Elsevier

Neuroscience

Volume 154, Issue 2, 23 June 2008, Pages 556-562
Neuroscience

Clinical neuroscience
Apocynin improves outcome in experimental stroke with a narrow dose range

https://doi.org/10.1016/j.neuroscience.2008.03.090Get rights and content

Abstract

Inflammation following ischemic stroke is known to contribute to injury. NADPH oxidase (NOX) is a major enzyme system originally studied in immune cells that leads to superoxide (O·) generation. Apocynin is a NOX inhibitor that has been studied as a potential treatment in experimental stroke. Here we explored the effect of different doses of apocynin in a mouse model of 2 h transient middle cerebral artery occlusion (tMCAO) followed by 22 h reperfusion. Apocynin, given i.v. at a dose of 2.5 mg/kg 30 min before reperfusion, improved neurological function (P<0.01), reduced infarct volume (P<0.05), and reduced the incidence of cerebral hemorrhage (P<0.05), but not at higher doses of 3.75 and 5 mg/kg, where it actually increased brain hemorrhage. Apocynin also tended to reduce mortality at the lower dose, but not at higher doses. Using hydroethine fluorescence to delineate O· in the brain, neurons and some microglia/macrophages, but not vascular endothelial cells were found to contain O·. Apocynin at protective doses markedly prevented ischemia-induced increases in O·. Our data suggested that apocynin can protect against experimental stroke, but with a narrow therapeutic window.

Section snippets

Experimental procedures

All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except where noted. Animals were obtained from Jackson Laboratories (Bar Harbor, ME, USA).

Apocynin penetrates the intact BBB

Texas Red–labeled apocynin administered to uninjured mice was readily detected 3 h later in the brain (Fig. 1A). In comparison, injection of Texas Red led to little detection of the fluorescent label (Fig. 1B). Thus, after systemic administration, apocynin is capable of crossing the BBB and entering the brain.

Apocynin reduces infarct size, but not at higher doses

Compared with vehicle-treated animals, apocynin 2.5 mg/kg significantly reduced infarct volume by about 40% (84.6±30.6 mm3 vs. 139.4±35.7 mm3, P<0.05). However, at higher doses, apocynin

Discussion

In this study, we show that apocynin at a dose of 2.5 mg/kg given just prior to reperfusion, or 1.5 h after ischemia onset, resulted in reduced infarct volume, BBB disruption and cerebral hemorrhage, and improved neurological outcome. We also found that in this model of stroke, O· is largely generated by neurons and some microglia/monocytes, with no generation in brain vascular endothelial cells. Apocynin at protective doses markedly reduced O· in the brain. Interestingly, apocynin at higher

Conclusions

In conclusion, our data showed that apocynin, a NOX inhibitor, reduces infarct volume, cerebral hemorrhage, and BBB disruption and improves neurological function following experimental stroke but with a narrow therapeutic window. We suggest that this is a relevant therapeutic target, but safer inhibitors should be developed.

Acknowledgments

This work was supported by NIH NINDS grant R01 NS40516 (M.A.Y.), P50 NS014543 (M.A.Y.), P01 NS37520 (M.A.Y.), and an American Heart Association Established Investigator Award (M.A.Y.).

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