs, a series of biotechnological techniques such as microarray, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) are being employed. Aim 1.2.: Assessing the efficacy of FA in ameliorating radiation-induced prenatal anomalies A possible link between prenatal irradiation and FA activity is being evaluated. FA will be administered during pregnancy and possible amelioration in radiation-induced anomalies at the height of the rostral NT and embryonic eye will be assessed. Custom made food is to be used as the vehicle to supply the animals with high FA intake. Initially, the FA content of the food has been validated with LCMS both directly by the manufacturer and indirectly, using LCMS of plasma/milk/brain samples of fortified pregnant mice. In addition, the impact of irradiation at E7.5 on the plasma content of various FA metabolites will be assessed, to better understand a possible link between X-irradiation and folate bioavailability. After food validation, a macroscopic study will be initiated where the prevalence of NTDs and EDs will be assessed in FA fortified pregnant females. Aim 2.1.: Role of prenatal ionizing radiation in the development of postnatal persistent defects The second aim will shift the focus towards postnatal anomalies and adult behavioral defects as a result of early prenatal exposure to radiation. Analyses will be performed both at the behavioral and morphological level. Animals irradiated at E7.5 will be subjected to an array of visual and behavioral tests to assess both visual acuity as well as cognition, learning and memory. The visual acuity tests will be performed first, to assess differences in visual ability that might be due to an abnormal, radiation-induced, eye development. In addition, Magnetic Resonance Imaging (MRI) and IHC will be employed to determine detailed eye and brain morphology, whereas optical coherence tomography (OCT) will be employed to assess the structure of the retina. Aim 2.2.: Assessing the efficacy of FA in ameliorating radiation-induced prenatal anomalies Finally, FA will be administered pre- and/or postnatally to assess its efficacy in counteracting radiation-induced anomalous adult eye and brain morphology and a possible behavioral decline. To keep in mind the prenatal experiments, FA will be implemented in the experimental design only if its beneficial role in preventing X-ray induced congenital defects has been established (see Aim 1.2.).