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

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-null mice (EC-HIF-1α-null). 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-null mice. Isoflurane-induced DCI protection was attenuated in 2ME2-treated WT mice and EC-HIF-1α-null 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.

Figure 1

Postconditioning eliminates SAH-induced vasospasm. Mice underwent sham surgery; subarachnoid hemorrhage (SAH) surgery; or SAH surgery followed by isoflurane postconditioning (2% for 1 h, SAH-postC) starting 15 min, 1 h, 3 h, or 6 h after surgery. On post surgery day 3, pressure-controlled cerebrovascular casting was performed with gelatin–India ink. (A) Representative images of the middle cerebral artery (MCA) ipsilateral to endovascular perforation. Scale bar = 500 μm. (B) Vessel diameter of the ipsilateral MCA was assessed. N = 6 sham, N = 10 SAH, N = 16 SAH-postC-15′, N = 12 SAH-postC-1 h, N = 14 SAH-postC-3 h, N = 15 SAH-postC-6 h. Data represent mean ± SEM. *P < 0.05 versus sham, #P < 0.05 versus SAH, by repeated measures ANOVA.

Figure 2

Postconditioning attenuates SAH-induced cortical microvessel thrombosis. Mice underwent sham surgery, subarachnoid hemorrhage (SAH) surgery, or SAH surgery followed 1 h later by isoflurane postconditioning (2% for 1 h, SAH-postC). On post surgery day 3, fixed brain sections were subjected to fibrinogen immunohistochemistry. (A) Representative images of parietal cortex ipsilateral to endovascular perforation. Scale bar = 100 μm. (B) Cortical microvessel thrombosis was determined as percent coverage of ipsilateral parietal cortex. N = 15 sham, N = 16 SAH, N = 12 SAH-postC. Data represent mean ± SEM. *P < 0.05, **P < 0.01 by ANOVA.

Figure 3

Postconditioning reverses SAH-induced microvascular dysfunction. Mice underwent sham surgery, subarachnoid hemorrhage (SAH) surgery, or SAH surgery followed 1 h later by isoflurane postconditioning (2% for 1 h, SAH-postC). On post surgery day 3, microvessel reactivity of the distal middle cerebral artery (MCA) was examined through an open cranial window. Pial arteriolar vasodilatory responses to hypercapnia (CO₂), the endothelium-dependent vasodilator acetylcholine (ACh), and the endothelium-independent vasodilator S-nitroso-N-acetyl-penicillamine (SNAP) were assessed. N = 7 per group. Data represent mean ± SEM. *P < 0.05 by ANOVA.

Figure 4

Postconditioning improves neurological outcome after SAH. Mice underwent sham surgery; subarachnoid hemorrhage (SAH) surgery; or SAH surgery followed by isoflurane postconditioning (2% for 1 h, SAH-postC) starting 15 min, 1 h, 3 h, or 6 h after surgery. Neurobehavioral assessment was performed on post surgery days 0–3 via sensorimotor scoring. N = 6 sham, N = 10 SAH, N = 16 SAH-postC-15′, N = 12 SAH-postC-1 h, N = 14 SAH-postC-3 h, N = 15 SAH-postC-6 h (the same animals as were assessed for vasospasm in Fig.1). Data represent mean ± SEM. *P < 0.05 versus sham, #P < 0.05 versus SAH, by repeated measures ANOVA and Newman–Keuls multiple comparison test.

Figure 5

Hypoxia-inducible factor (HIF)-1 mediates isoflurane-induced transcription and postconditioning-induced neurovascular protection after subarachnoid hemorrhage (SAH). (A) Mice were administered vehicle or the HIF-1 inhibitor 2-methoxyestradiol (2ME2), exposed to isoflurane (2% for 1 h), sacked at 3 h, 24 h, or 72 h, and cortical tissue was subjected to quantitative real-time PCR. Data represent mean ± SEM. *P < 0.05 versus naïve, #P < 0.05 versus time-matched isoflurane only by ANOVA. N = 6 mice per group. (B and C) Mice were administered vehicle and subjected to sham surgery; were administered vehicle and subjected to SAH surgery followed 1 h later by isoflurane postconditioning (2% for 1 h, SAH-postC); or administered 2ME2 and subjected to SAH-postC. On post surgery day 3, pressure-controlled cerebrovascular casting was performed with gelatin–India ink (B). Data represent mean ± SEM. *P < 0.05 by ANOVA. Neurobehavioral assessment was performed on post surgery days 0–3 via Neuroscore (C). N = 8 sham, N = 10 SAH, N = 10 SAH-postC, N = 9 SAH-postC-2ME2. Data represent mean ± SEM. *P < 0.05 versus sham, #P < 0.05 versus SAH, ‡P < 0.05 versus SAH-postC-veh by repeated measures ANOVA and Newman–Keuls multiple comparison test.

Figure 6

Endothelial hypoxia-inducible factor (HIF)-1 mediates isoflurane-induced transcription and postconditioning-induced neurovascular protection after subarachnoid hemorrhage (SAH). Endothelial cell HIF-1 null (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.