poster abstracts-session 2 · of medicine, philadelphia, pa, usa; 2 laboratory of neuroscience,...

Journal of Rehabilitation Research and DevelopmentVol. 40, No. 6, November/December 2003, Supplement 3Tenth International Symposium on Neural RegenerationPages 39–62

Abstracts of Poster Presentations—Session 2

P42 IMMUNE-PRIVILEGED SERTOLI CELLS AS GENE DELIVERYVEHICLES FOR SPINAL CORD INJURYAlpa Trivedi*, Takuji Igarashi†,Nathalie Compagnone†, Xiaoqing Fan*, Jung-Yu, C. Hsu†, Deborah E. Hall*, Constance M. John*, Linda J. Noble†

*MandalMed, Inc., 2645 Ocean Avenue, San Francisco, CA, USA; †Department of Neurosurgery, University of California at San Francisco, CA, USA

We have evaluated transplantation of immune-privileged Sertoli cells as a proprietary meansfor delivery of recombinant human neurotro-phin-3 (hNT-3) into the injured spinal cord.Sertoli cells infected with recombinant aden-ovirus expressing hNT-3 were found to producehigh amounts of biologically active hNT-3. Wetested the bioactivity of the secreted hNT-3from infected cells on embryonic neocorticalcultures and found increased axonal outgrowthin cells cultured in the presence of conditionedmedium from modified Sertoli cells. AdultSprague Dawley rats were implanted with allo-geneic Sertoli cells genetically modified to pro-duce both hNT-3 and green fluorescent protein(eGFP) or the eGFP alone. Cells wereimplanted into either the uninjured spinal cordor immediately after contusion injury at the T8vertebral level. Viable eGFP fluorescing cells,positive for hNT-3, were detected in intact spi-nal cord. Injured animals that were implantedwith NT-3 secreting Sertoli cells were found toproduce significantly large amounts of hNT-3as compared to animals that were injured butdid not receive any cell implants (untrans-planted control). Animals that received trans-plantation of Sertoli cells, expressing eithereGFP or eGFP and hNT-3, had significantimprovement in functional recovery, at earliertime points after injury, as compared to thoseanimals subjected to injury alone (untrans-planted control) as assessed by the BBB scale.

Moreover, the rate of functional recovery wasimproved in these animals. In addition, recov-ery of automatic reflex emptying of the bladderwas significantly improved in animals that hadbeen implanted with cells secreting hNT-3. Wefound that Sertoli cells significantly attenuatedearly macrophage infiltration based upon quan-titative assessment of inflammatory cells in theacutely injured spinal cord. Together, theseresults suggest that hNT-3 delivery by alloge-neic Sertoli cells is a promising strategy for thetreatment of spinal cord injuries.

P43 GENERATION OF MULTIPLE CELL TYPES WITHREGULATABLE TYROSINE HYDROXYLASE PRODUCTION: A POSSIBLE METHOD TO IMPROVE CELL BASED THERAPIES FOR PARKINSON’S DISEASEKerry Thompson, PhD 1,2;Jack Mottahedeh, PhD 21 Neurology Research, VAMC-GLAHS; 2 Neurology Department, UCLA, CA, USA

Effective treatment of neurological diseasessuch as Parkinson’ disease, Huntington’s dis-ease, and epilepsy, remains problematic. Thedevelopment of new therapies that capitalize onour growing understanding of molecular genet-ics and developmental cell biology providessome promise for clinical applications. Theunfortunate disappointments of the recentlyreported double-blind trials of cell transplanttherapy for Parkinson’s disease have necessi-tated a return to the bench, and a rethinking ofthe approach to cell based therapies. We havegenetically engineered three conditionallyimmortalized cell lines [one neuronal, and twoglial (astrocytic and oligodendrocytic)] with thecDNA coding for the dopamine synthesizingenzyme tyrosine hydroxylase (TH). The cDNAwas inserted downstream of a doxycyclineresponsive (tet-off) promoter in the LINX vec-tor, using a PCR-based cloning strategy. The

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Journal of Rehabilitation Research and Development Vol. 40, No. 6, 2003, Supplement 3

LINX vector produces a transactivating protein(tTA) that drives the expression of the gene ofinterest. Doxycycline binds the tTA and haltstranscription. The resulting construct was usedto transfect multiple cell types that had beenpreviously transfected with a temperature sensi-tive oncogene. Following transfection, the cellswere found to produce immunologically detect-able TH (western blots and immunocytochemis-try). Importantly, with the addition of doxycy-cline to the medium, the ability of the tTA todrive gene expression was suppressed by morethan 90%. Several cell lines of each type weregenerated using these means and they are cur-rently being evaluated for regulatable catechola-mine production. The ultimate goal of produc-ing transplantable cell populations that havecontrollable expression of therapeutic geneswill likely be achieved using the methods usedhere. Combining stem cell technology andmolecular genetics may provide a way to over-come the recently reported side effects pro-duced by cell replacement strategies for Parkin-son’s disease. These cells provide new tools forbasic exploration of improved cell based thera-pies for neurodegenerative disorders.

P44 HUMAN NEURONAL CELL LINES FOR THE TREATMENT OF PAIN AND SPASTICITY FOLLOWING SCIM.J. EatonVA RR&D Center of Excellence in Func-tional Recovery in Chronic Spinal Cord Injury, Miami VAMC, Miami, Fl, USA; The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami Medical School, Miami, Fl, USA

Chronic neuropathic pain and spasticity afterspinal cord injury (SCI) are common clinicalproblems. SCI pain has often proven difficult totreat and no pharmacologic approach has beenshown to be effective over time in a significantnumber of individuals. Spasticity is oftentreated with an implanted mechanical pump tosupply the agent baclofen, but such technologyneeds improvement. The possibility of trans-planting cultured cells, derived from expand-able cell lines, that release biologic agents intothe subarachnoid space offers a new approachto the treatment of chronic pain and spasticity

in SCI. Our laboratory has pioneered the devel-opment of cell lines bioengineered to expressspecific antinociceptive and antispastic agents,such as the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). GABA alsooccurs endogenously in the motor and pain-processing pathways of the spinal cord, but isnot likely to be present in sufficient concentra-tions after SCI to function effectively in damp-ening the sensory imbalance that induceschronic neuropathic pain and spasticity. Fromthe human NT2 cell line, we have derived twounique neuronal human cell lines: ThehNT2.19 GABA and hNT2.19 serotonin (5HT)lines. Each has a characteristic phenotype,including the synthesis and secretion of theirrespective neurotransmitters after as little as aweek of differentiation in vitro. Each cell linewas transplanted into the subarachnoid space attwo weeks after injury in two different modelsof SCI pain and spasticity. The SCI pain modeluses spinal injection of quisqualic acid (QUIS)to induce tactile allodynia and thermal hyperal-gesia in the rat hindlimbs. The S2 sacral spinalcord transection is used as the model for spas-ticity. Only the GABAergic hNT2.17 cell lineimproved spasticity behaviors; transplants ofeither cell line improved the behavioral sensi-tivity associated with neuropathic pain. Each ofthese cell lines is being developed for testing inclinical trials after SCI.

P45 TRANSPLANTATION OFMULTIPOTENTIAL ANDLINEAGE-RESTRICTEDPRECURSOR CELLS DERIVED FROM FETALSPINAL CORDINTO THE MATURE CENTRAL NERVOUS SYSTEM (CNS)A.C. Lepore 1, S.S.W. Han 1, C.J. Tyler-Polsz 1, M.S. Rao 2,I. Fischer 11 Department of Neurobiology &Anatomy, Drexel University Collegeof Medicine, Philadelphia, PA, USA;2 Laboratory of Neuroscience, NIA,Baltimore, MD, USA

Transplantation of neural precursor cells maybe an ideal therapeutic strategy for cellularreplacement and repair because of advantagesthey provide in achieving a desired phenotypic

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fate. Multiple classes of precursor cells havebeen isolated and characterized from the CNSincluding multipotential neural stem cells andlineage-restricted precursor cells. However,the optimal transplantation protocols for cellreplacement and repair in the mature CNS arenot known. We have previously reported thatin developing spinal cord multipotential neu-roepithelial (NEP) stem cells predominate atE10.5, whereas at E13.5, the caudal neuraltube consists mostly of precursor cells—neu-ronal restricted precursors (NRPs) and glialrestricted precursors (GRPs)—with lineagerestrictions for neurons and glia, respectively.In the current study, we examined the fate ofE10.5 multipotential NEP cells and a mixedpopulation of E13.5 lineage-restricted cellsafter transplantation into the adult spinal cord,striatum, and hippocampus. To track trans-planted cells reliably in vivo, cells were pre-pared from transgenic rats expressing thehuman placental alkaline phosphatase gene.Our results show that grafted NEP cells sur-vive poorly at 3 days, and no cells wereobserved at later time points in all three CNSregions. These results indicate that most CNSregions do not support survival of multipoten-tial cells derived from fetal CNS. In contrast,at 4 weeks post-graft, lineage-restricted pre-cursor cells showed selective migration alongwhite matter tracts and robust survival in allthree CNS regions. These cells also expressedthe mature neuronal markers NeuN andMAP2, as well as the oligodendrocyte markerRIP and astrocyte marker GFAP. We concludethat mixed populations of lineage-restrictedCNS precursors are well suited to the adultCNS and provide a promising transplant forcellular replacement because of their goodsurvival, robust differentiation and migrationproperties.

P46 USE OF TRANSGENIC RAT CELLS TO TRACK TRANSPLANTED SCHWANN CELLS WITHIN THE INJURED AND INTACT ADULT RAT SPINAL CORDC.E. Hill 1, L.D.F. Moon 1,M.B. Bunge 1,2,3, P.M. Wood 1,2

1 The Miami Project to Cure Paralysis;2 Department of Neurosurgery, 3 Depart-ment of Cell Biology and Anatomy,University of Miami School of Medicine, Miami, FL, USA

Transplantation of Schwann cells can promoteregeneration and myelination of axons afterspinal cord injury. However, the relative con-tribution of host and transplanted cells to thisrepair has been difficult to determine.Recently transgenic rats that ubiquitouslyexpress human placental alkaline phosphatase(PLAP) have been developed and their cellssubsequently demonstrated to express thetransgene in vitro and in vivo for up to sixweeks following transplantation of precursorcells. PLAP cells provide a useful tool forevaluating transplanted cell survival and theirinteractions with host tissue. The PLAP pro-tein is detectable via histochemistry andimmunohistochemistry, and the gene con-struct via PCR. In the present experiment,Schwann cells were isolated from transgenicPLAP rats and transplanted into the uninjured,contused or transected spinal cord. Prior totransplantation PLAP Schwann cells main-tained PLAP expression in vitro, continued toexpress Schwann cell antigenic markers (p75,S100, GFAP), and formed myelin when co-cultured with DRG axons. When transplantedinto the spinal cord, double labeled PLAP+/p75+ Schwann cells were initially observed atthe injection site. Over time double labeledcells decreased. Few double labeled cells wereobserved two weeks after transplantation,however many cells expressed p75. DecreasedPLAP staining corresponded with a decreasein PLAP DNA within the first 24 hours.Together this suggests that the decrease inPLAP staining is due to the death of the trans-planted cells and that host Schwann cells enterthe spinal cord following transplantation.These initial studies suggest that PLAP cells

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may be a useful marker for identifying trans-planted Schwann cells, and their interactionswith host cells, as well as a useful tool foroptimizing the conditions for cells survivalfollowing transplantation.

