Short term pre and post operative stress prolongs incision induced pain hypersensitivity without changing basal pain perception

Transcription

1 Co et l. Mol Pin () 11:73 DOI 1.11/s Moleulr Pin RESEARCH Open Aess Short term pre n post opertive stress prolongs inision inue pin hypersensitivity without hnging sl pin pereption Jing Co 1,, Po Ki Wng,3, Vino Tiwri, Lingli Ling, Brinn Mrie Lutz, Kun Ruey Shieh, Wei Dong Zng 1, Anrew G. Kufmn, Alex Bekker, Xio Qun Go 1 n Yun Xing To 1, Astrt Bkgroun: Chroni stress hs een reporte to inrese sl pin sensitivity n/or exerte existing persistent pin. However, most surgil ptients hve norml physiologil n psyhologil helth sttus suh s norml pin pereption efore surgery lthough they o experiene short-term stress uring pre- n post-opertive perios. Whether or not this short-term stress ffets persistent postsurgil pin is unler. Results: In this stuy, we showe tht pre- or post-surgil exposure to immoiliztion h ily for three onseutive ys i not hnge sl responses to mehnil, therml, or ol stimuli or pek levels of inision-inue hypersensitivity to these stimuli; however, immoiliztion i prolong the urtion of inision-inue hypersensitivity in oth mle n femle rts. These phenomen were lso oserve in post-surgil exposure to fore swimming min ily for 3 onseutive ys. Short-term stress inue y immoiliztion ws emonstrte y n elevtion in the level of serum ortiosterone, n inrese in swim immoility, n erese in surose onsumption. Bloking this short-term stress vi intrthel ministrtion of seletive gluoortioi reeptor ntgonist, RU3, or ilterl renletomy signifintly ttenute the prolongtion of inision-inue hypersensitivity to mehnil, therml, n ol stimuli. Conlusion: Our results inite tht short-term stress uring the pre- or post-opertive perio elys postopertive pin reovery lthough it oes not ffet sl pin pereption. Prevention of short-term stress my filitte ptients reovery from postopertive pin. Keywors: Short-term immoiliztion, Short-term fore swimming, Stress, Postsurgil pin, Inision Bkgroun Persistent postsurgil pin, pin synrome tht n evelop fter surgery, is signifint puli helth prolem. Approximtely % of surgil ptients suffer from persistent pin fter surgery, of whom t lest 1 % Corresponene: gxq@zzu.eu.n; yt11@njms.rutgers.eu Jing Co n Po-Ki Wng ontriute eqully to this work 1 Deprtment of Antomy, College of Bsi Meiine, Zhengzhou University, Zhengzhou 1, Henn, Chin Deprtment of Anesthesiology, New Jersey Meil Shool, Rutgers, The Stte University of New Jersey, 1 S. Ornge Ave., MSB, F, Newrk, NJ 713, USA Full list of uthor informtion is ville t the en of the rtile hve severe pin [1]. The onition ffets their qulity of life n hs importnt legl n meio-eonomi rmifitions. Phrmologil mngement of persistent surgil pin onitions re ominte y two lsses of meitions: opiois n nonsteroil nti-inflmmtory rugs. Mny of these pinkillers hve limite effetiveness or serious sie effets, suh s nuse/emesis, onstiption, tolerne or hyperlgesi [1 ]. Unerstning the ftors tht my use n/or ffet the evelopment n mintenne of persistent surgil pin my provie insight into novel prevention or tretment strtegies. Co et l. This rtile is istriute uner the terms of the Cretive Commons Attriution. Interntionl Liense ( retiveommons.org/lienses/y/./), whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me. The Cretive Commons Puli Domin Deition wiver ( zero/1./) pplies to the t me ville in this rtile, unless otherwise stte.

2 Co et l. Mol Pin () 11:73 Pge of 1 Although mny forms of injury n stress our spontneously, ptients know the preise timing of the eletive surgil insult n ensuing pin in vne. In ition to surgil ftors, psyhosoil, soio-environmentl, n ptient-relte ftors pper to moulte risk of eveloping persistent postsurgil pin. Severl psyhosoil risk ftors hve een ientifie, inluing nxiety, epression, pin tstrophizing, n fer of surgery []. Self-reporte sleep isturne-inue stress efore n fter surgery lso onstitutes the strongest eterminnt of postsurgil pin []. Sustntil eviene from linil oservtions hs emonstrte tht sleep eprivtion-use hroni stress inrese sl pin pereption in helthy sujets [, 7]. Some surgil ptients with hroni severe physil or psyhologil stress self-reporte inrese sl pin sensitivity or exerte existing pthologil pin efore surgery [, 9]. In prelinil stuies, hroni stress inue y immoiliztion exertes mehnil lloyni fter peripherl nerve injury [1]. Sleep isturne-inue hroni stress les to therml hyperlgesi n n inrese response to eletril stimultion in intt, experimentl nimls [, 11 13]. Nevertheless, most surgil ptients hve norml pin pereption efore surgery lthough they hve stress use y risk ftors suh s epression, nxiety, fer of pin n surgery, n/or sleep isturne for severl ys efore n/or fter surgery [, ]. Our reent finings suggest tht pre- n post-surgil short-term sleep isturne i not ffet sl pin pereption ut i ely postsurgil pin reovery [1]. Whether or not short-term stress use y other risk ftors ffets the reovery from postopertive pin is still elusive. To moel short-term stress use y epression, the present stuy utilize two