FTY720 Protects Against Ischemia-Reperfusion Injury by Preventing the Redistribution of Tight Junction Proteins and Decreases Inflammation in the Subacute Phase in an Experimental Stroke Model.
ABSTRACT: Injury due to brain ischemia followed by reperfusion (I/R) may be an important therapeutic target in the era of thrombectomy. FTY720, a widely known sphingosine-1-phosphate receptor agonist, exerts various neuroprotective effects. The aim of this study was to examine the protective effect of FTY720 with respect to I/R injury, especially focusing on blood-brain barrier (BBB) protection and anti-inflammatory effects. Male rats were subjected to transient ischemia and administered vehicle or 0.5 or 1.5 mg/kg of FTY720 immediately before reperfusion. Positron emission tomography (PET) with [18F]DPA-714 was performed 2 and 9 days after the insult to serially monitor neuroinflammation. Bovine and rat brain microvascular endothelial cells (MVECs) were also subjected to oxygen-glucose deprivation (OGD) and reperfusion, and administered FTY720, phosphorylated-FTY720 (FTY720-P), or their inhibitor. FTY720 dose-dependently reduced cell death, the infarct size, cell death including apoptosis, and inflammation. It also ameliorated BBB disruption and neurological deficits compared to in the vehicle group. PET indicated that FTY720 significantly inhibited the worsening of inflammation in later stages. FTY720-P significantly prevented the intracellular redistribution of tight junction proteins but did not increase their mRNA expression. These results suggest that FTY720 can ameliorate I/R injury by protecting the BBB and regulating neuroinflammation.
Project description:Neuroinflammation mediated by activation of microglia and interruption of the blood-brain barrier (BBB) is an important factor that contributes to neuron death and infarct area diffusion in ischemia reperfusion injury. Finding novel molecules to regulate neuroinflammation is of significant clinical value. We have previously shown that adjudin, a small molecule compound known to possess antispermatogenic function, attenuates microglia activation by suppression of the NF-?B pathway. In this study we continued to explore whether adjudin could be neuroprotective by using the transient middle cerebral artery occlusion (tMCAO) model. Adjudin treatment after reperfusion significantly decreased the infarction volume and neuroscore compared to the vehicle group. Staining of CD11b showed that adjudin markedly inhibited microglial activation in both the cortex and the striatum, accompanied by a reduction in the expression and release of cytokines TNF-?, IL-1? and IL-6. Concomitantly, adjudin noticeably prevented BBB disruption after ischemia and reperfusion, as indicated by the reduction of IgG detection in the brain cortex and striatum versus the vehicle group. This finding was also corroborated by immunofluorescence staining and immunoblotting of tight junction-related proteins ZO-1, JAM-A and Occludin, where the reduction of these proteins could be attenuated by adjudin treatment. Moreover, adjudin obviously inhibited the elevated MMP-9 activity after stroke. Together these data demonstrate that adjudin protects against cerebral ischemia reperfusion injury, and we present an effective neuroinflammation modulator with clinical potential.
Project description:<h4>Background and purpose</h4>Inflammatory reaction plays a crucial role in cerebral ischemia reperfusion (IR) injury. It has been shown that activated microglia long-term existed in cerebral ischemia and induced second injury. Therefore, we hypothesize that prepared phosphatidylserine (PS)-modified microbubbles (PS-MBs) combined with ultrasound-targeted microbubble destruction (UTMD) can safely open the blood-brain barrier (BBB) and target activated microglia for inflammatory area in the later stage of ischemia reperfusion.<h4>Methods</h4>To verify our hypothesis, rat model of IR was established, then the change of activated microglia/macrophage (M/M) and permeability of BBB at 1, 7, 14, and 21?days could be clearly observed post IR. And the activated M/M still can be observed during the whole experiment.<h4>Results</h4>The Evans blue extravasation of BBB gradually declined from day 1 to day 21. Compared to the control group, microbubbles containing PS were taken up more by activated M/M (approximately twofold) both in vitro and in vivo.<h4>Conclusions</h4>PS-MBs combined with ultrasound (US) exposure could safely open BBB, and the resulting PS nanoparticles (PS-NPs) could further target activated M/M in the neuroinflammation.
