The Protective Role Of Bexarotene In Traumatic Brain Injuryand Related Mechanism

ObjectiveTraumatic brain injury(TBI)can lead to tissue loss and severe neurological dysfuction.There is almost no effective and efficient therapy till now.Brain consists of gray matter and white matter.Gray matter is mainly composed of cell bodies of neurons,synapses and astrocytes,while whihe matter is composed of nerve fiers and myelin formed by oligodendrocytes.TBI can not only cause neuronal and synaptic damage,but also lead to axonal injury and oligodendrocytes death.Reactive astrocytes and microglia play important roles in TBI,but the molecular mechanisms of astrocytic and microglial regulation remain largely unknown.A study reported that A1 astrocyte,a detrimental phenotype of astrocyte,can be activated by cytokines secreted from activated microglia.White matter injury and repair can be also influenced by microglia polarization.As a nuclear transcriptional factor,peroxisome proliferator–activated receptor gamma(PPAR?)can regulate microgliapolarization.Retinoid X receptor(RXR),a nuclear receptor,can bind to PPAR? in the form of heterodimer and regulate the functions of target genes.Bexarotene,a highly selective RXR agonist,can penetrate the blood-brain barrier.T0070907 is a selective PPAR? inhibitor.In the present study,we established the model of TBI in mice,administered them with a RXR agonist bexarotene,investigated the protective role of bexarotene in gray mattier represented by cortex and hippocampus as well as white matter represented by corpus callosom,internal capsule and external capsule,detected its effects on astrocyte phenotype and microglia polarization,and explored PPAR?-dependent mechanisms.Methods1.A total of 72 C57bl/6J mice were divided into 4 groups randomly:sham group(n=18),vehicle group(n=18),bexarotene group(n=418)and bexarotene+T0070907 group(n=18).The controlled cortical impact(CCI)model was established to induce TBI in mice,followed by intraperitoneal administration of bexarotene or vehicle,a PPAR? antagonist T0070907 and5-Bromo-2-deoxyUridine(BrdU)for 14 consecutive days.Neurological severity scores(NSS)and Morris water maze test were performed to estimate the effect of bexarotene on sensorimotor function,spatial learning and memory ability of mice after TBI.On day 14 after CCI,immunofluorescence was used to test the neuronal suvival,synapse density,axon regeneration,myelin integrity and the number of oligodendrocyte precursor cell(OPC).Western blot was used to test the expressions of presynaptic protein marker synaptophysin,postsynaptic protein marker postsynaptic density protein 95(PSD95),growth associated protein 43(GAP43),and myelin basic protein(MBP).2.A total of 180 C57bl/6J mice were divided into 4 groups randomly:sham group(n=41),vehicle group(n=49),bexarotene group(n=49)and bexarotene+T0070907 group(n=41).The controlled cortical impact(CCI)model was established to induce TBI in mice,followed by intraperitoneal administration of bexarotene or vehicle,or T0070907 for 14 consecutive days.Immunofluorescence was used to evaluate the number of A1 astrocytes,cleaved caspase-3 positive cells,M1 microglia and M2 microglia as well as the expression of BDNF.Western blot was used to test the expressions A1 astrocyte markers,apoptosis associated proteins,microglia-derived tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),complement component 1 q subcomponent ? chain(C1q?)and BDNF,M1 microglia markers and M2 microglia markers.The mRNA levels of microglia-derived TNF-?,IL-1? and C1q? as well as the markers of M1 and M2 microglia were measured using quantitative real-time polymerase chain reaction(q PCR).Additionally,nuclear translocation of RXR? and PPAR? was measured using Western blot and PPAR?-dependenttranscription was tested using transcriptional activity assay to explore the mechanism of how bexarotene influence microglia polarization.3.A total of 45 C57bl/6J mice were divided into 5 groups randomly:PBS group(n=9),IgG group(n=9),anti-TNF-? group(n=9),anti--IL-1?group(n=9),anti-C1q? group(n=9).CCI model was established to induce TBI in mice,followed by intracerebroventricular injection of neutralizing antibodies to TNF-?,IL-1?,C1q?,PBS or IgG.To explore the effects of TNF-??IL-1? and C1q? on A 1 astrocytes,we tested A1 astrocytes and their markers using immunofuorescence and Western blot respectively.4.A total of 45 C57bl/6J mice were divided into 5 groups randomly:control group(n=5),day 1 group(n=5),day 3 group(n=5),day 7 group(n=5),day 14 group(n=5).CCI model was established to induce TBI in mice.Western blot was used to detect the expressions of RXR? and PPAR?in the whole cell,cytoplasm and nuclei.Results1.Bexarotene improved sensorimotor function and spatial memory in CCI mice,compared to vehicle-treated mice.On day 14 after TBI,bexarotene preserved neuronal survival,increased synapse density,promoted axon regeneration,preserved myelin integrity,and promoted OPC proliferation.These effects of bexarotene were attenuated by T0070907.2.Bexarotene administration significantly reduced the number of A1 astrocytes,cell apoptosis,as well as microglia-derived cytokines after TBI.In addition,bexarotene reduced M1 microglia polarization,increased M2 microglia polarization,and increased microglia-derived BDNF expression after TBI as compared with vehicle-treated mice.These benefits were partially abolished by T0070907.In addition,the number of A1 astrocytes were decreased in the anti-TNF-? group,anti-IL-1? group and anti-C1q?group when compared with the PBS group.3.The expressions of RXR? and PPAR? declined first and then elevated in plasma,while they increased first and then decreased in nuclei after TBI.On day 14 after TBI,the expressions of RXR? and PPAR?decreased in cytoplasm and increased in nuclei in the bexarotene group compared with the vehicle group.T0070907 did not influence the effect of bexarotene on RXR? but abolished its effect on PPAR?.The transcriptional activity of PPAR? increased after TBI.Bexarotene increased the transcriptional activity of PPAR? after TBI,which was attenuated by T0070907.Conclusion1.Bexarotene promotes neuronal survival,increases synapse density,promoted axon regeneration,preserves myelin integrity,and improves neurological function after TBI in mice.2.The neuroprotective effect of bexarotene may be through inhibiting A1 astrocytes and promoting microglia polarization from M1 toward M2.3.These effects of bexarotene may be partially dependent on the nuclear translocation and transcriptional activity of PPAR?.