BACKGROUND
injuries of the central nervous system  can lead to devastating and irreversible loss of function because adult mammalian cns neurons have a limited regenerative capacity  <cit> .
this is partially due to an age dependent reduction in the intrinsic regenerative potential of cns neurons  <cit> .
in contrast, some adult invertebrate neurons are capable of spontaneous regeneration following injury  <cit> .
lymnaea stagnalis  has served as a critical model system to study nerve regeneration because of its ability to spontaneously regenerate and restore function in the adult  <cit> .
specifically, identified adult lymnaea neurons can be isolated individually and maintained in culture, allowing for axonal outgrowth and functional synapse formation between appropriate synaptic partners  <cit> .
l. stagnalis neural genes include homologues of well known transcription factors, genes involved in neurotransmission, axon guidance and signalling pathways  <cit> .
taking advantage of this model system, a number of groups demonstrated the roles of neurotrophins  <cit> , and neurotransmitters  <cit>  on neurite outgrowth and regeneration.
moreover, retinoic acid  isomers have been identified in adult lymnaea neurons, and 9-cis-ra has a novel role in chemoattraction and growth cone guidance  <cit> .
since many molecular mechanisms are conserved across species, the identification of molecules involved in neuronal regeneration in l. stagnalis will aid our understanding of factors which may promote regeneration in the mammalian cns.
one main limitation of using l. stagnalis in genetic functional studies of neuronal regeneration has been a lack of large-scale screening tools, such as microarray analysis  <cit> .
to circumvent this limitation, we have recently sequenced more than  <dig>   <dig> ests  from the cns transcriptome of l. stagnalis  <cit>  and established the largest neuronal est database in lymnaea .
in combination with the availability of the microarray technology  <cit> , these gene sequences provide us with the opportunity to perform a high-throughput screening for altered gene expression levels following nerve injury in l. stagnalis.
in this study, we have designed the first microarray chip covering  <dig>   <dig> known l. stagnalis genes to profile the gene expression patterns following cns injury.
we identified  <dig> genes that were differentially regulated following cns crush.
using real-time pcr  analysis we confirmed that the gene expression level of ccaat enhancer binding protein , a transcription factor, is up-regulated following nerve injury.
knockdown of c/ebp following axotomy of cultured pea cells lead to retraction of the distal, proximal and intact neurites.
our data suggest that c/ebp plays a crucial role in neuronal regeneration of lymnaea neurons via both somatic and local mechanisms.
RESULTS
cns injury model of l. stagnalis
in order to profile the gene expression pattern following injury of the central nervous system, we modified an established  <cit>  nerve injury model by crushing the right parietal  and right cerebral nerves  .
in control groups, sham operations were performed without nerve injury.
identification of differentially expressed genes
to search for genes that may be involved in neuronal regeneration or degeneration, microarray analyses were carried out to compare the gene expression levels between the sham-operated and crush-operated cns models .
we used an unpaired t-test for comparison of the  <dig>  array signals between two groups: sham- and crush-operated groups.
this analysis resulted in the selection of  <dig> genes or ests  that were differentially regulated  <dig> hours following cns injury .
further screening for genes with a fold-change >  <dig> resulted in a final group of  <dig> differentially expressed genes.
figure 1b shows a heat map representing the hierarchical clustering of the  <dig> genes with significant differential expression levels between the control and injured groups.
overall, more genes were up-regulated  than that of which were down-regulated , suggesting that diverse regulatory pathways and biological processes are activated within the first  <dig> hours following injury .
among the  <dig> regulated genes,  <dig> have been identified with known functions related to development, survival or signal transduction; the functions of the majority of genes  have not yet been described .
gene expression levels of c/ebp change in a time-dependent manner following injury
the microarray analysis indicated that the mrna level of ccaat enhancer binding protein, c/ebp, was significantly increased  <dig> hours following cns injury, as compared to the sham control group.
a protein sequence alignment of c/ebp orthologues from l. stagnalis, aplysia, rat, and human revealed that c/ebp has a high homology in the dna-binding domain, suggesting that c/ebp may have similar biological functions in these species .
to confirm that c/ebp is indeed up-regulated following nerve injury at  <dig> hours, we performed conventional rt-pcr analysis.
we found that the expression level of c/ebp mrna significantly increased, compared to the control gene gapdh, in the injured cns group  over the sham groups .
to study its temporal expression pattern, we further measured the gene level of c/ebp at  <dig> hour,  <dig> hours and  <dig> hours post cns injury using qpcr analysis.
the standard curves for c/ebp are shown in figure 3b with a slope of - <dig> , indicating the pcr efficiency for the primers are sufficient .
to visualize the gene expression level between the injured and sham groups, the threshold cycles  of c/ebp  and gapdh  were plotted against each other, as described previously  <cit> .
figure 3c shows the representatives of ct-ct correlation plots between the c/ebp and gadph gene pair at the three different time points .
the slopes of the plots were consistent in all experimental samples, indicating the equality in amplification efficiencies between the primers of the gene pair.
the y-intercepts /log) of these plots were smaller in the injury group  than in the controls  at all the times tested, indicating the relative expression level of c/ebp mrna increased following injury.
specifically, c/ebp mrna was initially increased by  <dig>  ±  <dig> ,  at  <dig> hr after cns injury, but appeared to reduce to  <dig>  ±  <dig>   by  <dig> hrs and  <dig>  ±  <dig> ,  by  <dig> hrs, albeit at levels that are still significantly greater than in sham-operated  animal .
these results suggest that elevation of c/ebp mrna expression may be necessary for transcriptional induction of regeneration associated genes, and thus likely to be a direct target of injury induced signalling cascades that promote neuronal repair.
the role of c/ebp in axonal elongation following axotomy
the increased expression of c/ebp mrna following nerve injury in vivo suggests that this protein may be an upstream gene regulator of either pro-regenerative genes or responsive genes for cell injury.
to elucidate the functional significance of c/ebp mrna following nerve injury, we examined the effect of c/ebp on axonal outgrowth and regeneration of pea neurons in culture using a sirna gene silencing approach as previously described  <cit> .
axotomy was performed on cultured pea neurons after  <dig> hrs which had undergone adequate neurite outgrowth .
immediately following axotomy, cells were treated with either conditioned medium , control sirna, c/ebp sirna# <dig> or c/ebp sirna# <dig>  the lengths of the proximal and distal ends of the axotomized neurite, as well as an intact neurite were measured at various times over a period of  <dig> hrs .
the net changes of the neurites following treatments were compared, as previously described  <cit> .
figure 5a shows that the distal end axons did not show additional growth following treatments with either c/ebp sirna# <dig> , or c/ebp sirna# <dig>  treated cells, whereas that continuously elongated in control cm  or control sirna  treated cells following axotomy.
the net changes in the distal axons were significant between the c/ebp sirnas and the control cm or control sirna groups .
in contrast, the net increases in length of the proximal neurites over  <dig> hrs are  <dig>  ±  <dig>  μm  in control cm, and  <dig>  ±  <dig>  μm  in control sirna treated cells, whereas that was significantly reduced in c/ebp sirna # <dig>  or c/ebp sirna # <dig>  treated cells, although no significant difference was found at the early hours  .
similarly, the net growth of intact neurites was only significantly reduced in c/ebp sirna # <dig>  and c/ebp sirna # <dig>  treated cells as compared to control cm  and control sirna treated cells   <dig> hrs after treatment .
these findings suggest that neuronal c/ebp is involved in neurite outgrowth.
sufficient c/ebp levels are required for maintaining the integrity of the distal axon.
due to the application of sirnas following axotomy, the differences found in the distal axons may be associated with local protein synthesis  <cit> .
c/ebp sirna treatment hinders locomotion recovery in vivo following cns injury
in order to determine whether c/ebp sirna treatment has a role in an in vivo model, we compared the locomotory activity of sham-operated and crush-operated snails treated with saline, control sirna and c/ebp sirna# <dig>  the distance that the snail crawled in  <dig> min was measured before and after nerve crush , between all six groups.
as shown in figure  <dig>  all groups had severely movement deficit following the nerve crush operation, but slowly gained their ability to crawl over the course of  <dig> days.
however, after  <dig> days, the distance  measured from the crush-operated c/ebp sirna# <dig> group  was reduced as compared to that from the sham-operated saline , control sirna  or c/ebp sirna# <dig> , as well as to that from the crush-operated saline , or control sirna treated snails .
these data suggest that c/ebp sirna treatment disrupted the recovery of snails following nerve injury and support the notion that c/ebp knockdown impedes cns regeneration in vivo, potentially through its effects on neurite regeneration.
discussion
in this study, we designed the first lymnaea gene chip for microarray analysis and profiled the gene expression patterns during the early stages of the cns repair following injury.
we found that  <dig> genes were significantly regulated during the first three hours post injury, including the conserved gene transcription factor c/ebp.
we further described the temporal gene regulation pattern of c/ebp following cns injury using qpcr analysis.
reduction of c/ebp mrna level by c/ebp sirnas following axotomy suppressed outgrowth of the distal and somatic neurite in vitro at different time points, and disrupted the recovery of locomotory function of the snails following nerve injury in vivo.
our data further support the notion that c/ebp is required for nerve regeneration.
l. stagnalis has been used as a simple and reliable model to investigate genes involved in regeneration, due to its ability to functionally recover following cns injury in vivo.
for example, central ganglia cell  connections to buccal ganglia were crushed and as a result the feeding behaviour in animals was disrupted.
two weeks following the procedure the cgc regenerated, functional synapses formed between proper synaptic partners and the feeding behaviour was again observed  <cit> .
in vivo cns regeneration was also observed following axotomy of innervating axons to the pneumostome and surrounding areas which prevented the occurrence of lung respiration in 69% of snails.
several weeks following surgery, axonal regeneration leading to the reformation of functional synapses was observed and pneumostome opening returned to normal  <cit> .
in this study, we used the modified cns injury model in which both the right parietal  and the right cerebral nerves  were crushed .
we are interested in the gene regulation occurring at the early stages of cns trauma, thus our in vivo experiments were completed within  <dig> hours following injury.
the ability to profile the expression of thousands of genes simultaneously makes microarrays an invaluable biomedical research tool  <cit> .
a previous study used microarray approach to assess the genes involved in anti-inflammatory responses in a spinal cord injury model  <cit> .
the anti-inflammatory response is an important component of secondary tissue damage following spinal cord injury.
the microarray study allowed for identification of a conserved novel regulator of regeneration, and provided a new mechanism by which to initiate the regenerative response  <cit> .
however, shortcomings of microarray analysis are associated with poor reliability  <cit>  and specificity  <cit> .
due to the inherent sources of error in microarray technology, it is necessary to confirm the findings using an independent methodology from separate samples.
real-time quantitative pcr is a readily used technique to confirm microarray data  <cit> .
in our study, we conducted qpcr analysis using different samples and verified that the expression of c/ebp was indeed up-regulated following nerve injury.
recovery from nerve injury and neuronal regeneration are time-dependent events, from the initial inflammatory response to the development of the glial scar  <cit> .
inflammation, neurotransmitter dysfunction, ionic imbalance, cytoskeletal alteration and increased transcription occur as early as  <dig> hrs following spinal cord injury  <cit> .
nerve outgrowth and regeneration can be enhanced by steroids during a critical window within the first  <dig> hrs after nerve injury  <cit> .
in the c. elegans, axons regenerate following axotomy in vivo and by  <dig> hrs fully functional synapses are formed  <cit> .
we have recently demonstrated that l. stagnalis regeneration post injury in vitro can be seen as early as  <dig> hrs, and reformation of connections between the proximal and distal axon is observed by  <dig> hrs  <cit> .
deducing the gene regulation during the early response following neuronal injury is important in uncovering the molecular basis of neuronal regeneration.
in this study, our microarray gene expression analysis identified  <dig> genes that were significantly regulated following cns injury in l. stagnalis.
we examined c/ebp gene expression at three different time points  in order to understand how it is regulated temporally in the early stages of recovery and repair.
our results indicated that the c/ebp gene was up-regulated in a time-dependent manner following cns injury .
we further showed the locomotory activity of the control snails was severely disrupted immediately following nerve injury but recovered gradually over  <dig> days.
c/ebp sirna application prior to the nerve crush procedure disrupted the recovery of locomotory function of the snails , suggesting that c/ebp is required for nerve regeneration in vivo.
although our in vitro data suggest that sufficient c/ebp levels in neurons are required for axonal outgrowth, it is possible that an increased c/ebp expression in non-neuronal  cells involved in nerve regeneration and inflammatory responses also contributes to the observed recovery of locomotory activity in the injured snails.
c/ebps belong to the basic leucine zipper dna binding protein family and are transcription factors with diverse cellular roles, including regulating cell growth, differentiation, learning and memory, and apoptosis  <cit> .
c/ebps are also responsive to brain injury and ischemia  <cit> .
six isoforms of c/ebp have been found in mammals  <cit> .
c/ebpβ is highly homologous to lymnaea c/ebp   <cit> .
c/ebpβ was first identified as a mediator of the inflammatory response and il- <dig> signalling through its binding to il- <dig> response elements in the promoters of acute phase response genes tnf, il- <dig>  and g-csf  <cit> .
c/ebp expression is enriched in neurons.
it is up-regulated following brain injury in various neuronal regenerative animal models  <cit> .
its expression is regulated by transcription factors such as creb and nfil- <dig>  <cit> .
aplysia nerve injury activates axoplasmic kinase, risk- <dig>  which phosphorylates apc/ebpβ and initiates the binding of apc/ebpβ to the ere enhancer site in vitro.
increases in risk- <dig> and apc/ebp were detected in injured neurons  <cit> .
in leech, c/ebp mrna was increased during neuronal regeneration  <cit> .
in this study, we show that c/ebp mrna expression is increased following cns injury in a time-dependent manner.
the 7-fold increase in c/ebp mrna at  <dig> hr following injury is likely related to the inflammatory response.
the sustained  <dig> fold increase over the  <dig> hr period suggests that c/ebp may be essential for transcriptional induction of regeneration associated genes.
knockdown of c/ebp mrna expression following axotomy by target-gene sirna caused a reduction in neurite outgrowth in both distal and proximal axons at different time points.
due to that sirnas were applied following axotomy, our data of c/ebp sirnas causing suppression of distal neurites suggests that local c/ebp mrna is likely involved in neuronal regeneration/elongation.
in contrast to the distal section of neurites which may rely on local protein synthesis to grow, the cell somata may continuously provide complimentary mechanisms to sustain c/ebp levels and maintain neurite properties.
the delayed effect of c/ebp sirnas on the proximal regrowth  further supports the notion that c/ebp is required for axonal regeneration.
cebp- <dig>  a member of the c/ebp family in the c. elegans, is a direct effecter of the dlk- <dig> cascade  <cit> .
cebp- <dig> mrna has been found in axons and presynaptic regions, and is stabilized via its 3'utr by activation of the dual-leucine zipper kinase- <dig>  pathway  <cit> .
the role of c/ebp in axon regeneration is suggested via dual-leucine zipper kinase mapkkk -dependent pathway  <cit> .
neuronal regeneration associated proteins such as α-tubulin and gap- <dig>  are transcriptional targets of c/ebp  <cit> .
following nerve injury, up-regulation of c/ebpβ mrna and c/ebp phosphoprotein coincides with increases in the transcription of the α-tubulin promoter in the wild-type but not in c/ebpβ-/- mice  <cit> .
dlk- <dig> acts in a mapk cascade with mapmkkk- <dig> and p <dig> kinase  <cit> .
both the dlk- <dig> pathway and cebp- <dig> were necessary for regenerative growth of adult touch neurons  <cit> .
we thus proposed a model by which the dlk- <dig> pathway regulates c/ebp mrna stability in both distal and proximal axons.
both somatic and local c/ebp levels are required for the regenerative properties of snail neurons .
local protein synthesis has been shown to increase in injured axons and thus play an important role in the regeneration-degradation process  <cit> .
our study provided the first evidence that not only the somatic but also local regulation of c/ebp mrna in axons is important for outgrowth and regeneration in l. stagnalis.
CONCLUSIONS
this is the first microarray study in l. stagnalis, allowing us to identify genes that are differentially regulated following cns injury.
these genes may be involved in regeneration and provide a new database for the study of specific proteins and genes in response to cns injury.
we also identified the role of the transcription factor c/ebp in neuronal regeneration following axotomy of adult neurons in vitro, and provided new insights into the potential targets for cns injury.

 injuries of the central nervous system can lead to devastating and irreversible loss of function because adult mammalian cns neurons have a limited regenerative capacity. this is partially due to an age dependent reduction in the intrinsic regenerative potential of cns neurons. in contrast, some adult invertebrate neurons are capable of spontaneous regeneration following injury. lymnaea stagnalis has served as a critical model system to study nerve regeneration because of its ability to spontaneously regenerate and restore function in the adult. specifically, identified adult lymnaea neurons can be isolated individually and maintained in culture, allowing for axonal outgrowth and functional synapse formation between appropriate synaptic partners. l. stagnalis neural genes include homologues of well known transcription factors, genes involved in neurotransmission, axon guidance and signalling pathways. taking advantage of this model system, a number of groups demonstrated the roles of neurotrophins, and neurotransmitters on neurite outgrowth and regeneration. moreover, retinoic acid isomers have been identified in adult lymnaea neurons, and 9-cis-ra has a novel role in chemoattraction and growth cone guidance. since many molecular mechanisms are conserved across species, the identification of molecules involved in neuronal regeneration in l. stagnalis will aid our understanding of factors which may promote regeneration in the mammalian cns. one main limitation of using l. stagnalis in genetic functional studies of neuronal regeneration has been a lack of large-scale screening tools, such as microarray analysis. to circumvent this limitation, we have recently sequenced more than ests from the cns transcriptome of l. stagnalis and established the largest neuronal est database in lymnaea. in combination with the availability of the microarray technology, these gene sequences provide us with the opportunity to perform a high-throughput screening for altered gene expression levels following nerve injury in l. stagnalis. in this study, we have designed the first microarray chip covering known l. stagnalis genes to profile the gene expression patterns following cns injury. we identified genes that were differentially regulated following cns crush. using real-time pcr analysis we confirmed that the gene expression level of ccaat enhancer binding protein, a transcription factor, is up-regulated following nerve injury. knockdown of c/ebp following axotomy of cultured pea cells lead to retraction of the distal, proximal and intact neurites. our data suggest that c/ebp plays a crucial role in neuronal regeneration of lymnaea neurons via both somatic and local mechanisms.  cns injury model of l. stagnalis in order to profile the gene expression pattern following injury of the central nervous system, we modified an established nerve injury model by crushing the right parietal and right cerebral nerves. in control groups, sham operations were performed without nerve injury. identification of differentially expressed genes to search for genes that may be involved in neuronal regeneration or degeneration, microarray analyses were carried out to compare the gene expression levels between the sham-operated and crush-operated cns models. we used an unpaired t-test for comparison of the array signals between two groups: sham- and crush-operated groups. this analysis resulted in the selection of genes or ests that were differentially regulated hours following cns injury. further screening for genes with a fold-change > resulted in a final group of differentially expressed genes. figure 1b shows a heat map representing the hierarchical clustering of the genes with significant differential expression levels between the control and injured groups. overall, more genes were up-regulated than that of which were down-regulated, suggesting that diverse regulatory pathways and biological processes are activated within the first hours following injury. among the regulated genes, have been identified with known functions related to development, survival or signal transduction; the functions of the majority of genes have not yet been described. gene expression levels of c/ebp change in a time-dependent manner following injury the microarray analysis indicated that the mrna level of ccaat enhancer binding protein, c/ebp, was significantly increased hours following cns injury, as compared to the sham control group. a protein sequence alignment of c/ebp orthologues from l. stagnalis, aplysia, rat, and human revealed that c/ebp has a high homology in the dna-binding domain, suggesting that c/ebp may have similar biological functions in these species. to confirm that c/ebp is indeed up-regulated following nerve injury at hours, we performed conventional rt-pcr analysis. we found that the expression level of c/ebp mrna significantly increased, compared to the control gene gapdh, in the injured cns group over the sham groups. to study its temporal expression pattern, we further measured the gene level of c/ebp at hour, hours and hours post cns injury using qpcr analysis. the standard curves for c/ebp are shown in figure 3b with a slope of -, indicating the pcr efficiency for the primers are sufficient. to visualize the gene expression level between the injured and sham groups, the threshold cycles of c/ebp and gapdh were plotted against each other, as described previously. figure 3c shows the representatives of ct-ct correlation plots between the c/ebp and gadph gene pair at the three different time points. the slopes of the plots were consistent in all experimental samples, indicating the equality in amplification efficiencies between the primers of the gene pair. the y-intercepts /log) of these plots were smaller in the injury group than in the controls at all the times tested, indicating the relative expression level of c/ebp mrna increased following injury. specifically, c/ebp mrna was initially increased by ±, at hr after cns injury, but appeared to reduce to ± by hrs and ±, by hrs, albeit at levels that are still significantly greater than in sham-operated animal. these results suggest that elevation of c/ebp mrna expression may be necessary for transcriptional induction of regeneration associated genes, and thus likely to be a direct target of injury induced signalling cascades that promote neuronal repair. the role of c/ebp in axonal elongation following axotomy the increased expression of c/ebp mrna following nerve injury in vivo suggests that this protein may be an upstream gene regulator of either pro-regenerative genes or responsive genes for cell injury. to elucidate the functional significance of c/ebp mrna following nerve injury, we examined the effect of c/ebp on axonal outgrowth and regeneration of pea neurons in culture using a sirna gene silencing approach as previously described. axotomy was performed on cultured pea neurons after hrs which had undergone adequate neurite outgrowth. immediately following axotomy, cells were treated with either conditioned medium, control sirna, c/ebp sirna# or c/ebp sirna# the lengths of the proximal and distal ends of the axotomized neurite, as well as an intact neurite were measured at various times over a period of hrs. the net changes of the neurites following treatments were compared, as previously described. figure 5a shows that the distal end axons did not show additional growth following treatments with either c/ebp sirna#, or c/ebp sirna# treated cells, whereas that continuously elongated in control cm or control sirna treated cells following axotomy. the net changes in the distal axons were significant between the c/ebp sirnas and the control cm or control sirna groups. in contrast, the net increases in length of the proximal neurites over hrs are ± μm in control cm, and ± μm in control sirna treated cells, whereas that was significantly reduced in c/ebp sirna # or c/ebp sirna # treated cells, although no significant difference was found at the early hours. similarly, the net growth of intact neurites was only significantly reduced in c/ebp sirna # and c/ebp sirna # treated cells as compared to control cm and control sirna treated cells hrs after treatment. these findings suggest that neuronal c/ebp is involved in neurite outgrowth. sufficient c/ebp levels are required for maintaining the integrity of the distal axon. due to the application of sirnas following axotomy, the differences found in the distal axons may be associated with local protein synthesis. c/ebp sirna treatment hinders locomotion recovery in vivo following cns injury in order to determine whether c/ebp sirna treatment has a role in an in vivo model, we compared the locomotory activity of sham-operated and crush-operated snails treated with saline, control sirna and c/ebp sirna# the distance that the snail crawled in min was measured before and after nerve crush, between all six groups. as shown in figure all groups had severely movement deficit following the nerve crush operation, but slowly gained their ability to crawl over the course of days. however, after days, the distance measured from the crush-operated c/ebp sirna# group was reduced as compared to that from the sham-operated saline, control sirna or c/ebp sirna#, as well as to that from the crush-operated saline, or control sirna treated snails. these data suggest that c/ebp sirna treatment disrupted the recovery of snails following nerve injury and support the notion that c/ebp knockdown impedes cns regeneration in vivo, potentially through its effects on neurite regeneration. discussion in this study, we designed the first lymnaea gene chip for microarray analysis and profiled the gene expression patterns during the early stages of the cns repair following injury. we found that genes were significantly regulated during the first three hours post injury, including the conserved gene transcription factor c/ebp. we further described the temporal gene regulation pattern of c/ebp following cns injury using qpcr analysis. reduction of c/ebp mrna level by c/ebp sirnas following axotomy suppressed outgrowth of the distal and somatic neurite in vitro at different time points, and disrupted the recovery of locomotory function of the snails following nerve injury in vivo. our data further support the notion that c/ebp is required for nerve regeneration. l. stagnalis has been used as a simple and reliable model to investigate genes involved in regeneration, due to its ability to functionally recover following cns injury in vivo. for example, central ganglia cell connections to buccal ganglia were crushed and as a result the feeding behaviour in animals was disrupted. two weeks following the procedure the cgc regenerated, functional synapses formed between proper synaptic partners and the feeding behaviour was again observed. in vivo cns regeneration was also observed following axotomy of innervating axons to the pneumostome and surrounding areas which prevented the occurrence of lung respiration in 69% of snails. several weeks following surgery, axonal regeneration leading to the reformation of functional synapses was observed and pneumostome opening returned to normal. in this study, we used the modified cns injury model in which both the right parietal and the right cerebral nerves were crushed. we are interested in the gene regulation occurring at the early stages of cns trauma, thus our in vivo experiments were completed within hours following injury. the ability to profile the expression of thousands of genes simultaneously makes microarrays an invaluable biomedical research tool. a previous study used microarray approach to assess the genes involved in anti-inflammatory responses in a spinal cord injury model. the anti-inflammatory response is an important component of secondary tissue damage following spinal cord injury. the microarray study allowed for identification of a conserved novel regulator of regeneration, and provided a new mechanism by which to initiate the regenerative response. however, shortcomings of microarray analysis are associated with poor reliability and specificity. due to the inherent sources of error in microarray technology, it is necessary to confirm the findings using an independent methodology from separate samples. real-time quantitative pcr is a readily used technique to confirm microarray data. in our study, we conducted qpcr analysis using different samples and verified that the expression of c/ebp was indeed up-regulated following nerve injury. recovery from nerve injury and neuronal regeneration are time-dependent events, from the initial inflammatory response to the development of the glial scar. inflammation, neurotransmitter dysfunction, ionic imbalance, cytoskeletal alteration and increased transcription occur as early as hrs following spinal cord injury. nerve outgrowth and regeneration can be enhanced by steroids during a critical window within the first hrs after nerve injury. in the c. elegans, axons regenerate following axotomy in vivo and by hrs fully functional synapses are formed. we have recently demonstrated that l. stagnalis regeneration post injury in vitro can be seen as early as hrs, and reformation of connections between the proximal and distal axon is observed by hrs. deducing the gene regulation during the early response following neuronal injury is important in uncovering the molecular basis of neuronal regeneration. in this study, our microarray gene expression analysis identified genes that were significantly regulated following cns injury in l. stagnalis. we examined c/ebp gene expression at three different time points in order to understand how it is regulated temporally in the early stages of recovery and repair. our results indicated that the c/ebp gene was up-regulated in a time-dependent manner following cns injury. we further showed the locomotory activity of the control snails was severely disrupted immediately following nerve injury but recovered gradually over days. c/ebp sirna application prior to the nerve crush procedure disrupted the recovery of locomotory function of the snails, suggesting that c/ebp is required for nerve regeneration in vivo. although our in vitro data suggest that sufficient c/ebp levels in neurons are required for axonal outgrowth, it is possible that an increased c/ebp expression in non-neuronal cells involved in nerve regeneration and inflammatory responses also contributes to the observed recovery of locomotory activity in the injured snails. c/ebps belong to the basic leucine zipper dna binding protein family and are transcription factors with diverse cellular roles, including regulating cell growth, differentiation, learning and memory, and apoptosis. c/ebps are also responsive to brain injury and ischemia. six isoforms of c/ebp have been found in mammals. c/ebpβ is highly homologous to lymnaea c/ebp. c/ebpβ was first identified as a mediator of the inflammatory response and il- signalling through its binding to il- response elements in the promoters of acute phase response genes tnf, il- and g-csf. c/ebp expression is enriched in neurons. it is up-regulated following brain injury in various neuronal regenerative animal models. its expression is regulated by transcription factors such as creb and nfil-. aplysia nerve injury activates axoplasmic kinase, risk- which phosphorylates apc/ebpβ and initiates the binding of apc/ebpβ to the ere enhancer site in vitro. increases in risk- and apc/ebp were detected in injured neurons. in leech, c/ebp mrna was increased during neuronal regeneration. in this study, we show that c/ebp mrna expression is increased following cns injury in a time-dependent manner. the 7-fold increase in c/ebp mrna at hr following injury is likely related to the inflammatory response. the sustained fold increase over the hr period suggests that c/ebp may be essential for transcriptional induction of regeneration associated genes. knockdown of c/ebp mrna expression following axotomy by target-gene sirna caused a reduction in neurite outgrowth in both distal and proximal axons at different time points. due to that sirnas were applied following axotomy, our data of c/ebp sirnas causing suppression of distal neurites suggests that local c/ebp mrna is likely involved in neuronal regeneration/elongation. in contrast to the distal section of neurites which may rely on local protein synthesis to grow, the cell somata may continuously provide complimentary mechanisms to sustain c/ebp levels and maintain neurite properties. the delayed effect of c/ebp sirnas on the proximal regrowth further supports the notion that c/ebp is required for axonal