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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Mar 4;177(11):2466–2477. doi: 10.1111/bph.14989

C3a receptor antagonist therapy is protective with or without thrombolysis in murine thromboembolic stroke

Saif Ahmad 1, Chirayu Pandya 5, Adam Kindelin 1, Kanchan Bhatia 1, Rafay Chaudhary 7, Alok Kumar Dwivedi 8, Jennifer M Eschbacher 4, Qiang Liu 2,3, Michael F Waters 2,3, Md Nasrul Hoda 2,3,6, Andrew F Ducruet 1,✉
PMCID: PMC7205805  PMID: 31975437

Abstract

Background and Purpose

Intravenous thrombolysis (IVT) after stroke enhances C3a generation, which may abrogate the benefits of reperfusion. The C3aR antagonist SB290157 is neuroprotective following transient but not permanent middle cerebral artery occlusion (MCAo). SB290157 remains untested in thromboembolic (TE) models, which better approximate human stroke and also facilitate testing in combination with IVT. We hypothesized SB290157 would confer neuroprotection in TE stroke with and without “late” IVT.

Experimental Approach

We used two different models of TE stroke to examine the efficacy of SB290157 alone and in combination with late IVT. We evaluated the benefit of SB290157 in attenuating post‐ischaemic behavioural deficits, infarction, brain oedema and haemorrhage.

Key Results

Plasma C3a was elevated 6 hr after TE stroke alongside increased cerebrovascular C3aR expression, which was sustained to 4 weeks. Increased C3aR expression also was visualized in human ischaemic brain. In a photothrombotic (PT) stroke model, which exhibits rapid spontaneous reperfusion, SB290157 given at 1 hr post‐PT significantly improved neurofunction and reduced infarction at 48 hr. In an embolic (eMCAo) model, SB290157 administered at 2 hr improved histological and functional outcomes. Conversely, late IVT administered 4.5 hr post‐eMCAo was ineffective likely due to increased haemorrhage and brain oedema. However, SB290157 administered prior to late IVT ameliorated haemorrhage and oedema and improved outcomes.

Conclusions and Implications

We conclude that SB290157 is safe and effective with and without late IVT following TE stroke. Therefore, C3a receptor antagonist therapy represents a promising candidate for clinical translation in stroke, particularly as an adjuvant to IVT.

Abbreviations

ATR

adhesive tape removal

C3aR

C3a receptor

C3aRA

C3a receptor antagonist

eMCAo

embolic clot middle cerebral artery occlusion

ET

endovascular thrombectomy

HT

haemorrhagic transformation

IVT

intravenous thrombolysis

NDS

neurological deficit scoring

PT

photothrombotic

TE

thromboembolic

tMCAo

transient middle cerebral artery occlusion

What is already known

  • Acute administration of SB290157 is neuroprotective in a murine mechanical middle cerebral artery occlusion model.

What this study adds

  • Single‐dose treatment with SB290157 confers functional and histological neuroprotection in a photothrombotic cortical stroke model.

  • SB290157 remains protective in a murine embolic thrombus model, both with and without intravenous thrombolysis.

What is the clinical significance

  • Pharmacological inhibition of the C3a receptor represents a promising strategy for treatment of acute stroke.

1. INTRODUCTION

Stroke is the fifth major cause of mortality and the leading cause of disability among adults in the United States (Benjamin et al., 2018; Go et al., 2013). Despite the widespread application of the two Food and Drug Administration‐approved reperfusion therapies in ischaemic stroke (intravenous tissue https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2392, intravenous thrombolysis [IVT] and endovascular thrombectomy [ET]), post‐ischaemic brain injury results in significant long‐term disability and increased health care costs in the large population of stroke survivors (Ducruet et al., 2012; Hoda et al., 2014). Prolonged ischaemia may reduce or abolish the benefits of reperfusion therapy and delayed reperfusion engenders secondary reperfusion injury (Nour, Scalzo, & Liebeskind, 2013). Therefore, the STAIR committee and NINDS Stroke Progress Review Group have encouraged the development of adjuvant therapies to protect against reperfusion injury as well as to extend the therapeutic window of intravenous thrombolysis and endovascular thrombectomy in stroke (Saver et al., 2009).

The complement pathway is a critical component of innate immunity which promotes neurovascular dysfunction after stroke (Gorsuch, Chrysanthou, Schwaeble, & Stahl, 2012; Machalinska, Kawa, Marlicz, & Machalinski, 2012; Pushpakumar et al., 2011; Zipfel & Skerka, 2009). The complement cascade is activated across several different models of ischaemic stroke with varying dynamics of cerebral blood flow alteration and patterns of reperfusion (Ames et al., 2001; Mocco et al., 2006; Rynkowski et al., 2009; Zhao, Larkin, Lauver, Ahmad, & Ducruet, 2017). We previously reported that the central complement component C3 is cleaved following both transient mechanical occlusion of the middle cerebral artery occlusion (tMCAo) and permanent middle cerebral artery occlusion in mice. The circulating cleavage product of https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9414, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3641 https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3641, activates its cognate receptor, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=31, to exacerbate post‐stroke injury including haemorrhage and brain oedema (Ducruet et al., 2008; Ducruet et al., 2012).

