recently , a group of small - molecule compounds have been identified as tyrosine receptor kinase ( trk ) receptor agonists that had profound pro - neuronal effects on neurogenesis and neuronal regeneration [ 14 ] .
these compounds hold great clinical importance for neurodegenerative diseases as they may easily penetrate blood - brain - barrier , and have great potency with half - effective concentration ( ec50 ) lower than 1 m . among those small - molecule compounds , a new compound , 7,8,3-trihydroxyflavone ( 7,8,3-thf ) ,
was created as the derivative of 7,8-dihydroxyflavone but with better potency . in the peripheral auditory system ,
thf was shown to rescue noise - damaged or chemically - injured auditory nerves both in vitro and in vivo .
however , the role of thf in spinal cord dorsal root ganglion ( drg ) neurons has not been characterized .
spinal cord drg neurons are subject to local anesthetics - induced neurotoxicity , which may contribute to various types of neurologic complications in young patients with regional anesthesia .
specifically , bupivacaine , one of the commonly used local anesthetics , is shown to induce neuronal apoptosis , growth cone collapse , and neurite retraction in drg neurons in animal models .
studies also showed that several of the neuronal signaling pathways , such as mitogen - activated protein kinase ( mapk ) and phosphatase and tensin homolog ( pten ) pathways , were closely associated with the regulation of bupivacaine - induced neurotoxicity in drg neurons .
member of the trk receptors  trkb receptor , and its ligand brain - derived neurotrophic factor ( bdnf )  are both upregulated in drg neurons after spinal cord injury [ 1416 ] , suggesting that there might be functional roles of the trkb signaling pathway in regulating drg neuron injury , but the exact role of trkb receptor in regulating local anesthetics - induce neurotoxicity remains elusive . in this study
, we used an explant model to culture neonatal mouse drg neurons in vitro to mimic the biological conditions of young spinal cord sensory neurons .
we first investigated the pro - neuronal effect of thf on the growth of neonatal drg neurons by examining its effect on drg neuron neurite outgrowth .
the potential protective effect of thf and the involvement of trkb signaling pathways were examined by apoptosis assay , neurite outgrowth assay , and western blot assay .
the results of our study may further our understanding on the possible pro - neuronal mechanisms of small - molecule trkb agonists in spinal cord sensory neurons .
doral root ganglion ( drg ) neurons were prepared from postnatal 23-day-(p2~p3 ) old c57bl/6j mice ( jackson lab , usa ) .
briefly , drg clumps were quickly dissected into cold ( 4c ) 1x hbss ( invitrogen , usa ) .
they were then transferred into dissociation medium with warm ( 37c ) dulbecco s modified eagle medium ( dmem , invitrogen , usa ) , 10% fetal bovine serum ( fbs , invitrogen , usa ) , 1 x b27 neuronal supplement ( invitrogen , usa ) , and 0.25% trypsin ( invitrogen , usa ) for 20 min .
dissociated cells were prepared by continuously triturating drg clumps in - and - out of 1 ml pipette tips for 10 min .
they were then re - suspended in serum - free culture medium containing neurobasal medium ( invitrogen , usa ) , penicillin / streptomycin ( penstrep , invitrogen , usa ) , and 1 x b27 neuronal supplement ( invitrogen , usa ) in a tissue culture chamber with 5% co2 at 37c overnight . on the second day , the floating non - neuronal cells were removed .
drg neurons were maintained in culture medium for 2~7 days , depending on the requirement of designated experiments .
cultured drg neurons were fixed with 4% paraformaldehyde ( pfa , sigma - aldrich , usa ) and 0.3% triton ( sigma - aldrich , usa ) in 1x pbs ( invitrogen , usa ) for 30 min , then incubated with a rabbit anti - neurofilament 2000 ( nf-2000 ) polyclonal antibody ( santa cruz , usa ) at 37c over night . on the second day , the culture was incubated with alexafluor 488 anti - rabbit secondary antibody at room temperature for 2 h. the green fluorescent images were viewed under an upright fluorescent microscope ( bx51 , olympus , japan ) . for each experimental condition ,
the averaged length of the longest 50~80 neurites were measured among at least three repeats , and then normalized to control condition . in drg neuron culture , various concentrations ( 2 , 5 , 10 , 20 , 50 , 100 , 250 , 500 , 1000 , 1500 , 2000 nm ) of 7 , 8
the effect of thf on drg neuron growth was estimated by the neurite outgrowth assay .
