Combined transplantation of GDAs BMP and hr-decorin in spinal cord contusion repair****

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1 NEURAL REGENERATION RESEARCH Volume 8, Issue 24, August doi: /j.issn [ Wu L, Li JJ, Chen L, Zhng H, Yun L, Dvies SJ. Combined trnsplnttion of GDAs BMP nd hr-decorin in spinl cord contusion repir. Neurl Regen Res. 2013;8(24): Combined trnsplnttion of GDAs BMP nd hr-decorin in spinl cord contusion repir**** Ling Wu 1, 2, 3, Jinjun Li 1, 2, Ling Chen 1, 2, Hong Zhng 1, Li Yun 1, 2, Stephen JA Dvies 4 1 School of Rehbilittion Medicine, Cpitl Medicl University, Beijing , Chin 2 Deprtment of Neurl Functionl Reconstruction of Spine nd Spinl Cord, Chin Rehbilittion Reserch Center, Beijing , Chin 3 Rehbilittion Center, Beijing Xiotngshn Rehbilittion Hospitl, Beijing , Chin 4 Deprtment of Neurosurgery, University of Colordo Denver, th Street Denver, Colordo 80217, USA Reserch Highlights (1) This study first verified tht combined bone morphogenetic protein-4 nd humn recombinnt decorin trnsplnttion inhibited erly inflmmtory rections nd protected xons in rts with T 8 spinl cord contusion, inhibited strocyte prolifertion nd glil scr formtion, nd promoted xonl regenertion nd growth, ll of which contributed to the recovery of motor nd sensory functions below the level of spinl cord contusion. (2) This combined trnsplnttion provides potentil new therpy for experimentl reserch nd clinicl trnsformtion for the repir of spinl cord injury. Abstrct Following spinl cord injury, strocyte prolifertion nd scr formtion re the min fctors inhibiting the regenertion nd growth of spinl cord xons. Recombinnt decorin suppresses inflmmtory rections, inhibits glil scr formtion, nd promotes xonl growth. Rt models of T 8 spinl cord contusion were creted with the NYU impctor nd these models were subjected to combined trnsplnttion of bone morphogenetic protein-4-induced glil-restricted precursor-derived strocytes nd humn recombinnt decorin trnsplnttion. At 28 dys fter spinl cord contusion, double-immunofluorescent histochemistry reveled tht combined trnsplnttion inhibited the erly inflmmtory response in injured rts. Furthermore, brin-derived neurotrophic fctor, which ws secreted by trnsplnted cells, protected injured xons. The combined trnsplnttion promoted xonl regenertion nd growth of injured motor nd sensory neurons by inhibiting strocyte prolifertion nd glil scr formtion, with strocytes forming liner rrngement in the contused spinl cord, thus providing xonl regenertion chnnels. Key Words neurl regenertion; spinl cord injury; strocytes; glil scr; neurl stem cells; combined trnsplnttion; glil progenitor cells; glil cells; humn recombinnt decorin; brin-derived growth fctor; glil fibrillry cidic protein; grnts-supported pper; neuroregenertion Ling Wu, Ph.D., M.D. Corresponding uthor: Jinjun Li, Mster, Professor, Chief physicin, School of Rehbilittion Medicine, Cpitl Medicl University, Beijing , Chin; Deprtment of Neurl Functionl Reconstruction of Spine nd Spinl Cord, Chin Rehbilittion Reserch Center, Beijing , Chin, lcrrc2007@ yhoo.com.cn; Stephen JA Dvies, Ph.D., Associte professor, Deprtment of Neurosurgery, University of Colordo Denver, th Street Denver, Colordo 80217, USA, sdvies@bcm.edu. Received: Accepted: (N ) Acknowledgments: We would like to thnk the stff in the Acdemy of Militry Medicl Sciences, nd Beijing Institute of Neuroscience, Cpitl Medicl University in Chin for technicl support. Funding: This work ws supported by funding from the Ministry of Finnce People s Republic of Chin, nd Chin Rehbilittion Reserch Center Reserch Progrm grnts, No , , ,

2 Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): Author contributions: Li JJ prticipted in study design, experimentl supervision nd finncil support. Dvies SJ prticipted in study design nd technicl support. Wu L prticipted in study design, technicl support, dt cquisition, dt nlysis nd pper drfting. Chen L nd Yun L prticipted in the study. Zhng H prticipted in the collection nd trnsltion of literture. All uthors pproved the finl version of the pper. Conflicts of interest: None declred. Ethicl pprovl: The study ws pproved by the Ethics Committee of Rehbilittion Medicine, Cpitl Medicl University, Chin. Author sttements: The mnuscript is originl, hs not been submitted to or is not under considertion by nother publiction, hs not been previously published in ny lnguge or ny form, including electronic, nd contins no disclosure of confidentil informtion or uthorship/ptent ppliction/funding source disputtions. INTRODUCTION After centrl nervous system dmge, injured xons re unble to produce functionl connections becuse xons cnnot effectively regenerte nd n inhibitory microenvironment is formed [1]. A vriety of cells hve been exmined for trnsplnttion fter spinl cord injury, such s humn umbilicl cord blood stem cells [2-4], mrrow stroml cells [5-6], Schwnn cells [7-8], strocytes [9-10], olfctory enshething gli [11-14], nd oligodendrocyte progenitor cells [15-18]. Becuse of structurl dmge nd scr formtion in the white mtter of the spinl cord [19-25], s well s xonl growth inhibitors present in the glil scr tissue [26-28] nd myelin of the centrl nervous system [29-31], insufficient endogenous regenerted xons trversed the lesion site fter vrious cell trnsplnttion into or round the injured spinl cord [32-34]. After spinl cord injury, ctive strocytes cn induce migrtion of nerve cells. Mture strocytes minly mintin nerve morphology, but they lso ply n importnt role in providing nerve growth fctor nd communiction between cells [35]. However, mture strocytes lso ffect nerve regenertion nd hinder xonl extension through the secretion of hrmful fctors tht form chemicl glil brriers [36]. Therefore, it is very