Introduction

Hemorrhagic stroke (HS) refers to an intracerebral rupture of blood vessels leading to injury to the brain1. Spontaneous HS due to hypertension most commonly occurs in the putamen and caudate nucleus (50–60%)1. The overall prognosis is poor, with a 30-day mortality rate of 35–52%2. One-half of these deaths occur within the first two days2, stressing the importance of acute brain injury in pathogenesis. The mechanisms of injury within the first few hours include the hematoma mass effect and ensuing toxicity of red blood cell (RBC) components. The release of free hemoglobin, heme, and iron, followed by the hemolytic cascade, triggers a second wave of damage after the initial bleed3. As blood enters the normally blood-free brain parenchyma, reactive immune responses commence. The dormant immune cells within the brain, microglia, respond to injury within minutes to release chemokines and cytokines, propagating the inflammatory process and secondary brain insults4.

Microglia migrate caudally during embryonic development to stay within the brain parenchyma throughout life5. They are similar to bone marrow-derived macrophages morphologically and functionally, with the ability to phagocytose and present foreign antigens to T cells via major histocompatibility complex (MHC) class II molecules (MHC-II). Microglia, comprising 10–20% of the total cells within the brain, also surveil the cerebral immune environment and contribute to many major neuro-inflammatory actions of various diseases such as ischemic stroke, multiple sclerosis, traumatic brain injury, and infections6,7.

Following an HS, microglia first polarize via the classic polarization pathway, resulting in a phenotypic spectrum commonly deemed as M1 microglia8. This group is characterized by its pro-inflammatory actions that contribute to perilesional edema and exacerbate a cerebral injury. As time progresses, another polarization pathway, which results in a phenotype broadly classified as M2 microglia starts to assume dominance9. M2 microglia comprises a wide spectrum of cells that promotes cell regeneration (M2a), phagocytosis of hemolytic products (M2b and M2c), and removal of cell debris (M2b and M2c). This shift in polarization from M1 to M2 normally takes place on day 79. Although the dichotomization of microglia into either M1/M2 phenotype is overgeneralized, for the sake of simplicity we would refer to them as such within this study.

In the present study, we explored an incidental finding: mice that had undergone two separate episodes of HS performed significantly better on functional studies when compared with those without a previous HS. This phenomenon has never been discussed previously, and we aimed to determine the mechanisms underlying the above observation. Interestingly, we also observed the modulatory effects of a previous HS on subsequent strokes for the first time. Behavioral studies showed a significant trend, so we further performed MRI imaging, demonstrating attenuation of total lesion volume in animals with a previous HS. A recent study has put forth a concept of priming stimuli as mice that received multiple peripheral lipopolysaccharide injections showed attenuation of inflammation when exposed to Alzheimer’s disease and ischemic stroke10. As microglia are responsible for removing hematoma and cell debris7). In vitro studies revealed that microglia had retained innate immune memory during the previous stroke and that T cells were involved in this protective mechanism regarding earlier alternative microglia polarization. Our incidental discovery presented new evidence on a mechanism regarding the potential clinical impacts of successive hemorrhagic strokes which may pave the road to new, anticipatory therapeutic interventions that have never been seen before.

Fig. 7: Graphic summary.
figure 7

The first hemorrhagic stroke initiated a cascade of inflammatory actions downstream that corresponds to what others have observed in the past: M1 microglia take dominance initially before gradually declining to give way to an M2 predominant profile at day 7 post hemorrhagic stroke. Microglia then return to baseline levels. When a second hemorrhagic stroke was introduced, microglia no longer followed the former polarization profile but rather assumed an M2 predominant phenotype at an early stage. The group of mice with a previous stroke demonstrated improved survival rates, better functional outcomes, and rapid lesion resolution, compared to mice that were only encountering a hemorrhagic stroke for the first time.

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Methods

Hemorrhagic stroke animal model

All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research of our institution. Male C57 (C57BL/6 N, Charles River Lab, USA) mice 10–12 weeks old underwent all the described experiments unless otherwise specified (e.g., immune-deficient mice experiment design).

