1Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin 300052, China 2Department of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ 85013, USA
Dl-3-n-butylphthalide (NBP) is a synthetic compound that has been approved for the treatment of ischemic stroke in China. The mechanisms underlying the treatment efficacy of NBP have been reported in multiple studies and remain controversial. Here, we show that NBP treatment attenuated ischemic brain injury in mice subjected to transient middle cerebral artery occlusion or photothrombosis-induced permanent cerebral ischemia. NBP induced downregulation of intercellular adhesion molecule 1 and protease-activated receptor 1 in cerebrovascular endothelial cells after cerebral ischemia and reperfusion. This effect was associated with the reduced brain infiltration of myeloid cells and improved cerebral blood flow after reperfusion. The beneficial effects of NBP were diminished in mice subjected to the depletion of Gr1+ myeloid cells before brain ischemia. Therefore, the restriction of neurovascular inflammation is a key mode of action for NBP in ischemic stroke.
Figure 1. NBP treatment ameliorates neurological deficits and brain infarction after cerebral ischemia and reperfusion. (A) Experimental scheme to assess the impact of NBP on neurological function and infarct volume in mice subjected to 60-min MCAO. NBP (60 mg/kg) or vehicle (oil) were given immediately after reperfusion by oral gavage (i.g.) and repeated daily for three consecutive days. Neurological deficits were evaluated with the modified neurological severity score (mNSS) and corner turning test. Infarct volume was quantified on days 1 and 3 using 7T MRI combined with TTC staining. (B) Summarized results showing the mNSS scores and corner turning test results of MCAO mice receiving NBP or vehicle at the indicated times after reperfusion. The data are expressed as the mean ± SEM. n = 10 per group. *P < 0.05, two-way ANOVA. (C) Infarct volume was quantified with MRI (T2WI) on days 1 and 3 after ischemia. The images show the infarct areas (yellow dashed line). n = 5 mice per group. *P < 0.05, two-tailed unpaired Student’s t test. (D) TTC-stained brain slices showing the infarct areas (blue dashed lines) of mice receiving NBP or vehicle at the indicated times after reperfusion. n = 10 mice per group. *P < 0.05, two-tailed unpaired Student’s t test. The data are representative of three independent experiments. Mean ± SEM
Figure 2. NBP improves long-term outcomes after photothrombosis-induced permanent cerebral ischemia. Cerebral ischemia was induced by photothrombosis in groups of mice receiving NBP or vehicle. (A) Experimental scheme used to assess the impact of NBP on neurological function in mice subjected to photothrombosis. NBP (60 mg/kg) or vehicle was administered daily by i.g. for 21 days. A battery of neurological tests, including mNSS, the foot fault test and the rotarod test, was used to comprehensively evaluate the motor and sensory deficits of the mice 1, 7, 14 and 21 days after surgery. (B-D) Graphs showing the results of mNSS (B), the foot fault test (C), and the rotarod test (D) for the groups of mice receiving NBP or vehicle control until day 21 after photothrombosis. n = 8 per group. *P < 0.05, **P < 0.01, two-way ANOVA. The data are representative of three independent experiments. Mean ± SEM.
Figure 3. NBP treatment attenuates brain inflammation after cerebral ischemia and reperfusion. MCAO mice were given NBP (60 mg/kg) or vehicle i.g. immediately after 60 min MCAO and reperfusion. Mice received daily treatment until they were sacrificed. (A) RT-PCR detected cytokine levels in the brain tissue homogenates of the ipsilateral hemisphere from the indicated groups of mice. n = 4 per group. *P < 0.05, **P < 0.01, two-tailed unpaired Student’s t test. (B) Counts of CNS-infiltrating immune cell subsets and microglia were measured using flow cytometry on day 3 after reperfusion. The gating strategies of immune cell subsets, including macrophages (CD45highCD11b+F4/80+), neutrophils (CD45highCD11b+Ly6G+), microglia (CD11b+CD45inter), CD4+ T cells (CD45highCD3+CD4+), CD8+ T cells (CD45highCD3+CD8+), NK cells (CD45highCD3-NK1.1+), and B cells (CD45highCD3-CD19+). (C-D) Summarized results showing the cell counts of the indicated subsets. n = 6 mice per group. *P < 0.05, two-tailed unpaired Student’s t test. The data are representative of three independent experiments. Mean ± SEM.
