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GDNF modi ?es reactive astrogliosis allowing robust axonal regeneration through Schwann cell-seeded guidance channels after spinal cord injury

Ling-Xiao Deng a ,b ,c ,d ,e ,f ,Jianguo Hu a ,b ,c ,d ,e ,f ,Naikui Liu a ,b ,c ,d ,e ,f ,Xiaofei Wang a ,b ,c ,d ,e ,f ,George M.Smith g ,h ,Xuejun Wen i ,Xiao-Ming Xu a ,b ,c ,d ,e ,f ,?

Spinal Cord and Brain Injury Research Group,Stark Neurosciences Research Institute,Indiana University School of Medicine,Indianapolis,IN 46202,USA b

Department of Neurological Surgery,Indiana University School of Medicine,Indianapolis,IN 46202,USA c

Department of Anatomy and Cell Biology,Indiana University School of Medicine,Indianapolis,IN 46202,USA d

Kentucky Spinal Cord Injury Research Center,University of Louisville School of Medicine,Louisville,KY 40292,USA e

Department of Neurological Surgery,University of Louisville School of Medicine,Louisville,KY 40292,USA f

Department of Anatomical Sciences and Neurobiology,University of Louisville School of Medicine,Louisville,KY 40292,USA g

Spinal Cord and Brain Injury Research Center,University of Kentucky Chandler Medical Center,Lexington,KY 40536,USA h

Department of Physiology,University of Kentucky Chandler Medical Center,Lexington,KY 40536,USA i

Clemson-MUSC Joint Bioengineering Program,Charleston,SC 29425,USA

a b s t r a c t

a r t i c l e i n f o Article history:

Received 13October 2010Revised 28January 2011Accepted 3February 2011

Available online 21February 2011Keywords:

Axonal regeneration GDNF

Reactive astrocytes Schwann cells Spinal cord injury Transplantation

Reactive astrogliosis impedes axonal regeneration after injuries to the mammalian central nervous system (CNS).Here we report that glial cell line-derived neurotrophic factor (GDNF),combined with transplanted Schwann cells (SCs),effectively reversed the inhibitory properties of astrocytes at graft –host interfaces allowing robust axonal regeneration,concomitant with vigorous migration of host astrocytes into SC-seeded semi-permeable guidance channels implanted into a right-sided spinal cord hemisection at the 10th thoracic (T10)level.Within the graft,migrated host astrocytes were in close association with regenerated axons.Astrocyte processes extended parallel to the axons,implying that the migrated astrocytes were not inhibitory and might have promoted directional growth of regenerated axons.In vitro ,GDNF induced migration of SCs and astrocytes toward each other in an astrocyte –SC confrontation assay.GDNF also enhanced migration of astrocytes on a SC monolayer in an inverted coverslip migration assay,suggesting that this effect is mediated by direct cell –cell contact between the two cell types.Morphologically,GDNF administration reduced astrocyte hypertrophy and induced elongated process extension of these cells,similar to what was observed in vivo .Notably,GDNF treatment signi ?cantly reduced production of glial ?brillary acidic protein (GFAP)and chondroitin sulfate proteoglycans (CSPGs),two hallmarks of astrogliosis,in both the in vivo and in vitro models.Thus,our study demonstrates a novel role of GDNF in modifying spinal cord injury (SCI)-induced astrogliosis resulting in robust axonal regeneration in adult rats.

?2011Published by Elsevier Inc.

Introduction

Glial cell line-derived neurotrophic factor (GDNF)and its receptors are widely expressed in the developing (Oppenheim et al.,1995)and adult central nervous system (CNS)(Arenas et al.,1995;Buj-Bello et al.,1995).Two receptors for GDNF,i.e.GFR α1and/or c-Ret,are expressed not only in neurons,but also in Schwann cells (SCs)and astrocytes (Widenfalk et al.,2001).In addition to its effect on neuron survival (Kordower et al.,2000;Perrelet et al.,2002)and axonal regeneration (Iannotti et al.,2003;Mills et al.,2007),growing

evidence suggests that the GDNF effect on axon regeneration may be mediated through affecting the behavior of glial cells (Iwase et al.,2005;Paratcha et al.,2003).Whether GDNF plays a role in modi ?cation of astrogliosis and the subsequent promotion of axonal regeneration remains unclear.

Reactive astrogliosis,developed in response to injuries of the CNS,signi ?cantly impedes axonal regeneration.Following spinal cord injury (SCI),astrocytes at and near the injury border adopt a reactive hypertrophic phenotype;they express elevated levels of glial ?brillary acidic protein (GFAP),and release inhibitory extracellular matrix molecules chondroitin sulfate proteoglycans (CSPGs)(Chau et al.,2004;Predy and Malhotra,1989).It is the physical and chemical barrier formed by reactive astrogliosis that inhibits axonal regener-ation through and beyond injuries in the CNS (Fitch and Silver,2008;Reier et al.,1983).However,in the developing and mature CNS,astrocytes play multifaceted roles.Radial glial cells,precursors to

Experimental Neurology 229(2011)238–250

?Corresponding author at:Spinal Cord and Brain Injury Research Group,Stark Neurosciences Research Institute,Indiana University School of Medicine,950W.Walnut Street,R2-427,Indianapolis,IN 46202,USA.Fax:+13172785849.

E-mail address:xu26@https://www.wendangku.net/doc/d75165061.html, (X.-M.

Xu).0014-4886/$–see front matter ?2011Published by Elsevier Inc.doi:

10.1016/j.expneurol.2011.02.001

Contents lists available at ScienceDirect

Experimental Neurology

j o u r n a l h o me p a g e :w w w.e l s e v i e r.c om /l o c a t e /y e x nr

astrocytes,are generated alongside neurons and are important for supporting neuronal migration and axon guidance(Vaccarino et al., 2007).In the mature CNS,astrocytes regulate synaptic activity, modulate the extracellular ionic environment and maintain the blood-brain barrier(Abbott et al.,2006;Tanaka,2007;Walz,2000). Even after injury,reactive astrocytes may show adaptive plasticity by secreting many cytokines and neurotrophic factors(Aubert et al., 1995;Levison et al.,1996),restoring the extracellular ionic environ-ment(Sykova et al.,1992),and upregulating various cellular surface molecules and extracellular matrix molecules such as L1,laminin,and ?bronectin(Alonso and Privat,1993;Frisen et al.,1993;Le Gal La Salle et al.,1992).Indeed,reactive astrocytes were shown to protect tissue and preserve function after SCI(Faulkner et al.,2004).Thus,a repair strategy aimed at minimizing the inhibitory properties of astrocytes and simultaneously maximizing their growth-promoting properties would be extremely attractive.

