Protective Effect And Mechanism Of Minocycline On Blood-Brain Barrier Disruption And Neurological Damage In ICH Model

BackgroundIntracerebral hemorrhage(ICH)is a significant cause of morbidity and mortality throughout the world.However,due to the limited knowledge on the underlying mechanism,developing a targeted therapy or an effective drug to improve the outcomes of ICH remains a challenge.After ICH,perihematomal edema develops within 3h from symptom onset and peaks between 10 and 20 days after ictus.Brain edema has been demonstrated as a critical factor for the development of neurologic deficits by increasing intracranial pressure or causing a substantial midline shift.In this regard,multiple forms of edema could be present after ICH,but the major form is vasogenic cerebral edema resulted from the disruption of blood-brain barrier(BBB).Minocycline(Mino)is a second-generation,semi-synthetic tetracycline derivative with established antimicrobial effects and inhibition of microglial activation.During last twenty years,Mino has been wildly used in clinical practice as a neuroprotective agent.Due to the characteristic of high lipophilicity and tissue penetration,Mino could easily penetrate the BBB.Despite there are many studies focused on the physiological property and the protective role of this chemical,the underlying molecular mechanism that Mino protects the integrity and permeability BBB and reduces edema remains less known,especially in the progression of ICH.Wnt/?-catenin signaling has been demonstrated to protect against the edema of BBB and attenuate the symptoms of ICH through controlling the expression of multiple downstream targets such as Occludin.In contrast,inhibition of Wnt/?-catenin activity was reported to enhance the production of inflammatory mediators,which are detrimental to the progression of ICH.Dickkopf-1(DKK1)is a major upstream suppressor of Wnt/?-catenin pathway.Inhibition or deficiency of DKK1 leads to the enhancement of Wnt signaling activity and subsequent increase of?-catenin expression.Although the protective effects of Mino on the integrity of BBB and the outcomes of ICH,as well as its influences on a variety of signaling pathways including PI3K,MAPK,and NF-?B,were widely reported,there is still a large gap in current knowledge about if Mino protects against the brain damage in ICH through acting on the expression of DKK1,at least partially,and thus affecting activity of Wnt signaling-mediated anti-inflammatory responses.PurposeIn this study,we assess the effects of minocycline on BBB structure,neurological function,and inflammatory responses using collagenase-induced ICH model and rat microglia,and elucidate underlying molecular mechanisms.Methods(1)Collagenase-induced ICH model in rat was established.The integrity of the BBB was assessed through the extravasation of Evan's blue and the brain water content was also measured at different time points.(2)Minocycline was dissolved in saline,which were injected intraperitoneally(i.p.)at dose of 45mg/kg immediately and 12h after ICH,followed by 22.5mg/kg twice a day for 2 days.SiDKK1 was administered via intracerebroventricular(i.c.v.)injection 48h prior to ICH-induction.(3)Using western blot,we compared the difference of DKK1,Wnt1,?-catenin,Occludin protein among different groups(Sham;ICH+vehicle;ICH+siNC;ICH+siDKK1;ICH+Mino;ICH+siDKK1+Mino).(4)4.0×10~5 rat microglia were placed in 12 wells plates and treated with E.coli LPS(100ng/mL)for 24h,and then the cell-free supernatants were assayed for cytokine and MMP-9 levels by ELISA.For Western blot,the whole cell lysate was collected after 4h challenge with E.coli LPS and 15?g total protein was used for analysis.(5)To further characterize the function of DKK1,we examine the localization of DKK1 in perihematomal tissues through fluorescence staining with multiple molecular markers including CD11b/c,NeuN,and GFAP.(6)We used several neurologic function tests and neurologic deficit score including forelimb placing,forelimb use asymmetry test and corner turn test to evaluate the neurologic function outcome.Results(1)Evan's blue extravasation in basal ganglia is significantly enhanced in the rats that underwent ICH,as compared with controls.ICH induces most significant Evan's blue extravasation in basal ganglia at day 3,compared with that of day 1 and day 7(P<0.001).Moreover,brain water content is also significantly increased in the basal ganglia at day 1 and day 3 after ICH,but not at day 7.The amount of brain water content is maximized at day 3 after ICH,compared with that of day 1 and day 7(P<0.01).(2)Evan's blue extravasation in ipsilateral basal ganglia was reduced significantly at day 3 in the group with the treatment of Mino(P<0.05),as compared with the vehicle control group.Moreover,the brain water content in the ipsilateral basal ganglia is also attenuated in Mino treated group,as compared with vehicle control group(P<0.01).(3)We find that the expression of DKK1 is significantly reduced in brain tissues near the hemorrhagic region in Mino treated group,as compared with vehicle control group(P<0.01).In addition,immunohistochemistry showed the expression of DKK1remarkably increased after ICH,and this increase was abrogated by the treatment of Mino,which was exactly consistent with our western blot.To further characterize the function of DKK1,we next examine the localization of DKK1 in perihematomal tissues through fluorescence staining with multiple molecular markers including CD11b/c,NeuN,and GFAP.Fascinatingly,we find the DKK1 is co-localized with the CD11b/c-or NeuN-positive cells,but not GFAP cells.We next examine the influences of Mino on the expression of DKK1 and the production of inflammatory mediators in rat microglia.Mino robustly decreases the expression of DKK1,while substantially increases the expression of?-catenin,as compared with control.To further examine the anti-inflammatory effects of Mino,E.coli LPS,a prototypical agonist of TLR4,was used to challenge microglial cells in the presence and absence of Mino.We find Mino pre-treatment significantly decreases the production of LPS-induced TNF,IL-6 and MMP-9 in microglia.(4)Mino significantly enhanced the protein levels of Wnt1,?-catenin and Occludin,as compared to the vehicle group(Wnt1:P<0.01;?-catenin:P<0.01;Occludin:P<0.05),suggesting that Mino indeed affect the activation of Wnt1 and its downstream molecules.We find deficiency of DKK1 significantly enhanced the expression of Wnt1,?-catenin and Occludin,as compared with the control group with ICH only(Wnt1:P<0.01;?-catenin:P<0.01;Occludin:P<0.05).Moreover,deficiency of DKK1 combined with treatment of Mino exhibited synergistic effects compared with treatment with either of them(Wnt1:P<0.05;?-catenin:P<0.05;Occludin:P<0.01).In addition,we also assessed the effects of Mino and siRNA-mediated deficiency of DKK1 on the integrity and permeability of BBB.We find Evan's blue extravasation and brain water content were reduced after the treatment of Mino compared with vehicle group(both P<0.05)and that Mino plus siDKK1 attenuated significant more than either of them(both P<0.05).On the other hand,neurologic deficits examination at 3 days after ICH showed that treatment with Mino or siRNA of DKK1 significantly attenuates the neurologic deficits,including significant improvements in corner turn test,forelimb placing score,and forelimb using asymmetry score,as compared with vehicle-treated ICH rats(P<0.05),indicating the protective function of Mino and DKK1 inhibition.However,no significant difference is observed between Mino plus siDKK1-treated ICH rats and Mino-treated only group(P>0.05).ConclusionOur data demonstrate that minocycline constitutes a potential treatment value for ICH by preserving BBB and improving neurological function through decreasing DKK1 expression and invigorating Wnt1-?-catenin activity.