prostatic acid phosphatase ( pap ) , a glycoprotein synthesized by the prostate gland , is traditionally used to diagnose prostate cancer .
pap has two isoforms , secreted and transmembrane isoforms , and , more than in the prostate gland , is also extensively expressed in nonprostate tissue such as nervous tissues .. recent studies have identified pap is molecular identity to thiamine monophosphatase ( tmpase ) and , historically , tmpase , with the acid phosphatase characteristics , has been widely used for labeling nociceptive dorsal root ganglia ( drg ) neurons .
pap hydrolyzes adenine monophosphate ( amp ) , particularly in acidic environments , to adenosine that acts as an analgesic effector .
moreover , small nociceptive drg neurons , including peptidergic and nonpeptidergic neurons , coexpress pap , suggesting that pap profiles correlate with pain modulation .
for example , a growing body of evidence has shown that intrathecal injection of recombinant pap counteracts inflammatory pain and enhances neuropathic pain in pap - knockout mice .
previously , we have established a mouse model of small - fiber neuropathy , in which small sensory nerves were specifically depleted by resiniferatoxin ( rtx ) , a capsaicin analog acting on the transient receptor potential vanilloid receptor 1 ( trpv1 ) .
this rtx - induced neuropathy model mimics human small - fiber neuropathy ; that is , the model exhibits the degeneration of cutaneous sensory nerves and neuropathic pain .
furthermore , rtx - induced neuropathy with skin denervation markedly increases the expression of activating transcription factor 3 ( atf3 ) , a marker of neuronal injury , in nonpeptidergic p2x3 neurons , which is correlated with neuropathic pain .
whether the expression profiles of this subpopulation of pap neurons correlate with injury - induced neuropathic pain should be determined , and the mechanism responsible for modulating the analgesic potential in injury - induced neuropathic pain should be investigated .
the compound 4-methylcatechol ( 4mc ) enhances the synthesis of neurotrophins such as nerve growth factor ( ngf ) and promotes skin reinnervation .
cutaneous sensory nerves function as the peripheral receptors of small drg neurons ; thus , inducing atf3 expression in these neurons caused skin denervation and neuropathic pain in our rtx - induced neuropathy model .
ngf is trophic to small drg neurons and modulates neuropathic pain through its high - affinity receptor , trka ; a reduction in ngf and trka expression is accompanied by various peripheral neuropathies . whether 4mc modulates pap expression profiles through ngf - trka signaling is unclear .
rtx - induced neuropathy is an excellent model for determining how the loss of the analgesic effect of pap leads to injury - induced neuropathic pain . in the present study , we investigated the intervention of pap profiles to neuropathic pain and the effect of 4mc on reversing neuropathic pain through ngf - trka signaling .
this study investigated pap - trka signaling - mediated pain behavior in rtx - induced neuropathy by investigating whether pap neuropathology is correlated with pain development and whether 4mc reverses pain and pap profiles in rtx - induced neuropathy in an ngf - trka signaling - dependent manner .
administration of a single dose of rtx ( 50  g / kg , sigma , st . louis , mo ) ( the rtx group ) .
injections of 4mc ( 10  g / kg , wako , osaka , japan ) immediately after rtx treatment for seven consecutive days ( the 4mc group ) , or an equal volume of vehicle as a control ( the vehicle group ) . to confirm the effect of 4mc ,
. administered . briefly , 2.5s ngf ( alomone labs , jerusalem , israel ) was dissolved in dulbecco s modified eagle medium , and mice received ngf ( 1  g/10  g ) immediately after rtx treatment ( the ngf group ) .
some mice in the 4mc group received additional anti - ngf antisera ( 30  g / kg , sigma ) to neutralize ngf induced by 4mc ( the abngf group ) . to compare each experimental approach , 2.5s ngf and anti
after treatment , the mice were housed in plastic cages in a 12-h light / dark cycle and were given access to water and food ad libitum .
all procedures were conducted in accordance with ethical guidelines for laboratory animals , and the protocol was approved by the institutional animal care and use committee of kaohsiung medical university .
all experimental procedures were performed carefully , and all efforts were made to minimize animal suffering .
the changes in the mechanical threshold of each group were assessed using the up - and - down method with different calibers of von frey monofilaments ( somedic sales ab , hrby , sweden ) in accordance with our established protocol . briefly , a series of monofilaments was applied to the plantar region of the hindpaw .
if paw withdrawal occurred , a monofilament of a smaller caliber was applied . in the absence of paw withdrawal
four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses , and the mechanical thresholds were calculated using a formula published in a previous study .
