Ketogenic diet improves the spatial memory impairment caused by exposure to hypobaric hypoxia through increased acetylation of histones in rats

Abstract

Exposure to hypobaric hypoxia causes neuron cell damage, resulting in impaired cognitive function. Effective interventions to antagonize hypobaric hypoxia-induced memory impairment are in urgent need. Ketogenic diet (KD) has been successfully used to treat drug-resistant epilepsy and improves cognitive behaviors in epilepsy patients and other pathophysiological animal models. In the present study, we aimed to explore the potential beneficial effects of a KD on memory impairment caused by hypobaric hypoxia and the underlying possible mechanisms. We showed that the KD recipe used was ketogenic and increased plasma levels of ketone bodies, especially β-hydroxybutyrate. The results of the behavior tests showed that the KD did not affect general locomotor activity but obviously promoted spatial learning. Moreover, the KD significantly improved the spatial memory impairment caused by hypobaric hypoxia (simulated altitude of 6000 m, 24 h). In addition, the improving-effect of KD was mimicked by intraperitoneal injection of BHB. The western blot and immunohistochemistry results showed that KD treatment not only increased the acetylated levels of histone H3 and histone H4 compared to that of the control group but also antagonized the decrease in the acetylated histone H3 and H4 when exposed to hypobaric hypoxia. Furthermore, KD-hypoxia treatment also promoted PKA/CREB activation and BDNF protein expression compared to the effects of hypoxia alone. These results demonstrated that KD is a promising strategy to improve spatial memory impairment caused by hypobaric hypoxia, in which increased modification of histone acetylation plays an important role.

Fig 1. The effect of KD treatment on plasma levels of lipids and ketones.

A. Schematic of the experimental design. Rats were acclimated for 3 days and were then randomly divided into two groups (n = 20 each) fed with either the standard diet (STD) or ketogenic diet (KD). After 14 days of treatment, the rats were subjected to an open field test and biochemical analysis. Next, the rats were subjected to the Morris water maze test for 6 days and hypobaric hypoxia treatment (10 rats in each treatment group) between the 5th day of acquisition training and the probe test on the 6^th day. B. The rats body weights were recorded during whole experimental period. Data are presented as mean ± S.E. C-F. The plasma levels of total cholesterol (C), triglycerides (D), BHB (E), and AcAc (F) were detected by ELISA assay. Five rats in each group. Values are presented as the mean ± S.E. (**p<0.01).

Fig 2. KD treatment enhances spatial learning and memory in adult rats.

A. The total distance travelled and the number of entries into the central area in the open field test were evaluated. Values are presented as the mean ± S.E. (n = 10). B The mean of the escape latencies to find the hidden platform across the four trials are shown for the five days during the acquisition training period. Ten rats in each group.

Fig 3. KD treatment reversed the spatial impairment induced by exposure to hypobaric hypoxia.

A. The mean number of platform crossings, the mean time in the target quadrant, and the latency to first pass the location of the original platform during the probe test were evaluated. Values are presented as the mean ± S.E. (n = 10). B. Representative locomotion tracking plots showing the total path-length during the probe test. The red circle indicates the platform location.

Fig 4. Exogenous BHB has direct improving-effects on spatial impairment induced by hypobaric hypoxia.

A. The plasma levels of BHB were detected by ELISA assay. Five rats in each group. Values are presented as the mean ± S.E. (**p<0.01). B The mean of the escape latencies to find hidden platform across the four trials are shown for the five days during the acquisition training period. Ten rats in each group. C-E. The mean number of platform crossings (C), the mean time in the target quadrant (D), and the latency to first pass the location of the original platform (E) during the probe test were evaluated. Values are presented as the mean ± S.E. (n = 10, *p<0.05, **p<0.01)). F. Representative locomotion tracking plots showing the total path-length during the probe test. The red circle indicates the platform location.

Fig 5. KD treatment increases histone acetylation in the hippocampus.

Protein levels of acetyl-histone H3 (K9/K14), acetyl-histone H3 (K14) and acetyl-histone H4 (K12) in the hippocampus were detected by western blot. The levels of β-actin serve as an inner control. Three rats in each group. The right bar graph shows the relative band density of each protein in each group, and the data are shown as the means ± S.E. (** p<0.01).

Fig 6. KD treatment increases histone acetylation in the CA1 region of the hippocampus.

Representative images of immunohistochemistry analysis of acetyl-histone H3 (K9/K14) and acetyl-histone H4 (K12) in the CA1 region of the hippocampus. The insets show higher magnification of representative areas located in the dotted box.

Fig 7. KD treatment promotes the PKA/CREB pathway and increase BDNF protein levels.

A. Protein levels of PKA substrates, p-CREB, CREB and BDNF, in the hippocampus were detected by western blot. The levels of β-actin serve as an inner control. Three rats in each group. The right bar graph shows the relative band density of each protein in each group, and the data are shown as the means ± S.E. (* p<0.05, ** p<0.01). B. Quantitative realtime PCR of BDNF gene expression in the four groups. Data are expressed as means ± S.E, and normalized to the STD group (** p<0.01). C. ChIP-PCR were performed to measured the binding of acetylated histone H3 (Lys9/14) to the promoter I of BDNF gene. Threshold amplification cycle numbers (C_t) were used to calculate IP DNA quantities as % of input control. Data are expressed as means ± S.E, and normalized to the STD group (** p<0.01).