Mice were randomly divided into four groups. The sham group underwent two sham operations on days −14 and 0. The “past mini-stroke only” group underwent a single minor stroke on day −14. The “previous stroke group” underwent a minor HS at the left basal ganglia on day 14 and a major HS on the right on day 0. The control group received only one major HS at the right basal ganglia, with a sham operation on day −14. (Fig. 1a). For induction of the major HS, a replicable model was conducted as described before13. Mice were anesthetized via intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). A stereotactic device was used. Mice were secured in a prone position, and scalp fur was removed. A midline scalp single incision was made. Intracranial bleeding was induced by inserting a 26-gauge needle (10 uL Hamilton syringe) at 3.5 mm below the dura into the right basal ganglia at coordinates (x, y) = (+0.2 mm, +0.05 mm), with the bregma as (0,0), through a burr hole. One minute later, 0.1units of collagenase (0.25 U/µL, 0.4 µL) (Sigma-Aldrich, St. Louis, MI, United States) was injected at a rate of 0.1 µL/min with an infusion pump. After needle retraction, the burr hole was covered with bone wax, and the scalp was sutured. For a minor HS induction, the procedure was the same as mentioned above, with the only difference being that collagenase dosage was adjusted to 0.04 U (0.2 U/µL, 0.2 µL), and the injection site was at the left basal ganglia (−0.02 mm, +0.5 mm). Replicable hematomas were induced using this dosage and method for the minor HS (Supplementary Fig. 3). For a sham operation, the mice had a bur hole drilled at the same coordinates with needle insertion for 10 min before retraction.

In order to observe whether an absence of certain immune cells could interfere with functional recovery under the above study design, we used immune-modified mouse strains. Athymic nude mice (BALB/cAnN-nu, Charles River Lab, USA) and NOD-SCID mice (NOD.CB17-Prkdcscid/J, The Jackson Laboratory, USA) were used to repeat phenotypical experiments—similar sequences of HS for the “previous stroke group” and control group were performed on athymic nude mice, which lack mature T cells, and NOD-SCID mice, which lack mature B cells, T cell, and have defective NK cells.

Phenotypical observations

Mice were monitored on days 1, 3, 5, and 7 following the induction of a major hemorrhagic stroke. A blinded observer took the measurements.

Modified neurological severity score (mNSS)

The mNSS test, first described by Chen et al.36 and verified by validation studies37, quantifies motor, sensory, and balance deficits post-stroke. With a maximum score of 18 being the most severe, the gradual decline in neurological deficit scores traces the speed and extent of motor and sensory recovery after the initial injury37.

Rotarod

The Rotarod apparatus (AccuRotor, Omnitech electronics) rotates at an acceleration of 0.133 cm/s2. Results are recorded as latency-to-fall, which is the duration between commencement of rotation until the mouse falls off the rod36. Mice were first trained 7 days in three separate sessions prior to the major HS event. Subsequently, mice were assessed by the Rotarod test with the same acceleration profile. For each mouse, three runs were repeated, with 20-min rest intervals in-between.

Body weight

Mice were weighed individually every morning on days 1, 3, 5, and 7. Results were documented in grams.

Histopathologic examination

Mice were sacrificed on days 1, 3, 5, and 7 and perfused with 50 mL 4 °C normal saline. For the mini-stroke only and sham groups, mice were sacrificed 13, 9, 5, and 1 day before the major hemorrhagic stroke of other groups took place. Brains were sectioned into coronal slices of 2 mm thickness, and a photo was taken of each coronal section. Coronal images were imported to ImageJ, converted to grayscale, and a gradient-gated threshold was used to obtain the hematoma area. The sum of the sections from the cranial-caudal and caudal-cranial views were obtained, and the mean was multiplied by 2 mm (slice thickness) to obtain a final value of hematoma volume (expressed in mm3).

Magnetic resonance imaging (MRI) studies

A 7 T MRI scanner (PharmaScan 70/16, Bruder Biospin GmbH) was used to take 12 coronal plain scans, each of 0.5 mm thickness and positioned perpendicular to the axial and sagittal axes as previously described38. T2-weighted images were acquired as anatomical reference using a Rapid Acquisition with Refocused Echoes (RARE) sequence (FOV = 32 × 32 mm2, data matrix = 256 × 256, RARE factor = 8, TE/TR = 36/4200 ms). Images of the coronal plain were imported into ImageJ (version 1.8.0 for Windows, ImageJ Software). For each slice of the 12 coronal images, the total lesion area, defined as the hyperintense core, periphery, and surrounding rim39 was outlined manually. The sum of the combined lesion area from the 12 coronal slices was multiplied by slice thickness (0.5 mm) to obtain the total volume of the lesion. For statistical analysis, the total lesion volume was compared chronologically within the same mouse, with that on day 1 being presented as 100%. A subsequent decline in injury volume is expressed as a decrease in the percentage of residual volume compared to that on day 1.