drg neurons were collected from cultures and treated with a lysis buffer ( 50 mm tris at ph 7.6 , 150 mm nacl , 1 mm edta , 10% glycerol , 0.5% np-40 and protease inhibitor cocktail , millipore , usa ) . after checking with concentrations ,
proteins were separated by electrophoresis on 12% sds - page gel , and transferred to pvdf membranes .
the membranes were blocked with 5% non - fat dry milk and 1% bsa for 2 h , followed by incubation with primary antibodies of rabbit polyclonal anti - trkb ( 1:500 , novus biological , usa ) and rabbit polyclonal anti - p - trkb ( 1:200 , novus biological , usa ) overnight at 4c . on the second day
, membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit secondary antibody ( 1:5,000 , novus biological , usa ) for 2 h at room temperature .
the blots were then visualized by enhanced chemiluminescence ( pierce , usa ) according to the manufacturer s protocol . in drg neuron culture ,
briefly , 10 mm bupivacaine was added into culture for 2 h to induce substantial neuronal apoptosis .
the culture was washed with fresh medium 3 times ( 10 min / time ) , and maintained for another 24 h before further evaluation .
drg culture was quickly washed with pbs , and fixed by 4% paraformaldehyde ( pfa , sigma - aldrich , usa ) for 30 min .
the apoptosis of drg neurons was examined by a terminal deoxyribonucleotidyl transferase ( tdt)-mediated biotin-16-dutp nick - end labeling ( tunel ) apoptosis kit ( r&d systems , usa ) according to the manufacturer s protocol .
a mouse monoclonal neun antibody ( millipore , usa ) was used to identify the drg neurons .
fluorescent images were then examined under an upright fluorescent microscope ( bx51 , olympus , japan ) .
relative apoptosis was quantified as the percentage of tunel - positive cells among all neun - positive cells for each experimental condition .
statistical comparisons between means were examined by two - tailed unpaired student s t test on spss software ( version 13.0 , spss , usa ) .
doral root ganglion ( drg ) neurons were prepared from postnatal 23-day-(p2~p3 ) old c57bl/6j mice ( jackson lab , usa ) .
briefly , drg clumps were quickly dissected into cold ( 4c ) 1x hbss ( invitrogen , usa ) .
they were then transferred into dissociation medium with warm ( 37c ) dulbecco s modified eagle medium ( dmem , invitrogen , usa ) , 10% fetal bovine serum ( fbs , invitrogen , usa ) , 1 x b27 neuronal supplement ( invitrogen , usa ) , and 0.25% trypsin ( invitrogen , usa ) for 20 min .
dissociated cells were prepared by continuously triturating drg clumps in - and - out of 1 ml pipette tips for 10 min .
they were then re - suspended in serum - free culture medium containing neurobasal medium ( invitrogen , usa ) , penicillin / streptomycin ( penstrep , invitrogen , usa ) , and 1 x b27 neuronal supplement ( invitrogen , usa ) in a tissue culture chamber with 5% co2 at 37c overnight . on the second day , the floating non - neuronal cells were removed .
drg neurons were maintained in culture medium for 2~7 days , depending on the requirement of designated experiments .
cultured drg neurons were fixed with 4% paraformaldehyde ( pfa , sigma - aldrich , usa ) and 0.3% triton ( sigma - aldrich , usa ) in 1x pbs ( invitrogen , usa ) for 30 min , then incubated with a rabbit anti - neurofilament 2000 ( nf-2000 ) polyclonal antibody ( santa cruz , usa ) at 37c over night . on the second day ,
the culture was incubated with alexafluor 488 anti - rabbit secondary antibody at room temperature for 2 h. the green fluorescent images were viewed under an upright fluorescent microscope ( bx51 , olympus , japan ) .
for each experimental condition , the averaged length of the longest 50~80 neurites were measured among at least three repeats , and then normalized to control condition .
in drg neuron culture , various concentrations ( 2 , 5 , 10 , 20 , 50 , 100 , 250 , 500 , 1000 , 1500 , 2000 nm ) of 7 , 8 , 3-trihydroxyflavone ( thf ) were added for 2 days .
the effect of thf on drg neuron growth was estimated by the neurite outgrowth assay .
drg neurons were collected from cultures and treated with a lysis buffer ( 50 mm tris at ph 7.6 , 150 mm nacl , 1 mm edta , 10% glycerol , 0.5% np-40 and protease inhibitor cocktail , millipore , usa ) . after checking with concentrations ,
proteins were separated by electrophoresis on 12% sds - page gel , and transferred to pvdf membranes .
