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HIF-1α Mediates Isoflurane-Induced Vascular Protection in Subarachnoid Hemorrhage.

Milner E, Johnson AW, Nelson JW, Harries MD, Gidday JM, Han BH, Zipfel GJ - Ann Clin Transl Neurol (2015)

Bottom Line: Isoflurane postconditioning provides strong HIF-1α-mediated macro- and microvascular protection in SAH, leading to improved neurological outcome.These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF-1α as a critical mediator of this vascular protection.They also identify isoflurane postconditioning as a promising novel therapeutic for SAH.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, Washington University School of Medicine St. Louis, Missouri, 63108 ; Program in Neuroscience, Washington University School of Medicine St. Louis, Missouri, 63108.

ABSTRACT

Objective: Outcome after aneurysmal subarachnoid hemorrhage (SAH) depends critically on delayed cerebral ischemia (DCI) - a process driven primarily by vascular events including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction. This study sought to determine the impact of postconditioning - the phenomenon whereby endogenous protection against severe injury is enhanced by subsequent exposure to a mild stressor - on SAH-induced DCI.

Methods: Adult male C57BL/6 mice were subjected to sham, SAH, or SAH plus isoflurane postconditioning. Neurological outcome was assessed daily via sensorimotor scoring. Contributors to DCI including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction were measured 3 days later. Isoflurane-induced changes in hypoxia-inducible factor 1alpha (HIF-1α)-dependent genes were assessed via quantitative polymerase chain reaction. HIF-1α was inhibited pharmacologically via 2-methoxyestradiol (2ME2) or genetically via endothelial cell HIF-1α- mice (EC-HIF-1α-). All experiments were performed in a randomized and blinded fashion.

Results: Isoflurane postconditioning initiated at clinically relevant time points after SAH significantly reduced cerebral vasospasm, microvessel thrombosis, microvascular dysfunction, and neurological deficits in wild-type (WT) mice. Isoflurane modulated HIF-1α-dependent genes - changes that were abolished in 2ME2-treated WT mice and EC-HIF-1α- mice. Isoflurane-induced DCI protection was attenuated in 2ME2-treated WT mice and EC-HIF-1α- mice.

Interpretation: Isoflurane postconditioning provides strong HIF-1α-mediated macro- and microvascular protection in SAH, leading to improved neurological outcome. These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF-1α as a critical mediator of this vascular protection. They also identify isoflurane postconditioning as a promising novel therapeutic for SAH.

No MeSH data available.


Related in: MedlinePlus

Endothelial hypoxia-inducible factor (HIF)-1 mediates isoflurane-induced transcription and postconditioning-induced neurovascular protection after subarachnoid hemorrhage (SAH). Endothelial cell HIF-1  (EC HIF-1−/−) mice were bred using a Cre-lox system. (A) Tie2-Cre mice were bred to ROSA26 reporter mice. Note green fluorescence in cerebrocortical endothelial cells (indicating Tie2-Cre expression) but not in other cell types (red) in the brains of the offspring. Scale bar = 500 μm. (B) EC HIF-1−/− mice were subjected to normoxia (naïve) or isoflurane (2% for 1 h), sacked at 3 h or 24 h, and cortical tissue was subjected to quantitative real-time PCR. N = 5 mice per group. Data represent mean ± SEM. *P < 0.05 versus naïve by ANOVA. n.s. P > 0.05. (C–D) EC HIF-1−/− mice underwent sham surgery, SAH surgery, or SAH surgery followed 1 h later by isoflurane postconditioning (2% for 1 h, SAH-postC). On post surgery day 3, pressure-controlled cerebrovascular casting was performed with gelatin–India ink. (C) Vessel diameter of the ipsilateral middle cerebral artery was assessed. N = 21 sham, N = 20 SAH, N = 11 SAH-postC. Data represent mean ± SEM. *P < 0.05 by ANOVA. n.s. P > 0.05. (D) Neurobehavioral assessment was performed on post surgery days 0–3 via Neuroscore. Data represent mean ± SEM. *P < 0.05 versus sham by repeated measures ANOVA and Newman–Keuls multiple comparison test.
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fig06: Endothelial hypoxia-inducible factor (HIF)-1 mediates isoflurane-induced transcription and postconditioning-induced neurovascular protection after subarachnoid hemorrhage (SAH). Endothelial cell HIF-1 (EC HIF-1−/−) mice were bred using a Cre-lox system. (A) Tie2-Cre mice were bred to ROSA26 reporter mice. Note green fluorescence in cerebrocortical endothelial cells (indicating Tie2-Cre expression) but not in other cell types (red) in the brains of the offspring. Scale bar = 500 μm. (B) EC HIF-1−/− mice were subjected to normoxia (naïve) or isoflurane (2% for 1 h), sacked at 3 h or 24 h, and cortical tissue was subjected to quantitative real-time PCR. N = 5 mice per group. Data represent mean ± SEM. *P < 0.05 versus naïve by ANOVA. n.s. P > 0.05. (C–D) EC HIF-1−/− mice underwent sham surgery, SAH surgery, or SAH surgery followed 1 h later by isoflurane postconditioning (2% for 1 h, SAH-postC). On post surgery day 3, pressure-controlled cerebrovascular casting was performed with gelatin–India ink. (C) Vessel diameter of the ipsilateral middle cerebral artery was assessed. N = 21 sham, N = 20 SAH, N = 11 SAH-postC. Data represent mean ± SEM. *P < 0.05 by ANOVA. n.s. P > 0.05. (D) Neurobehavioral assessment was performed on post surgery days 0–3 via Neuroscore. Data represent mean ± SEM. *P < 0.05 versus sham by repeated measures ANOVA and Newman–Keuls multiple comparison test.

