C57BL/6J male mice (6-8 weeks, 20-25 g) were purchased from Charles River and housed in the Experimental Animal Center of Fudan University in a temperature and humidity-controlled specific-pathogen-free laboratory with a 12 h/12 h light/dark cycle. All procedures were performed in accordance with the Guide for the National Science Council of the People’s Republic of China, and the study was approved by the Ethics Committee of Fudan University, Shanghai, China (IRB approval number 20180972A259). This manuscript was written in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
BCAS model and experimental design
The BCAS model was created as previously described12. Briefly, mice were anaesthetized using 4% isoflurane in 30% O2 and 70% N2 and maintained on 2% isoflurane in 30% O2 and 70% N2 using a mask. A midline skin incision was made, and the bilateral common carotid arteries were isolated and subsequently stenosed using 0.18 mm steel micro coils (Wuxi Samini/Sawane Spring Co., Ltd.; Hamamatsu City, Japan). For sham-operated mice, a similar procedure was followed, but micro-coils were not used for BCAS induction.
All the experimental groups were randomized, and all the outcome analyses were carried out by independent investigators blinded to the treatment conditions and mouse types. Randomization of each experimental group was performed before the surgical procedure using the random number generator in GraphPad. For TEM and the eight-arm maze experiments, preliminary data indicated that six and twelve animals per group, respectively, would suffice to obtain 80% power at a significance level of <0.05 with a two-sided test.
Cerebellum slice culture and experimental design
Cerebellar organotypic slice culture from postnatal day 8-9 (P8-9) mice were used to investigate hypoperfusion-induced demyelination. Briefly, 400 μm P8-9 mouse cerebellum parasagittal slices were obtained using a Tissue Slicer (ZQP-86; Zhixin Co., Ltd.; Shanghai, China). The slices were placed on cell culture inserts (Millipore, Bedford, MA, USA) and cultured in 50% DMEM with 25% HBSS, 25% horse serum, and 5 mg/ml glucose (Invitrogen, Carlsbad, CA, USA) in cell culture chambers at 37 °C.
Oxygen and glucose deprivation is a classic model for studying tissue ischemic responses to mimic the whole brain hypoperfusion, we used modified low glucose and low oxygen (LGLO) treatment for 48 h. Experiments and data analyses were performed in a double-blinded manner.
SNPH shRNA and AAV. SNPH-shRNA construction and stereotactic injection
shRNA targeting SNPH (Nosc-41370-SH, Santa Cruz Biotechnology, USA) was transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. 72 h after transfection, slices were processed for LGLO treatment. Western-blotting and immunostaining were subsequently performed for further analysis.
We further used the shRNA bought from Santa Cruz Biotechnology to synthesize the AAV. SNPH-shRNA with the hSyn promoter (GeneChem, Shanghai, China), and the synthesized AAV. SNPH-shRNA or AAV. control-shRNA was stereoscopically injected into lateral ventricles. Briefly, mice were anaesthetized using 4% isoflurane in 30% O2 and 70% N2 and maintained on 2% isoflurane in 30% O2 and 70% N2 using a mask. AAV vectors were infused into the left lateral ventricle (coordinates from bregma: AP, −0.2 mm; ML, +1.0 mm; DV, −2.3 mm). 5 × 1011 genome copies were infused at a rate of 4 μl min-1, after which the needle was left in place for 2 min to prevent backflow before the withdrawal.
SNPH overexpression lentivirus construction and administration
SNPH overexpression plasmid and lentivirus was constructed by Genomeditech (Genomeditech, Shanghai, China). Lentivirus was added to the slice medium at 1*108TU/ml 72 h before LGLO treatment. Western blotting and immunofluorescence were subsequently performed for further analysis.
Time-lapse imaging using confocal microscopy
Mitochondria were labelled with CMXRos after brain slices were treated with LGLO. The distribution, size, and mean transport velocity of CMXRos-positive mitochondria in myelinated and demyelinated Purkinje cell axons were imaged using the Olympus inverted confocal microscope as described previously2. Briefly, 3 h before imaging, slice medium containing 100 nM Mito Tracker (M7512, ThermoFisher, USA) was added to the slice chambers. Then, slices were washed thrice in PBS and replenished with normal culture medium for further live imaging in an airstream incubator at 37 °C using a 60x 1.3 NA oil immersion objective with 512 x 512-pixel resolution.
Upon imaging, a total of 5 min with 5s intervals were imaged for each experiment. Total live imaging time was restricted to 20 min to minimize phototoxic damage. Lengths, areas, and diameters of axonal mitochondria were measured by ImageJ (NIH, USA). The number and mean velocity of motile mitochondria were analyzed by kymographs. Stationary sites in this study were defined as CMXRos-positive profiles that were stationary during a 5-min period. To measure the size of the stationary sites, a pair of image stacks, including all CMXRos-positive profiles of each axon, were obtained at time 0 and time 5 min.