Most cases of human stroke are thromboembolic (TE) in nature and remain untreated. As such, these untreated thromboembolic strokes undergo spontaneous yet slow and partial reperfusion due to endogenous clot lysis which does not often result in clinical benefit. Therefore, thromboembolic stroke models most closely approximate the varied dynamics of cerebral blood flow alterations, extent of spontaneous reperfusion, neurovascular degradation, infarct progression and neurological decline observed in clinical stroke cases (Chen et al., 2015; Hoda et al., 2012; Webb et al., 2018). Moreover, thromboembolic stroke models are ideal for testing adjuvant therapies for the prevention of reperfusion injury following intravenous thrombolysis, the first‐line gold‐standard treatment for ischaemic stroke (Garcia‐Culebras et al., 2017; Hoda et al., 2014; Ishrat et al., 2018). Interestingly, we reported the novel finding that plasmin generated during intravenous thrombolysis cleaves C3 via a non‐canonical complement activation pathway to produce the C3a anaphylatoxin (Zhao et al., 2017). The resulting hyperactivation of the C3a receptor due to the elevated level of its agonist C3a exacerbates post‐ischaemic brain injury (Zhao et al., 2017). In a mechanical model of transient middle cerebral artery occlusion in mice, we also reported that either pharmacological C3a antagonist administration using https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3529 or genetic deletion of C3 attenuates haemorrhagic transformation (HT), blood–brain barrier disruption and oedema progression following intravenous thrombolysis (Zhao et al., 2017). This suggests a potential pathological role for hyperactivation of the C3a/C3a receptor axis in limiting the safety, efficacy and therapeutic window of intravenous thrombolysis in stroke. However, the benefits of C3a antagonist therapy remain untested in thromboembolic stroke models that also facilitate evaluation of an adjuvant therapy in conjunction with intravenous thrombolysis. In the present study, we tested the hypothesis that SB290157 will confer neurovascular protection with or without late intravenous thrombolysis in thromboembolic stroke models. These results will encourage translation of SB290157 and the next generation of C3a antagonists for neurovascular protection in stroke.

2. METHODS

2.1. Mice, experimental plan and procedures

The approval of the Institutional Animal Care and Use Committees (IACUCs) of St. Joseph's Hospital and Medical Center (SJHMC) and Augusta University, Augusta, GA, were obtained prior to animal experiments. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology (McGrath, McLachlan, & Zeller, 2015). C57BL/6J wild‐type 8‐week‐old male mice (Jackson Laboratory, Bar Harbor, Maine; Stock# 000664) or 20‐week‐old middle‐aged male mice from our in‐house colony were used as needed. To evaluate SB290157 in stroke models, we used 8‐week‐old male mice (23 ± 1 g) for the photothrombotic (PT) model while 20‐week‐old middle‐aged male mice (30 ± 2 g) were used in the model of embolic clot middle cerebral artery occlusion (eMCAo). In light of the recommendations of the STAIR/Stroke Progress Review Group, evaluation in aged animals is necessary prior to clinical translation. As our embolic model represents the most clinically relevant model that we tested, we chose to use aged mice in this model instead of repeating the data with young mice. The animals were group housed in a controlled environment (12‐hr light/dark cycles; 23°C; 40–50% humidity) with no more than five mice to a cage bedded with aspen wood chip material and provided with free access to water and chow. The animals were acclimated for 7 days prior to the start of the experiment. Animals were killed by cervical dislocation after deep anaesthetization with isoflurane (2–3%) or transcardial perfusion under ketamine/xylazine (100 mg·kg−1/10 mg·kg−1, respectively). In Experiment I, mice (total = 18, n = 8 per group) were randomized to either sham operation or embolic clot middle cerebral artery occlusion stroke (see Section 2.2). Mouse blood and brain tissue samples (n = 6) were collected at 6 hr after surgical procedures to evaluate changes in C3a plasma level and brain tissue expression of C3a receptors. Additionally, a small cohort of mice was followed for 4 weeks after embolic clot middle cerebral artery occlusion for preliminary Western blot analysis of brain tissue (n = 2 per group). In Experiment II, mice were randomly assigned to either sham operation or photothrombotic stroke (see Section 2.3) and were either treated with vehicle or C3a receptor antagonist therapy (SB290157). Mice were evaluated for behavioural outcomes at 48 hr post‐procedure followed by sacrifice for the determination of infarction volume (n = 9). Additionally, a small cohort of animals was prepared for preliminary Western blot analysis (n = 4) and immunofluorescence staining (n = 2). In Experiment III, a 2 × 2 factorial design was adopted [2 C3a antagonist (NO vs. YES) × 2 intravenous thrombolysis (NO vs. YES)] as reported earlier by us (Hoda et al., 2012) such that all four group mice (total = 80, n = 20 per group) were subjected to embolic clot middle cerebral artery occlusion followed by randomization into four different cohorts: vehicle, C3a antagonist, intravenous thrombolysis and C3a antagonist + intravenous thrombolysis. From each group, brain tissue from surviving mice was collected and assayed for Hb and water content (n = 7 per group for each assay). The remaining equal numbers of surviving mice were used to assess neurological deficit and infarction volume (n = 6 per group).

2.2. Randomization and blinding

All treatments in this study were performed in a randomized manner. Data collection and analysis for all the experiments were performed in a blinded manner.

