stroke is one of the leading causes of death worldwide and results in disabilities in about 75% of its survivors.1 , 2 it is of paramount importance to develop novel neuroprotective therapies to get enhanced recovery . brain self - repair by neuronal replacement from endogenous neural stem cells and precursors is insufficient and results in irreversible loss of neurons . amplification of this self - repair mechanism by neuroremodeling could be a promising strategy for stroke treatment . tissue responds to injury and remodels after ischemia by altering their gene and protein expression . micrornas ( mirnas ) , a family of non - coding rnas of 2025 nt , play pivotal roles during this remodeling process by regulating target genes at post - transcriptional level . several studies have demonstrated significant alterations in the  cerebral  mirna - ome  following ischemia.4 , 5 these reports suggest that mirnas may act as innovative gene therapeutic candidates contributing to neurogenesis , angiogenesis , and neural plasticity . among numerous mirnas , microrna-124 ( mir-124 ) is most abundantly expressed in the central nervous system ( cns ) . it has been reported that attenuation of endogenous mir-124 in neural progenitor cells in subventricular zone ( svz ) can abolish neuronal differentiation , whereas overexpression leads to acquisition of neuronal identity.7 , 8 after ischemia , the expression of mir-124 was increased in ischemic penumbra . exogenous mir-124 , by using agomir or liposomated mimic , could reduce infarct area.9 , 10 recently , a study showed that viral vector expressing mir-124 yielded increased angioneurogenesis and significant neuroprotection against stroke . however , blood brain barrier ( bbb ) has been proven to be the major obstacle for delivery of drugs to the cns . it has been estimated that 98% of all small molecules do not cross the bbb . recently , accumulating evidences showed that exosomes , which are lipid membrane vesicles of 30 to 100  nm in diameter , could cross bbb and carry cargoes such as proteins , lipids , and nucleotides , mediating the intertwined brain remodeling events after stroke.13 , 14 in order to achieve neuron - specific targeting , rabies virus glycoprotein ( rvg ) has been engineered to exosomal surface via fused protein lysosome - associated membrane glycoprotein 2b ( lamp2b)-rvg . in this study , we wondered whether delivery of mir-124 utilizing rvg - modified exosomes could protect against cortical ischemia . our results showed that rvg - exosome - mediated mir-124 delivery could efficiently ferry mirna to the ischemic region and ameliorate brain injury by promoting neural progenitors to obtain neuronal phenotype at infarct site . to generate rvg - exos , we fused the rvg peptide to lamp2b and  introduced the pcdna3.1()-rvg - lamp2b plasmid into bm - mscs . then we loaded the rvg - exos with mir-124 mimics or scrambled mirnas via electroporation ( figure  1a ) . to validate whether the rvg - lamp2b plasmid was successfully electroporated into bm - mscs , we assessed the protein level of lamp2b ( figure  1b ) . compared with bm - mscs electroporated by pcdna3.1( ) plasmid , the western blotting showed that the rvg - lamp2b - modified cells  expressed a higher level of lamp2b . as previously described , modification did nt appear to affect the physical property of the modified exosomes observed by electron microscopy ( em ) ( figure  1c ) . , cd63 , alix , and golgi marker gm130 further confirmed the identity of the exosomes and inclusion of rvg - lamp2b on the exosome ( figure  1d ) . the results showed that the expression of mir-124 was significantly higher in exosomes loaded with mir-124 mimics ( rvg - exos - mir124 ) than that of exosomes loaded with scrambled mirnas ( rvg - exo - scr ) ( figure  1e ) . moreover , there were about 1% of the mirnas encapsulated into the exosomes , meaning about 500 mirnas per exosome . all of these data confirmed  the efficient loading and stability of mirna in the modified exosomes . to investigate the potential of rvg - exos to specially deliver mir-124 to the injured region , we established a focal ischemia model . unmodified exosomes ( unmodified - exos ) or rvg - exos were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate ( dii ) dye . to wash away free dii , exosomes