Male Swiss albino mice (10 weeks old weighing 30–32 g) were procured from National Institute of Biosciences (NIBS), Pune. 6 mice per cage were housed in opaque polypropylene cages and kept under standard laboratory condition i.e. 22 ± 2 °C temperature, 60–70% humidity, maintenance of 12 hours natural day and night cycle with food and water ad libitum. Prior to the testing, the mice were allowed to get habituated to the testing rooms for 1week.
The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Institute of Chemical Technology, Mumbai, India with protocol number: ICT/IAEC/2018/P21. All experimental procedures were performed as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.
Drugs, reagents and treatment schedule
PCA (≥98 % HPLC), Rivastigmine (RVS), D-gal and methylglyoxal were purchased from Sigma Aldrich Co. (St. Louis, MO). L-Reduced glutathione (GSH), Tris-buffer Thiobarbituric acid (TBA) and 5, 5’- dithiobis (2- nitrobenzoic acid) (DTNB) were procured from Himedia Laboratories, Mumbai, India. Aminoguanidine HCl was purchased from TCI Ltd., bovine serum albumin (BSA) was purchased from S.D. Fine chemicals, nitroblue tetrazolium and sodium azide were purchased from Loba chemie pvt ltd. Mouse ELISA kits for NFκB, BACE-1, Caspace-3 and AGE were purchased from KinesisDX, USA. All other chemicals used were of analytical grade. PCA was suspended in Tween 80 for oral administration. All other drugs were dissolved in 0.9% saline. At the end of study, the animals were sacrificed by carbon dioxide asphyxiation.
The dose of PCA was determined based on earlier studies (Sakamula and Thong-asa 2018). The total 40 mice were divided into five groups, each group having 8 mice:
1. Group 1: Vehicle control (0.2 ml of saline) for 42 days
2. Group 2: Negative control (Saline + D-gal (150 mg/kg, s.c.) for 42 days
3. Group 3: Positive control (RVS (0.25 mg/kg i.p.) + D-gal (150 mg/kg, s.c.) for 42 days
4. Group 4: PCA (80 mg/kg p.o.) + D-gal (150 mg/kg, s.c.) for 42 days
5. Group 5: PCA (100 mg/kg p.o.) + D-gal (150 mg/kg, s.c.) for 42 days
The work plan of the study is illustrated in Fig. 1.
In vitro bovine serum albumin (BSA) glycation study
In vitro protein glycation was carried out as described previously (Peng et al. 2008). For AGE formation 200 µl of BSA (35 mg/ml) containing sodium azide (0.1 g/ml) was incubated with 400 µl of glucose (175 mg/ml) at 37°C for 21 days, in absence or presence of PCA solution at different concentrations (10–200 µg/ml). Aminoguanidine (AG) (4 mg/ml) was used as a standard. AGEs, pentosidine and fructosamine compounds were assessed.
In vitro glycation assay with BSA-MGO
BSA-MGO assay was performed as described previously (Lunceford & Gugliucci, 2005). 200 µl of BSA was incubated with 400 µl of MGO (0.4 mg/ml) at 37 °C for 14 days in absence or presence of different concentrations of PCA (10-200 µl/ml). Blanks containing BSA-glucose but no test sample were kept at -80°C, until the measurement. Aminoguanidine (AG) (4 mg/ml) was used as a standard.
Determination of fructosamine (amadori) content
The fructosamine content was measured by nitroblue tetrazolium (NBT) reduction assay (Islam et al., 2014) . The reduction of NBT was recorded by increase in absorbance at 530 nm from 5 to 15 min at 37 °C and expressed as % inhibition.
Morris water maze (MWM) test
The spatial memory and learning in mice were assessed using MWM test. A plastic pool having 122 cm diameter and 50 cm height and divided into 4 quadrants was filled with water (22 ± 2 °C) up to 30 cm and rendered opaque by dissolving powdered milk. In one quadrant, a white platform was placed 1 cm below water level. 3 bright-coloured visual clues were fixed around the pool. Position of escape platform and visual cues were kept unchanged during entire experiment. The MWM test was divided into two phases as follows:
Maze acquisition phase
On day 20, mice underwent 4 consecutive training sessions, with 15 min intertrial interval. Each time, mice were carefully put in one of the four quadrants and allowed to explore the platform for 90 s. In case the mice could not locate the platform during this time, it was gently guided to the platform. The mouse was allowed to remain on the platform for 60 s. The latency period was recorded as initial acquisition latency (IAL).
