Michalis Papadakis

Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy

Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia and the tolerance conferred by ischemic preconditioning (IPC), would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

Neuroprotection by Dimethyloxalylglycine following Permanent and Transient Focal Cerebral Ischemia in Rats

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1α levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.

Improved regional cerebral blood flow is important for the protection seen in a mouse model of late phase ischemic preconditioning

INTRODUCTION Ischemic preconditioning (IPC) induces protection to cerebral ischemia. However, it was previously unclear whether this protection resulted from altered susceptibility to ischemia. The current study examines the effects of late phase ischemic preconditioning in a mouse model of middle cerebral artery occlusion (MCAO). Specific examination of the regional cerebral blood flow (rCBF) was conducted. EXPERIMENTAL PROCEDURE Intra-abdominal radiofrequency probes were implanted in animals and core temperature was regulated. Mice were subjected to MCAO: (1) brief 15 min duration (preconditioning ischemia) and (2) 45 min MCAO (injurious ischemia). Naive (i.e. not preconditioned) animals were compared with preconditioned animals (preconditioning ischemia plus injurious ischemia at 72 h reperfusion). rCBF was measured using laser Doppler flowmetry (LDF) and magnetic resonance cerebral perfusion (MRP) arterial spin labeling. Percentage of brain infarcted was compared between groups. RESULTS rCBF was significantly improved in the preconditioned cohorts of mice. Naive animals showed flow reductions to 16+/-3.59% (MCAO_45; injurious, unpreconditioned) and 17.1+/-8.6% (MCAO_15; preconditioning ischemia alone) of baseline, while preconditioned animals had flows 33.9+/-13.2% (IPC_45; preconditioned animals with injurious ischemia at 72 h reperfusion) of baseline (p=0.001). Percentage of brain infarcted was 17.2+/-6.2% in naive animals, while it was 5.1+/-4.6% in the preconditioned animals (p=0.003). MRP of the perfusion to the ischemic hemisphere, in a striatal coronal slice of the brain was 26.7+/-5.8% of the contralateral hemisphere in naive animals while preconditioned mice had flows of 38.7+/-6.8% of contralateral (p=0.04). CONCLUSIONS Improved rCBF is an important factor in the protection of IPC, during injurious MCAO in the mouse. Stringent monitoring of rCBF is required in future studies of IPC.

Cerebral blood flow alteration in neuroprotection following cerebral ischaemia

Abstract  The best neuroprotectant for acute ischaemic stroke would always be the rapid return of oxygen and glucose to physiological levels. This is currently provided by thrombolysis which restores blood flow to the ischaemic region. The attempt to confer neuroprotection by targeting the brain parenchyma has shown promise in experimental stroke models, but has unequivocally failed to translate to the clinic. Neuroprotective therapy primarily targets the biochemical cascade that produces cell death following cerebral ischaemia. However, these agents may also alter signal transduction that controls cerebral blood flow, for example glutamate, which may affect the outcome after ischaemia. In these cases, neuroprotection may potentially be due to the improved access to oxygen and glucose rather than biochemical prevention of cell death. Improvement in cerebral blood flow is an important but often overlooked effect of neuroprotective therapy, analogous to the protective effects of drug‐induced hypothermia. This short review will discuss cerebral blood flow alteration and protection of the brain in the context of ischaemic preconditioning, oxygen sensing and thrombolysis. Future neuroprotection studies in cerebral ischaemia require stringent monitoring of cerebral blood flow, plus other physiological parameters. This will increase the chances that any protection observed may be able to translate to human therapy.

Therapeutic hypothermia in experimental models of focal and global cerebral ischemia and intracerebral hemorrhage

Experimental evidence shows that therapeutic hypothermia (TH) protects the brain from cerebral injury in multiple ways. In different models of focal and global cerebral ischemia, mild-to-moderate hypothermia reduces mortality and neuronal injury and improves neurological outcome. In models of experimental intracerebral hemorrhage (ICH), TH reduces edema formation but does not show consistent benefi cial effects on functional outcome parameters. However, the number of studies of hypothermia on ICH is still limited. TH is most effective when applied before or during the ischemic event, and its neuroprotective properties vary according to species, strains and the model of ischemia used. Intrinsic changes in body and brain temperature frequently occur in experimental models of focal and global cerebral ischemia, and may have infl uenced studies on other neuroprotectants. This might be one explanation for the failure of a large amount of translational clinical neuroprotective trials. Hypothermia is the only neuroprotective therapeutic agent for cerebral ischemia that has successfully managed the transfer from bench to bedside, and it is an approved therapy for patients after cardiac arrest and children with hypoxic–ischemic encephalopathy. However, the implementation of hypothermia in the treatment of stroke patients is still far from routine clinical practice. In this article, the authors describe the development of TH in different models of focal and global cerebral ischemia, point out why hypothermia is so efficient in experimental cerebral ischemia, explain why temperature regulation is essential for further neuroprotective studies and discuss why TH for acute ischemic stroke still remains a promising but controversial therapeutic option.

