Animals
Young (8-12 weeks old) male mice were used in all experiments. Mice were kept on a 12 hour:12-hour light cycle at room temperature (22-25oC). Food and water were provided ad libitum. All experiments were done with the approval of the University of Virginia Animal Care and Use Committee.
Transgenic mice deficient in the enzymes elastase (B6.129X1-Elane tm1sds: elastase KO), myeloperoxidase (B6.129X1-Mpo tm1Lus: MPO KO) or neutrophil cytosolic factor 1(NCF- component of the NADPH oxidase complex (B6(Cg)-Bcf1 m1J: NADPH oxidase KO) (The Jackson Laboratories) were used. All transgenic mice used were on a C57BL/6 background; therefore, control experiments were conducted on C57BL/6J mice.
Subarachnoid hemorrhage (SAH)
SAH or sham surgeries were performed as previously reported(16). Briefly, mice were anesthetized using isoflurane, and placed in a prone position. A 3mm incision was made on the back of the neck along the midline, the atlanto-occipital membrane was entered, and a conserved subarachnoid vein punctured. Bleeding from the vein was allowed to stop on its own. For the sham experiment, the procedure was the same except the atlanto-occipital membrane was not entered and the vein was not punctured.
Myeloperoxidase (MPO) injection
MPO KO mice received intracisternal injections of biologically active MPO enzyme, H2O2 or MPO with H2O2 in the setting of SAH. Biologically active MPO (ab91116; Abcam, Cambridge, MA, US) was reconstituted in sterile phosphate buffer saline (PBS) to a stock concentration of 1 µg/µl. Per product sheet recommendation, MPO production of hydrochlorous acid (HOCl) requires a H2O2 concentration of 0.0012% per unit of activity. Mice were given a cisterna magna injection of either 0.8 µg of MPO, 0.8 µg MPO with 0.0012% H2O2 in PBS, or 0.0012% H202 in PBS at the time of surgery.
CD45-FITC injection
Mice were injected with 3 µg of anti-CD45-FITC antibody (Thermofisher, Waltham, MA, US) intravenously 30 minutes prior to euthanasia. Mice were then perfused with PBS and 4% paraformaldehyde. Meninges were dissected, stained for neutrophils with the anti-neutrophil antibody 7/4-FITC (Abcam, Cambridge, MA, US; 1:100), and imaged using an Olympus FV1200 confocal microscope and Fluoview software.
Flow cytometry
Flow cytometry was performed on both the brain parenchyma and meninges of WT and MPO KO naïve mice. To generate a single cell suspension for flow cytometry, the meninges and brains were collected and processed as previously described(17). Briefly, meninges and brain samples were dissected and processed in DMEM media with 10% bovine serum albumin (BSA). Samples were digested using 1 mg/ml DNAse and 1.4 U/ml collagenase in Hanks Balanced Salt Solution with magnesium and calcium. Samples were then incubated in a 37°C water bath, triturated and strained through a 70 µm cell filter. Meningeal samples were then centrifuged and resuspended in DMEM media with 10% BSA and kept on ice until staining. Brain samples were further processed using a 40% Percoll solution in PBS to remove the myelin debris. Following Percoll, samples were rinsed with DMEM with 10% BSA and kept on ice until staining. Once single cell suspensions were obtained, samples were blocked with Fc block and incubated with an antibody cocktail containing Ly6G-FITC, Ly6C-PeCy7, CD11b-efluor 780 (Life Technologies, Carlsbad, CA, US; 1:200), CD45-Pacific blue (Biolegend, San Diego, CA, US; 1:200), and fixable viability dye-506 (Life Technologies, Carlsbad, CA, US; 1:1000) for 30 minutes at 4ºC. Samples were then analyzed using a Gallios cytometer (Beckman Coulter, Brea, CA, US), and FlowJo software (FlowJo, LLC, Ashland, OR, US).
