NT-3 gene delivery elicits growth of chronically injured corticospinalaxons and modestly improves functional deficits

after chronic scar resection

Mark H. Tuszynski,a,b,* Ray Grill,a,1 Leonard L. Jones,a Adam Brant,a Armin Blesch,a

Karin Low,a Steve Lacroix,a and Paul Lua

a Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0626, USAb Veterans Administration Medical Center, San Diego, CA 92165, USA

Received 3 July 2002; revised 6 November 2002; accepted 18 November 2002

Abstract

Nervous system growth factors promote axonal growth following acute spinal cord injury. In the present experiment, we examinedwhether delivery of neurotrophic factors after chronic spinal cord injury would also promote axonal growth and influence functionaloutcomes. Adult Fischer 344 rats underwent mid-thoracic spinal cord dorsal hemisection lesions. Three months later, primary fibroblastsgenetically modified to express human neurotrophin-3 (NT-3) were placed in, and distal to, the lesion cavity. Upon sacrifice 3 months later(6 months following the initial lesion), NT-3-grafted animals exhibited significant growth of corticospinal axons up to 15 mm distal to thelesion site and showed a modest but significant 1.5-point improvement in locomotor scores (P � 0.05) on the BBB scale, compared tocontrol-grafted animals. Thus, growth factor gene delivery can elicit growth of corticospinal axons in chronic stages of injury and improvesfunctional outcomes compared to non-growth-factor-treated animals.© 2003 Elsevier Science (USA). All rights reserved.

Keywords: Growth factors; Neurotrophic factors; Spinal cord; Spinal cord injury; Regeneration; Neurotrophins; Axon growth

Introduction

A number of studies have reported that the growth ofacutely injured axons of the spinal cord can be promoted bygrowth factor delivery. Whether applied immediately afterinjury (Blesch et al., 1999; Blits et al., 2000; Grill et al.,1997b; Houle and Johnson, 1989; Houweling et al., 1998;Liu et al., 1999; Oudega and Hagg, 1996; Tuszynski et al.,1996, 1994; Xu et al., 1993). or within 2 weeks of injury(Coumans et al., 2001) various growth factors promote thegrowth of specific spinal axonal systems. In some cases,growth factor delivery to the acutely or subacutely injured

spinal cord has been reported to elicit functional recovery(Blits et al., 2000; Coumans et al., 2001; Grill et al., 1997b;Houweling et al., 1998; Liu et al., 1999). To date, however,functional recovery after chronic spinal cord injury has notbeen reported after growth factor delivery, although severalreports have indicated that chronically injured axons retainsensitivity to growth factors (Grill et al., 1997a; Houle andYe, 1997; Jin et al., 2000). For example, when administered3 months after spinal cord injury, nerve growth factor(NGF) gene delivery promoted extensive growth of su-praspinal coerulospinal axons and local sensory axons, in-dicating that chronically injured axons retain long-term sen-sitivity to exogenously supplied growth factors (Grill et al.,1997a). However, functional effects of these manipulationshave not been assessed.

In the present study, we examined whether the cortico-spinal tract growth factor neurotrophin-3 (NT-3) (Blits etal., 2000; Grill et al., 1997b; Houweling et al., 1998; Schnell

* Corresponding author. Department of Neurosciences-0626, Univer-sity of California, San Diego, La Jolla, CA 92093. Fax: �1-858-534-5220.

E-mail address: mtuszynski@ucsd.edu (M.H. Tuszynski).1 Present address: Department of Neurosurgery, University of Texas-

Houston, Houston, Texas 10101.

R

Available online at www.sciencedirect.com

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0014-4886/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S0014-4886(02)00055-9

et al., 1994) would influence axonal growth and functionalrecovery after chronic spinal cord injury. Grafts of primaryfibroblasts genetically modified to secrete NT-3 have pre-viously been reported to promote corticospinal axon growthup to 8 mm distal to an acute spinal cord lesion site and togenerate partial functional recovery of conditioned locomo-tion (Grill et al., 1997b). Such growth required the presenceof host ventral gray matter underlying the lesion site to actas a permissive growth substrate for host axons; the fibro-

blast graft itself did not support corticospinal growth. In thepresent study, genetically modified NT-3-secreting fibroblastswere grafted to a chronic spinal cord injury site. Further, topotentially extend the distance of axon growth distal to thelesion site, NT-3-secreting fibroblasts were also grafted as acell suspension 6 mm distal to the injury (Fig. 1). Outcomeson function and corticospinal axon growth were examined 6months after the original injury or 3 months after grafting ofNT-3-secreting genetically modified cells.

