PRX-2 Promotes Secondary Brain Injury After Intracerebral Hemorrhage By Regulating The Expression Of LCN-2

BackgroundsSpontaneous intracerebral hemorrhage(ICH)remains a common neurological emergency with high morbidity and mortality worldwide.The epidemiological investigation has found that non-traumatic ICH comprises 10-15%of all strokes and accounts for more than 5 million cases and over 3 million deaths reported worldwide every year.Only 12%-39%of patients live independently after an ICH,and the case-fatality rate ranges from 35%at seven days to 60%at one year.In addition,more than 60%of survivors were left with severe disabilities,a burden on society and families.Blood pressure management,coagulopathy reversal,intracranial pressure control,prevention of hematoma expansion,and minimally invasive hematoma evacuation are the mainstays of acute ICH treatment under investigation.However,these treatments are limited and do not significantly improve patient survival.Therefore,more effective therapies are urgently required to reduce ICH patients’ disability and mortality rate.Recent studies have indicated that neuroinflammation plays a vital role in ICH-induced brain injury.Components of red blood cell(RBC)lysis such as iron and hemoglobin contribute to neuroinflammation,related to poor prognosis.Neuroinflammation consists of a series of pathological processes,such as activation and polarization of astrocytes and microglia/macrophages,neutrophil infiltration,production of proinflammatory cytokines,and ultimately leading to cerebral edema,blood-brain barrier(BBB)disruption,neuronal death,and neurological deficits.Therapeutic strategies that reduce neuroinflammation can effectively alleviate brain damage after ICH,which has recently become a hot topic.Hemolysis in the hematoma occurs as early as the first day after ICH.Peroxiredoxin 2(PRX-2),a member of the peroxiredoxin family of antioxidant enzymes,is the third most abundant protein in RBC.Intracellular PRX-2 plays an important role in antioxidant progress in various cell types such as RBCs,neurons,and neutrophils.Recent studies have shown that extracellular PRX-2 released from dead brain cells can act as one of the damage-associated molecular patterns(DAMPs)to cause brain injury by inducing various pro-inflammatory factors,activating microglia/macrophages,and promoting neuronal apoptosis.However,the specific role of extracellular PRX-2 after ICH has not been elucidated.LCN-2 is an acute-phase protein secreted by brain astrocytes and neutrophils under inflammatory conditions.Evidence indicated that LCN2 significantly increased immediately after the ICH and played a vital role in neuroinflammation.LCN2 can injure neurons directly,activate and polarize glial cells,destroy the BBB’s integrity,enlarge bran edema,induce the infiltration of inflammatory cells,and promote the secretion of inflammatory cytokines,which finally cause long-term neurological deficits.Notably,the infarct volume,neurological deficits,immune infiltrates,and pro-inflammatory molecules(IL-1β,TNF-α,CCL2,CXCL1,CXCL10,ICAM-1)were attenuated after LCN-2 deficient following ICH.Our previous studies showed that the expression of LCN-2 and PRX-2 were upregulated after ICH.However,the correlation between LCN-2 and PRX-2 and their effects is still unknown.Therefore,this study explored the potential role of PRX-2 after ICH and the mechanism of LCN-2 in PRX-2-induced brain damage.MethodsPart 1This study explored the potential role of LCN-2 in PRX-2-induced neuroinflammation and brain injury.There were four experiments in this study.Experiment 1:Adult male Sprague-Dawley(SD)rats had an intracaudate injection of lysed RBCs or saline.Brains were harvested one hour after injection to measure PRX-2 levels.Experiment 2:Rats received an intracerebral injection of either recombinant rat PRX-2,PRX-2(-),or saline(15μl).All rats had T2-weighted magnetic resonance imaging(MRI)and behavioral testing 24 hours after injection.The rats were then euthanized for brain histology or Western