Element Changes Occurring in Brain Point at the White Matter Abnormalities in Rats Exposed to the Ketogenic Diet During Prenatal Life

Abstract

A large number of clinical studies demonstrate that the ketogenic diet (KD) may be an effective approach to the reduction of epileptic seizures in children and adults. Such dietary therapy could also help pregnant women with epilepsy, especially since most antiseizure drugs have teratogenic action. However, there is a lack of medical data, considering the safety of using KD during gestation for the progeny. Therefore, we examined the influence of KD used prenatally in rats on the elemental composition of the selected brain regions in their offspring. For this purpose, synchrotron radiation-induced X-ray fluorescence (SR-XRF) microscopy was utilized, and elements such as P, S, K, Ca, Fe, and Zn were determined. Moreover, to verify whether the possible effects of KD are temporary or long-term, different stages of animal postnatal development were taken into account in our experiment. The obtained results confirmed the great applicability of SR-XRF microscopy to track the element changes occurring in the brain during postnatal development as well as those induced by prenatal exposure to the high-fat diet. The topographic analysis of the brains taken from offspring of mothers fed with KD during pregnancy and appropriate control individuals showed a potential influence of such dietary treatment on the brain levels of elements such as P and S. In the oldest progeny, a significant reduction of the surface of brain areas characterized by an increased P and S content, which histologically/morphologically correspond to white matter structures, was noticed. In turn, quantitative elemental analysis showed significantly decreased levels of Fe in the striatum and white matter of 30-day-old rats exposed prenatally to KD. This effect was temporary and was not noticed in adult animals. The observed abnormalities may be related to the changes in the accumulation of sphingomyelin and sulfatides and may testify about disturbances in the structure and integrity of the myelin, present in the white matter.

Keywords: animal models; brain development; ketogenic diet; multielement analysis; prenatal exposure; synchrotron X-ray fluorescence microscopy.

Figure 1

Microscope images (a) and distribution maps of P, S, and K (b–d, respectively) in the representative brain tissue slices taken from the rats of all the examined stages of postnatal development (2-, 6-, 14-, 30-, and 60-days old), the mothers of which were fed during pregnancy with the ketogenic (K) or standard fodder (N). Color scales below the maps express the elemental mass deposits in μg/cm².

Figure 2

Distribution maps of Ca, Fe, and Zn (a–c, respectively) in the representative brain tissue slices taken from the rats of all the examined stages of postnatal development (2-, 6-, 14-, 30-, and 60-days old), the mothers of which were fed during pregnancy with the ketogenic (K) or standard fodder (N). Color scales below the maps express the element mass deposits in μg/cm².

Figure 3

Box-and-whisker plots presenting the median, minimal, and maximal values as well as interquartile spans of the mass deposits of P, S, K, Ca, Fe, and Zn in the cortex determined for the experimental (K) and control (N) groups. The 2-, 6-, 14-, 30-, and 60-days old rats were taken into account in the analysis. No statistically significant differences (Mann–Whitney U test, 95% confidence level) were found between the K and N groups of the rats at a given age.

Figure 4

Box-and-whisker plots presenting the median, minimal, and maximal values as well as interquartile spans of the mass deposits of P, S, K, Ca, Fe, and Zn in the striatum determined for the experimental (K) and control (N) groups. The 2-, 6-, 14-, 30-, and 60-day-old rats were taken into account in the analysis. Statistically significant difference(s) (Mann–Whitney U test, 95% confidence level) between the K and N groups at a given age was(were) marked with *.

Figure 5

Box-and-whisker plots presenting the median, minimal, and maximal values as well as interquartile spans of the mass deposits of P, S, K, Ca, Fe, and Zn in the corpus callosum determined for the experimental (K) and control (N) groups. The 2-, 6-, 14-, 30-, and 60-day-old rats were taken into account in the analysis. Statistically significant difference(s) (Mann–Whitney U test, 95% confidence level) between the K and N groups at a given age was(were) marked with *.

Figure 6

Box-and-whisker plots presenting the median, minimal, and maximal values as well as interquartile spans of the mass deposits of P, S, K, Ca, Fe, and Zn in the internal capsule determined for the experimental (K) and control (N) groups. The 30- and 60-day-old rats were taken into account in the analysis. Statistically significant difference(s) (Mann–Whitney U test, 95% confidence level) between the K and N groups at a given age was(were) marked with *.

Figure 7

Box-and-whisker plots presenting the median, minimal, and maximal values of the relative sizes of the areas characterized by the increased P and S accumulation for the 30- and 60-day-old experimental and control rats (K and N groups, respectively). Statistically significant difference(s) (Mann–Whitney U test, 95% confidence level) between the K and N groups at a given age were marked with *.

Figure 8

Typical cumulative X-ray fluorescence spectrum (black line) obtained for a brain tissue sample taken from a 60-day-old control rat. The fitted curve and its background are marked with blue and red line, respectively. The analytical Kα lines of the elements were marked in black, while Kβ lines were signed in gray.

Figure 9

Sensitivity curve calculated on the basis of the micromatter XRF calibration standards.

Figure 10

Comparison of the localization of selected regions (cortex, striatum, hippocampal formation and structures of white matter: corpus callosum, fimbria, internal capsule, and external capsule) in the microscopic image of an exemplary brain slice taken from 60-days old rat (A) with the graphics, based on the Paxinos and Watson anatomical atlas, presenting these brain areas in the corresponding coronal diagram (B). Blend of the microscopic image of the brain with the map of P distribution (C) and definition of the region taken into account in calculations of the relative size of the area characterized by the increased P content (D).