2.3. Photothrombotic and intravenous thrombolysis models of stroke

Photothrombotic occlusion was performed as previously described (Feng et al., 2017). Adult male mice (C57BL/6; total = 48, n = 9 per group for functional and infarct volume study; n = 4 per group for WB and n = 2 per group for immunofluorescence analysis) were anaesthetized by inhalation with isoflurane (3% for induction and 1.5% during surgical procedure, mixed with 30% O2/70% N2O). Mice were placed in a stereotaxic apparatus (Stoelting Co, Wood Dale, IL, USA) and body temperature was maintained at 37 ± 0.5°C. The skull was exposed by a midline incision to identify the bregma and a laser fibre optic cable (4‐mm diameter) integrated to a cold light source (Schott KL 1600 LED, Elmsford. NY, USA) with a green bandpass filter (Thor Labs, Newton, NJ, USA) was positioned 2–3 mm lateral to the bregma. Mice were then injected with Rose Bengal intraperitoneally (RB; 150 mg·kg−1, in 150‐μl saline, i.p.; Sigma‐Aldrich, St. Louis, MO, USA) and the illumination of the skull was performed for 20 min following 5 min of RB infusion. Sham‐operated animals underwent the same surgical procedures including incision and RB injection but without illuminating the skull with laser light. Following the illumination period, the surgical wound was sutured, and the animal was transferred for post‐op care.

The embolic clot middle cerebral artery occlusion stroke model was performed (total mice = 98) as we previously reported with a slight modification in clot size (Hoda et al., 2012). Briefly, a human fibrinogen‐supplemented clot was prepared as previously described (Hoda et al., 2012). A modified catheter containing a 9.0 ± 0.5‐mm‐long clot was inserted into the right external carotid artery, advanced into the internal carotid artery and the clot was gently delivered into the brain in 100 μl of 1× sterile PBS. The catheter was retracted, the external carotid artery and the wound were sutured before transferring the animal for post‐op care. Sham‐operated mice underwent a similar procedure in which 100 μl of 1× PBS was infused without a clot.

For all surgical procedures, post‐operative care included administration of antibiotic (covenia 8 mg·kg−1 body weight, s.c.) and analgesic (buprenorphine sustained release (SR) 0.05 mg·kg−1 body weight, s.c.) obtained from the SJHMC Pharmacy. The animals were allowed to recover in a pre‐warmed cage with access to food and water and monitored until they were conscious and ambulatory, whereupon they were returned to their home cage. Animal welfare was assessed daily for pain and discomfort until the experimental endpoints for each group.

2.4. C3a antagonist and intravenous thrombolysis therapy in photothrombotic and embolic clot middle cerebral artery occlusion stroke models

Stock solution of the C3a antagonist, SB290157, was prepared by dissolving the compound in a minimal volume of sterile DMSO (100 mg·ml−1). Wherever needed in experiments, SB290157 was further diluted with 1× sterile PBS for injection to a final dose of 20 mg·kg−1 body weight in 100‐μl volume. In the photothrombotic stroke model, SB290157 or vehicle was injected intraperitoneally 1 hr after stroke while in the embolic clot middle cerebral artery occlusion model, it was intravenously injected 2 hr post‐stroke. In the intravenous thrombolysis and C3a receptor + intravenous thrombolysis cohorts of Experiment III (embolic clot middle cerebral artery occlusion model), altepase (10 mg·kg−1 body weight in 200‐μl volume) was intravenously injected beginning at 4.5 hr after eMCAo as a 10% bolus dose with the remainder infused slowly over 20 min as previously reported (Hoda et al., 2012; Hoda et al., 2014). In groups subjected to embolic clot middle cerebral artery occlusion without intravenous thrombolysis, an equal volume of 1× sterile saline was infused as a vehicle.

2.5. Plasma C3a content by ELISA and spectrophotometry

Plasma samples (sham and embolic clot middle cerebral artery occlusion, n = 6) were collected at 6 hr post‐ embolic clot middle cerebral artery occlusion and diluted to quantify C3a by ELISA following the manufacturer's protocol (LS Bio, Seattle, WA).

2.6. Analytical flow cytometry for C3a receptor expression in mouse brain endothelial cells

Flow cytometry was performed on brain single cell suspension obtained from the ipsilateral hemisphere as previously reported (Ducruet et al., 2008; Khan et al., 2018), with slight modification as required for C3a receptor expression in brain endothelial cells. Briefly, fixed single cell suspension was immunostained using a cocktail of three antibodies for phenotypic recognition and functional expression. Brain endothelial cells were separated as CD31 (bs‐0468R, Bioss Antibodies, MA, USA) and VEGFR2 (VEGF receptor 2; bs‐10412R, Bioss Antibodies) double‐positive cells, and the expression of C3a receptor was determined using a fluorochrome‐conjugated anti‐C3a receptor monoclonal antibody (sc‐133172, Santa Cruz Biotechnology, USA; RRID:AB_2066736). The data obtained were presented as the mean fluorescence intensity and fold change in the expression of C3a receptor. Samples also were double‐stained with control IgG, and cell markers were used to assess any fluorochrome spillover signal. Proper compensation was set to ensure that the median fluorescence intensities of negative and positive cells were identical and were both gated populations.

2.7. Immunoblotting and immunofluorescent staining of C3a receptor in mouse and human brain tissue

Gel electrophoresis followed by immunoblot for C3a receptor expression in the brain tissue (perfused with 50 ml of chilled 1× sterile PBS) samples was performed as previously reported (Zhao et al., 2017). Briefly, mouse brain was homogenized in modified RIPA buffer (Upstate, Lake Placid, NY), supplemented with 40‐mM NaF, 2‐mM Na3VO4, 0.5‐mM phenylmethylsulfonyl fluoride, and 1:100 (v/v) of proteinase inhibitor cocktail (Sigma), electrophoresed and immunoblotted separately against antibodies for anti‐mouse C3aR, and anti‐β actin (β‐actin, Santa Cruz Biotechnology, USA) as loading control. Immuno‐densitometric signal for C3a receptor was quantified in arbitrary units using NIH ImageJ (RRID:SCR_003070) free software and was normalized with that of the β‐actin signal.