P47 ELEVATION OF CYCLIC AMP ENHANCES REGENERATIONAND IMPROVES BEHAVIORAL RECOVERY IN SCHWANNCELL-GRAFTED ANIMALS AFTER SPINAL CORD INJURYD.D. Pearse 1, F.C. Pereira 4,A.E. Marcillo 1, Y.A. Berrocal 1,M. Filbin 5, M.B. Bunge 1,2,3The Miami Project to Cure Paralysis 1; Depts of Cell Biology and Anatomy 2, and Neurological Surgery 3; University of Miami School of Medicine, Miami, Florida, USA; Dept of Anatomy, Univer-sity of Sao Paulo, Brazil 4; Biology Dept, Hunter College 5, New York, NY, USA

The search continues for interventions thatwill enable axons to regenerate across theinjury site and into the uninjured tissuebeyond after damage to the spinal cord. It isnow known that central neurons are able toregenerate if an appropriate environment isintroduced. Inhibition of axonal growth isdue, in part, to the abundance of myelin debrisand a glial scar containing proteoglycans andsemaphorins. Central neurons may also beunable to regenerate as a result of an imbal-ance between growth “stop” and “go” signalswithin the cell soma. A promising new strat-egy to stimulate neurons to overcome inhibi-tory signals is the activation of cAMPsignaling pathways. We have employed thisstrategy after thoracic (T8) moderate contu-sion in adult rats, transplanted with Schwanncells (SCs) 1 wk post-injury. Elevation ofcAMP in injured-only and SC grafted animalswas achieved by (i) a one-time db-cAMPinjection near the growth cones at the injurysite (transient elevation); (ii) inhibition ofcAMP hydrolysis globally by subcutaneousadministration of the phosphodiesterase IVinhibitor, Rolipram, immediately after injury(acute) or at 1 wk (delayed) for 2 wk (to pro-long the elevation of cAMP); or (iii) a combi-nation of these treatments. We show that

global inhibition of cAMP hydrolysis byrolipram can produce significant sparing ofsupraspinal and proprioceptive axons and pro-mote myelination when combined with SCgrafts. Furthermore, combination of rolipramwith local increases in cAMP at the growthcone increased the amount of sparing andmyelination, promoted regeneration of sero-tonergic fibers into and beyond SC grafts, andled to significant improvements in locomotorfunction. By using a combination strategy, wefound that cAMP levels are important for pro-tection and regeneration of injured centralnervous system axons in vivo and suggest thatthis treatment could be useful for human spi-nal injuries.Supported by the Christopher Reeve ParalysisFoundation, NINDS, Miami Project andBuoniconti Fund.

P48 THE NICHE FOR NEURAL STEM CELLS AND PROGENITOR CELLS IN THE ADULT RAT BRAINRose Laczko, Sebastien Mambie,Jason Schnack, Katalin Csiszar,Frederic MercierLaboratory of Matrix Pathobiology,University of Hawaii, Honolulu, HI, USA

Fractones are newly revealed extracellularmatrix structures associated with neural stemcells and progenitor cells, as well as astrocytesand macrophages, in the ventricle walls of theadult mammalian brain (Mercier et al., 2002;2003). We speculate that fractones, which arelabyrinthine protrusions of perivascular basallamina within the subependymal layer, are thesites for growth factor/extracellular matrixinteractions, and form the “niche” that governstem cell and progenitor cell proliferation, dif-ferentiation, and migration. Here, we identifiedprotein components of fractones, and estab-lished anatomical relationships between post-mitotic neural cells and fractones. Componentsof fractones were assessed by immunocy-tochemistry using Confocal Laser ScanningMicroscopy on frozen sections of adult ratbrains. Post-mitotic cells were identified bybromodeoxyuridine (BrDU) antibodies on fro-zen sections generated from rats treated withintracerebroventricular (ICV) injections of

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BrDU 1-5 days prior animal sacrifice. In theseexperiments, the animals were either controlanimals or animals treated with ICV perfusionof brain derived neurotrophic factor (BDNF).In all sections examined, we found that frac-tones were uniformly immunoreactive for col-lagen-IV and laminin-gamma-1, two basiccomponents expected in basal lamina struc-tures. Fractones were also immunoreactive forboth matrix metalloproteinase-2 (MMP-2) andMMP-9. Interestingly, some fractones wereintensely immunoreactive for these MMPs. Inaddition, some fractones exhibited collagen-1immunoreactivity. Because both collagen-1 andMMPs are involved in remodeling during bothdevelopment and adulthood, we suggest thatthe presence of these molecules in fractonesreflect remodeling activity associated withadult neurogenesis. Furthermore, we frequentlyobserved post-mitotic BrDU-labeled cells inclose proximity to- or directly in contact with-fractones. These associations were frequentwhen neurogenesis was stimulated via BDNFinjections. Together, these results suggest thatfractones may participate in morphogenicevents, including proliferation of neural stemcells and progenitor cells throughout adulthood.Supported by HCF 436 634 grant.

P49 FRACTONES AND THEIRCOMPONENTS IN ADULTBRAIN AND SPINAL CORDJason Schnack, Katalin Csiszar,Frederic MercierLaboratory of Matrix Pathobiology,University of Hawaii, Honolulu, HI, USA

Fractones are newly revealed extracellularmatrix structures protruding from perivascularbasal laminae, and directly associated withneural stem cells, progenitor cells, plus localmacrophages, microglial cells, and astrocytesin the ventricle walls of the adult mammalianbrain (Mercier et al., 2002; 2003). We previ-ously found that laminin and collagen IV, twoubiquitous components of basal laminae arepresent in fractones. Here, we extended ourinvestigation of fractone composition andextent in the central nervous system (CNS),using immunocytochemistry and ConfocalLaser Scanning Microscopy. First, we con-

firmed that fractones are ubiquitous special-ized basal laminae in adult mammals,characterizing these structures in the ventriclewalls of adult humans, rats, and mice. Thepresence of fractones was investigated in micein the entire CNS, using serial frozen sections,laminin and collagen-IV immunoreactivity. Inaddition to third and lateral ventricles, wherethey were initially characterized, fractoneswere identified in the walls of the olfactoryventricle, fourth ventricle, brain stem, and spi-nal cord. Series of fractones, as imaged byserial punctuation of laminin immunoreactiv-ity, were also found along the rostral migra-tory stream (RMS), the migrating pathway forneuroblasts generated in the dorsal portion ofthe lateral ventricle, en route towards theolfactory bulb. Immunoreactivity for perlecan,a major heparan sulfate proteoglycan (HSPG)involved in the binding, activation, and pre-sentation of growth factors involved in tissueremodeling, cell proliferation and differentia-tion such as FGF2, was specifically associatedwith fractones in all ventricle walls. Nidogen,a matrix molecule binding laminins to col-lagen-IV, was specifically associated with asubpopulation of fractones. These results sug-gest that fractones may form a functionalextracellular interface between connective tis-sue cells (such as macrophages always associ-ated with fractones), neural cells or theirprogenitors, contributing to the “niche” thatregulates growth factor and cytokine activity,and governs neural cell proliferation, differen-tiation, migration, and function throughoutadulthood.Supported by HCF 436 634 grant.

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P50 THE EFFECTS OF P75 AND RHO PATHWAY INHIBITION ON THE SPROUTING OF DESCENDING AMINERGIC FIBRES FOLLOWING DORSAL ROOT INJURYJaimie F. Borisoff, Angela L. Magel, Andrew D. Gaudet, Matt S. Ramer1ICORD (International Collaboration On Repair Discoveries) The University of British Columbia BiosciencesBuilding, 6270 University Blvd.Vancouver, BC, CANADA, V6T 1Z4

A dorsal rhizotomy and the subsequent degen-eration of distal axons and terminals results inthe deafferentation of the dorsal grey matter inthe spinal cord. The increase in deafferentedpost-synaptic targets may promote the sprout-ing of descending supraspinal axons and pos-sibly lead to changes in the sensorimotorfunction of injured animals. Previous workhas shown that a variety of inhibitory mole-cules and environments may be overcome bytreatment with pharmacological inhibitors ofthe Rho-GTPase pathway, leading to anincreased propensity for axonal regenerationand/or sprouting. Recent evidence indicatesthat inhibitory myelin proteins signal throughthe p75 neurotrophin receptor (p75NTR) andRho. Here, the Rho-kinase inhibitor Y-27632was applied intrathecally in rats for sevendays following septuple dorsal rhizotomy. Aswell, wild-type and p75NTR knockout micewere similarly treated with NGF or NT-3. Thesprouting of serotonergic (5-HT) and noradr-energic fibres in the spinal cord dorsal hornwas then assessed. The application of Y-27632, NGF, and NT-3 significantly increasedthe density of 5-HT- and tyrosine hydroxylase(TH)-immunolabled fibres in both the ipsi-and contralateral spinal cord. Increases in thedensity of both fibre types were also seen inp75NTR knockout mice compared to wildtypein the presence and absence neurotrophins.The data suggest that sprouting of aminergicfibres is influenced by neurotrophins and sig-naling through the p75/Rho pathway.Supported by the CRPF, NSERC, and theMSFHR.

P51 THE ROLE OF P75 INREGENERATION ACROSS THE DORSAL ROOT ENTRY ZONEAngela L. Magel, Jaimie F. Borisoff, Matt S. RamerInternational Collaboration of Repair Discoveries (ICORD), University of Brit-ish Columbia, Vancouver, B.C., CANADA

The biological function of the p75 neurotrophinreceptor (p75NTR) in axonal regeneration fail-ure is poorly understood. Although this receptorhas been implicated in the promotion of regener-ation through neurotrophin binding, p75NTRhas also been suggested to act as a transducer formyelin-derived inhibitory proteins, responsiblefor the activation of GTPase Rho, and the conse-quential collapse of growth cones. Followingseptuple dorsal rhizotomy in homozygoticp75NTR knockout mice, we have been able todirectly examine the effect of p75NTR on theregenerative capacity of sensory neurons, in boththe absence and presence of exogenous neu-rotrophic treatment in vivo. Immunohistochemi-cal analysis of the of the transganglionically-transported tracers (B-fragment of cholera toxinand wheat germ agglutinin), injected intraneu-rally, several days prior to sacrifice, revealed asignificant increase in p75-/- neural regenerationinto the central nervous system through the dor-sal root entry zone, compared with the wild-typestrain. Interestingly, topical treatment of neu-rotrophin-3 (NT3) or nerve growth factor (NGF)in the p75NTR knockout strain only provided asmall, limited advancement of this regenerativeability compared to a similar treatment in wild-type mice. In a similar model, poor regenerationof injured dorsal root sensory axons occurred inWistar rats treated intrathecally with a specificRho Kinase antagonist (Y-27632). These resultssuggest that p75NTR participates in the inhibi-tion of regeneration following dorsal root injuryin the absence of Rho Kinase activation. Basedon these results, we propose that the absence ofp75 in sensory neurons leads to an increasedavailability of Schwann-cell derived neurotro-phins for trk binding in the regenerating dorsalroot, and enhanced regeneration into the spinalcord.Supported by: Christopher Reeve Foundation,Michael Smith Foundation of Health Research,and the Natural Sciences and EngineeringResearch Council of Canada.

