| BackgroundTraumatic brain injury(TBI)is one of the main causes of disability and traumatic death.TBI can be divided into primary injury and secondary injury,the former injuries include lacerations,contusions,bleeding,and axonal injuries that are caused by external forces.The secondary injury develops on the basis of the primary injury,further aggravating brain injury and worsening TBI prognosis.Neuroinflammation-induced damage to the blood-brain barrier,cerebral edema,and loss of neurons is an important pathological mechanism of secondary brain injury.Therefore,research on how to regulate neuroinflammation has important clinical value for guiding the treatment of TBI.Ischemic preconditioning(IPC),also known as ischemic induced tolerance,is a phenomenon in which a transient,sublethal ischemic injury of an organ in the body can protect the organ from secondary and more severe ischemic injury.The protective roles of IPC have been proven in brain,liver,kidney and lung damages.Remote ischemic preconditioning(RIPC)refers to making an organ undergo a mild ischemic injury operation to protect the following severe ischemia-reperfusion injury of a distant organ.The ischemic preconditioning of the remote limb is widely studied because of its simple and easy operations.A lot of studies have shown that limb RIPC can protect cerebral ischemia-reperfusion injury,and some clinical studies have also shown that it can effectively improve the prognosis of brain injury such as cerebral infarction,cerebral hemorrhage,and brain surgery.Studies have shown that the mechanisms by which RIPC protects these brain injuries refer to reducing neuroinflammatory responses,alleviating blood-brain barrier disfunction,inhibiting oxidative stress,and reducing apoptosis.These injury mechanisms also play an important role in the pathogenesis of TBI.Currently,the effect of limb RIPC on TBI and the cellular and molecular mechanisms of action remain unknown.Microglia are important innate immune cells in the central nervous system.They are the first line of defense in the event of brain injury or disease.Microglia can produce neuroprotective factors,remove cell debris,and coordinate the neural repair process that is conducive to nerve recovery after traumatic brain injury.However,they may also be over activated and produce excess pro-inflammatory and cytotoxic mediators,which will induce apoptosis or death of nerve cells,hinder the repair of the central nervous system,and cause neurological dysfunction.The pro-inflammatory or anti-inflammatory effects of microglia are related to their cell subtypes in a polarized state,including M1 and M2 cell subtypes.M1 microglia can produce proinflammatory factors after injury such as IL-1?,IL-12,TNF-?,chemokines and ROS,and these cytokines are protective in many situations such as timely removal of injury or pathogens.However,deregulation of microglia or excessive activation will lead to excessive release of proinflammatory factors and producing neurotoxic effects.M2 microglia are characterized by high expression of anti-inflammatory cytokines such as IL-4,IL-10,IL-13 and CD206,which can promote nerve regeneration,neovascularization,axonal remodeling and regeneration of myelin sheath,and all of these will exceed the process of nerve repair.After brain injury,damaged nerve cells can release cytokines and chemokines,and recruit peripheral macrophages to infiltrate the brain tissue through the damaged BBB to participate in the inflammatory response.Microglia and peripheral macrophages share basically the same function and most of markers,thus,microglia/macrophages were used in this study.The activation of microglia is related to multiple pattern recognition receptors(PRRs)expressed on the surface or in the cells.PRRs can recognize pathogen associated molecular patterns or damage associated molecular patterns.NLRP3 is a member of the NOD-like family of receptors in PRRs.When damage occurs,NLRP3 can bind to an apoptosis-associated speck-like protein(ASC)and be activated,and then recruit Caspase-1 precursors to form NLRP3 inflammasome.The NLRP3 inflammasome can activate the Caspase-1 precursor to Caspase-1,which in turn activates the precursors of IL-1? and IL-18 into active inflammatory factors and participates in the inflammatory cascade.Studies have shown that microglia are important cells which can produce IL-1?,and its production is regulated by NLRP3 inflammasome.The ROS produced by NADPH oxidase in microglia is an important signal for activating NLRP3 inflammasome,and NOX2 is the main subtype of NADPH oxidase.Studies have shown that gene knockout or drug inhibition of NOX2 can promote the transformation of activated microglia into M2 cell subtypes,while NLRP3 inhibitors can also inhibit the transformation of microglia into M1 cell subtypes.At present,some studies have showed that the mechanism of RIPC in alleviating cerebral ischemia-reperfusion injury is related to its reduction in ROS.After the occurrence of TBI,whether the operation of RIPC would inhibit the production