Controlled Cortical Impact
All procedures that involved animal work were approved by the Institutional Animal Care and Use Committee of Rutgers-New Jersey Medical School. Transgenic mice were group housed during pre and postoperative procedures with temperature control (23 oC), 12-h light-dark cycle and ad libitum access to food and water. 10-week old male mice, weighing at least 25 g, were anesthetized using isoflurane (3 % induction and maintenance 2 %) administered via a nose mask. Local anesthesia (buprenorphine 0.1 mg/kg, subcutaneous injection) was given, followed by a head shave. Animals were then secured on a stereotaxic frame (Stoelting Co. Wood Dale, IL), and a 20-mm midline incision was made over the skull. A unilateral craniectomy was performed between Bregma and Lambda using a hand drill with a 5-mm diameter trephine. A special care was given to stop the drilling when the spongy bone appeared to prevent damage to the dura mater. The bone flap was removed carefully with a pair of Dumont forceps, and using the Impact One (Leica, Concord, Ontario), the animal was given an impact with a 4-mm stainless steel impactor tip, at coordinates AP - 2.26 mm, ML + 2.0 mm and depth of 0.65 mm at a rate of 4.0 m/s and a dwell time of 200 ms, at an angle of 0.4o. After the injury, the wound was cleaned and closed with 6-0 nylon sutures, the animal was removed from anesthesia and placed back in a cage over a heating pad, and the righting reflex was monitored for recovery. Sham animals went through the same procedures as CCI-injured animals, except for the craniectomy and the CCI, as it has been shown that craniotomy itself can cause inflammation 26.
Behavior analyses
Rotarod
To assess motor function after injury, a rotarod machine (IITC Life Sciences, Woodland Hills, CA) that has an accelerating rotating cylinder was used. Briefly, mice were trained on the apparatus 3 days (3 trials per day, ITI ~ 30 min) prior to CCI or sham surgery, and the last training day was considered the baseline. The test was performed 1, 3 and 5 days post injury, and the latency to fall was measured in each trial.
Barnes maze
On 7 – 11 days post injury, mice (n = 4 for shams and n = 8 for CCI) were trained on a spatial reference memory task in the Barnes maze 27-29. The maze consisted of a white circular platform 100 cm in diameter, elevated 85 cm from the floor with 16 equally spaced holes of 5 cm in diameter along the circumference of the maze. Three visual cues were located on the wall that surrounded the maze. Under one of the potential escape holes, a black plexiglass box was placed for mice to escape. The location of the escape box was consistent for a given mouse, but different across the group. The potential intra-maze cues were abolished by rotating the maze in each trial, while keeping the relative locations of the escape box to the visual cues constant. To remove olfactory cues, the maze was wiped with 70 % ethanol. An incandescent light was placed above the maze to lit up the maze with a light level of ~ 400 lux.
For habituation, mice were placed in an adaptation box (20 x 15 x 15 cm) in the center of the maze for 1 min, then the box was lifted. The animal was then gently guided to the escape box and when they entered, the escape box covered, and the light was turned off. Mice were kept in the escape box for 2 min, and then placed back in the cage. After the habituation period, mice went through the acquisition phase, where they explored the maze until they find the escape box or 3 min has passed, whichever came first. Each mouse performed 2 trials per day (30 min ITI) for 4 consecutive days. On the 5th day, reference memory function was measured by performing a 60 seconds long probe trial where the escape box was removed and the duration of time mice spent in each of 4 quadrant (target, east, north, south) was measured.
Western blot analysis
The injured brain tissues from the ipsilateral hemisphere were carefully dissected under a dissecting microscope, and then homogenized in buffer solution containing M-PER Mammalian protein extraction reagent 5 mM Na3VO4, 1 mM NaF, 1 mM Na2P2O7, 1 mM Benzamidine, 5 mM EDTA, and HALT Protease Inhibitor Cocktail (Thermo Fisher scientific, Waltham, MA). Total protein concentration was estimated using a BCA kit (Thermo Fisher scientific, Waltham, MA). Protein samples were separated by 4 – 20% SDS-PAGE and transferred to PVDF membrane (BioRad, Hercules, CA). The blots were incubated in Signal Enhancer HIKARI (Nacalai Tesque INC, Japan) containing primary antibodies against alpha II spectrin (Santa Cruz Biotechnology, Dallas, TX), MMP9 (NeuroMab, San Diego, CA), IgG (Thermo Fisher scientific, Waltham, MA) or GAPDH (Cell Signaling, Danvers, MA). Molecular mass was estimated with pre-stained protein marker what molecular weight range (BioRad, Hercules, CA). Blots were developed using an ECL kit (Thermo Fisher scientific, Waltham, MA) and visualized by LAS-3000 Imaging System (Fuji, Cambridge, MA). Densitometric analyses were performed using ImageJ.
