Date of Award
Doctor of Philosophy (PhD)
Vasospasm-induced delayed cerebral ischemia (DCI) remains a major source of morbidity in patients with aneurysmal subarachnoid hemorrhage (SAH). Moreover, cognitive dysfunction is the primary driver of poor long-term outcome in SAH survivors; modeling such deficits preclinically is thus key for mechanistic and translational investigation. We hypothesized that activating innate neurovascular protective mechanisms by conditioning may represent a novel therapeutic approach against SAH-induced DCI, short-term, neurological deficits, and long-term neurocognitive deficits; and, secondarily, that the neurovascular protection it provides is mediated by endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factor 1α (HIF-1α).
In Experiment 1, wild-type C57BL/6 mice were subjected to hypoxic preconditioning (PC) or normoxia followed 24 hours later by endovascular-perforation SAH. Neurological function was analyzed daily via sensorimotor scoring; vasospasm was assessed on post-surgery day 2. Nitric oxide availability, eNOS expression, and eNOS activity were also assessed. In a separate experiment, wild-type and eNOS-null mice were subjected to hypoxic PC or normoxia followed by SAH and assessed for vasospasm and neurological deficits. All experiments were performed in a randomized and blinded fashion.
PC nearly completely prevented SAH-induced vasospasm and neurological deficits. It also prevented SAH-induced reduction in nitric oxide availability and increased eNOS activity in mice with and without SAH. PC-induced protection against vasospasm and neurological deficits was lost in wild-type mice treated with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester and in eNOS-null mice.
From these results, we conclude that endogenous protective mechanisms against vasospasm exist, are powerful, and can be induced by PC. eNOS-derived nitric oxide is a critical mediator of this neurovascular protection. These “proof-of-principle” results suggest that conditioning represents a promising new strategy to mitigate SAH-induced neurovascular dysfunction.
In Experiment 2, we sought to determine whether these innate protective mechanisms are induced by a more clinically relevant conditioning paradigm, and whether the cerebrovascular protection extends to non-vasospasm contributors to DCI, microthrombosis and microvessel dysfunction.
Adult male mice were subjected to sham surgery, SAH surgery, or SAH and subsequently postconditioned with isoflurane (2% for 1h, starting 1h after surgery). Contributors to DCI – vasospasm of the ipsilateral middle cerebral artery, cortical microthrombosis as assessed by fibrinogen immunohistochemistry, and cerebrovascular vasodilatory function was assessed in pial vessels through a closed cranial window – were measured 3 days post-SAH. Neurological outcome was assessed daily. Moreover, isoflurane-induced changes in HIF-1α-–dependent genes (glucose transporter-1, GLUT1; BNIP3) and HIF-2α-driven erythropoietin (EPO) were assessed via quantitative-PCR. HIF-1α was inhibited either pharmacologically (2-methoxyestradiol, 2ME2) or genetically in endothelial cells (EC-HIF-1-null). All experiments were performed in a randomized and blinded fashion.
We found the following: first, isoflurane postconditioning markedly reduced SAH-induced DCI in wild-type mice: vasospasm was attenuated, microthrombosis was significantly reduced, and microvessel function was restored. Neurological deficits were also significantly attenuated. Second, isoflurane modulated HIF-1α- and HIF-2α-dependent genes; these changes were abolished in 2ME2-treated wild-type mice and in EC-HIF-1-null mice (HIF-1α-dependent genes only in the latter). Third, postconditioning-induced protection against vasospasm and neurological deficits was attenuated in 2ME2-treated wild-type mice and in EC-HIF-1-null mice.
In Experiment 3, we sought to assess whether the protection afforded by conditioning extended to long-term neurocognitive outcomes. Whereas rat SAH causes long-term deficits in learning and memory, it remains unknown whether similar deficits are seen in the mouse, a species particularly amenable to powerful, targeted genetic manipulation. We thus subjected mice to SAH and assessed long-term cognitive outcome via the Morris water maze (MWM), the most commonly used metric for rodent neurocognition. No significant differences in MWM performance (by either of two protocols) were seen in SAH versus sham mice. Moreover, SAH caused negligible hippocampal CA1 injury. These results undercut the potential of commonly used methods (of SAH induction and assessment of long-term neurocognitive outcome) for use in targeted molecular studies of SAH-induced cognitive deficits in the mouse.
From these results, we concluded that endogenous protective mechanisms against DCI exist, are powerful, and can be induced by hypoxic PC or isoflurane postconditioning. This protection depends critically on eNOS-derived nitric oxide and endothelial cell–derived HIF-1α. These studies provide strong evidence that conditioning – especially isoflurane postconditioning – represents a promising new strategy to reduce DCI after SAH. Future studies examining long-term neurocognitive deficits should utilize rat models of SAH.
Chair and Committee
Gregory J Zipfel
Jeffrey Gidday, Byung Han, Jin-Moo Lee, Colin Derdeyn, Jeffrey Arbeit
Milner, Eric, "Endogenous Protection in Subarachnoid Hemorrhage" (2015). Arts & Sciences Electronic Theses and Dissertations. 667.