Tracking the inflammatory response in stroke in vivo by sensing the enzyme myeloperoxidase

Abstract

Inflammation can extend ischemic brain injury and adversely affect outcome in experimental animal models. A key difficulty in translating animal studies to humans is the lack of a definitive method to confirm and track inflammation in the brain in vivo. Myeloperoxidase (MPO), a key inflammatory enzyme secreted by activated neutrophils and macrophages/microglia, can generate highly reactive oxygen species to cause additional damage in cerebral ischemia. We report here that a functional, enzyme-activatable MRI agent can accurately track the oxidative activity of MPO noninvasively in stroke in living animals. We found that MPO is widely distributed in ischemic tissues, correlates positively with infarct size, and is detected even 3 weeks postinfarction. The peak level of MPO activity, determined by activation of the MPO-sensing agent in vivo and confirmed by MPO activity and quantitative RT-PCR assays, occurred on day 3 after ischemia. Both neutrophils and macrophages/microglia contribute to secrete MPO in the ischemic brain, although neutrophils peak earlier (days 1-3) whereas macrophages/microglia are most abundant later (days 3-7). In contrast to the conventional MRI agent diethylenetriamine-pentatacetate gadolinium, which reports blood-brain barrier disruption, MPO imaging is able to additionally track MPO activity and confirm inflammation on the molecular level in vivo, information that was previously only possible to obtain on ex vivo brain sections and impossible to assess in living human patients. Our findings could allow efficient noninvasive serial screening of therapies targeting inflammation and the use of MPO imaging as an imaging biomarker to risk-stratify patients.

Fig. 1.

MPO-sensing agent activation. (A) Mechanism of the MPO agent activation: MPO oxidizes the 5-hydroxytryptamide (5HT) moiety of MPO-Gd that leads to oligomerization and the activated agent can further bind to proteins. This results in a large increase in longitudinal relaxation rate (R₁) and prolonged enhancement at sites of increased MPO activity. (B) Representative example at 90 min of an animal imaged with the 2 agents, on days 7 (DTPA-Gd) and 8 (MPO-Gd). The oval indicates the area used for the image analysis. (C) MPO imaging results in higher CNRs than those of conventional imaging on delayed time points. Direct comparison of the 2 agents in the same animals imaged 1 day apart shows a 15% higher enhancement level of MPO-Gd at 60 min (P = 0.013) and 35% higher at 90 min after agent injection (P = 0.001). One animal was imaged with MPO-Gd first, and 2 animals were given DTPA-Gd first. (D) Representative images of the increased enhancement from MPO activation at 60 min compared with 6 min in 2 different animals, showing different degrees of MPO activation. (E) Activation ratios of MPO-Gd at 60 and 90 min demonstrate significantly higher CNRs than those of DTPA-Gd. *, P < 0.05; **, P < 0.01. (F) MPO imaging in MPO KO mice. Representative images of MPO imaging in MPO KO mice at 6 and 60 min after MPO-Gd administration (day 7), demonstrating no obvious increased enhancement at 60 min compared with the 6-min image. Activation ratios on days 3 and 7 after cerebral ischemia revealed statistically significant differences between MPO-Gd imaging of WT and MPO knockout mice and between MPO-Gd and DTPA-Gd imaging of the WT mice. No statistically significant difference was found between MPO-Gd imaging of MPO knockout mice and DTPA-Gd imaging of WT mice. These findings confirm specificity of MPO-Gd imaging.

Fig. 2.

MPO imaging allows tracking of inflammation in stroke in vivo over time. (A) (Upper) Infarct development over time is shown on ADC and T₂-weighted images. (Lower) Corresponding sections in the same mouse imaged with MPO-Gd (at 60 min after agent injection) show the enhancement evolution over time. (B) MPO imaging of a different mouse. (C) Quantitative analysis over 3 weeks after infarct demonstrates that the absolute CNR enhancement, which represents both BBB breakdown and MPO activation of the agent, peaks on day 7 (P < 0.05 on all days compared with the sham-operated animals). (D) Activation ratio of the MPO agent reveals that the highest MPO activity occurs on day 3 after stroke and remains elevated on day 21 (P < 0.05 on all days compared with the sham operated animals). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

Biochemical analyses corroborate MPO imaging findings. (A) MPO activity of activated macrophages/neutrophils (act. Mac/Neu) show that both express ≈10-fold higher MPO levels compared with the nonactivated cells (n.act. Mac/Neu) (P = 0.044 for macrophages and P = 0.043 for neutrophils). (B) Western blots confirm elevated MPO levels in the ischemic brain. Western blots detect high levels of the MPO precursor protein (92 kDa) and the MPO heavy chain (60 kDa) in the ischemic hemisphere, whereas only faint background levels of MPO exist in the sham-operated animals. GAPDH (38 kDa) is shown as a loading control. (C) MPO activity assays correlate with the MR imaging results. The MPO activity assays results confirm that the peak of MPO expression is at day 3 and shows significantly higher MPO expression (P < 0.05) compared with the sham-operated animals on all days except day 21. The MPO assay results correlate well with the activation ratio (R² = 0.85). (D) qRT-PCR for MPO mRNA confirms the activation ratio (R² = 0.93) and the enzyme activity assays (R² = 0.83). *, P < 0.05; **, P < 0.01.

Fig. 4.

Histopathological analyses correspond to MPO imaging findings. (A) MPO imaging on day 3 after infarction shows a large area of enhancement in the basal ganglia and cerebral cortex. The infarct appears as pale areas in the cortex and basal ganglia (H&E). The corresponding MPO immunostaining demonstrates diffuse MPO expression predominately from macrophages/microglia in the infarct. Lower row shows high-resolution images. (B) MPO is expressed by both neutrophils and macrophages (day 3). Double immunofluorescence staining of macrophages (green) and MPO (red) (Upper) and neutrophils (green) and MPO (red) (Lower) show both cell types as a source of MPO (day 3). Nuclei are counterstained with DAPI (blue). (C) Quantification of the inflammatory cell influx over time. Neutrophils were detected on days 1 and 3, whereas macrophages/microglia were observed over the entire investigated period of 3 weeks, with the highest levels on days 3 and 7. MPO-positive cells were detected throughout the entire investigated period and exhibited the highest levels on days 3 and 7, similar to the MPO imaging and biochemical results.