animals were sacrificed by intracardiac perfusion with 0.1  m phosphate buffer ( pb ) followed by 4% paraformaldehyde ( 4  p ) in 0.1  m pb . after perfusion , the 4th and 5th ( l4 and l5 , respectively ) lumbar drgs were removed carefully and postfixed in 4p for another 6  h. the drgs were cryoprotected with 30% sucrose in 0.1  m pb overnight , and 8  -m - thick cryosections were obtained using a cryostat ( cm1850 , leica , wetzlar , germany ) . for adequate sampling , two ganglia ( l4/l5 ) per mouse and 5 to 8 sections per drg tissue ( at 80-m intervals ) were immunostained .
the following primary antisera were used in this study : anti - pap ( chicken , 1:600 , aves labs , tigard , or ) , anti - atf3 ( rabbit , 1:100 , santa cruz biotechnology , santa cruz , ca ) , anti - peripherin ( rabbit , 1:800 , chemicon , temecula , ca ) , anti - neurofilament smi32 ( mouse , 1:600 , covance , emeryville , ca ) , anti - p2x3 ( rabbit , 1:400 , neuromics , edina , mn ) , and anti - trka ( rabbit , 1:200 , santa cruz ) .
briefly , sections were incubated overnight at 4 with one of the primary antiserum combinations : p2x3/pap , pap / peripherin , pap / smi32 , atf3/pap , and pap / trka . thereafter
, the sections were incubated with texas red or fluorescein isothiocyanate - conjugated secondary antisera ( 1:100 , jackson immunoresearch , west grove , pa ) corresponding to the appropriate primary antisera for 1  h. sections were mounted with vectashield ( vector , burlingame , ca ) for quantification . to quantify the various phenotypic drg neurons ,
each drg section was systematically photographed at 200    under a fluorescence microscope ( axiophot microscope , zeiss , oberkochen , germany ) to produce a montage of the entire drg section according to an established procedure . to avoid density bias , only neurons with a clear nuclear profile
were counted , and only sections containing neuronal ganglia were measured using imagej version 1.44d ( national institutes of health [ nih ] , bethesda , md ) . for morphometric analysis of pap neuropathology , the diameters of pap(+ ) neurons and atf3(+)/pap(+ )
neurons were measured using image pro - plus ( media cybernetics , bethesda , md ) and plotted as a histogram . to minimize individual bias ,
five to seven mice were included in each group , and the coding information was masked during the behavioral tests and quantification procedures .
all data are expressed as mean    standard derivation ( sd ) of the mean .
a t test was performed to analyze the data following a gaussian distribution . for data which did not follow a gaussian distribution , a nonparametric mann 
this study investigated pap - trka signaling - mediated pain behavior in rtx - induced neuropathy by investigating whether pap neuropathology is correlated with pain development and whether 4mc reverses pain and pap profiles in rtx - induced neuropathy in an ngf - trka signaling - dependent manner .
administration of a single dose of rtx ( 50  g / kg , sigma , st . louis , mo ) ( the rtx group ) .
injections of 4mc ( 10  g / kg , wako , osaka , japan ) immediately after rtx treatment for seven consecutive days ( the 4mc group ) , or an equal volume of vehicle as a control ( the vehicle group ) . to confirm the effect of 4mc ,
. administered . briefly , 2.5s ngf ( alomone labs , jerusalem , israel ) was dissolved in dulbecco s modified eagle medium , and mice received ngf ( 1  g/10  g ) immediately after rtx treatment ( the ngf group ) .
some mice in the 4mc group received additional anti - ngf antisera ( 30  g / kg , sigma ) to neutralize ngf induced by 4mc ( the abngf group ) . to compare each experimental approach , 2.5s ngf and anti
after treatment , the mice were housed in plastic cages in a 12-h light / dark cycle and were given access to water and food ad libitum .
all procedures were conducted in accordance with ethical guidelines for laboratory animals , and the protocol was approved by the institutional animal care and use committee of kaohsiung medical university .
all experimental procedures were performed carefully , and all efforts were made to minimize animal suffering .
the changes in the mechanical threshold of each group were assessed using the up - and - down method with different calibers of von frey monofilaments ( somedic sales ab , hrby , sweden ) in accordance with our established protocol . briefly , a series of monofilaments was applied to the plantar region of the hindpaw .
if paw withdrawal occurred , a monofilament of a smaller caliber was applied . in the absence of paw withdrawal
four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses , and the mechanical thresholds were calculated using a formula published in a previous study .
