Extending injury- and disease-resistant CNS phenotypes by repetitive epigenetic conditioning

Significant reductions in the extent of acute injury in the CNS can be achieved by exposure to different preconditioning stimuli, but the duration of the induced protective phenotype is typically short-lasting, and thus is deemed as limiting its clinical applicability. Extending the period over which such adaptive epigenetic changes persist – in effect, expanding conditioning’s therapeutic window – would significantly broaden the potential applications of such a treatment approach in patients. The frequency of the conditioning stimulus may hold the key. While transient (1-3 days) protection against CNS ischemic injury is well established preclinically following a single preconditioning stimulus, repetitively presenting preconditioning stimuli extends the duration of ischemic tolerance by many weeks. Moreover, repetitive intermittent postconditioning enhances postischemic recovery metrics and improves long-term survival. Intermittent conditioning is also efficacious for preventing or delaying injury in preclinical models of chronic neurodegenerative disease, and for promoting long-lasting functional improvements in a number of other pathologies as well. Although the detailed mechanisms underlying these protracted kinds of neuroplasticity remain largely unstudied, accumulating empirical evidence supports the contention that all of these adaptive phenotypes are epigenetically mediated. Going forward, additional preclinical demonstrations of the ability to induce sustained beneficial phenotypes that reduce the burden of acute and chronic neurodegeneration, and experimental interrogations of the regulatory constructs responsible for these epigenetic responses, will accelerate the identification of not only efficacious, but practical, adaptive epigenetics-based treatments for individuals with neurological disease

Role of hypoxia-inducible factor-1α in preconditioning-induced protection of retinal ganglion cells in glaucoma

PURPOSE: We recently demonstrated in a mouse model of glaucoma that endogenous epigenetic mechanisms can be activated by a repetitive hypoxic preconditioning (RHP) stimulus to provide robust retinal ganglion cell (RGC) protection. Although we also provided evidence that RHP prevents or delays the apoptotic demise of the RGC soma, the mechanisms responsible for signaling this epigenetic response, as well as the effectors of the glaucoma-tolerant phenotype at the somatic and axonal levels, remain unidentified. In the present study, we used conditional mutant mice lacking hypoxia-inducible factor-1α (HIF-1α) in RGCs (HIF-1α RGC-knockout [KO] mice) to test the hypothesis that RHP-mediated activation of this transcription factor in these cells protects them from glaucomatous injury. METHODS: Adult HIF-1α RGC-KO mice, generated by mating floxed HIF-1α mice with math5-Cre mice, were used. Experimental glaucoma was induced unilaterally in the HIF-1α RGC-KO mice and matched wild-types by elevating the intraocular pressure to 16–20 mmHg for 3 consecutive weeks, secondary to episcleral vein ligation. Mice of each genotype were randomized to either an RHP protocol (six total exposures to systemic hypoxia [11% oxygen], interspersed over a 2-week period, completed 3 days before ligation surgery) or to an untreated group. RGC soma and axon injury was quantified with Neuronal Nuclei (NeuN) immunohistochemistry in retinal flat mounts and SMI32 immunohistochemistry in cross sections of the post-laminar optic nerve, respectively. RESULTS: HIF-1α RGC-KO mice exhibited normal retinal function and morphology, and crosses of math5-Cre mice with floxed ROSA26 reporter mice confirmed Cre recombinase activity was confined to the RGC axons and soma. Untreated wild-type mice exhibited a 30±2% loss of RGC soma and a 31±3% loss of RGC axons after 3 weeks of intraocular hypertension (both p<0.05 versus fellow eye). The 90% and 81% improvement in soma and axon survival, respectively, observed in the wild-type mice treated with RHP (both p<0.05 versus the glaucoma eye in the untreated mice) was still observed to a near identical extent in the RHP-treated HIF-1α RGC-KO mice. RHP had no effect on the magnitude of intraocular pressure elevation in either the KO or wild-type groups, indicating that protection was realized in both genotypes in the face of ongoing intraocular hypertension. CONCLUSIONS: These findings indicate that the robust, glaucomatous protection of the RGC soma and axons induced by RHP does not require HIF-1α-mediated transcription of survival genes and other adaptive responses within the RGCs themselves. Rather, we infer that RGC survival is augmented secondary to the activation of other hypoxia-sensitive transcription factors in RGCs and/or the action of diffusible HIF-1α target gene proteins released from neighboring retinal cells. Ideally, the involvement of such autocrine- and/or paracrine-based mechanisms would be confirmed in future studies, but distinct components of the integrated, pleiotropic, and multicellular basis of this endogenous epigenetic response may prove difficult to demonstrate experimentally, as we found in the present study