Formalin‐fixed de‐identified autopsy human brain specimens (ischaemic left temporal cortex and non‐ischaemic contralateral right cortex) were retrieved from the Department of Pathology's archives. Human brain samples were paraffin‐embedded and 3‐μm serial sections were obtained. Sections were deparaffinized for antigen retrieval prior to staining. For the animal experiments, mouse brains were perfusion fixed with 4% PFA, 10‐μm serial sections were obtained from their frozen blocks, and antigen retrieval was performed with citrate buffer method prior to immunostaining. Immunohistochemical method routine to our laboratory was adopted with certain modifications to stain for C3a receptor in both human and mouse brain tissues (Ahmad et al., 2019). Briefly, sections were blocked with 10% horse serum in TBST (TBS supplemented with 0.5% Triton X‐100) for 1 hr and were incubated overnight at 4°C with primary antibodies against either human or mouse C3a receptor, as relevant to the species (1:50 in 2% horse serum). Following overnight incubation, sections were washed 3× with TBS, followed by 1‐hr incubation at room temperature with species appropriate fluorescein‐conjugated secondary antibody (Alexa Fluor 488, Invitrogen, 1:200 in 2% horse serum). Sections were then washed three times, labelled with endothelial markers (lectin and agglutinin for mouse and human, respectively; Vector Lab, USA) and mounted with DAPI (Vector Lab, USA). Sections were visualized at different magnifications using a fluorescence microscope (Keyence, CA, USA), and images were captured and analysed. The Immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018).

2.8. Neurofunctional outcome assessments in photothrombotic and photothrombotic stroke models

Neurofunctional testing was performed at 48 hr post‐stroke by an investigator blinded to the identity of groups. For the photothrombotic model of cortical infarct, the corner test was performed to assess sensorimotor and postural asymmetries as described previously (Balkaya, Krober, Rex, & Endres, 2013; Zhang et al., 2002). Briefly, two boards were placed at a 30° angle to form a narrow walk and then mouse was placed in between the boards facing a corner. As the animal reaches to the corner, it turns either right or left to exit the corner. For each mouse, this test was repeated 10 times at 30‐s intervals and the % right turn was calculated. The adhesive tape removal (ATR) test was also performed in photothrombotic model to test the sensorimotor function and tactile extinction as we previously reported (Hoda et al., 2014). For the adhesive tape removal test, no significant differences were found between cohorts at baseline (pre‐stroke). In the embolic clot middle cerebral artery occlusion model, neurological deficit scoring (NDS) was assessed on the modified Bederson scale 48 hr after stroke as previously reported by us (Hoda et al., 2014; Rynkowski et al., 2009).

2.9. Semi‐quantitative measurement of corrected infarct volume in photothrombotic and stroke models

For the assessment of infarct volume at 48 hr post‐stroke, animals were anaesthetized and brains were harvested (n = 9 for photothrombotic and n = 6 for thromboembolic). Coronal sections (1 mm thick in photothrombotic and 2 mm thick in embolic clot middle cerebral artery occlusion) were cut using a mouse brain matrix (Roboz Surgical Instrument Co) and stained with 1% solution of 2,3,5‐triphenyl tetrazolium chloride (TTC; Sigma‐Aldrich) at 37 ± 0.5°C for 15–20 min. Corrected infarct volume was calculated as previously reported (Zhao et al., 2017).

2.10. Quantification of haemorrhagic transformation (HT)

Colorimetric assay of Hb content (n = 7 per group) in brain tissue collected at 48 hr post‐ embolic clot middle cerebral artery occlusion was performed as previously reported (Choudhri, Hoh, Solomon, Connolly, & Pinsky, 1997; Hoda et al., 2014). Briefly, each ipsilateral hemisphere was homogenized in 250 μl of 1× chilled PBS. After centrifugation, 20 μl of supernatant obtained was mixed with 80 μl of 1× Drabkin's reagent (Sigma Chemical Co.) in a 96‐well plate and incubated at room temperature in the dark. The plate was read at 540 nm precisely at 15 min, and the Hb content in brain samples was calculated from the standard curve.

2.11. Quantification of brain oedema

Oedema at 48 hr post embolic clot middle cerebral artery occlusion was determined as % water content (n = 7 per group) in the ipsilateral hemisphere as described by us and others using the equation: % Water content = 100 × (Wet weight − Dry weight) / Wet weight (Hoda et al., 2014; Rynkowski et al., 2009).

2.12. Data and statistical analysis

The power analysis was performed using PASS 14 Power Analysis and Sample Size Software (NCSS, LLC. Kaysville, Utah, USA, http://ncss.com/software/pass). Based on our historic data, we anticipated 20% improvement in infarct volume as well as neurological score and 20% SD. We set the sample size to achieve >80% power at 5% level of significance to detect main effects as well as interaction effect using a 2 × 2 factorial ANOVA analysis (F test). Quantitative data are presented as mean ± SD. In Experiments I (embolic clot middle cerebral artery occlusion model) and II (PT model), unpaired Student's t‐test was performed to determine statistical significance. A log transformation was used prior to statistical analysis as needed to stabilize variance across groups. Non‐normal data between groups were compared using nonparametric asymptotic two‐sided Wilcoxon rank sum test. In Experiment III (embolic clot middle cerebral artery occlusion model with or without intravenous thrombolysis), a 2 C3a antagonist therapy (NO vs. YES) by 2 intravenous thrombolysis (NO vs. YES), a factorial ANOVA with interaction between C3a antagonist and intravenous thrombolysis followed by post hoc comparisons using independent t‐tests was used to analyse neurological deficit scoring outcome, infarction volume, Hb content, and oedema. Bonferroni multiple corrections were applied in the post hoc analysis. In the absence of a significant interaction, the main effects are considered to be additive when combined. Statistical analysis was undertaken only for data with at least n = 5, and a P value <.05 was considered statistically significant. No outlying data were excluded from analysis, nor were any assigned animals excluded from the experiments. All statistical analyses were conducted using STATA 15 (RRID:SCR_012763; StataCorp LLC, USA). The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018).

