BACKGROUND
delayed repair of a peripheral nerve trunk after injury often results in impaired axonal regeneration and poor clinical outcome, although this depends on the duration of the delay  <cit> .
schwann cells are rapidly activated after a nerve injury and start to dedifferentiate and proliferate  <cit> .
insufficient nerve regeneration after delayed nerve repair has been attributed to an inability of schwann cells to support axonal outgrowth  <cit> .
to achieve a good clinical outcome after nerve repair it is important to clarify the molecular mechanisms by which schwann cell proliferation and apoptosis are orchestrated.
a few studies have focused on signal transduction mechanisms with respect to axonal outgrowth after immediate and delayed nerve repair.
however, signal transduction has sparsely been investigated after nerve injuries with and without accompanying repair.
for instance, the activation of extracellular signal-regulated kinase  and the induction of apoptosis in schwann cells have been observed after a nerve injury and after a long delayed nerve repair, respectively  <cit> .
in this study, we examined activation of erk1/ <dig> and induction of cleaved caspase  <dig> in schwann cells after immediate, delayed or no repair of transected rat sciatic nerves and how the different repair conditions related to axonal outgrowth.
RESULTS
p-erk 1/2
p-erk 1/ <dig> stained schwann cells were observed both at the site of the lesion  and in the distal nerve segment  with a higher intensity in all experimental groups than in the uninjured control nerve .
there were significant differences among the groups at the site of the lesion  and in the distal nerve segment .
immunopositive area for p-erk1/ <dig> was higher at the site of the lesion compared to the corresponding distal nerve segment when immediate repair  and no delayed nerve repair  was performed, but not after a delayed nerve repair  and when no repair  was done.
a large immunopositive area for p-erk1/ <dig> was observed in the group with immediate nerve repair both at the site of the lesion and in the distal nerve segment, where the p-erk1/ <dig> positive area at the site of the lesion was almost twice as large as observed in the delayed repair group .
in the distal nerve segment, there were no significant differences between the groups of immediate and delayed nerve repair .
the immunopositive area was lower, however, when no immediate nerve repair  was done than after immediate nerve repair at the two measured sites .
the corresponding values in the experiments where a delayed or no delayed repair was done were p =  <dig>  for snl and p =  <dig>  for snd.
no differences were found between the experiments where no nerve repairs were done , where the analyses of p-erk 1/ <dig> were done  <dig>  and  <dig>  days after injury.
there were no differences between the four groups in the contralateral uninjured nerves with respect to p-erk1/ <dig>  double staining with erk1/ <dig> and s- <dig> revealed that all cells with an elongated nucleus and located within the basal lamina  also stained for s- <dig>  indicating that all our selection criteria were correct, and they also stained for p-erk1/ <dig> .
cleaved caspase 3
cells containing cleaved caspase  <dig> could be identified as schwann cells based on their shape and location  <cit>  .
double staining with cleaved caspase  <dig> and s- <dig> confirmed this identification .
there were significant differences between the groups at the site of the lesion  and in the distal nerve segment  .
the number of cleaved caspase  <dig> stained cells was higher at the site of the lesion compared to further down in the corresponding distal nerve segment  in all groups , except when a delayed nerve repair was done .
the numbers of cleaved caspase  <dig> stained cells were significantly increased in the group with a delayed nerve repair compared to those in the group with immediate nerve repair both at the site of the lesion  and in the distal nerve segment .
the numbers of cells were not different at the two sites in the corresponding experiments where no nerve repairs were done of the transected nerve trunk compared to the nerve repair groups .
furthermore, when comparing the experiments where no nerve repairs were done and analyzed after  <dig> and  <dig> days , no difference in caspase  <dig> stained cells were detected at the site of the lesion , but was almost twice as high at  <dig> days in the distal nerve segment .
there were no significant differences between the four groups in the contralateral uninjured nerves.
length of axonal outgrowth
axonal outgrowth from the proximal nerve segment into the corresponding distal nerve segments after nerve repair or from the proximal nerve segment after no repair was evaluated at  <dig> days post surgery by neurofilament staining  and was found to be different among the groups .
in the groups where no repair was done most of the proximal stumps of the sciatic nerve formed a neuroma and with axonal outgrowth of only a few mm .
axonal outgrowth after immediate nerve repair was longer than in any of the other groups; thus longer than after delayed nerve repair .
correlations
the length of axonal outgrowth correlated to the expression of p-erk 1/ <dig>  both at the site of the lesion  and in the distal nerve segment , while the length did not correlate with numbers of caspase  <dig> stained cells .
however, if only the repaired groups were included for cleaved caspase  <dig>  the corresponding values were p =  <dig> , r <dig> =  <dig>   and p =  <dig>  .
