Folic Acid Fortification Prevents Morphological and Behavioral Consequences of X-Ray Exposure During Neurulation

Craenen, Kai; Verslegers, Mieke; Callaerts-Vegh, Zsuzsanna; Craeghs, Livine; Buset, Jasmine; Govaerts, Kristof; Neefs, Mieke; Gsell, Willy; Baatout, Sarah; D'Hooge, Rudi; Himmelreich, Uwe; Moons, Lieve; Benotmane, Mohammed Abderrafi

doi:10.3389/fnbeh.2020.609660

ORIGINAL RESEARCH article

Front. Behav. Neurosci., 08 January 2021
Sec. Pathological Conditions
Volume 14 - 2020 | https://doi.org/10.3389/fnbeh.2020.609660

Folic Acid Fortification Prevents Morphological and Behavioral Consequences of X-Ray Exposure During Neurulation

Kai Craenen^1,2^†

Mieke Verslegers¹^*^†

Zsuzsanna Callaerts-Vegh³^†

Livine Craeghs^1,2^†

Jasmine Buset¹

Kristof Govaerts⁴^†

Mieke Neefs¹

Willy Gsell⁴^†

Sarah Baatout¹^†

Rudi D'Hooge³^†

Uwe Himmelreich⁴^†

Lieve Moons^1,2^†

Mohammed Abderrafi Benotmane¹^†

¹Radiobiology Unit, Interdisciplinary Biosciences, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (Studiecentrum voor Kernenergie; Centre d'étude de l'énergie nucléaire), Mol, Belgium
²Laboratory of Neural Circuit Development and Regeneration, Animal Physiology and Neurobiology Section, Department of Biology, Faculty of Science, Katholieke Universiteit Leuven, Leuven, Belgium
³Laboratory of Biological Psychology, Faculty of Psychology and Educational Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
⁴Molecular Small Animal Imaging Center, Biomedical MRI Unit, Department of Imaging and Pathology, Katholieke Universiteit Leuven, Leuven, Belgium

Previous studies suggested a causal link between pre-natal exposure to ionizing radiation and birth defects such as microphthalmos and exencephaly. In mice, these defects arise primarily after high-dose X-irradiation during early neurulation. However, the impact of sublethal (low) X-ray doses during this early developmental time window on adult behavior and morphology of central nervous system structures is not known. In addition, the efficacy of folic acid (FA) in preventing radiation-induced birth defects and persistent radiation-induced anomalies has remained unexplored. To assess the efficacy of FA in preventing radiation-induced defects, pregnant C57BL6/J mice were X-irradiated at embryonic day (E)7.5 and were fed FA-fortified food. FA partially prevented radiation-induced (1.0 Gy) anophthalmos, exencephaly and gastroschisis at E18, and reduced the number of pre-natal deaths, fetal weight loss and defects in the cervical vertebrae resulting from irradiation. Furthermore, FA food fortification counteracted radiation-induced impairments in vision and olfaction, which were evidenced after exposure to doses ≥0.1 Gy. These findings coincided with the observation of a reduction in thickness of the retinal ganglion cell and nerve fiber layer, and a decreased axial length of the eye following exposure to 0.5 Gy. Finally, MRI studies revealed a volumetric decrease of the hippocampus, striatum, thalamus, midbrain and pons following 0.5 Gy irradiation, which could be partially ameliorated after FA food fortification. Altogether, our study is the first to offer detailed insights into the long-term consequences of X-ray exposure during neurulation, and supports the use of FA as a radioprotectant and antiteratogen to counter the detrimental effects of X-ray exposure during this crucial period of gestation.

Introduction

Exposure to ionizing radiation during embryonic development has been linked to an increased risk of birth defects. The type and severity of these defect are predominantly determined by the developmental stage during which exposure occurred (Craenen et al., 2017). Epidemiological studies on Ukrainian cohorts illustrated an increased prevalence of neural tube defects (NTDs) and eye defects (EDs) in regions severely contaminated with radioactive Cs-137 isotopes following the Chernobyl nuclear accident. Although there are no accurate dose estimates, uptake of radioactive isotopes is known to be particularly high in pregnant women living in these regions (Wertelecki et al., 2016). Initially, it was observed that the more recent Fukushima Daiichi nuclear power plant accident elicited no increase in birth defects and pre-natal mortality due to environmental radioisotope contamination (Fujimori et al., 2014), but subsequent papers debated this conclusion (Mangano and Sherman, 2015; Scherb et al., 2016). In contrast to these more recent observations, reports after the atomic bombings in Japan only mentioned an increased incidence of microcephaly and intellectual disability (Plummer, 1952; Neel and Schull, 1956). It is likely that the discrepancy in health effects of the nuclear accidents and atomic bombings stems from differences in dose, dose rate, exposure duration and radiation type. The above highlights the need to increase our knowledge about the effects of pre-natal irradiation on biological structures and functions.