prelinil niml moels of stress immoiliztion stress n fore swimming stress [ 17] to etermine the optiml urtion of immoiliztion or fore swimming tht i not lter sl pw withrwl responses to therml, ol, n mehnil stimuli. We then etermine whether the short-term immoiliztion or fore swimming uner optiml onitions enhne the mgnitue n/or urtion of therml, ol, or mehnil hypersensitivity inue y hin pw inision. Finlly, we exmine whether this short-term immoiliztion or fore swim proue other physiologil or ehviorl signs of stress. Results Time epenent hnges in sl pw withrwl responses to mehnil, therml, n ol stimuli fter repete immoiliztion stress in mle rts We first efine the optiml numer of ily repetitions of immoiliztion stress tht i not ffet sl pw withrwl responses in n estlishe niml moel of immoiliztion stress. Mle rts were expose to immoiliztion stress for h ily for onseutive ys. Pw withrwl responses to mehnil, therml, n ol stimuli were exmine 1 y efore immoiliztion stress n h fter immoiliztion stress ily for ys. Signifint ereses in pw withrwl threshols in response to mehnil stimultion on oth left n right hin pws were oserve only on y post-immoiliztion stress s ompre to the orresponing ontrol group (P <. in Fig. 1, ), n inition of mehnil lloyni. Mrke reutions in pw withrwl ltenies in response to therml stimultion were seen on y (P <.) n (P <.1) post-immoiliztion stress on oth left n right hin pws s ompre to the orresponing ontrol group (Fig. 1, ), n inition of therml hypersensitivity. Similrly, signifint reutions in pw withrwl lteny in response to ol stimultion were etete oth on y (P <.) n (P <.1) fter immoiliztion stress in the left hin pw s ompre to the orresponing ontrol group (Fig. 1e), n inition of ol lloyni. These t showe tht sl pw withrwl responses i not hnge signifintly uring 3 onseutive ys of exposure to immoiliztion stress. Thus, h immoiliztion for 3 onseutive ys ws efine s short-term immoiliztion stress n ws use in the following experiments. Effet of pre surgil exposure to short term immoiliztion stress on postsurgil pin in mle rts To exmine whether short-term immoiliztion stress efore surgery ffete the mgnitue or urtion of inision-inue hypersensitivity, we rrie out unilterl plntr inision on the left hin pw in mle rts fter they were expose to h immoiliztion for three onseutive ys. Consistent with previous reports [1 ], the inision lone le to persistent mehnil, therml, n ol pin hypersensitivities on the ipsilterl (ut not ontrlterl) sie of the inision plus ontrol group (Fig. ). Pin hypersensitivity rehe pek on y 1, lste for 7 ys, n h ompletely isppere on y 9 post-surgery (Fig.,, e). As expete, pre-exposure to 3 ys immoiliztion stress lone (shm plus immoiliztion trete rts) i not lter sl pw responses to mehnil, het, or ol stimuli uring the 9 ys oservtion perio (Fig. ). However, pre-exposure to 3 ys immoiliztion stress signifintly elye the reovery from surgil pin, lthough it i not lter pek levels of inision-inue hypersensitivity to mehnil, therml, or ol stimuli, on the ipsilterl sie in the inision plus immoiliztion stress group (Fig.,, e). The inision plus immoiliztion stress group h signifintly lower pw withrwl threshols to mehnil

3 Co et l. Mol Pin () 11:73 Pge 3 of 1 Left hinpw Right hinpw threshol (g) 1 Control Immoiliztion 1 e Dys fter immoiliztion Dys fter immoiliztion Fig. 1 Time-epenent hnges in sl pw withrwl responses in mle rts to mehnil, het, n ol stimuli fter immoiliztion h ily for onseutive ys., Mehnil stimuli., Het stimuli. e Col stimuli.,, e Responses of left pws., Responses of right pws. Signifint reutions were seen in ilterl pw withrwl threshols in response to mehnil stimultion on y post-immoiliztion (, ), in ilterl pw withrwl ltenies in response to het stimultion on y n post-immoiliztion (, ), n in pw withrwl lteny in response to ol stimultion on ys n post-immoiliztion (e) in the immoiliztion stress group. Men ± SEM. P <., P <.1 vs the orresponing time points in the ontrol group. N = /group stimultion on ys 7 (P <.) n 9 (P <.1) postsurgery thn the inision plus ontrol group (Fig. ). The inision plus immoiliztion stress group lso h signifintly shorter pw withrwl ltenies to therml stimultion on ys 7 n 9 thn the inision plus ontrol group (oth P <.; Fig. ). Aitionlly, the inision plus immoiliztion stress group h signifintly shorter pw withrwl ltenies to ol stimultion on ys 7 (P <.) n 9 (P <.1) thn the inision plus ontrol group (Fig. e). As expete, the shm plus ontrol group mintine the seline level of pw withrwl threshol n ltenies in response to the ifferent stimuli on oth sies t ll-time points. The inision group showe no effets on pw withrwl threshol n ltenies in response to the ifferent stimuli on the ontrlterl sie (Fig. ). Effet of post surgil exposure to short term immoiliztion stress on postsurgil pin in mle rts We next exmine whether short-term immoiliztion stress fter surgery ffete the mgnitue or urtion of inision-inue hypersensitivity in mle rts. One hour fter unilterl inision on the left hin pw, mle rts were expose to h immoiliztion stress ily for three onseutive ys. Immoiliztion stress mrkely elye the reovery of surgil pin, lthough it i not lter pek levels of inision-inue hypersensitivity to mehnil, therml, or ol stimuli, on the ipsilterl