Maze retention phase
After 24 hours of maze acquisition phase, the escape latency was again evaluated and first retention latency (FRL) was recorded. Similarly, second retention latency was calculated on day 40 of treatment and recorded as second retention latency (SRL).
The memory consolidation in mice was assessed by probe test performed 24 hours after recording of SRL. In probe test the platform was removed and mice were allowed to swim freely for 60 seconds. Time spent by individual mice in the target quadrant was recorded.
Y maze test
Y-maze test is an excellent tool for assessing exploratory behaviour, spatial recognition and working memory. In Y maze test, apparatus (8 cm x 30 cm x 15 cm) with each of the arms at 120° apart was used. The test was conducted on day 22 and day 42, one hour after the dosing and included two trials. During the first trial, the mouse was allowed to explore only two arms (arm A and arm B) for 5 min keeping the third one (arm C) closed. Arm C was considered a novel arm. Post 30 min interval, the second trial was conducted, allowing each mouse to explore all 3 arms for a period of 5 min. Percentage spontaneous alteration behaviour (%SAB) and the number of entries in the novel arm were analysed for individual mouse. Consecutive entries in three different arms were counted as one alteration. Following formula was used to calculate percentage spontaneous alterations (Ghods-Sharifi et al., 2008):
Brain tissue collection
Animals sacrificed by CO2 asphyxia were perfused with PBS. Isolated brains were immediately transferred in ice-cold isotonic saline and stored at –80ºC.
Brain tissue was homogenised in 0.1 M PBS (pH 7.4). The homogenate (10% w/v) was subjected to cold centrifuge (10 000 g for 15 min). The supernatant thus obtained was used for further estimations.
Evaluation of AChE
As described by Ellman et al. (Ellman et al., 1961), to 50 μl of the brain tissue homogenate, 3 ml of PBS (pH 8), 100 μl of acetyl thiocholine iodide (0.75 nM), and 100 μl of 10 mM Ellman’s reagent were added. Absorbance was measured spectrophotometrically at an interval of 30 s for 5 minutes at 412 nm.
Estimation of reduced glutathione
As described by Smith et al. (Smith et al., 1988), each 3 ml reaction mixture (2.9 ml of DTNB in PBS and 0.1 ml of supernatant) was incubated at 37°C for 15 min. Absorbance was measured spectrophotometrically at 412 nm. The level of GSH/mg of protein was estimated.
Estimation of lipid peroxidation
As described by Draper et al. (Draper et al., 1993), mixture of 0.5 ml Tris-HCl buffer and 0.1 ml of the brain homogenate, was incubated for 2 h. 1 ml of 10% w/v TCA solution was then added to above mixture and it was subjected to cold centrifuge (1000 g) for 10 min. The supernatant was collected and to it, an equal volume of TBA (0.67% w/v) was added. The mixture was then subjected to heating in a boiling water bath for 15 min. The mixture was cooled in an ice bath and to it, 1 ml distilled water was added. Absorbance was measured spectrophotometrically at 532 nm.
Estimation of Superoxide dismutase
As per method by Nandi and Chatterjee (Nandi & Chatterjee, 1988), to 2.8 ml of PBS, 0.1 ml supernatant and 0.1 ml pyrogallol solution (2.6 mM in 10 mM HCl) was added. Absorbance was measured spectrophotometrically at 325 nm. The level of SOD units/mg of protein was estimated.
Immunoassays for biomarkers
NFκB, AGE, BACE-1 and Caspase-3 were estimated using ELISA kits. The manufacturer’s protocol was strictly followed and reading were taken using ELISA plate reader (BIORAD).
GraphPad Prism Version 5, San Diego, CA was used for statistical analysis. Data are represented as mean ± SEM. Escape latency in MWM and % SAB in Y maze were analysed using repeated measure two-way ANOVA followed by post hoc Bonferroni's test. In other tests, one-way ANOVA followed by post hoc Tukey's multiple comparison test was used to calculate statistical significance.