of ROS by reducing the expression of NOX2,and then reduce the activity of NLRP3 inflammasome and reduce the expression of IL-1? will be studied.And whether down-regulation of NLRP3 and IL-1? would thereby promote the activated microglia to the protective M2 cells type also need to be studied.Part I:Effect of limb RIPC on neural cell death and apoptosis,blood-brain barrier disruption,and neurological recovery after TBIObjectiveTo clarify the protective effects of limb RIPC on the blood-brain barrier,neural cell necrosis and apoptosis after TBI,and the effect of it on the recovery of neural function.Methods1.C57BL/6 mice were randomly divided into Sham,Sham+RIPC,TBI,and TBI+RIPC groups.Both hind limb RIPCs were operated 24 hours before TBI.2.Detection of brain edema after TBI:The drying wet weight method was used to detect the brain water content of mice at 72h after TBI.3.Calculation of brain injury volume after TBI:CV staining was applied to brain tissue sections at 28 days after TBI,and brain injury volume was calculated by ImageJ software.4.Assessing the integrity of BBB after TBI:72 hours after TBI,Evans blue(EB)was injected through the tail vein of mice to detect brain tissue content;Western Blotting was used to detect tight junction proteins ZO-1,Occludin and metal matrix protein MMP-9 expression.5.Clarification of neuronal necrosis and apoptosis:PI staining was used to determine neuronal death;TUNEL was used to determine neuronal apoptosis and Western Blotting was used to detect apoptosis-related proteins.6.Neural function scores and behavioral experiments:mNSS criteria were used to evaluate the recovery of neurological function in mice;Morris water maze test and novel objective recognition test were used to evaluate mouse learning and memory,spatial memory and motor function.Results1.RIPC reduced brain water content after TBI:The wet and dry weight of brain water content showed that the water content in the ipsilateral hemisphere of the TBI group was significantly higher than that in the Sham group,while the water content in the RIPC-treated group was significantly less than that in the TBI group(P<0.05).There was no significant difference of brain water content between the contralateral hemispheres of TBI injury.2.RIPC reduced brain injury volume after TBI:Brain injury volume calculated by CV staining showed that compared with the TBI group,the injury volume of the RIPC-treated group was significantly reduced(P<0.05).3.RIPC reduced BBB damage after TBI:EB content in the TBI group was significantly higher,while EB content in the RIPC-treated group was significantly lower than that(P<0.05);Western Blotting results showed that the expression of ZO-1 and Occludin in the TBI group were lower,while the RIPC-treated group got higher result than that(P<0.05);The expression of MMP-9 in the TBI group increased,and the RIPC-treated group got a significantly decreased result than that(P<0.05).4.RIPC reduced the rate of neural cell death and apoptosis:PI staining results showed that the number of dead neural cells in the TBI group increased significantly,while the number of RIPC-treated groups decreased significantly(P<0.05);TUNEL staining showed that the number of apoptotic cells around the injury was obvious increased,while the RIPC-treated group got a significantly lower result than that(P<0.05);Western Blotting detected apoptosis-related proteins and found that BAX expression was increased in the TBI group,Bcl-2 expression was decreased of the TBI group,while BAX was significantly reduced and Bcl-2 significantly increased in the RIPC-treated group(P<0.05).5.RIPC improved neurological recovery after TBI:Results of mNSS showed that the scores of mice in the RIPC-treated group on day 7 and 14 were significantly lower than those in the TBI group(P<0.05);The results of the novel objective recognition test showed that the time and number of contact with novel objective in the RIPC-treated group increased significantly,and the novel objective recognition index increased significantly(P<0.05).The results of the Morris water maze test showed that compared with the TBI group,the escaped latency time of mice in the RIPC-treated group was shortened,the number of crossing platforms and the time stayed in the target quadrant increased significantly(P<0.05).Part II:Effects of limb RIPC on microglial/macrophage phenotypic transformation after TBI and its mechanism of actionObjectiveTo study the effects of limb RIPC on the degree of microglial/macrophage activation and cell phenotypic transformation after TBI;To study the expression of NLRP3/Caspase-1/IL-1? after TBI and the regulation of NOX2 on it;To determine the effect of RIPC on NOX2 and NLRP3/Caspase-1/IL-1? expression.Methods1.To clarify the effect of RIPC on the degree of microglial/macrophage activation and its phenotypic transformation after TBI:Immunofluorescence detection of the number of microglial/macrophage marker Iba-1 and the activation status,Western Blotting was used to detect its expression;Immunofluorescence and