4 Co et l. Mol Pin () 11:73 Pge of 1 e withrw Threshol (g) Ipsilterl Contrlterl Shm + ontrol Shm + immoiliztion Inision + ontrol Inision + Immoiliztion Fig. Effet of pre-surgil exposure to short-term immoiliztion on post-surgil pin in mle rts., Mehnil stimuli., Het stimuli. e Col stimuli.,, e Responses of ipsilterl (inision-stresse) pws., Responses of ontrlterl pws. Pre-surgil exposure to short-term immoiliztion mrkely elye reovery in the pw withrwl threshol to mehnil stimultion () n pw withrwl ltenies to het () n ol (e) stimuli on the ipsilterl sie on ys 7 n 9 post-surgery in the inision plus immoiliztion group, ompre to the inision plus ontrol group. The shm plus ontrol n the shm plus immoiliztion groups showe no signifint ifferenes in pw withrwl responses uring the oservtion perio. Men ± SEM. P <., P <.1 vs the orresponing time points in the inision plus ontrol group. N = /group sie in the inision plus immoiliztion stress group (Fig. 3,, e). The inision plus immoiliztion stress group h signifintly lower pw withrwl threshols to mehnil stimultion on ys 7 (P <.) n 9 (P <.1) post-surgery thn the inision plus ontrol group (Fig. 3). In ition, the inision plus immoiliztion group h shorter pw withrwl lteny to therml stimultion on ys 7 (P <.) n 9 (P <.1) post-surgery thn the orresponing inision plus ontrol group (Fig. 3). Likewise, the inision plus immoiliztion group h signifintly shorter pw withrwl lteny to ol stimultion on ys, 7 (oth P <.), n 9 (P <.1) post-surgery thn the inision plus ontrol group (Fig. 3e). As expete, sl ehviorl responses were not ltere on the ontrlterl sie in ll groups n on the ipsilterl sies in the shm plus ontrol group n the shm plus immoiliztion group uring the oservtion perio (Fig. 3). Effet of post surgil exposure to short term immoiliztion stress on postsurgil pin in femle rts Gener-relte ifferenes in pin n stress hve een esrie in experimentl settings n linil oservtions [1, ]. Thus, we exmine whether short-term immoiliztion stress fter surgery hs stronger effets on postopertive pin in femle rts thn in mle rts. Femle rts were expose to inision n susequently 3 ys immoiliztion stress in the sme mnner s mle rts. The mgnitue n urtion of inision-inue pin hypersensitivity on the ipsilterl sie in the femle rts ws similr to those in mle rts (Fig. 3,, e vs Fig.,, e). Similr to the responses of mle rts, the

5 Co et l. Mol Pin () 11:73 Pge of 1 Ipsilterl Contrlterl threshol (g) 1 1 e 1 1 Shm + ontrol Shm + immoiliztion Inision + ontrol Inision + Immoiliztion Fig. 3 Effet of post-surgil exposure to short-term immoiliztion on post-surgil pin in mle rts., Mehnil stimuli., Het stimuli. e Col stimuli.,, e Responses of ipsilterl (inision-stresse) pws., Responses of ontrlterl pws. Post-surgil exposure to short-term immoiliztion mrkely elye reovery of pw withrwl threshol to mehnil stimultion () n pw withrwl lteny to het () on ys 7 n 9 post-immoiliztion n pw withrwl lteny to ol (e) stimuli on ys, 7, n 9 post-immoiliztion on the ipsilterl sie in the inision plus immoiliztion group ompre to the inision plus ontrol group. The shm plus ontrol n the shm plus immoiliztion groups showe no hnges in pw withrwl responses uring the oservtion perio. Men ± SEM. P <., P <.1 vs the orresponing time points in the inision plus ontrol group. N = /group femle inision plus immoiliztion stress group h signifintly slower reovery from surgil pin n n unhnge mgnitue of inision-inue hypersensitivity to mehnil, therml, or ol stimuli, on the ipsilterl sie when ompre to the inision plus ontrol group (Fig.,, e). The femle inision plus immoiliztion stress group h signifintly lower men pw withrwl threshol to mehnil stimultion on ys 7 n 9 post immoiliztion thn the inision plus ontrol group (oth P <.1; Fig. ). Similrly, the femle inision plus immoiliztion stress group h signifintly shorter men pw withrwl lteny to therml stimultion on ys 7 (P <.) n 9 (P <.1) post immoiliztion (Fig. ) n shorter men pw withrwl lteny to ol stimultion on ys 7 n 9 post immoiliztion (oth P <.; Fig. e) thn the femle inision plus ontrol group. As expete, sl ehviorl responses were not hnge on the ontrlterl sie of ll groups n the ipsilterl sie of the shm plus ontrol group n the shm plus immoiliztion group uring the oservtion perio in femle rts (Fig. ). Effet of post surgil exposure to short term fore swimming stress on postsurgil pin in mle rts Our pilot work showe tht sl pw withrwl responses i not hnge mrkely uring three

