Neutrophils contribute to intracerebral haemorrhages after treatment with recombinant tissue plasminogen activator following cerebral ischaemia

Abstract

Background and purpose: Polymorphonuclear neutrophils (PMNs) contribute to the vascular damage caused by transient cerebral ischaemia. Here we have evaluated the role of PMNs in intracerebral haemorrhage (ICH) induced in a model of thrombolysis with recombinant tissue plasminogen activator (t-PA) during the acute phase of cerebral ischaemia.

Experimental approach: The middle cerebral artery (MCA) of male spontaneously hypertensive rats was occluded for 1 h followed by reperfusion and, 5 h later, infusion of thrombolytic products (generated in vitro by t-PA on autologous clots). Effects of pretreatment (before the MCA occlusion) with vinblastine (4 days before; 0.5 mg.kg(-1)), monoclonal anti-neutrophil antibody (mAbRP3; 12 h, 0.3 mg.kg(-1)) or saline on ICH, neutrophil infiltration, MCA vascular reactivity and brain infarct volume were assessed, 24 h after the beginning of reperfusion.

Key results: Depletion of circulating neutrophils significantly reduced t-PA-induced ICH (vinblastine, 4.6 +/- 1.0; mAbRP3, 5.2 +/- 1.0 vs. saline, 10.8 +/- 2.7 haemorrhages; P < 0.05). This depletion was associated with a decrease in cerebral infiltration by neutrophils and a decrease of endothelium-dependent, vascular dysfunction in isolated MCA, induced by the ischaemia/reperfusion and t-PA treatment. Brain infarct volume was significantly decreased after vinblastine treatment (159 +/- 13 mm(3) vs. 243 +/- 16 mm(3) with saline; P < 0.01) but not after depletion with mAbRP3 (221 +/- 22 mm(3)).

Conclusions and implications: Our results showed that pharmacological depletion of PMNs prevented t-PA-induced ICH, in parallel with a decrease in cerebral infiltration by PMNs and a decreased endothelial dysfunction in cerebral blood vessels.

Figure 1

Effect of i.v. administration of vehicle (NaCl 0.9%), vinblastine (0.5 mg·kg⁻¹) or mAbRP3 (0.3 mg·kg⁻¹) on neutrophil infiltration in rats submitted to ischaemia/reperfusion and TLP treatment. Infiltration was quantified by counting cells positive to anti-MPO antibody on six adjacent fields of 1 mm² in ischaemic zones. Values are mean ± SEM. *P < 0.05 vs. vehicle. Scale bar: 100 µm. MPO, myeloperoxidase; PMN, polymorphonuclear neutrophil; TLP, thrombolysis products.

Figure 2

Effect of i.v. administration of vehicle (NaCl 0.9%), vinblastine (0.5 mg·kg⁻¹) or mAbRP3 (0.3 mg·kg⁻¹) on haemorrhagic risk in rats submitted to ischaemia/reperfusion and thrombolysis products treatment. Intracerebral haemorrhages were confined to infarct areas as seen in vivo on T2-weighted MRI images (A). Petechial haemorrhages were macroscopically visible on histological sections (B).

Figure 3

Effect of i.v. administration of vehicle (NaCl 0.9%), vinblastine (Vb: 0.5 mg·kg⁻¹) or mAbRP3 (0.3 mg·kg⁻¹) on total, cortical and striatal infarct volume (corrected for oedema). All rats were submitted to ischaemia/reperfusion and thrombolysis products treatment. (*P < 0.05 vs. vehicle). Volumes are expressed in mm³ (mean ± SEM).