Project description:Lipocalin-2 (LCN2) is a secreted protein of the lipocalin family, but little is known about the expression or the role of LCN2 in the central nervous system. Here, we investigated the role of LCN2 in ischemic stroke using a rodent model of transient cerebral ischemia. Lipocalin-2 expression was highly induced in the ischemic brain and peaked at 24?hours after reperfusion. After transient middle cerebral artery occlusion, LCN2 was predominantly expressed in astrocytes and endothelial cells, whereas its receptor (24p3R) was mainly detected in neurons, astrocytes, and endothelial cells. Brain infarct volumes, neurologic scores, blood-brain barrier (BBB) permeabilities, glial activation, and inflammatory mediator expression were significantly lower in LCN2-deficient mice than in wild-type animals. Lipocalin-2 deficiency also attenuated glial neurotoxicity in astrocyte/neuron cocultures after oxygen-glucose deprivation. Our results indicate LCN2 has a critical role in brain injury after ischemia/reperfusion, and that LCN2 may contribute to neuronal cell death in the ischemic brain by promoting neurotoxic glial activation, neuroinflammation, and BBB disruption.
Project description:<b>Background:</b> The dysfunctional blood-brain barrier (BBB)-glymphatic system is responsible for triggering intracerebral amyloid-beta peptide (Aβ) accumulation and acts as the key link between ischemic stroke and dementia dominated by Alzheimer's disease (AD). Recently, pyroptosis in cerebral ischemia and reperfusion (I/R) injury is demonstrated as a considerable mechanism causing BBB-glymphatic dysfunctions and Aβ acute accumulation in the brain. Targeting glial pyroptosis to protect BBB-glymphatic functions after cerebral I/R could offer a new viewpoint to prevent Aβ accumulation and poststroke dementia. Yi-Zhi-Fang-Dai formula (YZFDF) is an herbal prescription used to cure dementia with multiple effects of regulating inflammatory responses and protecting the BBB against toxic Aβ-induced damage. Hence, YZFDF potentially possesses neuroprotective effects against cerebral I/R injury and the early pathology of poststroke dementia, which evokes our current study. <b>Objectives:</b> The present study was designed to confirm the potential efficacy of YZFDF against cerebral I/R injury and explore the possible mechanism associated with alleviating Aβ acute accumulation. <b>Methods:</b> The models of cerebral I/R injury in rats were built by the method of middle cerebral artery occlusion/reperfusion (MCAO/R). First, neurological function assessment and cerebral infarct measurement were used for confirming the efficacy of YZFDF on cerebral I/R injury, and the optimal dosage (YZFDF-H) was selected to conduct the experiments, which included Western blotting detections of pyroptosis, Aβ<sub>1-42</sub> oligomers, and NeuN, immunofluorescence observations of glial pyroptosis, aquaporin-4 (AQP-4), and Aβ locations, brain water content measurement, SMI 71 (a specific marker for BBB)/AQP-4 immunohistochemistry, and Nissl staining to further evaluate BBB-glymphatic functions and neuronal damage. <b>Results:</b> YZFDF obviously alleviated neurological deficits and cerebral infarct after cerebral I/R in rats. Furthermore, YZFDF could inactivate pyroptosis signaling via inhibiting caspase-1/11 activation and gasdermin D cleavage, ameliorate glial pyroptosis and neuroinflammation, protect against BBB collapse and AQP-4 depolarization, prevent Aβ acute accumulation and Aβ<sub>1-42</sub> oligomers formation, and reduce neuronal damage and increase neurons survival after reperfusion. <b>Conclusion:</b> Our study indicated that YZFDF could exert neuroprotective effects on cerebral I/R injury and prevent Aβ acute accumulation in the brain after cerebral I/R associated with inhibiting neuroinflammation-related pyroptosis and BBB-glymphatic dysfunctions.