Immunohistochemistry
Mice were transcardially perfused with 4% paraformaldehyde in PBS. Brains and meninges were dissected and processed separately for immunohistochemistry. Brains were post-fixed in 4% paraformaldehyde for 24 hours followed by cryoprotection in a 30% sucrose solution. Brains were then frozen and 30 µm sections collected using a cryo-microtome. Sections containing the hippocampus were selected, rinsed in PBS, and blocked in a 0.3% Triton-X with 5% normal goat serum solution. Sections were then incubated with rabbit anti-Iba1 (Wako Chemicals, Japan 1:500), anti-neutrophil antibody 7/4-FITC (anti-Ly6B) (Abcam Cambridge, MA, US; 1:100), chicken anti-GFAP (Abcam, Cambridge, MA, US; 1:1000), and rabbit anti-vimentin (Abcam, Cambridge, MA, US; 1:200) overnight at room temperature. The following day, sections were rinsed and incubated with AlexaFluor conjugated secondary antibody (ThermoFisher Scientific, Waltham, MA, US; 1:500) for 1 hour at room temperature, mounted on Superfrost Plus slides (ThermoFisher, Waltham, MA, US), and cover-slipped using Vectashield mounting medium with DAPI (Vector laboratories, Burlingame, CA, US).
Meninges were removed from the skullcap and kept in PBS until processing. Non-specific binding sites were blocked using PBS containing 1% BSA, 5% normal goat serum, 0.1% Triton-X, 0.2% tween, and Fc block(18). Meninges were then incubated in a primary antibody cocktail containing the anti-neutrophil antibody 7/4-FITC (Abcam, Cambridge, MA, US; 1:100) at 4ºC overnight.
Confocal image acquisition and analysis
All images were obtained on the Olympus FV1200 confocal microscope with Fluoview software. Each image was collected as a Z-stack. Prior to image analysis, all images were converted to a maximum intensity image by collapsing all stacks using ImageJ/Fiji (NIH, Bethesda, MD, US) and blinded for analysis. Image Analysis was then performed by a technician/student in the laboratory not aware of the treatment conditions.
Brain: Images of both the left and right CA1 regions of the hippocampus were obtained using a 20x oil immersion objective. Composite images containing the nuclear marker DAPI and the cellular stain of interest (Iba1 for microglia and GFAP/vimentin for astrocytes) were then generated. For cell counts, the multipoint tool was used to count all cell within an image. For fluorescent intensity, confocal images were converted to 8-bit images. Then using the measure tool, the fluorescence intensity was determined for each image. Counts for both left and right hippocampi were added and divided by 2 to generate an average, number of cells or fluorescence intensity, per case. Finally, using the ‘Coloc 2’ application for ImageJ/Fiji, a co-localization index for GFAP and Vimentin was obtained for each hippocampus. The values were also averaged for left and right hippocampi to generate a value for each case.
Meninges: In order to image both the meninges containing the transverse sinus and the meningeal parenchyma (fibrous portion), whole mounts of the meninges were stained for neutrophils (NT 7/4; Abcam, Cambridge, MA, US). Using 20x oil immersion objective, a 3 x 3 tile was obtained of the transverse sinus and the meninges. Each image was obtained as a Z-stack. Prior to analysis, a maximum intensity image was obtained for each stack, and a DAPI and NT 7/4 composite generated. To randomize data acquisition, a 9 x 9 grid was placed on each image within the tile and the number of neutrophils counted within every 6th square (starting from the left top square, 6 squares were counted (5 horizontally and 1 vertically)). As such, a total of 7 squares were counted per grid laid images for a total of 252 um2 area (36 um2 area/grid square) counted out of 4225 um2 area (image size). The number of cells was then added across all 9 images (from 3 x 3 tile) per case, divided by 252 (to generate # of cells/ um2), and multiplied by 4225 to generate a representative number of neutrophils present in each case.
Microglia Scholl analysis
Brain images of sections labelled with anti-iba1 antibody, were converted to 8-bit files. The iba1 fluorescence threshold of the images was set at 90% intensity, and a 10 x 10 grid was placed on each image. To randomize data acquisition and minimize bias, the fifth row of squares was cropped out and analyzed. Cells were only included in the analysis if fully located within the selected row. The ‘Scholl analysis’ application was then used to quantify the number of processes observed on selected cells using the protocol previously described (19).
Vasospasm
Previous studies from our lab show the presence of vascular spasm in the middle cerebral artery (MCA) of SAH mice early (at day 1) and, more importantly, in a delayed manner (6 days after the hemorrhage) corresponding to DCI in mice(5). Since the peak neutrophil infiltration occurs in our model 3 days post-hemorrhage and delayed vasospasm is correlated with DCI, we focused our vasospasm analysis on the delayed vasospasm occurring on the 6th day after SAH.