importnt to fvorbly regulte strocyte expression fter spinl cord injury. Glil-restricted precursor cells from the embryonic spinl cord, which re the precursors of oligodendrocytes nd strocytes [37-39], cn improve the microenvironment, regulte strocyte expression, inhibit glil scr formtion nd dely the expression of proteoglycns. Glil-restricted precursor-deri- ved strocytes induced by bone morphogenetic protein (BMP)-4 (GDAs BMP ) not only hve common chrcteristics of glil-restricted precursor cells, but cn lso fill the injured interfce, rerrnge tissues, protect injured xons, nd promote xonl regenertion nd functionl recovery fter cute spinl cord injury [40]. Decorin decreses fibrosis, s the core protein of decorin, cn inhibit collgen fiber prolifertion nd neutrlize trnsforming growth fctor-β [40-42]. Decorin within the nervous system not only regultes the extrcellulr collgen mtrix, it lso promotes xon fibronectin dhesion, Schwnn cell prolifertion nd neurl cell survivl [43]. Humn recombinnt decorin (hr-decorin) hs been shown to inhibit inflmmtion, glil scr formtion nd chondroitin sulfte proteoglycn expression, nd my promote xonl growth cross the injured interfce fter cute spinl cord injury [44-46]. After spinl cord injury, strocytes become ctivted nd begin to proliferte. It is very difficult to effectively control the prolifertion of strocytes nd inhibit the formtion of the glil scr through single method. To provide fvorble microenvironment for spinl cord regenertion, the sttionry, ctivted nd prolifertive sttes of the strocytes should be considered comprehensively fter spinl cord injury. In this experiment, rts with T 8 spinl cord contusion were subjected to GDAs BMP nd hr-decorin trnsplnttion. The response of trnsection model of spinl cord injury could not objectively mimic clinicl pthologicl chnges of spinl cord injury s spinl cord contusion ccounts for the vst mjority of clinicl cses. Hr-decorin inhibited strocyte prolifertion nd glil scr formtion, nd even degrded preformed glil scr. GDAs BMP trnsplnttion to contusive spinl cord promoted strocyte to form liner rrngement tht provided xonl growth chnnels, which promoted xonl connectivity nd recovery of sensory nd motor functions below the level of spinl cord contusion. To confirm tht combined GDAs BMP nd hr-decorin trnsplnttion could effectively promote the recovery of sensory nd motor functions below the level of spinl cord contusion, this study observed nd nlyzed the size of the cvity, the degree of spinl cord trophy, the expression of strocytes, nd the extent of motor nd sensory xonl regenertion in rts with T 8 spinl cord contusion fter tretment with GDAs BMP nd hr-decorin trnsplnttion. 2237

3 Men ngle (degree) Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): RESULTS Quntittive nlysis of experimentl nimls Before T 8 spinl cord contusion, 72 femle Sprgue-Dwley rts were rndomly divided into hr-decorin injection group, GDAs BMP trnsplnttion group, combined GDAs BMP nd hr-decorin trnsplnttion group, nd spinl cord injury group (ech group n = 18). The spinl cord injury group served s control nd did not receive ny tretment. According to the time fter spinl cord contusion, ech group ws rndomly divided into 3-, 7- nd 28-dy subgroups (ech subgroup n = 6). Five rts died of nesthetic ccident nd seven rts died of excessive bleeding during surgery. New rts were used to supplement the ded rts. Seventy-two rts were included in the finl nlysis. Effects of combined GDAs BMP nd hr-decorin trnsplnttion on the ngles between strocytelined xons in rts with spinl cord contusion The ngles between djcent glil fibrillry cidic proteinpositive strocyte-lined xons were mesured with Imge Pro Plus 6.0 softwre t 28 dys fter spinl cord contusion, nd the extent of liner rrngement ws estimted by compring the ngles (0 90 ; the smller the degree, the more liner the rrngement). The ngles in the hr-decorin tretment group nd spinl cord injury group were fr bigger thn the GDAs BMP tretment group nd combined GDAs BMP nd hr-decorin trnsplnttion group (P < 0.05; Figure 1), indicting tht GDAs BMP trnsplnttion lone or combined trnsplnttion could effectively promote liner rrngement of strocyte-lined xons but spinl injection of hr-decorin lone hd no such effect. Effect of combined GDAs BMP nd hr-decorin trnsplnttion on the cvity size nd crosssectionl re in the contusive spinl cord of rts The cvities nd cross-sectionl re of spinl cord in glil fibrillry cidic protein-positive strocyte fluorescence imges (Figure 2) were mesured nd nlyzed with Imge Pro Plus 6.0 softwre. At 3 dys fter spinl cord contusion, hr-decorin injection or GDAs BMP trnsplnttion lone inhibited strocyte prolifertion, resulting in lrge cvities in the spinl cord center. GDAs BMP lone or combined trnsplnttion prtilly filled the injured spinl cord, so the cross-sectionl res of spinl cord were reltively lrger thn the other two groups. Seven dys fter tretment, hr-decorin significntly inhibited strocyte prolifertion (P < 0.05), resulting in the enlrged cvities in the hr-decorin injection group nd thin spinl cord in the combined trnsplnttion group. Twenty-eight dys lter, the cvities were obviously reduced in the GDAs BMP lone or combined trnsplnttion groups, but were further enlrged in the spinl cord injury group (Figures 3 5) Blnk Hr-decorin GDAs BMP GDAs BMP +Hr-decorin Figure 1 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on the ngles between the strocyte-lined xons in the contusive spinl cord of rts t 28 dys fter injury. The ngles between djcent glil fibrillry cidic protein-positive strocyte-lined xons were mesured with Imge Pro Plus 6.0 softwre. Smll ngles indicte tendency for liner rrngement. P < 0.05, vs. blnk group nd hr-decorin injection group. Dt re expressed s men ± SD of six rts for ech group (two-smple t-test nd one-wy nlysis of vrince). Blnk: Spinl cord injury group; Hr-decorin: humn recombinnt decorin injection group; GDAs BMP : glil-restricted precursorderived strocytes induced by bone morphogenetic protein-4 trnsplnttion group; GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. Glil scr formtion in the dmged region of rts undergoing combined GDAs BMP nd hr-decorin trnsplnttion t 28 dys fter spinl cord contusion The integrted bsorbnce vlues (re verge bsorbnce) of glil fibrillry cidic protein-positive strocytes in the spinl cord injury group were significntly higher thn the three tretment groups (P < 0.05). Hr-decorin inhibition of strocyte prolifertion nd glil scr formtion ws slightly stronger thn in the GDAs BMP trnsplnttion group, nd combined trnsplnttion enhnced this effect but it ws not significnt (P > 0.05) between the three tretment groups (Figure 6). Effect of combined GDAs BMP nd hr-decorin trnsplnttion on sensory nd motor xons within contusive spinl cord of rts t 28 dys fter spinl cord contusion A rnge of injured xons spred to 1 mm wy from the injury center of the spinl cord. The injured xon terminls in the spinl cord injury group showed thinning, neurom formtion, bifurction, germintion, swelling, 2238

4 Cvity re (pixel, 10 4 ) Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): winding or disorgnized growth within the injured interfce t 28 dys fter spinl cord contusion (Figure 2). The growth bility of sensory nd motor xons ws reduced, nd pssing rtes of ll xons in ech point were significntly lower in the spinl cord injury group thn the three tretment groups (P < 0.05). Hr-decorin injection, GDAs BMP trnsplnttion lone, or combined trnsplnttion could effectively promote regenertion nd growth of sensory nd motor xons; nerly hlf of the sensory xons nd some of the motor xons pssed through the injury center; smll prt of the sensory xons nd motor xons reched rnge of 5 mm from the injury center (Figures 7 9). dys lter, Bsso-Bettie-Bresnhn scores of ll rts were no more thn 2 points, indicting tht there ws no significnt recovery of hindlimb motor function. However, 7 dys lter, Bsso-Bettie-Bresnhn scores in the tretment groups were significntly higher thn the spinl cord injury group (P < 0.05). Twenty-eight dys lter, the recovery level of hindlimb motor function in the tretment groups incresed significntly, especilly in the combined GDAs BMP nd hr-decorin trnsplnttion group (pproching 17 points; P < 0.05), while Bsso-Bettie- Bresnhn scores in the spinl cord injury group were round 10 points (Figure 10). Blnk Hr-decorin GDAs BMP GDAs BMP +Hr-decorin b 0 3 d 7 d 28 d Figure 2 Expression of strocytes, cvities nd chnge of xonl terminl morphology in the center of the injured spinl cord t 28 dys fter injury. Cross-sections (A D) under fluorescence microscope ( 50) show the presence of strocytes (fluoresceine isothiocynte, green) nd cvities in the center of the injured spinl cord: 28 dys fter spinl cord contusion, the holes were further enlrged in the blnk group, but were obviously reduced in the GDAs BMP lone or combined trnsplnttion groups. (A D) Scle br: 2 mm. Longitudinl sections (scle br: 125 μm; E) under fluorescence microscope ( 400) showed xons (red) (vidin-cy3 fluorescence stining fter biotinylted dextrn mine xonl trcing) terminl morphology in the blnk group: shows xonl terminl neurom formtion; b shows thinner xonl terminl; c shows xonl bifurction nd germintion; d shows thicker xonl terminl; e shows xon whipping growth within the injured interfce; f shows xonl flexion growth nd terminl neurom formtion. GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. Effects of combined GDAs BMP nd hr-decorin trnsplnttion on hindlimb motor function in rts with spinl cord contusion At 1 dy fter spinl cord contusion, the Bsso-Bettie- Bresnhn scores of rts in ech group were 0. Three Figure 3 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on the cvity re in the contusive spinl cord of rts. The cvity re of the spinl cord in the glil fibrillry cidic protein-positive strocyte fluorescence imge ws mesured nd nlyzed with Imge Pro Plus 6.0 softwre, nd clculted by pixels. The cvities of the blnk group were smllest t 3 nd 7 dys, but lrgest t 28 dys fter spinl cord injury ( P < 0.05, vs. blnk group t 3 nd 7 dys). The hr-decorin injection group nd combined GDAs BMP nd hr-decorin trnsplnttion group hd decresing trend, but there ws little chnge. The GDAs BMP trnsplnttion group decresed significntly t 28 dys fter spinl cord injury compred with the other groups t the sme time ( b P < 0.05). Dt re expressed s men ± SD of six rts for ech group (two-smple t-test nd one-wy nlysis of vrince). GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. DISCUSSION GDAs BMP trnsplnttion fter spinl cord contusion effectively filled the cvity in contusive spinl cord Glil-restricted precursor cells cn be directly isolted from embryonic spinl cord [47], nd neurl stem cells nd embryonic stem cells [48-49] cn lso be seprtely 2239