6 Co et l. Mol Pin () 11:73 Pge of 1 Pw withrw threshol (g) e Ipsilterl Contrlterl Shm + ontrol Shm + immoiliztion Inision + ontrol Inision + Immoiliztion Fig. Effet of post-surgil exposure to short-term immoiliztion on post-surgil pin in femle rts., Mehnil stimuli., Het stimuli. e Col stimuli.,, e Responses of ipsilterl (inision-stresse) pws., Responses of ontrlterl pws. Post-surgil exposure to short-term immoiliztion mrkely elye reovery of pw withrwl threshol to mehnil stimultion () n pw withrwl ltenies to het () n ol (e) stimuli on ys 7 n 9 post-immoiliztion on the ipsilterl sie in the inision plus immoiliztion group, ompre to the inision plus ontrol group. No hnges in pw withrwl responses were seen uring the oservtion perio in the shm plus ontrol n shm plus immoiliztion groups. Men ± SEM. P <., P <.1 vs the orresponing time points in the inision plus ontrol group. N = /group onseutive ys of exposure to fore swimming stress ( min ily; t not shown). Thus, we efine -min fore swimming for 3 onseutive ys s short-term fore swimming stress. To further explore the role of short-term stress on reovery from postsurgil pin, we exmine whether short-term fore swimming stress fter surgery ffete postopertive pin in mle rts. One hour fter unilterl plntr inision on the left hin pw, mle rts were expose to the fore swimming stress ( min) ily for three onseutive ys. Similr to the effets of the 3 ys immoiliztion stress esrie ove, post-exposure to fore swimming stress ily for 3 ys lso mrkely elye the reovery from surgil pin, lthough it i not lter the mgnitue of inisioninue hypersensitivity to mehnil, therml, or ol stimuli, on the ipsilterl sie in the inision plus fore swimming stress group (Fig.,, e) ompre to the reovery in the inision plus ontrol group. The inision plus fore swimming stress group showe lower men pw withrwl threshols to mehnil stimultion on ys 7 n 9 post surgery thn the inision plus ontrol group (oth P <.1; Fig. ). In ition, the inision plus fore swimming stress group showe shorter pw withrwl ltenies to therml stimultion on ys 7 n 9 post surgery (oth P <.1; Fig. ) n shorter pw withrwl ltenies to ol stimultion on ys 7 n 9 post-surgery (oth P <.1; Fig. e). As expete, no signifint hnges were etete in pw withrwl threshol n ltenies on the ontrlterl sie in ll groups uring the 9 ys. The ipsilterl sies of the shm

7 Co et l. Mol Pin () 11:73 Pge 7 of 1 Ipsilterl Contrlterl threshol (g) 1 1 e Shm + ontrol Shm + swimming Inision + ontrol Inision + swimming 3 Fig. Effet of post-surgil exposure to short-term fore swimming on post-surgil pin in mle rts., Mehnil stimuli., Het stimuli. e Col stimuli.,, e Responses of ipsilterl (inision-stresse) pws., Responses of ontrlterl pws. Post-surgil exposure to short-term fore swimming mrkely elye reovery in pw withrwl threshol to mehnil stimultion () n pw withrwl ltenies to het () n ol (e) stimuli on ys 7 n 9 post-fore swimming on the ipsilterl sie in the inision plus fore swimming group ompre to the inision plus ontrol group. No hnges in pw withrwl responses were seen uring the oservtion perio in the shm plus ontrol n the shm plus fore swimming groups. Men ± SEM. P <., P <.1 vs the orresponing time points in the inision plus ontrol group. N = /group plus ontrol n the shm plus swimming stress group lso mintine the seline levels of the pw withrwl threshols n ltenies uring the oservtion perio (Fig. ). Existene of stress in mle rts fter post surgil exposure to 3 y immoiliztion stress To ientify the existene of the stress uner our optimize onitions, we lso exmine post-surgil mle rts use for ehviorl stuies esrie ove in the following 3 tests. We first mesure the level of ortiosterone (CORT) in serum to etermine if their stresse ehvior orrelte with CORT levels. The loo ws ollete immeitely fter post-surgil exposure to h immoiliztion stress ily for three onseutive ys. The levels of serum CORT in the shm plus immoiliztion stress group n the inision plus immoiliztion stress group were signifintly higher thn those in the shm plus ontrol group (oth P <.1, Fig. ). No signifint ifferene ws oserve in serum CORT levels etween the inision plus ontrol group (P >.) n the shm plus ontrol group (Fig. ). We further rrie out swimming immoiliztion test, in whih rts uner stress isply longer urtion of immoility [3], 1 h fter loo withrwl. The rts in the shm plus immoiliztion stress group n in the inision plus immoiliztion stress group spent signifintly more time immoile thn rts in the shm plus ontrol group

8 Co et l. Mol Pin () 11:73 Pge of 1 Cortiosteroi level ng/ml Preferene for surose (%) 1 1 S + C S + Im In + C In + Im Immoility time (s) Cumultive oy weight on y 9 (%) 1 1 S + C S + Im In + C In + Im S + C S + Im In + C In + Im S + C S + Im In + C In + Im Fig. Existene of stress fter short-term immoiliztion. Cortiosterone serum levels. Fore swim test. Surose onsumption. Boy weight. Post-surgil exposure to short-term immoiliztion signifintly elevte the level of ortiosterone in serum (), inrese immoility time in fore swim test (), n erese surose onsumption in surose preferene test () in oth the shm inision plus immoiliztion (S + Im) group n the inision plus immoiliztion (In + Im) group ompre to the shm plus ontrol (S + C) group. These hnges were not oserve in the inision plus ontrol group ( ). No signifint ifferenes in hnges in oy weight etween efore surgery n on y 9 postsurgery were seen mong the four groups (). Men ± SEM. P <.1 vs the orresponing shm plus ontrol group. N = /group. C ontrol, Im 3 immoiliztion, In inision, S