Induction of aneurysms in MCA IA model

Formation of IAs was induced by local microinjection of elastase (1µL, 35 IU) behind the MCA bifurcation (Fig. 1a; Additional file 1: Figure 1a) and immediately followed by subcutaneous Angiotensin II (Ang II) osmotic pump (800 µg in 90 µL of saline solution; diffusion rate 37 ng/min/14 days) insertion (Fig. 1a; Additional file 1: Figure 1a). This model is a variation of the model previously described by Nuki et al. [37], inspired by models developed for abdominal aortic aneurysm studies [2]. The dose of elastase was chosen after a dose–response test where different parameters were studied (i.e., IA formation, occurrence of hemorrhage, survival rate etc.) (Additional file 1: Figure 2a).

To avoid including animals with potential surgically induced hemorrhages in the study, all animals were thoroughly assessed 24 h post-surgery by using the specific T2*-w sequence to visualize potential hemorrhages. Mice presenting spontaneous postoperative hemorrhages were excluded from further studies (Fig. 1a). Signal void appearing at the later stages (after aneurysm development) in T2*-w were considered aneurysm ruptures (Additional file 1: Figure 1e–h). To confirm that the vessel wall degradation was caused by the elastase micro-injection and was not due to potential micropipette-induced vascular damage, a control experiment was performed by injecting PBS (n = 5) or elastase (35 mU/µL) (n = 8). None of the PBS-injected mice developed aneurysms, and no hemorrhage was observed at any time throughout the protocol (Additional file 1: Figure 1i).

Human SCAA sample

The studied aneurysm sample was resected by Dr. Frösen at the Kuopio University Hospital, following clipping of the aneurysm neck. The sample was from a large middle cerebral artery aneurysm with a diameter of 2cm. A summary of the preoperative imaging studies is presented in Fig. 3. An informed consent was obtained from the patient prior to the procedure, and the study was approved by the Ethical committee of the Hospital District of Northern Savo (TKU 52 2014). Following resection, the tissue sample was fixed overnight in 4% paraformaldehyde and embedded in paraffin so that the original shape of the aneurysm dome was preserved.

Histology and immunofluorescence staining of human samples

The resected human MCA aneurysm sample was cut into 4 µm thick sections with a microtome, following which these sections were deparaffinized and rehydrated using a standard alcohol series. After rehydration, some of the sections were stained with standard hematoxylin–eosin staining. For those sections that were immunostained, an antigen retrieval procedure was performed by incubating the sections in close to boiling temperature in HIER buffer (ThermoFisher Scientific, Watham, MA, USA) for 2 × 5min. After that, a protein block in 3% normal horse serum (Biowest, Nuaillé, France) in 0.1% PBST for 30 min at room temperature was performed. Following the antigen retrieval and serum block, the sections were incubated overnight at 4 °C with the primary antibody (Table 1) diluted in 1.5% normal horse serum (Biowest) in 0.1% PBST. Following this, after 3 × 5 min washes in PBS, the sections were incubated with the secondary antibody (Vector Laboratories, Burlingame, CA) (dilution 1:200) in 1.5% normal horse serum in 0.1% PBST for 30 min at room temperature. Secondary antibodies were chosen according to the species where primary antibodies were produced. After the incubation with the secondary antibody, the sections were washed for 3 × 5 min in PBS and underwent an endogenous peroxidase block with 3% hydrogen peroxide (ThermoFisher Scientific) in PBS for 20 min at room temperature. This was followed by 3 × 5 min PBS wash, following which the sections were incubated with a horseradish peroxidase conjugated avidin–biotin complex (Vector Laboratories) for 30min in room temperature. After subsequent 3 × 5min PBS washes, the positive signal was detected with DAB (3′–5′-diaminobenzidine) (Vector Laboratories). Hematoxylin was used for counter staining and the stained sections dehydrated and mounted with Depex.

Table 1 Primary antibodies used for histology and immunofluorescence

Double immunofluorescence staining was performed to confirm the co-localization of markers CD11b and CD163. Protocol was similar to the one described above, except that normal goat serum (Vector Laboratories) in 0.1% PBST was used for protein blocking and antibody dilutions. Secondary antibodies were AlexaFluor 488 and 594 conjugated (dilution 1:200, ThermoFisher Scientific) for signal detection. Mounting was performed with Vectashield medium with DAPI (Vector Laboratories).