Microarray analysis of the global gene expression profile following hypothermia and transient focal cerebral ischemia

BACKGROUND Hypothermia is one of the most robust experimental neuroprotective interventions against cerebral ischemia. Identification of molecular pathways and gene networks together with single genes or gene families that are significantly associated with neuroprotection might help unravel the mechanisms of therapeutic hypothermia. MATERIAL AND METHODS We performed a microarray analysis of ischemic rat brains that underwent 90 min of middle cerebral artery occlusion (MCAO) and 48 h of reperfusion. Hypothermia was induced for 4 h, starting 1 h after MCAO in male Wistar rats. At 48 h, magnetic resonance imaging (MRI) was performed for infarct volumetry, and functional outcome was determined by a neuroscore. The brain gene expression profile of sham (S), ischemia (I), and ischemia plus hypothermia (HI) treatment were compared by analyzing changes of individual genes, pathways, and networks. Real-time reverse-transcribed polymerase chain reaction (RT-PCR) was performed on selected genes to validate the data. RESULTS Rats treated with HI had significantly reduced infarct volumes and improved neuroscores at 48 h compared with I. Of 4067 genes present on the array chip, HI compared with I upregulated 50 (1.23%) genes and downregulated 103 (3.20%) genes equal or greater than twofold. New genes potentially mediating neuroprotection by hypothermia were HNRNPAB, HIG-1, and JAK3. On the pathway level, HI globally suppressed the ischemia-driven gene response. Twelve gene networks were identified to be significantly altered by HI compared with I. The most significantly altered network contained genes participating in apoptosis suppression. CONCLUSIONS Our data suggest that although hypothermia at the pathway level restored gene expression to sham levels, it selectively regulated the expression of several genes implicated in protein synthesis and folding, calcium homeostasis, cellular and synaptic integrity, inflammation, cell death, and apoptosis.

Hemodynamic monitoring of intracranial collateral flow predicts tissue and functional outcome in experimental ischemic stroke

Intracranial collaterals provide residual blood flow to penumbral tissue in acute ischemic stroke and contribute to infarct size variability in humans. In the present study, hemodynamic monitoring of the borderzone territory between the leptomeningeal branches of middle cerebral artery and anterior cerebral artery was compared to lateral middle cerebral artery territory, during common carotid artery occlusion and middle cerebral artery occlusion in rats. The functional performance of intracranial collaterals, shown by perfusion deficit in the territory of leptomeningeal branches either during common carotid artery occlusion or middle cerebral artery occlusion, showed significant variability among animals and consistently predicted infarct size and functional deficit. Our findings indicate that leptomeningeal collateral flow is a strong predictor of stroke severity in rats, similarly to humans. Monitoring of collateral blood flow in experimental stroke is essential for reducing variability in neuroprotection studies and accelerating the development of collateral therapeutics.

Roles of individual prolyl-4-hydroxylase isoforms in the first 24 hours following transient focal cerebral ischaemia: insights from genetically modified mice

•  Cerebral ischaemia results in the activation of multiple pathways that can independently lead to neuronal death. Agents targeting a number of processes at one time are likely to be translated into stroke therapy. •  Hypoxia‐inducible factor (HIF) is a transcription complex which responds to changes in oxygen. HIF levels are tightly regulated by a group of prolyl hydroxylases (PHDs). •  In this study, we investigated the function of each of the HIF‐PHDs in the first 24 hours following transient focal cerebral ischaemia by using mice with each isoform genetically suppressed. •  We found that the PHD1−/−, PHD2+/−, PHD3−/− mice had different outcomes after inducing ischaemia. In particular, the PHD2+/− mice had an improved rCBF response post‐reperfusion with better behavioural scores. The PHD3−/− mice have worse rCBF but no behavioural change. •  The information gained enhances understanding of the biological processes involved and informs strategies for therapeutic targeting of the PHD enzymes.

Endovascular Stroke Treatment Today

SUMMARY: The purpose of this study was to review current treatment options in acute ischemic stroke, focusing on the latest advances in the field of mechanical recanalization. These devices recently made available for endovascular intracranial thrombectomy show great potential in acute stroke treatments. Compelling evidence of their recanalization efficacy comes from current mechanical embolectomy trials. In addition to allowing an extension of the therapeutic time window, mechanical recanalization devices can be used without adjuvant thrombolytic therapy, thus diminishing the intracranial bleeding risk. Therefore, these devices are particularly suitable in patients in whom thrombolytic therapy is contraindicated. IV and IA thrombolysis and bridging therapy are viable options in acute stroke treatment. Mechanical recanalization devices can potentially have a clinically relevant impact in the interventional treatment of stroke, but at the present time, a randomized study would be beneficial.

HIF prolyl hydroxylase inhibition prior to transient focal cerebral ischaemia is neuroprotective in mice

This study investigated the effects of 2‐(1‐chloro‐4‐hydroxyisoquinoline‐3‐carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia‐inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8‐ to 12‐week‐old C57/B6 mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) either immediately or 24 h after receiving IOX3. Mice receiving IOX3 at 20 mg/kg 24 h prior to the MCAO had better neuroscores and smaller blood–brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60 mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up‐regulated HIF‐1α, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up‐regulated HIF‐1α and delocalized ZO‐1. Pre‐treating IOX3 on RBE4 cells 24 h before oxygen–glucose deprivation had a protective effect on endothelial barrier preservation with ZO‐1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia.