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Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism
Philberta Y. Leung, Susan L. Stevens, Amy E.B. Packard, Nikola S. Lessov, Tao Yang, Valerie K. Conrad, Noortje N.A.M. van den Dungen, Roger P. Simon and Mary P. Stenzel-Poore

Stroke. 2012;43:1383-1389, originally published April 23, 2012
https://doi.org/10.1161/STROKEAHA.111.641522

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Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism
Philberta Y. Leung, Susan L. Stevens, Amy E.B. Packard, Nikola S. Lessov, Tao Yang, Valerie K. Conrad, Noortje N.A.M. van den Dungen, Roger P. Simon, Mary P. Stenzel-Poore

Stroke May 2012, 43 (5) 1383-1389; DOI: https://doi.org/10.1161/STROKEAHA.111.641522

Figure 1.
Gardiquimod (GDQ) preconditioning reduces ischemic injury. A, C57Bl/6 mice were preconditio...
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Gardiquimod (GDQ) preconditioning reduces ischemic injury. A, C57Bl/6 mice were preconditioned with escalating doses of GDQ (N=8; 10, 20, or 40 μg per mouse, subcutaneous) or saline (N=5) 72 hours before 60 minutes of middle cerebral artery occulusion (MCAO). Infarct size was determined 24 hours after MCAO. B, C57Bl/6 mice were pretreated with GDQ (N=8; 20 μg/mouse, subcutaneous) or saline (n=6) 72 hours before MCAO. Infarct size was determined 72 hours after MCAO. C, C57Bl/6 mice were pretreated with GDQ (N=5–6; 20 μg/mouse, subcutaneous) or saline (n=6) at various times before MCAO. Infarct size was determined 24 hours after MCAO. Two-way analysis of variance (ANOVA), Bonferroni post hoc, *P<0.01, **P<0.01, ***P<0.001 vs saline controls. D and E, C57BL/6 mice were treated with GDQ (N=5; 40 μg/mouse, subcutaneous) or saline (N=6) 72 hours before MCAO (60 minutes). Mice were then examined using the (D) neurological score (focal and general) and (E) corner test to determine neurological and sensorimotor deficits 24 hours after MCAO. Student t test: *P<0.01, **P<0.01, ***P<0.001 vs saline controls.
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Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism
Philberta Y. Leung, Susan L. Stevens, Amy E.B. Packard, Nikola S. Lessov, Tao Yang, Valerie K. Conrad, Noortje N.A.M. van den Dungen, Roger P. Simon, Mary P. Stenzel-Poore

Stroke May 2012, 43 (5) 1383-1389; DOI: https://doi.org/10.1161/STROKEAHA.111.641522

Figure 2.
Gardiquimod (GDQ)-induced neuroprotection is mediated through Toll-like receptor (TLR) 7. TLR7^+/+...
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Gardiquimod (GDQ)-induced neuroprotection is mediated through Toll-like receptor (TLR) 7. TLR7^+/+ (N=7) or TLR7^−/− (N=5–7) mice were preconditioned with GDQ (40 μg/mouse, subcutaneous) or saline 72 hours before middle cerebral artery occlusion (MCAO; 45 minutes). Infarct size was determined 24 hours after MCAO. Two-way analysis of variance (ANOVA), Bonferroni post hoc, **P<0.01 vs saline control for respective genotype.
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Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism
Philberta Y. Leung, Susan L. Stevens, Amy E.B. Packard, Nikola S. Lessov, Tao Yang, Valerie K. Conrad, Noortje N.A.M. van den Dungen, Roger P. Simon, Mary P. Stenzel-Poore

Stroke May 2012, 43 (5) 1383-1389; DOI: https://doi.org/10.1161/STROKEAHA.111.641522

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Table.
Interferon-Associated Genes in the Brains of Gardiquimod-Preconditioned Animals After Middle Cerebral Artery Occlusion (24 Hours)Show More

Interferon-Associated Genes in the Brains of Gardiquimod-Preconditioned Animals After Middle Cerebral Artery Occlusion (24 Hours)Show Less
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Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism
Philberta Y. Leung, Susan L. Stevens, Amy E.B. Packard, Nikola S. Lessov, Tao Yang, Valerie K. Conrad, Noortje N.A.M. van den Dungen, Roger P. Simon, Mary P. Stenzel-Poore

Stroke May 2012, 43 (5) 1383-1389; DOI: https://doi.org/10.1161/STROKEAHA.111.641522

Figure 3.
Tumor necrosis factor (TNF) is not required for Toll-like receptor (TLR)7-induced neuroprotection. A...
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Tumor necrosis factor (TNF) is not required for Toll-like receptor (TLR)7-induced neuroprotection. A, C57Bl/6 mice were injected with either Gardiquimod (GDQ; 40 μg/mouse, subcutaneous), lipopolysaccharide (LPS; 20 μg/mouse, subcutaneous), or saline. Blood was collected at various time points after injection and serum levels of TNF were determined via enzyme-linked immunosorbent assay (ELISA). N.D.=not detected (N=4 – 6). B, TNF^−/− (N=6 – 8) and TNF^+/+ mice (N=7) were injected with GDQ (40 μg/mouse, subcutaneous) or saline 72 hours before middle cerebral artery occlusion (MCAO; 50 minutes). Infarct size was determined 24 hours after MCAO. Two-way analysis of variance (ANOVA), Bonferroni post hoc: *P<0.05 vs saline controls.
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Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism
Philberta Y. Leung, Susan L. Stevens, Amy E.B. Packard, Nikola S. Lessov, Tao Yang, Valerie K. Conrad, Noortje N.A.M. van den Dungen, Roger P. Simon, Mary P. Stenzel-Poore