animals were sacrificed by intracardiac perfusion with 0.1  m phosphate buffer ( pb ) followed by 4% paraformaldehyde ( 4  p ) in 0.1  m pb . after perfusion , the 4th and 5th ( l4 and l5 , respectively ) lumbar drgs were removed carefully and postfixed in 4p for another 6  h. the drgs were cryoprotected with 30% sucrose in 0.1  m pb overnight , and 8  -m - thick cryosections were obtained using a cryostat ( cm1850 , leica , wetzlar , germany ) . for adequate sampling , two ganglia ( l4/l5 ) per mouse and 5 to 8 sections per drg tissue ( at 80-m intervals ) were immunostained .
the following primary antisera were used in this study : anti - pap ( chicken , 1:600 , aves labs , tigard , or ) , anti - atf3 ( rabbit , 1:100 , santa cruz biotechnology , santa cruz , ca ) , anti - peripherin ( rabbit , 1:800 , chemicon , temecula , ca ) , anti - neurofilament smi32 ( mouse , 1:600 , covance , emeryville , ca ) , anti - p2x3 ( rabbit , 1:400 , neuromics , edina , mn ) , and anti - trka ( rabbit , 1:200 , santa cruz ) .
briefly , sections were incubated overnight at 4 with one of the primary antiserum combinations : p2x3/pap , pap / peripherin , pap / smi32 , atf3/pap , and pap / trka . thereafter
, the sections were incubated with texas red or fluorescein isothiocyanate - conjugated secondary antisera ( 1:100 , jackson immunoresearch , west grove , pa ) corresponding to the appropriate primary antisera for 1  h. sections were mounted with vectashield ( vector , burlingame , ca ) for quantification .
to quantify the various phenotypic drg neurons , each drg section was systematically photographed at 200    under a fluorescence microscope ( axiophot microscope , zeiss , oberkochen , germany ) to produce a montage of the entire drg section according to an established procedure . to avoid density bias , only neurons with a clear nuclear profile were counted , and only sections containing neuronal ganglia were measured using imagej version 1.44d ( national institutes of health [ nih ] , bethesda , md ) . for morphometric analysis of pap neuropathology , the diameters of pap(+ ) neurons and atf3(+)/pap(+ )
neurons were measured using image pro - plus ( media cybernetics , bethesda , md ) and plotted as a histogram .
to minimize individual bias , five to seven mice were included in each group , and the coding information was masked during the behavioral tests and quantification procedures .
all data are expressed as mean    standard derivation ( sd ) of the mean .
a t test was performed to analyze the data following a gaussian distribution . for data which did not follow a gaussian distribution , a nonparametric mann 
in a previous study , we developed an rtx - induced small - fiber neuropathy mouse model , in which sensitized p2x3 purinociceptors - mediated mechanical allodynia . in the present study , rtx affected pap expression , as demonstrated by the coexpression of p2x3 and pap ( figure 1 ) .
notably , pap and p2x3 showed inverse patterns after neuropathy ; specifically , there was an increase in p2x3(+ ) and decrease in pap(+ ) neurons , resulting in an increased p2x3/pap ratio ( 67.4%    2.1% vs. 44.7%    3.4% , p  <  0.0001 ) and decreased pap / p2x3 ratio ( 87.1%    5.4% vs. 76.8%    2.3% , p  =  0.001 ) ( figure 1(e ) ) .
furthermore , pap expression was higher in peripherin(+ ) neurons than in neurofilament ( smi32)(+ ) neurons ( figure 2 ) .
the diameter histograms confirmed that the pap(+ ) neurons were small to medium drg neurons ( 25th75th percentile : 17.123.7  m ) ( figure 2(f ) ) .
figure 1.colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups .
( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d).bar , 50  m . * * p  <  0.01 , * * * p  < 
0.001 . 
 figure 2.expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons .
double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) .
( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) .
( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) .
( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons.bar , 50  m . * * p  <  0.01 colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups .
( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d ) .
bar , 50  m . * * p  <  0.01 , * * * p  < 
0.001 . expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons .
double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) .
( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) .
( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) .
( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons .
bar , 50  m . * * p  <  0.01 to understand the expression of pap and its relationship with atf3 , an injury marker , we performed double immunofluorescence staining of drg neuronal sections in rtx - induced neuropathy ( figure 3(a ) to ( e ) ) .
pap(+ ) neurons were mildly lower in the rtx group than in the vehicle group ( 190.6    12.2 vs. 269.5    6.9 neurons / mm , p  =  0.0003 ) ( figure 3(f ) ) .
by contrast , atf3(+ ) neurons were markedly higher in the rtx group than in the vehicle group ( 192.3    18.0 vs. 9.0    13.1 neurons / mm , p  < 
moreover , atf3 was induced preferentially in pap(+ ) neurons ( figure 3(a ) to ( e ) ) , resulting in an increased ratio of atf3(+)/pap(+ ) neurons in the rtx group ( 29.0%    5.6% vs. 0.2%    0.2% , p  =  0.0043 ) ( figure 3(h ) ) . 

