Myeloperoxidase propagates damage and is a potential therapeutic target for subacute stroke

Abstract

Few effective treatment options exist for stroke beyond the hyperacute period. Radical generation and myeloperoxidase (MPO) have been implicated in stroke. We investigated whether pharmacologic reduction or gene deletion of this highly oxidative enzyme reduces infarct propagation and improves outcome in the transient middle cerebral artery occlusion mouse model (MCAO). Mice were treated with 4-aminobenzoic acid hydrazide (ABAH), a specific irreversible MPO inhibitor. Three treatment regimens were used: (1) daily throughout the 21-day observational period, (2) during the acute stage (first 24 hours), or (3) during the subacute stage (daily starting on day 2). We found elevated MPO activity in ipsilateral brain 3 to 21 days after ischemia. 4-Aminobenzoic acid hydrazide reduced enzyme activity by 30% to 40% and final lesion volume by 60% (P<0.01). The MPO-knockout (KO) mice subjected to MCAO also showed a similar reduction in the final lesion volume (P<0.01). The ABAH treatment or MPO-KO mice also improved neurobehavioral outcome (P<0.001) and survival (P=0.01), but ABAH had no additional beneficial effects in MPO-KO mice, confirming specificity of ABAH. Interestingly, inhibiting MPO activity during the subacute stage recapitulated most of the therapeutic benefit of continuous MPO inhibition, suggesting that MPO-targeted therapies could be useful when given after 24 hours of stroke onset.

Figure 1

Figure summarizing the number of animals in different treatment groups and experiments performed. A subset of animals underwent specific tests such as MRI, NBT, or both. The number of animals in each group that underwent tests is provided in the bottom row, in brackets, next to the test(s) performed. ABAH, 4-aminobenzoic acid hydrazide; DWI, diffusion-weighted images (MRI); MPO, myeloperoxidase; MPO-Gd, activatable MPO specific MRI contrast agent; MPO-KO, MPO-knockout mice; MRI, magnetic resonance imaging; NBT, neurobehavior testing; T2w, T2-weighted images (MRI).

Figure 2

Temporal evolution of infarction and associated neuroinflammation using MPO-Gd molecular imaging. (A) Representative diffusion-weighted image (DWI) and apparent diffusion coefficient (ADC) map show the area of restricted diffusion on day 1 after stroke induction. (B) Pooled data from seven control (saline) treated mice show the mean infarct volume on day 1 based on the volume of area of restricted diffusion on the ADC map and the abnormal T2 hyperintense areas (days 1 to 21) representing a combination of infarcted tissue and edema. (C) Representative T2W images (top row) and MPO-Gd enhanced T1w images from a control mouse show the evolution of the T2 signal abnormalities and myeloperoxidase (MPO)-specific enhancement and neuroinflammation associated with cerebral infarction.

Figure 3

Partial inhibition of myeloperoxidase (MPO) activity after treatment of stroke mice with 4-aminobenzoic acid hydrazide (ABAH) beginning within half an hour after completion of stroke induction. (A) Semiquantitative calculation of in vivo MPO inhibition using the activation ratio (CNR 60 minutes/CNR 6 minutes) shows partial inhibition of MPO activity in vivo in ABAH-treated (n=7) animals compared with control (n=5) stroke mice. (B) Peroxidase assays performed on freshly prepared brain extracts confirm partial MPO activity inhibition on day 3 after induction (controls: n=3; ABAH: n=3). (C) Pseudocolor coded image of MPO-Gd imaging on brain MRI on day 3 after infarction shows reduction in activity in ABAH-treated mice compared with saline-treated controls. *P≤0.05, **P<0.01. CNR, contrast-to-noise ratio.

Figure 4

T2 lesion volumes on days 3 and 7 measured on T2w images. (A) Day 3 and (B) day 7 after infarct induction in wild-type mice treated with 4-aminobenzoic acid hydrazide (ABAH) (n=11), with saline control vehicle (n=7) or in myeloperoxidase (MPO)-knockout (MPO-KO) mice (n=9). **P <0.01, ***P<0.001.

Figure 5

T2 lesion volume on day 21 is significantly reduced after inhibition of myeloperoxidase (MPO) activity for the entire 21-day period after infarction and in MPO-knockout (MPO-KO) mice. Comparison of both (A) absolute volume on day 21 after infarction and (B) normalized volume (divided by the day 1 volume of restricted diffusion to correct for variations in infarct induction) shows significantly reduced final volume in mice treated with 4-aminobenzoic acid hydrazide (ABAH) (n=11) or in MPO-KO mice (n=9) compared with controls (n=7). (C) Illustrative examples. WT=wild-type mice treated with saline; ABAH=wild-type mice treated with ABAH; and MPO−/−=MPO-KO mouse. *P<0.05, **P<0.01, ns, not significant; DWI, diffusion-weighted images.

Figure 6

Neurobehavioral evaluation and survival analysis show beneficial effect of reduced myeloperoxidase (MPO) activity on stroke outcome. (A) Behavior and functional testing on day 21 after infarction (n: sham=8; saline vehicle=11; ABAH=10; MPO-KO saline=4, MPO-KO ABAH=3). Lower score indicates more deficits. All groups except sham were compared using two-way analysis of variance (ANOVA). The sham group was compared using one-way ANOVA. (B) Neurobehavioral testing results for days 3, 7, and 12. Statistical significance is shown for all groups compared with the saline vehicle control group, using the one-way ANOVA. (C) Survival analysis (n: ABAH=28; MPO-KO=10; saline-treated controls=25). *P<0.05, ****P<0.0001. ABAH, 4-aminobenzoic acid hydrazide; KO, knockout.

Figure 7

Myeloperoxidase (MPO) inhibition has the greatest impact when treatment was begun during the subacute stage of stroke (2 days) and continued for 21 days. (A) Comparison of normalized T2 lesion volume shows significantly smaller T2 lesion volume in the group treated during the subacute compared with the acute stage. There was only a mild decrease in the average T2 lesion volume in the group treated during the acute stage compared with controls (n: saline-treated controls=7, acute=5, subacute=8). (B) Neurobehavioral evaluation on day 21 after infarction. Treatment with 4-aminobenzoic acid hydrazide (ABAH) during the subacute stage confers the greatest benefit, similar to treatment during the entire (acute and subacute) periods as shown in Figure 5A. *P<0.05, **P<0.01, ****P<0.0001, ns=not significant (n: sham=8, saline=11, acute=5, subacute=8). Comparisons were made using the one-way ANOVA test.