were resuspended in 0.9% saline and centrifuged for three times before intravenous injection into c57bl/6 mice at 1  day post ischemia ( dpi ) . mice were sacrificed 2  hr after dii labeling for observation of exosome distribution ( figure  2b ) . serial coronal sections were made and images were taken by confocal microscopy . to test whether mirnas could get to the ischemic region fam could be scarcely detected 2  hr post injection ( figure  2c ) . this indicated that without encapsulation by exosomes , mirnas could be hardly endocytosed by the cell . then we injected dii - labeled unmodified - exos and rvg - exos , respectively . in unmodified - exos injected mice , few dii could be observed at the core of the injured region ( figure  2d ) . however , dii fluorescence was predominantly detectable in the ischemic region in rvg - exos injected mice , with relatively weak fluorescent signal scattered in the normal brain ( figure  2e ) . these data showed that compared with unmodified - exos , rvg - exos could efficiently promote targeting delivery . focal bbb disruption may also facilitate the cross of rvg - exos at the infarct site , making rvg - exos effective drug delivery tools for ischemia treatment . notably , most of the ischemic area was dii positive in the rvg - exosome delivery group , suggesting that besides neurons , astrocytes and endothelial cells might be also the recipient cells . besides the brain localization of the injected rvg - exosomes , we also observed abundant exosome distribution in liver , spleen , and lung , which should be attributed to the reticuloendothelial system function ( figure  s1 ) . moreover , we did not observe any significant change of food intake , body weight , and physical activity after exosomal injection , suggesting no obvious side effects of exosome injection . after loading modified exosomes with mir-124 , we examined whether rvg - exos could deliver mir-124 to the ischemic region . by qrt - pcr analysis , we compared the expression level of mir-124 in the contralateral and ipsilateral ( to the injured side ) cortex of rvg - exos - mir124 and rvg - exos - scr treated mice ( figure  3a ) . in the contralateral cortex , the expression of mir-124 is relatively higher in rvg - exos - mir124 treated mice than the control group , yet without statistical  significance . in the injured cortex , the  expression of mir-124 was significantly increased after rvg - exos - mir124 injection . these results demonstrated that mir-124 could be efficiently delivered to the infarct site by rvg - exos . mirnas target about 60% of mammalian genes and are involved in most biological and pathological processes . based on the gene regulatory role of mirnas , we suppose  that ectopic mir-124 expression may have dramatic effect on the gene network after stroke . we produced the ischemic model and injected rvg - exos - mir124 and rvg - exos - scr at 1 dpi . then we harvested the ischemic tissue at 3 dpi and examined the mrna expression of e2f1 , gli3 , stat3 , and tcf12 , which were previously  predicted under the regulation of mir-124 and participated  in  neural differentiation17 , 18 , 19 , 20 , 21 ( figure  3b ) . the results of  qrt - pcr showed that there was no significant difference of mrna expression of e2f1 and tcf12 , while significant decrease  of gli3  and stat3 upon exosomal mir-124 injection , indicating the functional regulation of target genes by exosomal mir-124 . since mir-124 is well known for its proneuronal role in both the  developing and mature brain , we further investigated the effect of mir-124 on neural differentiation and neurogenesis post ischemia . we injected rvg - exos with no mirnas , rvg - exos - scr , and rvg - exos - mir124 into ischemic mice at 1 dpi . we first examined the expression of neural progenitor marker sox2 in the ischemic cortex at 5 dpi ( figure  4a ) . in the rvg - exos and rvg - exos - scr group , sox2 was induced rapidly around the lesion area at 5 dpi as previously reported ( figures 4b and 4c ) . however , cell quantification in the ischemic region showed that the number of sox2-positive cells decreased significantly after rvg - exos - mir124 injection ( figures 4d and 4e ) . the expression of another neural progenitor marker nestin was also examined ( figures 5a and s2 ) . similarly , nestin exhibited the same expression change at 5 dpi . in the injured cortex , cells richly upregulated nestin in rvg - exo and rvg - exo - scr treated mice ( figures 5b and 5c ) , while there was a notable reduction of nestin - positive cells in rvg - exos - mir124 treated mice , suggesting the fate change of neural progenitors ( figures 5d and 5e ) . both sox2 and nestin are markers of the neural progenitor cells , and there are a robust percentage of the neural progenitor cells expressing both markers . immunohistochemistry of sox2 and nestin in the ischemic cortex at 5 dpi revealed that nearly all the sox2-positive neural progenitors around the injured area were nestin positive ( figure  6 ) , which suggests that exosomal mir-124 delivery might promote these progenitor cells toward neurogenesis . considering the expression of sox2 and nestin decreased significantly after ischemia , we wondered whether the fate of these  neural progenitors was changed into neuronal lineage under  the effect of mir-124 . then we examined neurogenesis  at  7  dpi ( figure  7a ) . the immature neuronal marker doublecortin  ( dcx ) scattered at an infarct site in  rvg - exos and rvg - exos - scr treated mice ( figures 7b and 7c ) . but the number  of  dcx - positive cells in rvg - exos - mir124  treated mice  was  approximately twice as much as that of control groups  ( figures 7d and 7e ) . our results showed that exosome - encapsulated mir-124 greatly promoted adult neurogenesis  post  ischemia via biasing neural progenitors into neuronal lineage . in the present study , we for the first time performed targeting delivery of mir-124 to the ischemic cortex using modified exosomes . the rvg - modified exosomes efficiently delivered mir-124 to the infarct region and promoted neurogenesis in the adult cortex after ischemia . our results demonstrate that systemic administration of engineered exosomes could be a superior candidate for specifically transporting gene drugs for the treatment of stroke . neuroremodeling by neurogenesis after stroke has been proven existing , although weak , especially after adult cortical ischemia.24 , 25 therapeutically enhancing neurogenesis would greatly benefit recovery . as mirnas regulate myriads of genes , a handful of studies target on mirnas therapeutic potentials . mirna-9 , mirna let-7b , mir-137 , and mir-184 have been reported to regulate neural stem cell proliferation and differentiation at different stages.27 , 28 , 29 , 30 mir-124 , the most brain - enriched mirna , is svz neuronal fate determinant and mediates stroke - induced neurogenesis in adult svz and striatum.7 , 11 , 31 in our study , we found that ectopic expression of mir-124 in cortex promoted neural progenitors to differentiate into neuronal lineage and further yielded the amelioration against ischemia via cortical neurogenesis . we carefully limited the infarct region in the cortex  and excluded all those corpus callosum injured cases . the  dcx - positive cells formed clusters and distributed along the  ischemic border . but we did nt observe the migrating route of new born neurons from svz , the well acknowledged neural stem cell niche . it will be interesting to investigate the cellular origin of these cortical neuroblasts and explore the underlying mechanisms . since the first report that exosomes contain both mrnas and  mirnas which can be delivered from parent cells to recipient  cells , more and more studies revealed the roles of  exosome - mediated intercellular genetic communication in cns  diseases . glioblastoma - derived exosomes vehicle mrnas / mirnas  to normal host cells to promote tumor growth . schwann cell - derived mirnas stimulated axonal growth and regeneration after nerve damage when taken up  by adjacent neurons . induced endothelial cell - derived exosomes modulate the cellular status of pericytes by transferring mirnas . compared with other kinds of cells , cultured msc is an efficient producer for exosomes , allowing the production on a  larger scale . msc - derived exosomes are capable of transferring mir-133b to neurons and subsequently contribute to neurite outgrowth after stroke.37 , 38 these indicate that msc - derived exosomes not only cross the bbb , but also deliver functional cargo to modulate gene expression in the recipient cells . due to  the