figure 3.phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a  e in red ) and anti - pap ( a  e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx  +  2.5s ngf ; d ) , and ( e ) abngf ( 4mc  +  anti - ngf antisera ) groups .
( f  h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) .
( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods .
the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups .
( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons .
( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups .
the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group.bar , 50  m . * p  <  0.05 , * * p  <  0.01 , * * * p  <  0.001 .
phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a  e in red ) and anti - pap ( a  e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx  +  2.5s ngf ; d ) , and ( e ) abngf ( 4mc  +  anti - ngf antisera ) groups .
( f  h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) .
( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods .
the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups .
( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons .
( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups .
the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group .
p  <  0.05 , * * p  <  0.01 , * * * p  <  0.001 .
mice in the rtx group showed mechanical allodynia compared with the vehicle group ( 747.1    156.0 vs. 299.6    70.6  mg , p  =  0.0025 ) ( figure 3(i ) ) . this behavioral change
correlated with the pap profiles ; that is , the densities of pap(+ ) neurons ( r  =  0.75 , p  <  0.0001 ; figure 3(j ) ) and the ratios of atf3(+)/pap(+ ) neurons ( r  =  0.60 , p  =  0.003 ; figure 3(k ) ) were inversely linear to the mechanical thresholds .
collectively , these findings indicate that pap expression had a strong influence in mediating pain development .
after neuropathy was induced using rtx , 4mc normalized pap expression and relieved mechanical allodynia ; specifically , the pap(+ ) neuronal density ( 269.7    48.8 neurons / mm , p  =  0.61 ) ( figure 3(c ) and ( f ) ) and mechanical allodynia ( 693.8    146.0  mg , p  =  0.54 ) ( figure 3(i ) ) in the 4mc group were comparable with those in the vehicle group .
furthermore , although 4mc reduced the atf3(+ ) neuronal density ( 102.4    27.8 neurons / mm , p  <  0.0001 ) and ratio of atf3(+)/pap(+ ) neurons ( 15.8%    5.9% , p  =  0.0095 ) , the ratios of atf3(+)/pap(+ ) neurons were still higher in the 4mc group than in the vehicle group ( p  =  0.016 ; figure 3(c ) , ( g ) , ( h ) ) .
morphometric analysis demonstrated that 4mc reduced the absolute number of atf3(+)/pap(+ ) neurons but did not have a significant effect on their mean diameter ( 15.5    2.2 vs. 16.5    2.8  m , p  >  0.05 ) ( figure 3(l ) vs. ( m ) ) . to understand the putative mechanisms of pain relief by 4mc
, we examined the coexpression of pap(+)/trka(+ ) neurons ( figure 4 ) . for each group , pap(+ ) neuronal density was similar to trka(+ ) neuronal density ( figure 3(f ) vs. 4(k ) ) .
the pap / trka ratios correlated linearly with the mechanical threshold ( r  =  0.62 , p  =  0.0062 ; figure 4(m ) ) . to confirm the effect of 4mc ,
the mice in the ngf group were systemically ( i.p . ) administered 2.5s ngf .
the ngf group exhibited the same effect as the 4mc group ; that is , pap and trka expression profiles were reversed , atf3 induction was normalized , and mechanical allodynia relieved .
those findings , however , were not observed in the abngf group ( administered anti - ngf antisera to neutralize ngf ) ( figures 3 and 4 ) .
figure 4.colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy .
( a  j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a  e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx  +  2.5s ngf ) , and ( e , j ) abngf ( 4mc  +  anti - ngf antisera ) groups . ( k , l )
the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) .
( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons.bar , 50  m . * p  <  0.05 , * * p  <  0.01 .
colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy .
( a  j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a  e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx  +  2.5s ngf ) , and ( e , j ) abngf ( 4mc  +  anti - ngf antisera ) groups .
( k , l ) the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) .
( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons .
in a previous study , we developed an rtx - induced small - fiber neuropathy mouse model , in which sensitized p2x3 purinociceptors - mediated mechanical allodynia . in the present study , rtx affected pap expression , as demonstrated by the coexpression of p2x3 and pap ( figure 1 ) .
notably , pap and p2x3 showed inverse patterns after neuropathy ; specifically , there was an increase in p2x3(+ ) and decrease in pap(+ ) neurons , resulting in an increased p2x3/pap ratio ( 67.4%    2.1% vs. 44.7%    3.4% , p  <  0.0001 ) and decreased pap / p2x3 ratio ( 87.1%    5.4% vs. 76.8%    2.3% , p  =  0.001 ) ( figure 1(e ) ) .
furthermore , pap expression was higher in peripherin(+ ) neurons than in neurofilament ( smi32)(+ ) neurons ( figure 2 ) .
the diameter histograms confirmed that the pap(+ ) neurons were small to medium drg neurons ( 25th75th percentile : 17.123.7  m ) ( figure 2(f ) ) .
figure 1.colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups .
( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d).bar , 50  m . * * p  <  0.01 , * * * p  < 
0.001 . 
 figure 2.expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons .
double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) .
( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) .
( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) .
( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons.bar , 50  m . * * p  <  0.01 colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups .
( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d ) .
bar , 50  m . * * p  <  0.01 , * * * p  < 
0.001 . expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons .
double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) .
( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) .
( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) .
( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons .
to understand the expression of pap and its relationship with atf3 , an injury marker , we performed double immunofluorescence staining of drg neuronal sections in rtx - induced neuropathy ( figure 3(a ) to ( e ) ) .
pap(+ ) neurons were mildly lower in the rtx group than in the vehicle group ( 190.6    12.2 vs. 269.5    6.9 neurons / mm , p  =  0.0003 ) ( figure 3(f ) ) .
by contrast , atf3(+ ) neurons were markedly higher in the rtx group than in the vehicle group ( 192.3    18.0 vs. 9.0    13.1 neurons / mm , p  < 
moreover , atf3 was induced preferentially in pap(+ ) neurons ( figure 3(a ) to ( e ) ) , resulting in an increased ratio of atf3(+)/pap(+ ) neurons in the rtx group ( 29.0%    5.6% vs. 0.2%    0.2% , p  =  0.0043 ) ( figure 3(h ) ) . 
 figure 3.phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a  e in red ) and anti - pap ( a  e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx  +  2.5s ngf ; d ) , and ( e ) abngf ( 4mc  +  anti - ngf antisera ) groups .
( f  h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) .
( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods .
the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups .
( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons .
( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups .
the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group.bar , 50  m . * p  <  0.05 , * * p  <  0.01 , * * * p  <  0.001 .
phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy .
( a  e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a  e in red ) and anti - pap ( a  e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx  +  2.5s ngf ; d ) , and ( e ) abngf ( 4mc  +  anti - ngf antisera ) groups .
( f  h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) .
( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods .
the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups .
( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons .
( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups .
the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group .
bar , 50  m . * p  <  0.05 , * * p  <  0.01 , * * * p  <  0.001 .
mice in the rtx group showed mechanical allodynia compared with the vehicle group ( 747.1    156.0 vs. 299.6    70.6  mg , p  =  0.0025 ) ( figure 3(i ) ) .
this behavioral change correlated with the pap profiles ; that is , the densities of pap(+ ) neurons ( r  =  0.75 , p  <  0.0001 ; figure 3(j ) ) and the ratios of atf3(+)/pap(+ ) neurons ( r  =  0.60 , p  =  0.003 ; figure 3(k ) ) were inversely linear to the mechanical thresholds . collectively , these findings indicate that pap expression had a strong influence in mediating pain development .
after neuropathy was induced using rtx , 4mc normalized pap expression and relieved mechanical allodynia ; specifically , the pap(+ ) neuronal density ( 269.7    48.8 neurons / mm , p  =  0.61 ) ( figure 3(c ) and ( f ) ) and mechanical allodynia ( 693.8    146.0  mg , p  =  0.54 ) ( figure 3(i ) ) in the 4mc group were comparable with those in the vehicle group . furthermore , although 4mc reduced the atf3(+ ) neuronal density ( 102.4    27.8 neurons / mm , p  <  0.0001 ) and ratio of atf3(+)/pap(+ ) neurons (
15.8%    5.9% , p  =  0.0095 ) , the ratios of atf3(+)/pap(+ ) neurons were still higher in the 4mc group than in the vehicle group ( p  =  0.016 ; figure 3(c ) , ( g ) , ( h ) ) .
