Post-ischemic Inflammation Research Articles

Influences of Ethanol on Inflammation and Apoptosis Following Transient Focal Cerebral Ischemia

BackgroundInflammation and apoptosis are two key elements involved in cerebral ischemia/reperfusion (I/R) injury. Ethanol is one of the most commonly and regularly used chemical substances. We determined the influences of chronic ethanol consumption on inflammation and apoptosis following transient focal cerebral ischemia.MethodsMale C57/BL6J mice were divided into 6 groups and gavage fed with 0.175, 0.35, 0.7, 1.4, 2.8 g/kg/d ethanol or volume‐matched water once a day for eight weeks. Cerebral I/R injury, adhesion molecules, microglial activation, neutrophil infiltration, pro‐ and anti‐inflammatory cytokines/chemokines, MMPs, cleaved caspase‐3‐positive neurons, and TUNEL‐positive neurons were evaluated at 24 hours after a 90‐minute unilateral middle cerebral artery occlusion (MCAO).ResultsCerebral I/R injury was only significantly reduced in 0.7 g/kg/d ethanol group (peak blood ethanol concentration: 9 mM) and increased in 2.8 g/kg/d ethanol group (peak blood ethanol concentration: 37 mM). Baseline and post‐ischemic upregulation of ICAM‐1 and E‐selectin were suppressed in all ethanol groups. Post‐ischemic microglial activation and neutrophil infiltration were significantly alleviated in 0.175–1.4 g/kg/d ethanol groups but dramatically exacerbated in 2.8 g/kg/d ethanol group. Ethanol tends to dose‐dependently increase both baseline pro‐ and anti‐inflammatory cytokines/chemokines, but dose‐dependently suppressed post‐ischemic increase in pro‐inflammatory cytokines/chemokines. Surprisingly, 2.8 g/kg/d ethanol strengthened post‐ischemic increase in anti‐inflammatory cytokines/chemokines. Furthermore, although baseline MMP‐9 activity was reduced in 0.7 g/kg/d ethanol group, the magnitude of post‐ischemic increase in MMP‐9 activity was significantly less in 2.8 g/kg/d ethanol group. Both cleaved caspase‐3‐positive neurons and TUNEL‐positive neurons were significantly less in 0.7 g/kg/d ethanol group.ConclusionsLight alcohol consumption protects against cerebral I/R injury by suppressing post‐ischemic inflammation and apoptosis, whereas heavy alcohol consumption may exacerbate cerebral I/R injury via an inflammation‐ and apoptosis‐independent mechanism.Support or Funding InformationThis study was supported by a National Institutes of Health Grant (AA023610) and funds from Louisiana State University Health Sciences Center‐Shreveport to HS, a postdoctoral fellowship to CL and a predoctoral fellowship to KDM from the Center for Cardiovascular Diseases and Sciences, Louisiana State University Health Science Center‐Shreveport.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mito-Tempo prevents nicotine-induced exacerbation of ischemic brain damage.

Nicotine may contribute to the pathogenesis of cerebrovascular disease via the generation of reactive oxygen species (ROS). Overproduction of ROS leads to brain damage by intensifying postischemic inflammation. Our goal was to determine the effect of Mito-Tempo, a mitochondria-targeted antioxidant, on ischemic brain damage and postischemic inflammation during chronic exposure to nicotine. Male Sprague-Dawley rats were divided into four groups: control, nicotine, Mito-Tempo-treated control, and Mito-Tempo-treated nicotine. Nicotine (2 mg·kg-1·day-1) was administered via an osmotic minipump for 4 wk. Mito-Tempo (0.7 mg·kg-1·day-1 ip) was given for 7 days before cerebral ischemia. Transient focal cerebral ischemia was induced by occlusion of the middle cerebral artery for 2 h. Brain damage and inflammation were evaluated after 24 h of reperfusion by measuring infarct volume, expression of adhesion molecules, activity of matrix metalloproteinase, brain edema, microglial activation, and neutrophil infiltration. Nicotine exacerbated infarct volume and worsened neurological deficits. Nicotine did not alter baseline ICAM-1 expression, matrix metallopeptidase-2 activity, microglia activation, or neutrophil infiltration but increased these parameters after cerebral ischemia. Mito-Tempo did not have an effect in control rats but prevented the chronic nicotine-induced augmentation of ischemic brain damage and postischemic inflammation. We suggest that nicotine increases brain damage following cerebral ischemia via an increase in mitochondrial oxidative stress, which, in turn, contributes to postischemic inflammation. NEW & NOTEWORTHY Our findings have important implications for the understanding of mechanisms contributing to increased susceptibility of the brain to damage in smokers and users of nicotine-containing tobacco products.