Previously,we co-administered recombinant human GDNF (rhGDNF or GDNF)and SCs in semi-permeable guidance channels grafted into hemisected spinal cords and found that GDNF alleviated astroglial reaction and modi?ed morphological properties of reactive astrocytes(Iannotti et al.,2003).However,the role and mechanism by which GDNF mediates such an action remains unclear.The goal of this study was to determine whether GDNF,over-expressed by SCs,would intensify this modi?cation which,in turn,would improve graft–host interfaces leading to enhanced axonal regeneration following SCI. Materials and methods

Generation of puri?ed Schwann cells(SCs)and astrocytes

SCs were puri?ed as described previously(Morrissey et al.,1991; Xu et al.,1995).Brie?y,SCs were harvested from the sciatic nerves of adult female Sprague–Dawley(SD)rats(Harlan,Indianapolis,IN) under aseptic conditions,then puri?ed and expanded in culture. Puri?ed SCs(purity N98%)at the third or fourth passage were collected for either in vitro experiments or seeding into mini-guidance channels for transplantation.Astrocytes were puri?ed from the cortex of neonatal rat brains(Muir et al.,2002).Cortices from postnatal day (P)0–1rats were minced in Hank's Buffered Salt Solution(HBSS)after the removal of meninges,digested in0.25%trypsin(Sigma,St.Louis, MO),triturated in DMEM with10%fetal bovine serum(FBS,Sigma), and centrifuged for5min at1000g.The cells were plated in Dulbecco's Modi?ed Eagle's Medium(DMEM)with10%FBS in tissue culture?asks.Once the cells had reached con?uence,they were shaken for170rpm for18h in an incubator shaker to remove microglia and oligodendrocyte progenitor cells,followed by a4day culture in20mM anti-mitogen AraC to eliminate the?broblasts,and then passaged and grown to con?uence.Astrocyte cultures were N98% GFAP positive and were maintained in DMEM containing10%FBS (D10).

Transduction of SC in vitro

SCs were seeded into6-well plates at a density of5×105cells/well for in vitro enzyme-linked immuno-sorbent assay(ELISA)or into a 25ml?ask at a density of1×106cells/?ask for transplantation.When cells were grown to over90%con?uence,they were pre-treated with 4–6μg/ml polybrene(Sigma)for30–60min,and then infected by lentiviruses expressing either green?uorescence protein(lenti-GFP) or GDNF(lenti-GDNF)for12h at a multiplicity of infection(MOI)of4, resulting in about50%infection of cells(Abdellatif et al.,2006). Infection media was then replaced with fresh media and,3days later, conditioned media in6well plates was collected for ELISA.Cells in 25ml?asks were prepared for transplantation.ELISA

The GDNF levels secreted by SCs after infection in vitro were measured by ELISA(Abdellatif et al.,2006).Three days after infection, the supernatant of SC was collected and centrifuged at20,000g for 10min at4°C.The procedure for ELISA followed the supplier's recommendations(G1620,Promega,Madison,WI).

Seeding SCs into mini-guidance channels

Semi-permeable60:40poly-acrylonitrile/poly-vinylchloride (PAN/PVC)copolymer guidance channels with an outer diameter of 1.25mm(Provided by Dr.Xuejun Wen,Clemson University,Charles-ton,SC)were cleaned and sterilized according to the established methods(Bamber et al.,2001;Xu et al.,1999).SCs were suspended in a60:40(v:v)of DMEM and Matrigel(MG,Collaborative Research, Bedford,MA)at a?nal density of120×106cells/ml and seeded into guidance channels as described previously(Xu et al.,1999).The channel contents include1)SCs alone(SCs),2)SCs infected with lenti-GFP(lenti-GFP SCs),3)SCs co-administered with GDNF protein (GDNF protein+SCs),and4)SCs infected with lenti-GDNF(lenti-GDNF SCs).In channels when GDNF was co-administered,an amount of DMEM was replaced with an equal volume of concentrated GDNF to achieve a?nal concentration of GDNF at5μg/μl(Iannotti et al.,2003). After seeding,the channel was closed at both ends with PAN/PVC glue and kept in DMEM for2–3h at37°C to allow polymerization of the MG.

Spinal cord hemisection and transplantation of SC-seeded guidance channels

Adult female SD rats(180–200g,Harlan)were randomly divided into four groups that received grafts of:1)SCs alone(n=10),2)lenti-GFP SCs(n=10),3)GDNF protein+SCs(n=10),and4)lenti-GDNF SCs(n=10).The procedures for spinal cord hemisection and mini-guidance channel implantation,as well as for pre-and post-operative animal care,were described in detail in previous publications (Bamber et al.,2001;Xu et al.,1999).Brie?y,a right-sided spinal cord hemisection was performed at the9th and10th thoracic(T) levels to create a2.8mm gap longitudinally followed by implantation of a3mm-long piece of SC-seeded guidance channel into the lesion site.In all groups,rats were sacri?ced at6weeks post-implantation. All animal handling,surgical procedures,and post-operative care were performed in accordance with the Guide for the Care and Use of Laboratory Animals(National Research Council,1996)and the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Care Committees of Indiana University.

Collection of Schwann cell conditioned medium(SCM)

When cultures of puri?ed SCs in T25?asks were con?uent,they were rinsed twice with DMEM and kept in D10without or with GDNF (100ng/ml)for24h.Then cultures were replaced with GDNF-free medium and maintained for an additional4days before medium collection.The medium was centrifuged and?ltrated through a 0.2μm?lter and stored(Millipore,Hertfordshire,UK).

Scratch wound healing migration assay

The scratch migration assay was used to measure two-dimensional cell movement(Boran and Garcia,2007).After astrocytes were grown to con?uence in24-well plates,a scratch was made on the monolayer using a sterile200μl pipette tip.Then the astrocyte cultures were exposed to the following?ve treatment groups:1)medium only (D10),2)GDNF(100ng/ml in D10),3)SC conditioned medium(SCM; at1:1ration to D10),4)SCM+GDNF(100ng/ml),and5)SCM

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pretreated with GDNF(GDNF-pretreated SCM,at1:1ration to D10). The GDNF concentration used(100ng/ml)was determined by a pilot dose–response study.At the beginning of the experiment(t=0h),a digital image of the scratch was taken at a magni?cation of10×. Twenty-four hours later(t=24h),the same region was imaged again.The images were quanti?ed using a NIH Image J program to determine the two-dimensional movements of the astrocytes by measuring the surface area of migrated cells at t=0h and comparing it with that at t=24h.Experiments were performed at least three times and measurements were made in triplicate.