5 Cvity re/spinl re (%) Spinl re (pixel, 10 5 ) Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): isolted from the centrl nervous system of mice nd humns [40, 50-54]. Blnk Hr-decorin GDAs BMP GDAs BMP +Hr-decorin d 7 d 28 d Figure 4 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on the cross-sectionl re in the contused spinl cord of rts. The cross-sectionl re of the spinl cord in the glil fibrillry cidic protein-positive strocyte fluorescence imge ws mesured nd nlyzed with Imge Pro Plus 6.0 softwre, nd clculted by pixels. The cross-sectionl re of combined GDAs BMP nd hr-decorin trnsplnttion group ws lest t 7 nd 28 dys ( P < 0.05, vs. the other groups t the sme time); the cross-sectionl re of hr-decorin group nd GDAs BMP trnsplnttion group ws lwys lrger thn the blnk group ( b P < 0.05). Dt re expressed s men ± SD of six rts for ech group (two-smple t-test nd one-wy nlysis of vrince). GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. b b After severe spinl cord contusion, lrge cvity grdully forms in the injury center tht seriously ffects regenertion nd growth. Smll cvities re initilly formed becuse of n erly serious inflmmtory response, intrmedullry hemorrhge, spinl cord edem, nd glil cell ctivtion nd prolifertion fter severe spinl cord contusion. However, in the lter stges, huge cvities re grdully formed in the spinl cord injury center due to gry nd white mtter necrosis; lrge glil scr forms surrounding the cvity; most xonl pths re disconnected; nd very smll number of motor xons cross the injury center. Mny methods hve been investigted for filling these cvities, but little success hs been chieved. After tretment with GDAs BMP from glil-restricted precursor cells induced with bone morphogenetic protein-4 [40, 55-57], lthough cvities formed in the first 7 dys fter spinl cord contusion, GDAs BMP inhibited strocyte prolifertion nd the inflmmtory response, nd drsticlly reduced cvity size fter dy 7, through the prolifertion of GDAs BMP nd strocytes, nd regenertion of nerve tissue. At 3 dys fter spinl cord contusion, the bility of hr-decorin to inhibit strocyte prolifertion nd the inflmmtory response reduced, resulting in obvious spinl cord edem. Subsequently, the spinl cord becme tpered s cute inflmmtion subsided. Following this, the cross-sectionl re of the spinl cord grdully recovered becuse most of the cvities were filled. The cvities decresed nd the recovery of cross-sectionl re of spinl cord provided crrier for xonl regenertion nd growth. Blnk Hr-decorin GDAs BMP GDAs BMP +Hr-decorin d 7 d 28 d Figure 5 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on the rtio between the cvity nd cross-sectionl res in the contusive spinl cord of rts. The cvity nd cross-sectionl res of spinl cord in the glil fibrillry cidic protein-positive strocyte fluorescence imge were mesured nd nlyzed with Imge Pro Plus 6.0 softwre, nd clculted by pixels. The rtio between the cvity re nd cross-sectionl re of the blnk group grdully incresed to the mximum compred with the tretment groups t 28 dys fter spinl cord contusion ( P < 0.05), while the hr-decorin tretment group nd the GDAs BMP trnsplnttion group slowly decresed. The combined GDAs BMP nd hr-decorin trnsplnttion group incresed nd then decresed compred with the blnk nd the GDAs BMP trnsplnttion groups t 28 dys fter spinl cord contusion ( b P < 0.05). Dt re expressed s men ± SD of six rts for ech group (two-smple t-test nd one-wy nlysis of vrince). GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. Formtion of strocyte liner rrngement nd reduction in glil scr The formtion of liner rrngement of strocytes in the contusive spinl cord creted the shortest pth for xonl growth, which enhnced the efficiency of xons pssing through the injury interfce [40]. After severe spinl cord contusion, serious glil scr, chrcterized by n incresed number nd density of strocytes, is formed within the injury interfce. This blocks xonl growth, resulting in xon terminl trophy, neurom formtion, nd buckling or winding growth. Although b b 2240

6 Integrted bsorbnce vlue ( 10 7 ) Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): mny methods hve been exmined previously, removl of the glil scr remins difficult issue Blnk Hr-decorin GDAs BMP GDAs BMP +Hr-decorin GDAs BMP produce brin-derived neurotrophic fctor, which cn promote survivl of neurons in the contusive spinl cord nd prevent xonl trophy [40, 58]. GDAs BMP cn lso dely the expression of chondroitin sulfte proteoglycns, thereby indirectly promoting xonl regenertion [40]. At 28 dys fter GDAs BMP trnsplnttion, number of motor xons crossed the injury center nd reched the distl end of the spinl cord by growing long nerve chnnels estblished by strocytes. The sensory xons in the spinl cord surfce were incresed, nd moved round the cvity or through the injury center to rech the distl end of the spinl cord. In ddition, GDAs BMP my lso hve promoted the growth of other motor-relted xons, such s the reticulr trct nd lterl corticospinl trct. Figure 6 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on glil fibrillry cidic protein-positive strocytes within the contusive spinl cord of rts t 28 dys fter injury. Integrted bsorbnce vlues (re verge bsorbnce) of glil fibrillry cidic protein-positive strocytes in immunofluorescence photogrphs were mesured with Imge Pro Plus 6.0 softwre. P < 0.05, vs. blnk group. Dt re expressed s men ± SD of six rts for ech group (two-smple t-test nd one-wy nlysis of vrince). GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. A B Blnk Hr-decorin GDAs BMP GDAs BMP + Hr-decorin Blnk Hr-decorin GDAs BMP Thus, numerous studies hve chnged strtegy to investigte mechnisms of preventing the formtion of the glil scr in the erly stges of spinl cord injury. After GDAs BMP trnsplnttion, the ngle of strocyte-lined xons significntly reduced, which denoted tht the glil scr ws significntly reduced nd strocyte-lined xons were evenly distributed. The results showed tht GDAs BMP trnsplnted into contusive spinl cord could effectively promote the liner rrngement of strocyte-lined xons, while tretment with PBS or hr-decorin injection hd no such effect. Promotion of xonl regenertion nd growth GDAs BMP derived from glil-restricted precursor cells re prticulr type of strocyte possessing numerous functions, such s support, nutrition, nd signl contct. After GDAs BMP trnsplnttion into the spinl cord, the vst mjority of the cvities were filled by strocytes nd GDAs BMP promoted