shm inision (P <.1; Fig. ). The immoility urtion ws similr etween the inision plus ontrol group n the shm plus ontrol group (Fig. ). Finlly, we exmine these nimls in the surose preferene test, in whih rts uner stress isply reue surose preferene [3]. The rts in the shm plus immoiliztion stress group n in the inision plus immoiliztion stress group showe signifintly less surose onsumption thn those in the shm plus ontrol group (P <.1, Fig. ). No ovious ifferene in surose onsumption ws oserve etween the inision plus ontrol group n the shm plus ontrol group (Fig. ). The effet of 3 ys immoiliztion stress on oy weight ws not signifint, s the oy weights were similr mong ll four groups on y 9 post-inision or -shm surgery (Fig. ). Effet of intrthel RU3 on exerte post surgil pin fter short term immoiliztion stress CORT proues its physiologil n ehviorl effets y ining to n tivting gluoortioi reeptors []. To etermine whether inrese CORT inue y immoiliztion uner our optimize onitions ontriute to the oserve prolongtion of inisioninue hypersensitivity, we intrthelly pre-ministere the ontrol vehile or seletive gluoortioi reeptor ntgonist RU3 1 h efore eh session of h immoiliztion stress ily for 3 ys. In greement with our forementione results, the inision-immoiliztion stress-vehile group showe the exerte pw withrwl responses to mehnil (Fig. 7), therml (Fig. 7), n ol (Fig. 7e) stimuli on the ipsilterl sie on y 9 post-inision ompre to the orresponing the inision-ontrol-vehile group (ll P <.). Intrthel pre-ministrtion of RU3 in the inision immoiliztion stress group ompletely olishe these exerte pw withrwl responses on the ipsilterl sie on y 9 (Fig. 7,, e). RU3 ministrtion t the ose use i not lter sl pw withrwl responses to mehnil n therml stimuli on the ontrlterl sie of the inision plus immoiliztion stress group n to mehnil, therml n ol stimuli on either ipsilterl or ontrlterl sie of the shm plus immoiliztion stress group (Fig. 7). Effet of ilterl ADX on exerte post surgil pin fter short term immoiliztion stress Serum CORT origintes minly from renl glns. To further sustntite the role of CORT n RU3 s phrmologil effet oserve ove in the exerte postsurgil pin, we rrie out ilterl ADX to eliminte the proution of serum CORT. Rts were given supplemente rinking wter to mintin sl CORT levels fter ADX surgery. The inision immoiliztion stress -shm ADX surgery group showe the exerte

9 Co et l. Mol Pin () 11:73 Pge 9 of 1 Ipsilterl Contrlterl Pw withrw Threshol (g) 1 1 e Shm + Con + V Shm + Im + RU3 In + Con + V In + Im + V In + Im + RU3 Fig. 7 Effet of intrthel pre-ministrtion of RU3 on exerte post-surgil pin inue y short-term immoiliztion stress., Mehnil stimuli., Het stimuli. e Col stimuli.,, e Responses of ipsilterl pws., Responses of ontrlterl pws. The inision plus immoiliztion plus intrthel vehile (In + Im + V) group h signifintly lower pw withrwl threshol to mehnil stimultion () n shorter pw withrwl ltenies to het () n ol (e) stimuli on the ipsilterl sie on y 9 post-inision thn the inision plus ontrol plus vehile group. In ontrst, RU3 ( μg/1 μl) 1 h efore eh h of immoiliztion for 3 ys ompletely olishe these reutions (,, e). RU3 i not lter sl pw withrwl responses to mehnil, het, n ol stimuli on the ontrlterl sie of the inision plus immoiliztion group (, ) n on either ipsilterl (,, e) or ontrlterl (, ) sie of the shm plus immoiliztion group. Intrthel vehile (V) i not ffet sl pw withrwl responses on either sie of ny group. Men ± SEM. P <.1 vs the orresponing shm plus ontrol group with intrthel vehile. N = /group. In inision, Im 3 immoiliztion, V vehile pw withrwl responses to mehnil, therml, n ol stimuli on the ipsilterl sie on y 9 post-inision (ll P <.; Fig.,, e, respetively). Bilterl ADX entirely reverse these exerte responses in the inision plus immoiliztion stress group (Fig.,, e). Bilterl ADX i not lter sl pw withrwl responses to mehnil n therml stimuli of the ontrlterl pws in ll groups (Fig., ) nor those on the ipsilterl of the remining tretment groups (Fig. ). Disussion Surgery-inue persistent pin is ommon linil symptom. Ientifying risk ftors tht exerte postsurgil pin uring the periopertive perio my help us to preit whih ptients my experiene elye reovery n offer n opportunity to mnge post-surgil pin effetively. Chroni stress inrese sl pin pereption n worsen existing pthologil pin [, 9]. However, most surgil ptients hve norml physiologil, psyhologil helth sttus n norml pin pereption efore surgery lthough they hve to some extent stress uring pre- n/ or post-opertive perio. Whether this short-term stress ffets persistent post-surgil pin is still elusive. We reently reporte tht pre- n post-surgil short-term sleep isturne i not ffet sl pin pereption ut i ely postsurgil pin reovery [1]. Here, we report tht pre- or post-surgil exposure to short-term stress prolongs the urtion of inision-inue mehnil, therml, or ol hypersensitivities even if it oes not lter sl noieption. Our finings suggest tht prevention of short-term stress uring pre- n post-opertive perio my help the ptients reover from postopertive pin.

