Chronic crocetin treatment of aged mice led to improvement in age-related deterioration of brain function
We treated aged mice for four months with crocetin to evaluate its effects on age-related cognitive behavior. However, for long-term treatment, we used a non-invasive and painless voluntary dosing method and trained the mice to take the drug as gelatin pellets containing crocetin (Supplementary video SV1). The uniformity of crocetin content in the gelatine-based formulation was confirmed by using RP-HPLC (Supplementary Table ST1).
Further, to confirm the penetration of crocetin into the brain while using a gelatin-based formulation, we performed an 8 h plasma and brain pharmacokinetic (pk) study with multiple points of oral dosing with 10 mg kg-1 of crocetin. Data revealed a significantly detectable quantity of crocetin is both plasma and the brain. (Figure 1A). Further, we found that after four months of treatment with crocetin, its level in the brain was less than the levels observed during eight-hour pk studies, which ruled out any accumulation (Figure 1B). Additionally, we measured the mice’s body weight at every 15 days during four months treatment and found no changes in the body weight (Supplementary Table ST2).
Further, the treated mice displayed significantly improved spatial memory, which was tested in an eight-armed radial arm maze. The animals treated with crocetin took a significantly less average time to search for their food reward as compared to the control group (Figure 1C). The treated animals also spent significantly longer time in the baited arm as compared to the control group (Figure 1C). Additionally, wrong entries into the non-baited arms were reduced to half in the crocetin group as compared to the untreated control group (Figure 1C). The crocetin group was also quick to find the baited arm, they travelled significantly less average distance in comparison to the control group (Figure 1C). Further, the crocetin group also displayed significantly improved working memory behavior in the radial arm maze, where alternate arms were baited. Improvement in the working memory was reflected in a number of tests that included improved latency to the first entry into the baited arm, where the crocetin group took less than half of the time than untreated group (Figure 1D). The Crocetin group also spent more average time in the baited arm as compared to the control group (Figure 1D). In contrast, the control group spent more time in the non-baited arms than the crocetin group (Figure 1D). The Crocetin group also showed a significantly higher number of entries into baited arms as compared to the control group (Figure 1D). The effect of crocetin was also observed clearly in the exploratory behavior of animals in the open field test. The Crocetin group spent more average time in the corners than the control group (Figure 1E). Crocetin treatment also improved the locomotor activity of mice, which were mobile for a significantly longer time (Figure 1E) and travelled significantly more distance (Figure 1E) at a higher speed (Figure 1E) while exploring the unfamiliar field as compared to the control group. The Crocetin group also displayed a significantly higher average number of rearing events as compared to the control group (Figure 1E), which appeared uninterested in exploring the unfamiliar environment. However, there was no significant difference in time spent in the centre between control and crocetin groups (Figure 1E).