Exposure to ionizing radiation during pregnancy most commonly occurs during clinical radiodiagnostic or therapeutic procedures (Mettler et al., 2009). Although medical practitioners advise against irradiation during pregnancy, it may be unavoidable in medical urgencies (Lazarus et al., 2009). In terms of radiation protection, conventional shielding methods are currently being used to partially mitigate the fetal dose (Chatterson et al., 2014; Moore et al., 2015; Owrangi et al., 2016). However, depending on the dose or the developmental stage during which exposure occurs, these conventional shielding strategies may not suffice. Animal studies have shown that the neurulation period in the early embryo is especially radiosensitive with regard to the pathogenesis of radiation-induced NTDs and ED (Russell, 1950, 1956; Di Majo et al., 1981; Heyer et al., 2000; Craenen et al., 2017, 2020b), but also in terms of cognitive disabilities and altered vision. Indeed, a decreased visual acuity in atomic-bomb survivors, irradiated in the first trimester, and born from mothers with acute radiation syndrome (≤ 2 km from hypocenter) has been reported (Burrow et al., 1964). Yet, most experimental work has focused on health risks after radiation exposure during neurogenesis, coinciding with the second trimester of human pregnancy (Plummer, 1952; Neel and Schull, 1956; Verreet et al., 2015, 2016a,b). Furthermore, there are currently no anti-teratogens or radio-protectants available to prevent (congenital) morphological and functional defects that arise from irradiation during brain development.

Folic acid (FA), a synthetic vitamin, is generally known to prevent NTDs [reviewed in Imbard et al. (2013)], in addition to other defects such as heart defects and some skeletal defects (Kappen, 2013). Besides, FA has been suggested to prevent the development of age-related neurodegenerative diseases and overall cognition (Craenen et al., 2020a). Several countries enforce staple food fortification, whereas others support FA supplementation during pregnancy (Imbard et al., 2013). Of note is that FA supplementation/fortification initiatives are currently lacking in high-risk areas, such as those severely contaminated with radioisotopes from the Chernobyl disaster. Although FA food fortification can prevent some defects such as NTDs, its efficacy depends on the causative teratogens or mutations. For example, BMS-189453 (a synthetic retinoid) causes anomalies such as NTDs and heart defects that can be prevented with FA fortification (Cipollone et al., 2009), whereas arsenate-induced NTDs do not appear to be responsive (Ferm and Hanlon, 1986). Interestingly, many of the hallmark consequences of ionizing radiation exposure, including oxidative stress, DNA damage, cell cycle arrest, cell death and epigenetic alterations, might be countered by FA (Heyer et al., 2000; Martin et al., 2014; Reisz et al., 2014).

This study is the first to offer an in-depth analysis of the morphological and behavioral consequences of irradiation during neurulation in mice. To this end, we used a multidisciplinary approach, including an extensive behavioral test battery and imaging techniques such as spectral domain optical coherence tomography (SD-OCT) and magnetic resonance imaging (MRI). In addition, we assessed the efficacy of FA food fortification in preventing fetal malformations as well as adult functional and morphological defects resulting from X-ray exposure.

Materials and Methods

Animals and FA Fortification

All animal experiments were conducted in line with the relevant guidelines and were approved by the Institutional Ethical Committees of SCK-CEN/VITO (ref. 02–012) and the Animal Welfare Committee of the KU Leuven University, and are in strict accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU). C57BL6/J mice (Janvier, Bio Services, The Netherlands) were housed in individually ventilated cages, under standard laboratory conditions (12-h light/dark cycle) and fed ad libitum. One week before coupling, animals designated for the macroscopic fetal study were placed on a control Teklad (Carfil Quality, Oud-Turnhout, Belgium) diet (3.5 mg/kg FA), a FA fortified diet (8 mg/kg FA) or an extra-FA fortified diet (12 mg/kg FA). The FA concentrations within the final customized food products were investigated in compliance with ISO 17025. We selected the dose of 8 mg/kg because it was observed that this is an effective concentration to achieve antiteratogenic effects in mice (Gray and Ross, 2009; Harris, 2009). A dose of 12 mg/kg was included based on the assumption that some teratogens require higher doses of FA (Gray and Ross, 2009; Harris, 2009).

Animals designated for the behavioral tests and MRI were limited to the control diet or the 8 mg/kg FA diet, and were kept on their respective diets until they were euthanized. Timed couplings were performed during a 2-h period at the start of the light phase (7:30 a.m.−9:30 a.m.) to attain synchronous timing of embryonic development. The day of coupling was identified as E0. At E7.5, animals were placed in a Plexiglas holder and transported to the irradiation installation. Mice intended for the macroscopic study were either sham-irradiated or irradiated with 1.0 Gy of X-rays. Animals used for behavioral testing and MRI were sham-irradiated or received a sub-lethal dose of 0.1 or 0.5 Gy of X-rays at E7.5. Irradiation was performed using an X-strahl 320 kV (0.14 Gy/min, inherent filtration: 0.21 mmAl, additional filtration: 3.8 mm Al + 1.4 mm Cu + DAP, tube voltage: 250 kV, tube current: 12 mA,) in accordance to ISO 4037. The number of animals used for the macroscopic, skeletal, behavioral and MRI experiments is depicted in Table 1, unless otherwise specified.

