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  • Animal studies were approved by the University of Texas at Dallas Institutional Animal Care and Use Committee (IACUC) and in accordance with the National Institutes of Health guidelines for animal care. 5xFAD mice overexpress a human form of mAPP-bearing mutations (SwFlLon) and PSEN1 mutations (M146L and L286V) linked to familial AD. 5xFAD mice (B6SJL-Tg(APPSwFlLon, PSEN1*M146L*L286V) 6799Vas/Mmjax) were obtained from Jackson Laboratory. 3.5 months old male non transgenic (nonTg) or 5xFAD mice were randomly allocated into four groups based on genotyping. The investigators performing the experiments did not select the mice allocation. The number of mice was determined by our previous data and power calculation. Statistical Analysis was performed to determine whether the combinations of different types of mice are statistically different for experimental designs. If the analysis indicated it was valid to continue, individual group and the combinations of group × treatment were compared by use of the Bonferroni corrected t-test. Based on our design, we performed power calculation to see whether we have enough power to detect the differences (http://www.stat.uiowa.edu/~rlenth/Power/). Transient occlusion of bilateral common carotid arteries: The procedure to induce transient cerebral ischemia was conducted as previously described [64, 65]. Briefly, surgical anesthesia was induced with isoflurane (3%) in a mixture of nitrous oxide and oxygen (70:30). Anesthesia was maintained throughout the procedure with isoflurane (1.2–1.7%) delivered via a face mask. Rectal temperature was monitored and the body temperature was maintained at 37±0.5°C by using a heating pad. An anterior midline incision was made in the neck, and both common carotid arteries were then exposed and loosely encircled with 3–0 silk to lift the vessels to facilitate later occlusion. The occlusion of both common carotid arteries was induced by applying microaneurysm clips (Surgipro Surgical Micro Vessel Clips, 50–80 g closing pressure) on each vessel for a period of 20minutes followed by the removal of the clips. Sham-operated animals were subjected to the same anesthetic and surgical interventions, with the exception that the carotid arteries remained unclipped. The skin incision was then sutured and the animals were maintained the body temperatures at 37°C until their recovery from the anesthesia. Acetated Ringer's solution (0.5 mL) was administered subcutaneously to all the animals 30 minutes and 24 h after ischemia. All mice were housed in a temperature-controlled facility. Fast blood glucose levels were measured at the following time points: before the operation, 3 days post operation, 7 days post operation and 14 days post operation (S1 Fig). 14 days after the surgery, the mice were subjected to experiments. Mice brain mitochondria were purified as we previously described [24, 66]. Briefly, brain cortex were dissected and placed in ice-cold isolation buffer [225 mM mannitol, 75mMsucrose, 2mM K2PO4, 0.1%BSA, 5mM Hepes, 1mMEGTA (pH 7.2)] followed by a homogenization for 10 strokes using a Dounce glass homogenizer. The resultant homogenate was centrifuged at 1,300 g for 5 min to remove cell debris, and the supernatant was layered on a 10mL discontinuous gradient of 15% (vol/vol) Percoll and centrifuged at 16,000 rpm on a Sorval RC5C (Rotor SS34) for 10 minutes. The resultant pellet was collected and subsequently resuspended in mitochondria isolation buffer followed by the treatment of 0.2% Digitonin to break synaptosome. After a centrifugation at 8,000 rpm for 10 minutes, the pellet was collected and resuspended in cold mitochondrial isolation buffer. Mitochondrial protein concentration was determined by Bio-Rad protein concentration kit with BSA as standard. The purity of mitochondria was determined as we previously described [25] by using immunoblotting. Mitochondrial respiration assays were performed following our previously published method [24]. Purified mitochondria were energized by glutamate (5mM) and malate (5mM) and subjected to respiration assays on a Clark electrode. Oxygen consumption was triggered by the addition of ADP (25μM). The mitochondrial respiratory control ratio was defined as the ratio of State III respiration/State IV respiration. Samples were prepared in 1x sample loading buffer [50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA and 0.02% bromophenol blue] and proteins were separated by SDS/PAGE (10% or 12% Bis-Tris gel; Life technology), and then transferred to a PVDF membrane for blotting (BioRad Laboratories). After blocking in TBS buffer (20 mM Tris-HCl, 150 mM sodium chloride) containing 5% (wt/vol) nonfat dry milk for 1 h at room temperature, the membrane was then incubated and gently shaken overnight (at 4°C) with primary antibodies. This was followed by the incubation with the appropriate secondary antibody for 1 h at room temperature. The following antibodies were used in this experiment: MFN2 (#9482, Cell Signaling Technology), OPA1 (#612606, BD Transduction Lab), DLP1(#611112, BD Transduction Lab), pDLP1 (#3455, Cell Signaling Technology), VDAC (#4866, Cell Signaling Technology), β-Amyloid (#8243, Cell Signaling Technology), HSP60(#4870, Cell Signaling Technology), Goat anti-mouse IgG HRP conjugated and goat anti-rabbit IgG HRP conjugated (Life technology). Images were collected on BioRad Chemidoc Imaging System. Image J software (National Institutes of Health) was used for data analysis. For paraffin-embedded brain section, the slides after deparaffinization and rehydration were subjected to antigen retrieval by boiling in citric acid buffer for 15 minutes. After blocking, the slices were incubated with antibodies against 4-HNE (ab48506, Abcam). After washing in PBS, the slices were probed with anti-rabbit IgG conjugated with Alexa 594 (Life technology). Images were collected on a Nikon confocal microscope and analyzed by using Nikon NIS Advanced Research software. A negative control using primary antibody preabsorbed with 4-hydroxy-2-nonenal-diethylacetal (OXIS Research, Inc.) was used to determine the threshold of positive 4-HNE staining. The