the liner rrngement of strocyte-lined xons, which provided chnnels for xonl regenertion nd growth in the host. GDAs BMP + Hr-decorin Figure 7 Pssing rtes of sensory (A) nd motor (B) xons t different points (1.5 mm nd 1 mm in front of the injury center [ 1.5 mm, 1 mm], the injury center nd 1 mm, 1.5 mm nd 5 mm posterior to the injury center [1.5 mm, 5 mm]) t 28 dys fter spinl cord contusion in ech group. The pssing rtes of sensory xons (t 1.5 mm nd 5 mm posterior to the injury center) nd motor xons (t 1, 1.5, 5 mm posterior to the injury center) in ll tretment groups were higher thn the blnk group (P < 0.05); the pssing rtes of sensory nd motor xons in the combined trnsplnttion group were significntly higher thn the other groups (P < 0.05). Dt re expressed s men ± SD of six rts for ech group (two-smple t-test nd one-wy nlysis of vrince). GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. Hr-decorin injection fter spinl cord contusion effectively inhibited glil scr formtion nd promoted glil scr degrdtion Decorin is smll leucine-rich chondroitin sul- 2241

7 0 Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): fte/dermtn sulfte proteoglycn, which is very rich in the vsculr border zone, the brin surfce, the ependym nd mesh tissue, s well s being expressed in the centrl nervous system fter injury. Hr-decorin cn promote endothelil cell formtion of blood vessels in vitro nd the genertion of cpillries in vivo, especilly during the inflmmtory stge. The degree of spinl cord blood stsis ws significntly reduced t 7 dys nd disppered t 28 dys fter hr-decorin injection by respectively compring gross specimens of contusive spinl cord in rts, indicting tht hr-decorin promoted the bsorption of stgnnt blood in the injured spinl cord. Hr-decorin hs lso been shown to inhibit inflmmtory fctors (trnsforming growth fctor-β) nd reduce the expression of I, III procollgen mrna in cells, so it cn effectively inhibit scr fibroblst prolifertion. Blnk 28 d Hr-decorin 28 d GDAs BMP 28 d GFAP+ GFAP+ GFAP+ BDA BDA BDA Hr-decorin+GDAs BMP 28 d GFAP+ BDA D1 GFAP+ BDA D2 Figure 8 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on sensory xonl regenertion in the contusive dorsl spinl cord of rts t 28 dys fter injury. Fluorescence microscopy (A1, B1, C1, 200) nd confocl fluorescence microscopy (A2, B2, C2, D1, D2, 200) show the expression of strocytes (fluoresceine isothiocynte, green) nd sensory xons (Cy3, red) pssing through the injury center nd interfce t 28 dys fter spinl cord contusion. Longitudinl sections show tht the sensory xonl pthwy ws dmged severely in the blnk group nd the Hr-decorin injection group, while the dmge ws less in the GDAs BMP trnsplnttion group nd the combined trnsplnttion group (A1, B1, C1, D1). Cross-sections show tht the number of sensory xons in the three tretment groups ws more thn the blnk group (A2, B2, C2, D2). GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. Scle br: 250 μm. d: Dy. A1 B1 C1 GFAP+ GFAP+ GFAP+ BDA BDA BDA A2 B2 C2 μ m 250 Figure 9 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on motor xonl regenertion within the contusive spinl cord of rts t 28 dys fter injury. Fluorescence microscopy ( 200) showed the presence of strocytes (fluoresceine isothiocynte, green) nd motor xons (CY3, red) pssing through the injury center nd interfce t 28 dys fter spinl cord contusion. Longitudinl sections showed tht the cvities of the spinl cord in the blnk group nd the combined trnsplnttion group were lrger thn the Hr-decorin injection group nd the GDAs BMP trnsplnttion group, but the pssing rte in the injury center in the combined trnsplnttion group ws higher thn the other groups (A1, B1, C1, D1). Cross-sections showed tht the number of the motor xons in the combined trnsplnttion group ws lrgest, with the hr-decorin injection group nd the GDAs BMP trnsplnttion group with lower vlues, nd the blnk group hving the lest (A2, B2, C2, D2). GDAs BMP + Hr-decorin: combined GDAs BMP nd Hr-decorin trnsplnttion group. Scle br: 250 μm. d: Dy. After hr-decorin tretment for 3 dys, the inflmmtory response nd the prolifertion of strocytes were effectively suppressed nd the product of the inflmmtory response ws degrded, which resulted in lrger cvities thn the spinl cord injury group. Following this, hr-decorin promoted plsminogen/plsmin synthesis, which not only inhibited synthesis of chondroitin sulfte proteoglycns, it lso promoted degrdtion of chondroitin sulfte proteoglycns. Hr-decorin injection gin t 14 dys helped to inhibit scr formtion nd lso degrde the glil scr tht hd formed in the erlier stges. Protection of xons nd promotion of xonl regenertion Hr-decorin within the nervous system regultes collgen 2242

8 BBB scores Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): extrcellulr mtrix, nd promotes the fibronectin dhesion of xons, Schwnn cell prolifertion nd neurl cell survivl [43]. Hr-decorin effectively inhibited the erly inflmmtory response nd promoted the formtion of tiny blood vessels, prevented further secondry dmge to injured xons, nd provided better microenvironment for xonl regenertion nd growth. Hr-decorin inhibited strocyte prolifertion (erly) nd degrded the glil scr (lte), resulting in reduction in the inhibition of xonl regenertion nd growth. Hr-decorin could not effectively induce liner rrngement of strocytes within the spinl cord, but could reduce the density of strocytes, llowing xons to extend into the cvity. Hr-decorin promoted the synthesis of plsmin, which ctivted neurotrophic fctors, degrded vriety of xonl growth inhibitors, nd promoted reconstruction of the centrl nervous system [44]. The experimentl results demonstrte tht hr-decorin not only effectively protected the injured spinl cord, it lso promoted xonl regenertion to certin extent Blnk Hr-decorin GDAs BMP GDAs BMP + Hr-decorin 3 d 7 d 14 d 21 d 28 d Figure 10 Effect of combined GDAs BMP nd hr-decorin trnsplnttion on the Bsso-Bettie-Bresnhn (BBB) scores in rts with contusive spinl cord. Motor function of the hindlimbs in rts ws divided into 22 grdes (BBB scores: 0 to 21 points). 