TABLE 1

Table 1. Sample sizes.

Macroscopic Scoring and Skeletal Stainings

The dissections, macroscopic scorings and alcian blue/alizarin red skeletal stainings were performed at E18 as previously described (Craenen et al., 2017). For the skeletal analyses, E18 fetuses were randomly selected from the macroscopic study. The axial skeleton was analyzed, with a focus on the vertebrae and the ribs. A subdivision was made between atlas, cervical, thoracic, lumbar, sacral and caudal vertebrae, whilst also differentiating between true, false and floating ribs and sternum.

Behavioral Tests

Starting at week (W)5 and ending at W14, behavioral tests were performed on male mice in the order described below (Table 2). All experiments were performed under blinded conditions. To assess visual acuity, optokinetic tracking was performed. We included cage activity to assess global activity, during both light and dark-phase, and assessed explorative and social behavior with the open field and social exploration tests. The elevated plus maze (EPM) was included to ascertain anxiety, whereas the accelerating rotarod was used to identify issues in motility. Next, to explore olfactory performance we used the odor habituation/dis-habituation assay. Finally, two tests for memory were included: the Morris water maze (MWM) and passive avoidance, to test spatial and fear-related memory, respectively.

TABLE 2

Table 2. Overview of test order and age at time of testing.

Optokinetic Tracking Response

Using a virtual-reality chamber (OptoMotry, Cerebral Mechanics, Medicine Hat, AB, Canada), the optokinetic tracking response was assessed (De Groef et al., 2016; Van Hove et al., 2016). The animal was placed on the center of an elevated platform within the optokinetic installation, where a vertical sine wave pattern was displayed on the monitors. Using a real-time camera system, visual acuity was scored manually using a staircase procedure, composed of random spatial frequencies (100% contrast, 12° per second speed).

Cage Activity

The impact of ionizing radiation exposure on ambulatory behavior was investigated over a 23 h time-period, starting at 4 p.m. until 3:30 p.m. the next day (Verreet et al., 2016a). During this period, animals were individually housed in transparent cages (20 × 26 cm) with minimal bedding, chow and water and placed in a laboratory-built activity logger with three infrared beams. Beam breaks were recorded over 30 min time bins.

Open Field and Social Exploration

To assess exploration and social interaction, a transparent Plexiglas arena (50 × 50 cm) was used (Stroobants et al., 2008; Bollen et al., 2015; Callaerts-Vegh et al., 2015). The arena was homogenously illuminated and equipped with an Any-maze (Dublin, Ireland) tracking system. For the open field test, animals were placed in the empty arena for 1 min of acclimatization, immediately followed by a 10 min test phase with active tracking. The social exploration experiment was identical to the open field test, except that in the center of the arena a small cage with two same-sex strange mice was placed.

Elevated Plus Maze

In order to investigate anxiety, animals were subjected to EPM testing as was previously described (Verreet et al., 2015). The cross-shaped EPM consisted of two perpendicular open and closed arms (21 × 5 cm). Five infrared detectors were installed on the EPM: 2 at the exits out of the closed arms and two at the entrances to the open arms (entries/exits) and one along the length of the open arms (time spent on the open section). The animal was placed in a closed arm and after 1 min of adaptation, beam breaks were recorded for 10 min.

Accelerating Rotarod

General motor function and balance following in utero X-ray exposure during neurulation were assessed using an accelerating rotarod (Ugo Basile, Italy), as was described previously (Verreet et al., 2015). Initially, the animals underwent two adaptation trials (2 min each), each at a constant speed of 4 rpm. In turn, the mouse was subjected to four subsequent test trials, where during each 5 min trial the rotation speed gradually increased from 4 to 40 rpm. Latency was recorded when the mouse lost its footing and fell off the rotating beam.

Odor Habituation and Dis-Habituation

To assess the interaction of mice with olfactory cues, i.e., habituation and dis-habituation, the animals were subjected to an odor discrimination test as was described previously (Yang and Crawley, 2009; Yang et al., 2012; Arbuckle et al., 2015). Animals were individually placed in a fresh cage with a small amount of bedding, followed by 30 min of acclimatization with a dry cotton swab fixed to the cover grid (tip ~5 cm from bottom). Next, the animals were exposed to a sequence of 15 subsequent odor exposures (2 min each): Three trials with water, three trials with grape (non-social odor 1 = NS1), three trials with banana (NS2), three trials with social odor one (S1) and three trials with social odor two (S2). For the preparation of the NS odor tests, respectively 1:100 diluted grape extract (SAFC, W26820-8-K methyl anthranilate ≥98%) and 1:100 diluted banana extract (Acros Organics, AC269481000 n-Butyl propionate >99%) on cotton tips was used. For the S odors, cotton tips were dipped in water and moved in a cross-pattern through the bedding of soiled cages of same-sex mice. During each trial, sniffing-time was recorded manually whenever the subject's nose was within a 2 cm radius of the cotton swab. The inter-session interval never exceeded 2 min.