Inverted coverslip migration assay

This assay has been used to detect the migratory ability of one type of cells above another type(Fok-Seang et al.,1995).In this experiment,astrocytes were pre-labeled with Di-I(20mM),a carbocyanine?uorescent tracer,for5min at37°C(Molecular Probes, Leiden,The Netherlands).Di-I-labeled astrocytes were plated onto coverslip fragments(≈2mm2)pre-coated with poly-lysine.After16–18h,the coverslips were washed to remove loose cells,inverted(with cells facing downward)onto a SC monolayer,and incubated in serum-free DMEM medium with or without the administration of GDNF (100ng/ml)for3days to allow cell migration.Cultures were then ?xed,and the maximum distance that cells migrated away from the edge of the fragment was measured.The number of cells that migrated out from the edge of the fragment was also counted at a distance of every100μm.Experiments were carried out in the presence of anti-mitogen Ara-C(5μg/ml)to make sure that the movement of cells away from the coverslip was solely caused by migration instead of proliferation.

Astrocyte and Schwann cell confrontation assay

The confrontation assay was performed according to a previously described method(Lakatos et al.,2003).Brie?y,10μl of two parallel strips of SCs and astrocytes(1×104/cell type),was seeded on a PLL-coated coverslip.The width of gap between two cell types was about 1mm.Cells were allowed to attach for1h before washing in DMEM to remove nonattached cells.Cultures were then maintained in D10and allowed to grow towards each other over a period of12–14days, giving time for cells to make contact and interact.In the GDNF treated group,GDNF(100ng/ml)was added to the cultures after the cells had been in contact with each other for two https://www.wendangku.net/doc/d75165061.html,ing the NIH Image J,a 300μm line was drawn along the interface between astrocytes and SCs.The numbers of SCs and astrocytes crossing this line were counted and averaged over?ve randomly chosen?elds.Experiments were performed at least three times and measurements were made in triplicate.

Astrocytes and Schwann cell co-culture assay

Co-cultures of astrocytes and SCs were performed as described previously(Lakatos et al.,2000).Brie?y,astrocytes and SCs were mixed at a ratio of1:3,respectively,with a total cell number of4×104 per well for24well plates.In order to create cell lysates,a total cell number of1.6×105was added to each well of six well plates,each containing2ml of D10.GDNF(100ng/ml)was added on the second day and fresh medium with or without GDNF was replaced every day. The cultures were maintained for14days by which time astrocyte responses had occurred.To assess whether GDNF affected the hypertrophic changes of astrocytes,we prepared astrocyte culture in the presence of vehicle(DMEM),GDNF(100ng/ml),SCM(1:1with DMEM),SCM+GDNF,SCM(pre-GDNF),SCs,and SCs+GDNF.The ratio of astrocytes and SCs was1:3.All culture media at various conditions were kept for7days before immunostaining for GFAP (astrocytes)and/or p75NTR(SCs).Astrocyte hypertrophy was assessed by measuring changes in individual astrocyte area using the NIH Image J software.

Western blotting

Western blotting followed procedures described previously(Liu and Xu,2006).Brie?y,protein samples(20μg)were electrophoresed on SDS-polyacrylamide gels,and transferred to polyvinylidene di?uoride membranes(Millipore,Bedford,MA).The blots were incubated with primary antibodies against GFAP(1:1000)or CS-56 (1:1000;an antibody recognize CSPGs)overnight followed by incubation with HRP-conjugated secondary antibody for1h at room temperature(1:5000).Blots were visualized using the enhanced chemiluminescence(ECL)plus detection system(GE Healthcare, Little Chalfont,UK).

Light and electron microscopy

The preparation for light and electron microscopy was described previously(Xu et al.,1999).Transverse1μm-thick semi-thin plastic sections through the mid-point of the guidance channel were stained in1%toluidine blue–1%sodium borate in order to quantify the mean number of myelinated axons(N),and tissue cable size(S)according to a previously published method(Xu et al.,1999).The density of axons (D)was calculated as D=N/S.For electron microscopy(EM),Ultra-thin sections were stained with uranyl acetate and lead citrate and examined with an electron microscope(FEI Tecnai G2F20,Hillsbora, OR).

Immunohistochemistry

Immunohistochemistry was performed as described previously (Iannotti et al.,2003;Liu et al.,2006).Brie?y,a10mm spinal segment containing the transplanted channel was removed,cryoprotected, sectioned on a cryostat at20–25μm,and mounted on microscope slides.The sections were incubated in primary antibodies overnight at 4°C.Polyclonal rabbit anti-GFAP antibody(GFAP;1:100,Chemicon, Temecula,CA)was used to identify astrocytes,while anti-NGFR P75 antibody(1:100;Sigma)was used to identify Schwann cells,and anti-P0antibody(1:100,a gift from Dr J.J.Archelos)was used to identify myelin formed by transplanted SCs.Monoclonal mouse anti-SMI-31 antibody(1:1000;Chemicon)was used to identify axons,and anti-chondroitin sulfate proteoglycans(CSPG)antibody(1:500,Chemi-con)was used to identify the glial scar.On the following day,cultures were incubated with the?uorescent nuclear dye,Hoechest33342 (10μg/ml,Sigma),and either rhodamine-conjugated goat anti-rabbit (1:100,ICN-Cappel,Aurora,OH)or AMCA-conjugated af?nipure donkey anti-mouse IgG(H+L)(1:100,Jackson

ImmunoResearch

Fig.1.Expression of GDNF in lentiviral infected Schwann cells(SCs)in vitro.Infection of lentiviruses overexpressing GDNF resulted in a signi?cant increase in SCs'production of GDNF as compared to the SCs alone or lenti-GFP SCs(***:p b0.001;n=5/group).M.

