FCF Application: The use of FCF (and other CFs) is possible by registering a request through the announcement section on the website for research center of Consciousness Fields (www.consmointel.com). For this purpose, each researcher, based on desired time and place and by introducing the generalities of the experiments, can allocate CFs interventions for the subject of his/her study. The Research and Development Department of the Science-fact research center is staffed to allocate the CFs treatments for studies under the direct supervision of Mr. Taheri, 24 hours a day, free of charge.
In this study, the FCF treatment was assigned to cellular and animal models of AD on a daily basis and for the entire duration of the experiment from the time the model was created to the ends of related assays. All executive processes and primary analyses of this study have been done by double-blind experts unfamiliar with the theory of how to use the FCF.
Antibodies. The primary antibodies were cis pT231-tau mAbs (gift from KPL, Harvard), β-actin mAb (Sigma, St Louis, MO), Caspase-3 (Abcam, Cambridge, MA), Tau5 (Biosource Camarillo, CA), and oligomeric tau T22 polyclonal antibodies (EMD Millipore, Billerica, MA).
Generating human embryonic neural progenitor cells (hESC-NPCs)
Induced Pluripotent Stem Cells (iPSCs) from two late onset AD patients and two healthy control age-matched subjects were donated from Royan Cell Bank; which were in turn generated from fibroblasts employing Yamanaka factors. The iPSCs were then differentiated to NPCs. The neuro-induction medium composed of DMEM/F12 medium (Gibco, 21331020) supplemented with 5% knockout serum (Gibco, 10828028), Glutamax (Gibco, 25030081), MEM-NEAA (Gibco, 11140050), 1% N2 (Gibco, 17502048), 3 µM SB431242 (Cyman, 13031), 5 µM Dorsomorphin (Stemgent, 04-0024), 3 µM CHIR99021 (Stemgent, 04-0004-10), and 0.5 µM SAG (Cayman, 912545-86-9). The rosette form structures were manually picked up after 7 days of the induction. The NPCs were then re-plated on 1mg/mL laminin and 15mg/mL poly-l-ornithine–coated tissue culture dishes (Sigma-Aldrich) in the neural expansion medium included DMEM-F12 medium supplemented with 5% knockout serum replacement (KOSR), 1% non-essential amino acid, 2 mM L-glutamine, 2% N2 (all from Invitrogen), 0.1 mM β-mercaptoethanol, 20 ng/ml basic fibroblast growth factor (bFGF, Royan Institute), 20 ng/ml additional epidermal growth factor (EGF, Sigma-Aldrich) and 0.2 mM ascorbic acid (Sigma-Aldrich). Medium was changed every other day for 7 days. After differentiation of neurons, the expansion medium was replaced with differentiation medium by eliminating growth factors (bFGF) and adding hBDNF (PR-1113), hGDNF (pr-1107) and cAMP (Sigma, D0627) for 35 days. The differentiation process was tracked and confirmed with SOX2, PAX6, NESTIN, NCAM, GFAP, MAP2, GAD65, GABA and S100 staining.
Nutritional starvation stress. We normally changed the culture medium every 4 days according to our differentiation protocol. However, in order to starve the cells, we didn’t change the medium for additional 96 hours. We initially confirmed tauopathy process time line in our cell culture model and found prominent tauopathy upon 96 hours starvation; consistent with previous findings .
Live and dead cell assay. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining was used for cell viability assessment. Aliquots of 20 µl of FDA stock solution and 50 µl of PI stock solution were diluted in 10 ml PBS . The cells were initially washed with cold PBS and then the FDA/PI solution was added to the cells and was inspected by a fluorescent microscope after 5 min incubation in room temperature with the solution.
Cell staining. Cells were harvested at different time points and fixed with 4% paraformaldehyde (PFA) for 20 minutes in dark. They were either directly subjected to microscopic observations or stained with antibodies. In case of further processing, they were permeabilized with 0.2 % TritonX 100 for 20 minutes and stained with primary antibodies overnight at 4 ºC. They were then incubated with Alexa Fluor 488 or 568 conjugated secondary antibodies for 1 hour at room temperature. The samples were visualized with a Zeiss confocal microscope (LSM 800).
