Neutrophil extracellular traps regulate ischemic stroke brain injury
Frederik DenormeIrina PortierJohn L. RustadMark J. CodyClaudia V. de AraujoChieko HokiMatthew D. AlexanderRamesh GrandhiMitchell DyerMatthew D. NealJennifer J. MajersikChristian C. YostRobert A. Campbell
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Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface-expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.Keywords:
Neutrophil Extracellular Traps
Stroke
HMGB1
Brain ischemia
Energy deficiency and oxidative stress are stages of the biochemical cascade in brain damage of ischemic origin. There is information in the literature about their changes in certain types of ischemia, but there are no data on the features and comparative characteristics in cerebral ischemia of varying severity. It was found that the most pronounced disturbances in the prooxidant-oxidant balance and energy metabolism were observed in total cerebral ischemia. Similar, however, less pronounced disorders were found in daily subtotal ischemia and in the subgroup of stepped subtotal ischemia with an interval between ligation of the common carotid arteries of 1 day. The least pronounced disorders were in the subgroup with an interval between ligation of the common carotid arteries 7 days The aim of the work is to assess the state of the energy and oxidative processes of the brain during its ischemia of varying severity.
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Cerebrovascular disease is the third leading cause of death in the industrialized world and is also a major cause of long-lasting disability. The development of gene deficient and transgenic mice has recently aided in research into and development of specific surgical procedures for brain ischemia in mice. The models of brain ischemia are divided into global ischemia and focal ischemia. Global ischemia is divided into two sub-models, forebrain ischemia and total ischemia. Forebrain ischemia is widely used to analyze delayed neuronal cell death including apoptosis, while total ischemia is used to clarify the neuronal cell death by the systemic ischemia such as cardiac arrest. On the other hand, focal ischemia is used to analyze pathological stroke. Pathophysiology of brain ischemia has been clarified by the use of these models. This review outlines the pathways that lead to cell death following ischemia. Moreover, we have reviewed these currently-used mouse experimental models of global and focal ischemia.
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The high-mobility group box 1 (HMGB1)/receptor for advanced glycation end products (RAGE) signaling pathway holds promise as a potential therapeutic target for ischemic brain injury. The effects of FPS-ZM1 and electroacupuncture (EA) on activation of the HMGB1/RAGE signaling pathway after cerebral ischemia remain uncertain.Middle cerebral artery occlusion (MCAO) model was established. Neurological function was assessed using Longa scores. Nissl staining was used to observe the morphology of neurons. The expression levels of HMGB1 and RAGE were assayed with immunofluorescence staining and western blot.The results showed that EA and FPS-ZM1 could reduce the neural function score and neurons cell injury in cerebral ischemia rats by inhibiting the expression of HMGB1 and RAGE in primary motor cortex (M1) region. In addition, EA combined with FPS-ZM1 had a better therapeutic effect.The HMGB1/RAGE pathway could be activated after cerebral ischemia. Both EA and FPS-ZM1 improved neurological deficits and attenuated neuronal damage in rats. They had synergistic effects. These interventions were observed to mitigate brain damage by suppressing the activation of HMGB1/RAGE.
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Abstract Neutrophil extracellular traps (NETs) has been recently found exacerbate sterile inflammatory injury during liver ischemia/reperfusion (I/R). High mobility group box-1 (HMGB1) released from damaged cells, mediates liver damage and inflammatory responses after I/R. However, the intracellular role of HMGB1 in NET formation remains unknown. We sought to determine the role of intracellular HMGB1 in NET formation. Myloid cells HMGB1 knockout (LysM-HMGB1 KO) and HMGB1-flox mice were subjected to a non-lethal warm liver I/R. Less NETs was identified in ischemic liver lobes of LysM-HMGB1 KO mice compared with flox mice by confocal immunofluorescence (IF) imaging. This was associated with significantly less NET markers in LysM-HMGB1 KO mice, serum level of myeloperoxidase (MPO)-DNA complexes, and tissue level of citrullinated histone H3. Absence of HMGB1 in myloid cells protected the live from I/R injury indicated by significantly decreased serum transaminases (sALT), less necrosis and inflammatory cytokines production compared to flox mice. In vitro, no NET formation was observed in HMGB1 KO neutrophils under stimulation of PMA (positive control) or extracellular HMGB1 or histones confirmed by IF imaging. Chromatin in HMGB1 KO neutrophils failed to de-condensate in response to PMA which is the key step of NET formation. Additionally, gene deletion of HMGB1 in neutrophils completely blocked citrullination of histone H3 as a key step of NET formation, compared with control neutrophils in response to various stimulations. Our study demonstrates the dominant role of intracellular HMGB1 in NET formation. Lack of HMGB1 in neutrophils leads to diminished NET formation protect the liver from organ damage and cells death after liver I/R.
