ABSTRACT
http://dx.doi.org/10.1590/1678-775720160150
The effect of capsaicin on expr ession pat t er ns
of CGRP in t r igem inal ganglion and t r igem inal
nucleus caudalis follow ing ex per im ent al t oot h
m ovem ent in rat s
Yang ZHOU, Hu LONG, Niansong YE, Lina LIAO, Xin YANG, Fan JIAN, Yan WANG, Wenli LAI
Sichuan University, West China Hospital of Stomatology, Department of Orthodontics, State Key Laboratory of Oral Diseases, Chengdu, China.
Corresponding address: Wenli Lai - Department of Orthodontics - State Key Laboratory of Oral Diseases - West China Hospital of Stomatology - Sichuan University - No. 14, Section 3, Ren Min South Road Chengdu - 610041 - China - Phone/fax: +86-28-85501442 - e-mail: wenlilai@scu.edu.cn
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bj ect ives: The aim of t his st udy was t o explor e t he effect of capsaicin on expr ession pat t er ns of calcit onin gene- r elat ed pept ide ( CGRP) in t he t r igem inal ganglion ( TG) and t r igem inal subnucleus caudalis ( Vc) follow ing exper im ent al t oot h m ovem ent . Mat er ial and Met hods: Male Sprague- Daw ley rat s w er e used in t his st udy and divided int o sm all- dose capsaicin+ force group, large- dose capsaicin+ force group, saline+ force group, and no force gr oup. Closed coil spr ings w er e used t o m im ic or t hodont ic for ces in all gr oups except for t he no for ce gr oup, in w hich spr ings w er e inact ivat ed. Capsaicin and saline w er e inj ect ed int o per iodont al t issues. Rat s w er e eut hanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d follow ing exper im ent al t oot h m ovem ent . Then, TG and Vc w er e obt ained for im m unohist ochem ical st aining and west ern blot t ing against CGRP. Result s: I m m unohist ochem ical result s indicat ed WKDW&*53SRVLWLYHQHXURQVZHUHORFDWHGLQWKH7*DQG&*53LPPXQRUHDFWLYH¿EHUVZHUH dist ribut ed in t he Vc. I m m unohist ochem ical sem iquant it at ive analysis and w est ern blot t ing analysis dem onst rat ed t hat CGRP expr ession levels bot h in TG and Vc w er e elevat ed at 12 h, 1 d, 3 d, 5 d, and 7 d in t he saline + for ce gr oup. How ever, bot h sm all- dose and lar ge- dose capsaicin could decr ease CGRP expr ession in TG and Vc at 1 d and 3 d follow ing exper im ent al t oot h m ovem ent , as com par ed w it h t he saline + for ce gr oup. Conclusions: These result s suggest t hat capsaicin could regulat e CGRP expression in TG and Vc follow ing exper im ent al t oot h m ovem ent in rat s.Ke y w o r d s: Capsaicin . Calcit on in gen e- r elat ed pept ide r ecept or s. Toot h m ov em en t . Tr igem inal ganglion. Tr igem inal caudal nucleus.
I N TROD UCTI ON
Or t hodont ic pain induced by t oot h m ovem ent is w idely consider ed as a negat ive consequence of or t hodont ic t r eat m ent s19. Alt hough t he per cept ion
of pain is a subj ect ive exper ience and m ight var y in in div idu als, alm ost all pat ien t s com plain t h e discom fort during ort hodont ic t reat m ent3. Therefore,
how t o alleviat e or t hodont ic pain and elucidat e it s underlying m echanism s clearly represent one of t he m aj or concerns for bot h pat ient s and ort hodont ist s. Or ofacial pain signals induced by t oot h m ovem ent are received by peripheral nocicept ors, t ransm it t ed WRWKH¿UVWRUGHUQHXURQVLQWKHWULJHPLQDOJDQJOLRQ
( TG) , co n v ey ed t o t h e t r i g em i n al su b n u cl eu s caudalis ( Vc) locat ed in t he caudal par t of m edulla oblongat a, t hen r elayed t o t he t hir d- or der neur ons in t h e t h alam u s, an d f in ally p er ceiv ed b y t h e cort ex19. Well- grounded experim ent al observat ions
have docum ent ed t hat bot h TG and Vc play a crucial r ole in t he t ransm ission of or t hodont ic pain16,30.
I t is well known t hat t he percept ion of ort hodont ic SDLQLVSUREDEO\GXHWRDQLQÀDPPDWRU\UHDFWLRQ localized in per iodont al t issues t hat involves t he UHOHDVH RI YDULRXV LQÀDPPDWRU\ PHGLDWRUV28. I n
of i n f l am m at or y p ai n2. Mor eov er, i t h as b een
dem onst rat ed t hat CGRP part icipat es in ort hodont ic pain f ollow in g ex per im en t al t oot h m ov em en t1 8.
Capsaicin , t h e m ain pu n gen t in gr edien t in h ot ch i l i p ep p er s, cou l d el i ci t h eat sen sat i on an d st im u lat e t h e r elease of sen sor y n eu r op ep t id e t hr ough select ively binding t o t ransient r ecept or p o t en t i al v an i l l o i d 1 ( TRPV1 , a n o n - sel ect i v e cat ion channel)6. CGRP could be synt hesized and
r eleased f r om a su b set of cap saicin - sen sit iv e pr im ar y affer ent neur ons in t he TG12. How ever, t he
r elat ionship bet w een capsaicin and CGRP r elease following t oot h m ovem ent is st ill poorly underst ood. Alt h ou g h p r ev iou s st u d ies h av e in d icat ed t h at capsaicin- sensit ized TRPV1 could evoke t he CGRP r elease fr om per ipheral ner ve axons13, t he effect
of capsaicin on CGRP ex pr essions in TG and Vc follow ing ex per im ent al t oot h m ovem ent r em ains lar gely unknow n. Fur t her m or e, adm inist rat ion of capsaicin does not affect t he rat e of or t hodont ic t oot h m ovem ent in rat s9.
