2.6. Sample preparation method
To 100 L of serum/ brain lysate, sample 100 L of internal standard
i.e., retinyl acetate (1 g/mL) was added and mixed for 1 min
and then 1 L of n-Hexane was added. The mixture was vortexed
for 1 min and centrifuged at 3000 rpm for 10 min. The upper hexane
layer was dispensed into another tube and the residue was
re-extracted with another 0.6 mL of n-hexane and centrifuged. The
upper hexane layer was pooled with the previous aliquot in the
tube. The contents of the tube were evaporated under nitrogen and
reconstituted in 100 L of methanol. An aliquot of 20 L is loaded
onto HPLC for analysis and the chromatogram show the peaks of
retinol and retinyl acetate according to their retention time points
and absorbance helps in quantify (Adapted from Bieri et al., 1979;
Coral et al., 1997) (Fig. 1).
3. Behavioural studies
3.1. Rotarod performance
Rota rod performance test is based on a rotating rod with
forced motor activity. The rota rod test evaluates balance and sensory
motor coordination of the subjects. We have evaluated the
alterations in rota rod performance in animal models of pathological
situations compared to control rats (Boix et al., 2010; Jones
and Roberts, 1968) the time of coordination between control and
experimental groups (Model Rota rod 4 compartments DolphinTM
instruments) were recorded.