John Thundyil

Toll-like receptors in ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injuries are implicated in a large array of pathological conditions such as myocardial infarction, cerebral stroke, and hepatic, renal, and intestinal ischemia, as well as following cardiovascular and transplant surgeries. The hallmark of these pathologies is excessive inflammation. Toll-like receptors (TLRs) are recognized as one of the main contributors to pathogen-induced inflammation and, more recently, injury-induced inflammation. Endogenous ligands such as low-molecular hyaluronic acid, fibronectin, heat shock protein 70, and heparin sulfate were all found to be cleaved in the inflamed tissue and to activate TLR2 and TLR4, initiating an inflammatory response even in the absence of pathogens and infiltrating immune cells. In this review, we discuss the contribution of TLR activation in hepatic, renal, cerebral, intestinal, and myocardial I/R injuries. A greater understanding of the role of TLRs in I/R injuries may aid in the development of specific TLR-targeted therapeutics to treat these conditions.

Pathophysiology, treatment, and animal and cellular models of human ischemic stroke

Stroke is the worlds second leading cause of mortality, with a high incidence of severe morbidity in surviving victims. There are currently relatively few treatment options available to minimize tissue death following a stroke. As such, there is a pressing need to explore, at a molecular, cellular, tissue, and whole body level, the mechanisms leading to damage and death of CNS tissue following an ischemic brain event. This review explores the etiology and pathogenesis of ischemic stroke, and provides a general model of such. The pathophysiology of cerebral ischemic injury is explained, and experimental animal models of global and focal ischemic stroke, and in vitro cellular stroke models, are described in detail along with experimental strategies to analyze the injuries. In particular, the technical aspects of these stroke models are assessed and critically evaluated, along with detailed descriptions of the current best-practice murine models of ischemic stroke. Finally, we review preclinical studies using different strategies in experimental models, followed by an evaluation of results of recent, and failed attempts of neuroprotection in human clinical trials. We also explore new and emerging approaches for the prevention and treatment of stroke. In this regard, we note that single-target drug therapies for stroke therapy, have thus far universally failed in clinical trials. The need to investigate new targets for stroke treatments, which have pleiotropic therapeutic effects in the brain, is explored as an alternate strategy, and some such possible targets are elaborated. Developing therapeutic treatments for ischemic stroke is an intrinsically difficult endeavour. The heterogeneity of the causes, the anatomical complexity of the brain, and the practicalities of the victim receiving both timely and effective treatment, conspire against developing effective drug therapies. This should in no way be a disincentive to research, but instead, a clarion call to intensify efforts to ameliorate suffering and death from this common health catastrophe. This review aims to summarize both the present experimental and clinical state-of-the art, and to guide future research directions.

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen–glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.

Adiponectin receptor signalling in the brain

Adiponectin is an important adipocyte‐derived hormone that regulates metabolism of lipids and glucose, and its receptors (AdipoR1, AdipoR2, T‐cadherin) appear to exert actions in peripheral tissues by activating the AMP‐activated protein kinase, p38‐MAPK, PPARα and NF‐kappa B. Adiponectin has been shown to exert a wide range of biological functions that could elicit different effects, depending on the target organ and the biological milieu. There is substantial evidence to suggest that adiponectin receptors are expressed widely in the brain. Their expression has been detected in regions of the mouse hypothalamus, brainstem, cortical neurons and endothelial cells, as well as in whole brain and pituitary extracts. While there is now considerable evidence for the presence of adiponectin and its receptors in the brain, their precise roles in brain diseases still remain unclear. Only a few research studies have looked at this facet of adiponectins in brain disorders. This brief review will describe the evidence for important functions by adiponectin, its structure and known actions, evidence for expression of AdipoRs in the brain, their involvement in brain disorders and the therapeutic potential of agents that could modify AdipoR signalling.

Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis

C5a receptors are found in the central nervous system (CNS), on both neurons and glia. However, the origin of the C5a, which activates these receptors, is unclear. In the present study, we show that primary cultured mouse cortical neurons constitutively express C5, the precursor of C5a, and express the classical receptor for C5a, CD88. With cell ischemia caused by 12 h glucose deprivation, or oxygen‐glucose deprivation (OGD), neurons demonstrated increased apoptosis, up‐regulation of CD88, and increased levels of C5a in the media. Exogenous murine C5a (100 nM) added to the neuronal cultures resulted in apoptosis, without affecting cell necrosis. Pretreatment of the cells with the specific CD88 receptor antagonist PMX53 (100 nM) significantly blocked ischemia‐induced apoptosis (~50%), and neurons from CD88–/– mice were similarly protected. In a murine model of stroke, using middle cerebral artery occlusion (MCAO), we found that C5a levels in the brain increased; this also occurred in cerebral slice cultures exposed to OGD. CD88–/– mice subjected to MCAO had significantly reduced infarct volumes and improved neurological scores. Taken together, our results demonstrate that neurons in the CNS have the capability to generate C5a following ischemic stress, and this has the potential to activate their C5a receptors, with deleterious consequences.—Pavlovski, D., Thundyil, J., Monk, P. N., Wetsel, R. A., Taylor, S. M., Woodruff, T. M. Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis. FASEB J. 26, 3680–3690 (2012). www.fasebj.org

Evidence that γ-Secretase-Mediated Notch Signaling Induces Neuronal Cell Death via the Nuclear Factor-κB-Bcl-2-Interacting Mediator of Cell Death Pathway in Ischemic Stroke

Notch-1 (Notch) is a cell surface receptor that regulates cell-fate decisions in the developing nervous system, and it may also have roles in synaptic plasticity in the adult brain. Binding of its ligands results in the proteolytic cleavage of Notch by the γ-secretase enzyme complex, thereby causing the release of a Notch intracellular domain (NICD) that translocates to the nucleus, in which it regulates transcription. Here we show that activation of Notch modulates ischemic neuronal cell death in vitro and in vivo. Specifically, our findings from the use of Notch-1 siRNA or the overexpression of NICD indicate that Notch activation contributes to cell death. Using modified NICD, we demonstrate an apoptosis-inducing function of NICD in both the nucleus and the cytosol. NICD transfection-induced cell death was reduced by blockade of calcium signaling, caspase activation, and Janus kinase signaling. Inhibition of the Notch-activating enzyme, γ-secretase, protected against ischemic neuronal cell death by targeting an apoptotic protease, cleaved caspase-3, nuclear factor-κB (NF-κB), and the pro-death BH3-only protein, Bcl-2-interacting mediator of cell death (Bim). Treatment of mice with a γ-secretase inhibitor, compound E, reduced infarct size and improved functional outcome in a model of focal ischemic stroke. Furthermore, γ-secretase inhibition reduced NICD, p-p65, and Bim levels in vivo. These findings suggest that Notch signaling endangers neurons after ischemic stroke by modulating the NF-κB, pro-death protein Bim, and caspase pathways.

The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice.

The development of the brain tissue damage in ischemic stroke is composed of an immediate component followed by an inflammatory response with secondary tissue damage after reperfusion. Fisetin, a flavonoid, has multiple biological effects, including neuroprotective and antiinflammatory properties. We analyzed the effects of fisetin on infarct size and the inflammatory response in a mouse model of stroke, temporary middle cerebral artery occlusion, and on the activation of immune cells, murine primary and N9 microglial and Raw264.7 macrophage cells and human macrophages, in an in vitro model of inflammatory immune cell activation by lipopolysaccharide (LPS). Fisetin not only protected brain tissue against ischemic reperfusion injury when given before ischemia but also when applied 3 hours after ischemia. Fisetin also prominently inhibited the infiltration of macrophages and dendritic cells into the ischemic hemisphere and suppressed the intracerebral immune cell activation as measured by intracellular tumor necrosis factor α (TNFα) production. Fisetin also inhibited LPS-induced TNFα production and neurotoxicity of macrophages and microglia in vitro by suppressing nuclear factor κB activation and JNK/Jun phosphorylation. Our findings strongly suggest that the fisetin-mediated inhibition of the inflammatory response after stroke is part of the mechanism through which fisetin is neuroprotective in cerebral ischemia.