Ther efor e, t he pur pose of t he pr esent st udy w as t o i n v est i g at e t h e ef f ect o f cap sai ci n o n expr ession pat t er ns of CGRP in TG and Vc follow ing experim ent al t oot h m ovem ent in rat s and t o explore it s under lying m echanism s.
M ATERI AL AN D M ETH OD S
All ex per im ent al pr ocedur es w er e conduct ed according t o t he Nat ional I nst it ut es of Healt h Guide for t he Car e and Use of Laborat or y Anim als, and w er e appr oved by t he Et hical Com m it t ee of West China School of St om at ology, Sichuan Univer sit y and St at e Key Laborat or y of Oral Diseases. Effor t s were m ade t o m inim ize bot h t he num ber of anim als and t heir discom for t .
An im a ls
Male Sp r ag u e- Daw ley r at s ( ag e: 8 w eek s; w eight : 200- 250 g) w er e used in t his st udy. These rat s w er e pr ov ided by t he Anim al Ex per im ent al Cent er, Sichuan Univer sit y, Chengdu, China. They w er e h ou sed in a t em per at u r e- r egu lat ed r oom at 25± 2°C w it h st andar d rat chow and wat er ad
libit um , and m aint ained on a 12/ 12 day- night cycle
LQDTXLHWHQYLURQPHQWIRUDWOHDVW¿YHGD\VSULRU t he exper im ent .
Ex pe r im e n t de sign
I n or der t o ex am ine t he effect s of capsaicin on CGRP expr ession follow ing exper im ent al t oot h m o v e m e n t , r a t s w e r e r a n d o m l y d i v i d e d i n t o sm all- d ose cap saicin + f or ce g r ou p, lar g e- d ose capsaicin+ for ce gr oup, saline+ for ce gr oup, and no for ce gr oup. Fixed and st ainless st eel closed- coil VSULQJVZHUHOLJDWHGEHWZHHQOHIWXSSHU¿UVWPRODUV and t he ipsilat eral upper incisors t o delivery a m esial
for ce of 40 g as pr eviously descr ibed23 in t he for ce
gr oups w hile spr ings inact ivat ed in t he no for ce group ( 0 g) . Rat s w ere fed w it h soft food aft er force applicat ion. They w er e eut hanized t hr ough cer vical dislocat ion follow ing general anest hesia at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d ( six rat s at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d in each gr oup) . I n par t icular, in all gr oups, t he rat s eut hanized at 0 h w it hout any int er vent ion w er e t aken as t he baseline cont r ol for each gr oup.
Th e r at s of t h e sm all- d ose cap saicin + f or ce JURXSZHUHLQMHFWHGZLWKNj/RIFDSVDLFLQVROXWLRQ ( 3× 10- 2 m ol/ L; Sigm a-Aldrich, St . Louis, MO, USA)15
int o per iodont al t issues ar ound upper left m olar s DWIRXUGLIIHUHQWVLWHVNj/VLWHZKLOHUDWVRIWKH ODUJHGRVHIRUFHJURXSZHUHLQMHFWHGZLWKNj/ Nj/VLWH7KHGRVDJHRIFDSVDLFLQXVHGLQWKLV st udy has been show n t o be effect ive in a pr evious
st u dy3 1 0RUHRYHU VDOLQH RU Nj/
was used in t he saline+ for ce gr oup. Per iodont al in j ect ion s w er e per for m ed 3 0 m in befor e for ce applicat ion.
Tissu e sa m ple pr e pa r a t ion s
Follow in g cer v ical d islocat ion , t h e m ax illar y p o r t i o n o f TG a t t h e i p si l a t er a l si d e o f f o r ce applicat ion and t he caudal part of m edulla oblongat a w er e ob t ain ed f or im m u n oh ist och em ist r y an d w est er n blot .
I m m u n oh ist och e m ist r y
The ex pr essions of CGRP in TG and Vc w er e det ect ed by im m unohist ochem ist ry at six t im e point s ( 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d) aft er experim ent al t oot h m ovem ent in rat s ( n= 6 per gr oup) . Tissue VDPSOHV ZHUH ¿[HG ZLWK SDUDIRUPDOGHK\GH w ash ed in ph osph at e- bu ffer salin e ( PBS) t h r ee WLPHVDQGWKHQHPEHGGHGLQSDUDI¿Q6HULDOWLVVXH VHFWLRQVZHUHFXWDWDWKLFNQHVVRINjPDQGWKHQ GHSDUDI¿QL]HG$IWHUDQWLJHQUHWULHYDOPLFURZDYH cit r at e acid bu f f er ) , t h e sect ion s w er e w ash ed in PBS, blocked w it h goat ser um , and incubat ed ZLWKDVSHFL¿FUDEELWDQWL&*53DQWLERG\ ab47027; Abcam , Cam br idge, MA, USA) at 37°C for 45 m in. Aft er t hat , sect ions w er e r insed w it h PBS f or 1 0 m in an d in cu bat ed w it h En VisionTM
( K500711; DAKO, Car pint er ia, CA, USA) at 37°C for 45 m in. Follow ing washing w it h PBS for 10 m in, t hey w er e v isualized w it h 3,3’- diam inobenzidine ( D AB) an d w ash ed w i t h d i st i l l ed w at er. Af t er count er st ained w it h hem at oxylin and dehydrat ed w i t h e t h a n o l , i m m u n o st a i n e d se ct i o n s w e r e m ount ed on cov er slips. All t issues follow ed t he sam e im m unohist ochem ical procedures t o m inim ize t he var iabilit y in laborat or y.