The sections chosen for the staining of human samples were adjacent sections from different depths of the IA, making it possible to compare co-localization of different inflammation markers. Immunoperoxidase stained sections were imaged with Olympus AX70 microscope (Olympus, Japan). For immunofluorescence staining, imaging was performed with LSM800 Zeiss confocal microscope system (Carl Zeiss Ag, Oberkochen, Germany) with 405/488/555 nm diode lasers and appropriate emission filters (20x/0.5 PlanApo objectives, 1024 × 1024 frame sizes). Image processing was performed by ImageJ (Rasband, W.S., National Institutes of Health, Bethesda, Maryland).

Animals

8 weeks old male Swiss (Janvier Labs) mice were housed (Centre Universitaire de Ressources Biologiques, Normandy University, Caen, France) at 21 °C in a 12 h light/dark cycle with food and water ad libitum. Animal care and manipulations complied with recommendations issued by the French and European guidelines for the care and use of laboratory animals (European directive 2010/63/UE) and were authorized by the ethical committee (authorization no. 27499). All experiments were performed following the ARRIVE guidelines (www.nc3rs.org.uk). All the procedures needing anesthesia were performed by an initial exposure to 5% isoflurane followed by a maintaining phase of 1.5–2% isoflurane 30% O2/70% N2O during experiments.

Depletion of CAMs

Depletion of CAMs was carried out 5 days before the MCA aneurysm induction surgery to minimize the pro-inflammatory effects of CLO per se, by injecting CLO intracerebroventriculary. Anesthetized mice were placed in a stereotaxic device, and after the skin was removed, a small craniotomy was performed (coordinates: − 0.2 mm anteroposterior; + 1 mm lateral; − 2 mm depth from the Bregma). With a glass micropipette, 10 µl PBS-liposomes (PBS group) or clodronate-encapsulated liposomes (CLO group) (purchased from clodronateliposomes.com) were gently injected into the left lateral ventricle for 20 min. CLO liposomes injected into the cerebral ventricles are transported by the cerebrospinal fluid to the perivascular spaces and phagocytized by CAMs. In the cytosol, CLO acts as a cytotoxic ATP analog, which impairs mitochondrial oxygen consumption and leads to cell death by apoptosis [29].

Blood pressure measurements

Blood pressure in mice was measured using the Visitech BP-2000 blood pressure analysis system to ensure accurate physiological data while minimizing stress on the animals. Measurements, including arterial blood pressure (systolic, diastolic, mean) and heart rate, were taken every third day using a volume pressure recording sensor and an occlusion tail-cuff system. The experimental setup involved activating the BP-2000 computer and control unit, setting the heating platform to 34 °C, and placing mice in a magnetic restraining device with their tails free. Mouse was gently placed into an integrated animal holder to ensure proper restraint during the procedure. The tail was inserted through the corresponding tail-cuff, taped down, and covered with an LED light sensor. During the analysis cycle, the blood pressure control unit pressurized and attempted to read a pulse, producing results at the end of each measurement cycle. Recording sessions, integrated with data acquisition, were repeated until experimental variability was minimized, typically requiring three sessions.

Tissue harvesting and fixation of mouse samples

Brain samples for IHC were isolated 5 and 15 days after the MCA aneurysm surgery. Terminally anesthetized mice were transcardially perfused with cold heparinized saline (15 mL/min) and fixed with 50 mL of 4% paraformaldehyde phosphate buffer (pH 7.4). Brains were post-fixed with 4% paraformaldehyde phosphate buffer (18 h; 4 °C) and cryoprotected (sucrose 20% in PBS; 24 h; 4 °C) before freezing in CryomatrixTM medium, frozen in isopentane cooled with liquid nitrogen, and stored at − 80 °C.

