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

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.

Figure 1

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)

Figure 2

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

Figure 3

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

Figure 4

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