A TSPO ligand attenuates brain injury after intracerebral hemorrhage

Abstract

Intracerebral hemorrhage (ICH) is a devastating disease without effective treatment. After ICH, the immediate infiltration of leukocytes and activation of microglia are accompanied by a rapid up-regulation of the 18-kDa translocator protein (TSPO). TSPO ligands have shown anti-inflammatory and neuroprotective properties in models of CNS injury. In this study, we determined the impact of a TSPO ligand, etifoxine, on brain injury and inflammation in 2 mouse models of ICH. TSPO was up-regulated in Iba1⁺ cells from brains of patients with ICH and in CD11b⁺CD45^int cells from mice subjected to collagenase-induced ICH. Etifoxine significantly reduced neurodeficits and perihematomal brain edema after ICH induction by injection of either autologous blood or collagenase. In collagenase-induced ICH mice, the protection of etifoxine was associated with reduced leukocyte infiltration into the brain and microglial production of IL-6 and TNF-α. Etifoxine improved blood-brain barrier integrity and diminished cell death. Notably, the protective effect of etifoxine was abolished in mice depleted of microglia by using a colony-stimulating factor 1 receptor inhibitor. These results indicate that the TSPO ligand etifoxine attenuates brain injury and inflammation after ICH. TSPO may be a viable therapeutic target that requires further investigations in ICH.-Li, M., Ren, H., Sheth, K. N., Shi, F.-D., Liu, Q. A TSPO ligand attenuates brain injury after intracerebral hemorrhage.

Keywords: etifoxine; hemorrhagic stroke; immune modulation; inflammation.

Figure 1.

Up-regulation of TSPO in the brains of ICH mice and patients with ICH. A) Single-cell suspensions were prepared from brain tissues of mice with ICH induced by collagenase injection at 24 h after surgery. Flow cytometry plots show gating of CD11b⁺CD45^int, CD11b⁺CD45^hi, and CD11b^-CD45^hi cell subsets that express TSPO. B) Bar graph shows the expression of TSPO in indicated cell subsets (n = 6 per group). *P < 0.05, **P < 0.01 vs. sham-treatment group of each cell subset. ^#P < 0.05, ^##P < 0.01 vs. ICH group of CD11b⁺CD45^int cell subsets. C, D) Immunostaining (C) and summarized results (D) of TSPO and Iba1 in brain sections from patients with ICH (<24 h after onset) or nonneurologic disease controls. Scale bars, 100 μm (n = 20 sections from 5 patients with ICH; n = 15 sections from 4 control subjects). Throughout, data are presented as means ± sem. **P < 0.01 vs. control group of each cell subset.

Figure 2.

Etifoxine attenuated brain injury in 2 mouse models of ICH. A) Flow chart illustrates the regimen of TSPO administration and experimental design. ICH was induced in mice by injection of autologous blood or collagenase, immediately followed by intraperitoneal injection of etifoxine (ETX; 50 mg/kg) or vehicle, which was continued daily until the end of the experiment. B) A battery of neurologic tests was performed to evaluate the motor, sensory, and balance functions in the mice given vehicle or ETX at d 1 and after ICH induced by collagenase injection or autologous blood injection. C, D) Sequential 7-T MRI was used to visualize and measure lesion, hematoma, and edema volumes 3 d after ICH. C) T2-weighted image (T2WI) sequences were scanned to assess lesion volume, as outlined with the red line. SWI sequences were assessed for hematoma lesion volumes, visible in yellow regions. Representative 7-T MRI imaging of these lesions and hematomas resulting from ICH were induced by injections of either collagenase or autologous blood. D) Quantification of lesion volume and edema volume in mice given vehicle or etifoxine treatment 3 d after ICH induced by injection of either collagenase or autologous blood. Data are means ± sem (n = 10 mice per group). *P < 0.05, **P < 0.01 vs. control group at indicated time points.

Figure 3.

Etifoxine reduced cellular components and microglial proinflammatory cytokine production in the brain after ICH. ICH was induced by collagenase injection and immediately followed by daily intraperitoneal injection of etifoxine (ETX, 50 mg/kg) or vehicle until the end of the experiment. Three days after injection, brain tissues were harvested to isolate single cells for flow cytometry analysis. A) The gating strategy for isolating microglia (CD11b⁺CD45^int), CD4⁺ T cells (CD45^hiCD3⁺CD4⁺), CD8⁺ T cells (CD45^hiCD3⁺CD8⁺), NK cells (CD45^hiCD3^-NK1.1⁺), neutrophils (CD45^hiCD11b⁺1A8⁺), and macrophages (CD45^hiCD11b⁺ F4/80⁺). B, C) Cell counts of microglia and CNS-invading leukocytes in the brain after ICH. D) Cell counts of microglia expressing IL-6, TNFα, IL-10, and TGF-β in brains from ICH mice given vehicle or etifoxine treatment (n = 6 per group). Data are means ± sem. *P < 0.05, **P < 0.01.

Figure 4.

Etifoxine attenuated BBB leakage and loss of tight junction proteins after ICH. ICH was induced by collagenase injection and immediately followed by daily intraperitoneal injection of etifoxine (ETX, 50 mg/kg) or vehicle until the end of the experiment. Three days after injection, sequential 7-T MRI scans were assessed for BBB leakage into the brain, and Western blot analysis was used to detect tight junction protein expression. A, B) Seven-tesla MRI (A) and quantification of data (B) show that etifoxine reduced Gd-DTPA extravasation to brain parenchyma. T1-weighted image (T1) sequences were scanned for this evaluation. Red dashed lines: region of Gd-DTPA leakage (n = 6 per group). C, D) Western blot analysis (C) and quantification of data (D) show that etifoxine preserves tight junction protein (claudin-5 and ZO-1) expression. Data are means ± sem (n = 4 per group). *P < 0.05, **P < 0.01.

Figure 5.

Etifoxine reduced cell death in the brain after ICH. ICH was induced by collagenase injection and immediately followed by daily injections of etifoxine (ETX, 50 mg/kg, i.p.) or vehicle until the end of the experiment. Three days after injection, brain tissues of ICH mice receiving etifoxine or vehicle treatment were harvested for cell death analysis. A) Brain tissue sections were stained with TUNEL to measure cell death. Representative images show TUNEL⁺ cells in ICH mice receiving etifoxine or vehicle control. Red rectangle indicates the measured region. Graphs show quantified data. Scale bars, 100 μm. B) Summarized results show reduced cell death in ICH mice receiving etifoxine treatment (n = 6 per group). C, D) Flow cytometry plots (C) and summarized results (D) show annexin V⁺ cells in brain tissues from the indicated groups (n = 6 per group). Data are means ± sem. *P < 0.05.

Figure 6.

The protective effect of etifoxine was abolished in ICH mice depleted of microglia. Mice were treated with PLX3397 (40 mg/kg) before ICH surgery for 21 d, and the treatment continued until the experiment ended. ICH was induced by collagenase injection. Etifoxine injections started immediately after ICH induction and continued daily until d 2. A) Flow chart illustrates the regimen of PLX3397 and etifoxine treatment and ICH induction. B) Neurodeficit assessment shows mNSS and corner-turning test score in ICH mice depleted of microglia subjected to etifoxine or vehicle treatment. C, D) MRI (C) and quantification of lesion volume and perihematomal edema volume in indicated groups of mice (D) at d 1 and 3 after ICH (n = 15 per group). Data are presented as means ± sem.