Mentions: To test our hypothesis that HIF-1α-driven transcriptional activation in ECs in response to isoflurane postconditioning drives the observed vasculoprotective phenotype, we generated EC-specific HIF-1α- mice. EC expression of Cre in our Tie2-Cre mice was verified by crossing them with ROSA26 reporter mice and examining cerebral microvascular fluorescence in the brains of their progeny. As shown in Figure6, green fluorescence – indicative of Cre expression – was seen throughout the cerebrovascular endothelium of Cre-positive mice (Fig.6Av–viii), but the endothelium of Cre-negative mice fluoresced red (Fig.6Ai–iv). In naïve EC HIF-1α−/− mice, isoflurane exposure did not significantly affect transcription of the HIF-1α targets GLUT1 and BNIP3; in contrast, transcription of EPO was significantly increased (Fig.6B; P < 0.05, ANOVA), which is consistent with a known role of vascular HIF-2α (retained in these mice) in regulating EPO.40 Genetic deletion of endothelial HIF-1α eliminated the protection afforded by isoflurane postconditioning against SAH-induced vasospasm (Fig.6C; P < 0.05, ANOVA) and neurological deficits (Fig.6D; P < 0.05, repeated measures ANOVA). Collectively, these results provide causal evidence that vascular endothelium-derived HIF-1α is critical for isoflurane's transcriptional effect and the neurovascular protection afforded by its use as a postconditioning treatment in SAH.


HIF-1α Mediates Isoflurane-Induced Vascular Protection in Subarachnoid Hemorrhage.

Milner E, Johnson AW, Nelson JW, Harries MD, Gidday JM, Han BH, Zipfel GJ - Ann Clin Transl Neurol (2015)

