Antioxidants have a rapid and long-lasting effect on neuritic abnormalities in APP:PS1 mice

Abstract

Senile plaques are a major pathological hallmark of Alzheimer's disease (AD). Compelling evidence suggests that senile plaques lead to structural alterations of neuronal processes and that local toxicity may be mediated by increased oxidative stress. Anti-oxidant therapy can alleviate the neuronal abnormalities in APP mice, but the time-course of this beneficial effect is unknown. We used multiphoton microscopy to assess in vivo the characteristics of antioxidant treatment on senile plaques and neurites in AD model mice (APPswe/PS1dE9). We observed that α-phenyl-N-tert-butyl nitrone (PBN), Ginkgo biloba extract (EGb 761) and Trolox had no effect on the size of existing senile plaques. However, all anti-oxidants had a straightening effect on curved neurites. This effect was detected as soon as 4 days after commencing the treatment, and was maintained after 1 month of daily treatment, with no further increase in the effect. The straightening of neurites persisted 15 days after stopping the treatment. These data indicate that neuronal plasticity is fast and still active in adult animals, and suggest that amelioration of the neuritic distortions associated with senile plaques with antioxidants is both rapid and long lasting.

Figure 1

Effect of PBN treatment on neuritic curvature when neurites up to 50 µm from plaque border were examined. Data shown are the means±SD of all measured neurites per condition (15–176 neurites from 3–6 animals). Animals were imaged on a daily basis before commencing the treatment (day 0) and for the next 4 consecutive days (days 1–4) as well as one last time 7 days after beginning the treatment (day 7). Individual, identified neurites were analyzed at each serial imaging session. The curvature ratio approaches 1.0 for straight neurites, and is less than 1 for “curvy” neurites. Significant differences were determined by Student T test. Statistically significant differences were observed between Control and PBN treated animals as soon as 4 days after commencing the treatment and differences were maintained until the end of the treatment (day7) (*p<0.05 vs. Control group).

Figure 2

Representative example of PBN treatment on neuritic curvature. In vivo images with multiphoton microscopy were taken before, as well as 4 and 7 days after commencing the treatment (A, Control day 0; B, Control day 4; C, Control day 7; D, PBN day 0; E, PBN day 4; F, PBN day 7). Neurons expressing YFP or GFP are green, blood vessels (red) contain Texas Red dextran and dense-core Aβ plaques are histochemically labeled by methoxy-XO4 (blue). Single neurites were identified and measured for curvature at each time point. Scale bar: 50 µm

Figure 3

Effect of long term treatment with Ginkgo biloba extract (EGb 761) (100mg/Kg/day p.o.) and Trolox (210 mg/Kg/day p.o.) on neuritic curvature when neurites up to 50 µm from plaque border were examined. Data shown are the means±SD of all measured neurites per condition (4–73 from 3–5 animals). Ratio of curvature for each group was analyzed by one way ANOVA followed by Tuckey B test, with distance to senile plaque as independent variable. There was no significant effect, of distance to the closest plaque, on the curvature ratio between groups {F_4,241=0.877, p=0.571}. Statistically significant differences were observed between animals treated with Ginkgo biloba (100 mg/Kg) and Trolox (210 mg/Kg) for 30 days or after 15 days of washing out and Control mice ({F_4,273=3.890, *p=0.004} vs. Control group).

Figure 4

Representative example of long term treatments with Ginkgo biloba extract (EGb 761) and Trolox on neuritic curvature. In vivo images with multiphoton microscopy were taken 30 days after commencing the treatment (A, Control; B, Ginkgo biloba extract; C, Trolox). Neurons are green, blood vessels (red) contain Texas Red dextran and dense-core Aβ plaques are histochemically labeled by methoxy-XO4 (blue). Scale bar: 25 µm