small size , exosomes can avoid phagocytosis by macrophages . they are naturally stable to escape endosomal - lysosomal degradation compared with polymeric nanoparticles and liposomes . collectively , with lack of immunogenicity , these features make exosomes promising candidates for gene drug delivery . in our study , the qrt - pcr showed that the expression of mirna-124 in the ischemic cortex was significantly increased at 3 dpi after exosome injection , indicating persistent and sufficient  mir-124  concentration  for treatment . considering mscs - derived exosomes package a repertoire of bioactive molecules and can promote the outcome of stroke without artificial loading , there may be other factors besides mir-124 contributing to the beneficial effects observed in  our experiment . to date , targeting drugs to brain cells is a troublesome challenge . in our study , dii fluorescence ferried by rvg - exos scattered in the normal brain , which was consistent with the distribution pattern reported by  alvarez - erviti in 2011 . however , compared with unmodified  exosomes , the concentrated fluorescence in the ischemic region showed high targeting efficiency of rvg - exos with bbb disruption . except the rvg system , modifying exosomal surface proteins with targeting ligands or receptor - specific antibodies has  also been used to achieve tailored delivery . considering the therapeutic application of exosomes , the better alternative may need a homing device that is specific for a target which is induced or upregulated under pathological conditions . in our study presented here , most of the ischemic area was dii positive in the rvg - exosome delivery group , suggesting that besides neurons , astrocytes and endothelial cells might be also the recipient cells . future studies identifying the targeting peptide specific for neural progenitors and with high bbb penetrating capacity are still needed . in summary , our results have shown that rvg - modified exosomes are efficient tools for gene drug delivery to the ischemic cortex . systemic administration of rvg - exosomes loaded with mir-124 can promote cortical neural progenitors to obtain neuronal identity and attenuate ischemic injury by cortical neurogenesis . considering the  intrinsic weak ability of neurogenesis in the adult cortex after  ischemia , our study suggests that treatment with rvg - exos - mir124 during the acute phase of stroke can be a promising therapeutic strategy for neuroprotection and neuroremodeling via promoting neurogenesis . all animal experiments were carried out under protocols approved by the animal care and use committee of fourth military medical university . focal cortical ischemia was induced as previously described . briefly , after the tail injection of rosa bengal ( sigma ) at 25  mg / kg , a skull window was made without hurting brain tissue , ranging from 0.8 to 2.4  mm posterior to the bregma and 0.5 to 2.1  mm right to the midline . the skull window was illuminated for 9.5  min by an optic fiber connected to a cold light source . lamp2b was cloned with cdna from mice cortex and was inserted between nhe1 and bamh1 into a pcdna3.1( ) vector . primers designed to encode rvg were used to introduce  the targeting ligand between xho1 and bspe1 . bm - mscs were electroporated with pcdna3.1()-rvg - lamp2b plasmid to produce rvg - positive exosomes ( rvg - exos ) . cells were washed by pbs and suspended in -modified minimum essential medium ( mem , hyclone ) , supplemented with 20% fetal bovine serum ( fbs , gibco ) and antibiotics . at 3  days later , cells tightly adhered to the plastic flasks were considered to be p0 bm - mscs . prior to culture medium collection , bm - mscs were washed twice with pbs , and the medium was switched to serum - free medium . after incubation for 48  hr , the supernatant was collected and went through sequential ultracentrifugation at 2,000   g for 30  min , 10,000   g for 30  min , and 100,000   g for 4  hr at 4c . to avoid contamination  by  the fbs - derived exosomes , the exosomes were washed once with pbs and resuspended for further characterization . for fluorescence labeling of exosomes , a 4  mg / ml solution of dii ( molecular probes ) was added to the pbs ( 1:200 ) and incubated following the manufacturer s instructions . after the isolation