the poor regenerative capability of the central nervous system ( cns ) , compared to the peripheral nervous system ( pns ) , limits recovery from a number of traumatic and degenerative conditions . on the other hand , central neuroregeneration has been observed in limited contexts ( e.g. , [ 1 , 2 ] ) , indicating a need to better understand mechanisms underlying regenerative capacity . a key advance in understanding mechanisms underlying the robustness of pns regeneration was the identification and characterization of a retrograde injury signaling ( ris ) pathway , which is hypothesized to require transport of injury signals from the injury site to the cell body . details of this pathway in peripheral neurons have been well summarized in several reviews [ 37 ] . briefly , rapid ion influx at the injury site generates a rapid response that propagates retrogradely to provide the first indication of lesion events [ 3 , 810 ] . a slower component of ris results in dynein - dependent transport of an injury - induced signaling complex from the site of injury to the nucleus [ 3 , 7 , 11 ] . importantly , injury also induces local axonal translation of several proteins required for ris complex transport , including importin 1 ( karyopherin 1 ) , ranbp1 , and vimentin [ 35 , 7 , 12 , 13 ] . importin 1 , among its diverse roles [ 1417 ] , is a key node in ris pathways , as its knockout attenuates transcriptional responses to nerve injury and delays functional recovery in vivo . interestingly , recent evidence suggests that importin 1 may also play a role in central neuronal regeneration . importin 1-associated stat3 signaling molecules were transported retrogradely after injury of hippocampal neurons , but only when the importin - stat association was intact [ 18 , 19 ] . despite these compelling advances , several key gaps remain in understanding ris mechanisms in central neurons . a key unknown is whether and by what mechanisms levels of importin 1 and other ris - associated transcripts are altered locally in the axon in response to injury . the goal of this study was to perform an initial examination of importin 1-dependent ris mechanisms in the cns , through the use of a new hippocampal explant system , which enables examination of axonal mrna and protein expression independent of neuronal cell bodies . our results suggest a biphasic axonal response , in which levels of several axonal transcripts , including those associated with ris , increase rapidly in axons after injury . importin 1-dependent activity at the nucleus then appears to modulate a second wave of ris - associated transcripts , which are likely to further support axonal outgrowth . all animal protocols were approved by the university of maryland institutional animal care and use committee ( iacuc ) . details and validation of the explant culture system , including assessment of axonal purity , brains were harvested and maintained in cold hank 's balanced salt solution ( hbss ) . hippocampi were detached from the surrounding tissue and seeded on lysine - coated glass cover slips in neurobasal media supplemented with 2% b-27 . media were changed carefully every 3 days , so as not to dislodge the explant . explants were allowed to grow for seven days , after which axons were severed with a needle at a distance ~2/3 of the longest axons away from the explant edge . injured or noninjured axons were collected using appropriate lysis buffer and a micropipette , through careful observation under a light microscope , and aspiration perpendicular to the axons . to avoid any cell body and dendritic contamination , we avoided regions at the explant edge ( figure 1(a ) ) . for severed axons , tissue was collected both proximal and distal to the injury site , to enable comparison with corresponding control axons . we performed additional analysis on each sample to assess the exclusive axonal nature of the preparation , as described previously . for rt - pcr assays , we confirmed the absence of -actin mrna by pcr , which resides only in the soma but is restricted from axons [ 2123 ] . for protein assays , we confirmed the absence of neun , a neuron - specific nuclear , and thus axon - excluded , protein by immunoblotting . any samples that were contaminated with cell body markers were not used for further analysis ; ~30% of samples used for pcr and ~10% of the samples used for western blot experiments were rejected due to cell body contamination . control or injured axonal samples were collected at 1 , 6 , 15 , and 24 hours after injury . for detection of axonal transcripts , ~100  ng of rna from axons was used as a template for reverse transcription ( rt ) with m - mlv reverse transcriptase ( invitrogen , carlsbad , ca ) and an oligo ( dt ) primer at 90c for 1  h. the rt reactions were diluted 10-fold and used for transcript - specific pcr . for primer sequence , we used primer 3 tool based on specific nucleotide sequence found on pubmed . negative controls were performed on each sample , and they consisted of rna processed without the addition of reverse transcriptase . for quantitative rt - pcr , the control and sample rt reactions above were amplified using the thermocycler detection system ( bio - rad , hercules , ca ) . these reactions were performed using the ssofast evagreen supermix ( bio - rad , hercules , ca ) for all transcripts . thermal cycling was initiated with an initial denaturation at 50c for 2  min and 95c for 10  min followed by 40 cycles at 95c for 15  s and 60c for 1  min . relative levels of individual transcripts were calculated by normalizing to the csf1 control using a comparative threshold ( ct ) value method . briefly , the ct for each transcript was determined using the automatic ct algorithm of the my iq software to calculate the optimal baseline range and threshold values . individual ct values were then determined by subtracting the csf1 ct value from the individual transcript ct values . from this , the calculation of ct was determined by subtracting ct sample ( injury to the axons ) from ct control ( no injury to axon ) . the fold difference was then expressed as 2 , with ct + sd and ct  sd , where sd is the standard deviation attained from ct values as described by livak and schmittgen in . transcript levels are expressed relative to ct values of the control ( uninjured ) axons . as some transcripts were expressed at very low levels in axons , primers were validated in whole explant lysate . actinomycin d ( amd ) dose was set at 5  g / ml , a dose that rapidly and persistently suppresses all classes of transcription in mammalian cells , based on h - uridine incorporation and reduction of a variety of transcripts differing in gc - richness of sequence and length . treatment duration was varied from 1 to 3  hrs to test amd efficacy in explants . as amd treatment resulted in substantial reduction of multiple transcripts within two hours , explants were treated with this dose for two hours before being injured ( or not , for controls ) . samples were collected one hour after injury and rt - pcr was performed as above , with amd treated uninjured axons used as controls . importazole ( ipz ; sigma - aldrich , st . louis , mo ) has been well - characterized in nonneuronal cells . we followed a similar approach to characterizing inhibition of importin - mediated nuclear import in our system . explants were transfected 12  hrs prior to ipz treatment with nfat - gfp expression plasmid ( pkw520 , a kind gift from dr . karsten weis ) , using effectene transfection reagent ( qiagen , valencia , ca ) as per manufacturer 's instructions . all transfections took place in neurobasal media supplemented with 2% b-27 ( life technologies ) . ipz was used at concentrations of 18  m for up to 24 hours , supplying fresh media with ipz every 12 hours . for controls , fresh media without ipz were used . at 22 hours ( 1.5 hours prior to 24-hour treatment ) , ionomycin was added at 15  m to induce intracellular calcium influx and trigger nuclear import . to assess import , 100200 cells for each condition were analyzed and nuclear accumulation of nfat - gfp was assessed using image j. to test cell viability , after transfection and ipz and ionomycin treatments , cultures were washed three times and assessed using live / dead reagent ( invitrogen , grand island , ny ) , as per manufacturer 's protocol . explants were incubated for 20 minutes , washed , and imaged via an inverted nikon te-2000e microscope . cell viability of explants containing 0.1 to 0.4% of dmso was performed as a control . hippocampal explants were fixed with 4% paraformaldehyde in pbs for 10 minutes and rinsed with pbs three times . following permeabilization with 0.2% triton x-100 in pbs , the cells were blocked with 10% fetal goat serum and 3% bsa for 30 minutes . 