&*53 H[SUHVVLRQ ZDV GH¿QHG DV \HOORZEURZQ st ained cyt oplasm for TG and yellow- brow n st ained r et icular for m at ion for Vc. For each rat , int egrat ed opt ical densit y ( I OD/ Ar ea) for TG and Vc w er e calculat ed ( I m age- Pro Plus 6.0, Media Cybernet ics, Rock v ille, MD, USA) in each of f iv e r an d om ly FRQVHFXWLYH¿HOGVLQHDFKRIWKUHHVOLGHVDQGWKH PHDQ RI WKHVH ¿HOGV ZDV HPSOR\HG DV &*53 expr ession level.
W e st e r n blot t in g
Th e CGRP p r ot ein ex p r ession in TG an d Vc w er e st udied by w est er n blot t ing at six t im e point s aft er exper im ent al t oot h m ovem ent in rat s ( n= 6
per gr oup) . Follow ing m echanical gr inding, t issue
sam ples w er e disint egrat ed w it h RI PA lysis buffer on ice for 30 m in and cent r ifuged at 4°C Then, t he super nat ant s w er e ext ract ed and st or ed at - 70°C. Aft er det er m ining t he t ot al pr ot ein concent rat ion, sam ples w er e separat ed by 15% SDS- PAGE and t r an sf er r ed t o p o l y v i n y l i d en e f l u o r i d e ( PVD F)
m em branes. They w er e washed four t im es and blocked w it h 5% defat t ed m ilk pow der for 2 h, WKHQ LQFXEDWHG ZLWK D VSHFL¿F UDEELW DQWL&*53 ant ibody ( 1: 200; ab47027, Abcam , Cam br idge, MA, USA) . The m em branes w er e t hen washed in PBS and incubat ed wit h secondary ant ibody, Dylight 6 8 0 conj ugat ed goat ant i- rabbit I gG ( 1 : 1 0 0 0 0 , .3/ ǃDFWLQ ZDV XVHG DV DQ LQWHUQDO FRQWURO pr ot ein. Band int ensit y was com put er analyzed by a densit om et er ( Quant it y One, Bio- Rad, Her cules, CA, USA) . Each band was analyzed repeat edly t hree WLPHV DQG WKH PHDQ UDWLR RI &*53ǃDFWLQ ZDV em ployed as t he CGRP expr ession level.
St a t ist ica l a n a ly se s
Dat a w er e expr essed as m ean± st andar d er r or ( SEM) . On e- w ay an aly sis of v ar ian ce ( ANOVA) ( Tu k ey p ost h oc t est ) w as ap p lied t o an aly ze d if f er en ces of CGRP ex p r ession lev els am on g differ ent t im e point s in each gr oup and t o com par e t he differences of CGRP expression levels am ong t he
four gr oups at each t im e point . P values less t han ZHUHFRQVLGHUHGWREHVWDWLVWLFDOO\VLJQL¿FDQW All t he st at ist ical analyses w er e per for m ed in SPSS 19.0 ( SPSS, Chicago, I L, USA) and GraphPad Pr ism 6.0 ( GraphPad soft war e, San Diego, CA, USA)
RESULTS
The e ffe ct s of ca psa icin on CGRP e x pr e ssion i n t h e T G f o l l o w i n g e x p e r i m e n t a l t o o t h m ov e m e n t
As displayed in Figur e 1A, bands of CGRP w er e VKRZQQHDUWRN'DDQGWKDWRIǃDFWLQQHDUWR 42 kDa.
For t h e salin e+ for ce gr ou p, w h en com par ed w it h t he baseline level ( 0.628± 0.041) , as show n i n Fi g u r e 1 B, e x p e r i m e n t a l t o o t h m o v e m e n t induced an up- r egulat ion of CGRP ex pr ession in t h e salin e+ f or ce gr ou p at 1 2 h ( 0 . 9 8 8 ± 0 . 0 1 9 , p< 0.001) and 1 d ( 1.216± 0.011, p< 0.001) , peaked at 3 d ( 1.599± 0.034, p< 0.001) , t hen decr eased gr adu ally at 5 d ( 0 . 9 6 2 ± 0 . 0 2 6 , p< 0 . 0 0 1 ) an d
Figure 2- CGRP expressions in trigeminal ganglion (TG) following experimental tooth movement. A and B: CGRP were mainly expressed in TG neurons at baseline and stained in yellow-brown, as indicated by the black arrows (100x and 400x); C and D: CGRP was expressed in TG (as indicated by the black arrows) in the saline+force group at 3 d (100x and 400x); E and F: CGRP was expressed in TG (as indicated by the black arrows) in the small-dose capsaicin+force group at 3 d (100x and 400x); G and H: CGRP was expressed in TG (as indicated by the black arrows) in the large-dose capsaicin+force group at 3 d (100x and 400x). CGRP-negative cell is indicated by the red arrow
7 d ( 0 . 8 2 2 ± 0 . 0 1 5 , p= 0 . 0 0 2 < 0 . 0 5 ) . For t he no for ce gr ou p, w h en com par ed w it h t h e baselin e l e v e l ( 0 . 7 4 5 ± 0 . 0 3 5 ) , t h e CGRP e x p r e s s i o n levels w er e only incr eased at 3 d ( 1.024± 0.046, p= 0.001< 0.05) .