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P52 VESICULAR GLUTAMATETRANSPORTER 1 AS ANANATOMICAL MARKER OF ADULT RAT DORSAL COLUMN AXON SYNAPSE FORMATIONJ.M. Massey 1,4; M.E. Bickford 2;X.P. Zhang 2,4; B. Giamarra 2;C.B. Shields 3,4; F.J. Roisen 2,4;D.S.K. Magnuson 2,4;S.M. Onifer 2,41 MD, PhD Program; 2 Departments of Anatomical Sciences and Neurobiology;3 Departments of Neurological Surgery; 4 Kentucky Spinal Cord Injury Research Center, University of Louisville Schoolof Medicine, Louisville, KY, USA

Several strategies have recently been reportedthat allow dorsal column axonal regenerationafter spinal cord injury. For this regeneration tobe meaningful, functional synapses must berestored with target neurons. Here we reportthat vesicular glutamate transporter 1 (vGluT1)may be useful as an anatomical marker of func-tional synapses made by adult rat dorsal col-umn (DC) axons with secondary cuneatenucleus (CN) neurons. At low intensity(1.5 mA) forepaw stimulation, short latencysomatosensory evoked potentials (SSEPs)were recorded from the contralateral soma-tosensory cortices of both normal and incom-plete cervical dorsal spinal cord hemisection(CDH) rats but not from complete CDH rats atvarying post-SCI times. The SSEPs recordedfrom incomplete CDH rats were of longerlatency and smaller amplitude compared tonormal SSEPs. After bilateral forepaw choleratoxin B subunit (CTB) injections, confocalimages of transverse sections through thebrainstem revealed vGlut1/CTB co-labeled ter-minals throughout the forepaw representationarea of the normal CN. These vGluT1/CTB-labeled terminals made apparent synapses withneuronal nucleus/microtubule associated pro-tein 2-labeled CN neurons. In contrast, fewervGluT1/CTB co-labeled terminals wereobserved in incomplete CDH rats and nonewere observed in a complete CDH rat. Addi-tionally, vGlut1-labeled terminals seen in acomplete CDH rat were largely restricted to theareas of the CN corresponding to the locationsof terminals from primary afferent axons origi-nating above the injury. Little vGlut1 was

observed at the end of CTB-labeled axons atthe DC injury site. These results suggest thatterminals of DC axons that have made func-tional synapses express vGlut1. Furthermore,SSEPs recorded from the somatosensory cor-tex reflect the number of vGlut1/CTB-labeledDC axon terminals observed in the CN.Supported by NIH NS40411, NIH RR15576,and KY NSF EPSCoR EPS-9874764.

P53 ATTENUATION OFRUBROPSPINAL ATROPHYBY LENTI-VIRAL VECTORMEDIATED BDNF EXPRESSIONIN THE RED NUCLEUSJie Liu, Clarrie Lam,Loren Oschipok, Armin Blesch*, Wolfram TetzlaffICORD, International Collaboration On Repair Discoveries University of British Columbia, Vancouver, BC, CANADA; *Department of Neuroscience, University of California, San Diego, CA, USA

Rubrospinal neurons undergo a massive atro-phy after axonal injury at the level of the cer-vical spinal cord. This atrophy occurs duringthe second week and reaches a plateau after1–2 months. In previous studies we haveshown that this atrophy can be fully pre-vented by the infusion of BDNF into thevicinity of the rubrospinal cell bodies (Koba-yashi et al. 1997, J. Neurosci 17:9583). More-over, even a delayed treatment with BDNF, aslate as one year post lesion, reverses this atro-phy and restores the regenerative capacity ofthese neurons (Kwon BK, Liu J et al. 2002,PNAS 99:3246). This suggests that regenera-tion after chronic spinal cord injury might befeasible eventually. Here we have askedwhether alternative methods of BDNF deliv-ery could replace the rather traumaticintraparenchymal infusion via osmoticminipumps. We injected a lentiviral vectorexpressing BDNF into the vicinity of therubrospinal neurons at the time of spinalinjury (dorsolateral funiculotomy, C4) and inanother group of animals at one months afterthe lesion. The expression of the viral con-struct was assessed by GFP fluorescence inand around the red nucleus as well as by

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immunohistochemical staining of the injec-tion site for BDNF. Inflammatory changes atthe injection site were minor as evidenced bysome mononuclear cell infiltration. Cell pro-file sizes were found around 71% in animalstreated with BDNF-virus versus 56% in GFP-virus controls (p=0.06; n=3) at one monthsafter axotomy. In situ hybridization for Ta1and GAP-43 are currently underway to testwhether this viral BDNF delivery is sufficientto sustain the regenerative mode of theseneurons. In addition, we will present data forrats that received the BDNF-virus treatmentas late as 2 months to test whether it canreverse chronic atrophy of rubrospinalneurons.Supported by CIHR and BC-Neurotrauma.

P54 INTEGRIN-LIGAND BINDING MODULATES NEURONAL BDNF AND TRKB EXPRESSIONChing-Yi Lin, Christine M. GallDepartment of Anatomy & Neurobiology, University of California, Irvine, CA, USA

Integrin class adhesion proteins influence sig-naling and surface expression of tyrosinekinase trophic factor receptors in various celltypes. Moreover, integrin binding to extracel-lular matrix (ECM) ligands regulates geneexpression in non-neural cells. Findings in ourlaboratory indicate that integrins also regulatetrophic activities of adult central nervous sys-tem (CNS) neurons. Studies of mature hippoc-ampal slices showed that treatment with theintegrin ligand peptide gly-arg-gly-asp-ser-pro(GRGDSP) increases gene expression for theneurotrophins BDNF and NGF, and for theBDNF receptor trkB. The present study evalu-ated mechanisms through which integrins reg-ulate neurotrophin expression and signaling.In situ hybridization and real time PCR analy-ses show that GRGDSP-induced increases ingene expression depend entirely on NMDAreceptors and, in part, on L-type voltage sensi-tive calcium channels (VSCCs). Western blotanalyses show that GRGDSP treatmentincreases tyrosine phosphorylation of NMDAreceptor subunits NR2A and NR2B and of thea1 L-type VSCC subunit, and that co-treat-ment with the NMDA receptor antagonist

APV blocks GRGDSP-induced increases in a1phosphorylation. Together these findings indi-cate that integrin-ligand binding upregulatesneurotrophin expression via sequential effectson NMDA receptor and L-type VSCC phos-phorylation and function. Current studies indi-cate that integrin ligation also influences cellsurface expression of codistributed receptorsincluding the BDNF receptor trkB. Togetherthese findings highlight the importance ofmatrix interactions in sustaining endogenoustrophic factor expression and signaling, andthe potential value of manipulating the ECMenvironment to facilitate neuronal growth andsurvival effects of endogenous neurotrophinsfollowing damage to the adult CNS.Supported by NINDS grant NS37799.

P55 NEUROTROPHIC FACTORS EXPRESSED IN BOTH THECORTEX AND THE SPINAL CORD INDUCE AXONAL PLASTICITY AFTER SPINAL CORD INJURYLijun Zhou1,2, H. David Shine1–4

1Department of Neurosurgery; 2Center for Cell and Gene Therapy; 3Divisionof Neuroscience; 4Department of Molec-ular and Cellular Biology, BaylorCollege of Medicine, Houston, TX, USA

We recently reported that overexpression ofneurotrophin-3 (NT-3) by motoneurons in thespinal cord of rats will induce sprouting of cor-ticospinal tract (CST) axons (Zhou et al., 2003).We now report that overexpression of brain-derived neurotrophic factor (BDNF) or glialcell-derived neurotrophic factor (GDNF) in therat sensorimotor cortex near the CST neuronalcell bodies together with overexpression of NT-3 in the lumbar spinal cord significantlyincreases axonal sprouting over that induced byNT-3 alone. Two weeks after unilaterallylesioning the CST at the level of the pyramids,we injected rats with saline or adenoviral vec-tors (Adv) carrying genes coding for BDNF(Adv.BDNF), GDNF (Adv.GDNF) or EGFP(Adv.EGFP) at six sites in the sensorimotorcortex, while delivering Adv.NT-3 to motoneu-rons in each of these four groups on thelesioned side of the spinal cord by retrogradetransport from the sciatic nerve. Four days later,

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biotinylated dextran amine (BDA) was injectedinto the sensorimotor cortex on the unlesionedside to mark CST axons in the spinal cord.Morphometric analysis of axonal sprouting3 weeks after BDA injection showed that thenumbers of CST axons crossing the midline inrats treated with Adv.BDNF or Adv.GDNFwere 46% and 52% greater, respectively, thanin rats treated with Adv.EGFP or PBS (p<0.05).These data demonstrate that sustained localexpression of neurotrophic factors in the sen-sorimotor cortex and spinal cord will promoteincreased axonal sprouting after spinal cordinjury, providing a basis for continued develop-ment of neurotrophic factor therapy for centralnervous system damage.

P56 DECORIN SUPPRESSESNEUROCAN, BREVICAN,PHOSPHACAN AND NG2EXPRESSION AND PROMOTES AXON GROWTH ACROSS ADULT RAT SPINAL CORD INJURIESJ.E. Davies 1, X. Tang 1,J.W. Denning 1, S.J. Archibald 3,S.J.A. Davies 1,21 Department of Neurosurgery; 2 Divi-sion of Neuroscience, Baylor Collegeof Medicine, Scurlock Tower Suite 944, 6560 Fannin Street, Houston, TX, USA; 3 Integra LifeSciences, 311C Enterprise Drive Plainsboro, NJ, USA

The formation of misaligned scar tissue by avariety of cells types expressing multiple axongrowth inhibitory proteoglycans presents aphysical and molecular barrier to axon regener-ation after adult spinal cord injuries (Tang etal., J. Neuro. Res. 71:427-444 [2003]). Decorinis a small, leucine rich proteoglycan that haspreviously been shown to reduce astrogliosisand basal lamina formation in acute cerebralcortex stab injuries (Logan et al., Exp. Neurol.159:504-10 [1999]). We have therefore testedwhether mini pump infusion of hr-decorin intoacute stab injuries of the adult rat spinal cord can not only inhibit formation of an astrogliallimitans but also deposition of the axon growthinhibitory proteoglycans neurocan, NG2, phos-phacan and brevican. Combined immunohis-tochemical and quantitative Western blotanalysis revealed major reductions in levels of

core protein expression (>80 for 130kD neuro-can, 145/80kD brevican, 300kD phosphacan)and immunoreactivity for all four CSPGswithin decorin treated injuries compared tountreated controls. Astrogliosis within lesionmargins and the accumulation of OX42+ mac-rophages/microglia within lesion centers werealso significantly reduced. These decorininduced changes in scar formation combined topromote the striking ability of axons frommicrotransplanted adult sensory neurons toenter, grow within and exit decorin infused spi-nal cord injuries, in sharp contrast to the com-plete failure of GFP+ axons to cross untreated,CSPG rich lesions. The ability of decorin topromote axon growth across acute spinal cordinjuries via a coordinated suppression ofinflammation, CSPG expression and astroglialscar formation make decorin treatment a prom-ising component of future spinal cord regenera-tion strategies.