blots.Experiment 3:Rats received an intracerebral injection of either recombinant rat PRX-2or saline(15μl.All rats had MRI and behavioral testing at 24 and 72 hours.The rats were then euthanized for brain histology.Experiment 4:Rats had an intracerebral injection of 15μl of either lysed RBCs with vehicle(DMSO)or lysed RBCs with conoidin A.All rats had MRI and behavioral tests 24 hours after injection.The rats were then euthanized for brain histology.Part 2This study explored the potential role of LCN-2 in PRX-2-induced neuroinflammation and brain injury.There were four experiments in this study.Experiment 1:Male WT mice received an intracerebral injection of 10μl saline,while male LCN-2 KO mice,LCN-2 HET mice,and WT mice received an intracerebral injection of 10 μl PRX-2.Following MRI and behavioral testing on day 1,mice were euthanized for brain histology and Western blotting.Experiment 2:Male LCN-2 KO mice,LCN-2 HET mice,and WT mice received an intracerebral injection of 10 μl PRX-2.Following behavioral testing on day 1,3,7,and 14 and an MRI on day 14 after injection,mice were euthanized for brain histology.Experiment 3:Male LCN-2 KO mice received an intracerebral injection of 10 μl PRX-2 with 1 μg recombinant mouse LCN-2 protein or vehicle.Male WT mice had only 10 μl PRX-2 with the vehicle.Behavioral tests and MRI were performed on day 1 before the mice were euthanized for brain histology.Experiment 4:Female LCN-2 KO mice,LCN-2 HET mice,and WT mice(n=14)received an intracerebral injection of 10 μl PRX-2.Following MRI and behavioral testing on day 1,6 mice in each LCN-2 KO,LCN-2 HET,and WT group were euthanized for brain histology.The remaining 8 mice in each group received behavioral tests on day 1,3,7,and 14.In addition,MRI was obtained on day 14.The mice were then euthanized for histology.ResultsPart 1No rats died in this study.One rat from the PRX-2 group and another from lysed RBCs+conoidin-A group were excluded from brain histology measurements because of poor brain perfusion.However,MRI images and behavioral results of the two rats were still used in this study.The level of PRX-2 in brain tissue increased significantly by 1 hour after an injection of red blood cell lysate.In addition,blood-brain barrier disruption,brain swelling,neutrophil infiltration,microglial activation,neuronal death,and neurological deficits were significantly increased after injection of 15μl PRX-2 at 24 hours.PRX-2 inhibitor conoidin A significantly reduced brain swelling,neutrophil infiltration,neuronal death,and neurological deficits caused by red blood cell lysate at 24 hours.Part 2A single male WT mouse died on day 7 after intracerebral injection of extracellular PRX-2 and was therefore excluded from this study.The expression of LCN-2 was significantly increased after injection of 10 μl PRX-2 at 1 hour.Immunofluorescence indicated that LCN-2 positive cells were mainly neutrophils and astrocytes,and only a few LCN-2 positive cells were Iba-1(microglia/macrophage marker)or NeuN(neuron marker)positive cells.Intracerebral PRX-2 injections resulted in increased expression of LCN-2 protein.PRX-2-induced brain swelling,neutrophil infiltration,microglia/macrophage activation,neuronal cell death,and neurological deficits were reduced in male LCN-2 HET,and KO mice compared to WT and were exacerbated by exogenous LCN-2 co-injection.Additionally,intracerebral PRX-2 injections caused brain injury and neurological deficits in female WT mice;effects were reduced in female LCN-2 KO mice.ConclusionPart 1These results suggest that extracellular PRX-2 released by hematoma contributes to neuroinflammation and brain injury after intracerebral hemorrhage(blood-brain barrier disruption,brain swelling,neutrophil infiltration,microglia/macrophage activation,neuronal death,and neurological deficit)and maybe a potential therapeutic target.Part 2Intracerebral injection of PRX-2 upregulates the expression of LCN-2.LCN-2 plays a critical role in PRX-2-induced neutrophil infiltration,microglia/macrophage activation,and brain injury.