Materials and Methods
Ethics Statement: All animal experiments have been conducted after the approval from the Institutional Animal Ethics Committee (UH/IAEC/PPB2014-I/68), University of Hyderabad, India.
2.1 Experimental groups:
Total animals of 30 male and female C57BL/6 mice of 3-4 weeks old (10-15 g) were purchased from the National Institute of Nutrition, Hyderabad. All animals were maintained at appropriate 12 hour light and dark condition and fed with animal feed and sterile water ad libitumin the animal house facility, at the University at Hyderabad. Animals were infected with Plasmodium berghei ANKA (PbA), intraperitoneally (i.p.) at a concentration of 106, a parasite strain that reiterates the human symptoms of CM on day 5-9. Based on the Rapid Murine Coma Behavior scale (RMCBS), we administered the dosage of 3mg/kg of polycyclitol dissolved in sterile water along with ARM (25mg/Kg) dissolved in Arachis oil to animals symptomatic to CM i.p., once per 7 days. Animals infected with PbA, treated and controls were subjected to rapid murine coma behavior score (RMCBS), which is a video based evaluation of symptoms of CM and assessment for rescue drug therapy based on 10 neurobehavioural parameters (Carroll et al., 2010). Out of 30 animals, 26 animals were symptomatic to CM and administered with ARM and SR4-(01-04) (n = 6 animals per group) for 6 days and observed for survivability period of 30 days.
Cognitive tests: We performed cognitive tasks relevant to working and reference memory in all the experimental animals after day 30 in all the treated and control groups. The experimental data was analyzed by a group of blinded researchers using ANYmaze software version 6.0.
Barnes maze
This experiment is performed to evaluate the spatial long-term memory in the rodents. This test consists of a circular platform with 20 equidistantly spaced holes along its perimeter (100 cm in diameter). An escape platform was placed under one of the holes leaving the rest empty. Each animal was guided from the center of the maze to detect escape platform for 4 minutes per session up to 4 days (acquisition phase). The maze and the escape platform were cleaned with 70 % alcohol following each trial. Animals were subjected to probe trial after removing the escape platform on day 5. The time to detect the escape platform (primary latency) and the number of holes entered before primary latency (primary error) were recorded. The mice were video-recorded and tested individually with the ANY-maze behavioural tracking software version 6.0, Stoelting Co, Wood Dale, USA.
T-maze test
Mice were subjected to T-maze consisting of three arms which are left and right sided goal arms measuring 30 cm (diameter) × 15 cm (height) and a start arm of 40 cm (diameter) × 15 cm (height). A forced choice of spontaneous alternation was selected where each mouse was gently placed in the start arm for 3 minutes for include references. The mouse was placed in the start arm of the maze after blocking any one side of the arm. The mouse is forced to explore the L-shaped maze for 5 minutes (acquisition phase). The mouse was placed back in its home cage for 15 minutes time duration. The maze was cleaned thoroughly with 70 % alcohol to remove olfactory cues in the area. During the test phase, the blockage in the arm was removed, and mouse was placed in the start arm and observed for its entry to the arm not visited previously (correct alternation) (test phase). Mouse exploring the arm visited previously during the test phase is considered as wrong alternation. Each mouse was subjected to 6 trials per day for four days to study the ”correct alternation” and ”wrong alternation.” The percentage of correct alternation per animal with side preference rate (actively adapt to one side of the arm) was calculated and compared among the groups.
One-trial novel object recognition test
On day 1, a mouse was placed in an empty square-shaped box made of transparent glass material (dimensions: 30 × 30 × 30 cm) for 20 minutes (habituation phase). The mouse was removed from the arena and placed back in its home cage. The box was cleaned with 70% alcohol. On day 2, two identical objects were placed 5 cm away from the walls. The same mouse was placed in the box for 5 minutes (familiarization phase). Mouse was placed back in its home cage. The walls of the box along with the identical objects were cleaned thoroughly with 70% alcohol. One of the identical objects was replaced with a novel object with a different shape and color in the same position. After 60 minutes, the same mouse was placed in the center of the arena for 5 minutes (test phase). The total time spent by the subject interacting with both the identical objects was recorded in the familiarization and novel object in test phase by sniffing, pawing within a distance of 2 cm.