P57 ROLE OF SULFATEDPROTEOGLYCANS INTHE GLIAL SCAR ASTROCYTEHang Wang, Yasuhiro Katagiri,Herbert M. GellerDevelopmental Neurobiology Group, NHLBI, NIH, Bethesda, MD, USA

It is our hypothesis that soluble mediators causechanges in astrocyte physiology which causethem to become reactive and inhibitory to neu-ronal regeneration, in large part to their synthe-sis of chondroitin sulfate proteoglycans(CSPGs). Here we present data using cell cul-ture models that 1) transforming growth factor-beta1 (TGF-B1) treated astrocytes are a modelof the glial scar and 2) proteoglycan glycosami-noglycans and sulfation are essential for theinhibitory activity of CSPGs. Treatment ofmonolayers of mouse astrocytes with TGF-B1reduced the ability of these astrocytes to supportneuronal growth and upregulated the synthesisof sulfated proteoglycans. Addition of inhibitorsof glycosaminoglycan (GAG) chain synthesisor sulfation reduced this inhibitory activity toneuronal growth, suggesting that sulfation is anessential feature. RT-PCR data indicated thatTGF-B1 also upregulated the transcriptionallevels of several sulfotransferases that produce4- and 6- sulfated proteoglycans. In order to

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more directly test the role of GAG chain sulfa-tion, we investigated the behavior of axons at a boundary between CS-A (a predominantly 4-sulfated sugar) and PLL. We found that axonsfrom neurons on PLL did not cross onto CS-A,but the crossing percentage is significantlyincreased after treatment with 4- and/or 6-sulfa-tase. FACE sugar structure analysis confirmedthat 4- or 6-sulfate is removed by these sulfa-tases. Furthermore, overexpression of chon-droitin-4-sulfotransferase in astrocytes reducedthe length of axons plated onto these astrocytes.In summary, our current observations demon-strate that TGF-B1 induces the upregulation ofastrocyte-associated sulfated proteoglycans andsulfotransferase mRNA, and this matrix inhibitsneuronal growth. Moreover, the sulfation com-position and pattern of the sugar moiety is verycritical to the inhibitory properties of CSPGs.

P58 LIBERATION OF HEAT SHOCK PROTEINS (HSPS) FROMSTEROID RECEPTORS IS A POTENTIAL MECHANISMOF STEROID-INDUCEDNEUROPROTECTION INMOTONEURONS IN VITROJ.E. Tetzlaff 1,3; K.J. Jones 2,31 Neuroscience Program, LoyolaUniversity, Maywood, IL, USA; 2 Department of CBN & Anatomy,Loyola University, Maywood IL,USA;3 R&D Services, Hines VA Hospital, Hines, IL, USA

Steroid hormone treatments such as testoster-one and estrogen have been found to be neuro-protective in both in vivo and in vitro injurymodels. The protective effects of both test-osterone and estrogen have been investigatedin our laboratory in vivo following variousperipheral nerve injuries. We have found thatphysiological levels of both steroid hormonesaccelerate regeneration and behavioral recov-ery following peripheral nerve injury in vivo.The mechanism of steroid-induced neuropro-tection is unknown. The present study investi-gated the ability and the potential mechanismof steroid hormone treatments to rescueinjured embryonic rat spinal motoneuronhybrid cells transfected with the human andro-gen receptor gene (a generous gift from

Dr. K.H. Fischbeck). In addition to expressingthe androgen receptor, these motoneuronshybrid cells may also express the estrogenreceptor. Steroid hormone treatment was foundto be protective in vitro following a 30-minuteheat shock at 11° above normal incubationtemperature. Future experiments have beenestablished to investigate the mechanism forsteroid-induced neuroprotection. It is hypothe-sized that the existence of protective HSPscomplex with steroid hormone receptors maybe the mechanism for steroid-induced neuro-protection. In vitro co-immunoprecipitationexperiments are planned to investigate whetheror not steroid treatment liberates reputedly pro-tective HSPs from the steroid receptor thusproviding a potentially protective pool ofHSPs following steroid treatment and injury.Supported by: NIH NS28238

P59 CADHERIN LOCALIZATION AND INJURY-INDUCED PLASTICITY WITHIN THE ADULT RAT SPINAL CORDJ.H. Brock, A.M. Elste, G.W. HuntleyFishberg Research Center forNeurobiology, The Mount Sinai Schoolof Medicine, New York, NY, USA

Different somatic sensations originating inperipheral tissues are conveyed via sensoryaxons that have highly precise, distinct centraltermination patterns within the dorsal horn ofthe spinal cord. Some large-diameter myeli-nated axons (light touch) terminate in laminaIII-V, while small-diameter non-myelinatedaxons (pain and temperature) terminate in lam-ina I/II. The mechanism of such laminar-spe-cific targeting is poorly understood. Variousfamilies of cell adhesion molecules have beenimplicated in this process during development,including the Ig superfamily, integrins, ephrins,and cadherins. Because of their involvement inneurite outgrowth, laminar targeting and synap-togenesis during development, we chose toinvestigate cadherin expression and localiza-tion with subpopulations of sensory afferentcentral terminations within the adult rat dorsalhorn. Sciatic nerve injections of cholera subunitB (CTB) and wheat germ agglutinin (WGA)label specific central terminations of distinct

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sensory afferent fibers. CTB labels termina-tions of large diameter myelinated fibers inlamina III and WGA labels terminals of smalldiameter non-myelinated fibers in lamina I/II.Confocal and immuno-gold EM analyses con-firm that E-cadherin is present specifically atWGA-labeled terminals, while N-cadherinassociates with CTB-labeled terminals. There-fore, cadherins may be involved in specifyingsynaptic targets of primary afferent fibers. Inaddition we are investigating the involvementof cadherins in injury-induced plasticity withinthe spinal dorsal horn. Peripheral nerve injurycauses tactile allodynia, where a normally non-noxious stimulus is increasingly perceived as nociceptive. It has been postulated that struc-tural reorganization of sensory afferents withinthe dorsal horn may be involved. Our resultssuggest a bidirectional change in E-cadherinand N-cadherin protein levels within the dorsalhorn after sciatic nerve transection. E-cadherinprotein levels are decreased specifically withinthe sciatic nerve termination zone of lamina IIinner, while N-cadherin protein levels isincreased within lamina I/II.Supported by NIH/NINDS: NS044868, NINDSNRSA: NS44718, Christopher Reeve ParalysisFoundation.

P60 THE THROMBIN PROTEASE ACTIVATED RECEPTOR (PAR-1) AFTER SPINAL CORD CONTU-SION INJURY: UPREGULATIONIN GFAP POSITIVE ASTROCYTESW.D. Whetstone 1, L.J. Noble 21 Division of Emergency Medicine,University of California San Francisco; 2 Department of Neurosurgery UCSF, CA, USA

Spinal cord injury produces prominent disrup-tion of the blood spinal cord barrier, an acuteinflammatory reaction and an astrocyticresponse. One of the earliest mediators of theseeffects following tissue injury is the serine pro-tease thrombin. It has previously shown thatthrombin, acting through the Protease Activat-ing Receptor (PAR-1) opens endothelial gapjunctions, causes neutrophil infiltration,directly modulates the shape of astrocytes andinduces neurite retraction. We used a spinal

cord contusion model to examine PAR-1 recep-tor expression following injury. PAR-1 receptorexpression was characterized by immunocy-tochemistry in both uninjured and 1,3,7,14,and21 day post injury tissue. In uninjured mice, thePAR-1 receptor appears to be weakly expressedprimarily throughout the spinal cord gray mat-ter in cells with a motoneuron phenotype. Fol-lowing injury, there appears to be a robustPAR-1 signal within GFAP positive astrocytesat the injury epicenter and rostral and caudal tothe injury site. Our findings demonstrate thatthe thrombin PAR-1 receptor appears to beupregulated in GFAP positive astrocytes fol-lowing contusion injury and may play animportant role in subsequent gliosis.Supported by K08-NS43302, and R01-NS39278, NS39847, the DANA Foundation,and the Roman Reed Spinal Cord InjuryResearch Fund of California.

P61 INVOLVEMENT OF N-END RULE PROTEIN DEGRADATION INNEURITE OUTGROWTH AND AXONAL REGENERATION WITH PARTICULAR REFERENCE TO THE UBIQUITIN-CONJUGATING ENZYME E2-14KDP. Kavakebi, A. Tomasino,L. KlimaschewskiUniversity of Innsbruck, Institute of Anatomy, Histology and Embryology, Department of Neuroanatomy, Mueller-strasse 59, A-6020 Innsbruck

We are investigating the role of the ubiquitin/proteasome system—the principal regulator ofintracellular protein levels—in neurite out-growth and axonal regeneration. Previous workdemonstrated that NGF-mediated neuronal dif-ferentiation of pheochromocytoma (PC12) cellsis accompanied by increased levels of highmolecular weight ubiquitin-conjugates in PC12nuclei and decreased levels of free ubiquitin incytoplasmic and nuclear extracts. The searchfor enzymes responsible for increased utiliza-tion of ubiquitin revealed an up-regulation ofE2-14kD mRNA in NGF-treated PC12 cells.E2-14kD mRNA is a component of the N-endrule pathway, which is implicated in the cleav-age of proteins with destabilizing N-terminalresidues that include bulky hydrophobic or

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basic amino residues. We show that E2-14kD ispresent in peripheral neurons and localizes tothe nucleus and plasma membrane. Down-reg-ulation of E2-14kD mRNA by small interferingRNA or treatment of cells with dipeptide-inhib-itors of the N-end rule pathway suppress NGF-induced neurite outgrowth and axonal elonga-tion of peripheral neurons obtained from adultrats. The present data indicate that E2-14kD isup-regulated during neuronal differentiationand is required for axonal regeneration inperipheral neurons in vitro.Supported by the FWF (P15376).

