Above. Below H&E. Above CD4. CD11b. Fluoromyelin BDA BDA BDA. Cerebral cortex. Focal lesion. Thoracic cord. Lumbar cord. Hindlimb placing score
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1 Tim tr lsion inution () wks wks Tim tr lsion inution () i Ltrl sor. Mil Aov Blow.. 1 wk On sgmnt ov g 3. Lsion Fluoromylin Fluoromylin h CD11 Hinlim pling sor CD Aov Blow Fol lsion Lumr or H&E Lsion Crrl ortx Thori or Ntur Miin oi:1.138/nm.93 Supplmntry Fig. 1. Dmg to orsl CST n rsultnt motor iit y trgt. () Shmti igrm o ortiospinl trt rorgniztion in trgt mi. A ol lsion ws introu into th orsl (min) CST in th thori spinl or (Th3 n Th). () Histopthology o lsions in trgt mi. Rprsnttiv hmtoxylin n osin-stin ross-stions o trgt spinl or t y 7. Mssiv inlmmtory ll iniltrtion ws osrv in ol lsions in th orsl olumn. () Rprsnttiv ross-stions o th thori spinl or 7 ys tr trgt inution n o th ontrol thori spinl or showing immunortivity or CD n CD11. () Rprsnttiv ross-stions or luoromylin stining. Ths stions wr otin rom th thori spinl or t n 1 sgmnt ov th injtion sit o ytokin mixturs svn ys tr trgt inution. () Rprsnttiv ross-stions o trgt spinl or ll with (ll hinlim CST irs) ov or low th ol lsion on y 8 tr inution. ( n g) Quntiition o sors () n hinlim-pling sors (g) ovr tim in trgt mi. Vlus rprsnt s mn ± SEM (n = 8). (h) High-mgniition imgs o Fig. 1. (i) Dinition o mil n ltrl rgions or quntiition o th -positiv xons in th spinl or. Sl rs in,,, i, µm;, h, 1 µm.
2 Ntur Miin oi:1.138/nm.93 Hinlim pling sor 3 1 Bor Atr Bor Atr Hinlim pling sor 3 1 Bor Atr Bor Atr Fluoromylin Fluoromylin NuN NuN g Numr o NuN+ lls ( 1 3 mm -3 ) Cul Bor Atr Bor Atr Lsion Mylin r (mm ) Rostrl Bor Atr Bor Atr h Lsion lngth (µm) 3 1 Supplmntry Fig.. Spinl intrnurons ontriut to rwiring o th injur motor ntwork. ( n ) Comprison o hinlim-pling sors or n 3 ys tr NMDA inusion into th thori spinl or in trgt () n ontrol () mi. NMDA ws injt 8 ys tr inution. Vlus rprsnt s mn ± SEM (: or, n = 1; tr, n = 11; ontrol: or, n = 9; tr, n = 11). P <.1 using on-wy nlysis o vrin ollow y Sh s tst. ( n ) Rprsnttiv ross-stions o th thori spinl or stin with NuN n luoromylin or n tr injtion o NMDA in trgt () n ontrol () mi. () Quntiition o th numr o NuN + lls in th spinl or o n ontrol mi or n tr NMDA injtion. () Quntiition o th luoromylin-positiv r in th orsl olumn o th spinl or in n ontrol mi. ( n ) Vlus rprsnt mn ± SEM (: or, n = ; tr, n = ; ontrol: or, n = ; tr, n = 7). (g) Nissl-stin spinl or stions showing th lsion prou y NMDA injtion in n ontrol mi. (h) Th grph shows th lngth o th lsion inu y NMDA injtion in n ontrol mi. Vlus rprsnt mn ± SEM (n = ). P <.1 using rpt msurs nlysis o vrin. Sl r in,, µm; g, µm.
3 Ntur Miin oi:1.138/nm.93 CD1 GFAP 1 wk wks wks Lngth o -positiv mirovssl (µm),,, 3,, 1, 1 wk wks wks * * CD1 CD1 Ki7 Supplmntry Fig. 3. Vsulr ormtion inu y trgt. () Rprsnttiv ross-stions o th thori spinl or immunostin or CD1 (ll with Alx Fluor 8) n GFAP (ll with Alx Fluor 88) 7 ys tr inution. () Rprsnttiv ross-stions o th thori spinl or immunostin or rom ontrol n mi t th init prio or (ontrol) n tr inution. () Quntittiv nlysis o th totl lngth o -positiv loo vssls pr stion. Vlus rprsnt s mn ± SEM (n = or h groups). P <.1, * P <. using on-wy nlysis o vrin ollow y Sh s tst. () Rprsnttiv ross-stions o th spinl or immunostin or CD1 (ll with Alx Fluor8) n (ll with Alx Fluor 88) rom mi 7 ys tr inution. Uppr n lowr pnls, high- n low-mgniition imgs, rsptivly. () Immunostining or CD1 (ll with Alx Fluor 88) n Ki7 (ll with Alx Fluor8) in th trgt spinl or. Sl rs:, µm;, 1 µm;, uppr, 1 µm; lowr, µm;, 1 µm.