Endothelial hypoxia-inducible factor (HIF)-1 mediates isoflurane-induced transcription and postconditioning-induced neurovascular protection after subarachnoid hemorrhage (SAH). Endothelial cell HIF-1  (EC HIF-1−/−) mice were bred using a Cre-lox system. (A) Tie2-Cre mice were bred to ROSA26 reporter mice. Note green fluorescence in cerebrocortical endothelial cells (indicating Tie2-Cre expression) but not in other cell types (red) in the brains of the offspring. Scale bar = 500 μm. (B) EC HIF-1−/− mice were subjected to normoxia (naïve) or isoflurane (2% for 1 h), sacked at 3 h or 24 h, and cortical tissue was subjected to quantitative real-time PCR. N = 5 mice per group. Data represent mean ± SEM. *P < 0.05 versus naïve by ANOVA. n.s. P > 0.05. (C–D) EC HIF-1−/− mice underwent sham surgery, SAH surgery, or SAH surgery followed 1 h later by isoflurane postconditioning (2% for 1 h, SAH-postC). On post surgery day 3, pressure-controlled cerebrovascular casting was performed with gelatin–India ink. (C) Vessel diameter of the ipsilateral middle cerebral artery was assessed. N = 21 sham, N = 20 SAH, N = 11 SAH-postC. Data represent mean ± SEM. *P < 0.05 by ANOVA. n.s. P > 0.05. (D) Neurobehavioral assessment was performed on post surgery days 0–3 via Neuroscore. Data represent mean ± SEM. *P < 0.05 versus sham by repeated measures ANOVA and Newman–Keuls multiple comparison test.
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fig06: Endothelial hypoxia-inducible factor (HIF)-1 mediates isoflurane-induced transcription and postconditioning-induced neurovascular protection after subarachnoid hemorrhage (SAH). Endothelial cell HIF-1 (EC HIF-1−/−) mice were bred using a Cre-lox system. (A) Tie2-Cre mice were bred to ROSA26 reporter mice. Note green fluorescence in cerebrocortical endothelial cells (indicating Tie2-Cre expression) but not in other cell types (red) in the brains of the offspring. Scale bar = 500 μm. (B) EC HIF-1−/− mice were subjected to normoxia (naïve) or isoflurane (2% for 1 h), sacked at 3 h or 24 h, and cortical tissue was subjected to quantitative real-time PCR. N = 5 mice per group. Data represent mean ± SEM. *P < 0.05 versus naïve by ANOVA. n.s. P > 0.05. (C–D) EC HIF-1−/− mice underwent sham surgery, SAH surgery, or SAH surgery followed 1 h later by isoflurane postconditioning (2% for 1 h, SAH-postC). On post surgery day 3, pressure-controlled cerebrovascular casting was performed with gelatin–India ink. (C) Vessel diameter of the ipsilateral middle cerebral artery was assessed. N = 21 sham, N = 20 SAH, N = 11 SAH-postC. Data represent mean ± SEM. *P < 0.05 by ANOVA. n.s. P > 0.05. (D) Neurobehavioral assessment was performed on post surgery days 0–3 via Neuroscore. Data represent mean ± SEM. *P < 0.05 versus sham by repeated measures ANOVA and Newman–Keuls multiple comparison test.
Mentions: To test our hypothesis that HIF-1α-driven transcriptional activation in ECs in response to isoflurane postconditioning drives the observed vasculoprotective phenotype, we generated EC-specific HIF-1α- mice. EC expression of Cre in our Tie2-Cre mice was verified by crossing them with ROSA26 reporter mice and examining cerebral microvascular fluorescence in the brains of their progeny. As shown in Figure6, green fluorescence – indicative of Cre expression – was seen throughout the cerebrovascular endothelium of Cre-positive mice (Fig.6Av–viii), but the endothelium of Cre-negative mice fluoresced red (Fig.6Ai–iv). In naïve EC HIF-1α−/− mice, isoflurane exposure did not significantly affect transcription of the HIF-1α targets GLUT1 and BNIP3; in contrast, transcription of EPO was significantly increased (Fig.6B; P < 0.05, ANOVA), which is consistent with a known role of vascular HIF-2α (retained in these mice) in regulating EPO.40 Genetic deletion of endothelial HIF-1α eliminated the protection afforded by isoflurane postconditioning against SAH-induced vasospasm (Fig.6C; P < 0.05, ANOVA) and neurological deficits (Fig.6D; P < 0.05, repeated measures ANOVA). Collectively, these results provide causal evidence that vascular endothelium-derived HIF-1α is critical for isoflurane's transcriptional effect and the neurovascular protection afforded by its use as a postconditioning treatment in SAH.

Bottom Line: Isoflurane postconditioning provides strong HIF-1α-mediated macro- and microvascular protection in SAH, leading to improved neurological outcome.These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF-1α as a critical mediator of this vascular protection.They also identify isoflurane postconditioning as a promising novel therapeutic for SAH.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, Washington University School of Medicine St. Louis, Missouri, 63108 ; Program in Neuroscience, Washington University School of Medicine St. Louis, Missouri, 63108.

ABSTRACT

Objective: Outcome after aneurysmal subarachnoid hemorrhage (SAH) depends critically on delayed cerebral ischemia (DCI) - a process driven primarily by vascular events including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction. This study sought to determine the impact of postconditioning - the phenomenon whereby endogenous protection against severe injury is enhanced by subsequent exposure to a mild stressor - on SAH-induced DCI.

Methods: Adult male C57BL/6 mice were subjected to sham, SAH, or SAH plus isoflurane postconditioning. Neurological outcome was assessed daily via sensorimotor scoring. Contributors to DCI including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction were measured 3 days later. Isoflurane-induced changes in hypoxia-inducible factor 1alpha (HIF-1α)-dependent genes were assessed via quantitative polymerase chain reaction. HIF-1α was inhibited pharmacologically via 2-methoxyestradiol (2ME2) or genetically via endothelial cell HIF-1α- mice (EC-HIF-1α-). All experiments were performed in a randomized and blinded fashion.

Results: Isoflurane postconditioning initiated at clinically relevant time points after SAH significantly reduced cerebral vasospasm, microvessel thrombosis, microvascular dysfunction, and neurological deficits in wild-type (WT) mice. Isoflurane modulated HIF-1α-dependent genes - changes that were abolished in 2ME2-treated WT mice and EC-HIF-1α- mice. Isoflurane-induced DCI protection was attenuated in 2ME2-treated WT mice and EC-HIF-1α- mice.

Interpretation: Isoflurane postconditioning provides strong HIF-1α-mediated macro- and microvascular protection in SAH, leading to improved neurological outcome. These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF-1α as a critical mediator of this vascular protection. They also identify isoflurane postconditioning as a promising novel therapeutic for SAH.

No MeSH data available.


Related in: MedlinePlus