discussion
activation of p-erk1/ <dig> and cleaved caspase  <dig> in schwann cells was different after immediate and delayed nerve repair in the rat sciatic nerve and axonal outgrowth was longer after immediate repair.
in addition, the length of axonal outgrowth correlated with erk 1/ <dig> activation, but not clearly correlated with cleaved caspase  <dig> stained cells.
thus, even a short delay of nerve repair as  <dig> days results in impaired axonal outgrowth.
the mitogen activating pathway kinase  erk1/ <dig> is one important signaling pathway and activation of erk 1/ <dig>  promotes schwann cell proliferation after nerve injury  <cit> .
schwann cells are removed by apoptosis during development before myelination and during regeneration after nerve injury  <cit> , which can be marked by caspase  <dig> or by tunel staining  <cit> .
nick labelling of apoptotic nuclei of sensory neurons  <cit>  after injury has also been detected.
the immunopositive area for evaluation of p-erk1/ <dig> was present more predominantly at the site of the nerve lesion after immediate nerve repair, where 3-thymidine incorporation is maximal after a nerve injury  <cit> .
the increased presence of p-erk1/ <dig> at site of lesion after immediate repair could be related to contact between schwann cells and regenerating axons  <cit>  and to a cellular response similar as suggested for stat- <dig> in the distal nerve segment far from site of lesion  <cit> .
notably, a recent report indicates that functional recovery of injured nerves does not require schwann cell proliferation  <cit> .
however, in the contralateral nerve erk 1/ <dig> is low in the presence of uninjured axons, indicating that the nature of axonal contact is important.
the positive correlation between length of axonal outgrowth and p-erk1/ <dig> stained area is reasonable since activation of erk 1/ <dig> is required for schwann cell proliferation and that axonal outgrowth positively correlates to number of schwann cells; thus, consistent with that schwann cell proliferation is crucial for nerve regeneration  <cit> .
the decreased p-erk1/ <dig> immunopositive area after a delayed nerve repair suggests that the responsiveness of schwann cells is weaker already after a  <dig> days delayed nerve repair; further supported by findings of no differences between site of lesion and distal nerve segment with respect to erk 1/ <dig> and cleaved caspase  <dig> after delayed nerve repair.
activation of erk 1/ <dig> was still observed after  <dig> days, which is consistent with a previous report observing erk 1/ <dig>  <dig> days after nerve transection  <cit> .
the present experimental design with a delayed nerve repair included two injuries to the nerve with a  <dig> day interval.
such a conditioning lesion, i.e.
first injury, can improve axonal outgrowth after a second lesion  <cit> .
however, conditioning by transection  <cit>  has a lower effect on axonal outgrowth than that of a conditioning crush lesion  <cit> .
of the counted total number of cells stained by dapi in the contralateral uninjured nerve, more than 90% had an elongated nucleus and were located inside the basal lamina .
the corresponding numbers at site of lesion and in distal nerve segment were  <dig> and 85%, indicating that most cells were schwann cells.
in addition, double staining of schwann cells with cleaved caspase  <dig> and s- <dig> also showed that most of the cleaved caspase  <dig> stained cells were schwann cells.
the number of cleaved caspase  <dig> stained cells was significantly higher after a delayed nerve repair in accordance with a previous study using longer time points before repair  <cit> .
a higher number of cleaved caspase  <dig> stained cells after a delayed nerve repair may contribute to impaired axonal outgrowth, since the extent of outgrowth is dependent on the number of schwann cells  <cit> .
a longer delay than  <dig> days severely impairs axonal outgrowth and correlates to expression of the transduction factor atf  <dig> and inversely to number of cleaved caspase  <dig> stained schwann cells  <cit> .
taken together with present findings, it strongly suggests that a prompt nerve repair should be performed after a nerve injury.
the neurobiological data support the clinical observation that a delayed nerve repair following transection or laceration may impair functional recovery  <cit> .
CONCLUSIONS
a decreased regenerative capacity after a delayed nerve repair is associated with an impaired activation of schwann cells and an increased death of schwann cells .
the regenerating axons influence the erk 1/ <dig> activation and the number of apoptotic schwann cells.