Brain slices were fixed overnight in PBS containing 4% formaldehyde and then in 30% sucrose for 2 days at 4 °C. For cultured neurons grown on polylysine (PLL) coated glass, cells were blocked with PBS containing 5% BSA for 1 h after three washes with PBS and permeabilization with 0.1% Triton X-100 in PBS for 15 min. Primary antibodies diluted in blocking buffer were added and incubated at 4 °C overnight. We used anti-NF (1:50, ab8135, Abcam, USA), anti-MBP (1:200, ab40390, Abcam, USA), anti-Caspr (1:100, ab252535, Abcam, USA), anti-Nav1.6 (1:200, ASC-009, Alomone Labs, Israel), and anti-panNfasc (1:100, MABN621, Sigma Aldrich, USA) antibodies. Slices were washed with PBS three times and labelled with a fluorescence-conjugated secondary antibody for 1 h (Alexa Fluor 488 and 594, 1:1000, Life Technologies). Nuclei were visualized with DAPI (28718-90-3; Sigma Aldrich, USA).
For ROS staining, MitoSOX™ Red mitochondrial superoxide indicator (M36008, ThermoFisher, USA) was used to label ROS in mitochondria. Briefly, MitoSOX was diluted according to the manufacturer’s instruction and incubated with the slices for 3 h. Thereafter, the slices were washed with PBS three times and replenished with the indicated culture medium. After LGLO treatment, slices were fixed with 4% formaldehyde overnight and 30% sucrose for 2 days at 4 °C. Subsequent immunostaining was performed as described above.
Brain slices were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) with protease inhibitor. Equal amounts of proteins measured using the BCA method were loaded and analyzed after 15% Bris-Tris NuPAGE and then transferred onto nitrocellulose membranes. The membranes were incubated overnight at 4 °C with following primary antibodies: anti-SNPH (ab69992, Abcam), anti-Miro1 (ab188029, Abcam), anti-Trak1 (ab28751, Abcam), anti-HSP60 (ab190828, Abcam), anti-Caspr (ab216144, Abcam), anti-MBP (ab77924, Abcam), anti-NF155 (ab186734, Abcam), and anti-β -actin (ab115777, Abcam) at a dilution of 1:1000.
Transmission Electron Microscope
Transmission electron microscope (TEM) was performed as previously described13. In brief, brain samples were perfused with 4% formalin and fixed in formalin overnight. Dissected tissues (1 mm in thickness) were post fixed in buffered OsO4, dehydrated in graded alcohol solutions and propylene, embedded in Epon, and examined by light microscopy after toluidine blue staining. Thin sections cut on formvar-coated slot grids and stained with uranyl acetate and lead citrate were examined using a JEOL 1200 electron microscope. G ratios were determined as the inner to outer axonal circumference ratio using ImageJ.
Eight-arm radial maze test
The eight-arm radial maze test was performed as previously described14. The maze consisted of a central platform (24 cm in diameter) with eight arms that extended radially. Rats were allowed to visit each arm to eat eight pellets in food cups placed near the end of each arm. Each test animal was trained once per day to memorize the apparatus. The performance of test animals in each trial was assessed using two parameters: number of correct choices in the initial eight chosen arms and number of errors (defined as choosing arms that had already been visited). When the test animals made seven or eight correct choices and no more than one error in three successive sessions, they were deemed to have memorized the maze.
Whole-cell patch-clamp electrophysiology
Cultured brain slices were transferred to the bath solution for 1 h prior to recording. The bath solution contained 126 mM NaCl, 2.5 mM KCl, 26 mM , 1.25 mM , 2 mM , 2 mM , and 11 mM glucose was bubbled with 95%+5%; the temperature of bath solution was maintained at 35 ℃. For mEPSC recordings, patch pipettes contained 126 mM K-gluconate, 4 mM KCl, 4 mM ATP-Mg, 0.3 mM GTP-, 10 mM PO creatine, 10 mM HEPES, and 0.2-0.5% biocytin (pH 7.3 adjusted using KOH, 300 mOsm maintained using sucrose) with a tip resistance of 6–8 MΩ. During mEPSC recording, TTX (0.5 μM), was administered to silence network activity through inhibition of voltage-sensitive sodium channels, and bicuculline (10 μM) was given to block the GABA-A-mediated inhibitory signalling.
For APs recording, patch pipettes contained 140 mM K-gluconate, 5 mM EGTA, 0.5 mM , 2mM ATP-Mg, 0.3 mM GTP-, 10mM sucrose, 10mM HEPES, and 0.2-0.5% biocytin (pH 7.3 adjusted using KOH, 300 mOsm maintained using sucrose) with a tip resistance of 6-8 MΩ.
Series resistance was monitored in 2 min intervals, and recordings were excluded if the series resistance and leak current changed significantly and/or exceeded 40 MΩ or 200 pA, respectively.
Cerebral blood flow measurement
Cerebral blood flow measurement was performed as previously described15. Briefly, mice were anaesthetized using 4% isoflurane in 30% O2 and 70% N2 and then maintained on 2% isoflurane in 30% O2 and 70% N2 using a mask. The temperature of the animals was maintained at 37 °C using a heating pad and a feedback control system (FHC; Bowdoin, ME, USA). A midline incision was made on the skin of the skull, and a laser Doppler probe was fixed in place (5 mm lateral and 2 mm posterior to the bregma). Each measurement was repeated three times.
Data were analyzed using SPSS Statistics 20 and graphed with GraphPad Prism 6.0. The sample size was calculated based on power as 0.95 and α as 0.05. All the data from mice are represented as the mean ± SD and data from brain slides are represented as mean ± SEM. Different treatment groups were evaluated using one-way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons or two-way ANOVA to determine differences among individual groups. The null hypothesis was rejected when P-value was <0.05.