P62 NEURON-INTRINSICLIMITATIONS TO AXONREGENERATION IN THEDEVELOPING SPINAL CORDMurray Blackmore, Paul LetourneauDept. of Neuroscience, University of Minnesota, MN, USA

Unlike adults, embryonic birds and mammalsshow a remarkable ability to regenerate axonsin the injured spinal cord. This regenerativeability is lost during a discrete developmentaltransition. Two main factors likely cause thistransition: The emergence of inhibitory mole-cules in the spinal cord environment and adecline in the intrinsic ability of neurons toregenerate injured axons. To quantify the con-tributions of environmental versus neuron-intrinsic factors we are using as a model theembryonic chicken, which loses the ability toregenerate spinal cord axons on embryonicday 13 (E13). We have developed a culturesystem in which explants of brainstem neu-rons are placed adjacent to spinal cord tissue,and axon regeneration is quantified as theages of brainstem neurons and spinal cordenvironment are varied independently. Wefind that as the spinal cord ages from E9 toE17, it supports 50% less axon regeneration.Thus changes in the spinal cord alone reducebut do not abolish axon regeneration. How-ever, as brainstem neurons age from E9 to E15they show a 90% reduction in axon regenera-tion, even in the permissive environment ofthe younger spinal cord. A similar reductionin axon growth from older brainstem neuronsis observed on substrates of laminin. TUNEL

staining reveals no difference in cell death incultured brainstem of different ages, suggest-ing that the reduction in axon regeneration isnot secondary to increased cell death. In addi-tion, video analysis of living axons shows thatolder brainstem neurons elongate axons 50%more slowly than younger neurons. Com-bined, these data suggest that neuron-intrinsicchanges contribute significantly to the failureof axon regeneration in the developing spinalcord.Supported by NIH, HHMI, and Morton CureParalysis Fund.

P63 VERTEBRAL COLUMN FIXATION ALLOWS BETTER STUMPSEALING AFTER COMPLETESPINAL CORD LESION C. Muñoz-Quiles, B. Piedrafita,A. Ramón-Cueto, F.F. Santos-BenitoLaboratory of Neural Regeneration, Insti-tute of Biomedicine, Spanish Council for Scientific Research, Valencia, SPAIN

A treatment for spinal cord injuries is still amajor challenge. In the past, a variety ofapproaches in rodents elicited regeneration ofspinal axons and partial functional recovery.Complete spinal cord transection, after multiplelaminectomy, seems the most proper model tostudy axonal regeneration in experimental ani-mals. However, after these extensive lesionsany vertebral column movement may increasethe gap between the cord stumps, change theirdorsoventral orientation or cause spinal cordbending and, consequently, additional damage.To provide spinal cord stability during the pro-cess of healing, we have designed in rats amethod to fix the vertebral column by using abridge made of inlay pattern resin (Duralay).This bridge was placed covering the gap cre-ated by laminectomy, immediately after lesion,and both ends were attached to the spinous pro-cesses and laminae of the adjacent intact verte-brae. Sealing effectiveness of the stumps wasanalyzed fifteen days, one and four monthspost-surgery. To evaluate possible side effectsof the bridge, we performed laminectomies,without spinal cord lesion, in sham operatedanimals. In all paraplegic rats, dense connectivetissue was formed beneath the bridge and

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above the spinal cord. Both stumps wereattached with no gap between them suggesting that in addition to stabilization, the bridge mayprotect the spinal cord lesion site. Our bridgesprevent the development of scoliosis, allowingcorrect walking and limb position. Moreover,placing a bridge seems essential to properlymaintain the cord stumps for cell transplanta-tion time after injury (chronic stage). In sum-mary, we have developed a reliable and simplemethod for vertebral column fixation in ratswith spinal cord injury, that may be advanta-geously used in combination with differentrepair strategies to reduce the variabilitybetween individuals and between differentregeneration studies.Supported by grants FIS 01/1134, SAF2001-2242, Foundations Ramón Areces and FIRSN.

P64 COURSE OF MOTOR RECOVERY FOLLOWING VENTROLATERAL SPINAL CORD INJURY IN THE RATAubrey A. Webb 1*, Gillian D. Muir 1,2Department of Veterinary Biomedical Sciences 1; Cameco MS and Neuro-science Research Center 2; University of Saskatchewan, Saskatoon, SK, CANADA. *Present affiliation: Department of Bio-medical Sciences 3, University of Prince Edward Island, Charlottetown, PE, CANADA

The purpose of this study was to determine theimportance of the pathways running in theventrolateral spinal funiculus for overgroundlocomotion in adult, unrestrained rats. Left-sided ventrolateral cervical spinal cord injurywas performed in adult female Long-Evansrats. The behavioural abilities of these animalswere analyzed at 2 days, and at 1.5, 2.5, 3.5,4.5 and 5.5 weeks following spinal cord injury.Behavioural testing consisted of von Frey fila-ment testing, ladder walking, a paw usage task,and the assessment of ground reaction forcesduring trotting. Animals with injury to the leftventrolateral cervical (C3) spinal cord did notdevelop enhanced sensitivity to pedal mechan-ical stimulation at 5.5 weeks following injury.At 2 days following injury, animals hadimpaired skilled locomotion as indicated byincreased number of footslips during ladder

walking. At 2 days, these animals also usedboth limbs together more often for supportwhile rearing and the forelimb ipsilateral to theinjury less than before spinal cord injury.Ground reaction force determination revealedthat animals tended to bear less weight on theforelimb ipsilateral to the spinal cord injuryand have substantially different footfall pat-terns during locomotion 2 days after injury. Allanimals returned to normal or near normal sen-sorimotor, including locomotor, abilities by 5.5weeks following spinal cord injury. Only verysubtle alterations in ground reaction forceswere detectable by 5.5 weeks following spinalcord injury. These results support the currentdogma that there is substantial functionalredundancy of pathways traveling in the ven-tral spinal cord of the adult rat. Presumably,therapies need not be directed at regeneratingpathways in the ventral spinal cord in theirentirety.

P65 BIODEGRADABLE POLYMER IMPLANTS AS A PLATFORMFOR OPTIMIZING SPINAL CORD INJURY REPAIR STRATEGIESS. Ameenuddin, A. Knight, B-K. Chen,M. Moore, A. Krych , H. Olson,K. Galvin, P. Sabharwal, L. Gross,R. Spinner, M. Yaszemski, B. Currier,A.J. WindebankSpinal Cord Injury Research Program, Mayo Clinic College of MedicineRochester, MN, USA

Spinal cord axons have the potential to regener-ate into a supportive microenvironment. Togain functional recovery, three questions mustbe answered: 1) What is the optimal micro-environment for axonal regeneration? 2) Howcan axons be guided to grow through residualcord towards their cellular targets? 3) What isrequired to re-establish interneuronal functionalconnections? Biodegradable polymer scaffoldsmay be used to gain answers to the first ques-tion and may contribute information to the sec-ond two. Using polymer chemistry and micro-tissue engineering techniques, we have usedbiodegradable polymer implants to begin tooptimize the structural, molecular and cellularmicro-environment to promote regeneration.Poly DL-lactic-co-glycolic acid (PLGA) and

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polycaprolactone fumarate (PCLF) scaffoldscontaining multiple parallel aligned channelshave been used to bridge a 2 mm gap in the ratspinal cord (T8-10). Channels of differentdiameter have been constructed (450 and660 µm). Scaffolds have been pre-loaded withSchwann cells (SC), a SC precursor line(SpL201), or fibroblasts in matrigel. The bridgehas also been implanted with or without con-current systemic treatment with prednisoloneand/or minocycline or tetracycline. In parallelexperiments we have studied the release kinet-ics of chondroitinase-ABC and a synthetic ana-log of pigment epithelium derived growthfactor (PEDF44). Microscopic magnetic reso-nance imaging, morphometry and three dimen-sional tissue reconstruction have been used todetermine the ability of different micro-envi-ronments to support regeneration. Morphomet-ric analysis of neurofilament stained axonsdemonstrated that axons and capillaries grewthrough the length of the channels. Wild-typeSC provide the best cellular support. Biologi-cally active PEDF44 and chondroitinase-ABCwere released from PLGA microspheres dem-onstrating that encapsulation does not degradethe peptides. Data will be presented to addressthe role of channel diameter and potential neu-roprotective strategies (minocycline and pred-nisolone). In the future, this model will providea valuable platform to rapidly assess the valueof different protective or supportive strategiesto repair function after spinal cord injury.

P66 AXONAL REGENERATION THROUGH NERVECOAPTATIONS IS ENHANCEDBY DEGRADATION OFCHONDROITIN SULFATEPROTEOGLYCAND. Muir, D. Neubauer, J.B. Graham,C.A. KrekoskiDepartments of Neuroscience and Pediat-ric Neurology, University of Florida Col-lege of Medicine, Gainesville FL, USA

Injury to peripheral nerve initiates a degenera-tive process that converts the denervated nervefrom a suppressive environment to one thatpromotes axonal regeneration. We investigatedthe role of matrix metalloproteinases (MMPs)in this degenerative process and whether effec-

tive predegenerated nerve grafts could be pro-duced in vitro. Rat peripheral nerve explantswere cultured for 1-7 days in various media andtheir neurite-promoting activity was assessedby cryoculture assay, in which neurons aregrown directly on nerve sections. The neurite-promoting activity of cultured nerves increasedrapidly and, compared to uncultured nerve, amaximum increase of 72% resulted by 2-dayculture in the presence of serum. Remarkably,the neurite-promoting activity of short-termcultured nerves was also significantly betterthan nerves degenerated in vivo. We examinedif in vitro degeneration is MMP-dependent andfound that the MMP inhibitor GM6001 largelyblocked the degenerative increase in neurite-promoting activity. In the absence of hematoge-nic macrophages, MMP-9 was trivial whereaselevated MMP-2 expression and activation par-alleled the increase in neurite-promoting activ-ity. MMP-2 immunoreactivity localized toSchwann cells and the endoneurium and colo-calized with gelatinolytic activity demonstratedby in situ zymography. Lastly, in vitro prede-generated nerves were tested as acellular graftsand, compared to normal acellular nerve grafts,axonal ingress in vivo was approximately dou-bled and preliminary findings indicateimproved functional recover with these graftsas well. We conclude that Schwann cell expres-sion of MMP-2 plays a principal role in thedegenerative process that enhances the regener-ation-promoting properties of denervatednerve. Combined with their low immunogenic-ity, acellular nerve grafts activated by in vitropredegeneration may be a significant advance-ment for clinical nerve allografting.

