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).