the membranes were blocked with 5% non - fat dry milk and 1% bsa for 2 h , followed by incubation with primary antibodies of rabbit polyclonal anti - trkb ( 1:500 , novus biological , usa ) and rabbit polyclonal anti - p - trkb ( 1:200 , novus biological , usa ) overnight at 4c . on the second day
, membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit secondary antibody ( 1:5,000 , novus biological , usa ) for 2 h at room temperature .
the blots were then visualized by enhanced chemiluminescence ( pierce , usa ) according to the manufacturer s protocol .
in drg neuron culture , treatment of bupivacaine was conducted according to the method described before .
briefly , 10 mm bupivacaine was added into culture for 2 h to induce substantial neuronal apoptosis .
the culture was washed with fresh medium 3 times ( 10 min / time ) , and maintained for another 24 h before further evaluation .
drg culture was quickly washed with pbs , and fixed by 4% paraformaldehyde ( pfa , sigma - aldrich , usa ) for 30 min .
the apoptosis of drg neurons was examined by a terminal deoxyribonucleotidyl transferase ( tdt)-mediated biotin-16-dutp nick - end labeling ( tunel ) apoptosis kit ( r&d systems , usa ) according to the manufacturer s protocol .
a mouse monoclonal neun antibody ( millipore , usa ) was used to identify the drg neurons .
fluorescent images were then examined under an upright fluorescent microscope ( bx51 , olympus , japan ) .
relative apoptosis was quantified as the percentage of tunel - positive cells among all neun - positive cells for each experimental condition .
statistical comparisons between means were examined by two - tailed unpaired student s t test on spss software ( version 13.0 , spss , usa ) .
we first investigated the possible pro - neuronal effect of thf on drg neuron growth .
the cultured mouse neonatal ( p2~p3 ) drg neurons were treated with various concentrations of thf ( 2 , 5 , 10 , 20 , 50 , 100 , 250 , 500 , 1000 , 1500 , and 2000 nm ) for 2 days .
images of green - fluorescence - positive drg neuron neurites showed that thf significantly promoted neurite growth in a dose - dependent manner ( figure 1a ) .
the relative lengths of drg neuron neurites , corresponding to different thf concentrations , were compared in neurite outgrowth assay and fit with a hill equation ( figure 1b ) .
the ec50 concentration was determined to be 67.4 nm we then investigated the molecular pathway associated with pro - neuronal effect of thf on promoting drg neurite outgrowth .
we used western blot assay to examine the protein level of trb receptor , as well as phosphorylated trkb ( p - trkb ) .
it showed that protein levels of trkb were unchanged by treatment of different concentrations of thf , but levels of p - trkb were significantly unregulated with the applications of higher concentrations of thf ( figure 1c ) .
thus , our data strongly suggests that thf promoted drg neuron growth by acting as a trkb agonist .
one of the commonly used anesthetics , bupivacaine , was shown to induce neurotoxicity in drg neurons in a concentration - dependent manner .
as we showed thf had pro - neuronal effect on drg neuron growth , we wondered whether thf could also rescue bupivacaine - induced neurotoxicity in drg neurons . to test this hypothesis , we maintained drg culture for 3 to 5 days , followed by pre - treatment of thf for 24 h. we then used a high concentration of bupivacaine  10 mm ( 2 h )  to induce significant neuronal apoptosis in drg neurons .
twenty - four hours after bupivacaine treatment , a tunel assay was conducted to evaluate the apoptosis among drg neurons .
a neun neuronal - antibody was used alongside tunel assay to precisely identify the neuronal population in the culture .
the immunohistochemical results demonstrated that while there was no thf pre - treatment ( control ) , most of the drg neurons ( neun - positive ) were apoptotic ( tunel - positive ) due to bupivacaine - induced neurotoxicity ( figure 2a , left column ) .
however , when drg neurons were pre - treated with 50 nm or 1 m thf , bupivacaine - induced neuronal apoptosis was markedly reduced ( figure 2a , right two columns ) . quantified measurement of tunel assay showed that the percentage of apoptotic drg neurons was significantly reduced , from 85.34.5% with no thf pre - treatment ( control ) , to 45.27.3% with 50 nm thf pre - treatment , then to 19.65.7% with 1 m thf ( figure 2b , 
 p<0.05 ) .
thus , our data suggest that thf pre - treatment was effective in reducing bupivacaine - induced neuronal apoptosis in drg neurons .