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Fig.2.GDNF induced migration of host astrocytes into the Schwann cell (SC)grafts.(A)Dorsal view of the brain and spinal cord of an adult rat subjected to a SC-seeded guidance channel transplantation.(B)A high magni ?cation image of transplanted area illustrated how tissues were processed.A 1mm segment at the graft mid-point was cut transversely and processed for EM.The remaining proximal and distal segments of the graft were cut longitudinally for immunohistochemistry (IH).Images shown in C –N are representative photomicrographs of the caudal graft –host interface from grafts that contained 1)SCs alone,2)lenti-GFP SCs,3)GDNF protein+SCs,and 4)lenti-GDNF SCs.(C –E)In the SC alone graft,a dense meshwork of hypertrophic astrocytes,labeled with GFAP,was seen at the host side of the caudal graft –host interface (yellow dashed line).Note that host astrocytes did not migrate into the SC graft in the absence of GDNF.(F –H)In the lenti-GFP SC graft,a similar dense meshwork of hypertrophic astrocytes was found.The survival of grafted SCs,evidenced by GFP-staining,with elongated processes extending along the axis of the graft was clearly seen.In this group,host astrocytes did not migrate into the SC graft.(I –K)In the GDNF protein+SC graft,numerous host astrocytes migrated into the graft environment.(L –N)In the lenti-GDNF SC graft,remarkably more host astrocytes migrated into the graft environment for considerable distances.(D,G,J and M)and (E,H,K and N)are high magni ?cations of boxed areas of the graft proper and caudal graft –host interface,respectively,shown in C,F,I and L.Yellow dashed lines indicate the graft –host interfaces.White dash lines in C,F,I and L depict the graft proper.(O)A comparison in migratory distances of astrocytes,represented as astrocyte index ,into the SC graft among the four transplantation groups.(P)A comparison in orientation of astrocyte processes at the distal graft –host interfaces among the four transplantation groups.0°is parallel and 90°is perpendicular to the channel.***:p b 0.001(lenti-GDNF SCs vs.GDNF protein+SCs);+++:p b 0.001(lenti-GDNF SCs vs.SCs alone);$$$:p b 0.001(lenti-GDNF SCs vs.lenti-GFP SCs);★★or ★★★:p b 0.01or p b 0.001(GDNF protein+SCs vs.SC alone);##or ###:p b 0.01or p b 0.001(GDNF protein+SCs vs.lenti-GFP SCs).Scale bars:C,F,I and L=400μm;D,E,G,H,J,K,M and N=100μm.

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Lab.,West Glove,PA).Slides were washed,mounted,examined,and photographed using an Olympus DX60?uorescent microscope. Primary antibody omission and mouse and rabbit isotype controls (Zymed Lab Inc.,San Francisco,CA)were used to con?rm the speci?city of the antibodies.

BrdU incorporation

Cell proliferation was assayed by measuring the incorporation of BrdU according to methods described previously(Hu et al.,2008). Brie?y,astrocytes were seeded at low density onto PLL-coated coverslips and left to adhere overnight.Cells were treated with DMEM for12h and then exposed to the following?ve treatment conditions:1)medium only(D10),2)GDNF(100ng/ml in D10),3) SCM(at1:1ration to D10),4)SCM+GDNF(100ng/ml),and5)SCM pretreated with GDNF(GDNF-pretreated SCM,at1:1ration to D10). For the astrocyte and SC coculture,control groups were kept in D10 and GDNF groups were treated with GDNF(100ng/ml)for16h.In all groups,10μM BrdU(Sigma)was added to label dividing cells and cultures were maintained for an additional16h.Then,cells were ?xed,immuno-labeled with mouse anti-BrdU antibody(1:80;Dako, Santa Barbara,CA)for40min,followed by the secondary IgG1-FITC antibody(1:100,ICN)for30min.The percentage of BrdU-positive cells was calculated by counting200DAPI-labeled nuclei at three random sites and averaged.These experiments were performed in triplicate.

Assessments of GFAP and CSPG immunoreactivity in vivo

Fluorescence intensity of GFAP and CSPG immunoreactivity(IR) was measured to estimate the fold increase in GFAP and CSPG levels at the lesion border over baseline levels of an uninjured spinal cord,as described previously(Iannotti et al.,2003).The GFAP-IR was used as a marker to outline the boundary between the grafted and host tissues. After outlining of the astrogliotic region at the graft–host interfaces, the intensity of GFAP-IR and CSPG-IR was determined using an Olympus digital camera and NIH Image J.For each animal,20μm serial sections at equal medial–lateral distances were used for analysis.The intensity of GFAP-IR and CSPG-IR at the rostral and caudal graft–host interfaces were measured from three longitudinal sections through the guidance channel and tissue cable(one section through the middle of the channel and the other two located100μm medially and laterally away from the central section).Sections from each group were processed simultaneously for GFAP-IR and CSPG-IR.The total intensity values were then averaged for each group.

Quanti?cation of astrocyte migration and process directionality In all cases quanti?cation was performed with the experimenter blind to the treatment group.For quanti?cation of GFAP-labeled astrocytes,three GFAP-stained longitudinal sections equidistant through the guidance channel and tissue cable were used for analysis. We de?ned the host–graft boundary as a starting point“0”to measure the distances of host astrocytes that migrated into the SC-seeded guidance channel at every200μm intervals.The number of GFAP-positive astrocytes is presented as astrocyte index at0.2,0.4,0.6,0.8, 1.0,and1.2mm position,relative to the graft–host interface which is indicated as“0”position.Astrocyte index is a ratio of GFAP astrocyte number at a speci?c position over the astrocyte number at“0”position,similar to a recent study(Liu et al.,2008).For the orientation of astrocyte processes,GFAP-IR processes were randomly selected within the interface area and‘best?t’lines were traced over them using the Image J software.Angles between the lines and longitudinal axis of the channel were calculated with the Image J.A0°designation is considered parallel and a90°designation is perpendicular to the channel axis.All angles ranging from0°to90°were measured.Thirty randomly chosen astrocytic processes in each section were analyzed, and the mean and median angles were determined.

Statistical analysis

Data were expressed as mean±standard deviation(S.D.)of the mean.One-way ANOVA with Tukey's post-hoc test was used to determine statistically signi?cance.A p value of b0.05was considered statistically signi?cant.

Results

SCs,infected by lenti-GDNF,expressed high levels of GDNF

We?rst determined whether infection of SCs by lenti-GDNF induced high expression of GDNF.The infection ef?ciency of SCs by lentiviruses was N60%.The ELISA results showed that,in the lenti-GDNF group,GDNF concentration was272.2±79.45(pg/ml/106cell/ 24h),as compared to the lenti-GFP group(53.00±48.00)or non-infected group(49.67±45.70).These results clearly showed a successful infection of SCs by lenti-GDNF resulting in signi?cantly higher levels of GDNF expression than the control groups lacking GDNF(Fig.1;one-way ANOVA,p b0.0001).

