Mice
C57BL/6 mice, 8–10 weeks old (18-20g), were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). Animals were housed under standardized light-dark cycle and provided with free access to food and water. QuickCalcs (GraphPad Software, La Jolla, CA, USA) was used to randomly assign animals into each treatment group. The coagulation time and bleeding time were measured in the same cohort of mice subjected to the measurement of flow velocity. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Animal Care and Use Committees of Tianjin Neurological Institute (Tianjin, China).
Animal preparation for in vivo imaging
Surgical preparation for in vivo imaging was performed as previously described[13, 14]. Briefly, mice were initially anesthetized by 4.5% isoflurane inhalation which was then maintained at 1-1.5% for the remainder of the study. Animals were placed in a stereotactic frame. Animal heads were immobilized and body temperature was recorded and maintained at a 37°Cusing circulating warm-water blanket. A midline scalp incision was made, and the periosteum was removed from the skull, exposing bone suture markings. Hemostasis was established in the soft tissues with monopolar cautery and in the bone by Gelfoam strips. A cranial window (diameter 6mm) was created over the right and left frontoparietal cortex with the use of a drill. The skull and dura were removed and covered with a removable cover glass (diameter 8.0 mm) that was glued to the skull using dental acrylic cement to cover the craniotomy window. The space between the exposed brain surface and the cover glass was filled with PBS.
2-photon laser scanning microscopy
A customized microscope attached to a laser generator with 680- to1020-nm tuning range (Model: Chameleon coherent, UK), a multiphoton laser scanning microscope (FVMPE-RS, Olympus, USA) operated by FluoView software (FV31S, Olympus, USA), and a 25X water-immersion objective lens (NA1.05, Olympus, USA) were used for in vivo imaging. Texas Red-dextran (MW: 70 kDa, 0.1 mL of 1% in saline; Invitrogen, USA) via the tail vein was used to visualize the vasculature and detect the motion of red blood cells (RBCs) using two-photon microscopy[15]. In each animal, cortical arterioles (diameter: 30–50µm, subsurface depth: 10–20 µm) were imaged via z-scans and flow velocity was determined by repetitive line-scans performed along the central longitudinal axis of the selected vessels[16]. Linear shadows as a result of nonfluorescent particles within the vessel lumen was used to compute RBC velocity, which was proportional to the slope Δx/Δt.
Localized vascular injury was produced by laser irradiation as previously described[14]. Arteriolar injury was induced by delivering multiple pulse laser-irradiation on the arteriole (~ 30 x 50 µm ellipse field) for ~ 6 minutes in a wavelength of 920 nm until thrombus formed and occupied the entire luminal cross-section. The onset of thrombosis was identified by the dilatation of the irradiated segment, the emergence of bright fluorescence along the vessel wall, and by a nonfluorescent mass within the irradiated arteriole. The laser pulse energy which ranges between 0.1 to 0.6 mW weak enough to avoid dye extravasation and vessel rupture. If there was occurrence of vascular rupture, the animal was not used.
Thrombus volume was measured by 3D image using two-photon microscope. Whole thrombus was scanned every 1 µm from top to bottom under two-photon microscope at 60 min after thrombosis and up to 90 min after vehicle or Ozanimod treatment. Thrombus volume were determined by measuring the area of thrombus in a series of scanned sections, and then multiplying the section thickness (1 µm).
Drug administration
Animals with successful arteriolar thrombosis were randomly treated with tPA, ozanimod or vehicle by intravenous tail vein injection. Ozanimod (MCE, Monmouth, NJ) was dissolved in 5% DMSO + 0.9% saline and stored at 4°C as we previously described[17]. Mice were treated with Vehicle (100µl), Vehicle (50µl) + low dose tPA (5mg/kg, 50µl) or Ozanimod (0.6mg/kg, 50µl) + low dose tPA (5mg/kg, 50µl) at 60 min after vascular injury induced by laser irradiation.
Assessments of bleeding time, coagulation time
Mouse tails were transected at 2 mm from the tip with a scalpel blade and immersed in a 15 ml clear tube with normal saline at 37°C. Bleeding time was determined as time to cessation of bleeding within a 20 min observation period[15]. Bleeding was considered stopped if no bleeding was observed for 30 s. For the coagulation time assessment, blood was drawn from the angular vein into a 1 mm diameter glass capillary after anesthesia. The capillary tube was filled completely full of blood, then laid flat on the bench top. Next, a small section of the end of the capillary tube was carefully snapped every 30 s until a fibrin thread appeared. Both bleeding time and coagulation time were measured at 90 min after treatment with Ozanimod (0.6 mg/kg, i.v.) or vehicle.
Immunostaining
After two-photon imaging, mice were sacrificed and perfused with 10 ml cold PBS, followed by 10 ml 4% paraformaldehyde perfusion solution. Brain tissues were harvested and fixed in 4% paraformaldehyde overnight, and then dehydrated with 30% sucrose. Frozen brain tissues were sectioned (20 µm thickness) and incubated for 30 min at room temperature in blocking buffer (5% goat serum, 1% BSA, 0.3% Triton X-100). Brain sections were then incubated with primary antibodies against mouse CD45 (1:200, 13917 Cell Signaling, Danvers, MA) or CD3 (10 µg/mL, MAB4841 R&D, Minneapolis, MN) at 4°C overnight. After washing in PBS, brain sections were incubated with appropriate fluorochrome-conjugated secondary antibodies at 4°C overnight. DAPI (H-1200, Vector, Burlingame, CA) was used to counterstain cell nuclei. Confocal Z stacks were acquired from thrombus area labeled by Texas-Red-Dextran using a Zeiss 710 microscope with ×20 objective, NA 0.8 (Oberkochen, Germany). Data were analyzed with Image J software (National Institutes of Health, Washington, DC).
Statistical analysis
Randomization was performed using GraphPad QuickCalcs. Measurements and data analysis were performed by investigators blinded to experimental treatments. Statistical significance was determined by two-tailed unpaired Student’s t-test for two groups, one-way analysis of variance (ANOVA) followed by Tukey post-hoc test for multiple groups, or two-way ANOVA accompanied by Bonferroni post hoc test for multiple comparisons. Statistical analysis was performed using Prism 8.0 software (GraphPad, San Diego, CA). Data are shown as mean ± SD. Values of p < 0.05 were considered significant.