Mice were randomly assigned to no treatment, a single dose of AZD5904 (180 μg/kg) through the intrathecal (IT) or intraperitoneal (IP) route 3 days after hemorrhage.

Mice were randomly assigned to receive either AZD5904 (20 µmol/kg) in dimethyl sulfoxide or phosphate buffered saline/dimethyl sulfoxide (placebo) IP. Mice began treatments 2 days after the hemorrhage/sham and continued daily for four doses. Daily doses were given 2 h after the completion of testing.

Six days after SAH, the mice were euthanized, the skull cap was removed, and the meninges were dissected. The meninges were stained with monoclonal antibodies against CD31 (ab182981; Abcam, Cambridge, UK) and Ly6G-FITC (AB53453; Abcam, Cambridge, MA). Tiled images were obtained on the Olympus FV1200 confocal microscope with Fluoview software (Olympus, Tokyo, Japan). Each image was collected as a Z-stack. All images were converted to a maximum intensity image, by collapsing all stacks, reconstructed using “surface” and “spots” features using the Imaris software (Oxford Instruments, Abingdon, UK). Using the “distance from surface” feature of Imaris, the number of neutrophils observed at graded distances from the nearest blood vessel was obtained. Specifically, the distance from the center of the nearest blood vessel (the center point in line with the target neutrophil and side walls of the blood vessel measured as the (distal side wall position-proximal side wall position/2) to the neutrophil was measured for all neutrophils and plotted on a distance-incidence plot.

The Barnes maze test is an elevated circular maze with 20 holes with a black escape box which did not change in location throughout the test. There are visual cues in the room to orient the test mice. The environment in the testing room included nondirect white light, high frequency (15,000 kHz) sound at 75 dB at a fixed source, and nondirect air movement. Starting each trial, a mouse is placed at the center of the maze. The trial was terminated at the point at which the mouse entered the box, stopped at the box for > 2 s, or did not find the box in 180 s. Mice were trained for 2 days before surgery to learn the placement of the escape box. Day 2 after surgery or sham, mice began daily testing until day 10 (Fig. 2a). Day 2 was considered a training day, and data collection started on day 3. Data were gathered using video tracking software (EthosVision; Noldus, Leesburg, VA). Parameters collected were latency to goal box, total distance traveled, heat maps of position, and mean velocity. Each animal underwent four trials each day. The mean performance on the trials each day was used for analysis.

Neutrophils do not migrate out of blood vessels into the meninges after SAH with MPOi. a Quantitative analysis of neutrophils in the meninges comparing three groups: SAH with placebo, SAH with MPOi administered by IT injection, and SAH with MPOi administered through IP injection (example image and rendering). b Number of neutrophils by proximity to the nearest blood vessel measured in Å. There is a significant decrease in the distance neutrophils travel away from the blood vessel (0Å) in the IP MPOi compared with the placebo. c, The percentage of neutrophils found inside blood vessel (0 Å) across the same three categories: SAH, SAH MPOi IT, and SAH MPOi IP show a difference between SAH and SAH AZD5904 IP. Å, angstroms, IP, intraperitoneal, IT, intrathecal, MPOi, myeloperoxidase inhibitor, SAH, subarachnoid hemorrhage

Graphpad Prism 8.1 (Graphpad, La Jolla, CA) software was used to analyze all the data obtained. Scholl analysis was adaptive to evaluate the distance from the blood vessel to each of the neutrophils in the meninges [23]. The Mann–Whitney U-test and two-way analysis of variance (ANOVA) were used to determine significant differences between treatment groups (p < 0.05). No outliers were identified in the dataset by the robust regression and outlier removal method (ROUT).

For behavioral studies, our previous studies have shown that significant behavioral changes on the Barnes maze test by on the 10th day of the trial in mice after SAH compared with control are seen with six animals. The assessment was based on the following: Effect size 127 s, standard deviation 64.5 s, α 0.05, 0.90 with an expected sample size of 6. Therefore, a minimum of six animals was used for each experimental condition.