Animals
A total of 80 C57BL/6 male and female mice of 3–4 weeks old of 15–20 g were purchased from the National Institute of Nutrition (NIN), Hyderabad. All the animals were fed with chow and sterile water ad libitum followed by 12 hours light / dark cycle in the animal house facility at the University of Hyderabad.
Parasite infection and evaluation of symptoms of CM
Mice were infected intraperitoneally (i.p.) with 100–350µl of PbA infected red blood corpuscles (iRBC’s) at a concentration of 106 was obtained from the National Institute of Malaria Research (NIMR), New Delhi and stored in liquid nitrogen. The PbA infected vials were thawed at 4 0C and diluted in 1x phosphate buffered saline (PBS) for administration into the mice (n = 15/group). All the infected mice were segregated into 3 groups (CM infected; ARM + APO adjunctive therapy; ARM monotherapy). The parasitemia from the caudal blood smears was monitored by Giemsa staining after 72 hours post-infection. Each mouse was subjected to Rapid Murine Coma Behavior Scale (RMCBS), a tool for identifying the subjects of CM and performing rescue treatment. RMCBS experiment consists of 10 behavioral parameters; gait, balance, motor performance, body position, limb strength, touch escape, pinna reflex, toe pinch, aggression, grooming. Each parameter was scored from 0–2 depending on the behavior exhibited by an infected mouse considering a total score of 20. Mice with scores ranging from 5–12 were considered as symptomatic to CM and subjected to treatment.
Experimental groups
Animals symptomatic to CM was administered (i.p.) immediately with ARM at a concentration of 25 mg/Kg body weight (b.w.) of ARM (dissolved in Arachis oil) and APO at 5mg/Kg (b.w.) (dissolved in 10 % of dimethyl sulphoxide (DMSO) once per day up to seven days. All the animals that received the adjunctive therapy were transferred to a separate cage labeled as ARM + APO group (n = 23). Another cohort of CM infected animals (n = 16) were administered i.p. with 25 mg/kg (b.w.) artemether and transferred to a cage labeled as ARM group (n = 27). Control mice (CON) (n = 15) were administered with saline. Animals symptomatic to CM were categorized as CM infected group (n = 11), which were euthanized humanely with ketamine (150 mg/kg) and xylazine (10 mg/kg) i.p. on day 6–11. All the treated animals were subjected to cognitive tests after a survival period of 30 days and euthanized for brain sampling.
Behavioral tests
Animals with mild CM symptoms (n = 11) were tested immediately for spontaneous activity (cylinder and adhesive removal test). Memory associated tests were performed in the treated animals (ARM n = 15. ARM + APO n = 12) in comparison to CON group after survivability of 30 days. All the tests were performed during the light cycle and analyzed by a group of blinded researchers.
Cylinder test
Each animal from all the experimental groups was placed in a clean, transparent plastic open-top cylinder (height: 26 cm; diameter: 16 cm) and video recorded for 3 minutes. The number of rears (vertical posture of the mouse, standing on its hind limbs) for each mouse was counted. The cylinder was periodically cleaned with 70 % alcohol after each trial. The average number of rears and time spent grooming were measured in all the animal groups.
Adhesive removal test
This test was performed to study the deficits in sensorimotor response to stimulation test adapted for rodents [23–26]. Small pieces of adhesive labels were stuck beneath the toe of the mouse and placed in the cage. Each animal was subjected to three alternative trials with a 2 minute time interval between the tests. If the animal fails to remove adhesive after 120 seconds, the trial was preceded with the next mouse. The average time to contact the adhesive, till its removal from the toe was recorded.
Beam balance test
Each animal (except CM group) was guided to walk from one end of the beam to another (40 cm height, 1 meter beam, 12 mm width) to study the motor deficits [27] for two days and one day for testing. Each mouse received three trials on the test day to analyze the rate of “slipping” (any foot coming off the beam) as a motor deficit with a scoring index of “1” (inability to cross the beam), “2” (crossing the beam with dragging limbs) and “7” (crossing the beam with fewer than 2-foot slips). The beam was cleaned with 70% alcohol, and droppings were removed with clean dry paper.
