| BackgroundSpinal cord injury(SCI)results in irreversible disorder of movement,sensation and autonomic function below the injury site with a high disability rate,causing heavy mental and economic burden on individuals,families and society.Up to now,spinal cord injury is an unsolved medical challenge all over the world,and there is still no effective therapeutic strategy in clinical practice.Spinal cord injury triggers complex pathological cascades,which result in destructive tissue damage,and culminate in incomplete tissue repair characterized by the formation of a scar.Although astrocyte scars have been proposed as the barrier to axonal regrowth,recent evidence indicated that astrocyte scar formation can beneficial for tissue repair and axon regeneration.Whereas the fibrotic scar impedes tissue regeneration.While the current research indicates that reducing scar formation is beneficial for axon regeneration and locomotion recovery,less is known about the intrinsic mechanisms that lead to scar formation after spinal cord injury.Thus,deciphering the underlying factors in scar formation is essential to develop potential interventions for spinal cord injury.Different from mature mammalian spinal cord injury,scar-free healing occurs after some peripheral tissue injuries and the central nervous injury(CNS)injuries in zebrafish,newts,even neonatal mice.Transient activation of macrophages plays a critical role in scar-free healing,including promote inflammation resolution,and mediating tissue regeneration and repair.Whereas activated bone marrow-derived macrophages(BMDM)persistently accumulate in the adult mammalian spinal cord lesions as a main component of fibrotic scar.However,less is known about the intrinsic mechanisms that lead to macrophage accumulation.The lipid content is extremely rich in CNS.Following spinal cord injury,the transcriptional profiles of macrophages are predominantly enriched in lipid catabolism in biological processes at 7 days post injury,which is a potential intervention target for spinal cord injury.Cholesterol is one of main components of CNS lipids,while excess cholesterol accumulation induces ubiquitous toxicity involved in the progression of numerous diseases,including atherosclerosis,neurodegenerative diseases and tumors.So far,little is known about cholesterol metabolism in spinal cord lesions,therefore,it is essential to elucidate the cholesterol metabolism and its potential consequence in CNS lesions.MethodsWe assessed cholesterol hemostasis in spinal cord lesions using confocal reflection microscopy and real-time PCR analyses.The appearance of cholesterol crystals was verified using transmission electron microscopy,and distribution of which was assessed combined with confocal fluorescence microscopy.The involvement of the proteins,which were predicted to promote cholesterol efflux in spinal cord lesions,were assessed with Liver X receptor(LXR)agonist and Apolipoprotein E(APOE)deficiency.We assessed cholesterol hemostasis and its role in injured peripheral nerves in wild type and APOE KO mice.For a better understanding of the different consequences in response to cholesterol accumulation between CNS and PNS,we performed in vitro experiment to study the role of serum in the clearance of myelin-derived cholesterol.We detected myelin in the neonatal spinal cord using luxol fast blue staining.We determined the consequence of excess cholesterol accumulation in CNS by transplantation of myelin and myelin-overloaded macrophages into neonatal spinal cord lesions.Results1.A large number of cholesterol crystals appeared as early as 7 days post injury(dpi)in spinal cord lesions in young-adult mice,and increased at 2 weeks post injury(wpi)and accumulated at the lesions at least for 6 weeks,while which were not detected in the spinal cord of 3 dpi and sham.In the lesion corn of mature scar,crystals were primarily present in bone marrow-derived macrophages(BMDMs).At the lesion border,crystals existed in both BMDMs and astrocytes.We detected that cathepsin D(CTSD)increased obviously from 7 dpi.Moreover,both cleaved caspase-1 and mature IL-1βwere elevated persistently from 7 dpi,indicating activation of the NLRP3 inflammasome,consistent with the appearance of cholesterol crystals.The injured spinal cord increased expression of the genes involved in cholesterol export.Moreover,oral administration of GW3965 did not reduce excess cholesterol accumulation.Unexpectedly,there were no significant differences in