regeneration. cebp- a member of the c/ebp family in the c. elegans, is a direct effecter of the dlk- cascade. cebp- mrna has been found in axons and presynaptic regions, and is stabilized via its 3'utr by activation of the dual-leucine zipper kinase- pathway. the role of c/ebp in axon regeneration is suggested via dual-leucine zipper kinase mapkkk -dependent pathway. neuronal regeneration associated proteins such as α-tubulin and gap- are transcriptional targets of c/ebp. following nerve injury, up-regulation of c/ebpβ mrna and c/ebp phosphoprotein coincides with increases in the transcription of the α-tubulin promoter in the wild-type but not in c/ebpβ-/- mice. dlk- acts in a mapk cascade with mapmkkk- and p kinase. both the dlk- pathway and cebp- were necessary for regenerative growth of adult touch neurons. we thus proposed a model by which the dlk- pathway regulates c/ebp mrna stability in both distal and proximal axons. both somatic and local c/ebp levels are required for the regenerative properties of snail neurons. local protein synthesis has been shown to increase in injured axons and thus play an important role in the regeneration-degradation process. our study provided the first evidence that not only the somatic but also local regulation of c/ebp mrna in axons is important for outgrowth and regeneration in l. stagnalis.  this is the first microarray study in l. stagnalis, allowing us to identify genes that are differentially regulated following cns injury. these genes may be involved in regeneration and provide a new database for the study of specific proteins and genes in response to cns injury. we also identified the role of the transcription factor c/ebp in neuronal regeneration following axotomy of adult neurons in vitro, and provided new insights into the potential targets for cns injury. 
in vivo knockdown of c/ebp postponed recovery of locomotory activity following nerve crush. to identify genes that are involved in the robust regenerative capacity observed in l. stagnalis, we designed the first gene chip covering ~ l. stagnalis cns est sequences. our qpcr analysis confirmed that ccaat enhancer binding protein was up-regulated following nerve injury in a time-dependent manner. we have recently conducted a partial neuronal transcriptome sequencing project and reported over est sequences which allowed us to develop and perform a large-scale high throughput microarray analysis. we conducted microarray analysis to compare the gene expression profiles of sham-operated and crush-operated central ganglia of adult l. stagnalis. our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult cns neurons. neuronal regeneration in the adult mammalian central nervous system is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. thus, l. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. taken together, our data suggest both somatic and local effects of c/ebp are involved in neuronal regeneration. we reported that genes were regulated following central nerve injury in adult l. stagnalis and provided the first evidence for the involvement of local c/ebp in neuronal regeneration. this is the first high-throughput microarray study in l. stagnalis, a model of axonal regeneration following cns injury. knockdown of c/ebp following axotomy prevented extension of the distal, proximal and intact neurites. in contrast, the cns of adult lymnaea stagnalis, a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. the expression levels of genes were found to be significantly altered following nerve injury.
 neuronal regeneration in the adult mammalian central nervous system is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. in contrast, the cns of adult lymnaea stagnalis, a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. thus, l. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. however, the usage of this model has been limited due to insufficient molecular tools. we have recently conducted a partial neuronal transcriptome sequencing project and reported over est sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.  to identify genes that are involved in the robust regenerative capacity observed in l. stagnalis, we designed the first gene chip covering ~ l. stagnalis cns est sequences. we conducted microarray analysis to compare the gene expression profiles of sham-operated and crush-operated central ganglia of adult l. stagnalis. the expression levels of genes were found to be significantly altered following nerve injury. from this pool, sequences showed a greater than 2-fold change: of which were up-regulated and down-regulated. our qpcr analysis confirmed that ccaat enhancer binding protein was up-regulated following nerve injury in a time-dependent manner. in order to test the role of c/ebp in regeneration, c/ebp sirna was applied following axotomy of cultured lymnaea pea neurons. knockdown of c/ebp following axotomy prevented extension of the distal, proximal and intact neurites. in vivo knockdown of c/ebp postponed recovery of locomotory activity following nerve crush. taken together, our data suggest both somatic and local effects of c/ebp are involved in neuronal regeneration.  this is the first high-throughput microarray study in l. stagnalis, a model of axonal regeneration following cns injury. we reported that genes were regulated following central nerve injury in adult l. stagnalis and provided the first evidence for the involvement of local c/ebp in neuronal regeneration. our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult cns neurons. 