Abstract 72: The Innate Immunity Receptor CD36 Contributes to Ischemic Brain Injury by Regulating Post-ischemic Caspase-1 Activity and Interleukin-1b Production in Microglia

CD36, an innate immunity receptor enriched in microglia, and IL-1β, a cytokine produced by the inflammasome, play a key role in ischemic brain injury by mediating the inflammatory component of the damage (J Neurosci, 35:4674, 2015; Nat Rev Immunol, 5:629, 2005). However, a link between post-ischemic CD36 activation and IL-1β has not been established. Microglial CD36 could contribute to ischemic brain injury by increasing the post-ischemic production of IL-1β. To test this hypothesis, male wild type (WT) and CD36 –/– mice were subjected to transient middle cerebral artery occlusion (tMCAO), and infarct volume was assessed in cresyl violet-stained brain sections 72 hrs later. At 18 hrs after tMCAO, the expression of IL-1β mRNA in microglia, isolated by cell sorting, increased equally in WT (3.4±0.9) and CD36 –/– mice (3.6±0.8, fold increase; p>0.05; N=3 pools, 3 mice/pool). However, the levels of mature IL-1β protein, measured by cytometric bead arrays in the ischemic hemisphere, were higher in WT mice (104±30 pg/g) than in CD36 –/– mice (59±14 pg/g; p<0.05 from WT; n=5/group). Since caspase-1 regulates active IL-1β levels by cleaving the mature form from its pro-peptide, we next analyzed microglial caspase-1 activity using a flow cytometry-based assay. The percentage of active caspase-1 was greater in WT (4.0±0.2%) than in CD36 –/– microglia (2±0.2 %; p= 0.001; n=5/group). Therefore, the reduction in infarct volume in CD36 –/– mice could be due to reduced caspase-1 and attendant IL1β signaling. Consistent with this hypothesis, pretreatment with recombinant IL-1β receptor antagonist (2 μg into the cerebral ventricles) decreased infarct volume in WT (47±5 vs. 32±5 mm 3 ; p<0.5; n=8-5), but not in CD36 –/– mice (23±6 vs. 21±5 mm 3 ; p> 0.5; n=5). We conclude that CD36 localized to microglia is a key determinant of post-ischemic IL-1β production by regulating caspase-1 activity. Although the CD36 ligand(s) and the molecular mechanisms linking CD36 to caspase-1 activation remain to be established, this study identifies microglial CD36 signaling as a previously unrecognized pathway for inflammasome activation and IL-1β production after cerebral ischemia, and a viable target to mitigate the damaging effects of post-ischemic inflammation.

Prevention of the Severity of Post-ischemic Inflammation and Brain Damage by Simultaneous Knockdown of Toll-like Receptors 2 and 4

Toll-like receptor 2 (TLR2) and TLR4 belong to a family of highly conserved pattern recognition receptors and are well-known upstream sensors of signaling pathways of innate immunity. TLR2 and TLR4 upregulation is thought to be associated with poor outcome in stroke patients. We currently show that transient focal ischemia in adult rats induces TLR2 and TLR4 expression within hours and shRNA-mediated knockdown of TLR2 and TLR4 alone and in combination decreases the infarct size and swelling. We further show that TLR2 and TLR4 knockdown also prevented the induction of their downstream signaling molecules MyD88, IRAK1, and NFκB p65 as well as the pro-inflammatory cytokines IL-1β, IL-6, and TNFα. This study thus shows that attenuation of the severity of TLR2- and TLR4-mediated post-stroke inflammation ameliorates ischemic brain damage.