2.13. Materials

All reagents used in this study were purchased from ThermoFisher (https://www.google.com/search?safe=active&q=Waltham,+Massachusetts&stick=H4sIAAAAAAAAAOPgE-LSz9U3MCooMTBJU-IAsTOqjE21tLKTrfTzi9IT8zKrEksy8_NQOFYZqYkphaWJRSWpRcWLWMXCE3NKMhJzdRR8E4uLE5MzSotTS0qKAbi_f6RdAAAA&sa=X&ved=2ahUKEwixv4aH_aHmAhVDqZ4KHboxBMEQmxMoATAkegQICBAh, USA) and Sigma‐Aldrich. SB290157 (CAS no. 1140525‐25‐2) was obtained from EMD Millipore (USA). Antibodies were procured from Bioss USA and Santa Cruz Biotechnology (TX, USA), Abcam (CA, USA), and R&D system (MN, USA). ELISA kit was purchased from LS Bio (USA).

2.14. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Fabbro et al., 2019; Alexander, Kelly 2019).

3. RESULTS

3.1. Concurrent increases in plasma C3a levels and brain endothelial C3a receptor expression following thromboembolic stroke

As the majority of clinical stroke cases occur following thromboembolic occlusion, an embolic clot middle cerebral artery occlusion model is the most clinically relevant preclinical stroke model. Therefore, we first evaluated whether the C3a/C3a receptor axis is altered after murine embolic clot middle cerebral artery occlusion in the acute phase. In Experiment I, we found that, in mice subjected to embolic clot middle cerebral artery occlusion, plasma C3a level was significantly and dramatically elevated as compared to sham‐operated animals (7,440.6 ± 3,384.4 vs. 2,160.8 ± 430.4; Figure 1a). Since increased C3a level causes endothelial dysfunction and haemorrhagic transformation likely via endothelial C3a receptor agonism (Ducruet et al., 2009), we next investigated changes in brain endothelial C3a receptor expression at 6 hr post embolic clot middle cerebral artery occlusion. As expected, brain endothelial C3 receptor expression also was significantly increased by ~2‐fold in the embolic clot middle cerebral artery occlusion group as compared to sham‐operated animals (5,688.3 ± 579.4 vs. 2,936.7 ± 437.9, Figure 1b–d). Subsequent immunoblot of brain tissue harvested 4 weeks after embolic clot middle cerebral artery occlusion provides preliminary evidence that C3a receptor expression remains chronically elevated after stroke (Figure 1e). Similarly, we found an elevated C3a receptor expression on cerebral vessels of human stroke brain tissue relative to non‐ischaemic brain specimens (Figure 1f). These findings in both a murine embolic clot middle cerebral artery occlusion model and human stroke establish that the C3a/C3a receptor axis is hyperactivated in ischaemic cerebral endothelium following thromboembolic stroke.

Figure 1.

Figure 1

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thromboembolic (TE) stroke triggers cleavage of complement component C3 to produce the C3a anaphylatoxin which exacerbates post‐ischaemic cerebral injury. (a) embolic clot middle cerebral artery occlusion (eMCAo) increased plasma C3a levels as early as 6 hr post‐ischaemia, as quantified by ELISA (*P < .05 vs. sham). (b–d) Simultaneously, C3a receptor (R) expression was increased in brain endothelial (CD31+VEGFR2+C3aR+) cells 6 hr post‐stroke, as assessed by flow cytometry. Panel (b) shows the change in brain endothelial C3a receptor expression, while Panels (c) and (d) show the representative flow cytometry panels (*P < .05 vs. eMCAO). (e) Western blot demonstrates that C3a receptor expression in the ipsilateral hemisphere remains elevated at 4 weeks post‐ischaemia. (f) Immunofluorescence of brain sections obtained from human ischaemic cortex showing elevated C3a receptor expression in endothelial cells using agglutinin as an endothelial marker. Comparison is made to cortex obtained from the contralateral hemisphere of the same patient, showing no significant C3a receptor expression. Comparisons of means were performed using Student's t‐test. Data were expressed as mean ± SD (n = 6 for (a)–(d), and n = 2 for (e) with no statistics)

3.2. Alterations in C3a receptor expression and efficacy of C3a antagonist therapy (SB290157) in photothrombotic stroke

To evaluate the efficacy of C3a antagonist treatment in the early post‐ischaemic time period, we used immunostaining to provide preliminary evidence that photothrombotic stroke also triggers an increase in the brain expression of C3a receptor (Figure 2a–c). Moreover, SB290157 treatment 1 hr after photothrombotic stroke significantly improved neurofunctional outcome assessed by the adhesive tape removal (Figure 2d) and corner turn (Figure 2e) tests. Finally, SB290157 significantly reduced the infarct volume relative to vehicle‐treated animals at 48 hr post‐photothrombotic (3.67 ± 0.59 vs. 5.33 ± 0.56; Figure 2f,g).

Figure 2.