Flow cytometry analysis of infiltrating leukocyte and microglia
To isolate microglia and infiltrating monocytes, brains were freshly dissected at 3 DPI and cells were dissociated to be analyzed by flow cytometry. Briefly, mice were anesthetized with ketamine and then transcardially perfused with ice-cold saline solution containing 20 U/mL heparin. Undamaged brain hemispheres from sham animals or ipsilateral hemispheres from CCI-injured brains were carefully dissected, minced (~1mm in diameter) in cold Hanks’ Balanced Salt Solution using scalpels, and then the tissue was passed through 70 µm cell strainer. Brain tissue was centrifuged at 300 – 400g at 4 oC for 5 min, followed by digestion in 2 U/mL Liberase TL (Sigma Aldrich, city, state) for 1 hour at 37 oC. Subsequently, cells were washed with 666 U/mL DNAse (Worthington Biochemical Corp., Lakewood, NJ), passed through 100µm cell strainer, followed by centrifugation at 300-400 g at 4 oC for 5 min. Cells were then identified by surface expression of CD11b, CD45, Ly6C and Ly6G. To block the FCγRs, cells were preincubated with anti-mouse CD16/CD32 antibody (BD Biosciences, San Jose, CA) for 15 minutes at 4oC, and then incubated with PerCP -Cy5.5-anti CD11b antibody (BD Biosciences, San Jose, CA), PE-Cy7-antiCD45 antibody (Thermo Fisher Scientific, Waltham, MA), BV510-anti Ly6C antibody (Biolegend, San Diego, CA) and APC-anti Ly6G antibody (Thermo Fisher Scientific, Waltham, MA) for 20 min at 4 oC in the dark. Cells were analyzed by LSRII flow cytometer (BD Biosciences, San Jose, CA), and then analyzed by FACS Diva software. Gating strategy is described in supplementary Fig 2 and 3.
Assessment of Panx1 channel activity via dye uptake
To isolate microglia and infiltrating monocytes, brains from Cx3cr1EGFP/Cre :: Panx1fl/fl mice were freshly dissected at 3 DPI and cells were dissociated to be analyzed by flow cytometry. Briefly, mice were anesthetized with ketamine and then transcardially perfused with ice-cold saline solution containing 20 U/mL heparin. Undamaged brain hemispheres from sham animals or ipsilateral hemispheres from CCI-injured brains were carefully dissected, minced (~1mm in diameter) in cold Hanks’ Balanced Salt Solution using scalpels, and then the tissue was passed through 70 µm cell strainer. Brain tissue was centrifuged at 300 – 400g at 4 oC for 5 min, followed by digestion in 2 U/mL Liberase TL (Sigma Aldrich, city, state) for 1 hour at 37 oC. Subsequently, cells were washed with 666 U/mL DNAse (Worthington Biochemical Corp., Lakewood, NJ), passed through 100µm cell strainer, followed by centrifugation at 300-400 g at 4 oC for 5 min. Cells were resuspended in 300µl DMEM/1%BSA, and then co-incubated with BzATP (300 μM, Sigma) and 1 μM To-Pro-3 for 30 minutes, followed by flow cytometry analysis. Cells were analyzed by LSRII flow cytometer (BD Biosciences, San Jose, CA), and then analyzed by FACS Diva software. Gating strategy is described in supplementary Fig 2 and 3. For the analysis by confocal microscopy, cells were co-incubated with BzATP (300 μM, Sigma), 1 μM To-Pro-3 and DAPI (1:10,000). Time-series acquisitions were obtained for 25 minute time period, every 5 minutes. Cells that were positive for DAPI were excluded from the analysis. To-Pro-3 dye fluorescence emission (642/661) was detected at 37oC using appropriate lasers.
Magnetic Resonance Imaging (MRI) analysis of Mouse Brain after CCI
MRI scanning was performed at 6 DPI using M2™ Compact High-Performance MRI (1T) scanner (Aspect, Israel). A custom-made MRI compatible head holder was used to secure the animal, which was anesthetized with 1.2 % isoflurane in oxygen for the duration of the procedure. Mice were initially scanned with a Fast Spin Echo to determine the brain, ventricle and wound size. To measure blood brain barrier leakage, mice were pre-scanned with Gradient Spin Echo, dosed with Magnevist (0.1 mmol/kg, tail vein), and then scanned again, followed by volumetric analysis to determine the volume of dye leaked into the parenchyma.
Statistical analysis: Values are represented as mean ± standard deviation. Comparisons between groups were made using linear regression analysis using treatment and the interaction between treatment and time as factors(Rotarod and Barnes maze). Paired Student´s t-test (Magnevist uptake), one-way ANOVA (western blot, flow cytometry) plus post hoc test were used as appropriate. The values were expressed as the means ± SD. The differences with p < 0.05 were considered statistically significant.