Human Adipose Mesenchymal Stem Cells Derived Exosomes Promote Recovery From Traumatic Brain Injury By Inhibiting Microglia/macrophages Activation In Rats

Background:Traumatic Brain Injury(TBI)is a leading cause of morbidity and mortality in young individuals under the age of 45,and survivors after TBI will face severe neurological deficits and behavioral disorders.Besides,TBI is the critical risk factor of developing a chronic neurodegenerative disease,such as Alzheimer’s disease,Parkinson’s disease.However,the traumatic brain injury is still lack of effective clinical treatment so far.Mesenchymal stem cells(MSCss)are a kind of adult stem cells derived from mesoderm tissue.They have multipotent differentiation potential and self-renewal ability,with low immunogenicity.They are considered to be ideal stem cells for the treatment of injury disease.Many studies have shown that MSCs can significantly improve neurological deficits after TBI.Recent studies show that the effect of neuronal remodeling and functional recovery promoted by MSCss transplantation in treating TBI in animal models seems to be related to the paracrine action,such as exosomes,but not cell replacement.Recent evidence suggests that exosomes can ameliorate multiple disease processes by suppressing inflammatory response in injured tissue,reducing cell apoptosis,promoting regeneration of functional cells,and angiogenesis.Compared with MSCss,exosomes are easily mass-produced and stored,without the risks of embolization and immune rejection.So,it will be a safer strategy for exosomes transplantation to treat TBI.Object:To study the therapeutic effect of exosomes derived from human adipose mesenchymal stem cells on TBI and its possible mechanisms.Methods:The rat TBI model was established by the Feeney’s free-fall method,followed 24 hours later by contralateral intracerebroventricular(ICV)injection with human adipose mesenchymal stem cell(hADSC),human adipose mesenchymal stem cell-derived exosome(hADSC-ex),or phosphate-buffered saline(PBS).The modified neurological Severity Scores(mNSS)and footfault test were performed to assess the recovery of sensorimotor function after treatment;the expression of inflammatory factors in damaged brain tissue and cerebrospinal fluid were detected by quantitative PCR and ELISAs;CD68 immunohistochemical staining was performed to observe the microglial/macrophage infiltration in the lesion boundary zone;Tunel-NeuN immunofluorescence staining was performed to evaluate neuronal apoptosis the lesion boundary zone,Brdu-NeuN and Brdu-GFAP immunofluorescence staining was used to assess the neurogenesis in dentate gyrus(DG)and astrocyte proliferation in the lesion boundary zone.Sequential in vivo imaging was performed to observe the biodistribution and migration of DiR labeled hADSC-ex(DiR-hADSC-ex)after ICV injection.After DiI-labeled hADSC-ex(DiI-hADSC-ex)ICV injection,the immunofluorescence staining for the neural cell markers CD11b or IBA1(microglia/macrophage),GFAP(astrocyte),NeuN or MAP2(mature neuron),and MBP(mature oligodendrocyte)were performed in brain coronal sections and discrete neural cells to elucidate the cellular uptake of hADSC-ex in vivo.The primary mixed neural cells were incubated with DiI-hADSC-ex for 24 hours,and the change of DiI fluorescence intensity with time in each type of neural cell was detected by Cytation5 cell imager.Finally,in the LPS+IFN-y-induced primary microglia inflammatory model,microglia were incubated with hADSC-ex for 12 hours,and the expression of M1 related factors was measured by quantitative PCR.The phosphorylation level of three subunits of NF-kB and p38 MAPK signaling pathway were detected by Western blot and the p65 nuclear translocation was determined by immunofluorescence.Results:ICV injection of hADSC-ex 24h after TBI promoted sensorimotor functional recovery,suppressed neuroinflammatory,reduced neuronal apoptosis,and increased neurogenesis and astrocyte proliferation in rats.The therapeutic effect was comparable to that of hADSC.In the meantime,sequential in vivo imaging displayed increasing aggregation of DiR-labeled hADSC-ex in the lesion area.Additionally,immunofluorescence staining for the neural cell markers in brain coronal sections and discrete neural cells showed distinct overlaps between DiI(hADSC-ex)and CD11b or IBA1-positive cells.In primary mixed neural cells,the DiI fluorescence signal was significantly enriched in microglia.In the LPS+IFN-γ-induced primary microglia inflammatory model,hADSC-ex inhibited the expression of M1-related factors and decreased the phosphorylation levels of p65,IKKα/β,IKBα and p38 and the nuclear translocation of p65.Conclusion:hADSC-ex could specifically target microglia/macrophages and suppress their activation during brain injury,reducing neuroinflammation,thereby improve injury microenvironment and facilitating functional recovery.