Morris Water Maze

In order to assess whether FA and sub-lethal pre-natal doses of X-rays during neurulation affected adult spatial learning, MWM was performed. Animals were tested in a circular pool (diameter 150 cm, height 30 cm), filled with opacified non-toxic water as previously described (Latif-Hernandez et al., 2016; Verreet et al., 2016a). For the acquisition trials, a see-through acrylic platform was consistently placed in the same quadrant, 1 cm below the water surface. The pool was located in the center of a homogeneously-lit room, with invariable visual cues. Acquisition training was performed over a period of 5 days, followed by a 2-day resting period, followed again by 5 days of training. During each training day, every mouse was subjected to four trials. The trial interval was approximately 15-min and the quadrant-starting positions varied in a semi-random order for every trial. If the animal was unable to find the platform within 120 s, it was placed on the platform for 10 s and subsequently removed from the basin. On day 5 of the acquisition trials and 2 days after the last acquisition trial, probe trials were performed. During these probe trials, the platform was removed from the basin and mice were subjected to a single probe trial of 100 s, where the starting position was opposite to the target quadrant. Using an automated video capture and tracking system (EthoVision, Noldus, The Netherlands), various parameters such as trajectory and swim speed were recorded. We observed floating behavior (swim velocity <5 cm/s, more than 30 s per swim) in all groups, except the control diet + 0.0 Gy group. However, for the path length analysis to determine if the animals covered the same track during learning, we included all animals due to the low animal numbers per group. Non-responders (floating >35% of test time) were excluded for the reference memory test. As such, a reduced number of animals was included for the reference memory test, as compared to Table 1. More specifically, for this analysis in particular we included under control diet condition 29 animals (9 for 0.0 Gy, 11 for 0.1 Gy and 9 for 0.5 Gy), and 27 under high FA condition (10 for 0.0 Gy, 9 for 0.1 Gy and 8 for 0.5 Gy).

Passive Avoidance

We investigated fear-aggravated learning and memory using a passive avoidance set-up (Lo et al., 2013). Animals were placed in a brightly lit compartment and the door leading into a dark adjacent chamber was opened after 5 s. Latency to enter the dark chamber was timed starting immediately after opening of the dark chamber and stopped when the animal had all four paws on the electric grid in the dark room. Next, the door separating the two compartments was closed and a shock (0.3 mA, 2 s) was administered. The next day, the procedure was repeated, albeit without the administration of an electric shock.

In vivo Imaging

Optical Coherence Tomography

To assess retinal development and thickness, SD-OCT was used as was previously discussed (Van Hove et al., 2016). The animal was anesthetized by intraperitoneal (ip) injection of 75 mg/kg body weight ketamine (Anesketin, Eurovet, Bladel, The Netherlands) and 1 mg/kg medetomidine (Domitor, Pfizer, NY, USA). Shortly before imaging, pupils were dilated using topical 0.5% tropicamide (0.5% Tropicol, Thea Pharma, Wetteren, Belgium). Next, SD-OCT was performed using an Envisu R2210 (Bioptigen, Morrisville, NC, USA) via 100 serial B-scan lines with each line consisting of 1,000 A-scans, in a 1.4 × 1.4 mm field. Afterwards, ip injection of atipamezol (1 mg/kg, Antisedan, Pfizer) was applied to reverse the anesthesia. Thickness of the retina was investigated using InVivoVue Diver software (Bioptigen).

Magnetic Resonance Imaging

For MRI we used female mice, which originated from the same litters as the behavioral test mice. When the female mice were on average W10, in vivo MR imaging of the brain was performed using a 7 T Bruker Biospec 70/30 MRI scanner (30 cm horizontal bore with actively shielded gradients (200 mT/m), Bruker Biospin, Ettlingen, Germany). All data were acquired using a quadrature volume coil (72 mm internal diameter, transmit, actively decoupled) in combination with a dedicated mouse brain surface receive coil (Bruker Biospin). To obtain high resolution 3D images of the entire mouse brain, image acquisition and animal anesthesia was performed similar to previously described experiments (Verreet et al., 2016a). In brief, after the acquisition of localizer scans morphological 3D MR imaging was performed using a rapid acquisition relaxation enhancement (RARE) T2-weighted sequence with a RARE factor of 16 and a repetition time and echo time of 1,000 ms and 67 ms, respectively. The field of view was 24 × 15 × 8.3 mm with a matrix of 256 × 160 × 88, resulting in an isotropic resolution of 94 μm. The total acquisition time was 16 min. The methodology of image post-processing and the labeled template was based on previously published work (Verreet et al., 2016a). Briefly, we first corrected for image intensity inhomogeneity using the N4 bias field correction algorithm (Tustison et al., 2010) using an in-house developed MeVislab pipeline (MeVis Medical Solutions, Germany). Images were affinely registered to the template used in Verreet et al. (2015, 2016a) to obtain brain masks for each animal, which were isotropically dilated by 2 voxels. These brain masks were applied to the raw data and the bias field correction was repeated. Finally, images were non-rigidly registered to the template using the Fast Free-Form Deformation algorithm implemented in Niftyreg (Modat et al., 2010). Template labels were propagated to the individual study images using the transformations obtained from this step, and quantified using an in-house developed Python script (Python 2.7, Python Software Foundation).