Western Blotting were used to detect the staining and expression of M1 phenotypic markers CD86 and iNOS,M2 phenotypic markers Argl and CD206.2.To determine the expression of NLRP3/Caspase-1/IL-1? after TBI and the regulation of RIPC on them:qRT-PCR and Western Blotting were used to detect the expression of NLRP3,Caspase-1,IL-1?,and IL-18 in brain tissue.3.To clarify cell types that express NLRP3:Immunofluorescence was used to detect the coincidence of the positive region of microglial/macrophage marker Iba-1 and astrocyte marker GFAP with the positive region of NLRP3.4.To determine the regulatory effect of NOX2 on NLRP3/Caspase-1/IL-1?:C57BL/6 mice were randomly divided into Sham+Vehicle,Sham+Apocynin,TBI+Vehicle,and TBI+Apocynin groups(Apocynin is a NOX2 inhibitor),qRT-PCR and Western Blotting were used to detect the expression of NOX2,NLRP3,Caspase-1,and IL-1?.5.To elucidate the regulation of NOX2 and ROS by RIPC:qRT-PCR and Western Blotting were used to detect the expression of NOX2 in Sham,Sham+RIPC,TBI,and TBI+RIPC groups;Immunofluorescence detection of NOX2 fluorescence area and the types of cells produced;ROS fluorescent probe-DHE staining to detect ROS production.Results1.RIPC reduced the degree of microglial/macrophage activation after TBI and promoted its transformation to the M2 phenotype:1.1 RIPC reduced the activation status of microglia/macrophages,and reduced the number of activated cells and their marker protein expression:Immunofluorescence staining showed that the number of Iba-1 positive cells was significantly increased,and the cells were in an activated state in the TBI group,while the number of cells were decreased significantly in RIPC-treated group(P<0.05),and the cell volume was relatively reduced;Western Blotting results showed that the expression of Iba-1 protein in the TBI group increased significantly,while that in the RIPC-treated group decreased significantly(P<0.05).1.2 RIPC reduced the number of M1 phenotype microglia/macrophages and their marker protein expression:Immunofluorescence staining showed that the number of CD86 and iNOS positive cells of the M1 phenotype in the TBI group increased significantly,while the RIPC-treated group significantly decreased(P<0.05);Western Blotting results showed that the CD86 and iNOS protein expression levels in the TBI group were significantly increased,while that in the RIPC-treated group was significantly lower(P<0.05).1.3 RIPC increased the number of M2 phenotype microglia/macrophages and their marker protein expression:Immunofluorescence staining showed that the number of CD206 and Argl-positive M2 phenotypic markers in the TBI group was greater than that in the Sham group,while that in the RIPC-treated group was further increased(P<0.05);Western Blotting results showed that the expression of CD206 and Argl protein in the TBI group was higher than that in the Sham group,and that in the RIPC-treated group was further increased than the TBI group(P<0.05).2.NLRP3/Caspase-1/IL-1? were highly expressed after TBI,and RIPC down-regulated their expression:qRT-PCR and Western Blotting results showed that mRNA and protein expression of NLRP3,Caspase-1,IL-1?,and IL-18 in the TBI group levels were significantly increased,while that in the RIPC-treated group were significantly lower(P<0.05).3.Both microglia/macrophages and astrocytes could produce NLRP3:Immunofluorescent staining showed that the TBI group got NLRP3 positive areas where were coincident with both Iba-1 and GFAP positive areas;Meanwhile,the number of Iba-1,GFAP-positive cells and NLRP3-positive regions in the RIPC-treated group were significantly lower than those in the TBI group(P<0.05).4.NOX2 could regulate the expression of NLRP3/Caspase-1/IL-1?:qRT-PCR results showed that the mRNA expression of NLRP3,Caspase-1,and IL-1? in the Apocynin-treated group were significantly lower than that in the TBI group(P<0.05);Western Blotting results showed that the protein expression of NOX2,NLRP3,Caspase-1,and IL-1? in the Apocynin-treated group were significantly lower than that in the TBI group(P<0.05).5.RIPC inhibits NOX2 expression and inhibits ROS production:5.1 Western Blotting results showed that the expression of NOX2 protein in the RIPC-treated group was significantly lower than that in the TBI group(P<0.05).Immunofluorescence staining results showed there were many NOX2 positive areas overlapped with Iba-1 positive areas,and the NOX2 fluorescence area of the RIPC-treated group was much less than that of the TBI group(P<0.05).5.2 ROS fluorescent probe-DHE staining results showed that the ROS-positive areas in the TBI group were significantly increased,and that in the RIPC-treated group was significantly reduced(P<0.05).ConclusionsLimb RIPC could reduce the production of ROS by down-regulating NOX2,thereby down-regulate the expression of NLRP3/Caspase-1/IL-1? pathway,and reduce the activation of microglia/macrophages and promote its transformation to M2 subtype and finally alleviate TBI damage. |
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