Figure 2

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Photothrombotic (PT) stroke triggers increased C3a receptor ® expression in cerebral vasculature. (a) Immunofluorescence analysis of mouse brain demonstrates increased expression of C3a receptor in endothelial cells in the ischaemic region. (b, c) Western blot at 48 hr showed increased C3a receptor expression in mouse PT stroke brain compared with sham. Densitometry analysis was performed using ImageJ (NIH). Data are expressed as mean ± SD (n = 4 with no statistical analysis). (d, e) Functional outcomes (adhesive tape removal and corner test) were improved in C3a antagonist (RA)‐treated animal following PT stroke (*P < .05 vs. sham; *P < .05 vs. PT). (f, g) C3a antagonist administration significantly reduces indirect infarct volume compared to vehicle‐treated animals (*P < .05 vs. PT‐C3aR‐A; n = 9). Comparisons of means were performed using Student's t‐test and one‐way ANOVA with Bonferroni multiple corrections post hoc test

3.3. C3a antagonist therapy following embolic clot middle cerebral artery occlusion improves neurological outcomes and suppresses infarct progression with and without late intravenous thrombolysis

We then assessed the benefits of SB290157 in improving neurological outcomes and preventing infarct progression with or without intravenous thrombolysis in an adhesive tape removal model (Figure 3). We did not observe any significant interaction between C3a antagonist and intravenous thrombolysis treatment groups. We found that C3a antagonist administration alone significantly improved neurological outcomes as compared to the vehicle‐treated group (2.13 ± 0.84 vs. 3.29 ± 0.49; Figure 3a). As expected, late intravenous thrombolysis therapy alone remained ineffective in improving neurological outcomes relative to vehicle‐treated animals (3.30 ± 0.76 vs. 3.29 ± 0.49). The neurological deficit scoring of C3a antagonist group was also significantly improved relative to the intravenous thrombolysis group (2.13 ± 0.84 vs. 3.30 ± 0.76). When tested in combination, the C3a antagonist + intravenous thrombolysis group exhibited a trend towards improvement in the neurologic outcomes as compared to both late intravenous thrombolysis and vehicle (2.28 ± 0.90 vs. 3.30 ± 0.76; and 2.28 ± 0.90 vs. 3.29 ± 0.49 respectively).

Figure 3.

Figure 3

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C3a antagonist (RA) treatment improves neurological outcome and protects against thromboembolic (TE) stroke with or without late intravenous thrombolysis (IVT). (a) C3aRA administration results in improved NDS assessed using a modified Bederson scale at 48 hr post‐stroke, although late IVT alone does not protect against post‐stroke neurological deficit (*P < .05 vs. Veh; $ P < .05 vs. C3aR‐A; # P < .05 vs. IVT). (b) Corrected infarct volume, calculated as per cent of the contralateral hemisphere, was also lower in C3aRA‐treated groups; late IVT alone was not protective (*P < .05 vs. Veh; $ P < .05 vs. C3aR‐A; # P < .05 vs. IVT; *P < .05 vs. Veh). (c) Representative TTC stained coronal sections showing reduced infarction in C3aRA‐treated groups, as well grossly visible signs of haemorrhagic transformation (HT) in the late IVT‐treated group. Data in (a) and (b) are expressed as mean ± SD (n = 6). Factorial ANOVA with post hoc t‐test with Bonferroni multiple corrections was utilized

We next analysed whether C3a antagonist therapy suppresses infarct progression with and without late intravenous thrombolysis (Figure 3b,c). No significant interaction between C3a antagonist and intravenous thrombolysis treatment groups was observed. C3a antagonist alone administered at 2 hr after stroke significantly reduced the infarction volume compared to vehicle‐treated animals (30.63 ± 4.95 vs. 47.26 ± 8.64). As expected, late intravenous thrombolysis therapy alone did not reduce the infarction relative to vehicle‐treated animals (47.06 ± 9.74 vs. 47.26 ± 8.64;). When administered 2.5 hr prior to late intravenous thrombolysis, infarction volume in the C3a antagonist + intravenous thrombolysis group was significantly reduced relative to both the vehicle‐treated group (33.63 ± 5.19 vs. 47.26 ± 8.6) and the intravenous thrombolysis alone group (33.8 ± 7.2 vs. 47.06 ± 9.74). Combination treatment C3a antagonist + intravenous thrombolysis group did not exhibit further reduction in the infarct volume relative to C3a antagonist group.

3.4. C3a antagonist therapy prevents haemorrhagic transformation and oedema with and without late intravenous thrombolysis following intravenous thrombolysis

Hyperactivation of the C3a/C3a receptor axis, especially following late intravenous thrombolysis in stroke, may induce severe cerebrovascular dysfunction resulting in haemorrhagic transformation and brain oedema. Therefore, we next evaluated whether C3a antagonist therapy was effective in preventing haemorrhagic transformation and oedema after thromboembolic stroke, particularly when combined with late intravenous thrombolysis treatment (Figure 4). There was no statistical interaction observed between C3a antagonist and intravenous thrombolysis (Figure 4a). C3a antagonist treatment alone reduced the brain Hb content in comparison to the Veh group (1.50 ± 0.66 vs. 3.31 ± 0.82). In contrast, late intravenous thrombolysis alone significantly increased the Hb content in the intravenous thrombolysis group as compared to the Veh group (6.66 ± 1.33 vs. 3.31 ± 0.82 ). Nevertheless, combination therapy of C3a antagonist prior to late intravenous thrombolysis significantly decreased the brain Hb content by a factor of 3 in the C3a antagonist + intravenous thrombolysis group as compared to the intravenous thrombolysis group (2.08 ± 0.97 vs. 6.66 ± 1.33).