Fig. 1. Nissl stain of lesioned and grafted spinal cords. (a) At time 0, 128 rats underwent spinal cord dorsal hemisection lesions. Three months later, animalsreceived either NT-3-secreting or �-gal-producing fibroblast grafts (G), embedded in collagen, into the lesion site (dashed lines indicate cord interface withlesion and graft; an NT-3-grafted animal is shown). In addition, suspension grafts of the same cell types (G-Dist) were injected into the central gray matterof the spinal cord 6 mm distal to the lesion site. Six months after the original lesion, animals were sacrificed and the number of BDA-labeled axons crossinga vertical line was quantified in each subject at distances of 0, 5, 10, 15, and 20 mm caudal to the lesion site. In addition, cord volume underlying the lesionzone was quantified in each subject (area measured between dotted lines) using NeuroLucida software. GM, gray matter; VWM, ventral white matter. Scalebar, 0.35 mm. (b, c) View of host/graft interface at the lesion site (dashed line) in an NT-3 grafted subject. The graft is closely apposed to host gray matterat the lesion site. Scale bar b, e � 30 �m, c, f � 12 �m. (e, f) �-Gal-producing graft in the lesion cavity is also closely apposed to host gray matter. (d) Arrowsindicate fibroblasts within NT-3-secreting and (g) �-gal-producing grafts. Scale bar d, g � 3 �m.

48 M.H. Tuszynski et al. / Experimental Neurology 181 (2003) 47–56

Methods

Experimental subjects

Adult Fischer 344 rats (128) weighing 160–200 g servedas experimental subjects. Animals were housed 3 per cageand had free access to food and water. Institutional guide-lines for animal safety and comfort were adhered to.

Preparation of autologous, genetically modified cells

Primary syngenic Fischer 344 rat fibroblasts were ge-netically modified to produce and secrete human NT-3, aspreviously described (Grill et al., 1997b; Rosenberg etal., 1988). Briefly, the 908-bp coding sequence for humanNT-3 was inserted in a Moloney leukemia virus retroviralvector lacking the gag, pol, and env genes, and 10 –15 �gof plasmid DNA was transfected into the PA317 ampho-tropic producer cell line (packaging line) by lipofection.Conditioned medium from these cultures was used toinfect primary fibroblasts. In vitro production of humanNT-3 mRNA was verified by Northern blot, and produc-tion of biologically active protein was verified by a sig-nificant increase in the number of the TH-immunolabeledneurons in cultures of E14 fetal anterior rhombencepha-lon by conditioned medium (CM) from cultures of NT-3-transfected cells compared to CM from control-transfected cells (P � 0.05). ELISA indicated that NT-3-transduced fibroblasts produced 21 ng of NT-3/106

cells/day, whereas �-gal fibroblasts produced no detect-able amounts of NT-3. Control fibroblasts were geneti-cally modified to express the reporter �-gal; these cellswere used in grafts in control-lesioned subjects. Thus,cells in control subjects differed from NT-3-transfectedcells by a single gene. Prior to surgical implantation, 2.5� 106 transduced fibroblasts (NT-3 or control) weresuspended into 2 ml of a chilled liquid solution of type Irat tail collagen (Sigma) as previously described(Tuszynski et al., 1996). After incubation for 48 h at37°C, the collagen/cell mixture was cut into small piecesand grafted into in vivo T7 spinal cord dorsal hemisectionchronic lesion cavities in adult Fischer 344 rats (n � 66),as described below. In addition, 3-�l suspension grafts ofNT-3-secreting cells at a concentration of 100,000cells/�l were grafted into the cord eipcenter 6 mm distalto the lesion site at a depth of 1.5 mm using a 10-�lHamilton blunt-ended needle. Control subjects receivedgrafts of primary �-gal-transduced fibroblasts (n � 62animals) to the lesion cavity and suspension grafts of�-gal-producing cells 6 mm distal to the lesion site, asdescribed for NT-3-grafted subjects. Previously, we havereported that these grafts sustain gene expression for timeperiods of least 3 months in vivo (Blesch et al., 1999;Grill et al., 1997b; Tuszynski et al., 1994).