morphometric analysis demonstrated that 4mc reduced the absolute number of atf3(+)/pap(+ ) neurons but did not have a significant effect on their mean diameter ( 15.5    2.2 vs. 16.5    2.8  m , p  >  0.05 ) ( figure 3(l ) vs. ( m ) ) . to understand the putative mechanisms of pain relief by 4mc
, we examined the coexpression of pap(+)/trka(+ ) neurons ( figure 4 ) . for each group , pap(+ ) neuronal density was similar to trka(+ ) neuronal density ( figure 3(f ) vs. 4(k ) ) .
the pap / trka ratios correlated linearly with the mechanical threshold ( r  =  0.62 , p  =  0.0062 ; figure 4(m ) ) . to confirm the effect of 4mc ,
the mice in the ngf group were systemically ( i.p . ) administered 2.5s ngf .
the ngf group exhibited the same effect as the 4mc group ; that is , pap and trka expression profiles were reversed , atf3 induction was normalized , and mechanical allodynia relieved . those findings , however , were not observed in the abngf group ( administered anti - ngf antisera to neutralize ngf ) ( figures 3 and 4 ) .
figure 4.colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy .
( a  j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a  e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx  +  2.5s ngf ) , and ( e , j ) abngf ( 4mc  +  anti - ngf antisera ) groups .
( k , l ) the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) .
( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons.bar , 50  m . * p  <  0.05 , * * p  <  0.01 .
colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy .
( a  j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a  e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx  +  2.5s ngf ) , and ( e , j ) abngf ( 4mc  +  anti - ngf antisera ) groups .
( k , l ) the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) .
( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons .
this is the first report documenting the intervention of pap and ngf - trka signaling , which mediates neuropathic pain .
we speculate one possible mechanism : neuropathic pain in rtx - induced small - fiber neuropathy develops because of pap neuropathology and the decrease in ngf - trka signaling .
rtx is specifically neurotoxic to trpv1(+ ) neurons ; it also induces small - fiber neuropathy , such as skin denervation , which results in thermal hypoalgesia , small - diameter neuron injury , and peripheral sensitization of p2x3 purinoceptors , leading to mechanical allodynia .
atf3 is both an injury marker and a marker of pain ; for example , the ratios of p2x3(+)/atf3(+ ) neurons correlate linearly with the degree of neuropathic pain .
pap is susceptible to rtx because of the high ratios of pap(+)/p2x3(+ ) and pap(+)/peripherin(+ ) neurons .
thus , injured pap neurons are critical for modulating neuropathic pain ; that is , the ratios of atf3(+)/pap(+ ) neurons are inversely linear to the mechanical threshold .
the balance of extracellular purine nucleotides such as adenine nucleotides is maintained by functional membrane - bound ectonucleotidase activity , which responded for the cellular pathophysiological homeostasis .
the ectonucleotidase activity of pap may facilitate the hydrolysis of amp to adenosine , which acts as an analgesic ligand .
pap downregulation leads to the imbalance of amp / adenosine ratios , inhibiting the analgesic effect of adenosine . taken together ,
these findings suggest the neuronal soma underlies the irritation and/or injury coincident with the metabolic imbalance of extracellular nucleotides , resulting in the loss of the analgesic effect of pap . additional studies should investigate the effects of altered purine metabolism to clarify the cellular responses to neuronal soma irritation .
a previous study demonstrated that pap - mediated neuropathic pain was trpv1-dependent . in our rtx - induced neuropathy model , trpv1
was completely depleted , and the present study provides evidence of an alternative nociceptive pathway to the trpv1-dependent nociceptive pathway ; the loss of the analgesic effect of pap is mediated by neuronal soma injury and/or irritation .
furthermore , pain development correlated with trka profiles ; for example , the ratios of pap(+)/trka(+ ) neurons were linearly correlated with the degree of mechanical allodynia .
this report demonstrates the amelioration of neuropathic pain by 4mc through the intervention of pap and ngf - trka signaling .
in addition to previous studies demonstrating that 4mc promotes skin reinnervation , the present study demonstrated that 4mc could facilitate the recovery irritated and/or injured small - diameter neuronal soma , as indicated by the reduced ratios of atf3(+)/pap(+ ) neurons .
conversely , ngf - sensitized sensory nerves resulted in pain perception and targeting of ngf - trka signaling is a new direction for pain therapy . coincidentally
, several studies have suggested that the interaction of ngf and trpv1 is critical for pain transmission .
the present study shows contrary outcomes ; for example , 4mc - promoted ngf played the role of a modulator in the recovery of pap and pap(+)/trka(+ ) neuronal profiles coinciding with the normalization of mechanical allodynia .