IMMUNOPATHOGENETIC ASPECTS OF ISCHEMIC STROKE

Immune system plays a key role in pathogenesis of ischemic stroke. Ischemic brain injury causes destruction of cells and extracellular matrix proteins, and the release of „danger“ molecules that cause rapid activation of innate immunity, and induce adaptive autoimmune reactions. The cells of innate immunity are responsible for elimination of cellular and extracellular debris and tissue repair, being, however, involved in post-ischemic inflammation causing brain injury. An adaptive immune response which develops within a few days is directed against self-antigens, being not related to nervous tissue damage in acute phase of stroke. The cells of adaptive immunity are able to regulate microglial activity and promote repair processes in the central nervous system. However, long-term persistence of lymphocytes with reactivity for brain-derived antigens may be involved into the atrophy of cerebral cortex, and development of dementia in remote post-stroke period. Immune response following ischemic brain stroke is manifesting by local and systemic reactions. Nevertheless, in contrast to prolonged local intracerebral inflammation in infarction area, and peri-infarction region, a systemic inflammatory response is quickly replaced by development of immunosuppression, as evidenced by lymphopenia, lymphoid organ atrophy and defective functions of immune cells. Immunosuppression is largely due to activation of sympathoadrenal system, aiming for limitation of inflammatory and autoimmune reactions. However, excessive immunosuppression leads to development of infectious complications, which may often cause lethal outcomes, and also may have a negative impact on efficiency of repair processes. The present review provides experimental and clinical data describing the innate and adaptive immune reactions in response to cerebral ischemia. Moreover, this review discusses possible pathogenetic importance of immune cells and their positive or negative effects, thus allowing further development of new therapeutic approaches for treatment of ischemic stroke.

Brain-gut axis after stroke.

Stroke leads to inflammatory and immune response in the brain and immune organs. The gut or gastrointestinal tract is a major immune organ equipped with the largest pool of immune cells representing more than 70% of the entire immune system and the largest population of macrophages in the human body. The bidirectional communication between the brain and the gut is commonly known as brain–gut or gut–brain axis. Stroke often leads to gut dysmotility, gut microbiota dysbiosis, “leaky” gut, gut hemorrhage, and even gut-origin sepsis, which is often associated with poor prognosis. Emerging evidence suggests that gut inflammatory and immune response plays a key role in the pathophysiology of stroke and may become a key therapeutic target for its treatment. Ischemic brain tissue produces damage-associated molecular patterns to initiate innate and adaptive immune response both locally and systemically through the specialized pattern-recognition receptors (e.g., toll-like receptors). After stroke, innate immune cells including neutrophils, microglia or macrophages, mast cells, innate lymphocytes (IL-17 secreting γδ T-cell), and natural killer T-cell respond within hours, followed by the adaptive immune response through activation of T and B lymphocytes. Subpopulations of T-cells can help or worsen ischemic brain injury. Pro-inflammatory Th1, Th17, and γδ T-cells are often associated with increased inflammatory damage, whereas regulatory T-cells are known to suppress postischemic inflammation by increasing the secretion of anti-inflammatory cytokine IL-10. Although known to play a key role, research in the gut inflammatory and immune response after stroke is still in its initial stage. A better understanding of the gut inflammatory and immune response after stroke may be important for the development of effective stroke therapies. The present review will discuss recent advances in the studies of the brain–gut axis after stroke, the key issues to be solved, and the future directions.

Molecular and cellular mechanisms underlying the sterile inflammation after ischemic stroke

Inflammation is an essential step for the pathology of ischemic stroke, and is also an important therapeutic target for developing novel therapeutic methods which have a wide therapeutic time window. Since there is no pathogen in the brain, the inflammation will be triggered by some endogenous molecules which are called as danger associated molecular patterns (DAMPs). So far two important DAMPs, high mobility group box 1 (HMGB1) and peroxiredoxin (PRX), have been recently identified in the ischemic brain. HMGB1 exaggerates the disruption of blood brain barrier; on the other hand, PRX activates mononuclear phagocytes and induces the inflammatory cytokine production through the activation of Toll-like receptor 2 (TLR2) and TLR4. Various inflammatory molecules produced from infiltrating immune cells have been known to exacerbate the neurological deficits of ischemic stroke patients. Recently, it has been paid attention that the inflammation after tissue injury also induces tissue repair, while its mechanisms remain to be clarified. Novel therapeutic methods will be established by clarifying detailed molecular mechanisms underlying the induction of neural repair after cerebral post-ischemic inflammation.