P67 EMBRYONIC SPINAL CORD MOTOR NEURONS EXHIBITPREFERENTIAL OUTGROWTHON PRELESIONED PERIPHERAL NERVE TISSUE SECTIONSIN VITROS.R. Chadaram 1, R.D. Madison 1,21 Neurosurgery, Duke University, Durham, NC, USA; 2 VA Medical Research Service, VAMC, Durham, NC, USA

Previous studies have shown that motor neu-rons from embryonic rat spinal cord explants

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exhibit preferential outgrowth on stripes ofmuscle membranes in vitro, according to theirappropriate rostrocaudal position (Wang et al.,J Neurosci. 19: 4984-93,1999; Chadaram et.al., J Neurobiol. 2003 Sep 15; 56(4): 347-59).In the present set of experiments, we exploredthe outgrowth of E-15 ventral spinal cord(motor neurons) on peripheral nerve tissuesections from normal or lesioned (7 days post-denervation) sciatic nerves of adult rats. Cry-ostat sections of alternating normal andlesioned nerves were plated onto a neucle-opore filter. Explants attached and extendedneurite bundles on both substrates, howevermore extensive outgrowth (measured both interms of length and number of neurite bun-dles) was evident on sections from prele-sioned nerve. Of the 121 individual culturesthat were examined, 77 explants extendedneurites on both substrates while 31 grew onlyon prelesioned nerve sections, the remaining13 grew only on normal nerve sections. Com-paring just the explants that grew on bothtypes of substrate, the overall average lengthof the neurite bundles was longer on the prele-sioned substrate (359 vs. 241 microns; p <0.005), as was the average number of neuritebundles per explant (15.5 on prelesioned vs.9.3 on normal; p < 0.05), Individually, 72 ofthe 77 explants that grew on both substrateshad longer and more neurite bundles on theprelesioned nerve section. These results showboth preferential outgrowth and more exten-sive outgrowth on the prelesioned tissue sub-strate. These findings are in agreement withour previous study looking at outgrowth onstripes of nerve membranes (Soc. Neurosci.245.6, 2003), and reports from other laborato-ries using embryonic sensory neurons platedonto tissue-section substrates of adult sciaticnerve. The current report is the first to showthat E-15 ventral spinal cord explants alsopreferentially extend neurites, most likelyfrom primary motor neurons, onto sectionsmade from prelesioned adult nerve.Supported by the NIH (HD41747 to RDM)and the Office of Research and Development,Medical Research Service, Department ofVeterans Affairs (RDM). RDM is a ResearchCareer Scientist for the Medical Research Ser-vice, Department of Veterans Affairs.

P68 REDUCTION OFPOSTOPERATIVE NEURONAL LOSS AND IMPROVEDRECOVERY OF VIBRISSAE MOTOR PERFORMANCE BY COPOLYMER-1 AFTER FACIAL NERVE REPAIR IN MICE*

M. Lenzen 1, T.L. Tomov 1,O. Guntinas-Lichius 2, W.F. Neiss 1,J. Kipnis 3, M. Schwartz 3,D.N. Angelov 11 Institut I für Anatomie; 2 HNO-Klinik der Universität zu Köln; 3 Weizmann Institute of Science, Rehovot

Although the etiology of motoneuron degenera-tive diseases varies, they all share similar path-ways which culminate in motoneuron death.Based on a recent discovery that the immunesystem protects from nerve insult, we show thatvaccination with Copaxone (Cop-1, glatirameracetate) protects mice motoneurons againstacute degeneration. Animals were distributed in3 groups (A, B, C). The mice of group A (n = 8)underwent transection and suture of the facialnerve (facial-facial anastomosis, FFA). BeforeFFA all animals of group B (n = 10) received asubcutaneous injection of Cop-1 plus CompleteFreund’s Adjuvant (CFA) and those of GroupC—phosphate buffer saline (PBS) and CFA(n = 9). Two months after surgery, alterationsin the vibrissal motor performance (2D/ManualAdvanced Video System PEAK Motus 2000)and the degree of motoneuron loss (counts afterretrograde labeling with 1% Fluoro-Goldinjected into the whisker pad musculature)were estimated. All animals that underwentFFA after Cop-1 sensitization showed a signifi-cantly better recovery than those without treat-ment. This was best demonstrated by theamplitude of whisking, which measured 38.9 ±10.6° (group B), 22.1 ± 9.9° (group C) and 11.0± 6.0° (group A). The mean value before sur-gery was 40.0 ± 14.0°. Cop-1 vaccination waseffective in inhibiting motoneuron death. Eightweeks after FFA, the number of motoneuronswhich survived and reinnervated the whiskerpad was 1172 ± 152 in group B, but 670 ± 178in the axotomized non-vaccinated (group A)and 766 ± 104 in the axotomized vaccinatedwith CFA only. In line with the experimentallybased concept of protective autoimmunity, the

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present findings suggest that Cop-1 vaccinationboosts local immune responses needed to com-bat destructive endogenous compounds associ-ated with motoneuron death.*Supported by the Köln-Fortune Programm.

P69 EXPRESSION OF SEMAPHORIN RECEPTORS: THE PLEXINS, AFTER PERIPHERAL VS. CENTRAL NERVOUS SYSTEM INJURYE.D. Spinelli 1,2; L.W. Oschipok 1,2; L.T. McPhail 1,2; J. Liu 1;W. Tetzlaff 1–31 ICORD (International Collaboration on Repair Discoveries), 2 Department of Zoology, 3 Department of Surgery;University of British Columbia,Vancouver, BC, CANADA

Semaphorins form a large family of moleculesimplicated in neuronal guidance events indevelopment, as well as in adulthood. Themostly inhibitory nature of Semaphorins hasled to the speculation that they may be involvedin the inability of injured central nervous sys-tem (CNS) neurons to regenerate. The Sema-phorin receptor complex is comprised ofNeuropilins: The ligand binding subunit, andPlexins, the signal transducing subunit, whichhas been shown to link to intracellular path-ways converging onto the cell cytoskeleton. Inthe present study, we show in injured, non-regenerating rubrospinal neurons CNS, Plexin-A2 mRNA expression increased, while Plexin-A1 mRNA did not change. In contrast, ininjured facial neurons (peripheral nervous sys-tem: PNS), which are able to regenerate,Plexin-A2 mRNA expression decreased whilePlexin-A1 mRNA did not change. We are pres-ently expanding our study by analyzing themRNA expression of other members of thePlexin receptor family: Plexins-A3, A4 and B1,in the injured rubrospinal CNS and facial(PNS) neurons. By understanding how Sema-phorins and their receptors respond to injury,we hope this may lead to potential target mole-cules usable in a therapeutic approach to pro-moting regeneration after injury in the CNS.Supported by: the Rick Hansen NeurotraumaInitiative and the Christopher Reeve ParalysisFoundation.

P70 REGENERATION OFSUPERNUMERARY AXONSIN THE ADULT RAT SPINAL CORD IN A CAUDA EQUINA INJURY AND REPAIR MODELT.X. Hoang, J.H. Nieto, L.A. HavtonDepartment of Neurology and Brain Research Institute, UCLA, Los Angeles, CA; Maimonides Medical Center, New York, NY, USA

We have previously shown that lumbosacralventral root avulsions lead to autonomic andmotor neuron death in an adult rat model ofcauda equina injury (Hoang et al., 2003, JCN,in press). However, implantation of theavulsed ventral root into the spinal cord canprotect these neurons against cell death andpromote reinnervation of the implantedavulsed ventral root. Here, we studied someof the long-term effects (6 weeks to 5 months)of this surgical neural repair strategy. We per-formed a unilateral avulsion of the L5-S2 ven-tral roots with an acute implantation of theavulsed L6 ventral root into the spinal cord.Using immunohistochemistry for cholineacetyltransferase (ChAT), we demonstrated inthe neuropil on the ipsilateral side of the cordseveral unusual and aberrant ChAT positivestructures with an axonal phenotype. Thesestructures exhibited a uniform diameter, anirregular course, occasional branching, andlarge terminal bouton-like swellings. Manysupernumerary axons demonstrated growthtowards the implanted avulsed ventral root.The atypical arbors were reminiscent ofregenerating supernumerary axons. Doubleimmunohistochemical labeling for ChAT andp75, a low affinity neurotrophic factor recep-tor, demonstrated in confocal microscopycolocalization of ChAT and p75 immunofluo-rescence in these atypical axon-like structures.The studies suggest that axotomized cholin-ergic neurons may generate ChAT positivesupernumerary axons, many of which extendtowards the implanted ventral root graft. Ourstudies also support recent suggestions forp75 to play a role in axonal elongation.Supported by: NIH/NINDS (NS042719), Stateof California Roman Reed Funds, ParalysisProject of America.

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P71 INCREASED NEURONAL NITRIC OXIDE SYNTHASE EXPRESSION PRECEDES PROGRESSIVE DEATH OF AUTONOMIC NEURONS IN A CAUDA EQUINA INJURY MODELB.T. Franchini 1, T.X. Hoang 1,J.H. Nieto 4, N.J.K. Tillakaratne 2,3;L.A. Havton 1,3Department of Neurology 1;Department of Physiological Science2; Brain Research Institute 3; UCLA, Los Angeles, CA, USA; Maimonides Medical Center 4, New York, NY, USA

We have previously demonstrated progressivedeath of autonomic preganglionic parasympa-thetic neurons (PPNs) and motoneurons in aunilateral L5-S2 ventral root avulsion injurymodel in the adult male rat. Here we report atransient increase in both the frequency andconcentration of neuronal nitric oxide syn-thase (nNOS) expression in these populationsthat precedes neural death. PPNs and moto-neurons were identified using retrogradely-transported FluoroGold, and nNOS wasdetected using immunohistochemistry. Stereo-logical counts were used to determine thenumber of nNOS-positive neurons in unin-jured control lumbosacral spinal cords, alongwith counts performed at 3-day, and 1, 2, 4,and 6-week post-injury survival times. Thesecounts indicate a marked increase in the fre-quency of nNOS-positivity in PPNs, mostnotably at 3-days post-injury, as compared tothe non-injured side. Densitometry and sizemeasurements suggest increased density ofnNOS immunoreactivity within PPN cell bod-ies at all post-injury time points. Qualitatively,PPNs on the injured side show much darkernNOS staining extending further into theirdendritic projections. We believe an increasednNOS concentration may mediate the progres-sive death of PPNs and motoneurons follow-ing ventral root avulsion, and may provide anavenue for therapeutic intervention followingtraumatic cauda equina injury.Supported by: NIH/ NINDS (NS042719), TheCalifornia State–Roman Reed Bill, The Paral-ysis Project of America.

P72 FILUM TERMINALE IN THE RAT, NEW MODEL FOR STUDIES OF AXONAL REGENERATION INTHE ADULT CENTRAL NERVOUS SYSTEM (CNS)J.M. Kwiecien, R. Avram, J. Tang,C. Hui, J. BainDepartments of Pathology and Molecu-lar Medicine and of Surgery, McMaster University, Hamilton, Ontario, CANADA

CNS myelin is inhibitory to regeneration ofinjured axons. Dysmyelination in the adultCNS, such as in the Long Evans Shaker (LES)rat coincides with spontaneous axonal plastic-ity observed as abundant sprouting and withregeneration of transected axons in the spinalcord. Massive injury of the spinal cord in theadult rat however, results in severe motor def-icits and loss of bladder function requiringintensive and expensive animal care. Wedetermined the anatomy and function of thefilum terminale (FT), a slender extension ofthe terminal spinal cord—the conus med-ullaris (CM)—towards the tail. FT is definedby a pia limitans, contains the central canalsurrounded by a narrow rim of axons inter-spersed by oligodendrocytes and astrocytesbut not neurons. FT is >3 cm long and nar-rows from 170 microns at the CM to50 microns at 3 cm distal to CM. In a studyaddressing axonal regeneration in the adultdysmyelinated CNS, FT was crushed 2 mmcaudal to the CM and rats allowed survival for2 weeks. Neurological deficits typical in thespinal cord injury such as paraplegia and uri-nary bladder dysfunction were absent. In crosssections of FT taken from the site of crush to3 cm caudal, there was abundant axonalregeneration observed in close associationwith ependymal cells up to 3 cm distal to thecrush. In a tracing experiment, a dextranetracer microinjected into CM as detected inthe FT >2 cm caudal to the crush site. Thisnew model of adult CNS injury allows fordetailed studying of axonal regeneration with-out the necessity of inflicting a cripplinginjury on the model animal.