Evidence that adiponectin receptor 1 activation exacerbates ischemic neuronal death

Background-Adiponectin is a hormone produced in and released from adipose cells, which has been shown to have anti-diabetic and anti-inflammatory actions in peripheral cells. Two cell surface adiponectin receptors (ADRs) mediate the majority of the known biological actions of adiponectin. Thus far, ADR expression in the brain has been demonstrated in the arcuate and the paraventricular nucleus of hypothalamus, where its activation affects food intake. Recent findings suggest that levels of circulating adiponectin increase after an ischemic stroke, but the role of adiponectin receptor activation in stroke pathogenesis and its functional outcome is unclear.Methods-Ischemic stroke was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAO) for 1 h, followed by reperfusion. Primary cortical neuronal cultures were established from individual embryonic neocortex. For glucose deprivation (GD), cultured neurons were incubated in glucose-free Lockes medium for 6, 12 or 24 h. For combined oxygen and glucose deprivation (OGD), neurons were incubated in glucose-free Lockes medium in an oxygen-free chamber with 95% N2/5% CO2 atmosphere for either 3, 6, 9, 12 or 24 h. Primary neurons and brain tissues were analysed for Adiponectin and ADRs using reverse transcriptase polymerase chain reaction (RT-PCR), immunoblot and immunochemistry methods.Results-Cortical neurons express ADR1 and ADR2, and that the levels of ADR1 are increased in neurons in response to in vitro or in vivo ischemic conditions. Neurons treated with either globular or trimeric adiponectin exhibited increased vulnerability to oxygen and glucose deprivation which was associated with increased activation of a pro-apoptotic signaling cascade involving p38 mitogen-activated protein kinase (p38MAPK) and AMP-activated protein kinase (AMPK).Conclusions-This study reveals a novel pathogenic role for adiponectin and adiponectin receptor activation in ischemic stroke. We show that cortical neurons express ADRs and reveal a pro-apoptotic role for ADR1 activation in neurons, which may render them vulnerable to ischemic death.

Intravenous immunoglobulin protects neurons against amyloid beta‐peptide toxicity and ischemic stroke by attenuating multiple cell death pathways

J. Neurochem. (2012) 122, 321–332.

Intestinal ischemia reperfusion injury leads to inflammatory changes in the brain

Intestinal ischemia-reperfusion (I/R) injury is a well-established animal model of systemic inflammation and can lead to multiple organ failure as well as severe and lasting morbidity and even death. It can occur in humans as a result of vascular surgery or as secondary sequelae to many common conditions including low blood pressure, myocardial infarction, and necrotizing enterocolitis. Systemic inflammation induced through kidney I/R injury has been shown previously to lead to encephalopathic adverse effects, and it was theorized that intestinal injury would also cause secondary central nervous system effects. This study presents evidence that over a 6-h time frame, mouse intestinal I/R injury does not cause neuronal cell death in the brain in vivo. However, at the genetic level, certain inflammatory mediators such as endothelial nitric oxide synthase, intercellular adhesion molecule 1, P selectin, TNF-&agr;, and IL-6 are significantly upregulated. There was a significant increase in brain edema observed in sham-operated animals as well as in fasted and nonfasted I/R groups, but neurons were not apoptotic, in the 6-h time period. Conversely, Iba1-expressing activated microglia cells and glial fibrillary acidic protein-expressing astrocytes were found to be markedly increased in fasted and nonfasted I/R mice compared with controls and sham-operated animals. These data demonstrate that intestinal I/R injury induces inflammatory changes in the brain.