 delayed repair of a peripheral nerve trunk after injury often results in impaired axonal regeneration and poor clinical outcome, although this depends on the duration of the delay. schwann cells are rapidly activated after a nerve injury and start to dedifferentiate and proliferate. insufficient nerve regeneration after delayed nerve repair has been attributed to an inability of schwann cells to support axonal outgrowth. to achieve a good clinical outcome after nerve repair it is important to clarify the molecular mechanisms by which schwann cell proliferation and apoptosis are orchestrated. a few studies have focused on signal transduction mechanisms with respect to axonal outgrowth after immediate and delayed nerve repair. however, signal transduction has sparsely been investigated after nerve injuries with and without accompanying repair. for instance, the activation of extracellular signal-regulated kinase and the induction of apoptosis in schwann cells have been observed after a nerve injury and after a long delayed nerve repair, respectively. in this study, we examined activation of erk1/ and induction of cleaved caspase in schwann cells after immediate, delayed or no repair of transected rat sciatic nerves and how the different repair conditions related to axonal outgrowth.  p-erk 1/2 p-erk 1/ stained schwann cells were observed both at the site of the lesion and in the distal nerve segment with a higher intensity in all experimental groups than in the uninjured control nerve. there were significant differences among the groups at the site of the lesion and in the distal nerve segment. immunopositive area for p-erk1/ was higher at the site of the lesion compared to the corresponding distal nerve segment when immediate repair and no delayed nerve repair was performed, but not after a delayed nerve repair and when no repair was done. a large immunopositive area for p-erk1/ was observed in the group with immediate nerve repair both at the site of the lesion and in the distal nerve segment, where the p-erk1/ positive area at the site of the lesion was almost twice as large as observed in the delayed repair group. in the distal nerve segment, there were no significant differences between the groups of immediate and delayed nerve repair. the immunopositive area was lower, however, when no immediate nerve repair was done than after immediate nerve repair at the two measured sites. the corresponding values in the experiments where a delayed or no delayed repair was done were p = for snl and p = for snd. no differences were found between the experiments where no nerve repairs were done, where the analyses of p-erk 1/ were done and days after injury. there were no differences between the four groups in the contralateral uninjured nerves with respect to p-erk1/ double staining with erk1/ and s- revealed that all cells with an elongated nucleus and located within the basal lamina also stained for s- indicating that all our selection criteria were correct, and they also stained for p-erk1/. cleaved caspase 3 cells containing cleaved caspase could be identified as schwann cells based on their shape and location. double staining with cleaved caspase and s- confirmed this identification. there were significant differences between the groups at the site of the lesion and in the distal nerve segment. the number of cleaved caspase stained cells was higher at the site of the lesion compared to further down in the corresponding distal nerve segment in all groups, except when a delayed nerve repair was done. the numbers of cleaved caspase stained cells were significantly increased in the group with a delayed nerve repair compared to those in the group with immediate nerve repair both at the site of the lesion and in the distal nerve segment. the numbers of cells were not different at the two sites in the corresponding experiments where no nerve repairs were done of the transected nerve trunk compared to the nerve repair groups. furthermore, when comparing the experiments where no nerve repairs were done and analyzed after and days, no difference in caspase stained cells were detected at the site of the lesion, but was almost twice as high at days in the distal nerve segment. there were no significant differences between the four groups in the contralateral uninjured nerves. length of axonal outgrowth axonal outgrowth from the proximal nerve segment into the corresponding distal nerve segments after nerve repair or from the proximal nerve segment after no repair was evaluated at days post surgery by neurofilament staining and was found to be different among the groups. in the groups where no repair was done most of the proximal stumps of the sciatic nerve formed a neuroma and with axonal outgrowth of only a few mm. axonal outgrowth after immediate nerve repair was longer than in any of the other groups; thus longer than after delayed nerve repair. correlations the length of axonal outgrowth correlated to the expression of p-erk 1/ both at the site of the lesion and in the distal nerve segment, while the length did not correlate with numbers of caspase stained cells. however, if only the repaired groups were included for cleaved caspase the corresponding values were p =, r = and p =. discussion activation of p-erk1/ and cleaved caspase in schwann cells was different after immediate and delayed nerve repair in the rat sciatic nerve and axonal outgrowth was longer after immediate repair. in addition, the length of axonal outgrowth correlated with erk 1/ activation, but not clearly correlated with cleaved caspase stained cells. thus, even a short delay of nerve repair as days results in impaired axonal outgrowth. the mitogen activating pathway kinase erk1/ is one important signaling pathway and activation of erk 1/ promotes schwann cell proliferation after nerve injury. schwann cells are removed by apoptosis during development before myelination and during regeneration after nerve injury, which can be marked by caspase or by tunel staining. nick labelling of apoptotic nuclei of sensory neurons