Mor eover, CGRP expr ession levels in t he sm all-d ose cap saicin + f or ce g r ou p w er e sig n if ican t ly h igh er t h an in t h e salin e+ f or ce gr ou p at 1 2 h ( 1.468± 0.019 vs. 0.988± 0.019, p< 0.0001) , t hen JUHDWO\GHFUHDVHGDQGEHFDPHVLJQL¿FDQWO\ORZHU t han in t he saline+ force group at 1 d ( 1.007± 0.026 vs. 1.216± 0.011, p< 0.05) , 3 d ( 0.974± 0.027 vs. 1.599± 0.034, p< 0.0001) , and 5 d ( 0.734± 0.025 vs. 0.962± 0.026, p< 0.05) , w her eas it incr eased again ( 1.082± 0.056 vs. 0.822± 0.015, p= 0.0001) at 7 d. Sim ilar ly, CGRP expr ession levels in t he lar ge- dose FDSVDLFLQJURXSZHUHDOVRVLJQL¿FDQWO\KLJKHUWKDQ in t he saline+ for ce gr oup at 12 h ( 1.934± 0.101 vs. 0.988± 0.019, p< 0.0001) , t hen decreased and were
VLJQL¿FDQWO\ORZHUWKDQLQWKHVDOLQHIRUFHJURXS at 1 d ( 0.982± 0.061 vs. 1.216± 0.011, p< 0.05) , 3 d ( 0.868± 0.048 vs. 1.599± 0.034, p< 0.0001) , and 5 d ( 0.602± 0.074 vs. 0.962± 0.026, p< 0.0001) ; KRZHYHUWKH&*53H[SUHVVLRQOHYHOV¿QDOO\EHFDPH sim ilar bet w een t he t w o gr oups ( 0.805± 0.022 vs. 0.822± 0.015, p= 0.9797> 0.05) at 7 d.
I nt erest ingly, regarding t he com parison bet ween t he t wo groups wit h different capsaicin dosages, we IRXQGWKDW&*53H[SUHVVLRQOHYHOVZHUHVLJQL¿FDQWO\ h igh er in t h e lar ge- dose capsaicin + for ce gr ou p t han in t he sm all- dose capsaicin+ for ce gr oup at 12 h ( p< 0.05) . Conver sely, follow ing t he sim ilar CGRP expr ession levels bet w een t hese t w o gr oups at 1 d, 3 d, and 5 d ( all p> 0.05) , CGRP expr ession OHYHOVZHUHVLJQL¿FDQWO\KLJKHULQWKHVPDOOGRVH cap saicin + f or ce g r ou p t h an in t h e lar g e- d ose capsaicin+ for ce gr oup ( p< 0.05) .
As sh ow n in Fig u r e 2 , CGRP w as b asically
e x p r e sse d i n t r i g e m i n a l n e u r o n s a n d CGRP-posit ive cells could also be obser ved at baseline ( as indicat ed by t he black ar r ow ) . As displayed in Fig u r e 3 , t r en d s in CGRP ex p r ession lev els ( q u an t if ied t h r ou g h im m u n ost ain in g in t en sit y ) for all t he four gr oups w er e sim ilar w it h t hose of ZHVWHUQ EORWWLQJ 6SHFL¿FDOO\ IRU WKH VPDOOGRVH capsaicin+ for ce gr oup, CGRP ex pr ession levels increased rapidly at 12 h, declined at 1 d, 3 d, and 5 d, and r ebounded at 7 d. I n cont rast , for t he lar ge-dose capsaicin+ force group, CGRP expression levels peaked at 12 h and decr eased t her eaft er w it hout r ebounding at 7 d. Mor eover, for t he saline+ for ce gr oup, CGRP expr ession levels st ar t ed t o incr ease at 12 h, peaked at 3 d, and gradually decr eased t hereaft er. I n cont rast , for t he no force group, CGRP expr ession levels only incr eased at 3 d.
The e ffe ct s of ca psa icin on CGRP e x pr e ssion i n V c f o l l o w i n g e x p e r i m e n t a l t o o t h m ov e m e n t
We could also obser ve t hat bands of CGRP w er e VKRZQQHDUWRN'DDQGWKDWRIǃDFWLQQHDUWR 42 kDa ( Figur e 4A) .
Com pared wit h t he baseline level ( 0.406± 0.004) , as displayed in Figur e 4B, CGRP expr ession levels i n t h e sa l i n e+ f o r ce g r o u p i n cr ea sed a t 1 2 h ( 1.187± 0.041, p< 0.001) and 1 d ( 1.511± 0.017, p< 0.001) , peaked at 3 d ( 1.737± 0.033, p< 0.001) , t hen gradually decr eased at 5 d ( 0. 823± 0. 008, p < 0 . 0 0 1 ) a n d 7 d ( 0 . 6 3 3 ± 0 . 0 2 4 , p < 0 . 0 0 1 ) . Sim ilar ly, for t he no for ce gr oup, w hen com par ed w i t h t h e b asel i n e l ev el ( 0 . 3 9 9 ± 0 . 0 0 7 ) , CGRP expr ession levels incr eased at 12 h ( 1.100± 0.061, p< 0.001) , r em ained at t he incr eased level at 1 d ( 1 . 1 2 2 ± 0 . 0 2 9 , p< 0 . 0 0 1 ) , 3 d ( 1 . 0 8 6 ± 0 . 0 1 2 ,
p< 0.001) , and 5 d ( 1.041± 0.019, p< 0.001) , t hen decr eased at 7 d ( 0.650± 0.068, p< 0.05) .