GDNF induced migration of host astrocytes into the SC grafts

We next evaluated the effects of GDNF on the migratory ability of astrocyte,stained with GFAP,in longitudinal sections through the channel(Fig.2).A total of4transplantation groups that contained1) SCs alone,2)lenti-GFP SCs,3)GDNF protein+SCs,and4)lenti-GDNF SCs(n=10/group)were analyzed.In the groups that received transplantation of SCs alone or lenti-GFP SCs,a dense meshwork of reactive astrocytes(GFAP-IR)was found on the host side of the spinal cord demarcating a clear interface between the host and grafted tissues(Figs.2C,E,F and H).Few astrocytes,if any,migrated into the graft region(Figs.2D and G).In contrast,in grafts when GDNF is co-administered with SCs(GDNF protein+SCs)or overexpressed in SCs by lentiviral-mediated gene transfer(lenti-GDNF SCs),robust migra-tion of astrocytes into the transplants was observed(Figs.2I–N).The migrated astrocytes contained elongated processes which extended in parallel to the longitudinal axis of the graft(Figs.2J and M). Accordingly,the astrogliotic response at the graft–host interface in the GDNF-treated groups was reduced(Figs.2K and N)as compared to the control groups lacking GDNF(Figs.2E and H).In the GDNF-treated groups,the astrocytes migrated throughout the entire length of the graft as they were seen in the rostral and caudal graft segments by immunohistochemistry(IH)as well as in the middle segment by EM.For example,in the caudal segment,astrocytes migrated for up to

Fig.3.GDNF reduced GFAP and CSPG expression at graft–host interfaces in vivo.(A–L)Representative photomicrographs show the caudal graft–host interface of channels that contained1)SCs alone(A–C),2)lenti-GFP SCs(D–F),3)GDNF protein+SCs(G–I),and4)lenti-GDNF SCs(J–L).In grafts containing either SCs alone(A–C)or lenti-GFP SCs(D–F), increased expression of GFAP(A and D)and CSPG(B and E)was found at the distal graft–host interface,which could be further appreciated in the merged images(C and F).In contrast,in grafts containing either GDNF protein+SCs(G–I)or lenti-GDNF SCs(J–L),the expression of both GFAP(G and J)and CSPG(H and K)was considerably reduced,as clearly seen in the merged images of I and L.Such reduction was correlated with signi?cant migration of host astrocytes into the graft environment in groups when GDNF was administered (I and L).White dash lines indicate the caudal graft–host interfaces whereas white asterisks in A–F indicate enhanced expression of GFAP and CSPG at the caudal graft–host interface. (M and N)Quantitative analyses show that the difference in GFAP(M)and CSPG(N)expressions in the four groups were statistically signi?cant(*:P b0.05;**:P b0.01).Scale bar: 100μm.

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1.2mm rostrally(the longest distance that was examined)within the channels from the caudal graft–host interface(Fig.2O).Moreover,the amount of GFAP-IR cells that migrated over distances within the channels,indicated as astrocyte index,was examined and signi?cant differences between the GDNF treated and non-treated groups were clearly seen(Fig.2O).Finally,GDNF treatments also altered astrocyte morphology so that these cells became less reactive and gave rise to elongated processes in parallel to the graft axis.This is in sharp contrast to the morphology of the reactive astrocytes with thicker and randomly extended processes in the non-GDNF treatment groups (Figs.2E and H vs.K and N).The astrocyte processes were more parallel to the graft axis in the GDNF-treated groups(lenti-GDNF SCs, 18.85±18.80°;GDNF protein+SCs,24.46±21.34°)as compared to the non-GDNF-treated groups(lenti-GFP SCs,60.25±20.33°;SCs alone,68.94±28.64°)with0°designated as complete parallel and90°perpendicular to the graft axis(Fig.2P).

GDNF reduced GFAP and CSPG expression at the graft–host interface Since GFAP is a hallmark of reactive astrogliosis and CSPGs are a major class of axon growth inhibitors associated with reactive astrocytes(Bradbury et al.,2002;Chau et al.,2004;Davies et al., 1999;Hsu and Xu,2005),we investigated the expression of GFAP and CSPG at the graft–host interfaces in the four groups that received either GDNF or non-GDNF treatments,as described above(Fig.3).In grafts that contained SCs alone or lenti-GFP SCs,increased expression of GFAP(Figs.3A and D)and CSPG(Figs.2B and E)was clearly seen.In contrast,in grafts that contained GDNF protein+SCs or lenti-GDNF SCs,both GFAP and CSPG expressions were signi?cantly reduced (Figs.3G–L),and the differences between the GDNF treated and non-treated groups were statistically signi?cant(Figs.3M and N).

GDNF induced parallel alignment between migrated astrocytes, regenerated axons and new myelin formed by grafted Schwann cells We next examined the association of migrated astrocytes with regenerated axons and new myelin formed by grafted Schwann cells under the in?uence of GDNF.In both GDNF-treated groups(Figs.4G and H),greater axonal growth into the guidance channels was found as compared to the non-GDNF-treated groups(Figs.4E and F).Toluidine blue stained cross sections taken from the graft mid-point showed that the total number and density of myelinated axons were all signi?-cantly increased in the GDNF-treated groups as compared to the non-GDNF-treated groups(Suppl.Fig.1),which was consistent with our previous results(Iannotti et al.,2003;Zhang et al.,2009).We further examined whether migrated astrocytes,after GDNF treatment,were disposed to axon growth and subsequent myelination by grafted SCs using immuno?uorescence double labeling of GFAP with SMI-31,an axon marker,or P0,a marker for SC myelin.In non-GDNF-treated groups(SCs alone or lenti-GFP SCs),astrocytes did not migrate into the graft environment and,therefore,no association of astrocytes with axons(Figs.4A,B,E and F)or myelin(Figs.4I,J,M and N)was found within the grafts.Interestingly,the lack of migrated astrocytes in the control groups correlated well with the reduced amount of SMI-31-IR axons within the channel(Figs.4E and F).At the graft–host interface,the astrocyte processes were misaligned with regenerated axons in the control groups(Figs.4E and F).In contrast,in the GDNF-treated groups,astrocytes vigorously migrated into the channel with their processes extended longitudinally and in close alignment with regenerated axons(Figs.4C,G,D and H).Furthermore,migrated astrocytes were also in close association with new myelin(P0-IR) formed by grafted SCs(Figs.4K,O,L and P).Note that GDNF treatments induced greater myelin formation than the non-GDNF treatments (Figs.4O and P vs.M and N).In contrast to the clear dissociation between myelinated SCs and host astrocytes in the non-GDNF-treated groups(Figs.4I and J),migrated astrocytes in the GDNF-treated groups showed parallel alignment of their processes with new myelin formed by grafted SCs(Figs.4O and P).At the EM level,the association of astrocytes or their processes with regenerated axons,both myelinated and unmyelinated,were clearly seen(Figs.4Q and R).

GDNF promoted interdigitative migration between astrocytes and SCs To determine the effect of GDNF on astrocyte–SC interaction,a confrontation assay was employed.Such an assay was used previously to demonstrate that SCs did not intermingle with astrocytes(Grimpe et al.,2005;Lakatos et al.,2000;Wilby et al.,1999).In the absence of GDNF,a sharp and straight boundary(Fig.5A,red line)was formed between two seeded cell populations.In contrast,GDNF administra-tion induced migration of both cell populations towards each other, resulting in an interdigitated boundary between the two(Fig.5B,red line).Such an interaction between astrocytes and SCs could be further appreciated when these cells were immunostained with their phenotypic markers GFAP and p75,respectively(Figs.5C and D). Quantitative analysis showed that the number of cells that crossed a 300μm line(Figs.5A and B,yellow line)in both SCs and astrocytes were signi?cantly increased in the GDNF treated group as compared to the control group(Fig.5E;p b0.01).Thus,GDNF induced interdigitative migration of both astrocytes and SCs towards their counterparts.