0 points ment complete prlysis of hindlimbs nd 21 points ment complete norml motor function of the hindlimbs. Three dys lter, BBB scores of ll rts were no more thn 2 points. At 7, 14, 21, 28 dys fter spinl cord contusion, P < 0.05, vs. blnk group. Dt re expressed s men ± SD of six rts for ech group (two-smple t-test nd one-wy nlysis of vrince). GDAs BMP + Hr-decorin: combined GDAs BMP nd hr-decorin trnsplnttion group. Combined GDAs BMP nd hr-decorin trnsplnttion fter spinl cord contusion more effectively inhibited the inflmmtory response nd reduced secondry xonl injury Three dys fter spinl cord contusion, hr-decorin nd GDAs BMP trnsplnttion inhibited the inflmmtory response nd my hve decresed strocyte prolifertion, with no obvious thinning of the spinl cord; the remining spinl cord tissue significntly incresed s trnsplnted GDAs BMP filled the cvity nd both hr-decorin nd GDAs BMP effectively protected the spinl cord. Seven dys lter, becuse the inflmmtory response nd spinl cord edem decresed long with the inhibition of strocyte prolifertion by hr-decorin nd GDAs BMP tretment, the spinl cord hd obviously thinned. At 28 dys fter spinl cord contusion, the inflmmtory response hd disppered; GDAs BMP nd hr-decorin hd constntly inhibited strocyte prolifertion nd glil scr formtion, while hr-decorin lone lso possibly inhibited GDAs BMP prolifertion. Nevertheless, becuse of GDAs BMP prolifertion, there were no significnt chnges in the res of residul spinl cords nd cvities, nd the spinl cord tended to be stble. Decresed glil scr nd promotion of strocyte liner rrngement Combined GDAs BMP nd hr-decorin trnsplnttion for contusive spinl cord constntly inhibited the erly inflmmtory response nd strocyte prolifertion, which substntilly reduced glil scr formtion. At 28 dys fter spinl cord contusion, tretment with either hr-decorin injection, GDAs BMP trnsplnttion or combined GDAs BMP nd hr-decorin trnsplnttion effectively suppressed strocyte prolifertion nd scr formtion. Although there ws no sttisticlly significnt difference between the tretment groups, the decresing trend of glil scr ppered in the combined trnsplnttion group. The ngles between strocyte-lined xons fter combined trnsplnttion nd GDAs BMP trnsplnttion were smller, suggesting tht both GDAs BMP trnsplnttion lone or combined trnsplnttion effectively promoted the liner rrngement of strocyte-lined xons in the host. Significntly improved bilities of xonl regenertion nd growth The results suggest tht combined trnsplnttion promoted regenertion nd growth of sensory nd motor xons more effectively thn hr-decorin injection or GDAs BMP trnsplnttion. First, combined trnsplnttion further strengthened inhibition of the inflmmtory response, promoted the formtion of spinl cord cpillries nd reduced chondroitin sulfte proteoglycn expression. Second, GDAs BMP promoted the liner rrngement of strocytes to provide chnnels for xonl growth. Third, GDAs BMP secreted brin-derived neurotrophic fctor to protect xons nd neurons from trophy in the erly 2243

9 Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): stges, nd promoted xonl growth in the lter stges. Finlly, hr-decorin nd GDAs BMP provided fvorble microenvironment for xonl regenertion nd growth t n erly stge, which extended the time window for xonl regenertion in the cute stge. Enhnced hindlimb motor function Previous studies demonstrted tht GDAs BMP trnsplnttion t 3 dys fter spinl cord promoted xonl regenertion nd plsticity of xonl connections, nd resulted in recovery of motor function [40]. At 28 dys fter spinl cord contusion, GDAs BMP trnsplnttion or hr-decorin injection significntly promoted recovery of hindlimb motor function in rts. However, the mechnisms of functionl recovery for both tretments were different. The inhibitory effects of hr-decorin on the erly inflmmtory response nd glil scr formtion were stronger thn GDAs BMP, wheres GDAs BMP trnsplnttion provided brin-derived neurotrophic fctor, which in turn prevented xonl nd neuronl trophy. GDAs BMP trnsplnttion induced strocytes to form liner rrngement within the host tht provided growth chnnels for xons pssing through the injury center nd interfces, wheres hr-decorin lone could not chnge strocyte rrngement. Becuse hr-decorin injection nd GDAs BMP trnsplnttion hd different mechnisms for the tretment of spinl cord contusion, combined trnsplnttion ws dopted. Hr-decorin inhibited the inflmmtory response in the erly stges, nd protected xons nd neurons together with GDAs BMP, thus creting better microenvironment for enhncing xonl regenertion nd growth nd significntly promoting the recovery of hindlimb function. Furthermore, the recovery of hindlimb motor function ws more obvious with time in the combined tretment group, indicting tht the speed nd extent of motor xonl regenertion were stronger in the lter stges. Together, these results indicte tht combined trnsplnttion estblished xonl regenertion chnnels, reduced the glil scr nd provided fvorble microenvironment for xonl regenertion nd growth, which obviously enhnced the speed nd degree of motor xon regenertion. MATERIALS AND METHODS Design A rndomized, controlled, niml experiment. Time nd setting The experiment ws performed t the Lbortory of Acdemy of Militry Medicl Sciences, Beijing Institute of Neuroscience, Cpitl Medicl University, Chin from 2009 to Mterils A totl of 72 femle Sprgue-Dwley rts weighing 200 ± 10 g nd one embryonic dy 13.5 rt were