Effects of remote limb ischemic conditioning on muscle strength in healthy young adults: A randomized controlled trial

Remote limb ischemic conditioning (RLIC) is a clinically feasible method in which brief, sub-lethal bouts of ischemia protects remote organs or tissues from subsequent ischemic injury. A single session of RLIC can improve exercise performance and increase muscle activation. The purpose of this study, therefore, was to assess the effects of a brief, two-week protocol of repeated RLIC combined with strength training on strength gain and neural adaptation in healthy young adults. Participants age 18-40 years were randomized to receive either RLIC plus strength training (n = 15) or sham conditioning plus strength training (n = 15). Participants received RLIC or sham conditioning over 8 visits using a blood pressure cuff on the dominant arm with 5 cycles of 5 minutes each alternating inflation and deflation. Visits 3-8 paired conditioning with wrist extensors strength training on the non-dominant (non-conditioned) arm using standard guidelines. Changes in one repetition maximum (1 RM) and electromyography (EMG) amplitude were compared between groups. Both groups were trained at a similar workload. While both groups gained strength over time (P = 0.001), the RLIC group had greater strength gains (9.38 ± 1.01 lbs) than the sham group (6.3 ± 1.08 lbs, P = 0.035). There was not a significant group x time interaction in EMG amplitude (P = 0.231). The RLIC group had larger percent changes in 1 RM (43.8% vs. 26.1%, P = 0.003) and EMG amplitudes (31.0% vs. 8.6%, P = 0.023) compared to sham conditioning. RLIC holds promise for enhancing muscle strength in healthy young and older adults, as well as clinical populations that could benefit from strength training

Reduction of leukocyte microvascular adherence and preservation of blood-brain barrier function by superoxide-lowering therapies in a piglet model of neonatal asphyxia

Background: Asphyxia is the most common cause of brain damage in newborns. Substantial evidence indicates that leukocyte recruitment in the cerebral vasculature during asphyxia contributes to this damage. We tested the hypothesis that superoxide radical (O2⋅_) promotes an acute post-asphyxial inflammatory response and blood-brain barrier (BBB) breakdown. We investigated the effects of removing O2⋅_ by superoxide dismutase (SOD) or C3, the cell-permeable SOD mimetic, in protecting against asphyxia-related leukocyte recruitment. We also tested the hypothesis that xanthine oxidase activity is one source of this radical.Methods: Anesthetized piglets were tracheostomized, ventilated, and equipped with closed cranial windows for the assessment of post-asphyxial rhodamine 6G-labeled leukocyte-endothelial adherence and microvascular permeability to sodium fluorescein in cortical venules. Asphyxia was induced by discontinuing ventilation. SOD and C3 were administered by cortical superfusion. The xanthine oxidase inhibitor oxypurinol was administered intravenously.Results: Leukocyte-venular adherence significantly increased during the initial 2 h of post-asphyxial reperfusion. BBB permeability was also elevated relative to non-asphyxial controls. Inhibition of O2⋅_ production by oxypurinol, or elimination of O2⋅_ by SOD or C3, significantly reduced rhodamine 6G-labeled leukocyte-endothelial adherence and improved BBB integrity, as measured by sodium fluorescein leak from cerebral microvessels.Conclusion: Using three different strategies to either prevent formation or enhance elimination of O2⋅_ during the post-asphyxial period, we saw both reduced leukocyte adherence and preserved BBB function with treatment. These findings suggest that agents which lower O2⋅_ in brain may be attractive new therapeutic interventions for the protection of the neonatal brain following asphyxia

HIF-1α mediates isoflurane-induced vascular protection in subarachnoid hemorrhage

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

Sirtuin 1 mediates protection against delayed cerebral ischemia in subarachnoid hemorrhage in response to hypoxic postconditioning

Background Many therapies designed to prevent delayed cerebral ischemia (DCI) and improve neurological outcome in aneurysmal subarachnoid hemorrhage (SAH) have failed, likely because of targeting only one element of what has proven to be a multifactorial disease. We previously demonstrated that initiating hypoxic conditioning before SAH (hypoxic preconditioning) provides powerful protection against DCI. Here, we expanded upon these findings to determine whether hypoxic conditioning delivered at clinically relevant time points after SAH (hypoxic postconditioning) provides similarly robust DCI protection. Methods and Results In this study, we found that hypoxic postconditioning (8%

SIRT1 mediates hypoxic preconditioning induced attenuation of neurovascular dysfunction following subarachnoid hemorrhage