The protective effect of 2-(2-benzonfu-ranyl)-2-imidazoline against oxygen-glucose deprivation in cultured rat cortical astrocytes

Astrocytes play a pivotal role in neuronal survival in the setting of post-ischemic brain inflammation, but the astrocyte-derived mediators of ischemic brain injury remain to be defined. 2-(2-Benzofu-ranyl)-2-imidazoline (2-BFI) is a newly discovered ligand for high-affinity imidazoline I2 receptors (I2Rs) mainly located on the mitochondrial outer membrane in astrocytes. We previously reported that in a rat model of cerebral ischemia-reperfusion injury, 2-BFI limits infarct volume, reduces neurological impairment scores, and inhibits neuronal apoptosis in the ischemic penumbra. This study was performed to clarify the underlying mechanism in an astrocyte oxygen-glucose deprivation (OGD) model. The results show that 2-BFI reduces lipid peroxidation and inhibits mitochondria apoptotic pathways.

Effect of Low-Dose Alcohol Consumption on Inflammation Following Transient Focal Cerebral Ischemia in Rats

Increasing evidence suggest that low-dose alcohol consumption (LAC) reduces the incidence and improves the functional outcome of ischemic stroke. We determined the influence of LAC on post-ischemic inflammation. Male Sprague-Dawley rats were divided into 3 groups, an ethanol (13.5% alcohol) group, a red wine (Castle Rock Pinot Noir, 13.5% alcohol) group, and a control group. The amount of alcohol given to red wine and ethanol groups was 1.4 g/kg/day. After 8 weeks, the animals were subjected to a 2-hour middle cerebral artery occlusion (MCAO) and sacrificed at 24 hours of reperfusion. Cerebral ischemia/reperfusion (I/R) injury, expression of adhesion molecules and pro- and anti-inflammatory cytokines/chemokines, microglial activation and neutrophil infiltration were evaluated. The total infarct volume and neurological deficits were significantly reduced in red wine- and ethanol-fed rats compared to control rats. Both red wine and ethanol suppressed post-ischemic expression of adhesion molecules and microglial activation. In addition, both red wine and ethanol upregulated expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), downregulated expression of proinflammatory cytokines/chemokines, and significantly alleviated post-ischemic expression of inflammatory mediators. Furthermore, red wine significantly reduced post-ischemic neutrophil infiltration. Our findings suggest that LAC may protect the brain against its I/R injury by suppressing post-ischemic inflammation.

Deletion of the P2X4 receptor is neuroprotective acutely, but induces a depressive phenotype during recovery from ischemic stroke

IntroductionAcute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. MethodsWe subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30days after occlusion) effects of receptor deletion. ResultsGlobal P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. ConclusionsP2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.

TREM2 protects against cerebral ischemia/reperfusion injury

Although post-ischemic inflammation induced by the innate immune response is considered an essential step in the progression of cerebral ischemia injury, the role of triggering receptor expressed on myeloid cells 2 (TREM2) in the pathogenesis of ischemic stroke remains to be elucidated. Here, we found that the transcriptional and post-transcriptional levels of TREM2 were increased in cultured primary microglia after oxygen-glucose deprivation and reoxygenation and in the ischemic penumbra of the cerebral cortex after middle cerebral artery occlusion (MCAO) and reperfusion in mice. TREM2 was mainly expressed in microglia, but not in astrocytes, neurons, or oligodendrocytes in mice subjected to MCAO. Manipulating TREM2 expression levels in vitro and in vivo significantly regulated the production of pro- and anti-inflammatory mediators after ischemic stroke. TREM2 overexpression markedly suppressed the inflammatory response and neuronal apoptosis. By contrast, TREM2 gene silencing intensified the inflammatory response, increased neuronal apoptosis and infarct volume, and further exacerbated neurological dysfunction. Our study demonstrated that TREM2 protects against cerebral ischemia/reperfusion injury through the aspect of post-ischemic inflammatory response and neuronal apoptosis. Pharmacological targeting of TREM2 to suppress the inflammatory response may provide a new approach for developing therapeutic strategies in the treatment of ischemic stroke and other cerebrovascular diseases.

