1. Animals and parasites
C57BL/6J mice aged 11/2 months were purchased from the National Institute of Nutrition, Hyderabad (India), housed under aseptic conditions at an Animal house facility in our institute, and provided with sterilized food and water Ad libitum. Plasmodium berghei ANKA parasites were acquired from the Parasite bank, National Institute of Malaria, New Delhi, India. All the experiments performed are as per the Institutional Animal Ethical Committee (IAEC) and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. All the experiments in the current study were repeated thrice. We did not randomize randomization to allocate subjects in the study, neither blinding nor predetermined sample calculation.
2. Ethics Approval
The use of animals in this study were approved by the Institutional Animal Ethical Committee (IAEC), animal protocol approval nos. UH/IAEC/PPB-2-E1/2009 from 27/05/2009-10 and LS/IAEC/PPB/2010/12 from 18/03/10-17/03/11 is approved by CPCSEA. The authors of this manuscript declare that they have no competing interests.
3. Induction of ECM
C57BL/6J mice were intraperitoneally injected with 106 PbA parasites in 100 µl of PBS, and the control mice injected with the same amount of PBS (n=10 per each group). Infection was monitored by staining the blood smears with Giemsa and JSB-I and JSB-II, and the cerebral symptoms observed through behavioural changes. The percentage of cerebral symptoms was around 80-90%, and the infected mice expired in 7-15 days. The mice that showed convulsions and partially coma (lethargic, weak, and abnormal to no gait or reflexes and cognitive deficits: lack exploration and sense of touch) between 6-9 days of p.i. were considered as cerebral malaria mice. Mice that expired at later stages showed no distinct cerebral malaria features and passed after the 10th day was regarded as Severe Anaemia (SA). These were excluded from the study. Depending on the behavioural symptoms that were succumbed to complete or partial paralysis by 6-9th day p.i. were sacrificed, and the cortical brain samples collected were used for further analysis. Control brain samples were also collected simultaneously.
Administration of experimental serum
The SE and control sera (CS) were further injected into naïve mice (n=6), and these mice were kept in observation for survival. Brains from SE treated mice were collected once they become paralyzed, and CS injected mice sacrificed at the same time were used as controls for this experiment.
Administration of recombinant protein/inhibitor
i) Recombinant Lt-α
100 ng recombinant Lt-α protein in 200µl PBS was injected to the mice (n=10 for each dose) intravenously, and control mice were given the same volume of PBS. These mice were kept in observation for survival. Samples from these mice (n=6) were collected by euthanasia when the Lt-α mice got the cerebral symptoms. The schematic design of the methodology is shown in Fig. 2A and 3B.
ii) Inhibition of Lymphotoxin-α
100 μl of antibody (dose ranging from 5 to 10 ng) against Lt-α was injected to PbA infected mice (n=40; 8 mice for dose) intravenously from 3rd-day p.i., twice a day up to 7th day. Samples from these mice were collected for further studies when the mice were terminally ill. Of the 40 mice, no less than two representatives from each dose were collected for expression, western blot, and histochemical analysis
4. Estimation of BBB integrity
200µl of 0.2% Evan’s blue dye was intraperitoneally injected to study the blood-brain barrier disruption. Mice were sacrificed after 1 hr of Evan’s blue injection by euthanization. Brains were dissected out and imaged using CAMEDIA C-7070 wide zoom camera (Olympus).
4. Estimation of oedema
Brain water content was measured as the difference between wet and dry weight and expressed as %. Brains of the PbA infected mice, Ab(T), and PBS injected control were weighed immediately after dissection (n=3 for each group) and weighed again after drying at 1100C for 24 hr.
5. Brain sectioning and analysis
All the mice from different groups were sacrificed by 0.1% pentobarbital anesthesia and perfused with 75 ml of 0.9% ice-cold saline to wash out circulating lymphocytes and monocytes. Washing was followed by injecting the mice with 75 ml of ice-cold 4% paraformaldehyde to fix the tissue. Brains were dissected out, postfixed in 4% paraformaldehyde for 12 hrs. These brains were sliced using brain matrix and allowed to fix for 24 hrs and processed in series of graded alcohol (75, 80, 95, and 100%) for 2-3 min and 3 hrs twice in chloroform to remove the entrapped air bubbles in the tissue slices. These slices were embedded in the molten wax, allowed to solidify at room temperature, and the blocks were cut into 3µ thin sections using Leica ultra-microtome. These sections were collected on to poly-L-lysine coated slides.
5.1. Haematoxylin and Eosin staining:
Histological studies of mice brain sections were done by staining with haematoxylin and eosin stains. Briefly, sections were deparaffinized and cleared in Sulfur-free xylene and xylene for 7 min, respectively, followed by acetone for 2 min and dehydrated in 95% ethanol. These sections were washed under running tap water for 5 min and double distilled water for 3 min to wash off chemical traces, stained with haematoxylin and eosin for 2 min. Washed in double-distilled water for 2 min, followed by dipping in 95% ethanol thrice for 1 sec. to remove excess eosin, acetone absolute for 2 min twice, and dehydrate with xylene 2 min twice. Mounted the sections with DPX and observed under the light microscope (Nikon ALPHAPHOT-2(YS2))
5.2. Immunofluorescence Analysis
Sections from control, ECM, and Ab(T) treated mice brains were processed in coupling jars for immunolabelling of Lt-α (TNFβ (H-171) Cat # sc-8302, Santa Cruz Biotechnology Inc.) and FITC conjugated anti-CD4 antibody (Cat # 11-0043-81) and PE-conjugated anti-CD8 antibody (Cat # 12-0083-81), eBiosciences. These sections were blocked with 10% goat serum for 30-45 min, incubated with anti-LT-α primary antibody (1:1000) at 40C overnight. Sections were then incubated with anti-Rabbit TRITC conjugated secondary antibody for 1½ hr. at room temp., washed three times in PBS, followed by incubated with DAPI for 10 min and washed. Images were taken using a Leica Laser Scanning confocal microscope. For anti-CD4 and anti-CD8 (0.125µg/100µl each antibody per 0.2% non-immunized goat serum in PBS) staining, we incubated the sections with respective antibodies at room temperature for 45 min in the dark and mounted, as mentioned above. Images were captured using a blue laser, Laser Scanning Confocal microscope (Leica TCS SP2).
