All experimental procedures for this study were approved by a Monash University Ethics Committee (MARP/2017/144) and (MARP/23298) and performed in accordance with the National Health and Medical Research Council of Australia and ARRIVE guidelines for the care and use of animals in research. A total of 65 male FVB/N and 47 male C57Bl/6 mice (8 to 12 weeks old) obtained from the Monash Animal Research Laboratory were included in this study (Monash University, Clayton campus). All mice were maintained on a 12-hour light/dark cycle with free access to water and food pellets and were housed in specific pathogen free cages.
Inclusion criteria and blinding:
To be included in the current study, mice needed to survive for the 7 days post stroke and to have a measurable infarct. In total, 26 mice were excluded as they did not meet this criteria or were euthanised at an earlier time point due to animal welfare.
Experimenters were blinded to all treatments.
Photothrombotic stroke surgery
The PT stroke surgery was performed as previously described by Labat-gest and Tomasi. However, to obtain a concentration-response to light exposure, the light source was turned on for either 15, 18, or 20 minutes in both the C57Bl/6 and FVB/N strains of mice. Sham mice were subjected to the same procedure but did not receive the intraperitoneal (i.p) injection of rose Bengal. For a full description of the PT surgical procedure, refer to the Online Resource.
The hanging wire and adhesive removal tests were performed as an indicator of functional outcome. These tests were performed the day prior to stroke induction, to establish baseline performance, and then repeated at 24 hours, 72 hours, & 7 days post-stroke.
Hanging wire test:
The hanging wire test was used to assess forelimb strength and grasping ability, providing an indication of motor function. The test was performed as described by Hattori, et al., where mice were suspended by their forelimbs on a wire between two posts 45cm above a foam pillow. The time in seconds (s) for the mice to fall from the wire was recorded with a maximum test period of 300s. The test was repeated three times per mouse, with a 5-minute break period between each test. An average of the 3 individual tests was calculated and recorded as the latency to fall for that time point.
The adhesive removal test:
The adhesive removal test was used to measure sensory function and motor coordination . Mice were placed in an empty cage and allowed to acclimate for 2 minutes. Following this time, an adhesive sticker (8 mm diameter) was placed on each forelimb paw. The mice were then placed back in the empty cage and the time for the mouse to remove both adhesives was recorded. The maximum test period was 300s. The test was repeated three times per mouse with a 5-minute rest period between tests. The average of the 3 individual test periods was calculated and recorded as the time to remove adhesive for that testing period.
Harvesting of tissues:
On day 7 post-stroke, mice were euthanised with inhaled anaesthetic isoflurane (in 2 - 4% oxygen; Issorane, Baxter, Baxter Healthcare Pty. Ltd). Brains were removed from the skull and snap frozen using liquid nitrogen. The spleen and thymus were then removed, weighed, and snap frozen using liquid nitrogen. Tissue samples were then stored at -80°C. Spleen and thymus weight ratio was calculated using the formula: organ weight(mg)/body weight(g)
Preparing tissue sections:
Brain sections were cut using a cryostat (Leica Biosystems) and were thaw-mounted onto Superfrost Plus glass slides (Thermo Scientific; 25 X 75 X 1 mm) and stored at -80°C. To assess infarct volume, 30µm thick coronal sections were collected every 240µm across the span of the infarct. For immunofluorescence, a series of twelve 10µm thick coronal sections were collected every 240µm within the infarct.
Infarct size analysis:
Thionin staining was used to measure infarct volume. Thionin stains Nissl substances in soma and dendrites, where neuronal bodies stain dark blue, neuropil stains purple, whilst the infarcted tissue does not retain stain and appears white . Brain tissue sections (30µm thick) were stained with 0.1% thionin (Sigma, Australia, diluted in acetic acid, Ajax Chemicals, Australia), for 2 minutes, rinsed in dH2O water for 5s, placed in 70% and 100% ethanol for 2 minutes each, and then coverslipped with xylene (Merck, Australia) and distyrene plasticiser (DPX; Merck, Australia).