Barnes maze
This task is performed to evaluate the spatial long-term memory in the rodents [28,29]. This test consists of a circular platform with 20 equidistantly spaced holes along its perimeter (100 cm in diameter). 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 behavioral tracking software version 6.0, Stoelting Co, Wood Dale, USA.
T-maze experiment
Mice were subjected to T-maze consisting of three arms measuring (diameter 30 x15 cm height) of left and right sided goal arms and 40 cm of the start arm. A forced choice of spontaneous alternation was selected where each mouse was gently placed in the start arm for 3 minutes for habituation [30–32]. 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, mouse was placed in the empty square shaped box made of transparent glass material (dimensions: 30 x 30 x 30cm) 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. 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 having a different shape and colour in 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 in the familiarization and novel object in test phase via. sniffing, pawing, within a distance of 2 cm was recorded manually [33]. The discrimination ratio was calculated to estimate the preference towards novel object (novel object interaction/ total interaction with both objects x 100.
Histological staining
After conducting behavioral assays, all the animals were euthanized and perfused intracardially with saline and chilled 4% paraformaldehyde solution (PFA). The whole brain samples were collected and stored in 4% PFA. Brain samples were placed in Golgi-Cox stain solution at room temperature for 17 days. The rest of the brain samples were dehydrated in 20–40 % sucrose solutions followed by cryomicrotomy. Hippocampal sections of 10 µm thickness were collected to perform immunofluorescence, Fluoro-Jade C and Hematoxylin and Eosin (H&E) staining, 100 µm sections for Golgi-Cox staining. We assessed the changes in the CA1 and Dentate gyrus regions of hippocampus in all the experimental groups.
Fluoro-Jade C staining
Hippocampal brain sections were subjected to the Fluoro-Jade C staining, an anionic dye (AG325 Millipore) which stains only the degenerated neurons [34]. All the sections were immersed in xylene for 45 minutes and dehydrated in 100% ethyl alcohol with 5% sodium hydroxide for 5 minutes. All the sections were washed in phosphate buffer saline (PBS) buffer for 2 minutes and incubated in 0.06% potassium permanganate solution for 10 minutes with gentle shaking. All the slides were placed in of Fluoro-Jade C stain (FJ-C) solution (0.05% of Fluoro-Jade C powder dissolved in distilled water with 50 µl of acetic acid) for 20 minutes. All the sections were washed with distilled water for 2 minutes, dried at 50 0C at dark for 15 minutes and mounted with DPX mounting medium. The number of FJ-C positive neurons was quantified by the cell counter tool of Image J software.
Hematoxylin and Eosin (H&E) staining
H&E staining is a standard and well established method for studying morphological changes in tissues during various pathological conditions [35]. All the brain sections were stained with the Harris hematoxylin stain according to the protocols mentioned previously [36–38]. All the images were captured using the Olympus BX-51 microscope at 1000X magnification.
Golgi-Cox staining
Sucrose dehydrated whole brains impregnated with Golgi-Cox stain was subjected to cryosectioning using Leica Cryomicrotome CM 1850. Hippocampal brain sections were collected at 100µm thickness for studying the dendritic spine density and 200 µm to analyze the neuronal arborization pattern by Sholl analysis. The cryosections were developed according to the protocol of Sami Zaquot et al. [39]. All the images were captured by the Olympus BX-51 microscope at 1000X and 400X magnification.
Quantification of dendritic spine density of hippocampal CA1 pyramidal neurons
Dendritic spines were quantified from the skeletonized images of Golgi impregnated CA1 neurons using the cell counter tool of ImageJ software. Spine projections per 10 µm dendrite length of eighteen distal dendrites were considered for quantification from Golgi impregnated CA1 neuronal images captured at 1000X magnification using Olympus BX-51 microscope.