by  sequential ultracentrifugation as mentioned above , dii - labeled exosomes ( dii - exos ) were resuspended in 0.9% saline and centrifuged at 10,000   g for 30  min . this procedure was repeated for three times to wash free dii away ( figure  2b ) . then dii - exos were injected intravenously through tail vein into ischemic mice at 1 dpi . for em , purified exosomes from bm - mscs were resuspended in pbs and imaged by transmission electron microscope as detailed before . exosomes at a total protein concentration of 12  g ( bca protein assay kit , thermo scientific ) were electroporated with 12  g mir-124 mimics ( rvg - exos - mir124 ) or scrambled mirnas ( genepharma ) ( rvg - exo - scr ) at 350  v and 150  f in 0.4  cm electroporation cuvetes . to remove the unincorporated free mir-124 , exosomes were washed with cold pbs twice by ultracentrifugation at 100,000   g for 90  min . efficiency of electroporation was validated by qrt - pcr for detection of mir-124 levels . for cell western blotting , control bm - mscs or bm - mscs electroporated with pcdna3.1-rvg - lamp2b plasmid were lysed by ripa buffer . the primary antibodies used were as follows : goat anti - lamp2b ( 1:1,000 , abcam ) , mouse anti--actin ( 1:5,000 , sigma ) , mouse anti - cd63 ( 1:1,000 , abcam ) , rabbit anti - alix ( 1:1,000 , abcam ) , rabbit anti - gm130 ( 1:1,000 , abcam ) , and mouse anti - gapdh ( 1:5,000 , abcam ) . corresponding hrp - conjugated anti - goat , anti - rabbit , or anti - mouse ( 1:10,000 , pierce ) secondary antibodies were incubated for 1  hr at room temperature . total rna was extracted from exosomes or ipsilateral / contralateral cortex using trizol reagent ( invitrogen ) according to the manufacturer s instructions . for analysis of mrna levels , reverse - transcription was performed using primescript first - strand cdna synthesis kit ( takara ) , and cdnas were used for qrt - pcr using primescript rt master mix ( takara ) . for analysis of mirna levels , total rna was reverse - transcribed  to  cdna using mircute mirna first - strand cdna synthesis kit ( tiangen biotech ) , and qrt - pcr was carried out using mircute mirna qpcr detection kit ( sybr green ) ( tiangen biotech ) . all pcr reactions were run in triplicate and mrna or mirna expression , relative to -actin or u6 snrna , was calculated using the 2 method . serial coronal sections of 14  m in thickness were prepared on a cryostat . after being incubated with 0.3% triton x-100 and 3% bovine serum albumin ( bsa ) in pbs for 1  hr , the following primary antibodies were incubated overnight at room temperature : rabbit anti - sox2 ( 1:200 , sangon biotech ) , goat anti - nestin ( 1:200 , santa cruz biotechnology ) , and rabbit anti - dcx ( 1:400 , abcam ) . corresponding secondary antibodies as alexa fluor 488 and alexa fluor 594 ( donkey anti rabbit or anti - goat igg , 1:800 , invitrogen ) were incubated for 3  hr at room temperature . cellular nuclei were stained by hoechst 33342 ( 1:100 , sigma ) . for cell quantification , the ischemic region was defined by the outer lining of nestin - positive cells as before . cells were counted from every eighth slice of the ischemic region and five mice were included for comparison in each group . a two - tailed student s t test or anova was used to analyze the relative expression of qrt - pcr and cell counting .
the intrinsic ability of neurogenesis after stroke has been proven weak , which results in insufficient repair of injury in the nerve system . 
 recent studies suggest multiple micrornas ( mirnas ) are involved in the neuroremodeling process . 
 targeted mirnas delivery for amplification of neurogenesis is promising in promoting the prognosis after ischemia . here 
 , we showed that modified exosomes , with rabies virus glycoprotein ( rvg ) fused to exosomal protein lysosome - associated membrane glycoprotein 2b ( lamp2b ) , could efficiently deliver mir-124 to the infarct site . 
 systemic administration of rvg - exosomes loaded with mir-124 promoted cortical neural progenitors to obtain neuronal identity and protect against ischemic injury by robust cortical neurogenesis . 
 our study suggests that rvg - exosomes can be utilized therapeutically for the targeted delivery of gene drugs to the brain , thus having great potential for clinical applications .