we then evaluated the protective effect of thf pre - treatment on bupivacaine - induced neurite retraction in drg neurons . at 24 h after bupivacaine treatment , immunohistochemical results with a neurite outgrowth assay demonstrated that there was no thf pre - treatment ( control ) , but bupivacaine introduced significant neurite retraction in drg neurons ( figure 3a , left column )
however , when drg culture was treated with either 50 nm or 1 m thf , neurite retraction was markedly rescued ( figure 3a , right two columns ) . quantified measurement by neurite outgrowth assay indicated that drg neurite lengths were increased by 37933% with 50 nm thf pre - treatment , and by 157179% with 1 m thf , compared to the neurite length under control condition ( figure 3b , 
 p<0.05 ) .
we also evaluated whether the protection of thf on drg neurite retraction was correlated with the activation of the trkb signaling pathway .
the result of western blot confirmed this hypothesis by showing that , in bupivacaine - injured drg neurons , phosphorylated trkb ( p - trkb ) was increased by thf pre - treatment in a concentration - dependent manner , whereas trkb protein levels were unaltered ( figure 3c ) .
thus , our data suggest that thf pre - treatment was also effective in protecting bupivacaine - induced neurite retraction in drg neurons , very likely through activation of the trkb signaling pathway .
we first investigated the possible pro - neuronal effect of thf on drg neuron growth .
the cultured mouse neonatal ( p2~p3 ) drg neurons were treated with various concentrations of thf ( 2 , 5 , 10 , 20 , 50 , 100 , 250 , 500 , 1000 , 1500 , and 2000 nm ) for 2 days .
images of green - fluorescence - positive drg neuron neurites showed that thf significantly promoted neurite growth in a dose - dependent manner ( figure 1a ) .
the relative lengths of drg neuron neurites , corresponding to different thf concentrations , were compared in neurite outgrowth assay and fit with a hill equation ( figure 1b ) .
the ec50 concentration was determined to be 67.4 nm we then investigated the molecular pathway associated with pro - neuronal effect of thf on promoting drg neurite outgrowth .
we used western blot assay to examine the protein level of trb receptor , as well as phosphorylated trkb ( p - trkb ) .
it showed that protein levels of trkb were unchanged by treatment of different concentrations of thf , but levels of p - trkb were significantly unregulated with the applications of higher concentrations of thf ( figure 1c ) .
thus , our data strongly suggests that thf promoted drg neuron growth by acting as a trkb agonist .
one of the commonly used anesthetics , bupivacaine , was shown to induce neurotoxicity in drg neurons in a concentration - dependent manner .
as we showed thf had pro - neuronal effect on drg neuron growth , we wondered whether thf could also rescue bupivacaine - induced neurotoxicity in drg neurons . to test this hypothesis , we maintained drg culture for 3 to 5 days , followed by pre - treatment of thf for 24 h. we then used a high concentration of bupivacaine  10 mm ( 2 h )  to induce significant neuronal apoptosis in drg neurons .
twenty - four hours after bupivacaine treatment , a tunel assay was conducted to evaluate the apoptosis among drg neurons .
a neun neuronal - antibody was used alongside tunel assay to precisely identify the neuronal population in the culture .
the immunohistochemical results demonstrated that while there was no thf pre - treatment ( control ) , most of the drg neurons ( neun - positive ) were apoptotic ( tunel - positive ) due to bupivacaine - induced neurotoxicity ( figure 2a , left column ) .
however , when drg neurons were pre - treated with 50 nm or 1 m thf , bupivacaine - induced neuronal apoptosis was markedly reduced ( figure 2a , right two columns ) . quantified measurement of tunel assay showed that the percentage of apoptotic drg neurons was significantly reduced , from 85.34.5% with no thf pre - treatment ( control ) , to 45.27.3% with 50 nm thf pre - treatment , then to 19.65.7% with 1 m thf ( figure 2b , 
 p<0.05 ) .
thus , our data suggest that thf pre - treatment was effective in reducing bupivacaine - induced neuronal apoptosis in drg neurons .
we then evaluated the protective effect of thf pre - treatment on bupivacaine - induced neurite retraction in drg neurons . at 24 h after bupivacaine treatment , immunohistochemical results with a neurite outgrowth assay demonstrated that there was no thf pre - treatment ( control ) , but bupivacaine introduced significant neurite retraction in drg neurons ( figure 3a , left column )
however , when drg culture was treated with either 50 nm or 1 m thf , neurite retraction was markedly rescued ( figure 3a , right two columns ) . quantified
measurement by neurite outgrowth assay indicated that drg neurite lengths were increased by 37933% with 50 nm thf pre - treatment , and by 157179% with 1 m thf , compared to the neurite length under control condition ( figure 3b , 
 p<0.05 ) .