Sholl analysis of hippocampal CA1 pyramidal neurons
Neuronal arborization pattern was quantified by selecting the widely used linear method of the Sholl analysis algorithm [40]. Sholl analysis draws imaginary concentric circles overlapping from soma of the neurons. A highly arborized neuron develops more intersections compared to an altered neuron. The number of intersections (Nm), radius at maximum Nm is critical radius (rc), number of primary dendrites (Np) were calculated using Sholl plugin of ImageJ software. The soma centered hippocampal pyramidal neurons (n = 16 per group) were converted to 8 bit binary, skeletonized and subjected to threshold. The image was subjected to Sholl plugin after drawing a line from the soma to the border of the image. Based on the linear Sholl plot, a graph was plotted for average values of Nm on Y-axis against rc on X-axis for each experimental group. Further, we measured the number and total length of apical and basal dendrites of Golgi-impregnated CA1 neurons (n = 16) in all experimental groups by the Neuron J plugin 1.4.3 tool in ImageJ software.
Evans Blue staining
Cohorts of ARM (n = 3), ARM + APO (n = 3) on day 13, CM infected (n = 3) on day 7 and CON (n = 3) mice were injected intravenously (i.v.) with 2 ml/kg of 2% Evans Blue dye (SRL Chemicals 46650). Mice were anesthetized with ketamine (150 mg/kg), xylazine (10 mg/kg) and perfused intracardially with 1x PBS buffer after an hour. Brains were surgically removed, weighed and immersed in 1 ml of formamide solution for 48 hours at 37 0C. The samples were homogenized and centrifuged at 14,000 rpm for 20 minutes at 20 0C. Supernatants were collected carefully and absorbance was measured at 620 nm using UV-visible spectrophotometer (Hitachi–U2900). The amount of the dye (microgram per milligram of tissue) extravasated was quantified by measuring the absorbance of the dye from the brain samples compared to the standard values of Evans blue dye.
Semi-quantitative PCR
Total RNA was isolated from the whole brain samples of all the experimental groups (n = 4 per group) using Trizol reagent (T9424 Sigma Aldrich) and quantified using NanoDropTM 2000 UV-Visible spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized from the 1µg concentration of RNA using PrimeScript™ 1st strand cDNA Synthesis kit (6110A Takara Bio). cDNA at 0.5µg/µl (0.5 µl) concentration, 10 picomolar concentration of forward, reverse primers (1µl) were added to 5 µl of 2x Dreamtaq green PCR master mix (K1081 Thermo Fisher Scientific), made up to 10µl reaction with 3.5 µl of nuclease-free water and subjected to semi-quantitative PCR (Applied Biosystems Veriti 96 well Thermal cycler) at 25 cycles respectively. PCR protocol included denaturation step at 95 0C for 30 seconds (stage-1), 95 0C for 2 minutes (stage-2), annealing at respective melting temperature (Tm) for 45 seconds and extension at 72 0C for 5 minutes. Primers were procured from Integrated DNA Technologies (IDT). Primer sequences specific to NOX2 and GAPDH genes are listed in table 1.
Estimation of ROS by 2, 7-dichlorodihydrofluorescein diacetate (DCFDA) method
2′,7′-Dichlorofluorescin diacetate (Sigma Aldrich D6883) is a non-fluorescent probe used to estimate the cellular ROS levels involving hydrogen peroxide (H2O2), hydroxyl radicals (OH−) and peroxyl radicals (ROO−). De-esterification of DCFDA intracellularly leads to the formation of a highly fluorescent compound, 2′,7′-dichlorofluorescein. Whole brain samples (100 mg) (n = 6) were homogenized in 1x PBS buffer in a Dounce homogenizer and centrifuged for 6000 rpm at 4 0C for 15 minutes. 55 µl of HEPES buffer was mixed to 10 µl of the supernatant. 20 µM of DCFDA was dissolved in the culture grade dimethylsulphoxide (DMSO) solution was added to the above mixture in a 96 well plate and kept for incubation for 30 minutes at room temperature at dark condition. Hydrogen peroxide (100µM in PBS) was used as a positive control. Mean Relative Fluorescence Units (RFU) were measured at an excitation wavelength of 485 nm and emission wavelength of 515 nm of Tecan Infinite 200 PRO spectrofluorometer.
