Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice

Abstract

Background and purpose: Reactive oxygen species (ROS) derived from Nox2-containing reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is reportedly detrimental in cerebrovascular disease. However, ROS generation by other Nox isoforms may have a physiological role. No Nox2-selective inhibitors have yet been identified, and thus it is unclear whether isoform non-selective Nox inhibitors would necessarily improve outcome after stroke. We assessed the effect of apocynin on cerebrovascular ROS production and also on outcome following cerebral ischaemia when administered either before ischaemia or after cerebral reperfusion. The involvement of Nox2-containing NADPH oxidase in the effects of apocynin was assessed using Nox2(-/-) mice.

Experimental approach: Transient cerebral ischaemia was induced by 0.5 h middle cerebral artery occlusion followed by 23.5 h reperfusion. Mice received apocynin (2.5 mg.kg(-1), i.p.) either 0.5 h before ischaemia or 1 h after reperfusion. In situ superoxide production after cerebral ischaemia-reperfusion was measured in brain sections of wild-type mice at 24 h using dihydroethidium fluorescence.

Key results: Treatment with apocynin 0.5 h before ischaemia reduced total infarct volume, neurological impairment and mortality in wild-type but not Nox2(-/-) mice. Conversely, treatment with apocynin 1 h after initiation of reperfusion had no protective effect. Cerebral ischaemia and reperfusion increased superoxide production in the brain at 24 h, and pretreatment but not posttreatment with apocynin reduced superoxide levels.

Conclusions and implications: Apocynin improves outcome following stroke when administered before ischaemia in wild-type but not Nox2(-/-) mice.

Figure 1

The effect of apocynin 0.5 h pre-ischaemia in wild-type mice. (A) Regional cerebral blood flow (rCBF) decreased by ∼80% upon insertion of the monofilament, remained low throughout the ischaemic period (0.5 h) and increased upon reperfusion to a similar extent in mice treated with either vehicle (0.1% DMSO, n = 23) or apocynin (2.5 mg·kg⁻¹, n = 10). (B-C) Representative coronal brain sections from mice treated with vehicle (B) or apocynin (C) showing a smaller infarct area in apocynin (2.5 mg·kg⁻¹)-treated mice outlined in white. (D-E) Mice treated with apocynin (2.5 mg·kg⁻¹; n = 8, *P < 0.05) had smaller total, cortical and subcortical infarct volumes following MCAO than vehicle-treated mice (0.1% DMSO, n = 10). DMSO, dimethyl sulphoxide; MCAO, middle cerebral artery occlusion.

Figure 2

The effect of apocynin 0.5 h pre-ischaemia in Nox2^−/− mice. (A) Regional cerebral blood flow (rCBF) during MCAO in Nox2^−/− mice treated with either vehicle (0.1% DMSO, n = 6) or apocynin (2.5 mg·kg⁻¹, n = 5). (B-C) Representative coronal brain sections from Nox2^−/− mice treated with either vehicle (B) or apocynin (C) showing similar infarct areas outlined in white. (D-E) Mice treated with either vehicle (0.1% DMSO, n = 5) or apocynin (2.5 mg·kg⁻¹, n = 5) had similar total, cortical and subcortical infarct volumes following MCAO. DMSO, dimethyl sulphoxide; MCAO, middle cerebral artery occlusion.

Figure 3

The effect of apocynin 1 h post-reperfusion in wild-type mice. (A) Regional cerebral blood flow (rCBF) during MCAO in Mice treated with either vehicle (0.1% DMSO, n = 6) or apocynin (2.5 mg·kg⁻¹, n = 6). (B-C) Representative coronal brain sections from mice treated with either vehicle (B) or apocynin (C) showing similar infarct areas outlined in white. (D-E) Mice treated with either vehicle (0.1% DMSO, n = 6) or apocynin (2.5 mg·kg⁻¹, n = 6) had similar total, cortical and subcortical infarct volumes following MCAO. DMSO, dimethyl sulphoxide; MCAO, middle cerebral artery occlusion.

Figure 4

In situ cerebral superoxide production. Representative images of coronal brain sections (+1.0 mm from bregma) from mice infused with dihydroethidium. (A) Sham MCAO, (B) vehicle-treated MCAO, (C) 2.5 mg·kg⁻¹ apocynin 0.5 h pre-ischaemia and (D) 2.5 mg·kg⁻¹ apocynin 1 h post-reperfusion. (D) and (E) Semi-quantitative group data indicating relative fluorescence units (RFU; average of n = 5 slices per brain from n = 3 mice per group) presented as mean ± s.e.mean from the total ipsilateral hemisphere (E) and the ipsilateral cortex (F). MCAO, middle cerebral artery occlusion.