4mc appears to have a pleiotropic effect in modulating neuronal activity ; for example , it also increases the synthesis of the brain - derived neurotrophic factor ( bdnf ) and enhances the phosphorylation of the trk family and activation of the mapk / erk cascade . regarding the targeted signaling pathway of 4mc in rtx - induced neuropathy , bdnf modulates the function of the motor and myelinated sensory neurons , but no effect was observed in this current neuropathic model because ( 1 ) rtx specifically affects small - diameter sensory neurons with no pathologic alternations in the motor and myelinated sensory neurons , and ( 2 ) 4mc has no effect on trkb and/or trkc signaling because there are no changes in bdnf and neurotrophic 3 ( nt3 ) expression in the peripheral tissues , as determined in our previous study .
regarding the possible mechanism of the effect of 4mc on pain - relief in rtx - induced neuropathy , this report provides immunohistochemical evidence of the potential effect of 4mc , as demonstrated by the pap / trka ratios of approximately 70% .
collectively , these data reinforce our speculations : 4mc regulates pap pathology through ngf - trka signaling , which was confirmed through alternative ngf administration and ngf neutralization experiments . combining with the 4mc reduced the atf3(+)/pap(+ ) neurons ; this study suggests that 4mc has a pleiotropic effect on both the recovery of irritated and/or injured neuronal soma and restores ngf - trka signaling . in summary
, atf3 might be an upstream molecule initiating the loss of the analgesic effect of pap , and pap is a downstream molecule of ngf - trka signaling , restoring the analgesic effect of pap in a trka - dependent manner ( figure 5 ) . 

figure 5.diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy .
this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) .
( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology .
( 3 ) the pap pathology reduces the amp hydrolysis . ( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . ( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology . ( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis .
diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy .
this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) .
( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology .
( 3 ) the pap pathology reduces the amp hydrolysis . ( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . ( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology .
( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis .
rtx is specifically neurotoxic to trpv1(+ ) neurons ; it also induces small - fiber neuropathy , such as skin denervation , which results in thermal hypoalgesia , small - diameter neuron injury , and peripheral sensitization of p2x3 purinoceptors , leading to mechanical allodynia .
atf3 is both an injury marker and a marker of pain ; for example , the ratios of p2x3(+)/atf3(+ ) neurons correlate linearly with the degree of neuropathic pain .
pap is susceptible to rtx because of the high ratios of pap(+)/p2x3(+ ) and pap(+)/peripherin(+ ) neurons .
thus , injured pap neurons are critical for modulating neuropathic pain ; that is , the ratios of atf3(+)/pap(+ ) neurons are inversely linear to the mechanical threshold .
the balance of extracellular purine nucleotides such as adenine nucleotides is maintained by functional membrane - bound ectonucleotidase activity , which responded for the cellular pathophysiological homeostasis .
the ectonucleotidase activity of pap may facilitate the hydrolysis of amp to adenosine , which acts as an analgesic ligand .
pap downregulation leads to the imbalance of amp / adenosine ratios , inhibiting the analgesic effect of adenosine . taken together , these findings suggest the neuronal soma underlies the irritation and/or injury coincident with the metabolic imbalance of extracellular nucleotides , resulting in the loss of the analgesic effect of pap .
additional studies should investigate the effects of altered purine metabolism to clarify the cellular responses to neuronal soma irritation .
a previous study demonstrated that pap - mediated neuropathic pain was trpv1-dependent . in our rtx - induced neuropathy model , trpv1
was completely depleted , and the present study provides evidence of an alternative nociceptive pathway to the trpv1-dependent nociceptive pathway ; the loss of the analgesic effect of pap is mediated by neuronal soma injury and/or irritation .
furthermore , pain development correlated with trka profiles ; for example , the ratios of pap(+)/trka(+ ) neurons were linearly correlated with the degree of mechanical allodynia .
this report demonstrates the amelioration of neuropathic pain by 4mc through the intervention of pap and ngf - trka signaling .
in addition to previous studies demonstrating that 4mc promotes skin reinnervation , the present study demonstrated that 4mc could facilitate the recovery irritated and/or injured small - diameter neuronal soma , as indicated by the reduced ratios of atf3(+)/pap(+ ) neurons .
conversely , ngf - sensitized sensory nerves resulted in pain perception and targeting of ngf - trka signaling is a new direction for pain therapy . coincidentally
, several studies have suggested that the interaction of ngf and trpv1 is critical for pain transmission .
the present study shows contrary outcomes ; for example , 4mc - promoted ngf played the role of a modulator in the recovery of pap and pap(+)/trka(+ ) neuronal profiles coinciding with the normalization of mechanical allodynia .