Golgi cox staining
Golgi cox staining is one of the gold standard methods to study neuronal structure and its arborization. The brains collected from treated and control group and CM were subjected to Golgi cox stain (5% Potassium Dichromate, 5% Mercuric Chloride (sublimate) and 5% Solution of Potassium Chromate were added to distilled water). All the brain samples were stored in Golgi cox solution at room temperature in dark condition for 17 days. All the brains were sectioned at 200 μm and developed according to the protocol designed by Zaquot and Kaindl (Zaqout and Kaindl, 2016). Further, we quantified dendritic length of cortical and hippocampal neurons from Golgi impregnated neurons. The length of eighteen proximal and distal dendrites were measured in hippocampal and cortical Golgi impregnated neurons (n =13 neurons per group) of all the experimental groups. The broken line tool of Image J software was used to measure the dendritic length.
Preparation of brain tissue lysates
Tissues (100 mg) of all the experimental groups were homogenized using Dounce homogenizer with 8 to 10 strokes at 4 °C in Radio-Immunoprecipitation Buffer (RIPA) (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and pH 8). Protease and Phosphatase inhibitors (P8340, P0044 Sigma-Aldrich) (10 µl per 1 ml of lysate) and 1mM phenylmethylsulphonyl fluoride (PMSF) were added to the homogenate. The homogenate was centrifuged at 12,000 rpm for 15 minutes at 4 °C using refrigerated centrifuge. The supernatant was frozen upon collection and stored at – 80 °C in a freezer.
Western blotting
Protein estimation was carried out by Bradford reagent (B6916 Sigma-Aldrich). 50 µg of the protein was resolved by 12% sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE). The gel was transferred onto nitrocellulose membrane (Protran Amersham GE) in Transfer buffer (Tris-HCl- 3 g, glycine- 14.4 g, deionized water- 800 ml, methanol- 200 ml, pH 8.3) overnight for 4 °C. The membrane was washed with Tris-buffered saline (TBS) containing 0.05% Tween 20 for 5 minutes and blocked with 5% skimmed milk for 1 hour followed by incubation with the primary antibodies of pCDK5 (sc-377558) from Santa Cruz Biotechnology, CDK5 (sc-6247) from Santa Cruz Biotechnology, p35/25 (C64B10) from Cell signal technology, phospho Tau Ser396 (9632S) from Cell Signal Technology and GAPDH (D16H11) from Cell signal technology overnight at 4 °C. The membrane was washed with TBS and incubated with the anti-mouse and anti-rabbit secondary antibody conjugated to HRP for 2 hours at room temperature. Chemiluminescence signal against HRP conjugated secondary antibodies were analysed by adding Luminol and peroxide. Densitometric analysis of all the protein expression data, normalised with the loading control (GAPDH) was performed using Image J software (version, NIH, USA) and the graphs were plotted using Graph pad prism software.
Immunohistochemistry
Brain samples perfused with 4% paraformaldehyde were subjected to sectioning at 10-15 μm thickness. Brain sections were quenched using 3% hydrogen peroxide in methanol for inhibiting endogenous peroxidase activity. All the sections were blocked by 5% normal goat serum followed by primary antibody Phospho-Tau (Ser396) at a concentration of 1:1000 overnight at 4 oC. Polydector solution provided in the kit was added to the sections for an hour and covered in DAB (3′-3′ diaminobenzidine) buffer for 5 minutes. Hematoxylin was used as a counter stain for observing the nuclear staining and sections were mounted with DPX mountant. Images were captured at 400 X magnification using Leica trinocular DM6B microscope with Leica Application Suite X (LAS X) software.
Statistical Analysis
All the densitometric analysis for Western blots and immunohistochemistry images were quantified using Graph Pad Prism version 5.03 software. The dendritic lengths from Golgi-impregnated images were quantified using Image J software. Statistical differences of all the experimental groups were calculated by the one-way ANOVA with Student Newman-Kuels test using Graph Pad Prism software 5. The ***p values (p <0.001 & p<0.005) were considered as significant. All the results were represented as mean ±standard error mean.