1  :  1000 dilution of smi-31 ( abcam inc . , cambridge , ma ) and/or 1  :  500 dilution of map2 ( abcam inc . , cambridge , ma ) in bsa was applied for 1 hour at room temperature , followed by three washes in pbs . fluorescently labeled secondary antibody ( alexafluor-488 and alexafluor-594 , life technologies ) was then applied for 1  hr at 37c , followed again by three washes in pbs . after 1 , 3 , and 6 hours of injury , control ( uninjured ) axons and positive control ( whole explant ) samples were lysed using np40 lysis buffer mixed with protease inhibitor ( fisher scientific , houston , tx ) and phosphatase inhibitor ( roche diagnostics , indianapolis , in , usa ) . the homogenate was further lysed in liquid nitrogen , and supernatant was stored at 80c . protein concentration was measured using a bicinchoninic acid ( bca ) protein assay kit ( pierce ; thermo fisher scientific , rockford , il , usa ) . an equal amount of protein ( 60  g ) was loaded into each lane , run on 415% mini - protean precast gel ( bio - rad , hercules , ca ) , and transferred to pvdf membranes ( millipore , billerica , ma ) . the membranes were blocked overnight using casein - blocking buffer ( vector lab , burlingame , ca ) and incubated for 2  h at rt either with ranbp1 antibody ( diluted 1  :  100 ; santa cruz , dallas , tx ) or with vimentin antibody ( diluted 1  :  100 ; abcam , cambridge , ma ) . alternately , membranes were incubated for 35 minutes at rt with importin 1 antibody ( diluted 1  :  300 ; abcam , cambridge , ma ) , gapdh ( diluted 1  :  60000 ; fitzgerald , acton , ma ) , or neun ( diluted 1  :  500 ; millipore , billerica , ma ) . appropriate secondary antibodies and duolux chemiluminescent were used for detection following the protocol provided in vectastain abc - amp kit ( vector lab , burlingame , ca ) . the membranes were detected using chemidoc xrs+ ( bio - rad , hercules , ca ) and analyzed using image lab software ( bio - rad , hercules , ca ) , which accounted for regional background subtraction . bright - field and phase contrast imaging were performed on an inverted te-2000e microscope ( nikon , melville , ny ) outfitted with lumen - pro2000 ( prior scientific , rockland , ma ) illumination system . a custom built chamber ( precision plastics , beltsville , md ) maintained temperature , humidity , and co2 levels during imaging . leica sp5x confocal microscope ( buffalo grove , il ) was used for explant imaging . the confocal system was equipped with multiple laser lines , including a 405 diode , an argon laser ( 458 , 476 , 488 , 496 , and 514  nm ) , and a white light laser ( 470670  nm in 1  nm increments ) ; samples were imaged using either a 10x or 40x objective with filtering appropriate to visualize hoechst ( excitation 350  nm and emission 451  nm ) , smi-31 ( excitation 579  nm and emission 599  nm ) , and map2 ( 490  nm and emission 530  nm ) markers . all pcr experiments were assayed in triplicate for at least three independent experiments ( axonal injury experiments : n = 3 for 1 and 24  hrs and n = 4 for 6 and 15  hrs time points ; amd and ipz experiments : n = 3 , except for ranb1 , in which only a single sample amplified this transcript , possibly due to ipz effects on both control and injured populations , unrelated to injury response ) . quantitative pcr data were compared statistically using the relative expression software tool ( rest , qiagen , valencia , ca ) , as described previously . for importazole characterization , 100200 cells were quantified from two different explants for each condition , control , and ipz treatment for 12  hrs and 24  hrs . two - way student 's t - test , with bonferroni 's adjustment for multiple comparisons , was used to compare means . for immunoblots , due to lack of normality within data sets , data were reciprocally transformed and means of transformed data were compared using 1-way anova followed by dunnett 's test . for all experiments , type i error  was set to 0.05 . to examine the axonal expression and regulation of transcripts involved in cns ris , we developed a mouse hippocampal explant system , which enables injury and analysis of isolated axons . for this study , we cultured p1 explants for seven days and cut the axons on the seventh day to study axonal response to injury at time points up to 24 hours ( figures 1(a ) and 1(b ) ) . immunofluorescence evaluation of an axonal marker , phosphorylated neurofilament ( smi-31 ) , and a dendritic marker , microtubule - associated protein 2 ( map-2a ) , confirmed the axonal nature of long projections from the explant ( figure 1(c ) ; cf . ) . the exclusion of nuclear or cell body markers was assessed in the axonal lysate of each sample , and contaminated samples were excluded from analysis . transcripts of interest present and absent in cell lysate and axons are shown in table 2 . based on the retrograde transport of ris complexes observed in both the pns and cns [ 3 , 4 , 7 , 12 , 18 , 19 ] , we hypothesized that levels of transcripts encoding ris complex components would too be increased in axons . to test this hypothesis , we first performed rt - pcr at 1 , 6 , 15 , and 24  hrs after injury . injury caused a significant increase in axonal levels of importin 1 ( 2.82  1.7 , 4.5 ; p < 0.002 ) and ranbp1 ( 2.70  1.5 , 4.9 ; p < 0.009 ) , but not vimentin ( p = 0.46 ) , 1  hr after axotomy relative to control ( no injury ) . transcripts encoding -actin , which is a well - described locally synthesized protein not believed to play a role in ris , also significantly increased 1 hour after axotomy compared to controls ( 4.73  2.1 , 10.6 ; p < 0.003 ) , serving as a  positive control  ( figure 2 ) . axonal levels of all four transcripts were not significantly different between injured and uninjured axons 615 hours after axotomy ( p > 0.05 ) . interestingly , 24  hrs after axotomy , we observed a second wave of increased levels of importin 1 ( 2.74  0.7 , 10.6 ; p < 0.06 ) and ranbp1 ( 2.48  0.8 , 7.5 ; p < 0.05 ) mrna compared to controls . -actin mrna levels also trended higher at 24 hours ( p = 0.45 ) , but differences with controls did not reach statistical significance ( figure 2 ) . the above results support the hypothesis that an injury - induced signal causes elevation in axonal levels of importin 1 , ranbp1 , and -actin at an early stage , within 1  hr after axotomy . we next assessed whether this increase in transcript levels required de novo transcription in the cell body . we used the well - characterized antibiotic actinomycin d ( amd ) to inhibit transcription and performed rt - pcr to measure changes in transcript levels at 1  hr after injury . to identify conditions under which amd reduced transcript levels in our culture system , we quantified total mrna in response to a range of times . a dose of 5  mg / ml of amd for two hours resulted in ~50% total transcript reduction , and so this dosage was used for subsequent experiments . amd treatment suppressed the injury - induced increase in mrna levels of ranbp1 ( 0.98  0.34 , 2.74 ) and -actin ( 1.29  0.42 , 3.79 ) observed in untreated cells , suggesting that these genes were newly transcribed and transcripts were rapidly recruited to axons . however , surprisingly , amd treatment did not inhibit the injury - induced increase in axonal importin 1 mrna levels ( 2.64  0.60 , 11.5 ; p < 0.001 ) . these results suggest that increased axonal importin 1 transcript levels reflect contributions from preexisting transcript populations in the cell body or axons proximal to the level of harvest ( figure 3 ; black bars are replotted from figure 2 , for comparison ) . to test whether and over what time frame early increases in transcript levels ultimately resulted in changes in protein expression and thus the possible formation of ris , we explored changes in axonal levels of ris proteins in response to injury . we evaluated the expression of importin 1 , ranbp1 , and vimentin at 0  hrs , 1  hr , 3  hrs , and 6  hrs after axotomy . as was also the case for mrna levels , each ris protein responded uniquely to injury . anova revealed a significant effect of time on importin 1 levels ( p < 0.01 ) ; consistent with local axonal translation ( though not excluding some contribution from the cell body ) , post hoc testing revealed that importin 1 levels increased significantly at 1 hour ( 62% increase , p < 0.05 ) and again at 6 hours ( 84% increase , p < 0.005 ) but not at 3 hours ( figure 4 ) . ranbp1 protein levels fluctuated with time but maintained levels comparable to control at all time points ( figure 4 ) . vimentin levels also did not appear to change with time ; however , results were too variable to be formally compared with sufficient power ( data not shown ) . having established an increase in both importin  transcript and protein at 1 and 6 hours , we next tested whether importin 1-mediated nuclear import was required to transcribe new importin  , ranbp1 , and -actin mrna observed in the second phase of increased transcript levels at 24  hrs . to minimize nonspecific perturbation of the diverse functional roles of importin 1 , we specifically disrupted nuclear import mediated via importin  using importazole ( ipz ) . ipz 's role in disrupting importin -mediated nuclear import in nonneuronal cells was extensively characterized . however , ipz inhibition of nuclear import in an explant system and specifically in neurons has not yet been characterized . we therefore tested for such a role by tracking the localization of the transcription factor nfat fused to gfp ( nfat - gfp ) in response to ipz treatment . studies in nonneuronal cells have revealed that nfat - gfp shuttles between the nucleus and cytoplasm in calcium regulated manner and is imported by importin / and exported by crm1 [ 2932 ] . as