4mc appears to have a pleiotropic effect in modulating neuronal activity ; for example , it also increases the synthesis of the brain - derived neurotrophic factor ( bdnf ) and enhances the phosphorylation of the trk family and activation of the mapk / erk cascade . regarding the targeted signaling pathway of 4mc in rtx - induced neuropathy , bdnf modulates the function of the motor and myelinated sensory neurons , but no effect was observed in this current neuropathic model because ( 1 ) rtx specifically affects small - diameter sensory neurons with no pathologic alternations in the motor and myelinated sensory neurons , and ( 2 ) 4mc has no effect on trkb and/or trkc signaling because there are no changes in bdnf and neurotrophic 3 ( nt3 ) expression in the peripheral tissues , as determined in our previous study .
regarding the possible mechanism of the effect of 4mc on pain - relief in rtx - induced neuropathy , this report provides immunohistochemical evidence of the potential effect of 4mc , as demonstrated by the pap / trka ratios of approximately 70% . collectively , these data reinforce our speculations : 4mc regulates pap pathology through ngf - trka signaling , which was confirmed through alternative ngf administration and ngf neutralization experiments . combining with
the 4mc reduced the atf3(+)/pap(+ ) neurons ; this study suggests that 4mc has a pleiotropic effect on both the recovery of irritated and/or injured neuronal soma and restores ngf - trka signaling . in summary
, atf3 might be an upstream molecule initiating the loss of the analgesic effect of pap , and pap is a downstream molecule of ngf - trka signaling , restoring the analgesic effect of pap in a trka - dependent manner ( figure 5 ) . 

figure 5.diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy .
this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) .
( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology . ( 3 ) the pap pathology reduces the amp hydrolysis .
( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect .
( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology .
( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis .
diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy .
this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) .
( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology . ( 3 ) the pap pathology reduces the amp hydrolysis .
( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect .
( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology .
( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis .
for pain development , this study provides evidence of an alternative pathway to conventional small nociceptor sensitization - induced neuropathic pain ; the loss of analgesic effect of pap is mediated by neuronal soma irritation and/or injury .
alternatively , restoring the analgesic ligand may be a new direction for targeting painful neuropathy , that is , maintenance of steady - static adenosine content and functional pap ectonucleotidase activity at the neuronal soma level .
the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article .
the author(s ) disclosed receipt of the following financial support for the research , authorship , and/or publication of this article : this work was supported by grants from the national science council ( nsc100 - 2320-b-037 - 018 and nsc102 - 2320-b-037 - 009 ) ; ministry of science and technology ( most103 - 2320-b-037 - 015-my3 ) , taiwan ; and kaohsiung medical university hospital ( kmuh104 - 4m19 ) .
 backgroundneuropathic pain in small - fiber neuropathy results from injury to and sensitization of nociceptors . 
 functional prostatic acid phosphatase ( pap ) acts as an analgesic effector . however , the mechanism responsible for the modulation of pap neuropathology , which leads to loss of the analgesic effect after small - fiber neuropathy , remains unclear.resultswe used a resiniferatoxin ( rtx)-induced small - fiber neuropathy model to examine whether functional pap(+ ) neurons are essential to maintain the analgesic effect . 
 pap(+ ) neurons were categorized into small to medium neurons ( 25th75th percentile : 17.123.7  m ) ; these neurons were slightly reduced by rtx ( p  =  0.0003 ) . by contrast , 
 rtx - induced activating transcription factor 3 ( atf3 ) , an injury marker , in pap(+ ) neurons ( 29.0%    5.6% vs. 0.2%    0.2% , p  =  0.0043 ) , indicating pap neuropathology . 
 moreover , the high - affinity nerve growth factor ( ngf ) receptor ( trka ) colocalized with pap and showed similar profiles after rtx - induced neuropathy , and the pap / trka ratios correlated with the degree of mechanical allodynia ( r  =  0.62 , p  =  0.0062 ) . 
 the ngf inducer 4-methylcatechol ( 4mc ) normalized the analgesic effects of pap ; specifically , it reversed the pap and trka profiles and relieved mechanical allodynia . administering 2.5s ngf showed similar results to those of administering 4mc . 
 this finding suggests that the analgesic effect of functional pap is mediated by ngf - trka signaling , which was confirmed by ngf neutralization.conclusionsthis study revealed that functional pap(+ ) neurons are essential for the analgesic effect , which is mediated by ngf - trka signaling . 