6. FluoroJade B staining
Paraffin sections of brains from different experimental groups were dewaxed in xylene and rehydrated in 100% and 70% alcohol for 5 and 2 min, respectively. Slides were rinsed twice in ddH2O for 1 min, sections were then incubated with freshly prepared 0.06% KMNO4 for 17 min and rinsed for 1 min twice in dH2O, and further were incubated for 30 min in 0.001% Fluoro-Jade B solution at room temperature in the dark and rinsed twice in ddH2O for 1 min. Slides were air-dried for 10 min, mounted with permanent mounting media and the slides were stored in dark Images were captured using FITC filter under a fluorescent microscope (Olympus BX51)
7. PCR
We usedTRIzol reagent to isolate genomic DNA (Cat No # 15596026) (ref). A 10 µl PCR reaction was set up by taking 10ng of DNA template, 1 µl (10 pM) forward and reverse primers, 5 µl of PCR master mix (DreamTaqTM, Thermo Fischer Scientific Green PCR Master Mix # K9021), and water. The amplified product was resolved on the 1% agarose gel against the ladder (100 bp). Three different mice brain cortical samples (n=3) were analysed. Gel band intensities were quantified by densitometry analysis using ImageJ software (NIH).
8. Real-time PCR
TotalRNA was extracted using TRIzol Reagent (Invitrogen Cat No# 15596-026) method as per manufacture’s protocol, further quantified by NanoDrop200(Thermo SCIENTIFIC) and converted into cDNA using cDNA synthesis kit (BluePrintTM 1st Strand cDNA Synthesis Kit # 6115A). Expression levels of cell adhesion molecules and chemokine receptors was studied by using Power SYBR® Green PCR Master Mix (Applied Biosystems # 4367659). Quantitative PCR (qPCR) was performed in Applied Biosystems 7900HT Fast-Real Time System (Applied Biosystems) in triplicates. The relative amounts of target genes in control, ECM, and Ab (T) treated samples were compared by analysing the data by 2-∆∆CT Method to respective GAPDH expression levels.
9. Isolation of brain lipid rafts and Western blotting
Brain lipid rafts fractions were isolated by the detergent[28][28][28]_ENREF_28-free method [1][28] and stored at -800C until use. The total protein in the samples was estimated by the Lowry method. A total of 50μg of protein sample was resolved using 10% PAGE and transferred to the nitrocellulose membrane. The transferred nitrocellulose membrane was probed with anti-caspase-3 (cell signaling and technology Cat. No 9662), anti-calpain1 (Chemicon International, Temecula, USA (MAB3164), anti-Granzyme-b (Calbiochem, Cat. No AM52), anti-perforin (Cell Signalling and Technology, Cat: 3693, Danvers, MA, USA), anti-Neurofilament light (NF-L, Sigma, Cat No. N5139), anti-Synaptophysin (Cat. No. sc-6926; Santa Cruz Biotechnology, Santa Cruz CA) and anti-B-tubulin antibodies at a concentration of 1:1000 overnight at 40C after blocking with 0.5% skimmed milk powder solution in Tris-buffer saline (TBS). Membranes were washed with TBS and TBST (Tris-Buffer Saline with 0.1% tween) and incubated the membranes with anti-mouse secondary antibodies for 1 hr. at room temperature, washed with TBS and TBST before they were developed using the BCIP-NBT substrate or ECIL method. We performed the dot blots of serum and brain lipid rafts samples by allowing the samples to dry on the nitrocellulose membrane for 1 hr. at room temperature and followed the steps described for western blotting using anti-Lt-α (Santa Cruz, Temecula, USA) antibody and developed by BCIP NBT using ALP tagged anti-mouse secondary antibody.
Statistics:
The band intensities of the three individual western blots and PCR gels was determined by the densitometry analysis (Image J software (NIH)), and one-way ANOVA performed the statistical significance. Parasitemia of the infected and Ab(T) mice was calculated by counting the Giemsa or JSB-I/II stained caudal blood smears (at least five reading from each slide and averaged) from the day of infection till the mice succumbed to death/sacrificed. Survival graphs were plotted using GraphPad Prism survival analysis or Microsoft excel by calculating mean and standard error mean. Similarly, the graphs for the number of dying neurons in mice brain samples were plotted by counting the FluoroJadeB positive cells on each section (n=6) from three individual mice brain sections. The significance was performed by using one-way ANOVA. Real-time PCR analysis was performed by taking the mean ΔCT values of each gene (n=3, repeated thrice) using the Δ-2CT method and the graphs plotted using Graph Pad Prism v6.2 software.