Neutralizing anti-interleukin-1? antibodies modulate fetal blood-brain barrier function after ischemia.
ABSTRACT: We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1? monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1? protein. This antibody also neutralizes the effects of interleukin-1? protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1? monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1? antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1? monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1? protein and anti-interleukin-1? monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with ?-aminoisobutyric acid in multiple brain regions. Interleukin-1? protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1? protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1? monoclonal antibody infusions, plasma anti-interleukin-1? monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1? monoclonal antibody levels were higher (P<0.03), and interleukin-1? protein concentrations (P<0.03) and protein expressions (P<0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (P<0.04), and Ki showed an inverse linear correlation (r= -0.65, P<0.02) with anti-interleukin-1? monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1? monoclonal antibody infusions after ischemia result in brain anti-interleukin-1? antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1? protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1?, contributes to impaired blood-brain barrier function after ischemia in the fetus.
Project description:Ischemic injury can be alleviated by the judicious use of hypothermia. However, the optimal regimens and the temporal kinetics of post-stroke neurovascular responses to hypothermic intervention have not been systematically studied. These gaps slow the clinical translation of hypothermia as an anti-stroke therapy. Here, we characterized the effects of transient selective brain hypothermia (TSBH) from the hyperacute to chronic stages of focal ischemia/reperfusion brain injury induced by transient middle cerebral artery occlusion in mice. A simple cooling device was used to induce TSBH during cerebral ischemia. This treatment reduced mortality from 31.8% to 0% and improved neurological outcomes for at least 35 days post-injury. TSBH mitigated blood-brain barrier leakage during the hyperacute and acute injury stages (1-23?h post-reperfusion). This early protection of the blood-brain barrier was associated with anti-inflammatory phenotypic polarization of microglia/macrophages, reduced production of pro-inflammatory cytokines, and less brain infiltration of neutrophils and macrophages during the subacute injury stage (three days post-reperfusion). TSBH elicited enduring protective effects on both grey and white matter for at least 35 days post-injury and preserved the long-term electrophysiological function of fiber tracts. In conclusion, TSBH ameliorates ischemia/reperfusion injury in the neurovascular unit from hyperacute to chronic injury stages after experimental stroke.
Project description:Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the ?3 and ?4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.
Project description:Integrity of the blood-brain barrier structure is essential for maintaining the internal environment of the brain. Development of cerebral infarction and brain edema is strongly associated with blood-brain barrier leakage. Therefore, studies have suggested that protecting the blood-brain barrier may be an effective method for treating acute stroke. To examine this possibility, stroke model rats were established by middle cerebral artery occlusion and reperfusion. Remote ischemic postconditioning was immediately induced by three cycles of 10-minute ischemia/10-minute reperfusion of bilateral hind limbs at the beginning of middle cerebral artery occlusion reperfusion. Neurological function of rat models was evaluated using Zea Longa's method. Permeability of the blood-brain barrier was assessed by Evans blue leakage. Infarct volume and brain edema were evaluated using 2,3,5-triphenyltetrazolium chloride staining. Expression of matrix metalloproteinase-9 and claudin-5 mRNA was determined by real-time quantitative reverse transcription-polymerase chain reaction. Expression of matrix metalloproteinase-9 and claudin-5 protein was measured by western blot assay. The number of matrix metalloproteinase-9- and claudin-5-positive cells was analyzed using immunohistochemistry. Our results showed that remote ischemic postconditioning alleviated disruption of the blood-brain barrier, reduced infarct volume and edema, decreased expression of matrix metalloproteinase-9 mRNA and protein and the number of positive cells, increased expression of claudin-5 mRNA and protein and the number of positive cells, and remarkably improved neurological function. These findings confirm that by suppressing expression of matrix metalloproteinase-9 and claudin-5 induced by acute ischemia/reperfusion, remote ischemic postconditioning reduces blood-brain barrier injury, mitigates ischemic injury, and exerts protective effects on the brain.