10 Co et l. Mol Pin () 11:73 Pge 1 of 1 Pw withrw Threshol (g) 1 Ipsilterl 1 Contrlterl e Shm-inision + Con + Shm-ADX Shm-inision + Control + ADX Inision + Control + ADX Inision + Im + Shm-ADX 3 Shm-inision + Im + ADX Inision + Im + ADX Fig. Effet of ilterl renletomy (ADX) on exerte post-surgil pin inue y short-term immoiliztion stress., Mehnil stimuli., Het stimuli. e Col stimuli.,, e Responses of ipsilterl pws., Responses of ontrlterl pws. In the inision plus immoiliztion group, shm surgery of ADX (shm-adx) efore immoiliztion showe mrke reutions in pw withrwl threshol to mehnil stimultion () n pw withrwl ltenies to het () n ol (e) stimuli on the ipsilterl sie on y 9 post-inision. Bilterl ADX efore immoiliztion entirely reverse these reutions (,, e). ADX n shm ADX showe similr pw withrwl responses to mehnil, het, n ol stimuli on the ontrlterl sies of ll groups (, ). ADX n shm ADX i not ffet the responses of the ipsilterl pws to the stimuli (,, e) of the shm-inision plus ontrol group, the inision plus ontrol group, n the shm-inision plus immoiliztion group. Men ± SEM. P <.1 vs the orresponing shm-inision plus ontrol group with shm-adx. N = /group. Im immoiliztion Immoiliztion n fore swimming re estlishe stressors in roents s efine y their effetiveness to tivte the hypothlmi pituitry renl (HPA) xis n proue ehviorl signs of epression [ 17]. We estlishe two regimens of immoiliztion n fore swimming tht i not lter sl responses to mehnil, therml, n ol stimuli in nïve rts. These exposures to immoiliztion n fore swimming my moel linil onitions in most ptients tht hve norml pin pereption efore surgery, lthough they my experiene trnsient HPA xis tivtion in ssoition with short-term fer of pin, nxiety, epression, n pin tstrophizing uring pre- n post-opertive perios. Eviene of stress in our urrent moels is emonstrte y inrese immoility time in fore swim test, erese preferene for surose, n elevte serum CORT levels. Interestingly, short-term immoiliztion or fore swimming stress either efore or fter surgery prolonge post-surgil pin, lthough it i not lter the mgnitue of inision-inue hypersensitivity to mehnil, therml, or ol stimuli. Bloking spinl gluoortioi reeptor or removing the renl glns (tht re the primry soure of CORT) ompletely olishe the prolongtion of inisioninue hypersensitivity fter immoiliztion stress. Given tht gluoortioi reeptor is expresse preominntly in the neurons of spinl or n orsl root gnglion [ 7], these results inite tht CORT-triggere tivtion of gluoortioi reeptors in spinl or n orsl root gnglion (rther thn in rin) neurons my meite this prolongtion. Intrthel exogenous CORT woul e expete to ely the reovery of surgil pin. This expettion is strongly supporte y previous stuy showing tht systemi exogenous ministrtion

11 Co et l. Mol Pin () 11:73 Pge 11 of 1 of CORT reue the pin threshol in the ipsilterl hin pw fter peripherl nerve injury [1]. It shoul e note tht the ADX nnot rule out the involvement of other hormones (e.g., teholmines) in short-term stress-inue prolongtion of post-surgil pin s oth renl ortex n meull re remove. Unexpetely, oth mle n femle rts exhiite similr mgnitues n urtions of postsurgil pin in either presene or sene of immoiliztion stress in our oservtions. Sex ifferenes in response to pin n stress hve een reporte [1, ]. No sex ifferene in our oservtion my e relte to the short-term immoiliztion stress n no gener istint in inisionl pin []. The istint mehnisms ffete y short-term stress tht o not lter sl pin pereption ut o prolong inision-inue hypersensitivity re unknown, ut my involve one or more of the following signling moleules n proesses. Nitri oxie synthse-ontining mgnoellulr neurons of the rt hypothlmus, whih ontriute to the esening projetion to the spinl or, express Fos following stress [9]. Repete stress reues ADP hyrolysis [3], inreses -nuleotise tivity [3], n proues poptosis [31] in the spinl or. More interestingly, immoiliztion stress not only exertes nerve injury-inue mehnil lloyni ut lso enhnes nerve injury-inue expression of extrellulr signl-regulte kinse phosphoryltion in the superfiil orsl horn of spinl or [1]. Bloking spinl or NMDA reeptor prevents stress-inue exertion of lloyni fter spre nerve injury [1]. It ws reporte tht spinl or NMDA reeptor is not involve in entrl mehnisms unerlying postopertive pin in non-stresse nimls [3, 33]. Whether the role of NMDA reeptors n the signling omponents esrie ove prtiipte in the short-term stressinue prolongtion of inisionl pin in the present stuy remins to e further efine. Not without sying, other potentil mehnisms nnot e rule out. Conlusions In onlusion, the present stuy showe tht short-term stress oes not ffet sl noieption ut oes prolong post-surgil hypersensitivity to mehnil, ol, n therml stimuli pplie to the inision site. Although the etile mehnisms unerlying this effets re still unler, our finings inite tht prevention of short-term CORT signling uring pre n post-opertive perios my filitte reovery from postopertive pin for ptients. Methos Animl preprtion All mle n femle Sprgue Dwley rts weighing 3 g were otine from Chrles River Lortories (Wilmington, MA, USA) n were house in n niml fility tht ws kept in stnr -h light/rk yle, with stnr lortory wter n foo pellets ville liitum. Rt experiments were onute with the pprovl of the Animl