Stroke May 2012, 43 (5) 1383-1389; DOI: https://doi.org/10.1161/STROKEAHA.111.641522

Figure 4.
Gardiquimod (GDQ)-preconditioning induces IFNα and requires IRF7 for neuroprotection. A, C5...
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Gardiquimod (GDQ)-preconditioning induces IFNα and requires IRF7 for neuroprotection. A, C57Bl/6 mice were injected with GDQ (40 μg/mouse, subcutaneous), unmethylated cytosine-phosphate-guanine rich oligonucleotide (40 μg/mouse, subcutaneous), lipopolysaccharide (LPS; 20 μg/mouse, subcutaneous), or saline. Blood was collected at indicated times and serum interferon (IFN)α levels were measured (N=4–10). Two-way analysis of variance (ANOVA), Bonferroni post hoc: *P<0.05, ***P<0.001 vs saline controls. B, IRF7^−/− and IRF7^+/+ mice were injected with 40 μg GDQ or saline. Blood was collected at 2 hours and serum IFNα was measured (N=3 – 8). One-way ANOVA, Bonferroni post hoc: *P<0.05 vs saline controls. C, IRF7^+/+ (N=8–9) or IRF7^−/− mice (N=7–9) were preconditioned with GDQ (40 μg/mouse, subcutaneous) or saline 72 hours before MCAO (45 minutes). Infarct size was determined 24 hours after middle cerebral artery occlusion (MCAO). Two-way ANOVA, Bonferroni post hoc: **P<0.01 vs saline controls.
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Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism
Philberta Y. Leung, Susan L. Stevens, Amy E.B. Packard, Nikola S. Lessov, Tao Yang, Valerie K. Conrad, Noortje N.A.M. van den Dungen, Roger P. Simon, Mary P. Stenzel-Poore

Stroke May 2012, 43 (5) 1383-1389; DOI: https://doi.org/10.1161/STROKEAHA.111.641522

Figure 5.
Type I interferon receptor (IFNAR) signaling is required for TLR7-mediated neuroprotection. IFNAR^+/+...
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Type I interferon receptor (IFNAR) signaling is required for TLR7-mediated neuroprotection. IFNAR^+/+ and IFNAR^−/− mice were injected with Gardiquimod (GDQ; N=6 – 8), unmethylated cytosine-phosphate-guanine rich oligonucleotide (N=8–11), lipopolysaccharide (LPS; N=8–11), or saline (N=6 – 9) 72 hours before middle cerebral artery occlusion (MCAO; 45 minutes). Infarct size was determined 24 hours after MCAO. The data are presented as percent damage normalized to saline plus MCAO. Two-way analysis of variance (ANOVA), Bonferroni post hoc: ***P<0.001 vs saline control for respective genotype; #P<0.05 vs IFNAR^+/+ for respective treatment.
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Extracellular Zinc Protects Against Acidosis-Induced Injury of Cells Expressing Ca²⁺-Permeable Acid-Sensing Ion Channels
Jessica G. Hey, Xiang-Ping Chu, Joshua Seeds, Roger P. Simon and Zhi-Gang Xiong

Stroke. 2007;38:670-673, originally published January 29, 2007
https://doi.org/10.1161/01.STR.0000251443.68897.99

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Extracellular Zinc Protects Against Acidosis-Induced Injury of Cells Expressing Ca²⁺-Permeable Acid-Sensing Ion Channels
Jessica G. Hey, Xiang-Ping Chu, Joshua Seeds, Roger P. Simon, Zhi-Gang Xiong

Stroke February 2007, 38 (2) 670-673; DOI: https://doi.org/10.1161/01.STR.0000251443.68897.99

Figure 1. Lowering extracellular pH activates ASIC1a currents in HEK293 cells and these currents are potentiated by reducing Zn2+ concentrations. A, R...Show More

Figure 1. Lowering extracellular pH activates ASIC1a currents in HEK293 cells and these currents are potentiated by reducing Zn2+ concentrations. A, Representative current traces demonstrating the activation of ASIC1a-like current in HEK293 cells. The current is largely inhibited by PcTX venom (150 ng/mL), a specific blocker for homomeric ASIC1a channels. Reducing the concentration of Zn2+ by perfusion with 10 μmol/L TPEN dramatically increased the amplitude of the ASIC current. In contrast, perfusion of the cells with 10 μmol/L ZnCl2 slightly inhibited the ASIC current. B, Summary bar graph demonstrating the effect of changing the concentration of Zn2+ on ASIC current in HEK293 cells. n=5. **P<0.01 compared with control group.Show Less
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Extracellular Zinc Protects Against Acidosis-Induced Injury of Cells Expressing Ca²⁺-Permeable Acid-Sensing Ion Channels
Jessica G. Hey, Xiang-Ping Chu, Joshua Seeds, Roger P. Simon, Zhi-Gang Xiong

Stroke February 2007, 38 (2) 670-673; DOI: https://doi.org/10.1161/01.STR.0000251443.68897.99

Figure 2. Acid incubation induces injury of HEK293 cells. A, Representative black and white images showing the acid-induced injury of HEK293 cells. Ce...Show More

Figure 2. Acid incubation induces injury of HEK293 cells. A, Representative black and white images showing the acid-induced injury of HEK293 cells. Cells were incubated with extracellular solutions with pH buffered at either 7.4 or 5.5 for 3 hours before switching back to neurobasal medium. Photographs were taken 24 hours after the acid incubation. B, Bar graph summarizing time-dependent injury of HEK293 cells as indicated by increase in LDH release, after the acid incubation. LDH release at various time points has been normalized to the maximal releasable amount obtained by incubating cells with 1% Triton X-100 for 15 minutes. *P<0.05 and **P<0.01 compared with pH 7.4 treated group at the same time points.Show Less