Figure 4.

Figure 4

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C3a anatagnist (RA) therapy following thromboembolic (TE) stroke prevents haemorrhagic transformation (HT) and cerebral oedema with or without late intravenous thrombolysis (IVT). (a) Quantification of Hb content by spectrophotometric Hb assay. C3aRA therapy in both groups (with or without late IVT) reduced the Hb content in ischaemic brain. Late IVT exacerbated HT, and this was abrogated by prior C3aRA treatment (*P < .05 vs. Veh; $ P < .05 vs. C3aR‐A; # P < .05 vs. IVT; *P < .05 vs. Veh). (b) C3aRA therapy prevents secondary brain oedema assessed by the wet–dry method following TE stroke. Late IVT increased brain oedema; however, C3aRA therapy remained protective in both groups with or without late IVT (* P < .05 vs. Veh; $ P < .05 vs. C3aR‐A; # P < .05 vs. IVT; *P < .05 vs. Veh). Data in (a) and (b) are expressed as mean ± SD (n = 6). Factorial ANOVA with post hoc t‐test with Bonferroni multiple corrections was utilized

Increased haemorrhagic transformation, specifically as a result of late intravenous thrombolysis treatment, promotes brain oedema after stroke (Hoda et al., 2012). C3a antagonist administration alone after intravenous thrombolysis attenuated oedema in C3a antagonist group relative to the Veh group (76.60 ± 4.80 vs. 82.44 ± 2.90 Figure 4b). As expected, intravenous thrombolysis group exhibited the significantly higher oedema among all four groups (89.74 ± 2.33), likely due in part to increased haemorrhagic transformation as a result of late intravenous thrombolysis. The cohort receiving the combination therapy in the C3a antagonist + intravenous thrombolysis group exhibited lesser oedema (81.17 ± 3.86) relative to the intravenous thrombolysis group receiving late intravenous thrombolysis alone, thus demonstrating that C3a antagonist treatment attenuates the detrimental side effects of late thrombolysis.

4. DISCUSSION

We have previously reported that C3a antagonist confers robust neurovascular protection in a mechanically induced transient middle cerebral artery occlusion model that mimics a large vessel clinical stroke treated using endovascular thrombectomy. Since the majority of human stroke cases are thromboembolic in nature and intravenous thrombolysis remains the first line of defence, the critical question remains unanswered whether C3a antagonist therapy will be protective in thromboembolic stroke models and in combination with intravenous thrombolysis therapy. To the best of our knowledge, we now report the first preclinical validation of any C3a antagonists with and without intravenous thrombolysis in thromboembolic stroke models. These key findings support that C3a antagonist therapy is protective in thromboembolic stroke and reduces haemorrhagic transformation and oedema as a result of late intravenous thrombolysis. C3a antagonist therapy may therefore help to extend the window of thrombolysis after stroke.

Endothelial dysfunction triggers blood–brain barrier breakdown and exacerbates brain oedema after cerebral ischaemia (Krueger et al., 2017). We have reported the novel finding that intravenous thrombolysis‐generated plasmin acts as the major non‐canonical activator of C3 cleavage. Elevated plasma C3a in stroke, particularly after intravenous thrombolysis, hyperactivates the C3a receptor leading to endothelial dysfunction, which translates into increased haemorrhagic transformation and aggravated oedema progression (Wu et al., 2016). The present study demonstrates that plasma levels of C3a are increased during the acute phase of thromboembolic stroke (Figure 1a), which is in agreement with a prior report in human ischaemic stroke (Mocco et al., 2006). An abrupt increase in the level of endogenous ligands/agonists can accentuate transcriptional regulation, expression and functional activities of their cognate receptors (Gu et al., 2016). Along these lines, we also found that cerebrovascular expression of C3a receptor after thromboembolic stroke was increased in both mouse and human tissues and co‐localized with the markers of brain endothelial cells and blood vessels (Figures 1b–f and 2a–c). Furthermore, C3a receptor in mouse remained elevated out to 4 weeks following TE stroke (Figure 1e). The role of the chronic expression of C3a receptor post‐stroke requires further exploration. Indeed, a prior report demonstrates that C3a receptor inactivation reverses Alzheimer's disease‐type pathology (Litvinchuk et al., 2018). Since the progression of dementia is an important pathological sequela that increases dramatically among stroke survivors, it will be of invaluable translational interest in the future to investigate the potential role of C3a receptor hyperactivation in post‐stroke dementia and to validate if plasma levels of C3a can serve as a potential biomarker (Stokowska et al., 2011; Stokowska et al., 2013). The evaluation of the effects of the C3a receptor/C3a axis in the chronic phase of stroke recovery is also critical given additional reports of a potential beneficial role through increased post‐stroke synaptogenesis and axonal plasticity (Stokowska et al., 2017).

Thromboembolic stroke occurs either secondary to a vascular occlusion from a locally generated platelet‐rich thrombus or from a fibrin‐rich embolus that travels from a remote region to obstruct a cerebral artery (Chen et al., 2015). Therefore, we first tested SB290157 in a photothrombotic stroke model, which causes a smaller cortical infarct induced with locally generated thrombi in the middle cerebral artery territory. SB290157 therapy after photothrombotic stroke significantly improved sensorimotor impairment and postural asymmetries due to this cortical injury (Figure 2d,e), a finding that was further supported by reduced infarct volume (Figure 2f,g). Thus, our data derived from the photothrombotic model validate the efficacy of SB290157 in an early yet narrow therapeutic window following thrombotic occlusion.