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P73 SEROTONIN 1A RECEPTORACTIVATION OF A LATENT MOTOR PATHWAY AFTERSPINAL HEMISECTIONM.B. Zimmer, H.G. GoshgarianWayne State University, Department of Anatomy and Cell Biology, Detroit, MI, USA

Cervical spinal cord injury (SCI) (C2 hemisec-tion) results in paralysis of the ipsilateralhemidiaphragm, and subsequent alteration ofthe breathing pattern; breathing frequencyincreases and tidal volume decreases. A latentneural pathway, which runs down the cord, con-tralateral to hemisection and crosses distal tothe site of injury, can be activated under condi-tions of elevated respiratory drive, thus, restor-ing activity to the paralyzed hemidiaphragm.Since, activation of serotonin 1A receptorsincreases respiration and can reverse the alteredbreathing pattern induced by SCI, we hypothe-sized that activation of serotonin 1A receptorswould activate the latent motor pathway(crossed phrenic response) in paralyzed rats. C2hemisection was performed under sterile condi-tions 24 hours prior to experimentation. Phrenicnerve activity (respiration) was recorded inanesthetized, vagotomized, paralyzed, venti-lated Sprague Dawley rats, and the serotoninagonist, 8-OH-DPAT, was administered (IV).We found that SCI alone did not increase respi-ratory frequency in SCI rats (n=10) comparedto controls (n=7). Since central drive was main-tained constant and peripheral feedback wasremoved, the changes in breathing patternobserved in vivo by others must occur throughmodification of peripheral feedback mecha-nisms, but not at the central respiratory rhythmgenerator. 8-OH-DPAT increased frequency andpeak height of phrenic activity in controls,whereas frequency increased only in 50% ofSCI rats. While the peak height of phrenicactivity on the contralateral side increased morein SCI rats than controls, the latent phrenicpathway was only activated in SCI rats that alsoshowed an increase in frequency. This suggeststhat increasing the frequency component ofneural impulses rather than total neural activitymay preferentially drive activation of this latentpathway.Supported by NIH grant HD31550.

P74 SPONTANEOUS FUNCTIONAL RECOVERY IN A PARALYZED HEMIDIAPHRAGM FOLLOWING CERVICAL SPINAL CORD INJURY AND CAROTID BODY DENERVATION IN ADULT RATSKwaku Nantwi, Han Bae,Harry GoshgarianDepartment of Anatomy and CellBiology, Wayne State University,School of Medicine, Detroit, MI, USA

Previous studies have demonstrated that in ananimal model of spinal cord injury, a latentrespiratory motor pathway can be activated torestore function to a hemidiaphragm paralyzedby upper cervical (C2) spinal cord hemisec-tion. It has also been demonstrated that func-tional recovery of respiratory activity canoccur spontaneously in C2 hemisected ani-mals following prolonged post hemisectionperiods (2–4 months) without any therapeuticintervention. It is presently unknown whetherperipheral respiratory chemoreceptors locatedin the carotid body are involved in spontane-ous functional recovery after hemisection. Theobjective in the current investigation was toassess the effects of carotid body denervationon the onset of spontaneous functional recov-ery in C2 hemisected animals. Briefly, animalswere anesthetized with ketamine, 70mg/kgand xylazine, 7mg/kg and were then subjectedto bilateral carotid body denervation andallowed to recover for 5–6 days. All animalswere monitored closely post surgery. Follow-ing recovery from carotid body denervation,all animals were reanesthetized and subjectedto a cervical (C2) hemisection (HCBD group).Control animals were not subjected to carotid body denervation. Electrophysiologicalassessment of respiratory function was con-ducted 2 weeks after hemisection, i.e. approxi-mately 3 weeks after carotid body denerva-tion. In experiments thus far (n=8), recoveryof respiratory activity in the hemidiaphragmparalyzed by C2 hemisection occurred in themajority (n=6) of cases at only 2 weeks posthemisection. Two animals did not survive thecarotid body denervation and hemisection.Our findings thus far contrast markedly fromthe delayed onset of spontaneous recovery inC2 hemisected animals (with the carotid body

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intact) noted in our previous study. Our resultssuggest that carotid body denervation acceler-ates mechanisms that underlie spontaneousrecovery of the paralyzed hemidiaphragm fol-lowing C2 hemisection.Supported by NIH grant (HD 35776).

P75 INJURY-INDUCED SPINAL CORD PLASTICITY IN THE MOUSE—THE CROSSED PHRENICPHENOMENONL.K. Akison, H.G. Goshgarian*,N.W. SeedsDepartment of Biochemistry & Molecu-lar Genetics, University of Colorado Health Sciences Center, Denver CO, USA; *Department of Anatomy & Cell Biology, Wayne State University,Detroit MI, USA

The crossed phrenic phenomenon (CPP)describes respiratory functional plasticity thatarises following spinal cord injury. Cervicalspinal cord hemisection rostral to the phrenicnucleus paralyzes the ipsilateral hemidia-phragm by interrupting the descending flowof respiratory impulses from the medulla tophrenic motoneurons in the spinal cord. Thisloss of activity converts some synapses onphrenic motoneurons from a “functionallyineffective” state pre-hemisection to a “func-tionally latent” state post-hemisection. If theanimal is subjected to respiratory stress bytransecting the contralateral phrenic nerve,this latent respiratory pathway is activated andfunction is restored to the paralyzed hemidia-phragm. The mechanisms underlying this syn-aptic plasticity are not well-defined. Ourultimate aim is to understand the underlying molecular mechanisms of this functionalrecovery using a mouse model amenable to amolecular genetic approach. Although thephenomenon has been shown in dogs, cats,rabbits, guinea pigs and rats, this is the firstreport of CPP in mice. We have shown theCPP qualitatively in mice using electromyo-graphic (EMG) recordings from the dia-phragm. In particular, we examined the inter-operative delay time between the hemisectionand contralateral phrenicotomy required for aresponse. For each animal, a spinal cord

hemisection was performed on the left side atC2. A contralateral phrenicotomy was per-formed the next day (overnight animals), 6–8hours, 4–5 hours, or 1–2 hours post-hemisec-tion. An abdominal laparoscopy was per-formed to expose the diaphragm and bipolarelectrodes were placed on the left and righthemidiaphragms. EMG recordings wereamplified and filtered to reduce spurious sig-nals and electrocardiographic activity. Whilerecording, the animals were observed toensure that the EMG signal was respiratory-related inspiratory activity. As the interopera-tive delay was reduced, the proportion of micedisplaying the CPP decreased from 100% forovernight animals, 94% in 6–8h, 85% in 4–5h,to 74% for 1–2h mice, and <30% for animalsreceiving a phrenicotomy under 1 hour.Supported by NIH—NS44129 (NWS).

P76 MK-801 ACCELERATESRECOVERY OF THE IPSILAT-ERAL HEMIDIAPHRAGMFOLLOWING A LEFT C2 HEMISECTION IN THE RATWarren J. Alilain 1,Harry G. Goshgarian 1,21 Department of Psychiatryand Behavioral Neurosciences,Cellular and Clinical NeurobiologyProgram; 2 Department of Anatomyand Cell Biology, Wayne StateUniversity School of Medicine Detroit, MI, USA

A left cervical (C2) hemisection disrupts thebulbospinal projections from the rostral ventralrespiratory group to the phrenic motor nucleusand results in paralysis of the ipsilateral hemid-iaphragm. Spontaneous recovery of the hemidi-aphragm does occur, however, starting at sixweeks post injury. Studies have demonstratedthat increased mRNA levels for the 2A subunitof the N-methyl-D-aspartate (NMDA) recep-tor following a contusion injury at the thoraciclevel (T8) of the spinal cord correlate withimproved hindlimb function in chronicallyinjured rats. MK-801, a non-competitiveNMDA receptor antagonist, has demonstratedthe pharmacological ability to preferentiallyupregulate the mRNA levels of the 2A subunit

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in neonatal rats. In the present study, wehypothesized that pharmacological upregula-tion of the 2A subunit would facilitate recoveryof diaphragmatic function after C2 hemisec-tion. Female Sprague-Dawley rats underwent aleft C2 hemisection and were assessed for com-plete paralysis of the ipsilateral hemidia-phragm. One week post lesion, MK-801 wasadministered at a dosage of 0.50 mg/kg i.p.once per day for two days to upregulate the 2Asubunit. Two days following the last adminis-tration of MK-801 (9 days post injury) the dia-phragm was evaluated by electromyogram(EMG) recordings. Diaphragmatic EMG activ-ity was observed in the initially paralyzedhemidiaphragm and was similar to spontaneousrecovery observed at much later post hemisec-tion periods in our earlier studies. The resultssuggest that administration of MK-801 acceler-ates the spontaneous recovery of diaphragmaticfunction following a left C2 hemisectionthrough upregulation of the NMDA receptor2A subunit.Supported by NIH Grant 35501.

P77 ROLE OF ATP/P2 RECEPTORSIN THE ACTIVATION OF ASTRO-CYTIC PROTEIN KINASE B/AKT BY TRAUMATIC INJURYJ.T. Neary, Y. KangResearch Service, Miami VA Medical Center and Departments of Pathology, Biochemistry & Molecular Biology; Neu-roscience Program, University of Miami School of Medicine, Miami, FL, USA

Traumatic brain injury (TBI) activates proteinkinase B (Jenkins et al., 2002), also known asAkt, a key signaling molecule that promotes cellsurvival and inhibits apoptosis. Akt phosphory-lation has been implicated in neuronal survivalafter TBI (Noshita et al., 2002), but little isknown about injury-induced Akt activation inastrocytes, cells that exhibit hypertrophic andhyperplastic responses to central nervous sys-tem injury. Here we have investigated the effectof mechanical strain on Akt activation in pri-mary cultures of rat cortical astrocytes with thein vitro TBI model developed by Ellis et al.(1995). We have also examined the mechanismsthat lead to trauma-induced Akt activation. Akt

activation was determined by probing immuno-blots with an antibody that recognizes Akt phosphorylated at serine 473; total Akt wasmeasured as a loading control. We found thatwhen astrocytes were subjected to mechanicalstrain (50 msec; 7.5 mm displacement), Aktphosphorylation was increased at 3 min postin-jury, was maximal from 5 to 10 min anddeclined gradually thereafter. Akt activationwas dependent on the severity of the injury.Stretch-induced Akt phosphorylation was atten-uated by blocking phosphoinositide 3-kinase(PI3K), an upstream activator of Akt, withwortmannin. Because ATP is released afterstrain-induced injury (Ahmed et al., 2000), weexamined the role of extracellular ATP in theactivation of Akt. We observed that P2 receptorantagonists such as iso-pyridoxal-5’phosphate-6-azophenyl-2’5’-isulfonate (PPADS) attenuated trauma-inducedAkt activation. In uninjured astrocytes, wefound that extracellular ATP stimulated Aktphosphorylation via P2 receptors (Broch et al.,2003). We conclude that mechanical straincauses activation of Akt in astrocytes via stimu-lation of P2 receptors and PI3K signaling. Thissuggests that the P2 receptor/Akt pathway pro-motes astrocyte survival and may play a role inthe generation of reactive gliosis after TBI.Supported by the Department of VeteransAffairs.