Immunofluorescence (mouse samples)

For IF analysis 10 μm thick cryostat-cut sections were adhered and collected on poly-lysine coated microscope slides and stored at − 80 °C before processing. Sections were randomly selected and incubated overnight at room temperature, with primary antibodies listed in Table 1. Primary antibody solutions were prepared with PBS-Triton (25% v/v). To reveal primary antibodies, after rinsing three times in PBS (1x, pH = 7.4), brain sections were incubated with appropriate Donkey Fab’2 fragments conjugated with Alexa Fluor® 488, Alexa Fluor® 594 or Alexa Fluor® 647 (1:1000, Jackson ImmunoResearch, West Grove, USA).

After another set of rinsing in PBS (1x, pH = 7.4) and deionized water, slides were left to dry. Finally, sections were coverslipped with an antifade medium containing DAPI. Images were captured using Leica DM6000 epifluorescence microscope coupled camera and visualized with Leica MM AF 2.2.0. software (Molecular Devices, USA). Additionally, confocal images were taken on Leica TCS SP5 MP microscope using the 40X oil immersive objectives. Images were taken using LAS AF Leica Software.

Automated microglial morphology analysis

Following transcardial perfusion with 4% PFA, 100 µm thick brain sections were cut with vibratome and stained for microglia (Iba1, Ancam Ab5076) and cell nuclei (DAPI, Sigma 10236276001). Confocal stacks with 0.2 µm/pixel size and Z-step of 0.4 µm were acquired using Nikon A1R confocal microscope with 60 × water immersion objective (Plan Apo VC NA = 1.2 WD = 0.31–0.28 mm FOV = 215.04 µm; Nikon Instruments Europe B. V., Amsterdam, the Netherlands). Images were acquired both on the ipsi- and contralateral side of the aneurysm. Stacks were then analysed using the Microglia Morphology Quantification Tool [20]. This automated analysis provides an unbiased, 3D analysis of microglia morphology.

Magnetic resonance imaging

Imaging was performed on a Pharmascan 7T/12 cm system using surface coils (Bruker, Germany). Mice were deeply anesthetized with 5% isoflurane and maintained with 1.5–2% isoflurane 30%O2/70%N2O during the acquisitions. T2-weighted images were acquired using a multislice sequence: TE/TR 33/2500 ms. Hemorrhage volumes were quantified using the ITK software. Mice showing lesions < 6mm3 at 24 h post-surgery were considered as surgical failure and excluded from the analyses.

For molecular MRI (see below) three-dimensional T2*-weighted gradient echo imaging with flow compensation (field-of-view (FOV) 70 × 70 × 70 mm3 interpolated to an isotropic resolution of 70 mm), TE/TR 13.2 ms/200 ms and a flip angle of 21° was performed to visualize MPIOs. T2*-weighted sequences were used to verify if IAs ruptured.

Molecular MR Imaging of vascular inflammation and quantification

Micro-sized particles of iron oxide (MPIOs) (1.08 μm diameter; Dynabeads MyOne Tosyl Activated, Invitrogen) covalently conjugated to purified polyclonal goat anti-mouse antibodies for P-selectin (R&D Systems, clone AF737) and purified polyclonal mouse anti-rat antibodies for VCAM-1 (Clone MR106, BD Pharmingen) were prepared as previously described [32]. In brief, MPIOs were covalently conjugated to polyclonal antibodies in borate buffer (pH 9.5) at 37 °C for 48 h. The coating of the beads was made using 20 μg of VCAM-1 or 40 μg of P-selectin antibody per 1 mg of reactive MPIOs. After, beads were washed and incubated with PBS containing 0.5% BSA for 24 h at room temperature to block remaining active groups. MPIO solution is stored under continuous rolling at 4 °C until usage.

MRI acquisitions started immediately after the intravenous injection of MPIO-solution (200 µl of 2 mg Fe/kg of conjugated MPIOs). All T2*-weighted images presented are minimum intensity projections of six consecutive slices. The qualitative analysis and the distribution of the MPIO signal voids on 3D T2*-weighted images was assessed by ITK Snap Software [49].

Semi-automatic signal void quantification was performed similar to previously described [15]. Briefly, signal voids corresponding to MPIO presence were quantified in subject space within region-of-interest (ROI) masks of the aneurysm lesion and the ipsilateral hemisphere and their contralateral homologues. In MATLAB, histograms of voxel signal intensities in each T2*-weighted image in contralateral ROIs were generated and fitted to a Gaussian of certain peak center and peak width. Voxels in the brain with signal intensities lower than the value at the contralateral peak center minus its half width (i.e., signal intensity values lower than 6.25% of the value of the majority of contralateral reference voxels) were subsequently labeled as signal void. Per time point, signal voids were counted per ROI and expressed as a percent-difference of the count in the corresponding contralateral ROI.