Statistics

Statistical analyses were performed with GraphPad Prism 7.02 (GraphPad Software, San Diego, CA, USA). To analyze the data on the macroscopic and skeletal defects, the Kruskal-Wallis methodology was used, in combination with Dunn's post-hoc testing. Data on pre-natal viability were assessed using two-way ANOVA and Dunnet testing for multiple comparisons. For most behavioral tests, MRI and SD-OCT, we used two-way ANOVA (with pairing where required) in combination with Dunnet (inter-dose comparisons) and (Holm-)Sidak (inter-diet comparisons) post-hoc tests. To assess dishabituation, paired t-testing was done, whilst two-way ANOVA + Sidak was utilized to investigate habituation. To perform inter-dose and inter-diet comparisons, one-way ANOVA + Dunnet was used in conjunction with the first trial of the different odors. For all statistical tests, a p-value of 0.05 was considered statistically significant. All values are represented as mean ± SEM.

Results

FA Reduces the Prevalence of Radiation-Induced Anophthalmos, Exencephaly and Agnathia

First, we examined the prevalence of radiation-induced EDs and the prevention thereof with FA fortification. The prevalence of left-eye anophthalmos (Figure 1A), microphthalmos (Figure 1B) and iris anomaly (Figure 1C) was significantly increased following X-irradiation (respectively, 28.26 ± 4.72, 23.56 ± 4.28, and 17.92 ± 3.39 %). In contrast, X-irradiation did not affect the prevalence of the left eye open phenotype (3.03 ± 1.40%) (Figure 1D). Similar observations were made for the right eye (respectively 42.41 ± 5.93, 29.32 ± 3.93, and 18.59 ± 4.35%) (Figures 1E–H). Here, the right eye also showed an increase of the open phenotype (4.55 ± 1.89%). Of interest, we revealed a partial prevention of radiation-induced left-eye anophthalmos with both the 8 mg/kg FA (9.02 ± 3.40%) and 12 mg/kg FA (10.62 ± 2.74%) diets (Figure 1A). No such rescue was observed for the right eye (Figure 1E).

FIGURE 1

Figure 1. Prevalence of various eye defects observed at E18, following 1.0 Gy X-ray exposure at E7.5, and prevention with FA fortification. Radiation significantly increased the risk for left eye anophthalmos; a defect that could in turn be prevented by both FA fortified diets (8 mg/kg and 12 mg/kg FA) (A). Although both left eye microphthalmos (B) and iris anomaly (C) were induced by X-irradiation, no rescue effect of FA was observed on these phenotypes. The left eye open phenotype was not increased in prevalence following irradiation (D). Although defects of the right eye, including anophthalmos (E), microphthalmos (F), iris anomaly (G) and open eye (H) were more prevalent following irradiation, we observed no significant prevention of these defects by FA. Data are represented as mean ± SEM, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001.

In addition, we determined the number of fetuses with exencephaly, agnathia, gastroschisis and cleft palate. X-irradiation increased the prevalence of exencephaly (15.26 ± 3.95%) and agnathia (17.88 ± 4.17%) when the animals were fed the control diet, whilst 8 and 12 mg/kg FA provided significant prevention of both exencephaly (respectively, 4.89 ± 2.10 and 4.43 ± 2.03%) (Figure 2A) and agnathia (respectively, 5.41 ± 1.86 and 1.56 ± 1.56%) (Figure 2B). Furthermore, irradiation also increased the number of fetuses affected by gastroschisis in mothers on the control diet (11.3 ± 2.83%), but here no rescue was observed with the FA fortified diets (Figure 2C). Finally, X-ray exposure at E7.5 did not affect the occurrence of cleft palate in the fetuses, regardless of the diet (Figure 2D).

FIGURE 2

Figure 2. Prevalence of radiation-induced birth defects at E18, affecting the head and abdomen, and the prevention with FA. Irradiation at E7.5 significantly increased the risk for exencephaly (A), agnathia (B) and gastroschisis (C). The first two of these radiation-induced defects could be partially prevented by FA food fortification (both 8 and 12 mg/kg) (A,B). Radiation had only minimal impact on the prevalence of cleft palate (D). Data are represented as mean ± SEM, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001.