Figure 4. NBP treatment preserves BBB integrity and improves cerebral blood flow after brain ischemia. Mice were subjected to 60-min MCAO. NBP treatment by i.g. (60 mg/kg daily for three consecutive days) was initiated immediately after reperfusion. MCAO mice receiving vehicle were used as controls. The cerebral blood flow (CBF) and blood brain barrier leakage of the MCAO mice were measured using laser speckle Doppler and Evans blue extravasation, respectively, on day 3 after reperfusion. The brains of the mice were then harvested for pathology or flow cytometry analysis. (A) Quantification of Evans blue dye leakage on day 3 after MCAO in the indicated groups. EB content in brain tissue (μg / mg wet brain) = EB concentration (μg/ml) x formamide (ml) / wet weight (mg). n = 3 per group, two-tailed unpaired Student’s t test. (B) Flow cytometry analysis of the expression of tight junction proteins (ZO-1 and claudin-5) in endothelial cells from mice receiving NBP or vehicle. FMO, fluorescence minus one control. n = 6 per group. *P < 0.05, two-tailed unpaired Student’s t test. (C-D) H&E images showing occluded vessels in the ischemic brains of MCAO mice from the indicated groups. The bar graph shows the occlusion index of occluded vessels in the ischemic brains of MCAO mice. The occlusion index was calculated as the percentage of occluded vessels among the total observed vessels in the ischemic brain. Scale bars: 50 µm, 10 µm in the insets of C. n = 4 per group. *P < 0.05, two-tailed unpaired Student’s t test. (E) Images of CBF in sham-operated and MCAO mice that received NBP or vehicle treatment. (F) Quantification of blood perfusion in the ipsilateral hemisphere. n = 6 per group. *P< 0.05, **P < 0.01, one-way ANOVA. The data are representative of two independent experiments. Mean ± SEM.
Figure 5. NBP modulates the activity of cerebrovascular endothelial cells during brain ischemia. (A) Flow cytometry analysis of the expression of thrombin receptors (PAR-1), ICAM-1 and MMP-9 in brain endothelial cells from MCAO mice treated with NBP or vehicle control on day 3. n = 6 per group. *P < 0.05, two-tailed unpaired Student’s t test. (B) The effect of NBP treatment on the expression of the indicated mRNA in bEnd3 cells (a mouse brain endothelial cell line) after exposure to HGD in vitro. bEnd3 cells were exposed to HGD for 5 h in the presence of 100 µM NBP or vehicle (20% cyclodextrin in saline). After 24 h in culture, the cells were harvested for RT-PCR analysis. n = 4 per group. *P < 0.05, **P < 0.01, one-way ANOVA. The data are representative of three independent experiments. Mean ± SEM.
Figure 6. The benefit of NBP on ischemic stroke involves its effects on Gr1+ cells. (A) Schematic diagram illustrating the drug administration and experimental design. C57BL/6 mice received anti-Gr-1 mAb or IgG control 1 day prior to and 1 day after the MCAO procedure. NBP or an equal volume of vehicle were administered by i.g. (60 mg/kg daily for three consecutive days) immediately after reperfusion. On day 3 after MCAO, neurological deficits and the infarct volume were assessed. (B-C) Flow cytometric determination of absolute numbers of circulating Ly6G+ and Ly6Chigh cells from the mice that received anti-Gr-1 mAb or IgG control on day 3 after MCAO. n = 4 mice per group, **P < 0.01, two-tailed unpaired Student’s t test. (D) Summarized results of the mNSS and the corner turning test of MCAO mice treated with NBP or vehicle control with or without Gr1+ cell depletion. n = 6 mice per group. (E) Representative coronal brain sections stained with TTC and the quantification of the infarct size of mice receiving NBP or vehicle with or without Gr1+ cell depletion on day 3 after MCAO. n = 6 mice per group. *P < 0.05, **P < 0.01, one-way ANOVA. The data are representative of three independent experiments. Mean ± SEM.
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