The STAIR recommendations endorse evaluating adjuvant therapies to improve the safety and efficacy of Food and Drug Administration‐approved reperfusion therapies by reducing the risk of reperfusion injury, haemorrhagic transformation and oedema progression (Liebeskind, Derdeyn, Wechsler, & S.X.C.*, 2018). Our goal was also to utilize different stroke models to simulate a variety of clinical stroke syndromes and demonstrate the broad efficacy of C3a antagonist across different settings. The photothrombotic model, in our hands, is a spontaneously fast‐reperfusing stroke model with platelet‐rich clots, resulting in a shorter duration of ischaemia and smaller infarction that is not suitable for evaluation of combination therapy with intravenous thrombolysis administered in the late time window. Therefore, we utilized the photothrombotic model to evaluate C3a antagonist therapy (SB290157) in a spontaneously reperfusing “thrombotic” model without late intravenous thrombolysis. To test the effect of “late” intravenous thrombolysis, we utilized our well‐established thromboembolic model using a fibrin‐rich clot, which produces stable occlusion and longer period of significant ischaemia to test in combination with “late” intravenous thrombolysis (i.e. beyond 4.5 hr post‐stroke; Hoda et al., 2012). It is evident from our data that delayed SB290157 therapy given alone at 2 hr after embolic clot middle cerebral artery occlusion remains effective not only in improving neurologic outcomes and reducing infarction but also in reducing the detrimental effects of late intravenous thrombolysis (Figures 3 and 4). Moreover, we purposely administered late intravenous thrombolysis at 4.5 hr after embolic clot middle cerebral artery occlusion to mimic detrimental side effects in the setting of late thrombolysis. As expected, late intravenous thrombolysis was ineffective in improving outcomes likely due to its well‐known detrimental effects including haemorrhagic transformation and oedema (Hoda et al., 2012). On the other hand, SB290157 therapy prior to late intravenous thrombolysis attenuated post‐thrombolysis haemorrhagic transformation and the progression of oedema. These data collectively establish that C3a antagonist therapy is beneficial in thromboembolic stroke and safe with late intravenous thrombolysis.

We have extensively reported that C3a/C3a receptor axis is integral to the pathophysiology of ischaemic stroke independent of the stroke subtype and pattern of reperfusion (Ducruet et al., 2008; Ducruet et al., 2009; Ducruet et al., 2012). Herein, we provide further evidence supporting the efficacy, safety and therapeutic potential of C3a antagonists such as SB290157 in thromboembolic stroke with and without late intravenous thrombolysis. Nevertheless, the present study has certain limitations that must be addressed in the future to strengthen our preclinical safety data prior to further clinical translation. For example, stroke is prevalent in aged individuals and post‐stroke pathophysiology could be sexually dimorphic (Ahnstedt, McCullough, & Cipolla, 2016; Brann, Dhandapani, Wakade, Mahesh, & Khan, 2007; Davis, Fairbanks, & Alkayed, 2013; Kim, Chelluboina, Chokkalla, & Vemuganti, 2019) and we have yet to validate the sex‐independent benefits of C3a antagonist therapy in animal models of stroke. We did, however, utilize aged mice in our embolic clot middle cerebral artery occlusion model and we have previously reported that ageing abolishes this sexual dimorphism at least in the embolic clot middle cerebral artery occlusion model of thromboembolic stroke (Hoda et al., 2014). Further work evaluating both young and aged mice in the embolic clot middle cerebral artery occlusion model is planned for the future. We also did not utilize co‐morbid (diabetic and hypertensive) animals that often exhibit higher haemorrhagic transformation, oedema and mortality (Ergul, Kelly‐Cobbs, Abdalla, & Fagan, 2012; Martini & Kent, 2007). Therefore, C3a antagonist therapy needs to be tested in co‐morbid stroke models. Furthermore, we previously reported that long‐term treatment with SB290157 promotes neurogenesis at the same dose that we used in the present work (Ducruet et al., 2012). However, we acknowledge a few reports regarding off‐target effects of high‐dose and chronic SB290157 treatment (Mathieu et al., 2005; Therien, 2005), a phenomenon seen with numerous pharmacological agents.

5. CONCLUSIONS

In conclusion, chronic administration of SB290157 and other next‐generation C3a antagonists after embolic clot middle cerebral artery occlusion needs to be validated in rodents at risk of post‐stroke VaD/VCI. Most importantly, as our data show a sustained chronic increase in the expression of brain endothelial C3a receptor, future studies are warranted for the possible development and translation of brain–endothelium targeting genetic and pharmacological tools to manipulate the C3a/C3a receptor axis for the prevention of small vessel disease among stroke survivors.

AUTHOR CONTRIBUTIONS

S.A. performed experiments, analysed data, and drafted the manuscript; C.P., A.K., K.B., and R.C. performed experiments; A.K.D. performed statistical analysis; J.M.E. provided the human stroke samples and edited the manuscript; Q.L. and M.F.W. critically reviewed the manuscript; M.N.H. designed the experiments and edited the manuscript; and A.F.D. designed and oversaw the whole project, assisted in data analysis, and wrote and edited the manuscript.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14206, and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

ACKNOWLEDGEMENTS

This work was supported in part by the Barrow Neurological Foundation.

Ahmad S, Pandya C, Kindelin A, et al. C3a receptor antagonist therapy is protective with or without thrombolysis in murine thromboembolic stroke. Br J Pharmacol. 2020;177:2466–2477. 10.1111/bph.14989

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