Project description:Acute ischemia-reperfusion injury (IRI) of the extremities leads to local and systemic inflammatory changes which can hinder limb function and can be life threatening. This study examined whether the administration of the T-cell sequestration agent, FTY720, following hind limb tourniquet-induced skeletal muscle IRI in a rat model would attenuate systemic inflammation and multiple end organ injury. Sprague-Dawley rats were subjected to 1?hr of ischemia via application of a rubber band tourniquet. Animals were randomized to receive an intravenous bolus of either vehicle control or FTY720 15?min after band placement. Rats (n = 10/time point) were euthanized at 6, 24, and 72?hr post-IRI. Peripheral blood as well as lung, liver, kidney, and ischemic muscle tissue was analyzed and compared between groups. FTY720 treatment markedly decreased the number of peripheral blood T cells (p < 0.05) resulting in a decreased systemic inflammatory response and lower serum creatinine levels and had a modest but significant effect in decreasing the transcription of injury-associated target genes in multiple end organs. These findings suggest that early intervention with FTY720 may benefit the treatment of IRI of the limb. Further preclinical studies are necessary to characterize the short-term and long-term beneficial effects of FTY720 following tourniquet-induced IRI.
Project description:<h4>Background</h4>Systemic thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the standard of acute stroke care. Its potential to increase the risk of secondary intracerebral hemorrhage, especially if administered late, has been ascribed to its proteolytic activity that has detrimental effects on blood-brain barrier (BBB) integrity after stroke. FTY720 has been shown to protect endothelial barriers in several disease models such as endotoxin-induced pulmonary edema and therefore is a promising candidate to counteract the deleterious effects of rt-PA. Besides that, every putative neuroprotectant that will be eventually forwarded into clinical trials should be tested in conjunction with rt-PA.<h4>Methods</h4>We subjected C57Bl/6 mice to 3 h filament-induced tMCAO and postoperatively randomized them into four groups (n?=?18/group) who received the following treatments directly prior to reperfusion: 1) vehicle-treatment, 2) FTY720 1 mg/kg i.p., 3) rt-PA 10 mg/kg i.v. or 4) FTY720 and rt-PA as a combination therapy. We measured functional neurological outcome, BBB disruption by quantification of EB extravasation and MMP-9 activity by gelatin zymography.<h4>Results</h4>We observed a noticeable increase in mortality in the rt-PA/FTY720 cotreatment group (61%) as compared to the vehicle (33%), the FTY720 (39%) and the rt-PA group (44%). Overall, functional neurological outcome did not differ significantly between groups and FTY720 had no effect on rt-PA- and stroke-induced BBB disruption and MMP-9 activation.<h4>Conclusions</h4>Our data show that FTY720 does not improve functional outcome and BBB integrity in large hemispheric infarctions, neither alone nor in conjunction with rt-PA. These findings stand in contrast to a recently published study that showed beneficial effects of FTY720 in combination with thrombolysis in a thrombotic model of MCAO leading to circumscript cortical infarctions. They might therefore represent a caveat that the coadministration of these two drugs might lead to excess mortality in the setting of a severe stroke.
Project description:<h4>Objective</h4>Ischemia-reperfusion injury (IRI) produces systemic inflammation with the potential for causing organ failure in tissues peripheral to the initial site of injury. We speculate that treatment strategies that dampen inflammation may be therapeutically beneficial to either the initial site of injury or peripheral organs. To test this, we evaluated the impact of FTY720-induced sequestration of circulating mature lymphocytes on renal IRI and secondary organ injury.<h4>Methods</h4>A microvascular clamp was surgically placed around the left renal pedicle of anesthetized male Sprague-Dawley rats with either vehicle or FTY720 treatment (0.3 mg/kg) intravenously injected after 15 min of ischemia. Blood flow was restored after 60 min. Cohorts of anesthetized rats were euthanized at 6, 24, or 72 hrs with tissue samples collected for analysis.<h4>Results</h4>FTY720 treatment resulted in profound T lymphocyte reduction in peripheral blood. Histopathologic examination, clinical chemistries, and gene transcript expression measurements revealed that FTY720 treatment reduced hepatocellular degeneration, reduced serum markers of liver injury (ALT/AST), and reduced the expression of gene targets associated with IRI.<h4>Conclusion</h4>These findings support an anti-inflammatory effect of FTY720 in the liver where the expression of genes associated with apoptosis, chemotaxis, and the AP-1 transcription factor was reduced. Findings presented here provide the basis for future studies evaluating FTY720 as a potential therapeutic agent to treat complications resulting from renal IRI.