Project description:Ischemic stroke accounts for nearly 80% of stroke cases. Recanalization with thrombolysis is a currently crucial therapeutic strategy for re-building blood supply, but the thrombolytic therapy often companies with cerebral ischemia-reperfusion injury, which are mediated by free radicals. As an important component of free radicals, reactive nitrogen species (RNS), including nitric oxide (NO) and peroxynitrite (ONOO(-)), play important roles in the process of cerebral ischemia-reperfusion injury. Ischemia-reperfusion results in the production of nitric oxide (NO) and peroxynitrite (ONOO(-)) in ischemic brain, which trigger numerous molecular cascades and lead to disruption of the blood brain barrier and exacerbate brain damage. There are few therapeutic strategies available for saving ischemic brains and preventing the subsequent brain damage. Recent evidence suggests that RNS could be a therapeutic target for the treatment of cerebral ischemia-reperfusion injury. Herein, we reviewed the recent progress regarding the roles of RNS in the process of cerebral ischemic-reperfusion injury and discussed the potentials of drug development that target NO and ONOO(-) to treat ischemic stroke. We conclude that modulation for RNS level could be an important therapeutic strategy for preventing cerebral ischemia-reperfusion injury.
Project description:Phosphoinositide 3-kinase (PI3K)-? is linked to inflammation and oxidative stress. This study was conducted to investigate the role of the PI3K? in the blood-brain barrier dysfunction and brain damage induced by focal cerebral ischemia/reperfusion.Wild-type and PI3K? knockout mice were subjected to middle cerebral artery occlusion (60 minutes) followed by reperfusion. Evans blue leakage, brain edema, infarct volumes, and neurological deficits were examined. Oxidative stress, neutrophil infiltration, and matrix metallopeptidase-9 were assessed. Activation of nuclear factor-?B and expression of proinflammatory and pro-oxidative genes were studied.PI3K? deficiency significantly reduced blood-brain barrier permeability and brain edema formation, which were time-dependently correlated with preventing the degradation of the tight junction protein, claudin-5, and the basal lamina protein, collagen IV, and the phosphorylation of myosin light chain in brain microvessels. PI3K? deficiency suppressed ischemia/reperfusion-induced nuclear factor-?B p65 (Ser536) phosphorylation and the expression of the pro-oxidant enzyme NADPH oxidase (Nox1, Nox2, and Nox4) and proinflammatory adhesion molecules (E- and P-selectin, intercellular adhesion molecule-1) at different time points. These molecular changes were associated with significant inhibition of oxidative stress (superoxide production and malondialdehyde content), neutrophil infiltration, and matrix metallopeptidase-9 expression/activity in PI3K? knockout mice. Eventually, PI3K? deficiency significantly reduced infarct volumes and neurological scores at 24 hours after ischemia/reperfusion.Our results provide the first direct demonstration that PI3K? plays a significant role in ischemia/reperfusion-induced blood-brain barrier disruption and brain damage. Future studies need to explore PI3K? as a potential target for stroke therapy.
Project description:Background Systemic innate immune priming is a recognized sequela of post-ischemic neuroinflammation and contributor to delayed neurodegeneration. Given mounting evidence linking acute stroke with reactive lung inflammation, we asked whether enhanced expression of the endogenous antioxidant extracellular superoxide dismutase 3 (SOD3) produced by alveolar type II pneumocytes would protect the lung from transient global cerebral ischemia and the brain from the delayed effects of ischemia-reperfusion. Methods and Results Following 15 minutes of global cerebral ischemia or sham conditions, transgenic SOD3 and wild-type mice were followed daily for changes in weight, core temperature, and neurological function. Three days after reperfusion, arterial and venous samples were collected for complete blood counts, flow cytometry, and SOD3 protein blotting, and immunohistochemistry was performed on lung and brain tissue to assess tissue injury, blood-brain barrier permeability, and neutrophil transmigration. Relative to ischemic controls, transgenic SOD3 mice performed better on functional testing and exhibited reduced peripheral neutrophil activation, lung inflammation, and blood-brain barrier leak. Once released from the lung, SOD3 was predominantly not cell associated and depleted in the venous phase of circulation. Conclusions In addition to reducing the local inflammatory response to cerebral ischemia, targeted enrichment of SOD3 within the lung confers distal neuroprotection against ischemia-reperfusion injury. These data suggest that therapies geared toward enhancing adaptive lung-neurovascular coupling may improve outcomes following acute stroke and cardiac arrest.