P78 DIETARY FACTORS CANMODULATE THE CAPACITY OF THE BRAIN TO COMPENSATE FOR TRAUMATIC BRAIN INJURY (TBI)A. Wu, Z. Ying, F. Gomez-PinillaNeurosurgery, Div. UCLA Brain Injury Research Center, Dept. Physiological Sciences, Los Angeles, CA, USA

We have previously shown that a diet high insaturated fat (HF) decreases levels of brain-derived neurotrophic factor (BDNF), to theextent that compromises cognitive perfor-mance and aggravates the outcome of TBI.Here we present evidence that antioxidanttherapy with vitamin E compensates for dam-age incurred by this diet. These results showthat oxidative stress can interact with theBDNF system to modulate synaptic plasticity

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and cognitive function. These studies suggesta mechanism by which events classicallyrelated to the maintenance of energy balanceof the cell, such as oxidative stress, can inter-act with molecular events that modulate neu-ronal and behavioral plasticity. In addition,here we show that good diets such as a dietsupplemented with omega-3 fatty acids (e.g.DHA) can help to compensate for TBI. DHAplay a crucial role in signal transduction, regu-lation of gene expression, and neuronal pro-tection from neuronal apoptotic death. Wehave examined the possibility that omega-3fatty acids supplements may produce benefi-cial effects on the outcome of traumatic braininjured animals. Rats were fed a regular dietor experimental diet containing 8% fish oil,for 4 weeks before a mild fluid percussioninjury (FPI) was performed. FPI resulted inimpairment of learning ability in the Morriswater maze and reduction in BDNF and itsdownstream effectors on synaptic plasticitysuch as synapsin I and CREB. The DHA dietcompensated for the reduced learning capac-ity, and for the decrease in BDNF, synapsin I,and CREB, resulting from FPI.Supported by NINDS 39522, and UCLABrain Injury Research Center.

P79 CHANGES IN EXPRESSION OF NOGO, NOGO-66 RECEPTOR AND SMALL PROLINE-RICH REPEAT PROTEIN 1A FOLLOWING EXPERIMENTAL TRAUMATIC BRAIN INJURY IN RATSNiklas Marklund*, Saori Shimizu,Rishi Puri, Diego Morales,Asenia McMillan, Kristie Soltesz,Deborah Castelbuono, Carl T. Fulp, Tracy K. McIntoshAll at Head Injury Center, Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA*Presenting author

Axonal injury is a common finding followingexperimental and clinical traumatic braininjury (TBI). The post-traumatic expression ofinhibitors of axonal growth such as Nogo, act-ing on the Nogo-66 receptor (NgR), maypotently prevent axonal outgrowth and con-

tribute to the poor functional recovery seenfollowing TBI. The small proline-rich repeatprotein 1A (SPRR1A) was shown to be animportant promoter of axonal outgrowth fol-lowing peripheral nerve injury, but has notpreviously been detected in brain. Nogo, NgRand SPRR1A expression was evaluated 1, 3,and 7 days following fluid percussion (FP)brain injury of moderate severity in rats. Therats were perfused with 4% paraformalde-hyde, and primary antibodies against Nogo(1:8000), NgR (1:3000) and SPRR1A(1:1000; all antibodies a generous gift fromDr. Strittmatter, Dept. of Neurology, Yale)were used on 40 µm brain sections. Additionalsections were double labeled with the neu-ronal marker microtubule-associated protein(MAP)-2. In all injured rats (n=8), there wasan increased expression of Nogo in ipsilateralcortex, hippocampal subfield CA3 and thereticular thalamus as compared to shaminjured animals, and the increased expressionwas predominately seen in MAP-2 positivecells. In addition, increased expression ofNogo was seen in ipsilateral external capsuleand fimbriae. Increased SPRR1A expressionwas seen in MAP-2 positive cells in ipsilat-eral cortex and CA3 from one day post-injury,with the most marked increase at 7 days post-injury. Changes in NgR were less prominent,although a modest increase was seen in ipsi-lateral cortex in occasional animals. Ourresults show that FPI causes a changedexpression of proteins promoting (SPRR1A)and inhibiting (Nogo/NgR) axonal outgrowth.These changes may be important contributorsto the cognitive and neurological motorsequelae of TBI.Supported by NIH NS RO140978, a MeritReview Grant and a VA-DOD ConsortiumGrant from the Veterans Administration, andNIH NS P5008803.

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P80 REDUCED CENTRAL NERVOUS SYSTEM (CNS) SCAR FORMA-TION AND LESS NEUROMOTORDEFICIT WITH ENRICHEDENVIRONMENT COMBINED WITH MULTIMODAL STIMULATION AFTER TRAUMATIC BRAIN INJURY IN RATS*

M. Maegele, T. Ester-Bode,D.N. Angelov, T.K. McIntosh,E.A.M. Neugebauer,M. Lippert-GruenerUniversity of Cologne/Merheim Clinic, Cologne, Germany, University of Penn-sylvania, PA, USA

The present study was designed to investigatepossible effects of enriched environment (EE)combined with multimodal early onset stimu-lation (MEOS) versus standard housing (SH)without stimulation on neuromotor functionand lesion volume after experimental trau-matic brain injury (TBI). Sprague-Dawley rats,randomized to the following groups:(i) injured/EE+MEOS; (ii) sham/EE+MEOS;(iii) injured/SH; (iv) sham/SH, underwentmoderate fluid-percussion or sham injury.Thereafter EE+MEOS groups were placed intomodified cages containing various types ofbedding and stimulating objects. Along withEE animals underwent motor, olfactory, audi-tory and visual stimulation (MEOS). In con-trast, injured and sham SH groups were housedindividually without stimulation. Neuromotorfunction was assessed using a composite Neu-roScan (NS) test battery at 24h, 7, and 15 dayspost injury (DPI). On 15 DPI animals weretranscardially perfused and brains were har-vested for single coronal section immunocy-tochemistry for NSE, Caspase 3 active, andGFAP. Nissl counter-staining allowed demar-cation of zones containing reactive astrocytesenabling a quantification of the projection areaoccupied by GFAP-positive astrocytes in eachimmunoreacted section. Values for total areaof all sections and the section thickness servedfor calculation of the lesion volume in eachrat's brain. Neuromotor function was markedlyreduced in both injured groups at 24h post-injury being non-significant. However,injured/EE+MEOS animals performed signifi-cantly better when tested for neuromotor func-

tion compared to injured/SH animals on DPI 7(p < 0.05) and DPI 15 (p < 0.5). Better neuro-motor function in EE+MEOS animals at 15DPI was associated with a significantly smallerlesion volume compared to SH animals (14.28+/- 6.83 mm3 vs. 23.46 +/- 4.31 mm3). Thisfirst report on combined EE+MEOS post-trau-matic treatment indicates that exposure toEE+MEOS may reverse neurological deficitsand reduce CNS scar formation after TBI inrats.*Supported by the Koeln-Fortune Program/Faculty of Medicine, University of Cologne(Germany), No. 47/2002 (26800597).

P81 DIFFERENTIAL RESPONSESOF AXONS TO TRANSECTION: INFLUENCE OF NG2R. de Castro Jr., R. Tajrishi,J. Fukushi, W.B. StallcupDevelopmental Neurobiology Program, The Burnham Institute, N. Torrey Pines Road, La Jolla, CA, USA

On the basis of in vitro experiments, the NG2chondroitin sulfate proteoglycan has been pro-posed as an inhibitor of central nerve regener-ation. To test this possibility in vivo, we haveused wildtype and NG2 null mice to examinethe response of three types of axons to spinalcord transection. Corticospinal tract axonsdescending through the ventral region of dor-sal white matter do not regenerate in bothwildtype and NG2 knockout animals. In con-trast, CGRP-positive sensory afferents areobserved with equal frequency within thetransection scars of wildtype and NG2 nullanimals. Limited sprouting of descendingserotonergic axons is also seen within the scartissue of both groups of animals, but is morecommon in the presence of NG2. These stud-ies therefore do not support the concept ofNG2 as a general inhibitor of axon regenera-tion in the central nervous system. In somecases NG2 may even be stimulatory to axonre-growth. Our results suggest that nerveregeneration is a heterogeneous process thatdepends on the specific responses of differentclasses of axons to molecular cues present inthe injured nervous system.

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P82 CHEMOKINE EXPRESSIONFOLLOWING SPINAL CORD INJURY IN DIFFERENT STRAINS OF MICEM. Palmer, O. StewardReeve-Irvine Research Center, University of California at Irvine, CA, USA

Spinal cord injury in mice triggers a differentset of cellular responses than is seen in rats andother species. The present study was under-taken to asses expression of chemoattractantcytokines in different strains of mice. C57BL/6,C57BL/10, FVB/n, CD-1 and BALB/c micereceived complete crush injuries at theT8 level, and mRNA was isolated from a 8mmsection surrounding the lesion. Expression ofRantes, Eotaxin, MIP1alpha, MIP1beta, MIP2,IP10, and MCP1 were assessed using ribonu-cleotide protection assays at 0, 6, 12, 24, 48,72 hours, 7 days and 10 days after injury. Therewas a dramatic induction of chemokine expres-sion in all strains, with peak expression occur-ring at 6-12hrs post-injury. BALB/c miceexhibited lower levels of induction of lympho-cyte attractant MIP1alpha, MIP1beta, andRantes than other strains. C57BL/6 and

C57BL/10 strains exhibited higher levels ofIP10 at 12hrs post-injury than all other strains(p < 0.0001), whereas there were no strain dif-ferences in IP10 expression by 24 hr post-injury. C57BL/6, C57BL/10 and FVB/n strainsexhibited a second phase of expression ofMIP1alpha, MIP1beta, and Rantes that beganat 3 days post-injury and continued to increaseup to 10 days. This second peak was not seen inBALB/c mice; instead, cytokine expressionremained low after the initial peak. CD-1 miceexhibited continuously elevated levels ofMIP1alpha and MIP1beta up to 10 days afterinjury. The higher initial peak levels, and con-tinued expression of Rantes, MIP1beta,MIP1alpha, and IP10 expression suggest thatthere may be a persistent signal for T-cellrecruitment in C57BL/6, B57BL10, and FVB/nstrains that is not present in BALB/c. The con-sequences of these differences in chemokineinduction on inflammatory cell recruitmentremain to be defined.Supported by the Roman Reed Spinal CordInjury Research Fund of California, Researchfor a Cure.

poster abstracts-session 2 · of medicine, philadelphia, pa, usa; 2 laboratory of neuroscience,...

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