obtined from the Acdemy of Militry Medicl Sciences, Beijing, Chin (license No. SCXK (Militry) ). Methods Glil-restricted precursor cell isoltion nd GDAs BMP genertion After intrmusculr injection with 1% pentobrbitl sodium solution (45 mg/kg), spinl cords of embryonic dy 13.5 rt embryos were isolted under n ntomicl microscope [40, 48]. After the embryonic spinl cords were cut into 1 mm 1 mm pieces nd digested with 0.25% trypsin/ethylene dimine tetrcetic cid (Invitrogen, Crlsbd, CA, USA) solution, residul trypsin/ethylene dimine tetrcetic cid solution ws neutrlized by Dulbecco s modified Egle s medium (DMEM; Hyclone, Logn, UT, USA) with stndrd 10% fetl bovine serum (Hyclone Co.). The digested spinl cord tissues were gently pipetted with Psteur pipette, nd the cells were then collected nd diluted to cells/ml. After dding nti-a2b5 (Sigm-Aldrich Co., St. Louis, MO, USA) (0.1 mg/ml) nd phycoerythrin nti-mouse IgM (ebioscience Co., Sn Diego, CA, USA; 1:100) in sequence, A2B5- positive cells ccounted for 33.18% by flow cytometry nlysis, then the primry A2B-positive glil- restricted precursor cells (90.65%) were ultimtely sorted by fluorescence-ctivted cell sorting. The primry glil- restricted precursor cells were mintined on fibronectin/lminin substrte (Sigm-Aldrich) in DMEM/ F12 (Hyclone Co.) Sto-medium with 20 ng/ml rt bsic fibroblst growth fctor (Sigm-Aldrich) in vitro. Culture medi contining DMEM/F12 nd rt bsic fibroblst growth fctor were chnged every 2 dys. After serilly pssging for no more thn three genertions, more glil-restricted precursor cells were obtined. To differentite glil-restricted precursor cells into GDAs BMP (A2B5- negtive/glil fibrillry cidic protein-positive), glil-restricted precursor cells were seeded on fibronectin/ lminin substrte in DMEM/F12 Sto-medium with 20 ng/ml bsic fibroblst growth fctor nd 10 ng/ml of humn recombinnt bone morphogenetic protein-4 (R&D Systems Co., Ltd., Minnepolis, MN, USA) for 7 dys. GDAs BMP were collected by centrifugtion nd diluted by dding n pproprite volume of PBS. Subsequently, the nti-a2b5 nd rbbit nti-rt glil fibrillry cidic protein polyclonl ntibody (Sigm-Aldrich) ws dded. After incubtion for 1 hour in 2244

10 Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): C CO 2 incubtor, the secondry ntibody including phycoerythrin nti-mouse IgM nd got nti-rbbit IgG-fluoresceine isothiocynte (FITC) fluorescent ntibody (Sigm-Aldrich) were dded for 1 hour. A2-positive cells were removed by fluorescence-ctivted cell sorting, nd purified A2B-negtive glil fibrillry cidic protein-positive GDAs BMP were obtined. The purified GDAs BMP were collected by centrifugtion, nd diluted into cell suspensions ( cells/μl) with Dulbecco s modified Egle s medium/f12 for trnsplnttion. Preprtion of T 8 spinl cord contusion model After intrperitonel injection with 1% pentobrbitl sodium (Merck Co., Drmstdt, Hessen, Germny; 35 mg/kg), femle rts (body weight 200 ± 10 g) were fixed on the operting tble in the prone position. The T 8 9 spinous process nd lmin were exposed long the posterior midline of the spine (T 7 10 ), T 8 nd the upper prt of T 9 spinous process nd lminr were crefully removed to fully expose the dur. Subsequently, the T 8 spinl cord ws bruised with n NYU device (New York University, New York, NY, USA; 10 g 50 mm). Contusive spinl cord suffered from swelling nd congestion, nd motor nd sensory function in rts ws lost completely [59]. The Bsso-Bettie-Bresnhn scores of ll rts were 0 the next dy nd no more thn 2 points t 3 dys fter spinl cord contusion, indicting tht the combt strength fully met the requirements of the experiment. Lter, rts with spinl cord contusion were injected with penicillin ( U/kg per dy) for 3 dys, nd urinted mnully t lest twice dy until utonomic micturition reflex ppered. Drug injection or GDAs BMP trnsplnttion The strtegies of drug injection or GDAs BMP trnsplnttion to rt spinl cord were divided into three cross-sections (Figure 11A), two injection points on ech cross-section (Figure 11A, B) nd two levels on ech point (Figure 11A). The rts with T 8 spinl cord contusion were fixed in the prone position under rt brin stereotxic pprtus with micropipe instlled with 100 μm tiny glss needle. According to the design, the needle with drug or GDAs BMP ws slowly injected to 1.5 mm depth from the spinl cord dorsl surfce directly for 1 minute, then withdrwn to 0.5 mm depth for 10 minutes. Tretment of T 8 spinl cord contusion in rts Rts in the hr-decorin injection group, GDAs BMP trnsplnttion group, combined hr-decorin nd GDAs BMP trnsplnttion group nd spinl cord injury group were respectively injected with hr-decorin (R&D Systems Co., Ltd.;1 μg/μl), GDAs BMP suspension ( cells/μl), hrdecorin nd GDAs BMP suspension (the sme concentrtion nd density s bove), nd PBS solution (0.1 mol/l) into the 12 injection sites within the dmged re (ech site 0.5 μl; totl of 6 μl). Two weeks lter, rts in the 28-dy subgroup with hr-decorin injection received intrperitonel injection of 0.5 ml hr-decorin solution (12 μg/ml). The rts undergoing GDAs BMP trnsplnttion received intrmusculr injection of cyclosporine A (Novrtis AG, Bsel, Switzerlnd;10 mg/kg) every dy. A Figure 11 Schemtic illustrtion of PBS injection or humn recombinnt decorin (Hr-decorin) injection or bone morphogenetic protein-4 (GDAs BMP ) trnsplnttion mode. (A) Lterl view of spinl cord: three cross-sections (respectively locted in the midpoint of spinl cord injury center, the surfce bout 1 mm length from the injury center to the rostrl nd the surfce bout 1 mm length from the injury center to the cud), six injection points locted in the dorsl surfce of the spinl cord (two injection points in ech cross-section; respectively 0.5 mm lterl to the spinl cord medin