4 Ntur Miin oi:1.138/nm.93 1 IP rptor IP rptor 1 IP rptor α-tuulin α-tuulin Rltiv intnsity o xprssion Rltiv intnsity o IP rptor protin IP rptor 1 IP rptor Supplmntry Fig.. Knokown o IP rptor n in vitro. () Uppr pnls: Wstrn lots showing xprssion o n α-tuulin in ultur rin nothlil lls trnst with th init s. Th grph shows th rltiv xprssion o. Vlus rprsnt s mn ± SEM (n = 3). () Uppr pnls: Wstrn lots showing xprssion o IP rptor n α-tuulin in ultur ortil nurons trnst with th init s. Th grph shows th rltiv xprssion o th IP rptor. Vlus rprsnt s mn ± SEM (n = 3). (, ) P <.1 y Tuky s tst.
5 Ntur Miin oi:1.138/nm.93 NuN IP rptor IP rptor 1, 8 NuN NuN GFAP GFAP α-tuulin Rltiv intnsity o protin xprssion Rltiv intnsity o xprssion Rltiv intnsity o xprssion IP rptor -kto-pgf1α (pg/mg protin) GFAP 1, 1, CD1 COX-... Supplmntry Fig.. Exprssion o IP rptor n in th CNS. () Doul immunostining or th ttion o IP rptor (ll with Alx Fluor 88) in omintion with NuN, GFAP, n PDGF rptor α (ll with Alx Fluor 8) in th motor ortx rom ontrol mi. () Quntiition o th proution o -kto PGF1α in th thori spinl or rom ontrol or mi (on wk tr inution). Vlus rprsnt s mn ± SEM (n = 3). () Rprsnttiv ross-stions o th thori spinl or o-immunostin or (ll with Alx Fluor 88) in omintion with NuN, GFAP,, or PDGF rptor α (ll with Alx Fluor 8) rom ontrol n mi (on wk tr inution). () Th grphs show th rltiv xprssion o in + lls (uppr grph) n PDGF rptor α+ lls (lowr grph). ; 1 wk tr inution, n = 3 3 lls or h mous n mi or h group. * P <.1 using Stunt s t- tst. () Lt pnls: Wstrn lots showing xprssion o n α-tuulin in vsulr nothlil lls purii rom th rin n spinl or o ontrol n mi (1 wk tr inution). Th grph shows th rltiv xprssion o. Vlus rprsnt mn ± SEM (n = 3). () Th stions o th spinl or immunostin or CD1 (ll with Alx Fluor 8) n COX- (ll with Alx Fluor 88). Sl rs in n, µm or low- n 1 µm or high-mgniition imgs; sl r in, 1 µm.
6 Alx Fluor ll IP rptor- IP rptor.8. IP rptor IP rptor IP rptor 1 IP rptor 1 IP rptor IP rptor 1 g wks IP rptor 1 PKCγ IP rptor IP rptor 1 i.1 Mil.1. IP rptor 1 wks Numr o ll irs pr stion h 1 wks Numr o ll irs pr stion wks Rltiv intnsity o IP rptor protin IP rptor 1. IP rptor Rltiv intnsity o IP rptor protin 1. PKCγ r (mm) Rltiv intnsity o IP rptor protin DAPI IP rptor Alx IP rptor 1 α-tuulin IP rptor IP rptor 1 Ntur Miin oi:1.138/nm Ltrl. IP rptor 1 wks Supplmntry Fig.. Knokown o IP rptor n in vivo. () Rprsnttiv stion o rrl ortx stin with DAPI (lu) n Alx Flour -ll IP rptor s; mgnt) 7 ys tr in vivo trnstion. () Wstrn lots showing xprssion o th init protins in th motor ortx 8 ys tr in vivo trnstion. Th grph shows th rltiv xprssion o th IP rptor in th hinlim motor ortx. ( ) Immunohistohmil nlysis showing ttnution o IP rptor immunortivity in IP rptor -trnst lls in th motor ortx 1 ys tr in vivo trnstion () n th -ll CST irs in th thori spinl or rom trgt mi (). Vlus rprsnt s mn ± SEM (som: ontrol, n = ;, n = 1; CST ir: ontrol, n = 1;, n = 9). () High-mgniition imgs o Fig. 3. () Rprsnttiv PKCγ immunostining o ross-stions o th thori spinl or rom ontrol n mi trnst with or without ontrol or IP rptor s. (g) Th grph shows th PKCγ-positiv r in th orsl olumn o th thori spinl or rom h group o mi. Th tissus wr xmin t 1 ys tr inution. Vlus rprsnt mn ± SEM (n = 3 or h group). (h) Rprsnttiv ross-stions o th rvil spinl or showing ll hinlim CST irs (, ll with Alx Fluor 88). (i) Quntittiv nlysis o hinlim CST ir innrvtion in th rvil spinl or 1 ys tr inution. Vlus rprsnt mn ± SEM (n = or h groups). P <.1 ompr with ontrol y on-wy nlysis o vrin ollow y Tuky s tst or n i, n y Stunt s t tst or n. Sl r in, h (low mgniition), µm;,, 1 µm;,, h (high mgniition), 1 µm.
