Inflammatory Response Mediated White Matter Fiber Bundle Injury Around Hematoma In Mice After Intracerebral Hemorrhage And Intervention Strategies

BackgroundIntracerebral hemorrhage(ICH)served as a leading cause of death around the world.Even survivors may undergo long terms of disability and neurological dysfunctions,causing huge payments in health care and social burden.ICH most probably happens on the basal ganglia,which is rich in white matter fiber bundle.White matter injury(WMI)happens after ICH and contributes to neurological dysfunctions and long-term poor prognosis.By now,however,there are still limited strategies to tackle this issue.Studies showed that neuroinflammation happens in the early stage of ICH,and is one of the contributing factors to WMI.Inflammation factors and microglia activation caused by the degradation of hemoglobin are the leading causes of WMI.Moreover,inflammation-induced brain blood barrier damage would also cause infiltration of macrophage and edema in brain,exaggerating the inflammatory responses.However,the interventions for WMI damaged by inflammation response are limited.Here,we introduced a potassium channel Kv1.3,which was previously reported as an inflammation mediator,while blocking Kv1.3,usually by 5-(4-Phenoxybutoxy)psoralen(PAP-1),is related to inflammation inhibition and the microglia transition from M1 subtype to M2 subtype.The Kv1.3 blockage has yet to be tested in mice models of neuroinflammation in the early stage of ICH and inhibition of Kv1.3 may be an underlying target to boost the prognosis of ICH mice.Besides,hemoglobin is rich in iron.As hemoglobin degradation proceeds in the middle or late stage of ICH,iron from hemoglobin is released to induce the production of reactive oxygen species(ROS),which will lead to the degradation of the myelin sheath.Iron released from hemoglobin can also become a component of the lipid-peroxidation enzyme,causing oligodendrocyte ferroptosis and WMI.Targeting ferroptosis after ICH is a newly founded way to save mice from WMI.Here we introduce dexpramipexole(DPX),a federal drug administrator(FDA)sanctified drug used in neuropathy,which can probably decrease ferroptosis of oligodendrocytes and attenuate WMI.Mechanistically,DPX may make its way from saving the depletion of glutathione peroxidase 4(GPX4),a ferroptosis marker,and ferroptosis suppressing protein(FSP1),which was found closely related to ferroptosis.Part Ⅰ Potassium channel Kv1.3 blockage attenuates WMI through neuroinflammation inhibition in the early stage of mice ICH model.Object To explore neuroinflammation caused WMI after ICH and the effect of Kv1.3blockage by PAP-1.Methods ICH models were established by 15μL blood injection into the basal ganglia of C57BL/6J mice(aged 9-10 weeks,24-26g).Afterwards,mice were randomly divided into three groups: Sham,ICH,and ICH+PAP-1.Real-time polymer chain reactions(PCR),western blot(WB),and immunofluorescent(IF)were used to detect Kv1.3 expression change caused by ICH with time.The open field test(OFT),basso mouse score(BMS),and weight change of mice were taken for behavior evaluation.WB,IF,and transmission electronic microscopy(TEM)were used to detect white matter injury.PCR,WB,and enzyme-linked immunosorbent assay(ELISA)were used to detect inflammatory factors.PCR,WB,and IF were used for the evaluation of M1-M2 microglia composition.Results1.Kv1.3 expression increased around hematoma significantly on D3 of ICH compared with Sham group.2.Body weight of mice was decreased after ICH especially on D1-D3 compared with Sham group.Kv1.3 blockade PAP-1 abrogated this phenomenon.BMS score in mice of ICH group was decreased on D3 compared with Sham group.Mice in ICH+PAP-1 groups show increased BMS compared with ICH group.Open field test shows the same trends on D3 dosage of PAP-1 with 40 mg/kg/d seems to best reserve mice behavior prognosis.3.White matter injury in ICH group D3 was obvious,and the injury can be diminished by PAP-1 40mg/kg/d.Both IF and WB showed decreased MBP expression in ICH,while the increased MBP can be found in ICH+PAP-1 group compared with ICH group.TEM shows demyelination and increased G-ratio in D3 compared with Sham group,and PAP-1 shows significant protective effects in terms of demyelination and G-ratio.4.Inflammatory factors like tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were increased in WB,PCR,and ELISA,while decreased expressions were found in PAP-1 treatment group.5.Anti-inflammatory factors like interleukin-4 and interleukin-10 were increased in the ICH group.However,in ICH+PAP-1 group,the expression is even higher.6.The number of M1-like microglia increased around hematoma after ICH,compared with Sham group,while the application of PAP-1 decreased the M1-like microglia.7.The number of M2-like microglia increased around hematoma after ICH,compared with Sham group,while the application of PAP-1 increased the M2-like microglia even higher than ICH group.8.NF-κB signal is activated after ICH,which can be abolished by PAP-1 usage.Conclusion Kv1.3 blockade using PAP-1 reduced the accumulation of pro-inflammatory cytokines and upregulated the deposition of anti-inflammatory and neurotrophic factors by facilitating microglia polarization into M2-like microglia via the NF-κB signaling pathway.The present study provides evidence Kv1.3 blockade is suitable to mitigate WMI by facilitating microglia transformation into M2-like phenotype after ICH.Par Ⅱ Pramipexole facilitates white matter recovery through oxidation inhibition in middle to late-stage of mice ICH modelObject To detect the effect of dexpramipexole in white matter protection and ferroptosis inhibition in mice ICH model.Methods Survival rate and weight change were used for DPX’s effect and toxicity evaluation.Behavior tests like BMS,OFT,beam walking test(BWT),and forelimb strength were used for prognosis evaluation with or without DPX administration.WB,IF,TEM,and Hematoxylin and Eosin(HE)stains were taken for white matter bundle evaluation.Perl’s staining and Dihydroethidium(DHE)stain were used to evaluate the oxidation level around the hematoma.TEM was used for the illustration of ferroptosis-characteristic mitochondria.Wb,IF was used for the detection of the intercellular molecular mechanism of ferroptosis after ICH.Results1.Mice’s behavior conditions in BMS,mean velocity of mice in OFT,forelimb strength,and foot faults in BWT were worsen in ICH group than Sham group.And DPX with each dosage group saved the behavior loss of mice.DPX with 5mg/kg/d showed better outcomes among each dosage group.2.Survival rate and weight declined in ICH group.ICH+DPX groups show an increased survival rate and weight compared with ICH group.3.White matter injury is obvious in the ICH group,with decreased MBP expression,decreased injury size,and increased demyelination.DPX saved the WMI on D7 of ICH.4.Both iron and ROS were increased in ICH group on D7.DPX abolished the iron and ROS accumulation on D7 of ICH compared with ICH group.5.Percentage of shrunk mitochondria in ICH group increased on D7,DPX decreased the shrunk mitochondria compared with ICH group.6.DPX decreased the ferroptosis related genes expression,which is increased in ICH on D7 compared with Sham group.Conclusion ICH can cause long-term WMI,ferroptosis takes part in middle to late stage of ICH.DPX with 5mg/kg/d can protect white matter from ferroptosis after ICH.