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The general failure of neuroprotectants in clinical trials of ischemic stroke points to the possibility of a fundamental blind spot in the current conception of ischemic brain injury, the "ischemic cascade". This is the first in a series of four papers whose purpose is to work towards a revision of the concept of brain ischemia by applying network concepts to develop a bistable model of brain ischemia. This first paper sets the stage for developing the bistable model of brain ischemia. Necessary background in network theory is introduced using examples from developmental biology which, perhaps surprisingly, can be adapted to brain ischemia with only minor modification. Then, to move towards a network model, we extract two core generalizations about brain ischemia from the mass of empirical data. First, we conclude that all changes induced in the brain by ischemia can be classified as either damage mechanisms that contribute to cell death, or stress responses that contribute to cell survival. Second, we move towards formalizing the idea of the "amount of ischemia", I, as a continuous, nonnegative, monotonically increasing quantity. These two generalizations are necessary precursors to reformulating brain ischemia as a bistable network.
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AIM:Platelet-activating factor(PAF) bas been reported to be an active mediator of neuronal damage in regional cerebral ischemia on the basis of indirect pharmacological data from PAF antagonists. The direct measurement of PAF has not been reported previously in regional cerebral ischemia. It prompted us to investigate the change of PAF in unilateral ischemia in gerbils and regional ischemia in rats.METHODS:Thin layer chromatography was combined with biological methods for the determination of PAF in brain after cerebral ischemia.RESULTS:(1) The PAF content of ischemic brain was increaesd significantly in gerbils subjected to 3 h unilateral cerebral ischemia followed by 2h reperfusion respectively(4.40±0.90) and (1.30±0.40) pg/g, P 0.05. (2)The PAF content of ischemic brain was increased significantly also in rats subjected to 48 h regional ischemia respectively(5.74±0.75) and (0.76±0.05)pg/g, P 0.05. CONCLUSION:PAF could contribute to secondary neuronal damage after regional cerebral ischemia.
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The general failure of neuroprotectants in clinical trials of ischemic stroke points to the possibility of a fundamen-tal blind spot in the current conception of ischemic brain injury, the “ischemic cascade”. This is the first in a se-ries of four papers whose purpose is to work towards a revision of the concept of brain ischemia by applying network concepts to develop a bistable model of brain ischemia. This first paper sets the stage for developing the bistable model of brain ischemia. Necessary background in network theory is introduced using examples from developmental biology which, perhaps surprisingly, can be adapted to brain ischemia with only minor mod-ification. Then, to move towards a network model, we extract two core generalizations about brain ischemia from the mass of empirical data. First, we conclude that all changes induced in the brain by ischemia can be classified as either damage mechanisms that contribute to cell death, or stress responses that contribute to cell survival. Second, we move towards formalizing the idea of the “amount of ischemia”, I, as a continuous, non-negative, monotonically increasing quantity. These two generalizations are necessary precursors to reformulat-ing brain ischemia as a bistable network.
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Objective To study the effect of β aescinate on NO metabolism in acute ischemia and ischemia reperfusion model of rat brain. Methods The acute ischemia and ischemia reperfusion model of rat brain was induced by blocking of four vessels. The rats were divided into seven groups: control group; 30 minute global ischemia group; 30 minute global ischemia+drug group; 30 minute global ischemia and 30 minute reperfusion group; 30 minute global ischemia and 30 minute reperfusion +drug group; 30 minute global ischemia and 5 day reperfusion group; 30 minute global ischemia and 5 day reperfusion group. β aescinate (10 mg/kg, iv) was injected before operation. The changes of the contents of NO, MDA and SOD in the cortex and serum were observed. Results Compared with the rats in the drug group, those in the ischemia group and ischemia reperfusion group showed a significant increase in concentrations of NO and MDA in the cortex and serum ( F=18.51-123.66, q=5.41- 29.59 , P 0.01), but a significant decrease in the level of SOD ( F=5.87, q=4.81-26.02, P 0.05). Conclusion β aescinate may influence NO metabolism and protect the neuron cells by means of anti free radicals.
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Although neutrophil extracellular traps (NETs) form to prevent dissemination of pathogenic microorganisms, excessive release of DNA and DNA-associated proteins can also perpetuate sterile inflammation. In this study, we found that the danger-associated molecular pattern protein high-mobility group box 1 (HMGB1) can induce NET formation. NET formation was found after exposure of wild-type and receptor for advanced glycation end products-deficient neutrophil to HMGB1, whereas deficiency of Toll-like receptor (TLR)4 diminished the ability of neutrophils to produce NETs. Incubation of neutrophils with HMGB1 significantly increased the amount of DNA and histone 3 released as well as intracellular histone 3 citrullination, a signaling event that precedes chromatin decondensation. In vivo, neutrophils isolated from bronchoalveolar lavages of mice exposed to LPS and HMGB1 showed consistently greater ability to produce NETs compared with pulmonary neutrophils from mice that received LPS alone. In contrast, mice treated with LPS and neutralizing antibody to HMGB1 had decreased amounts of the inflammatory cytokines TNF-α and macrophage inflammatory protein 2, as well as of free DNA and histone 3 in bronchoalveolar lavage fluids. Airway neutrophils from LPS-exposed mice that had been treated with anti-HMGB1 antibodies showed decreased citrullination of histone 3. These results demonstrate that interactions between HMGB1 and TLR4 enhance the formation of NETs and provide a novel mechanism through which HMGB1 may contribute to the severity of neutrophil-associated inflammatory conditions.
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