Quantitative polymerase chain reaction (qPCR)

Mice were sacrificed 1, 3, 5, and 7 days after HS and perfused with 50 mL 4 °C normal saline. Brains were sectioned into slices of 2 mm thickness. Starting from the olfactory bulbs, the fourth and fifth slices were those with the largest hematoma area. These slices were harvested and subsequently divided into left and right sections. The right (injured) sections and enzymatically digested with RIPA buffer and PI. RNA was extracted from digested tissue, and cDNA was synthesized using a commercially available kit (Takara PrimeScript RT Reagent Kit). qRT-PCR using SYBR Green (QIAGEN) was performed to detect the subsequent genes: TNF-a, iNOS, CD206, and Arginine-1. The threshold cycle (CT) was normalized to GAPDH (ΔCT) of the same mouse and then to that of the sham mouse (ΔΔCT). Mean fold changes are depicted as 2(−ΔΔCT). Data acquisition was conducted in technical triplets. Primers are presented in Table 1.

Table 1 Primer sequences for the qPCR analysis conducted on in vivo brain section samples on days 1, 3, 5, and 7 after a major hemorrhagic stroke.
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Immunofluorescence staining

Mice were sacrificed and perfused with 50 mL cold PBS, followed by 50 mL 4% paraformaldehyde. Cryosections were obtained of the coronal plain at 5 μm thickness. Non-specific staining was blocked by 1% BSA for 1 h, followed by incubation with primary antibodies against microglia surface marker CD11b (1:100 Cell Signaling, Danvers, MA, USA), M2 marker CD163 (1:120 Cell Signaling) for 2 h at 4 °C. Slides were washed thrice before staining with secondary antibodies: anti-rabbit (Cell Signaling) and anti-mouse (Cell Signaling). DAPI nuclear staining (Sigma-Aldrich) was added to the mounting solution. Primary-cultured microglia were stained as follows: cells were fixed with 4% paraformaldehyde for 1 h, washed thrice with cold PBS, and stained with antibodies against Iba-1(1:100, Cell Signaling) for 2 h with nuclear staining DAPI (Sigma-Aldrich) added at the last 15 min. Cells were washed twice before incubation with secondary antibodies: anti-mouse (Cell Signaling) and anti-rabbit (Cell Signaling). All images were taken by the Invitrogen EVOS FL Color imaging system.

Flow cytometry

Mice from the previous stroke group and control group were sacrificed on days 1, 3, 5, and 7. Mice from the sham group and “past mini-stroke only” group were sacrificed on days −13, −9, −5, and −1. Peri-hematomal brain tissue was collected and mechanically homogenized by cutting with scissors, then digested by 5 mL 4% collagenase (Sigma-Aldrich, St. Louis, MI, United States) for 15 min at 37 °C. Digestion was halted by adding a 5 mL culture medium and single-cell suspension obtained by filtering through a 75 µm cell strainer. Cells were centrifuged at 400 × g for 5 min and resuspended in PBS. Cells were then washed again and resuspended in Flow Cytometry buffer (Cell signaling, Danvers, MA, USA) at a concentration of 105 cells per mL. For in vitro studies, microglia were scraped from the flask by a cell scraper at the experimental endpoint of each group. Cells were washed twice by centrifuging at 400 × g for 5 min and suspended in flow cytometry buffer. Each mL of suspended cells was incubated with conjugated flow antibodies (APC/Cy7 anti-mouse CD16/32 antibody, Biolegend #101327, 1:100; FITC anti-mouse CD206 antibody, Biolegend #141703, 1:80; Alexa Fluor 700 anti-mouse CD45.2 antibody, Biolegend #109822, 1:70; APC anti-mouse/human CD11b, Biolegend #101212, 1:100) for 1 h in darkness. For M2 verification, cells were additionally stained with PE anti-mouse CD163 antibody, Biolegend #156703. Cells were then washed twice by centrifuging at 400 × g for 5 min and resuspended in Flow Cytometry buffer. Analysis was conducted by CytoFLEX Flow Cytometer (Beckman Life Sciences, Indianapolis, IN, USA).