after injury has also been detected. the immunopositive area for evaluation of p-erk1/ was present more predominantly at the site of the nerve lesion after immediate nerve repair, where 3-thymidine incorporation is maximal after a nerve injury. the increased presence of p-erk1/ at site of lesion after immediate repair could be related to contact between schwann cells and regenerating axons and to a cellular response similar as suggested for stat- in the distal nerve segment far from site of lesion. notably, a recent report indicates that functional recovery of injured nerves does not require schwann cell proliferation. however, in the contralateral nerve erk 1/ is low in the presence of uninjured axons, indicating that the nature of axonal contact is important. the positive correlation between length of axonal outgrowth and p-erk1/ stained area is reasonable since activation of erk 1/ is required for schwann cell proliferation and that axonal outgrowth positively correlates to number of schwann cells; thus, consistent with that schwann cell proliferation is crucial for nerve regeneration. the decreased p-erk1/ immunopositive area after a delayed nerve repair suggests that the responsiveness of schwann cells is weaker already after a days delayed nerve repair; further supported by findings of no differences between site of lesion and distal nerve segment with respect to erk 1/ and cleaved caspase after delayed nerve repair. activation of erk 1/ was still observed after days, which is consistent with a previous report observing erk 1/ days after nerve transection. the present experimental design with a delayed nerve repair included two injuries to the nerve with a day interval. such a conditioning lesion, i.e. first injury, can improve axonal outgrowth after a second lesion. however, conditioning by transection has a lower effect on axonal outgrowth than that of a conditioning crush lesion. of the counted total number of cells stained by dapi in the contralateral uninjured nerve, more than 90% had an elongated nucleus and were located inside the basal lamina. the corresponding numbers at site of lesion and in distal nerve segment were and 85%, indicating that most cells were schwann cells. in addition, double staining of schwann cells with cleaved caspase and s- also showed that most of the cleaved caspase stained cells were schwann cells. the number of cleaved caspase stained cells was significantly higher after a delayed nerve repair in accordance with a previous study using longer time points before repair. a higher number of cleaved caspase stained cells after a delayed nerve repair may contribute to impaired axonal outgrowth, since the extent of outgrowth is dependent on the number of schwann cells. a longer delay than days severely impairs axonal outgrowth and correlates to expression of the transduction factor atf and inversely to number of cleaved caspase stained schwann cells. taken together with present findings, it strongly suggests that a prompt nerve repair should be performed after a nerve injury. the neurobiological data support the clinical observation that a delayed nerve repair following transection or laceration may impair functional recovery.  a decreased regenerative capacity after a delayed nerve repair is associated with an impaired activation of schwann cells and an increased death of schwann cells. the regenerating axons influence the erk 1/ activation and the number of apoptotic schwann cells. 
in the present study, axonal outgrowth, activation of erk1/ by phosphorylation and immunoreactivity of cleaved caspase were examined after immediate, delayed, or no repair of transected rat sciatic nerves. axonal outgrowth, detected by neurofilament staining, was longer after immediate repair than after either the delayed or no repair conditions. axonal outgrowth correlated to p-erk 1/ but not clearly with cleaved caspase contact with regenerating axons affected schwann cells with respect to p-erk 1/ and cleaved caspase after immediate nerve repair only. extracellular-signal regulated kinase is activated by nerve damage and its activation precedes survival and proliferation of schwann cells. in contrast, activation of caspase a cysteine protease, is considered as a marker for apoptosis in schwann cells. immediate repair also showed a higher expression of p-erk 1/ and a lower number of cleaved caspase stained schwann cells than after delayed nerve repair. the decreased regenerative capacity that has historically been observed after delayed nerve repair may be related to impaired activation of schwann cells and increased schwann cell death. outgrowing axons influence erk 1/ activation and apoptosis of schwann cells.
 extracellular-signal regulated kinase is activated by nerve damage and its activation precedes survival and proliferation of schwann cells. in contrast, activation of caspase a cysteine protease, is considered as a marker for apoptosis in schwann cells. in the present study, axonal outgrowth, activation of erk1/ by phosphorylation and immunoreactivity of cleaved caspase were examined after immediate, delayed, or no repair of transected rat sciatic nerves.  axonal outgrowth, detected by neurofilament staining, was longer after immediate repair than after either the delayed or no repair conditions. immediate repair also showed a higher expression of p-erk 1/ and a lower number of cleaved caspase stained schwann cells than after delayed nerve repair. if the transected nerve was not repaired a lower level of p-erk 1/ was found than in either the immediate or delayed repair conditions. axonal outgrowth correlated to p-erk 1/ but not clearly with cleaved caspase contact with regenerating axons affected schwann cells with respect to p-erk 1/ and cleaved caspase after immediate nerve repair only.  the decreased regenerative capacity that has historically been observed after delayed nerve repair may be related to impaired activation of schwann cells and increased schwann cell death. outgrowing axons influence erk 1/ activation and apoptosis of schwann cells. 