FA Counteracts the Effects of X-Ray Exposure on Pre-natal Survival

In the next part of our study, we investigated the impact of X-irradiation during neurulation on the number of implants, pre-natal deaths and fetal weight. Neither X-irradiation nor FA fortification affected the total number of conceptuses per pregnant female (Figure 3A). In terms of late fetal deaths (E18 fetuses with no signs of life), an increase was observed after irradiation in mothers on the control diet, while this increase was prevented with 8 and 12 mg/kg FA diets (Figure 3B). Furthermore, we found an increase in resorptions (implantation site at E18, which holds no developed fetus, and shows evident embryonic-stage death) after 1.0 Gy irradiation, with a notable rescue after 8 mg/kg, but not after 12 mg/kg FA fortification (Figure 3C). Finally, irradiation resulted in a marked fetal weight loss at E18 (Figure 3D), which was not rescued following FA fortification. Of note, sham-irradiated fetuses gained weight when placed on the 12 mg/kg FA diet, as compared to sham-irradiated animals on the control diet (Figure 3D). Altogether, we were able to demonstrate a preventive role of FA for radiation-induced late fetal deaths and resorptions.

FIGURE 3

Figure 3. Impact of 1.0 Gy X-ray exposure at E7.5 and FA on the number of conceptuses (A), late fetal deaths (B), resorptions (C) and fetal weight (D). Neither radiation nor FA diet (8 and 12 mg/kg FA) had an impact on the number of conceptuses at E18 per litter (A). Irradiation strongly increased the rate of late fetal deaths when mothers were fed the control diet, whilst FA fortification prevented this (B). A significant increase in resorptions was observed for irradiated mice on the control diet, which was in turn prevented only by the 12 mg/kg FA diet (C). FA fortification significantly increased fetal weight, whilst irradiation decreased fetal weight (D). Data are represented as mean ± SEM, ^*p ≤ 0.05, ^**p ≤ 0.01, ^****p ≤ 0.0001.

Axial Skeletal Defects and Prevention With FA

To assess general teratogenicity of X-ray exposure on the axial skeleton, alcian blue/alizarin red staining was utilized, a common methodology to assess the sub-macroscopic teratogenicity of chemical and physical agents (Young et al., 2000). X-irradiation at E7.5 increased the number of defects within the vertebrae, specifically in the atlas, the cervical vertebrae and the thoracal vertebrae when the animals were fed the control diet (Figure 4A, atlas, cervical and thoracal vertebrae). Within the cervical region, radiation primarily resulted in fused vertebrae and excessive cartilage (Figures 4B,C). At the thoracic level, the most common vertebral defects included fusions and excessive cartilage, whereas ribs were often missing (Figures 4D,E). Here, we also observed impaired ossification of the ribs (Figure 4F) and split ossification centers within the vertebrae (Figure 4G). Of note, irradiation also increased the incidence of a tilted sternum (Figures 4H,I). To a lesser extent, radiation lead to tilted vertebrae, displaced ribs, hooked (i.e., bent) ribs and short-length ribs (Supplementary Figures 1A–C). 8 mg/kg FA fortification prevented the occurrence of radiation-induced defects in the cervical region, whilst the 12 mg/kg diet group also showed a strong trend (henceforth defined as P = 0.05–0.08) toward prevention (Figure 4, cervical vertebrae). Surprisingly, a combination of 8 mg/kg FA and 1.0 Gy increased the number of defects within the caudal vertebrae, as compared to the 1.0 Gy irradiated animals that were fed the control diet (Figure 4A, caudal vertebrae). Furthermore, a trend was observed for the rescue of defects within the true and false ribs following fortification with the 8 mg/kg and 12 mg/kg diets (Figure 4A true and false ribs). Overall, FA fortification partially prevented skeletal defects within the cervical and thoracic vertebrae, whilst a trend toward prevention could be observed within the true and false ribs.

FIGURE 4

Figure 4. Prevalence and categories of axial skeletal defects at E18, following 1.0 Gy at E7.5 and prevention by FA. (A) Radiation significantly increased the number of defects in the atlas, cervical and thoracal vertebrae, while an insignificant trend could be observed in the ribs. FA fortification with 8 mg/kg prevented defects within the cervical vertebrae, with a trend toward prevention apparent in the true and false ribs. (B–I) In control animals, the arches of the cervical vertebrae only sporadically demonstrated an anomaly (B), whereas irradiation lead to a notable presence of vertebral fusions (affecting two or three arches, shown by an arrow ← and arrowhead ◂, respectively) and excessive cartilage ≪ (C). In controls, both the thoracic vertebrae and ribs never showed any anomalies (D), but irradiated fetuses often lacked ribs (arrowhead ◂) and depicted excessive cartilage (double arrowhead ≪) and fusions (arrow ←) in the vertebrae (E). In addition, the ribs also showed delayed ossification (arrowhead ◂) (F) and the vertebral bodies showed split ossification centers (arrowhead ◂) (G). Finally, radiation also promoted the presence of a tilted sternum (H,I). Data are represented as mean ± SEM, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001. Asterisks indicate a significant change as compared to the control FA + 1.0 Gy group of the respective skeletal region.