The arterial tree of each mouse was labeled using the vascular dye Microfil (Flow Tech, Carver, MA), and the cross-sectional MCA diameter was measured. Briefly, animals were anesthetized using sodium pentobarbital, transcardially perfused with 20 ml of cold PBS followed by 20 ml of cold 1% paraformaldehyde in PBS, then injected with 10 ml of Microfil dye. Brains were dissected, rinsed in PBS, then cleared using methyl salicylate (Sigma-Aldrich, St Louis MO US). The ventral surface of each brain was then imaged using a Leica DM 2000 LED light microscope. Because vasospasm leads to non-uniform constriction of arteries, there are affected and unaffected areas. The percent constriction was calculated as a ratio of the smallest diameter to the largest cross-sectional diameter of the MCA within a 2 mm segment distal to the posterior wall of the internal carotid using ImageJ and converted to a percentage.
Behavior analysis
Prior to surgery, mice were randomly assigned to treatment group using a random number generator. In addition, treatment conditions were blinded to the person administering the behavioral test.
Barnes Maze
Previous studies show that mice with SAH develop deficits in performance on the Barnes maze task 6-8 days after the hemorrhage(5). Therefore, in the present study, this task was used to assess the presence of cognitive deficits after SAH. Briefly, mice were habituated to the maze for 2 days before the surgery. Habituation was performed by placing mice in the middle of the maze and leaving them to explore for 5 min. Testing started on day three post-surgery and continued for 7 consecutive days thereafter. During testing, mice were placed in the middle of the maze and given a total of 300 seconds to find the escape hole. Each trial ended when the mouse entered the escape hole, stayed in the entry zone of the escape hole (defined by a circle of 5 cm diameter around the escape hole) for 5 consecutive seconds, or the 300 second maximum had elapsed. All trials were recorded and analyzed using the tracking software Ethovision 13 (Noldus, Leesburg, VA, US).
Rotarod
To determine whether the naïve MPO KO and WT mice showed different motor coordination and learning, mice motor performance was analyzed using the rotarod test. During this test, only the latency to fall was analyzed. In brief, mice were placed on the rod at low speed and left to acclimate for 60 seconds. Mice were then tested with a continuous increased speed on the rod for 5 consecutive minutes. Experiments ended when mice fell of the rod or the 5 min have elapsed. Each mouse was given 3 unique trials separated by a 30 minutes rest period in the home cage.
Primary cultures
Primary cortical neurons
Neuronal cultures were established using Thy1-GCaMP3 neonates as previous described(20,21). Briefly, cortices of P0 neonates were dissociated, in minimal essential medium (MEM) with 10% heat inactivated FBS and penicillin/streptomycin, using a 1000ul micropipette. Cells were filtered using a 70um cell filter and seeded on sterile 12 well plates coated with poly-D-lysine (0.1 mg/ml). Two hours after seeding, media was changed to neurobasal A media with B27, glutamax and penicillin/streptomycin. 25% of cultures media was subsequently exchanged every two days. Seven days in vitro (DIV) cultures were incubated with either 0.8 ug of MPO with or without H2O2 (0.0012%) or H2O2 alone. All cultures conditioned were imaged using a widefield Leica DM6000B microscope outfitted with a CCD camera. Before stimulation, a 5-minute time-lapse baseline video (at a rate of 2 frames per second) was obtained for each culture. Following the 5 min baseline recoding, neurons were stimulated with either MPO with or without H2O2 or H2O2 alone, followed by a 10minutes time-lapse recording. Cultures were left to acclimate to the stimulus for 2.5 hours. Cells were then stimulated with 10mM potassium chloride (KCl) solution. The first 10 min of the KCl stimulation were also recorded. Prior to analysis, all time-lapse recordings were blinded. Using Imaris (Bitplane, Concord, USA), 8-10 cells were chosen at random and a maximum fluorescent intensity value was generated for each cell at each frame acquired in the time lapse video. The values were plotted on excel and firing rate (peaks) were counted before and after stimulation in each experimental group (control, MPO, MPO+H2O2, and H2O2). Percent baseline values were then determined by dividing the firing rate of each cell after the stimulation to the firing rate before the stimulation. Values were averaged across each cell to generate a single value for that time lapse recording.