On t h e ot h er h an d, CGRP ex pr ession lev els w e r e s i g n i f i c a n t l y h i g h e r i n t h e l a r g e - d o s e capsaicin + for ce gr ou p t h an in t h e salin e+ for ce gr ou p at 1 2 h ( 1 . 4 7 4 ± 0 . 0 3 4 v s. 1 . 1 8 7 ± 0 . 0 4 1 , p < 0 . 0 5 ) , t h en g r eat ly d ecr eased an d b ecam e VLJQL¿FDQWO\ORZHUWKDQLQWKHVDOLQHIRUFHJURXSDW 1 d ( 0.880± 0.012 vs. 1.511± 0.017, p< 0.0001) , 3 d ( 1.061± 0.028 vs. 1.737± 0.033, p< 0.0001) , and 5 d ( 0.466± 0.002 vs. 0.823± 0.008, p< 0.0001) . Fu r t h er m or e, CGRP ex pr ession lev els in sm all-d ose cap saicin + f or ce g r ou p w er e sig n if ican t ly low er t h an in t h e salin e+ f or ce g r ou p at 1 2 h ( 0 . 9 1 5 ± 0 . 0 0 8 v s. 1 . 1 8 7 ± 0 . 0 4 1 , p < 0 . 0 5 ) , 1 d ( 0.842± 0.003 vs. 1.511± 0.017, p< 0.0001) , and 3 d ( 1.023± 0.019 vs. 1.737± 0.033, p< 0.0001) , EXW VLJQL¿FDQWO\ KLJKHU WKDQ WKDW LQ VDOLQHIRUFH g r ou p at 5 d ( 0 . 9 0 9 ± 0 . 0 2 1 v s. 0 . 8 2 3 ± 0 . 0 0 8 , p< 0 . 0 5 ) . I n addit ion , CGRP ex pr ession lev els bot h in t he lar ge- dose capsaicin+ for ce and sm all-dose capsaicin+ for ce gr oups gradually incr eased an d b ecam e si g n i f i can t l y h i g h er t h an t h at i n t he saline+ for ce gr oup at 7 d ( 1.075± 0.006 vs. 0 . 6 3 3 ± 0 . 0 2 4 , p < 0 . 0 0 0 1 ; 1 . 2 6 1 ± 0 . 0 0 9 v s . 0.633± 0.024, p< 0.0001, r espect ively) .
As shown in Figure 5, CGRP- like im m unoreact ivit y was obser ved in Vc at baseline, w hich displayed UHWLFXODUIRUPDWLRQDQGQHUYH¿EHUVVWDLQHGEURZQ w er e t he m ain st r uct ur es. As pr esent ed in Figur e 6 , follow ing ex per im ent al t oot h m ov em ent , t he chr onological changes in CGRP expr ession levels w er e si m i l a r w i t h t h o se o f w est er n b l o t t i n g . 6SHFL¿FDOO\ IRU WKH VPDOOGRVH FDSVDLFLQIRUFH gr oup, CGRP expr ession levels incr eased st eadily fr om 12 h and peaked at 7 d. I n cont rast , for t he
large- dose capsaicin+ force group, CGRP expression levels peaked at 12 h, declined t her eaft er unt il 5 d, an d r ebou n ded at 7 d. Mor eov er, f or t h e saline+ for ce gr oup, CGRP expr ession levels began t o incr ease at 12 h, peaked at 3 d, and dr opped t hereaft er. For t he no force group, CGRP expression
levels incr eased and r eached a plat eau fr om 12 h t o 5 d, and r et ur ned t o baseline at 7 d.
D I SCUSSI ON
Figure 7- When orthodontic force was exerted on a tooth, nociceptive stimulus would be induced and perceived by peripheral nociceptors in periodontal tissues, where it could then generate pain signals that propagate to the TG. Once receiving these signals, TG would synthesize and release CGRP to periodontal tissues and Vc via bidirectional transport. After local injection of capsaicin, small-dose capsaicin could recruit more CGRP released from TG into periodontal tissues than Vc compared with large-dose capsaicin
I n t his st udy, w e found t hat capsaicin could r egulat e CGRP r elease bot h in TG and Vc follow ing ex per im en t al t oot h m ov em en t . Mor eov er, bot h sm all- dose and lar ge- dose capsaicin could cause dow nr egulat ion of CGRP expr ession at 1 d and 3 d, w her eas a lar ge CGRP r elease was obser ved in t he saline+ for ce gr oup.
I t is well- known t hat m ost pat ient s could perceive or t hodont ic pain fr om 1 d t o 3 d and pain sensat ion gradually subsides t her eaft er22, w hich has been
t he m ain adver se effect of or t hodont ic t r eat m ent . Dur ing ex per im ent al t oot h m ovem ent , t he for ce exer t ed on per iodont al t issues could init iat e local LQÀDPPDWLRQDQGFDXVHWKHUHOHDVHRILQÀDPPDWRU\ PHGLDWRUV%HLQJWKH¿UVWRUGHUVHQVRU\QHXURQV i n t h e o r o f aci al r eg i o n , TG co u l d r ecei v e t h e nocicept ive signal fr om per ipheral ner ve t er m inals locat ed in per iodont al t issues, and t ransm it it t o t he Vc. CGRP is exclusively synt hesized in pr im ar y affer ent neur ons of t he TG12. Pr evious st udies have
indicat ed t hat CGRP plays an im por t ant r ole in t he under lying pat hogenesis of pain condit ions4. Our
r esult s show ed t hat CGRP expr ession levels bot h LQ7*DQG9FWKHVDOLQHIRUFHJURXSVLJQL¿FDQWO\ incr eased at 1 d and peaked at 3 d, t hen gradually decr eased at 5 d and 7 d. This change t r end was consist ent w it h a pr evious st udy, w hich indicat ed t hat CGRP was involved in t he t ransm ission and m odulat ion of pain follow ing ex per im ent al t oot h m ovem ent18. Accordingly, we suggest t hat increased &*53PLJKWUHÀHFWWKHHIIHFWRIQRFLFHSWLYHVWLPXOL on act ivat ion of t r igem inal syst em and subsequent t ransm ission and pr ocessing of t his infor m at ion i n t h e b r ai n st em2 5. How ev er, i r on i cal l y, CGRP
expr ession levels incr eased in t he no for ce gr oup bot h in TG and Vc. Act ually, since no for ce was exert ed in t his group, CGRP expression levels should be sim ilar w it h t hose at baseline. We at t r ibut e it t o discom for t s and t he buccal peculiar sense t hat m ight be caused by inact ivat ed int raoral spr ings in rat s17.