images were binarized before analysis and the intensity was measured as previously described [67]. ELISA assay for brain and mitochondrial Aβ Measurement: Mouse cortex or mitochondrial fractions were incubated in 5 M guanidine HCl and 50 mM Tris HCl (pH 8.0) overnight and then subjected to Aβ concentration detection using human Aβ1–40 and Aβ1–42 ELISA kits (Life technology) following the manufacturer’s instructions [24, 25]. The level of Aβ was presented as ng/mg protein. Histological Assessment of Aβ Plaque: Mice after anesthesia (Isofluorane) were subjected to intracardiac perfusion[68] using saline followed by 4% paraformaldehyde. Brain tissues were dissected and equilibrated in 30% sucrose. Sections were cut at 20μm on a cryostat (Thermo scientific). After 3 times washing in PBS for 5 minutes each, free-floating tissue sections were incubated with 0.3% H2O2 in PBS for 10 minutes at room temperature to block endogenous peroxidase. After 3 times washiing in PBS, sections were blocked with blocking buffer (5% goat serum, 0.3% Triton-X in PBS) for 60 minutes at room temperature. Sections were then incubated with polyclonal rabbit anti-Aβ (#8243, Cell Signaling Technology) at a 1:1,000 dilution overnight at 4°C followed by the incubation with a biotinylated secondary goat anti-rabbit antibody (Sigma) in a 1:1000 dilution for 1 hour at room temperature. After washing in PBS, the sections were incubated in HRP-conjugated Extraevidin (Sigma) at 1:1,000 for 1 hr at room temperature followed by washing in PBS, then developed with developed with 3,3′-Diaminobenzidine (#D5905, Sigma-Aldrich). Images were collected on an Olympus microscope. The percentage of occupied brain area and the intensity of the plaque were determined for each sample using the Image J program (NIH). Data from 3 slices per brain sample, spaced every 1 mm were averaged. In situ detection of mitochondrial superoxide levels: We performed In situ detection of mitochondrial superoxide levels by using Mitosox Red as we previously described [24]. Briefly, MitoSox Red (Life Technology) at 1mg/ kg body weight was intravenously injected via the mouse tail vein. 30 min after the injection, the mice were anesthetized the mice and sacrificed by transcardial perfusion with cold saline and then cold, freshly prepared 3.7% paraformaldehyde. The brain tissue was quickly dissected and frozen in 2-methyl butane (Sigma) with dry ice. Coronal frozen brain sections were prepared followed by a blocking using 5% BSA. The slices were incubated with antibody against NeuN (Millipore). After washing in PBS, the slices were probed with anti-mouse IgG conjugated with Alexa 488 (Life technology). We then examined sections under a fluorescence microscope immediately after the mounting. The images were binarized for the analysis of MitoSox Red intensity. Brain sections from mice were blindly coded and processed in parallel. Codes were broken after the analysis was complete. Morris water maze was conducted to access the mice spatial learning and reference memory according to previously described protocol [69]. Briefly, mice were trained to find a submerged escape platform in an open swimming arena. Repeated trials (n = 4) were performed each day for 9 days by starting the mice at different non-congruent start locations (NW, N, NE, E, SE) while keeping the platform at a single location (SW). Each trial lasted 60 seconds with an additional 30 seconds learning time where mice were allowed to remain on the platform. After 9 days learning, mice were subjected to a probe test in which the platform was removed. Mice were analyzed for number of times they passed previous learning time platform location (SW). Behavior data was analyzed using HVS Image 2015 software (HVS Image). Mouse neuron culture and treatment: Neurons were cultured as previously described [25]. Briefly, mouse primary neurons were cultured in neuron culture medium (neurobasal A with 2% B27 supplement, 0.5mM L-glutamine, 50U/ml penicillin, and 50μg/ml streptomycin) with an appropriate density. Neurons at Div 7 days were exposed to vehicle or oligomeric Aβ (200 nM) in the absence of presence of mitotempo (10 μM, Sigma) for a co-incubation of 24 hours followed by glucose- and oxygen-deprivation (OGD) [70]. Neurons were washed with PBS and the culture medium was replaced with glucose-free Earle’s balanced salt solution (EBSS) with vehicle or oligomeric Aβ treatment in the absence of presence of mitotempo. Cultured neurons were then placed in an anoxic chamber (NAPCO, Precision Scientific) filled with 5% CO2, 5% H2 and 90% N2 at 37°C for 1 h. Oxygen levels were maintained below 1% O2. Control neurons were washed with PBS and the culture medium was replaced with glucose-containing EBSS with vehicle or oligomeric Aβ treatment in the absence or presence of mitotempo in a regular 5% CO2 cell culture incubator. After the treatment, the neurons were then maintained in regular culture medium with or without Mitotempo in a regular 5% CO2 incubator. TUNEL assay was performed by using in situ colorimetric cell death assay kits from Promega following the manufacturer's instruction. The extent of brain damage was represented as number of TUNEL-positive cells per mm2 and the cell death of cultured neurons was evaluated by the percentage of TUNEL-positive neurons. Oligomeric Aβ1–42 was prepared as previously described [71]. Mitochondrial superoxide was determined as previously described by using Mitosox Red (Life technologies)[22–24]. Neurons were incubated with 2 μM MitoSox Red and 200 nM mitotracker green (Life Technology) for 30 minutes followed by washing. The images of Mitosox Red staining were collected on a Nikon inverted confocal microscope with on stage incubator. The intensity was subsequently analyzed by using Nikon NIS Advanced Research software. Two-way ANOVA followed by Bonferroni post hoc analysis or Student t tests wherever appropriate were used for repeated measure analysis on SPSS software (IBM software). The distribution and variance were normal and similar in all groups. P < 0.05 was considered significant. All data were expressed as the mean ± SEM.
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