Primary astrocyte culture
Primary astrocyte cultures were established as previously described(22). In brief, brain cortex was collected from P0 pups, dissociated in 2.5% Trypsin in HBSS solution, and vigorously pipetted using a 10ml serological pipette to generate a single cell suspension. Cells were then spun and resuspended in astrocyte media (DMEM, high glucose, 10% heat inactivated FBS, and penicillin/streptomycin). Cells were then platted on T75 culture flask and incubated at 37 degrees. 2 day after platting, media was changed and every 3 days thereafter. On the 7th day DIV, culture flask was shaken and rinsed to remove microglia and oligodendrocyte progenitor cells. The generated pure astrocyte culture was further incubated for 12-14 days, after which the culture was split. 2 days after the split, cultures were detached, rinsed and transferred to 15mm culture dish for testing. As with the primary neuronal culture, astrocytes cultures were imaged, exposed to 3 experimental conditions, MPO, MPO with H2O2, or H2O2 alone, then incubated for 4 hours. At the end of the incubation period, cultures were imaged. Cells were counted in the before and after images to determine the toxic/morphological effect of each experimental condition on astrocytes.
Of note, for both neuron and astrocyte cultures, each mouse brain was plated on 12 culture dishes (or 1 12-well culture plate). Triplicates of each experimental condition (control, MPO, MPO+H2O2, and H2O2) were performed for each mouse. For analysis, data was collected from each triplicate, and averaged to generate a single data point for a given mouse brain.
2-photon imaging of neuronal calcium activity
All imaging experiments were performed on a Olympus FVMPE-RS system equipped with sensitive GaAsP detectors and a resonant scanner. A cranial window was prepared on Thy1-GCaMP3 mice as previously described(23). In brief, mice were anaesthetized, mounted on the stereotactic instrument, and the skin on the scalp removed to expose the skull. A 3mm diameter circle was made in the parietal bone with a microdrill 2 mm lateral and 2 mm caudal to the bregma. The edge of the circle was further thinned by drilling until the circled bone cap was able to be flipped and removed with fine-tipped forceps. A few drops of 0.9% NaCl saline solution was applied on top of the exposed dura mater. A small area of dura was carefully cut with a fine needle and a solution of drug (MPO or saline) applied directly to the cortex. The cranial window was covered with a glass coverslip, sealed at the edges with super glue. To facilitate the injection of KCl into the cortex, a 1mm diameter burr hole was prepared 3 mm caudal and lateral to the cranial window. A flattened nail (used as a handle to orient mouse head during imaging) was horizontally glued the contralateral occipital bone.
The cranial window implanted mice were kept under anesthesia for the duration of the imaging session. 3 unique regions were imaged within the cranial window separated by a 10 minutes interval. Images were acquired using a Olympus 25X NA 1.05 objective lens with an 920 nm infrared laser. The line averaging on the resonant scanner was set to 6 to obtain images with 512X512 resolution and 2.5 Hz acquisition speed. A target field of view was selected at a depth between 100 nm and 200 nm and continuous 5 minutes-long T-series imaging session was recorded.
To induce KCl-triggered spreading depolarization, a 20 um diameter pulled micropipette was filled with 1 M KCl, mounted on a nanoliter injector (WPI) and inserted into the burr hole. For imaging, a 1-minute baseline was first recorded. Followed by cortical spreading depression generated by the application of 100 nL of 1 M KCl to the cortex though the burr hole, and 4 additional minutes of recording.
Each generated file was blinded and then transferred to the Imaris software (Bitplane, Concord, USA). Within each image, 10 individual neurons were selected at random. Maximum fluorescent intensity was collected for each cell at every frame during the recording. Values were averaged across all cells and the 3 unique field of view for each mouse to generate a single data point for each given mouse. Finally, increase fluorescence from baseline (peak and depression) were calculated across groups to determine whether the addition of the MPO, saline or SAH affected the neuronal response in cortical spreading depression.
Statistics
Graphpad Prism 8.1 (Graphpad, La Jolla,CA, US) software was used to analyze all of the data obtained. Student’s t-test or analysis of variance (ANOVA) were used to determine whether differences between treatment groups were statistically significant. Significance was attributed to p values less than 0.05.