Project description:Positron emission tomography (PET) is widely used in clinical and animal studies, along with the development of diverse tracers. The biochemical characteristics of PET tracers may help uncover the pathophysiological consequences of cardiac arrest (CA) and ischemic stroke, which include cerebral ischemia and reperfusion, depletion of oxygen and glucose, and neuroinflammation. PubMed was searched for studies of the application of PET for "cardiac arrest," "ischemic stroke," and "targeted temperature management." Available studies were included and classified according to the biochemical properties involved and metabolic processes of PET tracers, and were summarized. The mechanisms of ischemic brain injuries were investigated by PET with various tracers to elucidate the pathological process from the initial decrease of cerebral blood flow (CBF) to the subsequent abnormalities in energy and oxygen metabolism, to the monitoring of inflammation. In general, the trends of cerebral blood flow and oxygen metabolism after ischemic attack are not unidirectional but closely related to the time point of injury and recovery. Glucose metabolism after injury showed significant differences in different brain regions whereas global cerebral metabolic rate of glucose (CMRglc) declined. PET monitoring of neuroinflammation shows comparable efficacy to immunostaining. The technology of PET targeting in brain metabolism and the development of tracers provide new tools to track and evaluate the brain's pathological changes after ischemic brain injury. Despite no existing evidence for an available PET-based prediction method, discoveries of new tracers are expected to provide more possibilities for the whole field.
Project description:Oxidative stress and inflammation are two critical pathological processes of cerebral ischemia-reperfusion injury. Myeloperoxidase (MPO) is a critical inflammatory enzyme and therapeutic target triggering both oxidative stress and neuroinflammation in the pathological process of cerebral ischemia-reperfusion injury. MPO is presented in infiltrated neutrophils, activated microglial cells, neurons, and astrocytes in the ischemic brain. Activation of MPO can catalyze the reaction of chloride and H2O2 to produce HOCl. MPO also mediates oxidative stress by promoting the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), modulating the polarization and inflammation-related signaling pathways in microglia and neutrophils. MPO can be a therapeutic target for attenuating oxidative damage and neuroinflammation in ischemic stroke. Targeting MPO with inhibitors or gene deficiency significantly reduced brain infarction and improved neurological outcomes. This article discusses the important roles of MPO in mediating oxidative stress and neuroinflammation during cerebral ischemia-reperfusion injury and reviews the current understanding of the underlying mechanisms. Furthermore, we summarize the active compounds from medicinal herbs with potential as MPO inhibitors for anti-oxidative stress and anti-inflammation to attenuate cerebral ischemia-reperfusion injury, and as adjunct therapeutic agents for extending the window of thrombolytic treatment. We highlight that targeting MPO could be a promising strategy for alleviating ischemic brain injury, which merits further translational study.
Project description:The aim of the present study was to identify the effect of heme oxygenase (HO)-1 gene on cerebral ischemia-reperfusion injury. Sprague-Dawley rats were divided randomly into four groups: Sham group, vehicle group, empty adenovirus vector (Ad) group and recombinant HO-1 adenovirus (Ad-HO-1) transfection group. Rats in the vehicle, Ad and Ad-HO-1 groups were respectively injected with saline, Ad or Ad-HO-1 for 3 days prior to cerebral ischemia-reperfusion injury. Subsequently, the middle cerebral artery occlusion method was used to establish the model of cerebral ischemia-reperfusion injury. Following the assessment of neurological function, rats were sacrificed, and the infarction volume and apoptotic index in rat brains were measured. Furthermore, the protein expression levels of HO-1 in brain tissues were detected using western blot analysis. Results indicated that the neurological score of the Ad-HO-1 group was significantly increased compared with the Ad or vehicle groups, respectively (P<0.001). The volume of cerebral infarction and the index score of neuronal apoptosis in the vehicle and Ad groups was significantly increased compared with the Ad-HO-1 group (P<0.01). The death of neuronal cells following cerebral ischemia-reperfusion injury reduced remarkably induced by over-expression of HO-1. These findings suggest a neuroprotective role of HO-1 against brain injury induced by transient cerebral ischemia-reperfusion injury.