A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window

Abstract

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 +/- 22% when the treatment is started 12 h before ischemia and by 63 +/- 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1beta-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E(2) production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

Figures

Figure 1

(a) Minocycline treatment started 12 h before ischemia does not alter body temperature (T), mean arterial blood pressure (MABP), arterial PO2, PCO2, pH, or plasma glucose, as determined by measurements taken during the ischemia or 15 min after the ischemia (n = 5 in each group). (b) Minocycline treatment started 12 h before (pretreated) or 2 h after (posttreated) the onset of ischemia reduces the infarction area seen in triphenyltetrazolium chloride-stained brain slices 3 days after ischemia compared with saline-treated animals. (c) The infarction area is significantly reduced in the cortex and striatum compared with saline-treated animals when the minocycline treatment is started 12 h before ischemia (Pre, n = 10), 2 h after the onset of ischemia (Post, n = 10), or 4 h after the onset of ischemia (Post 4 h, n = 12). Values are means ± SD. *, P < 0.01 (one-way ANOVA followed by the Bonferroni test).

Figure 2

(a) Minocycline treatment inhibits ischemia-induced activation of microglia. Amoeboid-shaped CD11b-immunoreactive cells are seen around the infarction core 24 h after ischemia in saline-treated, but not in minocycline-treated, animals. (Bar = 20 μm.) (b) The ischemia-induced expression of ICE mRNA is prevented by minocycline treatment. The numbers and letter C in the brain section (B Upper Left) show the site of samples: (1) infarcted core in the striatum, (2) infarcted core in the cortex, (3) the cortical area adjacent to ischemia core (penumbra), and (C) the contralateral cortex used as control tissue. The arrowhead in the gel (B Upper Right) points to RT-PCR amplification products of ICE mRNA detected in the samples of the contralateral cortex (control) and in the penumbra (area 3) of saline- and minocycline-treated animals 12 h after ischemia. The histograms (B Lower) show quantitation of ICE bands derived from the three different regions of the ischemic hemisphere. Minocycline treatment (M) significantly reduced expression of the ICE message in the penumbra compared with expression after saline treatment (S; n = 4; ★, P < 0.01, one-way ANOVA followed by Bonferroni test; C refers to control tissue). (c) Minocycline-treatment prevents ischemia-induced COX-2 immunoreactivity and PGE2 production in the penumbra. COX-2 immunoreactivity (arrows in C Upper Left and at higher magnification in C Upper Right) is seen in cortical neurons 24 h after ischemia. At the same time point, the increase in PGE2 concentration (C Lower Right) is reduced by 51% (★, P < 0.01, one-way ANOVA followed by Bonferroni test). The results are from three different experiments (n = 5 + 5 in each). PGE2P/C, ratio of PGE2 concentrations in penumbra and the contralateral cortex.