Ou r r e su l t s sh o w e d t h a t b o t h sm a l l - d o se capsaicin and lar ge- dose capsaicin had an obvious im pact on CGRP expr ession levels in TG and Vc f ollow in g ex p er im en t al t oot h m ov em en t w h en com par ed w it h t he saline+ for ce gr oup. I t has been w ell- docum ent ed t hat capsaicin could select ively DFWRQXQP\HOLQDWHG&¿EHUVDQGWKLQP\HOLQDWHG $į¿EHUVLQSULPDU\VHQVRU\QHXURQVE\ELQGLQJWR TRPV16$FWLYDWLRQRI7539OHDGVWRDQLQÀX[RI
calcium and CGRP r elease fr om per ipheral ner ve axons13,29. I n our st udy, local per iodont al inj ect ions
of capsaicin caused a lar ge CGRP r elease bot h in TG and Vc at 12 h follow ing exper im ent al t oot h PRYHPHQW7KHLQFUHDVHG&*53PLJKWSDUWO\UHÀHFW t he st im ulat or y effect of capsaicin, w hich is know n t o act ivat e sensor y neur ons and st im ulat e CGRP
r elease5. I t is w or t h m ent ioning t hat t hese t w o
gr oups w it h differ ent capsaicin dosages show ed som e differ ences in t he CGRP expr ession levels at 12 h. The m echanism behind t his effect m ight be t he dose- dependent r elease of CGRP induced by excit at ion of TRPV124. This could also be suppor t ed
by t he obser vat ion t hat applicat ion of capsaicin cau sed a d o se- d ep en d en t r el ease o f sal i v ar y &*53 ZKLFK PLJKW UHÀHFW WKH DFWLYDWHG VWDWH RI t r igem inal sy st em27. Mor eover, in our st udy, w e
found t hat following experim ent al t oot h m ovem ent , CGRP expr ession levels bot h in TG and Vc at 1 d DQGGZHUHVLJQL¿FDQWO\ORZHUWKDQWKDWLQWKH saline+ for ce gr oup. One possible explanat ion for t his phenom enon is based on t he ev idence t hat t her e is anot her effect of capsaicin, w hich is t he inhibit or y effect .20 t hat w hen r ecur r ent applicat ion
of capsaicin or adm inist rat ion of a single high dose, it cou ld d esen sit ize t h e v olt ag e- g at ed calciu m channels on sensor y neur ons10 and t her eby could
reduce t he release of neurot ransm it t ers. ( This m ight be explained by t hat when recurrent or high dose of capsaicin is applied, it could desensit ize t he volt age-g at ed calciu m ch an n els on sen sor y n eu r on s1 0.
Su bsequ en t ly, t h e r elease of n eu r ot r an sm it t er s w ou ld be in h ibit ed. An ot h er ex plan at ion m igh t be t he lar ge deplet ion of CGRP bot h in TG and Vc follow ing inj ect ions of capsaicin and exper im ent al t oot h m ovem ent .
I n addit ion, CGRP expr ession levels began t o increase bot h in TG and Vc at 7 d aft er experim ent al t oot h m ovem ent . We at t r ibut e t his phenom enon t o t he beginning of CGRP synt hesis in TG neur ons and subsequent CGRP r elease in Vc. Regar ding t he differ ence of CGRP expr ession levels bet w een sm all- d ose+ f or ce g r ou p an d lar g e- d ose+ f or ce gr ou p at t h e af or em en t ion ed t im e poin t , t h er e ar e som e ev idence t hat m ight account for t his. Previous st udies have report ed t hat aft er prolonged e x p o su r e t o ca p sa i ci n o r h i g h - co n ce n t r a t i o n cap sai ci n ap p l i ed , i t m i g h t i n i t i at e a p r o cess descr ibed as defunct ionalizat ion of volt age- gat ed calciu m ch an n els1, w h ich is m ain ly du e t o t h e
act ivat ion of calcium - dependent pr ot eases7. This
pr ocess could induce degenerat ion of capsaicin-sensit ive nocicept ive ner ve endings8. We her eby
hypot hesized t hat large- dose capsaicin m ight cause a slow er r ecover y of t he TRPV1 act ivit y, r esult ing in low er CGRP expr ession levels t han sm all- dose capsaicin at 7 d.
fr om t he TG11, w hich could also be suppor t ed by
infer ior alveolar ner ve r esect ion14. On t he ot her
hand, cent ral processes of TG cells exhibit ing CGRP-im m unoreact ivit ies alm ost com plet ely t erm inat e at t he Vc21, w hich m eans t hat t he r eleased CGRP in Vc
der ives fr om t he ner ve ext ending fr om t r igem inal n e u r o n s2 6. B a s e d o n t h i s , w e h y p o t h e s i z e d
t h at f ollow in g ex p er im en t al t oot h m ov em en t , CGRP synt hesized in TG m ight be bidir ect ionally t r an spor t ed t o bot h per iodon t al t issu es an d Vc ( Figur e 7) . Fur t her m or e, w e suggest t hat sm all-dose capsaicin was likely t o st im ulat e CGRP release fr om TG m or e per ipherally t han cent rally, t her eby leading t o t he differ ence in CGRP expr ession levels bet w een TG and Vc.