GDNF had no direct effect on astrocyte migration but promoted their migration on the monolayer of SCs

To determine the effect of GDNF and/or SC-conditioned medium (SCM)on astrocyte migration,we used a scratch wound-healing model(Boran and Garcia,2007).Administration of GDNF failed to reduce the scratch gap at24h,compared to the vehicle control (Figs.6A and B),suggesting that GDNF had no direct effect on astrocyte migration.In contrast,the size of the wound was signi?cantly reduced in astrocytes treated with SCM(Figs.6A and B; p b0.01)indicating that factors secreted by SCs accelerated astrocyte migration.Interestingly,SCM from GDNF-pretreated SCs[SCM(pre-GDNF)]reduced the scratch gap and enhanced astrocyte migration (Figs.6A and B),suggesting that GDNF may affect astrocyte migration indirectly through modi?cation of SCs or their production of SCM.To determine whether SCM or GDNF had any effect on astrocyte proliferation which may affect their migration,a BrdU incorporation assay was performed.Results showed that SCM but not GDNF stimulated astrocyte proliferation(Suppl.Fig.2),similar to a previous

Fig.4.GDNF induced parallel alignment between migrated astrocytes,regenerated axons and SC myelin.(A–P)Representative photomicrographs show astrocyte–axon relationship at the caudal graft–host interface(A–H)as well as astrocyte–myelin association within the graft proper(I–P)in channels that contained1)SCs alone(1st row),2)lenti-GFP SCs(2nd row),3)GDNF protein+SCs(3rd row),and4)lenti-GDNF SCs(4th row).(A–H)In both GDNF treated groups,signi?cantly more axons(SMI-31-positive)grew into the graft environment(G and H)as compared to the grafts containing no GDNF(E and F).Such robust axonal regeneration in the GDNF treated groups was concomitant with vigorous migration of astrocytes(GFAP-positive)into the grafts within which astrocytic processes aligned in parallel to regenerated axons(G and H;arrows).In contrast,in the absence of GDNF,no astrocytic migration into the grafts was found(E and F).(I–P)Within the grafts of both GDNF treated groups,close association between migrated astrocytes(K and L;GFAP-positive)and SC myelin(O and P;P0-positive)was found,which was in high contrast to the lack of astrocyte migration(I and J)and limited SC myelination(M and N)in control grafts in the absence of GDNF.Dashed line in A–H indicates the caudal graft–host interface.Arrows in rows3and4indicate migrated astrocytes and their association with regenerated axons(G and H)or SC myelin(O and P).(Q and R)At the EM level,close association of an astrocytic process(Q;white asterisk)or astrocyte cell body(R;white asterisk) with regenerated myelinated(white arrows)or unmyelinated(black arrows)axons was clearly seen.Black asterisks indicate SC nuclei.Bars:A–P=100μm;Q=2μm;R=0.5μm. 244L.-X.Deng et al./Experimental Neurology229(2011)238–250

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report (Santos-Silva et al.,2007).Our experiment was done in the presence of an anti-mitogen Ara-C (5μg/ml)to eliminate the compound effect of cell proliferation on the cell migration assay.

To test whether cell –cell contact mediated GDNF-induced astro-cyte migration,we used an inverted coverslip migration assay (Fig.6C).In this experiment,the anti-mitogen Ara-C was also used for the reason mentioned above.At 3days after the GDNF treatment,both migratory distances and the number of astrocytes per migratory distances on the SC monolayer increased signi ?cantly,as compared to the control group (Figs.6C –E;p b 0.01).

GDNF reversed SC-induced hypertrophy of astrocytes and promoted their process extension

To assess whether GDNF affected the hypertrophic changes of astrocytes,we prepared astrocyte cultures at various culture condi-tions (Fig.7).Results showed that the GDNF did not have a direct effect on astrocyte area as compared to the vehicle group (Figs.7B and I).SCM alone (Fig.7C)or with the addition of GDNF (Fig.7D)or pretreated (Fig.7E)with GDNF,increased the astrocyte area or hypertrophy as compared to the vehicle control (Fig.7I,p b 0.05).The

same effect was observed when astrocytes were co-cultured with SCs compared with when astrocytes were cultured alone (Figs.7F and I;p b 0.01),indicating that the average size of individual astrocytes was signi ?cantly increased in the presence of SCs.Interestingly,when GDNF was added to the astrocyte –SC co-culture system,it reduced astrocyte size and promoted elongation of astrocyte processes (Figs.7G –I;p b 0.01),a phenomenon which was also observed in vivo (Fig.2).To rule out the possibility that GDNF reversed the hypertrophy of astrocytes by inducing SCs'secretion of other factors,we pretreated SCs with GDNF,and collected such GDNF-pretreated SCM [SCM (pre-GDNF)]as an additional control.The SCM (pre-GDNF)did not reverse the hypertrophic change of astrocytes in the SCM group indicating that GDNF did not have an effect on the production of secondary trophic factors as examined previously (Zhang et al.,2009).Thus,GDNF may reverse SC-induced astrocyte hypertrophy through the interaction between the two cell populations.

GDNF reduced expression of GFAP and CSPG in astrocyte –SC co-cultures Lastly,we examined the effect of GDNF on expressions of GFAP and CSPG,two hallmarks of astrogliosis,in astrocyte single cultures or astrocyte –SC co-cultures.Neither GDNF nor SCM alone had an effect on the expression of GFAP or CS-56in astrocyte single cultures as compared to the vehicle group (Figs.8A and B).Administration of a combination of GDNF and SCM in astrocyte single cultures had no effect on GFAP expression but signi ?cantly reduced that of CS-56.One-way ANOVA showed statistically signi ?cant differences in GFAP (Fig.8A;p =0.0016)and CS-56(Fig.8B;p b 0.0001)expressions among different treatment groups.Interestingly,only in the group when GDNF was administered to the astrocyte –SC co-cultures did it signi ?cantly reduce GFAP and CS-56expression (Figs.8A and B;p b 0.01).These results correlate well with the observation that GDNF reduced astrocytic hypertrophy in vitro (as shown in Fig.6)and gliosis in vivo (as shown in Fig.3)in the presence of both astrocytes and SCs.Together,these results suggest that the effects of GDNF on reducing astrogliosis must be mediated through the interaction between astrocytes and SCs.Discussion