Cre n Use Committee t New Jersey Meil Shool n were onsistent with the ethil guielines of the US Ntionl Institutes of Helth n the Interntionl Assoition for the Stuy of Pin. All efforts were me to minimize niml suffering n to reue the numer of nimls use. To minimize intr- n inter-iniviul vriility of ehviorl outome mesures, nimls were hitute h ily for ys efore ehviorl testing ws performe. The experimenters oul not e line to inision, ut were line to rug/stress tretments or renletomy uring ehviorl testing. Inisionl pin moel The inisionl surgery ws rrie out s esrie [1] with the following minor moifitions. After nimls were nesthetize with % isoflurne elivere vi nose one, the plntr spet of the left hinpw ws prepre sterilely (1 % povione-ioine solution) n inise longituinlly (1 m; numer 11 le through the skin n fsi) from. m from the proximl ege of the heel towr the toes. The plntris musle ws elevte n inise longituinlly. After hemostsis with gentle pressure, the skin ws suture with nylon. After surgery, the nimls were llowe to reover in their ges. Typilly, the woun hele well within ys. Immoiliztion stress Immoiliztion stress, strong non-invsive physil stressor with psyhologil omponents, ws rrie out for h (etween m n pm) without foo n wter ily in the ge on 3 onseutive ys s esrie [, 1]. Briefly, the rts were immoilize with metl mesh restriners seure t the he n til ens with lips to restrit the motion of the he n oy. Control rts were house in their usul ges uner norml onitions. Fore swimming stress Fore swimming for min in the morning of 3 onseutive ys ws rrie out iniviully in vertil Plexigls yliner (imeter 3 m, height m) fille with ± 1 C wter t m epth s esrie [7]. Briefly, rts, unle to touh the ottom with their hin pws, were onsiere to e swimming when they move roun the ontiner with ll four pws. Rts from the shm group were sujete to shm swimming session y llowing them to we in the yliner tht ontine only ± m of wter t ± 1 C. The rts were rie

12 Co et l. Mol Pin () 11:73 Pge of 1 efore eing returne to their home ges. The wter ws hnge fter eh session. Intrthel theter implnttion n rug ministrtion Intrthel theters (polyethylene 1) were implnte into the surhnoi spe etween L n L vertere n vne. m into the lumr enlrgement of the spinl or s esrie [3] efore rug ministrtion. The resiul theter ws tunnele uner skin to the nek re n the outer prt of the theter ws expose, refully plugge, n fixe onto the skin. Animls reeive U of peniillin to prevent infetion. The rts were llowe to reover for 7 ys. None of the nimls exhiite postopertive neurologil efiits (e.g., prlysis) or poor grooming hits fter theter insertion surgery. Mifepristone (RU3, μg, Sigm Alrih, St. Louis, MO, USA) issolve in 1 % ethnol solution (vehile) or vehile lone (1 % ethnol) ws ministere intrthelly in 1 μl volume followe y 1 μl sline flush 1 h prior to immoiliztion stress ily for 3 ys. The osge of RU3 use ws se on previous report []. Bilterl renletomy Bilterl renletomy (ADX) ws performe through two orsolterl miflnk skin n musulr inisions s esrie [3]. The ADX rts were supplemente with μg/ml CORT (Sigm Alrih, St. Louis, MO, USA) in the rinking sline to presumly mintin sl levels of CORT n its irin rhythmiity. Fresh solution ws prepre every ys. The shm-adx (Shm) rts unerwent the sme proeure exept for the removl of the renl glns. The shm rts reeive rinking sline (without CORT) inste of rinking wter fter surgery. All rts were llowe to reover for 1 week efore the experiments. Behviorl nlysis All ehviorl tests were rrie out in the fternoons. threshols in response to mehnil stimuli were mesure with the up own testing prigm esrie previously [3, 3]. Briefly, rts were ple in Plexigls hmers on n elevte mesh sreen. Von Frey filments in log inrements of fore (.7,.9, 1.,.1, 3.3,.9,.11,.1 g) were pplie to the plntr surfe of the rts left n right hin pws. The.1-g stimulus ws pplie first. If positive response ourre, the next smller von Frey hir ws use; if negtive response ws oserve, the next lrger von Frey hir ws use. The test ws terminte when (1) negtive response ws otine with the.1-g hir or () three stimuli were pplie fter the first positive response. threshol ws etermine y onverting the pttern of positive n negtive responses to the von Frey filment stimultion to % threshol vlue with formul provie y Dixon [37]. ltenies to noxious het were mesure with Moel 33 Anlgesi Meter (IITC In./ Life Siene Instruments, Wooln Hills, CA, USA) s esrie previously [3, 37]. Briefly, em of light tht provie rint het ws ime t the mile of the plntr surfe of eh hin pw. When the niml lifte its foot, the light em turne off. lteny ws efine s the numer of seons etween the strt of the light em n the foot lift; n llowe mximum of s voie tissue mge to the hin pw. Eh tril ws repete five times t -min intervls for eh sie. ltenies to noxious ol ( C) were mesure with ol plte s esrie [3, 3]. Briefly, the pw withrwl lteny ws efine s the numer of seons etween plement of the hin pw on the C plte n the rpi withrwl of the hin pw, with or without pw liking n iting. An llowe mximum of s voie pw tissue mge. Eh tril ws repete three times t 1-min intervls for the pw on the ipsilterl sie. Swimming immoiliztion test ws rrie out s esrie ove in the fore swimming stress. Briefly, swimming immoiliztion for eh rt ws efine s the miniml movement