The sections were then photographed using a charge coupled device (CCD) camera (Cohu Inc., San Diego, CA, USA) mounted above a lightbox (Biotec-Fischer Colour Control 5000, Reishkirchin, Germany) using IS capture imaging software. Infarct volume was analysed using image analysis software (Image J, NIH, Bethesda, MD, USA). The area of infarct for each section was measured and the sum of the total infarct area for all the stained sections was multiplied by the distance between the sections to give an approximation of the total infarct volume. Oedema was corrected for using the following formula: Corrected infarct area = [left hemisphere area – (right hemisphere area – right hemisphere infarct area) .
For immunohistochemical analysis, 10µm thick tissue sections were taken from mice subjected to either sham surgery or 18 minutes of photothrombosis. The 18-minute light exposure was selected as it produced a sufficient functional deficit in both strains, with the area of damage still being conserved to the cortex. Cellular architecture was assessed using a: mouse anti-NeuN antibody (MAB377, 1:500 dilution; Merck Millipore, USA) to detect the number of viable neurons; goat anti-GFAP antibody (SAB2500462, 1:500 dilution; Sigma) to detect astrocytes; rat anti-F4/80 antibody (MCA497G, 1:100 dilution, Bio-Rad) to detect macrophages; rabbit anti-CD3 antibody (Ab16669, 1:200 dilution, Abcam) to detect T cells; goat anti-Iba1 antibody (Ab5076, 1:400 dilution, Abcam) to detect microglia in the brain following stroke.
NeuN, GFAP, and Iba1 images were captured at 400X magnification using an Olympus fluorescent microscope and CellSens software (Version 1.15; Olympus, USA), 3 images were taken from the infarct region, peri-infarct region, and undamaged region of the stroked hemisphere, and also from the non-stroke hemisphere, in the region corresponding to the infarcted area. For mice subjected to sham-surgery, the region that would be subjected to infarct was duplicated as the peri-infarct region as they are in the same brain region. The number of NeuN positive (+) cells per region were counted using Image J and averaged per region of the brain for each animal. The percentage area of positive staining of GFAP or Iba1 was measured using Image J and averaged per region of the brain for each animal.
For CD3 staining, each hemisphere of the brain was imaged and the total number of CD3 positive cells was counted per hemisphere due to a low number of cells in the brain.
For F4/80, the stroked and non-stroked hemisphere was imaged at 40X magnification, and the percentage area of positive staining was measured with Image J and averaged per region for each animal.
For detailed immunofluorescence protocols and an image of the different brain regions analysed, refer to Supplementary Fig. S1 online. Representative images can be found in the online Supplementary material, Fig. S2 to S5.
To assess permeability of the BBB, mice were subjected to 18 mins of PT stroke or sham surgery and were then injected with 2% Evans Blue dye (Sigma) in saline (4mL/KG) at 4 hours post-stroke (i.v). The dye was allowed to circulate for 30 minutes prior to culling . The animals were then euthanised and transcardially perfused with heparinised PBS (10u/mL; Heparin sodium, Hospira, Australia). The brain was removed and imaged.
To quantify the amount of evans blue staining, the % area of blue dye in the hemisphere was measured by tracing the area around the ipsilateral hemisphere and the area of evans blue extravasation. The % area was calculated by: area evans blue extravasation/ area of ipsilateral hemisphere X 100.
All data was expressed as mean ± standard error of the mean (SEM). All statistical analyses were conducted using GraphPad Prism (version 9, GraphPad Software Inc., USA). Functional tests and weight measurements were analysed using two-way repeated measures (RM) ANOVA, followed by Tukey’s and Sidak’s post hoc tests. To assess evans blue extravasation, a one-way ANOVA test was used with Tukey’s post hoc test. To assess infarct volume and immunofluorescence, a two-way ANOVA was used, with Tukey’s and Sidak’s post hoc tests. Probability values of P<0.05 were considered as statistically significant.