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. 2014 Feb 27;5(2):e1083.
doi: 10.1038/cddis.2014.26.

High-dose of Vitamin C Supplementation Reduces Amyloid Plaque Burden and Ameliorates Pathological Changes in the Brain of 5XFAD Mice

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High-dose of Vitamin C Supplementation Reduces Amyloid Plaque Burden and Ameliorates Pathological Changes in the Brain of 5XFAD Mice

S-Y Kook et al. Cell Death Dis. .
Free PMC article

Abstract

Blood-brain barrier (BBB) breakdown and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Besides vitamin C being as one of the important antioxidants, recently, it has also been reported as a modulator of BBB integrity and mitochondria morphology. Plasma levels of vitamin C are decreased in AD patients, which can affect disease progression. However, investigation using animal models on the role of vitamin C in the AD pathogenesis has been hampered because rodents produce with no dependence on external supply. Therefore, to identify the pathogenic importance of vitamin C in an AD mouse model, we cross-bred 5 familial Alzheimer's disease mutation (5XFAD) mice (AD mouse model) with ι-gulono-γ-lactone oxidase (Gulo) knockout (KO) mice, which are unable to synthesize their own vitamin C, and produced Gulo KO mice with 5XFAD mice background (KO-Tg). These mice were maintained on either low (0.66 g/l) or high (3.3 g/l) supplementation of vitamin C. We found that the higher supplementation of vitamin C had reduced amyloid plaque burden in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration. These results suggest that intake of a larger amount of vitamin C could be protective against AD-like pathologies.

Figures

Figure 1
Figure 1
Amyloid plaque burden is decreased in the cortex and hippocampus of the high-supplementation group of 5XFAD mice. (a) Coronal serial sections of brains from 6-month-old mice (n=4 for each of the groups) were stained with anti-Aβ antibody (green; 4G8) and imaged by confocal microscopy. There were no signals in the frontal cortex of KO-WT mice in either of the two treatment. 4G8-positive areas were decreased in the frontal cortex of KO-Tg on the high-dose vitamin C treatment. Scale bar=600 μm. The section shown is at the interaural level, 4.78 mm; Bregma, −0.98 mm (Fig. 23 in Franklin and Paxinos). (b) Frontal cortex of KO-Tg mice of both treatment groups was magnified. (a and d) Somatosensory region; (b and c) motor region. Scale bar=100 μm. (c) Coronal serial sections of brains from 6-month-old mice (n=4 for each of the groups) were stained with anti-Aβ antibody (green; 4G8) and imaged by confocal microscopy. There were no signals in the hippocampus of KO-WT mice in either treatment groups. Plaques were particularly decreased in the hippocampus of KO-Tg on the high-dose vitamin C treatment. Scale bar=600 μm. The section shown is at the interaural level, 1.86 mm; Bregma, −1.94 mm (Fig. 47 in Franklin and Paxinos). (d) Hippocampus of KO-Tg mice of both treatment groups was magnified. Nuclei were stained with 4′-6-diamidino-2-phenylindole. Scale bar=100 μm. (e) Compared with the low-dose supplemented KO-Tg mice, the high-dose supplementation KO-Tg mice exhibited significantly less plaque burden in the cortex and hippocampus. Both P values (***P<0.001 and *P<0.05) mean the high-dose versus the low-dose supplemented KO-Tg group
Figure 2
Figure 2
Cerebral capillaries are less impaired in the brains from the high-supplementation group of 5XFAD mice. (A) Coronal serial sections of brains from 6-month-old mice (n=4 for each from standard and decreased groups) were co-immunostained with anti-GLUT1 (red) and anti-Aβ (green; 4G8) antibodies and imaged by confocal microscopy. Capillaries stained with anti-GLUT1 antibody (red) showed long tubular-like form in KO-WT mice of both treatment groups. (B) KO-Tg mice displayed amyloid plaque deposition (green) and cut capillary forms. Capillaries adjacent to the amyloid plaques displayed disconnected tubular-like form in KO-Tg mice (shown in the yellow dotted circle). Decreased impairment was observed in the high-dose supplemented KO-Tg mice than the low-dose supplemented KO-Tg mice (shown in the white dotted circle). Scale bar=50 μm. (C) 3D-SIM images of the brains from the KO-WT mice in both treated groups. (D) 3D-SIM images of the brains from both the low-dose supplemented KO-Tg (a–d) and the high-dose supplemented KO-Tg mice (e–h). Brain slices were recorded on 3D-SIM images along z axis with a thickness of 0.15 μm, reconstructed and 3D volume images were created with the alpha blending function. Axial directions are represented on each image. Capillaries stained with anti-GLUT1 antibody (red) and amyloid plaque stained with anti-Aβ (green; 4G8) antibody. Arrow, sectioned z axis image; arrow head, damaged micro-vessel. Scale bars=2 μm. 3D depth, 2.8–4.5 μm in the high-dose supplemented KO-Tg and 3.9–7.6 μm in the low-dose supplemented KO-Tg mice
Figure 3
Figure 3
Alteration of cerebral tight junctions (TJs) decreased in the high-supplementation group of 5XFAD mice. (a) EM pictures revealed that TJs were altered in the brains of KO-Tg mice as compared with KO-WT mice. TJs of KO-Tg mice were shorter than that of KO-WT mice in the low-dose supplemented groups, and TJs of the high-dose supplemented KO-Tg mice were longer than that of standard vitamin C-treated-KO-Tg mice. n=4 for each cortex from all four groups. Arrow, TJs; RBC, red blood cells. Scale bar=2 μm. (b) Length of eight TJs from all four groups was examined and the high-dose supplemented KO-Tg mice had longer (1,225.6 nm) TJs as compared with standard vitamin C-treated-KO-Tg mice (624.2 nm). Graph shows an average of eight capillaries. *P<0.05 means the high-dose versus the low-dose supplemented KO-Tg and ###P<0.001 means the low-dose supplemented KO-Tg versus the low-dose supplemented KO-WT mice
Figure 4
Figure 4
Gliosis decreased in the brains from the high-supplementation group of 5XFAD mice. (a) Coronal sections stained with anti-GFAP antibody (green) in the cortex of each KO-Tg group. Scale bar=600 μm. (b) Boxed areas of a were magnified. Compared with standard vitamin C-treated KO-Tg mice, the high-dose supplemented KO-Tg had 54.5% less GFAP-positive cells. Nuclei were stained with 4′-6-diamidino-2-phenylindole. Scale bar=50 μm. (c) Quantification of GFAP immunoreactivity. GFAP-positive cells were significantly decreased in the high-dose supplemented KO-Tg mice than standard vitamin C-treated KO-Tg mice (*P<0.05)
Figure 5
Figure 5
Abnormal mitochondrial morphology was prevented in the brains from the high supplementation group of 5XFAD mice. Representative EM from the brains of all four groups shows mitochondrial morphology. Boxed areas of KO-Tg image were magnified for each figure. KO-WT mice showed no alterations of mitochondrial structure. The high-dose supplemented KO-Tg mice had avoided impaired mitochondria than standard vitamin C-treated KO-Tg mice. Scale bar=1 μm

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