neessry to sty flot. The urtion of immoiliztion ws reore uring the -min testing perio. Surose preferene test Surose preferene test ws performe s two-ottle hoie prigm (wter or 1 % surose) esrie previously [3]. After rts were trine for ys with the two rinking ottles, the iniviully house rts h ess to rnomly ple (left vs right sie of the ge) ottles from one tril to nother. After stress, eh rt s onsumption of wter n surose uring h perio ws etermine using stnr weight sle. Surose preferene ws lulte using the following formt: surose intke (g)/[surose intke (g) + wter intke (g)]. Bloo olletion n ortiosterone levels To mesure the level of CORT in the loo fter the stress test without interferene from the stress of niml hnling, the loo ( μl) ws ollete vi retrooritl leeing proeure s esrie [3, 39] in lightly nesthetize rts (isoflurne) within 3 min s the CORT levels were elevte fter 3 min of niml hnling. After the smples were entrifuge for 1 min t rpm t

13 Co et l. Mol Pin () 11:73 Pge 13 of 1 C, the superntnt plsm ws ollete, liquote, n store t C. The CORT levels were etete using ortiosterone ELISA kit (Enzo Life Sienes, In. Frmingle, NY, USA). Mesurement of oy weight Rts were weighe efore stress or inision n on y 9 fter inision. Sttistil nlysis Results were ollete y sientists line to the tretments n reporte s men ± SEM. The t were nlyze with one-wy or two-wy ANOVA. When n ANOVA test showe signifint ifferene, pirwise omprisons etween mens were teste y the post ho Tukey metho (SigmStt, Sn Jose, CA, USA). Signifine ws set t p <.. Arevitions ADX: renletomy; C: ontrol; CORT: ortiosterone; HPA: hypothlmi pituitry renl; In: inision; Im: immoiliztion stress (3 onseutive ys of h of immoiliztion); NMDA: N-Methyl--sprtte; V: vehile. Authors ontriutions YXT n XQG oneive the projet n supervise ll experiments. JC, PKW, VT, n YXT esigne the projet. JC, PKW n VT performe the niml moels n surgery n onute ehviorl experiments. JC, PKW, n LL rrie out the surose preferene test, loo olletion, n ortiosterone level mesurement. JC, PKW, VT, BML, KRS, WDZ, AGK, AB, XQG, n YXT nlyze the t. JC, BML, n YXT wrote the mnusript. All uthors re n pprove the finl mnusript. Author etils 1 Deprtment of Antomy, College of Bsi Meiine, Zhengzhou University, Zhengzhou 1, Henn, Chin. Deprtment of Anesthesiology, New Jersey Meil Shool, Rutgers, The Stte University of New Jersey, 1 S. Ornge Ave., MSB, F, Newrk, NJ 713, USA. 3 Deprtment of Anesthesiology, Buhist Tzu Chi Generl Hospitl, Institute of Meil Sienes, Shool of Meiine, Tzu Chi University, Hulien, Tiwn. Deprtment of Anesthesiology n Critil Cre Meiine, Johns Hopkins University Shool of Meiine, Bltimore, MD, USA. Deprtment of Physiology, Shool of Meiine, Institute of Meil Sienes, Tzu Chi University, Hulien, Tiwn. Aknowlegements This work ws supporte y Grnts from the NIH (NS7, HL117, n DA3339) n the Rit Allen Fountion. 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14 Co et l. Mol Pin () 11:73 Pge 1 of 1 ortiosteroi hormone reeptors in erly humn fetl life. J Clin Enorinol Met. 199;3:9 7.. Bnik RK, Woo YC, Prk SS, Brennn TJ. Strin n sex influene on pin sensitivity fter plntr inision in the mouse. Anesthesiology. ;: Htkeym S, Kwi Y, Ueym T, Sen E. Nitri oxie synthseontining mgnoellulr neurons of the rt hypothlmus synthesize oxytoin n vsopressin n express Fos following stress stimuli. J Chem Neuront. 199;11:3. 3. Torres IL, Buffon A, Silveir PP, Durte MZ, Bssni MG, Oliveir SS, et l. Effet of hroni n ute stress on etonuleotise tivities in spinl or. Physiol Behv. ;7: Jllvn E, Jvn M, Heri-Rohni A, Ahmini A. Stress- n nonstress-meite mehnisms re involve in pin-inue poptosis in hippompus n orsl lumr spinl or in rts. Neurosiene. ;7:. 3. Zhn PK, Brennn TJ. Lk of effet of intrthelly ministere N-methyl--sprtte reeptor ntgonists in rt moel for postopertive pin. Anesthesiology. 199;: Pogtzki EM, Niemeier JS, Sorkin LS, Brennn TJ. Spinl glutmte reeptor ntgonists ifferentite primry n seonry mehnil hyperlgesi use y inision. Pin. 3;: Xu JT, Zhou X, Zho X, Ligons D, Tiwri V, Lee CY, et l. Opioi reeptortriggere spinl mtorc1 tivtion ontriutes to morphine tolerne n hyperlgesi. J Clin Invest. 1;: Rees SL, Pnesr S, Steiner M, Fleming AS. The effets of renletomy n ortiosterone replement on mternl ehvior in the postprtum rt. Horm Behv. ;: Zho X, Tng Z, Zhng H, Atinjoh FE, Zho JY, Ling L, et l. A long nonoing RNA ontriutes to neuropthi pin y silening Kn in primry fferent neurons. Nt Neurosi. 13;1: Chpln SR, Bh FW, Pogrel JW, Chung JM, Yksh TL. Quntittive ssessment of ttile lloyni in the rt pw. J Neurosi Methos. 199;3: Hui YH, Hung NH, Eert L, Bin H, Ching A, Mples C, et l. Phrmokineti omprisons of til-leeing with nnul- or retro-oritl leeing tehniques in rts using six mrkete rugs. J Phrmol Toxiol Methos. 7;:. 39. Shrm A, Fish BL, Mouler JE, Mehor M, Bker JE, Mer M, et l. Sfety n loo smple volume n qulity of refine retro-oritl leeing tehnique in rts using lterl pproh. L Anim (NY). 1;3:3. Sumit your next mnusript to BioMe Centrl n we will help you t every step: We ept pre-sumission inquiries Our seletor tool helps you to fin the most relevnt journl We provie roun the lok ustomer support Convenient online sumission Thorough peer review Inlusion in PuMe n ll mjor inexing servies Mximum visiility for your reserh Sumit your mnusript t