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.