To our knowledge,this is the ?rst study demonstrating an important role of GDNF on modifying astrogliotic responses at graft –host interfaces allowing robust axonal regeneration into SC grafts and subsequent remyelination following SCI.A key ?nding was that GDNF,administered with or over-expressed by grafted SCs,induced active migration of host astrocytes into SC grafts concomitant with enhanced axonal regeneration and remyelination.GDNF admin-istration or overexpression also signi ?cantly reduced GFAP and CSPG induction at the graft –host interfaces.Such a GDNF effect on astrogliotic responses in vivo was further con ?rmed in vitro and was considered to be mediated through interactions between astrocytes and SCs.Importantly,these studies indicate that the inhibitory properties of reactive astrocytes,induced by a CNS injury,can be readily modi ?ed by a trophic factor GDNF which leads to enhanced axonal regeneration and remyelination.They also indicate that modifying the inhibitory properties of reactive astrocytes may represent a novel and attractive strategy to promote greater axonal regeneration and recovery of function following SCI.

Astrocytes and SCs normally reside separately in the PNS and CNS,respectively,and do not interact with each other.At the peripheral nerve entry zone,astrocytes contribute to the formation of glial limitans that prevent SCs from migrating into the CNS and generate barriers to axonal regeneration after injury (Fraher,1997;Golding et al.,1997).Such a glial limitans or barrier is similar to the one that we observed between grafted SCs and reactive astrocytes in this study.At this interface,astrocytes prevented the migration of SCs into the

host

Fig.5.GDNF induced interdigitative migration between astrocytes and Schwann cells (SCs).(A and B)Phase contrast images show that,in a confrontational assay,a sharp boundary was formed between the SC and astrocyte (AC)monolayers in a control group receiving no GDNF (A;red line).In contrast,GDNF administration induced vigorous migration of both cell populations towards each other,resulting in an interdigitative boundary between the two (B;red line).(C and D)The migratory abilities of astrocytes and SCs in the absence (C)or presence (D)of GDNF could be further appreciated when the cultures were immunostained with their phenotypic markers GFAP and p75,respectively.(E)Quantitative analysis showed that the number of cells that crossed a 300μm line (A and B;yellow lines)in both SCs and astrocytes was signi ?cantly increased in the GDNF treated group as compared to the non-treated group (**:p b 0.01).Bars:A –D=100μm.

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spinal cord as they normally do at the glial limitans.In fact,very few grafted SCs can break through the astrocytic barrier to migrate into the host tissue (Baron-Van Evercooren et al.,1992;Sims et al.,1999),and only when the astrocytes were depleted could SCs migrate into and intermingle with the host tissue (Sims et al.,1999).The presence of GFP-SCs in the graft region of the present study clearly indicates that these cells survived SC bridge transplantation and that they did not migrate into the host spinal cord in the absence of GDNF.Our in vitro data also showed that,in the presence of GDNF,migration of SCs into the astrocyte monolayer occurred,supporting the possibility that GDNF may facilitate SC migration into the host tissue if the appropriate environment is provided.

Similarly,astrocytes do not migrate into the SCs environment and,in fact,they form a strong inhibitory barrier at the host side of

the

Fig.6.Effects of GDNF on astrocyte migration in the absence or presence of Schwann cell (SC)monolayers.(A)Live cell time-lapse imaging of astrocyte (AC)monolayers under different treatment conditions after a scratch https://www.wendangku.net/doc/d75165061.html,posite phase contrast photomicrographs were captured at 0and 24h after the scratch.Movement of cells into the scarred region resulted in a decrease in the surface area of the scar.Bar =100μm.(B)Quanti ?cation of the percentage of AC migration area to the total wound area showed that SC conditioned medium (SCM)induced astrocyte migration (**:p b 0.01).However,GDNF,used alone or in combination with SCM,did not induce or enhance SCM-mediated astrocyte migration.Interestingly,SCM from GDNF-pretreated SCs [SCM (pre-GDNF)]reduced the scratch gap and enhanced astrocyte migration.(C)DiI pre-labeled astrocytes were seeded on a SC monolayer using an inverted cover-slip assay.Merged phase contrast and immuno ?uorescent images showed that both migratory distances and the number of astrocytes per migratory distance on the SC monolayer increased signi ?cantly as compared to the control group.Bar=100μm.(D and E)Quantitative analysis showed that both the number of migrated astrocytes over distances away from the edge of inverted coverslip (D)and the maximum migratory distances (E)were signi ?cantly increased in the GDNF-treated group as compared to the non-treated group indicating that GDNF signi ?cantly promoted astrocyte migration on the SC monolayer.*:p b 0.01;**:p b 0.01.

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spinal cord,as reported previously(Xu et al.,1997;Xu et al.,1995;Xu et al.,1999).In vitro,astrocytes and SCs form separate territories with sharp boundaries between them(Lakatos et al.,2000;Wilby et al., 1999).Previously,we demonstrated that co-administration of rhGDNF in the SC-seeded mini-channel transplantation model reduced the extent of reactive astrogliosis and cavity formation as well as induced mild migration of astrocytes into the SC grafted areas (Iannotti et al.,2003).In the present study,the remarkable effect of GDNF on the migration of astrocytes into the lenti-GDNF SC grafts may result from long-lasting overexpression of GDNF from lentiviral-transfected SCs.Contrarily,the strong effect of GDNF on astrocyte migration was not found in a recent study(Tom et al.,2009).The difference between these two studies may be related,in part,to the difference in GDNF delivery,concentration,or expression.In our case, GDNF was slowly released from lentiviral infected SCs so that a focused and extended release effect of GDNF may be achieved. Migrating astrocytes were observed dispersed among and in close association with grafted SCs indicating that SCs and astrocytes are less repellent to each other under the in?uence of GDNF.Such an optimal interaction between the two cell types that previously repelled each other was also con?rmed in our in vitro assays.

Two possible interpretations exist to explain the effect of GDNF on astrocyte migration:a direct effect of GDNF on astrocytes or an indirect effect of GDNF through its action on SCs.Since GDNF did not have a direct effect on astrocyte migration in our in vitro assays,we favor the second possibility.For example,in the scratch wound healing assay,SC conditioned medium(SCM)induced astrocyte migration in astrocyte cultures indicating that SCM promotes astrocyte migration.However,such migration was not enhanced when GDNF was co-administered with SCM,indicating that GDNF has no additional effect on astrocyte migration.Interestingly,when SCs were pre-stimulated with GDNF,their conditioned medium,i.e.SCM-(pre-GDNF),enhanced astrocyte migration.The strongest migration of astrocytes,however,was found only when astrocytes were in direct contact with SCs,as was shown in both the inverted coverslip assay in vitro and SC-seeded bridge transplantation experiment in vivo.These results collectively indicate that the GDNF effect on astrocyte migration requires the presence of and interaction with SCs.Although SCM alone can induce astrocyte migration to a certain extent,when astrocytes and SCs encounter each other physically this effect is not suf?cient to induce astrocyte migration into the SC territory.

