Live imaging of platelets and neutrophils during antibody-mediated neurovascular thrombosis

Key Points Live imaging of cellular events in the blood circulation reveals firm and transient interactions of platelets with neutrophils. In response to immune complexes, platelets aggregate and neutrophils adhere to the cerebrovascular endothelium of FcγRIIATGN mice.


Complete blood analysis
Blood samples from mandibular veins of C57BL/6J and Ly6gCre +/-::Rosa26-TdT +/-::CD41-YFP +/mice were collected in EDTA tubes. Complete blood analysis was performed by the animal facility services (Centre Hospitalier Universitaire de Québec-Université Laval) with the scil Vet abc Plus+ hematology analyzer (Heska). GraphPad Prism 9 software was used for the statistical analysis. Groups were compared using Two-way ANOVA with Sidak's multiple comparison test.

Immune complexes injection model
Purified human IgG (Purified Human IgG, MP Biomedicals, Solon, OH, USA) were heat aggregated for 60 minutes at 62°C and used as a surrogate for immune complexes as previously described. 1 Heat aggregated IgG were injected intravenously (600 μg and 750 μg) in males and females, respectively.

Intravital Microscopy
Platelet and neutrophil interactions in the vasculature of the brain were examined using high-speed widefield fluorescence microscopy. All videos were acquired with an Olympus BX51WI workstation equipped with a Fluorescence Excitation Illumination System (Lumen 300-LED, Prior Scientific) using an Olympus 20 x water objective with a numerical aperture of 0.50. The microscope was equipped with a dual emission image splitter (Optosplit II system) and a fast acquisition camera (Digital CMOS camera ORCA-Flash4.0 V2, C11440-22CU, Hamamatsu) that permitted a high capture rate of 100 frames per second. Nirvana software (BliQ Photonics) was used to acquire the videos.
Cranial windows were installed by the animal facility services (Centre Hospitalier Universitaire de Québec-Université Laval) 4 to 6 weeks before the first intravital imaging period. Prior to the intravital imaging session, mice were anesthetized with 1,5-2%% isoflurane (vol/vol) with oxygen (0.5-1 L/min), restrained with a custom-built cranial stereotaxic apparatus (Davis Kopf Instruments) and then placed under the microscope (Fig.S2A). A heat-pad was used to maintain the body temperature of the mice. Ophthalmic ointment was applied to avoid ocular dryness (at the beginning of the imaging session and every 30 minutes thereafter). Gelseal (Cytiva/Global Life Sciences Solutions Canada, Mississauga, Ontario, Canada) was used around the cranial window to form a watertight rim, and the imaging cavity was filled with water. A brief overview of the imaging region of the brain was performed before the intravenous injection.
For the first imaging period, the mice were injected in the tail vein only with the diluent (sterile phosphate-buffered saline (PBS) solution (Wisent BioProducts inc.)). After the injection, different regions in the imaging zone (approximately 19.5 mm 2 ) were filmed for a total of 60 minutes. The mice were awakened after the imaging session and returned to their cages. A second imaging period was performed 1 week after the first session, during which heat-aggregated IgG (immune complexes) were injected in the tail vein instead of the diluent. As for the first imaging period, different regions in the imaging zone were filmed for a total of 60 minutes post-injection for each animal.

Platelet aggregate analysis
Platelet aggregates were counted and measured with FIJI/ImageJ software. The number of neutrophils in each platelet aggregate were counted by hand. Data are presented as means ± SEM. OriginPro 2022 software was used for the statistical analysis. Groups were compared using Twoway ANOVA with repeated measures followed by a post-hoc Holm-Bonferroni test.

Computer analysis
On a total of 1326 videos recorded (>2 TB of data), two videos were selected for each mouse and each condition to quantitatively assess neutrophil interactions with the blood vessel wall. Intravital microscopy videos of the mouse brain microcirculation were stabilized for motion artifact 3,4 and fluorescent neutrophils were tracked over time. Bitplane® Imaris 7.6 software was used to track neutrophil positions. Recorded trajectories were then filtered with a Mathworks® MATLAB 2018b script and classified into the following categories: 'stationary' for neutrophils moving in area of less than 10 µm diameter, 'rolling' when neutrophils slowed down to 200 µm/s or less over a 10 µm distance or more, and 'circulating' in other cases (algorithm described in Figures S2C-D). The MATLAB program is available on the GitHub platform (hyperlink provided here prior publication). Results are presented as means ± SEM. OriginPro 2020 software was used for the statistical analysis. Animal groups were compared using Two-way ANOVA with repeated measures followed by a post-hoc Holm-Bonferroni test.

Video treatment for publication
After analysis, videos were processed with both Bitplane® Imaris 9.7 software and FIJI/ImageJ software for the publication's figures and videos. Deconvolution and background subtraction were used to improve the platelet and neutrophil signals (Fig.S2B). Brain autofluorescence was subtracted by eliminating the signal outside blood vessels to improve the quality of figures and videos. videos. The acquired images with the videomicroscope were used, without any treatment, to perform the platelet aggregates analysis and the computer analysis. Following the analysis, the images were treated with the plug-in "subtract background" with the FIJI/ImageJ software to reduce the noise observed in the blood vessels. The images were crop to the region of interest (with the best focus) and the deconvolution plug-in in Imaris was used to optimize the signal of platelets and neutrophils. The autofluorescence of the brain tissue was subtracted with FIJI/ImageJ manually. C. The video files are selected and processed througth the steps (A) to (I) of an algorithm briefly described as followed. Its purpose is to classify the neutrophils among three states: stationary, rolling, or circulating. Video S8. Neutrophils (red) adhere to blood vessels in response to immune complexes in the brain of FcγRIIA TGN ::Ly6gCre +/-::Rosa26-TdT +/-::CD41-YFP +/mice. Scale bars, 50 µm. Time following the injection of immune complexes is indicated in the inferior right corner. Real time speed. White arrows indicated examples of circulating platelets interacting with adhered neutrophils (kiss and fly interactions).