expected , at steady state , nfat - gfp was predominately cytoplasmic in cells within and at the edge of the explant . also , as expected , an increase in cytoplasmic calcium induced by ionomycin led to accumulation of nfat - gfp in the nucleus ( figure 5(a ) ) . explants were then treated with 18  m ipz for 24  hrs followed by 1.5  hrs of ionomycin treatment in the continued presence of ipz . in stark contrast to control cells , there was no import of nfat - gfp in ipz treated cells ( figures 5(b ) and 5(c ) ) ; specifically , ipz treatment resulted in nuclear exclusion of nfat - gfp in 75% of cells after 12  hrs ( p < 0.015 ) and 86% of cells after 24  hrs ( p < 0.003 ) ( figure 5(c ) ) , demonstrating effectiveness of ipz . because explants contain both neuronal and nonneuronal cells , we performed immunofluorescence labeling with smi-31 ( an axonal marker ) to verify that nfat - gfp was indeed expressed in neuronal cells , and thus neuronal cells were among the cells potentially affected by ipz ( figure 5(f ) ) . following ipz and ionomycin treatment of cells expressing nfat - gfp , we performed a live / dead assay , which indicated over 86% survival of cells treated with dmso ( control ) , as well as transfected cells treated with ipz followed by ionomycin treatment ( figures 5(d ) and 5(e ) ) . we then evaluated changes in axonal mrna levels 24  hrs after injury followed by ipz treatment . ipz treatment suppressed the second wave of increased axonal mrna for all ris - related and -actin transcripts , as indicated by the lack of significant changes in axonal mrna levels of all transcripts after ipz treatment at 24  hrs after injury relative to control ( figure 6 ; grey bars are replotted from figure 2 , for comparison ) . when viewed in combination with the transcriptional and translational upregulation of several key ris proteins at earlier time points , these results support a model whereby ris feedback mechanism is required for secondary amplification of both ris - associated and unassociated mrna at later time points . in this study , we examined axonal levels of ris - associated transcripts , which will ultimately be translated to activate ris pathways . our findings suggest that axotomy results in elevation of axonal mrna , at 1 and 24 hours after injury . regulation of this biphasic response appears transcript - specific at the early time point , with importin -mediated nuclear import required for the second wave of increased axonal transcript levels for all transcripts . inhibition of protein synthesis in axons significantly impairs growth cone activity and axonal extension [ 33 , 34 ] , emphasizing the importance of local protein synthesis for regeneration . among the many axonally synthesized proteins , translation of importin 1 , ranbp1 , and/or vimentin following axonal injury suggests a role for local protein synthesis in ris . in particular , local translation of ranbp1 following injury results in rangtp dissociation from importins , allowing binding of newly synthesized importin  to importin  and dynein - bound ris complexes that are transported to neuronal cell bodies [ 3 , 4 , 13 , 19 ] . to execute these retrograde signaling pathways , required transcripts must be in place beforehand or the observed rapid increase in mrna transcripts within the axon implies rapid localization to the injury site ( figure 2 ) . this response is consistent with the rapid retrograde propagation of an electrophysiological response , possibly mediated by calcium , that provides the first indication of a lesion event [ 3 , 7 , 8 , 10 , 11 , 36 ] . on the other hand , electrical activity alone is not sufficient to initiate regeneration [ 12 , 37 ] , and thus our observed secondary elevation in mrna transcripts 24 hours after injury is consistent with both ris and the requirement for an additional signal for effective regeneration . the dip in axonal transcript levels at time points between 1 and 24 hours , as opposed to steadily increasing or stably increased levels , also supports the notion of two different mechanisms for transcript recruitment to the axon . a number of transcripts have been shown to alter their axonal levels following axotomy of central neurons . as expected , mrna levels for -actin , whose local synthesis has been extensively characterized , increased after axotomy , serving as a sort of positive control . consistent with ris , we also observed rapid increases in both importin  and ranbp1 transcript levels but not vimentin . transcriptional inhibition also revealed differences in the response of evaluated transcripts . the early increase in axonal levels of ranbp1 and -actin transcripts , like gap-43 , depended on newly synthesized mrnas ( figure 3 ; [ 38 , 39 ] ) . conversely , importin  , like several other proteins , such as cgrp , moved into axons [ 38 , 40 ] , indicating an additional nontranscriptional contribution to axonal synthetic capacity . differential regulation of importin 1 transcript levels compared to ranbp1 ( or vimentin ) may in part be a consequence of the functional diversity of importin . in addition to nuclear transport in the cell body , such roles for importin  include quality control of er proteins , assembly of mitotic spindles and centrosomes [ 14 , 15 ] , and learning - related plasticity . on the other hand , importin isoform localization is dictated by its 3 utr , with short 3 utr variant of importin more prominent in cell bodies and longer variant more prominent in axons . thus , any preexisting recruitable pool of importin 1 mrna must be predesignated for axonal localization . candidates for such a transport - ready pool are ribonucleoprotein particles ( rnps ) , which can migrate to axons and dendrites in response to a given stimulus [ 42 , 43 ] . such activity has been noted in response to a variety of stimuli , for example , zipcode binding protein 1- ( zbp1- ) mediated axonal transport of -actin mrna following the application of neurotrophins to chick cortical neurons [ 44 , 45 ] . the identity of zipcode - like mrna binding proteins that regulate axonal transport of importin 1 is yet unknown . however , recent clip - seq data suggests that fused in sarcoma ( fus ) and serine arginine - rich splicing factor 1 and factor 2 ( srsf1 and srsf2 ) are strong candidates for such a role , based on high numbers of target sites ( starbase 2.0 ; [ 46 , 47 ] ) . several lines of evidence suggest that importin  plays an important , even essential , role in the response to axonal injury . in peripheral neurons , importin - associated ris complexes transport signaling proteins , including transcription factors such as jnk , erk , atf2 , and atf3 , from the injury site [ 4 , 5 , 48 ] , and the depletion of importin  results in suppressed gene transcription and delayed functional recovery following nerve injury . importin  also stimulates axotomy - induced axonogenesis in the cns , in part by transporting the transcription factor stat3 [ 18 , 19 ] . our data support an important role for importin 1 after injury and reveal novel and intriguing temporal dynamics of importin - mediated neuronal response . based on ris complex transport rates , transcription rates , and mrna transport rates [ 49 , 50 ] , it is less likely that importin -mediated nuclear import plays a role in transcriptional changes at 1  hr after axotomy . though we can not exclude a rapid signal ( e.g. , an electrical signal ) triggering an initial wave of nuclear import mediated by existing pools of importin  in the cell body , insufficient time would have elapsed for the transport of newly synthesized importin  into the cell body , import processes to occur , and transcripts to appreciably accumulate . however , our data indicate that importin  plays a critical role in the secondary wave of axonal transcript elevation upon injury . increased importin 1 expression over 6 hours ( figure 4 ) suggests that early increases in importin 1 transcript are translated . in addition , axonal elevation of importin 1 and ranbp1 transcripts was suppressed following specific pharmacological inhibition of nuclear import , with other importin functions , including ris complex formation , presumably intact ( figure 6 ) . together , these data raise the possibility that rapidly synthesized importin 1 could feed back to further upregulate the axonal localization of ris - associated transcripts . it is important to note that we did not suppress nuclear import ( ipz treatment ) at early time points . as a consequence , we do not have insight into the time frame over which nuclear import contributed to upregulation of ris - associated transcription , nor can we unequivocally state that nuclear import was not involved in the early phase of transcriptional activation . it is possible that suppression of nuclear import and transcription ( i.e. , amd and ipz treatment ) at earlier time points may be superposed to additionally inactivate ris - associated transcriptional pathways . conversely , we also did not combinatorially suppress transcriptional and nuclear import pathways at the 24-hour time point ; as nuclear import precedes transcriptional upregulation , we would expect such an experiment to be uninterpretable . thus , though overall transcription of ris - associated proteins dips at