excess cholesterol accumulation between APOE KO and WT mice after spinal cord injury.2.We observed the accumulation of cholesterol crystals and lipid droplets in the sciatic nerve distal to the crush site at 7 dpi.Subsequently,cholesterol crystals and lipid droplets were eliminated at 6 wpi.In APOE KO mice,excess cholesterol deposited in injured nerves distal to the injury site and maintained for at least 6 wpi,suggesting limited clearance of myelin-derived cholesterol.Corresponding to cholesterol crystals deposition,active caspase-1 and mature IL-1β increased firstly after injury and then decreased at 6 wpi.However,compared to WT mice,both cleaved caspase-1 and mature IL-1β increased at 6 wpi in injured nerves of APOE KO mice.Interestingly,the injured nerves distal to the crush site were enlarged at 6 wpi in APOE KO mice.In the meantime,a large amount of CD68 positive macrophages accumulated in the injured nerves of APOE KO mice,while much less was present in WT mice.Furthermore,fibrosis was increased in APOE KO mice injured nerves.3.We first incubated the BMDMs with myelin debris for sufficient endocytosis of cholesterol.After that,we cultured the myelin-loaded cells in culture medium for hours.In the absence of FBS or high-density lipoprotein(HDL),excessive cholesterol accumulated in BMDMs after 48 hours.But in the appearance of FBS,accumulated cholesterol was effectively removed.In response to excess cholesterol accumulation,BMDMs up-regulated the expression of pro-inflammatory factors Il6 and Inos,and the up-regulation was reversible with removal of accumulated cholesterol.4.We observed that luxol fast blue stained myelin lipids are not present in the postnatal day 2(P2)mouse spinal cord,suggesting that P2 spinal cords are devoid of myelin-derived lipids.We found that spinal cord lesions are repaired without excess cholesterol accumulation at 2 weeks post P2 crush injury.At 2 weeks post myelin injection,we detected cholesterol crystals deposited in macrophages at the lesion core,and a scar was formed.Meanwhile,we observed fibrosis after myelin debris injection.At 2 weeks post transplantation of myelin-overloaded BMDMs,cholesterol-overloaded BMDMs clustered closely in the lesion site,similarly a scar was formed.ConclusionsWe found that excess cholesterol accumulates in phagocytes and is inefficiently removed in spinal cord lesions in young-adult mice,which is associated with late-onset NLRP3 inflammasome activation.Although the injured spinal cords do not lack the cholesterol exporter(ATP-binding cassette transporter A1,ABCA1)and cholesterol carrier(APOE),but excess cholesterol accumulation is unresolved in spinal cord lesionsPeripheral nerve injury leads to Wallerian degeneration,which results in excess cholesterol accumulation,bit is subsequently removed spontaneously.Moreover,blocking reverse cholesterol transport(RCT)limits cholesterol clearance in injured peripheral nerves.Meanwhile clearance of excessive cholesterol by RCT prevents NLRP3 activation,macrophage accumulation and fibrosis.In vitro model suggests that myelin-derived cholesterol cannot be efficiently removed from macrophages unless an effective acceptor of cholesterol is present in the culture medium.Moreover,clearance of excessive cholesterol reverses the upexpression of pro-inflammatory factors Il6 and Inos.However,quickly repairing of blood spinal cord barrier,which limits the efflux of cholesterol to blood through RCT,should be the barrier that leads to inefficient cholesterol clearance after spinal cord injury.Cholesterol derived from myelin debris leads to excessive cholesterol accumulates in macrophages and scar-forming in neonatal lesions,characterized by non-neural lesion core,gliosis and fibrosis,similar to the adult lesions.Altogether,our results suggest that myelin-derived cholesterol results in not only excess cholesterol deposition but also impaired scar-free healing with activated macrophage accumulation and fibrosis in spinal cord lesions.Moreover,quickly repairing of blood spinal cord barrier should be the barrier that limits cholesterol clearance in spinal cord lesions.Since efficient cholesterol clearance by RCT prevents macrophage accumulation and fibrosis in injured peripheral nerves,this study suggests that promoting cholesterol clearance and reestablishing homeostasis of cholesteroloverloaded macrophages are potential strategies to facilitate scarless healing in the CNS. |
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