BACKGROUND AND PURPOSE: Vasospasm and delayed cerebral ischemia (DCI) contribute significantly to the morbidity/mortality associated with aneurysmal subarachnoid hemorrhage (SAH). While considerable research effort has focused on preventing or reversing vasospasm, SAH-induced brain injury occurs in response to a multitude of concomitantly acting pathophysiologic mechanisms. In this regard, the pleiotropic epigenetic responses to conditioning-based therapeutics may provide an ideal SAH therapeutic strategy. We previously documented the ability of hypoxic preconditioning (PC) to attenuate vasospasm and neurological deficits after SAH, in a manner that depends on the activity of endothelial nitric oxide synthase. The present study was undertaken to elucidate whether the NAD-dependent protein deacetylase sirtuin isoform SIRT1 is an upstream mediator of hypoxic PC-induced protection, and to assess the efficacy of the SIRT1-activating polyphenol Resveratrol as a pharmacologic preconditioning therapy. METHODS: Wild-type C57BL/6J mice were utilized in the study and subjected to normoxia or hypoxic PC. Surgical procedures included induction of SAH via endovascular perforation or sham surgery. Multiple endpoints were assessed including cerebral vasospasm, neurobehavioral deficits, SIRT1 expression via quantitative real-time PCR for mRNA, and western blot for protein quantification. Pharmacological agents utilized in the study include EX-527 (SIRT1 inhibitor), and Resveratrol (SIRT1 activator). RESULTS: Hypoxic PC leads to rapid and sustained increase in cerebral SIRT1 mRNA and protein expression. SIRT1 inhibition blocks the protective effects of hypoxic PC on vasospasm and neurological deficits. Resveratrol pretreatment dose-dependently abrogates vasospasm and attenuates neurological deficits following SAH – beneficial effects that were similarly blocked by pharmacologic inhibition of SIRT1. CONCLUSION: SIRT1 mediates hypoxic preconditioning-induced protection against neurovascular dysfunction after SAH. Resveratrol mimics this neurovascular protection, at least in part, via SIRT1. Activation of SIRT1 is a promising, novel, pleiotropic therapeutic strategy to combat DCI after SAH

Clinical practice guidelines of remote ischemic conditioning for the management of cerebrovascular diseases

Remote ischemic conditioning (RIC) using transient limb ischemia and reperfusion has been shown in small clinical studies to reduce myocardial injury and infarction in cardiac patients, although larger clinical outcome studies have been neutral. Experimental and emerging clinical studies have also reported beneficial effects of limb RIC in a number of different settings of cerebrovascular disease including stroke (ischemic and hemorrhagic), carotid artery stenosis, intracranial artery stenosis, aneurysms, small vessel disease, and vascular cognitive impairment. Although limb RIC has many advantages, in that it is non-invasive, easy to administer, relatively innocuous, cost-effective, has few or no contraindications, and may be deployed under various circumstances (e.g., home, ambulance, and hospital), several questions remain regarding its clinical application for cerebrovascular disease. Therefore, in this document, we aim to provide practicing clinicians with a coherent synthesis of the latest scientific evidence, and we propose several recommendations to help facilitate the clinical application of limb RIC for the management of cerebrovascular disease

Cerebrovascular ischemic protection by pre- and post-conditioning

Stroke and cardiac arrest involve injury to all the brain′s resident cells and their respective progenitors, including neurons, all glial subtypes, vascular smooth muscle, vascular endothelium, and pericytes, resulting either in the death of the individual or in a lesion that likely manifests as long-term impairments across a number of cognitive and functional domains. Thousands of studies in experimental animals and results from a few clinical trials in humans have demonstrated that the mechanisms responsible for ischemic brain injury can be blocked or slowed by survival-enhancing epigenetic responses induced by "conditioning" the brain with a stress stimulus paradigm before or even after ictus. The resultant reduction in lesion size and functional deficits are often termed endogenous "neuroprotection," but this in fact involves cytoprotective responses on the part of all the aforementioned resident brain cells and the circulating immune cells as well. The present review will summarize findings demonstrating conditioning-induced protection of the cerebral vasculature, that in turn manifests as reductions in vascularly targeted inflammatory responses; less endothelial injury and improvements in structural integrity of the circulation across all levels of organization; enhanced perfusion with less thrombosis; reductions in vascular dysregulation and reactivity impairments; and, over the longer term, more robust angiogenesis and vascular remodeling. Advancing the mechanistic basis for these innately vasculoprotective phenotypes may provide therapeutic targets for limiting cerebral circulatory injury and dysfunction following stroke and cardiac arrest