CON CLUSI ON
Th e r e f o r e , CGRP e x p r e s s i o n l e v e l s w e r e elevat ed follow ing exper im ent al t oot h m ovem ent bot h in TG and Vc, suggest ing t heir involvem ent in t he t ransm ission of nocicept ive infor m at ion in t oot h m ovem ent . Bot h sm all- dose and lar ge- dose capsaicin cou ld r edu ce CGRP ex pr ession lev els bot h in TG and Vc follow ing ex per im ent al t oot h m ovem ent in rat s at 1 d and 3 d. CGRP synt hesized in TG neur ons could be bidir ect ionally t ranspor t ed t o p er ip h er al t issu es an d Vc. Tak en t og et h er, w e suggest t hat capsaicin could r egulat e CGRP ex pr ession in TG and Vc follow ing ex per im ent al t oot h m ovem ent in rat s. How ever, t he under lying m ech an ism w h er eb y cap saicin r eg u lat es CGRP e x p r e s s i o n s a n d t h e e f f e c t o f c a p s a i c i n o n or t hodont ic pain should be fur t her elucidat ed.
ACKN OW LED GEM EN T
This st udy was suppor t ed by Nat ional Nat ural Science Foundat ion of China ( NSFC, no. 81571004, 8 1 5 0 0 8 8 4 , an d 8 1 4 0 0 5 4 9 ) an d by Or t h odon t ic Nat ional Key Clinical Specialt y Const ruct ion Program of Ch in a, West Ch in a Hospit al of St om at ology, Sichuan Univer sit y.
REFEREN CES
1 - Anand P, Bley K. Topical capsaicin for pain m anagem ent : t herapeut ic pot ent ial and m echanism s of act ion of t he new high-concent rat ion capsaicin 8% pat ch. Br J Anaest h. 2011: 107( 4) : 490-502.
2- Benem ei S, Nicolet t i P, Capone JG, Geppet t i P. CGRP r ecept or s
LQ WKH FRQWURO RI SDLQ DQG LQÀDPPDWLRQ &XUU 2SLQ 3KDUPDFRO
2009: 9( 1) : 9- 14.
3- Brown DF, Moerenhout RG. The pain experience and psychological a d j u st m e n t t o o r t h o d o n t i c t r e a t m e n t o f p r e a d o l e sce n t s, ad olescen t s, an d ad u lt s. Am J Or t h od Den t of acial Or t h op . 1991: 100( 4) : 349- 56.
4- Cady RJ, Glenn JR, Sm it h KM, Dur ham PL. Calcit onin gene-r elat ed pept ide pgene-r om ot es cellulagene-r changes in t gene-r igem inal neugene-r ons and glia im plicat ed in per ipheral and cent ral sensit izat ion. Mol Pain. 2011: 7: 94.
5- Car pent er SE, Ly nn B. Vascular and sensor y r esponses of hum an skin t o m ild inj ur y aft er t opical t r eat m ent w it h capsaicin. Br J Phar m acol. 1981: 73( 3) : 755- 8.
6- Cat er ina MJ, Schum acher MA, Tom inaga M, Rosen TA, Levine JD, Julius D. The capsaicin r ecept or : a heat - act ivat ed ion channel in t he pain pat hway. Nat ur e. 1997: 389( 6653) : 816- 24.
7 Ch ar d PS, Bleak m an D, Sav idge JR, Miller RJ. Capsaicin -induced neur ot oxicit y in cult ur ed dor sal r oot ganglion neur ons: in v olv em en t of calciu m - act iv at ed p r ot eases. Neu r oscien ce. 1995: 65( 4) : 1099- 108.
8- Chung JM, Lee KH, Hori Y, Willis WD. Effect s of capsaicin applied t o a per ipheral ner ve on t he r esponses of pr im at e spinot halam ic t ract cells. Brain Res. 1985: 329( 1- 2) : 27- 38.
9- Davila JE, Miller JR, Hodges JS, Beyer JP, Lar son BE. Effect of neonat al capsaicin t r eat m ent on or t hodont ic t oot h m ovem ent in m ale Sprague- Daw ley rat s. Am J Or t hod Dent ofacial Or t hop. 2011: 139( 4) : e345- 52.
1 0 - Doch er t y RJ, Rob er t son B, Bev an S. Cap saicin cau ses pr olon ged in h ibit ion of v olt age- act ivat ed calciu m cu r r en t s in adult rat dor sal r oot ganglion neur ons in cult ur e. Neur oscience. 1991: 40( 2) : 513- 21.
11- Eber har dt M, Hoffm ann T, Sauer SK, Messlinger K, Reeh PW, Fischer MJ. Calcit onin gene- r elat ed pept ide r elease fr om int act isolat ed d or sal r oot an d t r ig em in al g an g lia. Neu r op ep t id es. 2008: 42( 3) : 311- 7.
12- Eft ek har i S, Salvat or e CA, Calam ar i A, Kane SA, Taj t i J, Edvinsson L. Differ ent ial dist r ibut ion of calcit onin gene- r elat ed pept ide and it s r ecept or com ponent s in t he hum an t r igem inal ganglion. Neur oscience. 2010: 169( 2) : 683- 96.