line) nd 12 injection sites within the spinl cord (two injection sites ech injection point; respectively 0.5 mm nd 1.5 mm depth to dorsl surfce) for drug injection or GDAs BMP trnsplnttion. (B) Posterior view of the spinl cord: six injection points locted on both sides of the spinl cord for drug injection or GDAs BMP trnsplnttion. Motor function of rts scored by Bsso-Bettie- Bresnhn scoring At 3, 7, 14, 21 nd 28 dys fter spinl cord contusion, behviorl chnges nd hindlimb motor function of rts were observed to obtin Bsso-Bettie-Bresnhn function scores [60]. These included the number nd scope of hindlimb joint ctivities, lod level of hindlimbs, coordintion of forelimbs nd hindlimbs, clws nd til ctivities. Bsso-Bettie-Bresnhn function scores of rts were divided into 22 grdes (0 to 21 grdes): zero indicted complete prlysis nd 21 indicted norml function. Trcking of biotinylted dextrn mine nterogrde motor or sensory xons The bility of xonl regenertion or growth fter spinl cord contusion ws observed with biotinylted dextrn mine (Moleculr Probes Co., Ltd., Junction, OR, USA) nterogrde motor or sensory xonl trcking. At 2 weeks before rts were executed, 10% biotinylted dex- B 2245

11 Wu L, et l. / Neurl Regenertion Reserch. 2013;8(24): trn mine ws injected into both sides of the cerebrl motor cortex in rts for nterogrde corticospinl trct trcking [61]. At 8 dys before rts were executed, scending endogenous xons were trcked by injecting 10% biotinylted dextrn mine into cunete nd grcile white mtter t the T 10 spinl level in both sides of the spinl cord [40]. For biotinylted dextrn mine nterogrde motor xonl trcking, the rts were fixed under stereotxic pprtus (Stoelting, Chicgo, IL, USA) in the prone position. A totl of 16 holes (eight holes in ech side; dimeter of 0.5 mm) locted in the 16 points were respectively drilled. Biotinylted dextrn mine ws slowly (0.1 μl/min) injected into the 16 points (Figure 12A, 1.0 mm depth to the cerebrl motor cortex surfce; ech point 0.5 μl) for 5 minutes. For biotinylted dextrn mine nterogrde sensory xonl trcking, T 10 dur mter ws fully exposed, nd then the rts were fixed under stereotxic pprtus in prone position. Biotinylted dextrn mine ws slowly (0.1 μl/min) injected into two points (Figure 12B); 0.5 mm depth to the spinl cord surfce; ech point 0.5 μl) for 10 minutes. A B structure of the spine ws stripped off; the spinl cord ws completely removed nd protected in 30% sucrose/pbs solution (4 C) overnight. The spinl cord ws embedded in optiml cutting temperture medium (SAKURA, Tokyo, Jpn) for seril frozen sectioning. Seril longitudinl sections (50 μm) were obtined from the dorsl to spinl medullry cnl. Cross-sections (50 μm) were respectively obtined t 1.5 mm, 1 mm, 0 mm, +1 mm, +1.5 mm nd +5 mm points ( 0, nd + indicting the spinl cord injury center, pproching nd withdrwing from the biotinylted dextrn mine injection point). After ntigen repiring, sections were immersed in sodium citrte buffer with 0.1% Triton X-100 for 5 minutes, treted with 5% stndrd got serum nd closed for 30 minutes t room temperture. Subsequently, rbbit glil fibrillry cidic protein polyclonl ntibody (Sigm-Aldrich; 1:100) ws dded t 4 C overnight. The next dy, sections were incubted with Avidin-Cy3 (Sigm-Aldrich;1:50; mrked biotinylted dextrn mine) nd got nti-rbbit IgG-FITC (Zymed Lbortories Co., Sn Diego, CA, USA) (1:50) fluorescent ntibody for 2 to 3 hours t room temperture, nd then immersed in PBS with 0.1% Tween-20. Finlly, biotinylted dextrn mine (red) nd glil fibrillry cidic protein (green) expression of the spinl cord sections were respectively observed with DMLA 4000B fluorescence microscope (Leic, Wetzlr, Germny) nd TCS SP5 confocl microscope (Leic), nd mesured with the Imge Pro Plus 6.0 softwre (Medi Cybernetics, Rockville, MD, USA). Figure 12 Biotinylted dextrn mine (BDA) nterogrde motor or sensory xonl trcking in rts. (A) BDA nterogrde motor xonl trcing: BDA ws slowly (0.1 μl/min) injected into the 16 injection points (front bregm 1.0 mm, bregm center, behind bregm 1.0 mm nd 2.0 mm s the center, s well s 1.5 mm nd 2.5 mm lterl to the spinl cord medin line) within the cerebrl corticl motor re; (B) BDA nterogrde sensory xons trcing: BDA ws slowly (0.1 μl/min) injected into the two injection points in the distl dorsl spinl cord (locting T 10 spinl level in both sides) bout 5 mm from the spinl cord injury center. Double immunofluorescence histochemistry A 3, 7 nd 28 dys fter spinl cord contusion, six rts from ech group were executed for double immunofluorescence histochemistry to observe the bility of motor nd dorsl scending sensory xonl regenertion (biotinylted dextrn mine mrked), nd strocyte expression within the contusive spinl cord (glil fibrillry cidic protein expression). After crdic perfusion, the spine ws completely removed nd post-fixed in 4% prformldehyde solution for 2 hours. The osseous Sttisticl nlysis Dt were expressed s men ± SEM nd nlyzed using two-smple t-test nd one-wy nlysis of vrince with SPSS 11.5 softwre (SPSS, Chicgo, IL, USA). A P vlue < 0.05 ws considered sttisticlly significnt. REFERENCES [1] Fry EJ. Centrl nervous system regenertion: mission impossible? Clin Exp Phrmcol Physiol. 2001;28(4): [2] Prk DH, Lee JH, Borlongn CV, et l. Trnsplnttion of umbilicl cord blood stem cells for treting spinl cord injury. Stem Cell Rev. 2011;7(1): [3] Liu J, Götherström C, Forsberg M, et l. Humn neurl stem/progenitor cells derived from embryonic stem cells nd fetl nervous system present differences in immunogenicity nd immunomodultory potentils in vitro. Stem Cell Res. 2013;10(3): [4] Kim JY, Kim DH, Kim JH, et l. Umbilicl cord blood mesenchyml stem cells protect myloid-β42 neurotoxi- 2246

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