Cleaning up after ICH: the role of Nrf2 in modulating microglia function and hematoma clearance

Abstract

As a consequence of intracerebral hemorrhage (ICH), blood components enter brain parenchyma causing progressive damage to the surrounding brain. Unless hematoma is cleared, the reservoirs of blood continue to inflict injury to neurovascular structures and blunt the brain repair processes. Microglia/macrophages (MMΦ) represent the primary phagocytic system that mediates the cleanup of hematoma. Thus, the efficacy of phagocytic function by MMΦ is an essential step in limiting ICH-mediated damage. Using primary microglia to model red blood cell (main component of hematoma) clearance, we studied the role of transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), a master-regulator of antioxidative defense, in the hematoma clearance process. We showed that in cultured microglia, activators of Nrf2 (i) induce antioxidative defense components, (ii) reduce peroxide formation, (iii) up-regulate phagocytosis-mediating scavenger receptor CD36, and (iv) enhance red blood cells (RBC) phagocytosis. Through inhibiting Nrf2 or CD36 in microglia, by DNA decoy or neutralizing antibody, we documented the important role of Nrf2 and CD36 in RBC phagocytosis. Using autologous blood injection ICH model to measure hematoma resolution, we showed that Nrf2 activator, sulforaphane, injected to animals after the onset of ICH, induced CD36 expression in ICH-affected brain and improved hematoma clearance in rats and wild-type mice, but expectedly not in Nrf2 knockout (KO) mice. Normal hematoma clearance was impaired in Nrf2-KO mice. Our experiments suggest that Nrf2 in microglia play an important role in augmenting the antioxidative capacity, phagocytosis, and hematoma clearance after ICH.

Keywords: Nrf2; hematoma; intracerebral hemorrhage; microglia; phagocytosis; sulforaphane.

Conflict of interest statement

The authors have no conflicts of interest to declare.

© 2014 International Society for Neurochemistry.

Figures

Fig 1

(A) Phagocytosis of RBC by microglia. Upon exposure to RBC the rat microglia (green; CD11b staining) became much bigger in size and filled out with RBC, as shown at 1h after starting phagocytosis. The RBC and the nuclei were labeled with a rabbit anti-rat RBC antibody (red) and DAPI (blue). Note that RBC are engulfed by microglia. Phagocytosis of CFDA-RBC by microglia in culture (B) was associated with the increased H2O2 in the culture media (C), as determined at 2h after initiation of phagocytosis. Pre-treatment of microglia with SF (2μM) increased engulfment of RBC (B; red bar) and reduced accumulation of H2O2 in the culture media (C; red bar). The data are expressed as mean ± SEM (n=3 independent experiments). *p ≤ 0.05, vs. RBC alone. (D) Nrf2 Western blot showing Nrf2 protein in the cytoplasm (CYT) and nuclear fractions (NE) at 1min and 5min after adding 2μM SF to rat primary microglia. Note very fast nuclear translocation/activation of Nrf2 with SF. (E) Representative photograph of Nrf2 immunofluorescence in rat microglia at the baseline (CONT) and at 2 min after exposure to 2μM SF, RBC or SF+RBC. Note increased nuclear presence of Nrf2 upon activation with SF. (F) DNA binding activity of nuclear Nrf2 in microglia upon adding RBC or RBC plus SF, checked by Nrf2 EMSA. (G) Photograph of representative gels showing RT-PCR products to demonstrate the gene expression profile - indicatives of Nrf2 activation in rat microglia at 6h after exposure to 2μM SF, RBC or SF+RBC.

Fig 2

Phagocytosis index indicating efficacy of RBC engulfment by rat brain microglia at 2h after adding CFDA-RBC with Nrf2-decoy (ARE-Decoy) or mutant decoy (Mut-Decoy), and absence or presence of 2μM SF. Microglia were pre-treated with the Nrf-2 decoy or mutant-decoy for 24h before the assessment of phagocytosis, while SF was added 16h before the exposure to CFDA-RBC. Results are expressed as mean ± SEM (n=3). *p

Fig 3

(A) Photograph of RT-PCR gels showing CD36 and GAPDH gene expression profile and (B) CD36, HO-1 and GAPDH protein expression levels in rat microglia upon exposure to RBC or RBC+2 μM SF for 6h. GAPDH is used as an internal control – the same GAPDH band was visualized using alkaline phosphatase (brows; upper band) and ECL (lower band). (C) Phagocytosis Index of rat microglia at 2h after exposure to CFDA-RBC in presence of 2μM SF in microglia treated with or without 10 μg/ml of anti-CD36 or anti-FITC antibodies (isotope control). The data are expressed as mean±SEM (n=3 independent experiments). *p ≤0.05, compared with all other groups.

Fig 4

(A) Photomicrograph of hematoma in rat brain on day 7 after ICH, double-stained with anti-RBC (red) and anti-CD68 (green) antibodies. The panel A″ and B shows the close-up morphology of CD68+-microglia/macrophages at the hematoma-affected brain at 7 and 3 days after ICH, respectively. (C) Hematoma size on day 10 after ICH in the SD rats treated with vehicle (10% corn oil in PBS) or SF (2.5 mg/kg at 30min and 24h after ICH). The data (mean±SEM, n=7) are expressed as the blood volume in the ICH-affected brain tissue. *p ≤0.05, compared with the vehicle control. (D) Hematoma size on day 7 after ICH in WT and Nrf2-KO mice treated with vehicle (10% corn oil in PBS) or SF (5 mg/kg at 30min and 24h after ICH). The data (mean±SEM, n=5–7) are expressed as the blood volume in the ICH-affected brain tissue.