1 3 - Flor es CM, Leon g AS, O Du ssor G, Har gr eav es KM, Kilo S. Capsaicin - ev ok ed CGRP r elease f r om r at bu ccal m u cosa: d e v e l o p m e n t o f a m o d e l sy st e m f o r st u d y i n g t r i g e m i n a l m ech a n i sm s o f n eu r o g en i c i n f l a m m a t i o n . Eu r J Neu r o sci . 2001: 14( 7) : 1113- 20.
14- Jacobsen EB, Hey eraas KJ. Effect of capsaicin t r eat m ent or infer ior alveolar ner ve r esect ion on dent ine for m at ion and calcit onin gene- relat ed pept ide- and subst ance P- im m unoreact ive
QHUYH¿EUHVLQUDWPRODUSXOS$UFK2UDO%LRO
31.
15- Lacr oix JS, Buvelot JM, Polla BS, Lundber g JM. I m pr ovem ent of sym pt om s of non- aller gic chr onic r hinit is by local t r eat m ent w it h capsaicin. Clin Exp Aller gy. 1991: 21( 5) : 595- 600.
16- Liao L, Hua X, Long H, Ye N, Zhou Y, Wang S, et al. Expr ession p at t er n s of n ocicep t in in r at s f ollow in g ex p er im en t al t oot h m ovem ent . Angle Or t hod. 2013: 83( 6) : 1022- 6.
17- Liao L, Long H, Zhang L, Chen H, Zhou Y, Ye N, et al. Evaluat ion of pain in rat s t hr ough facial expr ession follow ing exper im ent al t oot h m ovem ent . Eur J Oral Sci. 2014: 122( 2) : 121- 4.
18- Long H, Liao L, Gao M, Ma W, Zhou Y, Jian F, et al. Per iodont al CGRP cont r ibut es t o or ofacial pain follow ing exper im ent al t oot h m ovem ent in rat s. Neur opept ides. 2015: 52: 31- 7.
19- Long H, Wang Y, Jian F, Liao LN, Yang X, Lai WL. Cur r ent advances in or t hodont ic pain. I nt J Oral Sci. 2016: 8( 2) : 67- 75. 2 0 - Lou YP, Fr an co- Cer eced a A, Lu n d b er g JM. Dif f er en t ion channel m echanism s bet w een low concent rat ions of capsaicin an d h igh con cen t r at ion s of capsaicin an d n icot in e r egar din g pept ide r elease fr om pulm onar y affer ent s. Act a Physiol Scand. 1992: 146( 1) : 119- 27.
21- Sam sam M, Coveñas R, Ahangar i R, Yaj eya J, Nar váez JA, Tram u G. Sim ult aneous deplet ion of neur okinin A, subst ance P and calcit onin gene- r elat ed pept ide fr om t he caudal t r igem inal nucleus of t he rat dur ing elect r ical st im ulat ion of t he t r igem inal ganglion. Pain. 2000: 84( 2- 3) : 389- 95.
22- Scheur er PA, Fir est one AR, Bür gin WB. Per cept ion of pain
DVDUHVXOWRIRUWKRGRQWLFWUHDWPHQWZLWK¿[HGDSSOLDQFHV(XU-Or t hod. 1996: 18( 4) : 349- 57.
23- Shirazi M, Khosr ow shahi M, Dehpour AR. The effect of chr onic
UHQDOLQVXI¿FLHQF\RQRUWKRGRQWLFWRRWKPRYHPHQWLQUDWV$QJOH
24- Sim one DA, Baum ann TK, LaMot t e RH. Dose- dependent pain and m echanical hyperalgesia in hum ans aft er int raderm al inj ect ion of capsaicin. Pain. 1989: 38( 1) : 99- 107.
2 5 - Tepper SJ, St illm an MJ. Clin ical an d pr eclin ical r at ion ale for CGRP- r ecept or ant agonist s in t he t r eat m ent of m igraine. Headache. 2008: 48( 8) : 1259- 68.
2 6 - Ulr ich - Lai YM, Flor es CM, Har din g- Rose CA, Goodis HE, Har gr eav es KM. Capsaicin- ev ok ed r elease of im m unor eact iv e calcit onin gene- r elat ed pept ide fr om rat t r igem inal ganglion: e v i d e n c e f o r i n t r a g a n g l i o n i c n e u r o t r a n s m i s s i o n . Pa i n . 2001: 91( 3) : 219- 26.
27- Van Oost er hout WP, Schoonm an GG, Gar r elds I M, Danser AH, Ch an KY, Ter w in dt GM, et al. A h u m an capsaicin m odel t o quant it at ively assess salivar y CGRP secr et ion. Cephalalgia. 2015: 35: 675- 82.
9DQGHYVND5DGXQRYLF91HXUDOPRGXODWLRQRILQÀDPPDWRU\
react ions in dent al t issues incident t o ort hodont ic t oot h m ovem ent . A r eview of t he lit erat ur e. Eur J Or t hod. 1999: 21( 3) : 231- 47. 2 9 - W i n t e r J, Be v a n S, Ca m p b e l l E. Ca p sa i ci n a n d p a i n m echanism s. Br J Anaest h. 1995: 75: 157- 68.
30- Yang Z, Cao Y, Wang Y, Luo W, Hua X, Lu Y, et al. Behavioural r esponses and expr ession of P2X3 r ecept or in t r igem inal ganglion af t er ex p er im en t al t oot h m ov em en t in r at s. Ar ch Or al Biol. 2009: 54( 1) : 63- 70.