Traumatic brain injury. The mouse TBI model was used as previously described . Briefly, 18 male C57BL/6 mice (2–3 months old), obtained from Royan Animal Facility, were randomized to undergo injury or sham-injury. The mice were anaesthetized for 45 seconds using 4% isoflurane in a 70:30 mixture of air: oxygen. Anaesthetized mice were placed on a delicate task wiper (Kimwipe, Kimberly-Clark, Irving, TX) and positioned such that the head was placed directly under a hollow guide tube. The mouse’s tail was grasped. A 54-gram metal bolt was used to deliver an impact to the dorsal aspect of the skull, resulting in a rotational acceleration of the head through the Kimwipe. Mice underwent single severe injury (ssTBI, 60-inch height). Sham-injured mice underwent anesthesia but not concussive injury. All mice were recovered in room air. Anesthesia exposure for each mouse was strictly controlled to 45 seconds. Subsequent behavioral and histopathological testing was conducted in a blinded manner.
Immunohistochemistry. Mice were perfused with 4% PFA at various time points after injury and brains were harvested for further analysis. Serial 8 µm coronal sections from sham and injured brains were cut on a cryostat (Leica) and were collected on slides.
Immunoblotting analysis. Immunoblotting analysis was carried out as described . Briefly, brain tissues or cultured cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP 40, 0.1% SDS, 0.5% Na-deoxycholate, 50 mM NaF) containing proteinase and phosphatase inhibitors and then mixed with the SDS sample buffer and loaded onto a gel after boiling. The proteins were resolved by polyacrylamide gel electrophoresis and transferred to PVDF membrane and block stained with 5% milk in TBST (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 hour. The membrane was then incubated with primary antibodies in 5% milk in TBST overnight at 4 ºC. Then, the membranes were incubated with HRP-conjugated secondary antibody in 5% milk in TBST. The signals were detected using chemiluminescence reagent (Perkin Elmer, San Jose, CA). The membranes were washed 6 times with TBST after each step. Immunoblotting results were quantified using Quantity One from BioRad.
Immunostaining analysis. Immunofluorescence staining of mice brains was done essentially as described . After treatment with 0.3% hydrogen peroxide, slides were briefly boiled in 10 mM sodium citrate, pH 6.0, for antigen retrieval. The sections were incubated with primary antibodies overnight at 4 ºC. Then, the sections were incubated with an Alexa Fluor 488 or 568 conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature. The sections were washed 3 times with TBS after each step. The sections were visualized with a Zeiss confocal microscope (LSM 800). The gain of the confocal laser was set at the level where there are no fluorescence signals including autofluorescence in sections without primary antibody but with secondary antibody.
Elevated plus maze. The elevated plus maze was used to assess anxiety two months after injury and carried out as described. In brief, the maze consists of two closed and open arms (30 x 5 cm) extended out opposite from each other from a central platform (decision zone). The entire apparatus is raised 100 cm above the floor. Mice were placed on the center platform of the maze, facing a closed arm, and allowed to explore the apparatus for 5 min. The maze was thoroughly cleaned between subjects with a weak ethanol solution. We track-recorded the total time spent in the open center (decision zone), and the two closed arms and the two open arms. The percent time spent in the open arms is presented as a surrogate measure of risk-taking behavior.
Experiments were repeated at least three times. We didn’t exclude any animals or samples from the analysis. For all behavioral tests, experimenters were blinded to injury and treatment status. The hitmap data was collected with Python & OpenCV. All data are presented as mean ± standard deviation (SD) followed by two-way analysis of variance and multiple comparisons with a 95% confidence interval, and significant p-values less than 0.05. All other analyses were carried out with GraphPad Prism version 8.