Splenic Ly6Chi monocytes contribute to adverse late post-ischemic left ventricular remodeling in heme oxygenase-1 deficient mice

Heme oxygenase-1 (Hmox1) is a stress-inducible protein crucial in heme catabolism. The end products of its enzymatic activity possess anti-oxidative, anti-apoptotic and anti-inflammatory properties. Cardioprotective effects of Hmox1 were demonstrated in experimental models of myocardial infarction (MI). Nevertheless, its importance in timely resolution of post-ischemic inflammation remains incompletely understood. The aim of this study was to determine the role of Hmox1 in the monocyte/macrophage-mediated cardiac remodeling in a mouse model of MI. Hmox1 knockout (Hmox1−/−) and wild-type (WT, Hmox1+/+) mice were subjected to a permanent ligation of the left anterior descending coronary artery. Significantly lower incidence of left ventricle (LV) free wall rupture was noted between 3rd and 5th day after MI in Hmox1−/− mice resulting in their better overall survival. Then, starting from 7th until 21st day post-MI a more potent deterioration of LV function was observed in Hmox1−/− than in the surviving Hmox1+/+ mice. This was accompanied by higher numbers of Ly6Chi monocytes in peripheral blood, as well as higher expression of monocyte chemoattractant protein-1 and adhesion molecules in the hearts of MI-operated Hmox1−/− mice. Consequently, a greater post-MI monocyte-derived myocardial macrophage infiltration was noted in Hmox1-deficient individuals. Splenectomy decreased the numbers of circulating inflammatory Ly6Chi monocytes in blood, reduced the numbers of proinflammatory cardiac macrophages and significantly improved the post-MI LV function in Hmox1−/− mice. In conclusion, Hmox1 deficiency has divergent consequences in MI. On the one hand, it improves early post-MI survival by decreasing the occurrence of cardiac rupture. Afterwards, however, the hearts of Hmox1-deficient mice undergo adverse late LV remodeling due to overactive and prolonged post-ischemic inflammatory response. We identified spleen as an important source of these cardiovascular complications in Hmox1−/− mice.

A single-chain antibody-CD39 fusion protein targeting activated platelets protects from cardiac ischaemia/reperfusion injury.

CD39 is a cell membrane NTPase with anti-inflammatory and anti-platelet effects. However, its clinical use is limited by its bleeding side effect. With the goal of harnessing its therapeutic potential while avoiding haemostatic problems, we designed a fusion protein consisting of the extracellular domain of CD39 and a single-chain antibody (Targ-CD39) that specifically binds to activated glycoprotein (GP)IIb/IIIa and thus to activated platelets. Through this enrichment at activated platelets, the required systemic dose is below the dose impairing haemostasis. Using an ischaemia/reperfusion mouse model (left anterior descending artery ligated for 1 h) we achieved remarkable protection of the reperfused tissue with Targ-CD39 compared with Non-targ-CD39 (mutated, non-binding version of Targ-CD39) and PBS control. Targ-CD39 restored ejection fraction and fractional shortening to a level indistinguishable from pre-injury status, while controls showed functional deterioration. Employing advanced clinically relevant methods of ultrasound analysis, we observed that both radial and longitudinal strain and strain rate showed infarct-typical changes of myocardial deformation in controls, but not in Targ-CD39 treated mice. Histological assessment confirmed strong reduction of infarct size and increase in neovascularization. Furthermore, attenuation of post-ischaemic inflammation was seen in cytokine profiling. Overall, we demonstrate that Targ-CD39 holds promise for treatment of myocardial infarction.