Abnormal Adult Brain Morphology Following Pre-natal X-Ray Exposure

We performed volumetric MRI analyses to assess whether the adult brain is structurally affected following irradiation at E7.5. Here we also assessed whether FA fortification could prevent any radiation-induced anomalies with inclusion of the 8 mg/kg FA diet, which was based on the rescue effect we observed in view of the radiation-induced fetal defects. We observed no differences in the volumes of whole brain, the olfactory system, the frontal cortex, the corpus callosum, the amygdala, the cerebellum and the corpora quadrigemina in response to radiation and/or FA (Supplementary Table 1). In contrast, other brain regions were affected by the radiation dose and the diet. Ventricles appeared significantly enlarged following irradiation [F_{(2, 37)} = 6.125; P = 0.0050] (Figure 5A), although no significance was reached when comparing individual radiation doses. We also found a radiation-induced reduction in volume of the hippocampus (Figure 5B), striatum (Figure 5C), thalamus (Figure 5D), midbrain (Figure 5E) and pons (Figure 5F) when the mothers were irradiated with 0.5 Gy. Of interest, an interaction effect between irradiation and the diet could be established for the hippocampus [F_{(2, 37)} = 4.654; P = 0.0157) (Figure 5B), midbrain [F_{(2, 37)} = 4.654; P = 0.0157] (Figure 5E) and the pons [F_{(2, 37)} = 3.792; P = 0.0318] (Figure 5F), which supports an FA-dependent rescue of radiation-induced size decrease. Furthermore, X-irradiation resulted in a trend toward a volumetric decrease of the posterior cerebral cortex [F_{(2, 37)} = 2.731; P = 0.0783] (Figure 5G) and the basal ganglia [F_{(2, 37)} = 2.768; P = 0.0758] (Figure 5H). Unexpectedly, FA food fortification reduced the size of the basal ganglia [F_{(1, 37)} = 4.961; P = 0.0321) (Figure 5H) and the striatum [F_{(1, 37)} = 7.067; P = 0.0115] (Figure 5C). A trend toward FA-induced size decrease was also observed for the anterior commissure [F_{(1, 37)} = 3.796; P = 0.0590] (Figure 5I).

FIGURE 5

Figure 5. Volumetric analyses of various brain regions after pre-natal irradiation at E7.5. Ventricles were significantly increased after irradiation (A), whereas the hippocampus (B), striatum (C), thalamus (D), midbrain (E) and pons (F) were significantly smaller following a dose of 0.5 Gy in animals on the control diet. According to two way ANOVA, the radiation factor was significant in decreasing size of the posterior cerebral cortex (G). FA by itself decreased the size of the basal ganglia (H) and the anterior commissure (I). Data are represented as mean ± SEM, ^*p ≤ 0.05, ^**p ≤ 0.01.

Irradiation Impairs Vision and Olfaction, Which Is Ameliorated by FA Fortification

In order to determine whether X-irradiation during neurulation can affect visual acuity later in life, and whether these effects could be countered by FA, a virtual optokinetic drum was used. Here, we observed that radiation decreased visual acuity [Figure 6A, F_{(2, 64)} = 10.02; P = 0.0002], whilst FA increased visual performance as compared to animals on the control diet [Figure 6A, F_{(1, 64)} = 6.565; P = 0.0128]. Furthermore, the impairment in acuity elicited by 0.1 Gy was alleviated by FA (Figure 6A). SD-OCT analysis did not show any changes in total retinal thickness following X-ray exposure or FA fortification (Figure 6B). Yet, a more detailed investigation revealed that the nerve fiber and retinal ganglionic cell layer (NF + GCL) thickness was decreased following 0.5 Gy irradiation [Figure 6C, F_{(2, 63)} = 7.618; P = 0.0011], which was not alleviated following the FA-rich diet. On the other hand, the high FA diet was shown to elicit a protective effect on the radiation-induced decrease in eye diameter (Figure 6D). Altogether, we showed a radiation-induced decrease in visual acuity starting from a low dose of 0.1 Gy onward, together with a reduced NF and GCL layer thickness and eye diameter following 0.5 Gy. FA prevented the decreased visual acuity elicited by 0.1 Gy, and rescued the 0.5 Gy-induced decrease in axial eye size.

FIGURE 6

Figure 6. Visual acuity, retinal morphology, eye size and the impairment thereof by X-irradiation during neurulation. Radiation decreased visual acuity in adult mice (W5-W7) starting from 0.1 Gy, according to the optomotor test (A). 8 mg/kg FA increased the irradiation dose required for a significant effect on optokinetic response to 0.5 Gy (A). Irradiation or FA diet did not affect total retinal thickness, according to OCT analysis (B), but a more detailed analysis showed that 0.5 Gy significantly reduced thickness of the nerve fiber + ganglion cell layer (C). Using the MRI images for eye-size measurements, we identified a reduced axial length following 0.5 Gy, which was in turn ameliorated by FA fortification (D). Data are represented as mean ± SEM, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001.