we also evaluated whether the protection of thf on drg neurite retraction was correlated with the activation of the trkb signaling pathway .
the result of western blot confirmed this hypothesis by showing that , in bupivacaine - injured drg neurons , phosphorylated trkb ( p - trkb ) was increased by thf pre - treatment in a concentration - dependent manner , whereas trkb protein levels were unaltered ( figure 3c ) .
thus , our data suggest that thf pre - treatment was also effective in protecting bupivacaine - induced neurite retraction in drg neurons , very likely through activation of the trkb signaling pathway .
in the present study we discovered that , 7 , 8 , 3-trihydr - oxyflavone , a potent trkb agonist small molecule , had a profound pro - neuronal effect on neuronal growth , as well as protecting against local anesthetic - induced neurotoxicity in spinal cord drg neurons .
we demonstrated that thf promoted neurite growth in neonatal drg neuron culture in a concentration - dependent manner .
the calculated ec50 was calculated to be 67.4 nm , in line with other studies showing low thf ec50 .
this finding was particularly encouraging as it suggests that thf can promote spinal cord neuronal development with great potency .
we also demonstrated that the thf - activated trkb signaling pathway works by phosphorylating trkb in drg neurons , possibly acting as a trkb agonist .
recently , a relative of thf  7,8-dihydroxyflavone ( dhf )  was shown to promote spinal cord motoneuron embryonic development , as well as inducing functional recovery in myotrophic lateral sclerosis .
interestingly , dhf seemed to act through the akt signaling pathway rather than directly on the bdnf / trkb signaling pathway in motoneurons .
therefore , it seems that complex signaling pathways may be differentially associated with thf ( or dhf ) in regulating spinal cord drg neurons and motoneurons .
studies have shown that epigenetic regulation of microrna , as well as pten / akt signaling pathways , are involved in local anesthetics - induced neurotoxicity in drg neurons .
in addition , p38 mitogen - activated protein kinase ( mapk ) was reported to be a critical component contributing to lidocaine - induced neurotoxicity in adult drg neurons . in the present study we demonstrated that thf had a protective effect on bupivacaine - induced neurotoxicity in drg neurons by rescuing neuronal apoptosis and neurite retraction .
also , we showed that the trkb signaling pathway was activated in accordance with thf treatment in bupivacaine - injured drg neurons .
although our data suggest that thf likely acts as a survival factor through a trkb - dependent manner in bupivacaine - injured drg neurons , future experiments would help to elucidate the direct involvement of the bdnf / trkb signaling pathway , as well as its association with other molecular pathways in anesthetics - induced neurotoxicity in drg neurons .
overall , in the present study we revealed new mechanism of 7 , 8 , 3-trihydroxyflavone as a pro - neuronal small molecule in promoting neuronal growth and protecting against bupivacaine - induced neurotoxicity in spinal cord drg neurons .
the associated signaling pathway of thf in drg neurons is very likely through trkb activation .
these findings may help to identify novel molecular targets to be applied in future clinical settings to benefit patients with spinal cord disorders or injuries .
 background7 , 8 , 3-trihydroxyflavone ( thf ) is a novel pro - neuronal small molecule that acts as a trkb agonist . in this study 
 , we examined the effect of thf on promoting neuronal growth and protecting anesthetics - induced neurotoxicity in dorsal root ganglion ( drg ) neurons in vitro.material/methodsneonatal mouse drg neurons were cultured in vitro and treated with various concentrations of thf . 
 the effect of thf on neuronal growth was investigated by neurite outgrowth assay and western blot . 
 in addition , the protective effects of thf on bupivacaine - induced neurotoxicity were investigated by apoptosis tunel assay , neurite outgrowth assay , and western blot , respectively.resultsthf promoted neurite outgrowth of drg neurons in dose - dependent manner , with an ec50 concentration of 67.4 nm . 
 western blot analysis showed thf activated trkb signaling pathway by inducing trkb phosphorylation . 
 thf also rescued bupivacaine - induced neurotoxicity by reducing apoptosis and protecting neurite retraction in drg neurons . 
 furthermore , the protection of thf in bupivacaine - injured neurotoxicity was directly associated with trkb phosphorylation in a concentration - dependent manner in drg neurons.conclusionsthf has pro - neuronal effect on drg neurons by promoting neurite growth and protecting against bupivacaine - induced neurotoxicity , likely through trkb activation . 