Flow cytometry

After transcardiac perfusion with PBS, brains were roughly minced and homogenized with a potter tissue grinder in Hanks’ balanced salt solution (HBSS) containing 15 mM HEPES buffer and 0.54% glucose. Whole brain homogenate was separated by 37% Percoll gradient centrifugation at 800g for 30 min at 4 °C (no brake). The pellet containing CNS leukocytes at the bottom of the tube was then collected and washed once with PBS containing 2% FCS before staining.

Fc receptors were blocked with CD16/32 (553142, BD Biosciences) for 10 min at 4 °C before incubation with the primary antibodies. Cells were stained with antibodies directed against CD11b (M1/70, BioLegend), CD45 (30-F11, BD Biosciences), Ly6G (1A8, BD Biosciences), CD3e (145-2C11, BD Biosciences) and CD206 (C068C2, BioLegend) for 45 min at 4 °C. After washing, samples were analyzed by a FACSVerse flow cytometer or sorted by a FACSAria (BD Biosciences). Appropriate isotype control antibodies were used to establish sorting parameters. Data were analyzed with the FlowJo 7.6.5 software (TreeStar Inc.). Data are expressed as percentages.

Elastica van Gieson staining

For histological examination of brain tissue sections and visualization of connective tissue made up of elastic fibers Elastica van Gieson staining kit was used (Merck, Darmstadt, Germany). Slides with 10 μm thick brain sections were incubated for 13 min in resorcin fuchsin solution and then for 5 min in alcoholic hematoxylin solution and hydrochloric acid iron(III)nitrate solution in ratio 1:1. After each incubation slides were rinsed under running tap water for 1 min. Slides were then incubated in pirofuchsin solution (picric acid/acid fuchsin solution) for 2 min and rinsed in 70%, 96% and 100% ethanol (two times for 1 min in each percentage). Finally, slides were incubated in toluene for 10 min and coverslipped with mounting medium. Images were captured using Leica DM6000 epifluorescence microscope coupled camera and visualized with LAS X Leica Software (Molecular Devices, USA).

Measurements of the vessels and cell quantification

All images were analyzed using ImageJ 1.51k software. To determine the intima-media thickness and wall-to-lumen ratio, first, the diameters of both vessel and vessel lumen were measured. Intima-media thickness was calculated as a half value of the vessel diameter subtracted by the diameter of the lumen. The results are expressed in μm. To calculate wall-to-lumen ratio, the value of intima-media thickness was divided by the lumen diameter.

CD206+, Ly6G+, and CD3+ cells were quantified in a nonrestricted area around the aneurysms in the ipsilateral hemisphere and control arteries in the contralateral hemisphere. DAPI staining for cell nuclei was used to confirm that signal comes from the cell. Results for each cell type are presented as a number of cells around the aneurysm and contralateral artery. Microglial activation and proliferation were analyzed on images taken using 20X objective of Leica DM6000 epifluorescence microscope coupled camera, in an area of 400 × 400 μm. All quantifications were done manually, blinded to the treatment.

Image visualization

Images presented in the in Fig. 1; Figs. 2 and 3 and Additional file 1: Fig. 1 were adapted from BioRender with postprocessing using PowerPoint.

Statistical analysis

All data are expressed as the mean ± standard error from the mean (s.e.m.) with the analysis being conducted using two-way ANOVA with Dunnett’s post hoc multiple-comparisons or Mann–Whitney U test. Two-Way ANOVA was used to compare the values between ipsi- and contralateral hemispheres in CLO and PBS-treated groups. Fisher’s exact test was used to compare the aneurysm rupture rate between the PBS and CLO treated groups. The Mann–Whitney U test was used for the comparison between two groups. Significance was set at P < 0.05. N values refer to the number of replicates. All statistical analysis and plotting were performed in GraphPad 9 (GraphPad). All quantifications were done manually, blinded to the treatment.