Hydrogen sulfide improves intestinal recovery following ischemia by endothelial nitric oxide-dependent mechanisms.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that has vasodilatory properties. It may be a novel therapy for intestinal ischemia-reperfusion (I/R) injury. We hypothesized that 1) H2S would improve postischemic survival, mesenteric perfusion, mucosal injury, and inflammation compared with vehicle and 2) the benefits of H2S would be mediated through endothelial nitric oxide. C57BL/6J wild-type and endothelial nitric oxide synthase knockout (eNOS KO) mice were anesthetized, and a midline laparotomy was performed. Intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using laser Doppler. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery. Following ischemia, the clamp was removed, and the intestines were allowed to recover. Either sodium hydrosulfide (2 nmol/kg or 2 µmol/kg NaHS) in PBS vehicle or vehicle only was injected into the peritoneum. Animals were allowed to recover and were assessed for mesenteric perfusion, mucosal injury, and intestinal cytokines. P values < 0.05 were significant. H2S improved mesenteric perfusion and mucosal injury scores following I/R injury. However, in the setting of eNOS ablation, there was no improvement in these parameters with H2S therapy. Application of H2S also resulted in lower levels of intestinal cytokine production following I/R. Intraperitoneal H2S therapy can improve mesenteric perfusion, intestinal mucosal injury, and intestinal inflammation following I/R. The benefits of H2S appear to be mediated through endothelial nitric oxide-dependent pathways.NEW & NOTEWORTHY H2S is a gaseous mediator that acts as an anti-inflammatory agent contributing to gastrointestinal mucosal defense. It promotes vascular dilation, mucosal repair, and resolution of inflammation following intestinal ischemia and may be exploited as a novel therapeutic agent. It is unclear whether H2S works through nitric oxide-dependent pathways in the intestine. We appreciate that H2S was able to improve postischemic recovery of mesenteric perfusion, mucosal integrity, and inflammation. The beneficial effects of H2S appear to be mediated through endothelial nitric oxide-dependent pathways.

Interleukin 15 activates Akt to protect astrocytes from oxygen glucose deprivation-induced cell death

Astrocytes play a pivotal role in neuronal survival under the condition of post-ischemic brain inflammation, but the relevant astrocyte-derived mediators of ischemic brain injury remain to be defined. IL-15 supports survival of multiple lymphocyte lineages in the peripheral immune system, but the role of IL-15 in inflammatory disease of the central nervous system is not well defined. Recent research has shown an increase of IL-15-expressing astrocytes in the ischemic brain. Since astrocytes promote neuron survival under cerebral ischemia by buffering excess extracellular glutamate and producing growth factors, recovery of astrocyte function could be of benefit for stroke therapy. Here, we report that IL-15 is the pro-survival cytokine that prevents astrocyte death from oxygen glucose deprivation (OGD)-induced damage. Astrocytes up-regulate expression of the IL-15/IL-15Rα complex under OGD, whereas OGD down-regulates the levels of pSTAT5 and pAkt in astrocytes. IL-15 treatment ameliorates the decline of pAkt, decreases the percentage of annexin V+ cells, inhibits the activation of caspase-3, and activates the Akt pathway to promote astrocyte survival in response to OGD. We further identified that activation of Akt, but not PKCα/βI, is essential for astrocyte survival under OGD. Taken together, this study reveals the function of IL-15 in astrocyte survival via Akt phosphorylation in response to OGD-induced damage.

Glutamine metabolism drives succinate accumulation in plasma and the lung during hemorrhagic shock.