To investigate whether X-ray exposure during neurulation has an impact on olfactory performance and discrimination, and whether radiation-induced differences can be rescued by FA, we performed an olfaction-dependent habituation and dis-habituation test. When presented with a novel odor, mice will show specific approach and sniffing behavior (dishabituation, see Table 3), which increases further in the presence of S odors, and diminishes over the three time bins of 2 min (habituation). To compare the rate of habituation and dishabituation between the groups, we calculated the difference in sniffing time between (a) within the same odor of trial 1 and trial 3 (habituation) and (b) between odors from old to new odor (dishabituation). Habituation was observed in all conditions for all odors, indicating a normal loss of interest for odors over time (Figures 7A,B, Table 3). Similarly, approach behavior to a novel odor was observed in both sham and irradiated animals and was not affected by FA enrichment (Figures 7A,B, Table 3). However, irradiation reduced the total amount of time spent sniffing NS odors under control diet conditions compared to sham-irradiated animals (Figure 7C). Two-way ANOVA for factor diet (control diet or FA) and dose (sham, 0.1 and 0.5 Gy) during the NS odor presentation, indicated a significant effect for diet [F_{(1, 63)} = 4.078; P = 0.048] and for dose [F_{(2, 63)} = 3.908; P = 0.025] without significant interaction. Post-hoc analysis revealed a significant difference between sham- and 0.5 Gy-irradiation in the control diet group, indicating a reduced approach time to NS odors after irradiation. This reduced approach was alleviated when given the high FA diet. Of note, this reduced sniffing time is not due to an inability to approach, since presentation of S odors increased the sniffing time, but is possibly due to a decrease in attractiveness or detection of the odor itself. High FA diet normalized the sniffing time and approach to novel odors to baseline levels (Figures 7B,C), which is indicative of a protective role for FA. A similar trend was observed for S odors, however, the two way ANOVA did not indicate a significant effect of either factors. To conclude, irradiation in conjunction with the control diet resulted in hyposmia (i.e., a decreased sense of smell) for the NS odors, or a reduced interest in NS odors. These anomalies were alleviated when the diet was fortified with FA.

TABLE 3

Table 3. Differences in sniff time used to assess habituation and dishabituation.

FIGURE 7

Figure 7. Odor habituation and dis-habituation in adult mice (W9–W11) following pre-natal irradiation at E7.5, under control or FA fortified diet. All animals show a typical approach behavior toward novel odors (dishabituation) and reduction in sniffing over time toward the same odor (habituation) (A,B). Under control diet condition, irradiated animals show reduced sniffing time to novel odors (A). In contrast, under high FA diet, sniffing time is at sham level (B). The total amount of sniffing time toward a non-social odor, was reduced in irradiated animals under control diet, while high FA fortification increased the sniffing time to sham levels (C). Data are represented as mean ± SEM, ^*p ≤ 0.05 vs. sham (post hoc), $ p ≤ 0.05 control vs. FA diet (two-way ANOVA).

No Changes in Activity and Motor Performance Following Irradiation of Animals on the Control Diet

General arousal and changes in circadian activity was assessed in the 23 h cage test. Under control diet conditions, radiation had no effect on the spontaneous activity, and all animals displayed the typical increase in night-time activity. Here, repeated measures (RM) ANOVA indicated a significant effect for time [F_{(47, 1504)} = 38.34; P < 0.0001], but not for radiation dose (Figure 8A). Animals on the high FA diet also showed a typical increase in night-time activity [F_{(47, 1504)} = 50.38; P < 0.0001). In addition, the high FA diet increased night-time activity in mice exposed to a low dose of 0.1 Gy [F_{(2, 32)} = 5.063; P = 0.0123) (Figures 8B,C). When animals were fed the high FA diet, repeated-measures (RM) ANOVA revealed a significant interaction effect between radiation and diet during the dark period [F_{(2, 64)} = 3.485; P = 0.0366], and post-hoc analysis indicated that only 0.1 Gy was significantly different from the sham-irradiated group (P = 0.0191). This interaction effect was also observed in the overall duration of the experiment [F_{(2, 64)} = 4.127; P = 0.0206] (Figure 8D).

FIGURE 8

Figure 8. Cage activity in adult mice (W7–W9) and the impact of X-irradiation at E7.5 and FA food fortification. When the control diet was fed, no effect of pre-natal radiation exposure on cage activity was observed (A). When animals were fed the FA diet, irradiation with 0.1 Gy at E7.5 significantly increased activity during the dark period, as tested in adult 7–9 week old mice (B). (C,D) A summarized total of beam breaks during the dark period (C) and the overall experiment (D) confirmed the observations made in (A,B). Data are represented as mean ± SEM, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001.

Balance and coordination was tested on the accelerating rotarod, but radiation had no effect on motor coordination, and we also saw no effect of FA diet (Supplementary Figure 2).