Microglia isolation and cell culture

In vitro experiments were conducted with primary-cultured microglia harvested from newborn pups of 0–3 days old, as previously described40. Pups were sacrificed by intraperitoneal injection of phenobarbital, then sprayed with 70% alcohol and decapitated with sterile equipment. The decapitated heads were rinsed in 4 °C PBS and immersed in dissection media. Under a microscope, the scalp was removed and the skull dissected along the midline up to the bregma, so that the brain could be scooped out using curving forceps. 5–6 brains were pooled and mechanically homogenized by cutting with scissors, then digested with 0.25% trypsin at 37 °C for 15 min. To end the digestion process, a 1.2 ml 1 mg/ml trypsin inhibitor was added and incubated for 1 min. The homogenized products were then centrifuged at 400 × g for 5 min and resuspended in warmed culture media (90% Dulbecco’s modified Eagle medium, Thermo Fisher Scientific, Gibco™; 10% fetal bovine serum, GE Healthcare, Hyclone™) twice, before being plated onto PDL coated T75 flasks at a density of 50,000 cells/cm2. Culture media was changed the next day, and subsequently every 5 days. This first step yields a confluent layer of astrocytes and microglia after 7 days.

To separate microglia from strongly adherent astrocytes, the flasks were shaken at 200 rpm for 2 h in a cell shaker maintained at 37 °C. The floating cells were collected and transferred to a newly coated T75 flask. This process yielded high quantity and high-quality microglia, which are ready for experimental use the next day.

Red blood cell (RBC) coculture

In order to mimic an HS in the cell culture setting, we added whole RBCs collected from the tail vein of mice at a ratio of 10:1 (RBC to microglia), as described before41. The coculture system was maintained at 37 °C for 1 h, and RBCs were subsequently removed via changing of cell medium. RBCs were non-adherent, while microglia were adherent to the flask wall.

Isolation of T cells

Splenic cells were obtained from adult C57 mice by homogenizing the whole spleen mechanically with scissors. RBCs were removed via lysis medium (Thermo Fisher, Waltham, MA, USA). Pan-naïve T cells were extracted via column-free magnetic cell isolation: first by positive selection (CD3 + CD45RA + CD197 + ), then negative selection (CD45RO−) using magnetic beads and a commercial kit (EasySep™ Human Naïve Pan T Cell Isolation Kit).

The collected pan-naïve T cells were added to Group F immediately after the first RBC coculture event (without direct contact with the RBCs) at a ratio of 1:1 (microglia to T cell). T cells were added to group C at the same time.

Erythrophagocytosis study

Whole RBCs were labeled with PKH-26 fluorescent probe (Sigma-Aldrich) and cocultured with primary microglia at a ratio of 10:1, as previously described33. After incubation at 37 °C for 1 h, the unengulfed RBCs were removed by washing thrice with PBS. Microglia were then harvested from the flask wall. The engulfed, intra-microglial RBCs were detected by flow cytometry via the FITC channel. The level of phagocytosis was quantified by the positive FITC percentage of each measurement.

Statistics and reproducibility

Phenotypical studies that include multiple measurements of the same mouse over time (body weight, mNSS, Rotarod, MRI total lesion volume) were analyzed by repeated-measurements two-way ANOVA with matched values of the same mouse across time (n = 10 for the previous stroke and control groups, n = 5 for the mini-stroke only and sham groups). Additionally, Sidak’s multiple comparisons test were conducted on the mean value obtained on each date between experimental groups (n = 25). Mechanistic studies that require sacrificing mice at the time of measurement (flow cytometry, qPCR) were analyzed by regular two-way ANOVA (no repeated measures) and Sidak’s multiple comparisons test (n = 7 for the previous stroke and control groups; n = 4 for the mini-stroke only and sham groups). Cell line studies involving more than two groups of importance to compare were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Cell line experiments were repeated three times within each group. The Gehan–Breslow–Wilcoxon test was used in the Kaplan–Meier survival curve analysis. A p value < 0.05 was considered statistically significant. GraphPad Prism version 6.04 for Windows, GraphPad Software was used.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.