Project description:Inflammation and proteolysis crucially contribute to myocardial ischemia and reperfusion injury. The extracellular matrix metalloproteinase inducer EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) may be involved in both processes. The aim of the study was to characterize the role of the CD147 and CyPA interplay in myocardial ischemia/reperfusion (I/R) injury.Immunohistochemistry showed enhanced expression of CD147 and CyPA in myocardial sections from human autopsies of patients who had died from acute myocardial infarction and from mice at 24 hours after I/R. At 24 hours and 7 days after I/R, the infarct size was reduced in CD147(+/-) mice vs CD147(+/+) mice (C57Bl/6), in mice (C57Bl/6) treated with monoclonal antibody anti-CD147 vs control monoclonal antibody, and in CyPA(-/-) mice vs CyPA(+/+) mice (129S6/SvEv), all of which are associated with reduced monocyte and neutrophil recruitment at 24 hours and with a preserved systolic function at 7 days. The combination of CyPA(-/-) mice with anti-CD147 treatment did not yield further protection compared with either inhibition strategy alone. In vitro, treatment with CyPA induced monocyte chemotaxis in a CD147- and phosphatidylinositol 3-kinase-dependent manner and induced monocyte rolling and adhesion to endothelium (human umbilical vein endothelial cells) under flow in a CD147-dependent manner.CD147 and its ligand CyPA are inflammatory mediators after myocardial ischemia and reperfusion and represent potential targets to prevent myocardial I/R injury.
Project description:Risk of intracerebral hemorrhage is the primary factor limiting use of tissue plasminogen activator (tPA) for stroke. Clinical studies have established an association between admission hyperglycemia and the risk of hemorrhage with tPA use, independent of prior diabetes. Here we used an animal model of tPA-induced reperfusion hemorrhage to determine if this clinical association reflects a true causal relationship.Rats underwent 90 minutes of focal ischemia, and tPA infusion was begun 10 minutes prior to vessel reperfusion. Glucose was administered during ischemia to generate blood levels ranging from 5.9 ± 1.8mM (normoglycemia) to 21 ± 2.3mM. In some studies, apocynin was administered to block superoxide production by nicotinamide adenine dinucleotide phosphate (NADPH). Brains were harvested 1 hour or 3 days after reperfusion to evaluate the effects of hyperglycemia and apocynin on oxidative stress, blood-brain barrier breakdown, infarct volume, and hemorrhage volume.Rats that were hyperglycemic during tPA infusion had diffusely increased blood-brain barrier permeability in the postischemic territory, and a 3- to 5-fold increase in intracerebral hemorrhage volumes. The hyperglycemic rats also showed increased superoxide formation in the brain parenchyma and vasculature during reperfusion. The effects of hyperglycemia on superoxide production, blood-brain barrier disruption, infarct size, and hemorrhage were all attenuated by apocynin.These findings demonstrate a causal relationship between hyperglycemia and hemorrhage in an animal model of tPA stroke treatment, and suggest that this effect of hyperglycemia is mediated through an increase in superoxide production by NADPH oxidase.
Project description:Blood-brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring.Here, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1?-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-?B in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation.Taken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke.
Project description:BACKGROUND:Platelet activation at sites of vascular injury is essential for hemostasis, but it is also a major pathomechanism underlying ischemic injury. Because anti-inflammatory therapies limit thrombosis and antithrombotic therapies reduce vascular inflammation, we tested the therapeutic potential of 2 proresolving endogenous mediators, annexin A1 N-terminal derived peptide (AnxA1Ac2-26) and aspirin-triggered lipoxin A4 (15-epi-lipoxin A4), on the cerebral microcirculation after ischemia/reperfusion injury. Furthermore, we tested whether the lipoxin A4 receptor formyl-peptide receptor 2/3 (Fpr2/3; ortholog to human FPR2/lipoxin A4 receptor) evoked neuroprotective functions after cerebral ischemia/reperfusion injury. METHODS AND RESULTS:Using intravital microscopy, we found that cerebral ischemia/reperfusion injury was accompanied by neutrophil and platelet activation and neutrophil-platelet aggregate formation within cerebral microvessels. Moreover, aspirin-triggered lipoxin A4 activation of neutrophil Fpr2/3 regulated neutrophil-platelet aggregate formation in the brain and inhibited the reactivity of the cerebral microvasculature. The same results were obtained with AnxA1Ac2-26 administration. Blocking Fpr2/lipoxin A4 receptor with the antagonist Boc2 reversed this effect, and treatments were ineffective in Fpr2/3 knockout mice, which displayed an exacerbated disease severity, evidenced by increased infarct area, blood-brain barrier dysfunction, increased neurological score, and elevated levels of cytokines. Furthermore, aspirin treatment significantly reduced cerebral leukocyte recruitment and increased endogenous levels of aspirin-triggered lipoxin A4, effects again mediated by Fpr2/3. CONCLUSION:Fpr2/lipoxin A4 receptor is a therapeutic target for initiating endogenous proresolving, anti-inflammatory pathways after cerebral ischemia/reperfusion injury.