intermediate time points , we can not conclude whether and when pathways contributing to initial transcriptional increases are turned off . finally , it will be interesting to evaluate the persistence of these putative mechanisms at later time points in future studies . our work points to interesting similarities and differences between ris in the pns and cns . on one hand , we also observe that axonal levels of the ris - associated transcripts importin 1 and ranbp1 indeed increase following central axonal injury , and importin  plays a key role in regulating this increase . this is consistent with the observation in optic neurons ( retinal ganglion cells ) that hyperactivated stat3 , which interacts with importin  , enhanced the axonal regenerative response [ 51 , 52 ] . our observed increase in axonal levels of importin 1 mrna after 6  hrs of axotomy ( figure 4 ) is also similar to that in the pns , with maximum increases observed 68  hrs after axotomy . a recent study by ohara and colleagues suggested that , unlike our results , several axonal proteins , including importin 1 , slightly increased 10  mins after axotomy of dissociated cortical neurons , before returning to control levels 1  hr after injury . however , in their study , isolated axons were harvested only from regions distal to the injury site , which were presumably degenerating , and axonal regions proximal to the injury site were not separated from cell bodies [ 18 , 19 ] , precluding direct comparison with our results . additionally , no further quantification was performed beyond 1  hr . on the other hand , in contrast to studies in the pns , which show a gradual increase in axonal protein levels of vimentin and ranbp1 over period of 6  hrs after axotomy in the pns [ 5 , 13 ] , we observed neither . in fact , our study on cns neurons indicated unchanged vimentin transcript levels over 24 hours ( figure 2 ) and no significant change in vimentin or ranbp1 proteins within 6  hrs after axotomy ( figure 4 ) . while , in the pns , calpain - cleaved vimentin binds to phosphorylated erks ( perk ) , linking perk to dynein via importin 1 , this regulatory pathway does not appear to be conserved in our cns model . it is not clear why axonal levels of ranbp1 were unchanged after injury ; the fact that protein levels did not rise and fall with transcript levels suggests a temporal decoupling of these processes for this protein . our observed biphasic increase in axonal transcript levels suggests tightly and differentially regulated control of local protein synthesis in hippocampal neurons after injury , including a key role for importin -mediated nuclear import ( figure 7 ) . while our focus was on the regulation of ris - associated transcripts , additional questions regarding ris signaling , local protein synthesis , and , ultimately , axonal outgrowth in cns axons remain , including the exact set of transcription factors responsible for de novo mrna synthesis and how acute transcriptional regulation impacts long - term neuronal outgrowth . our findings confirm intrinsic regenerative capabilities in the cns and have important implications for exploiting ris and local protein synthetic pathways towards enhanced cns repair .
axons in the peripheral nervous system respond to injury by activating retrograde injury signaling ( ris ) pathways , which promote local axonal protein synthesis ( lps ) and neuronal regeneration . 
 ris is also initiated following injury of neurons in the central nervous system ( cns ) . 
 however , regulation of the localization of axonal mrna required for lps is not well understood . 
 we used a hippocampal explant system to probe the regulation of axonal levels of ris - associated transcripts following axonal injury . 
 axonal levels of importin 1 and ranbp1 were elevated biphasically at 1 and 24  hrs after axotomy . 
 transcript levels for -actin , a prototypic axonally synthesized protein , were similarly elevated . 
 our data suggest differential regulation of axonal transcripts . at 1  hr 
 after injury , deployment of actinomycin revealed that ranbp1 , but not importin 1 , requires de novo mrna synthesis . at 24  hrs after injury , use of importazole 
 revealed that the second wave of increased axonal mrna levels required importin -mediated nuclear import . 
 we also observed increased importin 1 axonal protein levels at 1 and 6  hrs after injury . 
 ranbp1 levels and vimentin levels fluctuated but were unchanged at 3 and 6  hrs after injury . 
 this study revealed temporally complex regulation of axonal transcript levels , and it has implications for understanding neuronal response to injury in the cns .