It is well accepted that reactive astrogliosis,developed in response to injuries of the CNS,is a major impediment for axonal regeneration(Fitch and Silver,2008;Predy and Malhotra,1989;Silver and Miller,2004).One effective strategy to remove this impediment is to enzymatically remove inhibitory CSPGs produced by reactive astrocytes at the site of injury (Bradbury et al.,2002;Chau et al.,2004;Houle et al.,2006).An alternative approach,as presented in this study,is to modify the properties of reactive astrocytes making them less inhibitory and more permissive to axonal growth,as they are during development.If reactive astrocytes can be modi?ed to create a directionally-oriented permissive environment, then this typically growth-inhibitory cell population can be transformed into a growth-promotive cell population to enhance axonal regeneration. Indeed,GDNF treatment in the present study reversed the inhibitory properties of reactive astrocytes,induced migration and aligned extension of their processes from the host into the SC graft,and reduced both GFAP and CSPG reactivities at the graft–host interfaces.Enzymatic removal of CSPG with ChABC was associated with migration of astrocytes into the caudal graft environment(Bradbury et al.,2002;Chau et al., 2004;Houle et al.,2006)which may provide the mechanism for

GDNF-

Fig.7.GDNF reduced hypertrophy of astrocytes(AS)in vitro and promoted their processes

extension.(A and B)GDNF alone did not have an effect on astrocyte morphology(I).(C–E)

Schwann cell(SC)-conditioned medium(SCM)alone(C),SCM+GDNF(D),or SCM

pretreated with GDNF[SCM(pre-GDNF)](E),increased the astrocyte area as compared to

the vehicle control(I,p b0.05).(F)A similar effect was observed when astrocytes were co-

cultured with SCs(compared with astrocyte cultured alone;H,p b0.01).(G,H)When GDNF

was added to the astrocyte-SC co-cultures,it reduced astrocyte hypertrophy and promoted

elongation of astrocyte processes(white arrows)as were shown in both the single staining

of GFAP(G)and double staining of GFAP(red)and p75(green),a SC marker(H).All

sections were counterstained with Hochest33342(blue),a?uorescent nuclear dye.(I)

Quanti?cation of astrocyte area was performed using a NIH Image J software.*:p b0.05;**:

p b0.01.Bar=100μm.

248L.-X.Deng et al./Experimental Neurology229(2011)238–250

mediated astrocyte migration observed in the current study.These effects may collectively contribute to the conversion of reactive astrocytes from growth-inhibitory to growth-promotive to axonal regeneration.Within the SC graft,migrated astrocytes aligned along regenerated axons implying that these astrocytes facilitated directional growth of regenerated axons.Moreover,the astrocytic processes were in close contact with regenerated axons which provides a morphological basis for astrocyte –axon interaction that could be important for exchanging metabolites and supplying nutrients to regenerated axons (Clatterbuck et al.,1996;Logan et al.,1994;Nieto-Sampedro et al.,1988).The evidence that migrated astrocytes were widely dispersed among grafted SCs and that they were in close association with SC myelin indicates that the astrocyte –SC interaction,induced by GDNF,may enhance SC-mediated axonal regeneration and remyelination.

One interesting ?nding is that,in astrocyte –SC co-cultures,GDNF treatment signi ?cantly reduced astrocyte hypertrophy and induced their process extension.Such a GDNF effect was also observed in vivo at the graft –host interface concomitant with reduced production of GFAP and CSPG and enhanced axonal regeneration into the SC graft.Thus,GDNF not only affects the biochemical changes of reactive astrocytes but also their morphological changes in response to the injury.

Although the present study has identi ?ed a novel role of GDNF on modifying astrocyte responses to injury and regeneration,the role of GDNF on CNS injury and regeneration may be more complicated than we thought.Previously,we demonstrated that GDNF reduced lesion volume and increased white matter sparing in a contusive SCI model (Iannotti et al.,2004).We also demonstrated that co-administration of GDNF protein with SCs increased axonal regeneration and reduced astrogliosis,cavity formation and in ?ltration of in ?ammatory cells at graft –host interfaces in a SC-seeded mini-channel implantation model (Iannotti et al.,2003).We further demonstrated that,in the same SC transplantation model,GDNF enhanced both the number and caliber of regenerated axons (Zhang et al.,2009).The GDNF effect is mediated primarily through a direct effect on neurons.However,the possibility of its effect on SCs cannot be ruled out.GDNF,however,had no effect on the proliferation of isolated SCs or their secretion of neurotrophins nerve growth factor (NGF),neurotrophin-3(NT3),or brain-derived trophic factor (BDNF)(Zhang et al.,2009).These results collectively

suggest that GDNF-enhanced axonal regeneration and SC myelination is mediated through multiple factors.Taken together,the previous and current results indicate that GDNF may orchestra a multi-faceted response of both neurons and glial cells,including astrocytes and SCs,that eventually results in synergistic and bene ?cial effects on axonal regeneration and remyelination following SCI.

Contrary to the rapid degradation of GDNF protein,the expression of GDNF by lentiviral infected SCs is relatively long-lasting and highly localized at the site of transplantation,which may enhance its effect on modi ?cation of astrocytes.However,long-term and high levels of GDNF expression can be a double-edged sword.For example,regenerated axons can be trapped within the graft preventing them from growing back into the host tissue.Additionally,elevated expression of a single growth factor may provide an unfavorable environment for those who are not responsive to this particular factor.Finally,transplantation of viral-infected SCs may trigger in ?ammation at the site of injury.These disadvantages need to be considered when considering lenti-GDNF as a tool or therapeutic agent in the repair of SCI.

In summary,here we report a novel function of GDNF on modifying reactive astrogliosis,a classically considered inhibitory barrier to axonal regeneration,at the SC graft –host interface following SCI.Reversing the inhibitory properties of reactive astrocytes may open a new avenue to foster axonal regeneration,remyelination,and recovery of function following SCI.

Supplementary materials related to this article can be found online at doi:10.1016/j.expneurol.2011.02.001.Acknowledgments

This work was supported by NIH grants NS036350,NS052290,NS050243,and NS059622,the Daniel Heumann Fund for Spinal Cord Research,and Mari Hulman George Endowments.We thank Amgen Inc.(Thousand Oaks,CA)for providing recombinant human GDNF.References

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