Metabolomic investigations have consistently reported succinate accumulation in plasma after critical injury. Succinate receptors have been identified on numerous tissues, and succinate has been directly implicated in postischemic inflammation, organ dysfunction, platelet activation, and the generation of reactive oxygen species, which may potentiate morbidity and mortality risk to patients. Metabolic flux (heavy-isotope labeling) studies demonstrate that glycolysis is not the primary source of increased plasma succinate during protracted shock. Glutamine is an alternative parent substrate for ATP generation during anaerobic conditions, a biochemical mechanism that ultimately supports cellular survival but produces succinate as a catabolite. We hypothesize that succinate accumulation during hemorrhagic shock is driven by glutaminolysis. Sprague-Dawley rats were subjected to hemorrhagic shock for 45 minutes (shock, n = 8) and compared with normotensive shams (sham, n = 8). At 15 minutes, animals received intravenous injection of C5-N2-glutamine solution (iLG). Blood, brain, heart, lung, and liver tissues were harvested at defined time points. Labeling distribution in samples was determined by ultrahigh-pressure liquid chromatography-mass spectrometry metabolomic analysis. Repeated-measures analysis of variance with Tukey comparison determined significance of relative fold change in metabolite level from baseline. Hemorrhagic shock instigated succinate accumulation in plasma and lungs tissues (8.5- vs. 1.1-fold increase plasma succinate level from baseline, shock vs. sham, p = 0.001; 3.2-fold higher succinate level in lung tissue, shock vs. sham, p = 0.006). Metabolomic analysis identified labeled glutamine and labeled succinate in plasma (p = 0.002) and lung tissue (p = 0.013), confirming glutamine as the parent substrate. Kinetic analyses in shams showed constant total levels of all metabolites without significant change due to iLG. Glutamine metabolism contributes to increased succinate concentration in plasma during hemorrhagic shock. The glutaminolytic pathway is implicated as a therapeutic target to prevent the contribution of succinate accumulation in plasma and the lung-to-postshock pathogenesis.

Mannan binding lectin-associated serine protease-2 (MASP-2) critically contributes to post-ischemic brain injury independent of MASP-1.

BackgroundComplement activation via the lectin activation pathway (LP) has been identified as the key mechanism behind post-ischemic tissue inflammation causing ischemia-reperfusion injury (IRI) which can significantly impact the clinical outcome of ischemic disease. This work defines the contributions of each of the three LP-associated enzymes—mannan-binding lectin-associated serine protease (MASP)-1, MASP-2, and MASP-3—to ischemic brain injury in experimental mouse models of stroke.MethodsFocal cerebral ischemia was induced in wild-type (WT) mice or mice deficient for defined complement components by transient middle cerebral artery occlusion (tMCAO) or three-vessel occlusion (3VO). The inhibitory MASP-2 antibody was administered systemically 7 and 3.5 days before and at reperfusion in WT mice in order to assure an effective MASP-2 inhibition throughout the study. Forty-eight hours after ischemia, neurological deficits and infarct volumes were assessed. C3 deposition and microglia/macrophage morphology were detected by immunohistochemical, immunofluorescence, and confocal analyses.ResultsMASP-2-deficient mice (MASP-2−/−) and WT mice treated with an antibody that blocks MASP-2 activity had significantly reduced neurological deficits and histopathological damage after transient ischemia and reperfusion compared to WT or control-treated mice. Surprisingly, MASP-1/3−/− mice were not protected, while mice deficient in factor B (fB−/−) showed reduced neurological deficits compared to WT mice. Consistent with behavioral and histological data, MASP-2−/− had attenuated C3 deposition and presented with a significantly higher proportion of ramified, surveying microglia in contrast to the hypertrophic pro-inflammatory microglia/macrophage phenotype seen in the ischemic brain tissue of WT mice.ConclusionsThis work demonstrates the essential role of the low-abundant MASP-2 in the mediation of cerebral ischemia-reperfusion injury and demonstrates that targeting MASP-2 by an inhibitory therapeutic antibody markedly improved the neurological and histopathological outcome after focal cerebral ischemia. These results contribute to identifying the key lectin pathway component driving brain tissue injury following cerebral ischemia and call for a revision of the presently widely accepted view that MASP-1 is an essential activator of the lectin pathway effector component MASP-2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0684-6) contains supplementary material, which is available to authorized users.

Role of microglia in ischemic focal stroke and recovery: focus on Toll-like receptors