7 Ntur Miin oi:1.138/nm.93 Rltiv luorsn intnsity o xprssion Rltiv luorsn intnsity o xprssion Alx-ll oligonuloti Lngth o -positiv mirovssl (µm),,, 3,, 1, +iloprost Supplmntry Fig. 7. Moultion o prostylin h no t on th vsulr ormtion inu y trgt. () Rprsnttiv stions o th spinl or oul-ll or (ll with Alx Fluor 88) in omintion with (lt-si pnls) or PDGF rptor ntiois (right-si pnls) (ll with Alx Fluor 8). () Th grphs show th rltiv xprssion o in + lls (lt) n PDGF rptor α+ lls (right). Vlus rprsnt mn ± SEM (n = ). P <.1 y on-wy nlysis o vrin ollow y Stunt s t tst. () Rprsnttiv stions o spinl or immunostin with PDGF rptor α ntioy (ll with Alx Fluor 88) n Alx Flour -ll oligonuloti (r) 7 ys tr in vivo trnstion. () High-mgniition imgs o Fig.. () Rprsnttiv ross-stions o th thori spinl or rom th trgt mi with th init trtmnt immunostin or t 1 ys tr inution. () Quntiition o th totl lngth o + loo vssls pr stion. Vlus rprsnt mn ± SEM (n = 3). Sl r in,, 1 µm;,, 1 µm.
8 Ntur Miin oi:1.138/nm IP rptor 11 CD11 high Lngth o -positiv mirovssl (µm). Numr o CD11+ lls pr stion 1 Numr o CD+ lls pr stion Mylin r (mm). CD11 low APP CD low CD high 11 Fluoromylin 11,,, 3,, 1, 11 Supplmntry Fig. 8. Moultion o prostylin h no t on mylintion or immun rtions in th trgt mi. () High-mgniition imgs o Fig.. () Doul immunostining or th ttion o th IP rptor (ll with Alx Fluor 88) in omintion with PDGF α rptor (ll with Alx Fluor 8) in th spinl or rom th ontrol mi. () Rprsnttiv ross-stions o th spinl or rom th trgt mi with th init trtmnt immunostin or myloi prursor protin (APP) (ll with Alx Fluor 8; or tting mg xons) n luoromylin t 1 ys tr inution, n or CD (ll with Alx Fluor 88) n CD11 (ll with Alx Fluor 88) 7 ys tr inution. Th stions wr otin 1 sgmnt ov th lsion pintr. Low n high, low- n high-mgniition imgs o CD, rsptivly. () Quntiition o th luoromylin-positiv r n th numr o CD- n CD11-positiv lls pr stion in th spinl or t 1 sgmnt ov th lsion pintr rom th trgt mi with th init trtmnt. Vlus rprsnt mn ± SEM (n = or h group). () Rprsnttiv ross-stions o th spinl or immunostin or rom th trgt mi with th init trtmnt. () Th grph shows th totl lngth o -positiv loo vssls pr stion. Vlus rprsnt mn ± SEM (n = or h group). Sl r in,,, 1 µm; in, µm or CD high, µm or APP, 1 µm or othrs.
9 Ntur Miin oi:1.138/nm.93 1, 8 Rtio o BrU inorportion MBP MIP-1α (pg mg -1 ) 11 8 RANTES (pg mg -1 ) IFN-γ (ng ml -1 ) IL- (pg ml -1 ) IL-17 (pg ml -1 ) IL- (pg ml -1 ) IL- (pg ml -1 ) MCP-1 (pg ml -1 ) MIP-1α (pg ml -1 ) RANTES (pg ml -1 ) Supplmntry Fig. 9. Th ts o prostylin moultors on ytokin proution n prolirtiv rsponss. () Th grph shows th rtio o BrU inorportion into spln lls tr stimultion with iloprost or 11. Spln lls wr otin rom th trgt mi, rstimult with MBP or unstimult (ontrol), n susquntly trt with th init gnts. Vlus rprsnt mn ± SEM (n = 3 or h group). * P <. using on-wy nlysis o vrin ollow y Tuky s tst. () Quntittiv nlyss o th ytokins in th thori spinl or o mi with th init trtmnt 7 ys tr inution. Vlus rprsnt mn ± SEM (n = or h group). () Quntittiv nlyss o ytokin proution rom spln lls o mi with th init trtmnt 7 ys tr inution. Vlus rprsnt mn ± SEM (n = 3 or h group).
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