Western blotting
The whole brain samples were homogenized in sucrose radio-immunoprecipitation assay buffer (RIPA) buffer (10 mM Tris-HCl, 0.32 M sucrose, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, and pH-7.2) with 10 µl per 1 ml of protease inhibitor (P0044 Sigma-Aldrich) using a Dounce homogenizer at 4 0C. The protein extracts were estimated by Bradford method and 50 µg of proteins were resolved in 10 % Sodium-Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) gel. The resolved gel were blotted on the nitrocellulose membrane soaked in Towbin buffer (Tris-HCl- 3 g, glycine-14.4 g, deionized water – 800 ml, methanol − 200 ml pH-8.3) overnight at 4 0C. The membranes were blocked in 5 % skimmed milk buffer for 1 hour at room temperature and washed with Tris-buffered saline containing 0.05 % Tween 20 (TBST) for 5 minutes. Membrane was incubated with primary antibodies of NOX2 rabbit raised polyclonal antibody (Abcam ab80508) and GAPDH (#5174, Cell Signaling Technology) at dilution of 1:1000 overnight at 4 0C. The membrane was washed with TBST and probed with anti-rabbit IgG (1:30,000) (whole molecule) (A3687 Sigma-Aldrich) alkaline phosphatase conjugated secondary antibody for 2 hours at room temperature. Immunoreactivity was detected by adding 120 µl of BCIP solution (5-bromo-4-chloro-3-indolyl-phosphate) (75531 SRL chemicals), 120 µl of NBT solution (Nitro Blue Tetrazolium) (73654 SRL chemicals) to 4ml of alkaline phosphatase buffer (Tris-2.422 g, NaCl- 1.68 g and MgCl2- 0.203 g dissolved in 200 ml of distilled water) on the membrane for 5–10 minutes at room temperature in dark condition.
Double immunofluorescence experiment
Hippocampal brain sections were permeabilized with 0.2 % Tween for 20 minutes. All the sections were blocked using blocking buffer (5 % normal goat serum (Cell Signaling Technology, #5425) dissolved in 1x PBS) for 2 hours at room temperature. Sections were incubated with the primary antibody cocktail of rabbit generated anti-NOX2/gp91phox antibody (1:100) (Abcam, ab80508) and mouse raised NeuN (neuronal marker) antibody (1:100) (Cell Signaling Technology, #94403) overnight for 16 hours at 40C. Sections were rinsed in 1x PBS and incubated with a secondary antibody cocktail of anti-rabbit IgG (1:1000) (4412S Alexa Fluor® 488 Conjugate) and anti-mouse IgG (1:1000) (4409S Alexa Fluor® 555 Conjugate) from Cell Signaling Technology for 2 hours at room temperature in dark condition. After washing the sections in 1x PBS, the sections were mounted with Prolong Gold Anti-Fade reagent with DAPI (8961S Cell Signaling Technology). Images were captured by the Carl Zeiss LSM 710 confocal microscope using ZEN Blue software. The fluorescence intensities of images were quantified by the ImageJ software (n = 8 images per group).
Statistical analysis
The statistical differences among the experimental groups were calculated by one-way ANOVA (Analysis of Variance) with post-hoc Student-Newman-Keuls test (multiple comparisons) using the GraphPad Prism software version 5.03. The p-values less than 0.05 and 0.001 were considered significant.