Stroke is the leading cause of disability in adults. Drug treatments that target stroke-induced pathological mechanisms and promote recovery are desperately needed. In the brain, an ischemic event triggers major inflammatory responses that are mediated by the resident microglial cells. In this review, we focus on the microglia activation after ischemic brain injury as a target of immunomodulatory therapeutics. We divide the microglia-mediated events following ischemic stroke into three categories: acute, subacute, and long-term events. This division encompasses the spatial and temporal dynamics of microglia as they participate in the pathophysiological changes that contribute to the symptoms and sequela of a stroke. The importance of Toll-like receptor (TLR) signaling in the outcomes of these pathophysiological changes is highlighted. Increasing evidence shows that microglia have a complex role in stroke pathophysiology, and they mediate both detrimental and beneficial effects on stroke outcome. So far, most of the pharmacological studies in experimental models of stroke have focused on neuroprotective strategies which are impractical for clinical applications. Post-ischemic inflammation is long lasting and thus, could provide a therapeutic target for novel delayed drug treatment. However, more studies are needed to elucidate the role of microglia in the recovery process from an ischemic stroke and to evaluate the therapeutic potential of modulating post-ischemic inflammation to promote functional recovery.

The Anti-inflammatory Effects of Agmatine on Transient Focal Cerebral Ischemia in Diabetic Rats.

In the previous study, we observed agmatine (AGM) posttreatment immediately after 30 minutes of suture occlusion of the middle cerebral artery (MCAO) reduced the infarct size and neurological deficit in diabetic rats. The aim of the present study was to investigate the anti-inflammatory effect of AGM to reduce cerebral ischemic damage in diabetic rats. Normoglycemic (n=20) and streptozotocin-induced diabetic rats (n=40) were subjected to 30 minutes of MCAO followed by reperfusion. Twenty diabetic rats were treated with AGM (100 mg/kg, intraperitoneal) immediately after 30 minutes of MCAO. Modified neurological examinations and rotarod exercises were performed to evaluate motor function. Western blot and immunohistochemical analysis were performed to determine the expression of inflammatory cytokines in ischemic brain tissue. Real-time polymerase chain reaction was performed to measure the mRNA expression of high-mobility group box 1, receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, and TLR4 RESULTS AND CONCLUSIONS:: AGM posttreatment improved the neurobehavioral activity and motor function of diabetic MCAO rats at 24 and 72 hours after reperfusion. Immunohistochemical analysis showed that AGM treatment significantly decreased the expression of inflammatory cytokines in diabetic MCAO rats at 24 and 72 hours after reperfusion (P<0.01). Western blotting and real-time polymerase chain reaction results indicated that AGM treatment significantly decreased the expression of high-mobility group box 1, RAGE, TLR2, and TLR4 in diabetic rats at 24 hours after reperfusion (P<0.05). This neuroprotective effect of AGM after MCAO was associated with modulation of the postischemic neuronal inflammation cascade.

Donepezil, an acetylcholinesterase inhibitor, attenuates LPS-induced inflammatory response in murine macrophage cell line RAW 264.7 through inhibition of nuclear factor kappa B translocation

We have previously demonstrated that the pharmacotherapy with donepezil, an acetylcholinesterase inhibitor, suppresses cardiac remodeling in a mouse model of ischemic heart failure after myocardial infarction (MI). However, the precise mechanisms of the cardioprotective effect of donepezil have not been completely delineated. Because post-ischemic inflammation is a pathological key event in the cardiac remodeling process following MI, we investigated the hypothesis that donepezil acts as an inhibitor of inflammatory mediators. RAW 264.7 murine macrophage cells were pretreated with donepezil (100µM) prior to a pro-inflammatory stimulation by administration of lipopolysaccharide (LPS, 10ng/ml). Donepezil significantly reduced intra- and extracellular levels of various kinds of inflammatory mediators such as TNF-α, IL-1β, IL-2, IL-6 and IL-18 after the LPS stimulation, and attenuated LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB). These results indicate that donepezil possesses an anti-inflammatory property. However, the inhibitory effect of donepezil on the macrophage inflammatory responses was never reproduced by ACh, nor was disrupted by ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to inhibit the inflammatory responses in LPS-stimulated macrophage cells. These results suggest that a cholinergic anti-inflammatory pathway would not be involved in the anti-inflammatory effect of donepezil and that the specific characteristics of donepezil in suppressing the LPS-induced cytokine release and the NF-κB activation would be independent of its acetylcholinesterase inhibition. The present study showed that donepezil exerts an anti-inflammatory effect independently of acetylcholinesterase inhibitory action, thereby donepezil may contribute to cardioprotection during cardiac remodeling process in an ischemic heart failure after MI.