Spinal cord surgery and cell injection

Rats were deeply anesthetized with a mixture (2 ml/kg)of ketamine (25 mg/ml), rompun (1.3 mg/ml), andacepromazine (0.25 mg/ml). Three-millimeter-long spinalcord dorsal hemisection lesions were made at the T7midthoracic level in 128 Fischer-344 rats using a fine-tippedglass aspiration device (Fig. 1), as previously described(Grill et al., 1997b; Weidner et al., 2001). This lesiontransected the entire dorsal corticospinal tract, dorsal col-umn sensory axons, and axons of the dorsolateral spinalcord, but spared host gray matter underlying the lesion sitein order to present a potential growth substrate to injuredaxons. Lesion extent was verified by direct visual guidance.Complete transection of the dorsal corticospinal tract wasensured by observation of total removal of dorsal columnwhite matter under microscopic guidance, leaving only re-maining ventral gray matter. Postoperatively, animals werekept warm, placed on beds of sawdust, received manualbladder evacuation for a period of approximately 10 days,and received im ampicillin (25 mg twice per day) to preventand treat urinary tract infections. Animals regained auto-matic neurogenic bladder function after 5 to 10 days.

Three months later, animals were reanesthetized and theprevious lesion site was reexposed. Scar tissue was resectedat both the rostral and caudal aspects of the lesion site untilnormal spinal cord tissue could be observed (typically adistance of less than 0.5 mm). A piece of type I rat tailcollagen matrix (Sigma) containing 2.5 � 106 geneticallymodified fibroblasts/2 ml of collagen solution was thenpositioned in, and filled, the lesion cavity. Once again, thespinal cord was covered and the surgical site was closed; 66subjects received NT-3-secreting fibroblasts and 62 subjectsreceived �-gal-producing control fibroblasts. To provide apotential neurotropic stimulus for eliciting axonal growthover distances greater than the previously reported 8 mm(Grill et al., 1997b) (Fig. 1), a second partial dorsal lami-nectomy was performed in the same surgical session 6 mmcaudal to the original lesion site. The spinal cord was ex-posed, and 3-�l cell suspensions were injected at a depth of1.5 mm from the dorsal spinal cord surface. Subjects thatreceived NT-3-secreting cells in the lesion site also receivedsuspension grafts of NT-3-secreting cells distal to the lesionsite; subjects that received �-gal grafts in the lesion sitereceived �-gal grafts distal to the lesion site. In this and allsubsequent aspects of the study, experimenters were blindedto group identity.

Functional testing

After an additional 3-month period (6 months after theinitial injury), locomotion was assessed on the 21-pointmodified Basso-Beattie-Bresnehan (BBB) scale (Table 1)over 5 sequential days of testing (Basso et al., 1995). Thecriteria for rating on the 21-point scale are indicated inTable 1. Two different observers blinded to group identity

49M.H. Tuszynski et al. / Experimental Neurology 181 (2003) 47–56

independently evaluated rat performance. Interobservervariability on the BBB scale was no greater than 0.5, andvalues of the two observers were pooled into a single dataset. Repeated measures ANOVA was used to examinegroup difference across days, with significance set at the95% level, using StatView II software.

Histology

Upon completion of functional testing, animals receivedinjections of the anterograde tracer biotinylated dextranamine (BDA) to trace the corticospinal tract, as previouslydescribed (Weidner et al., 2001). Briefly, 100 nl of 10%BDA (Mr 10,000, Molecular Probes) were injected into eachof 12 sites per hemisphere spanning the rostrocaudal extentof the rat sensorimotor cortex (�2.5 �l of 10% BDA peranimal) using a PicoSpritzer II (Weidner et al., 2001). Threeweeks later, animals were transcardially perfused with a 4%solution of paraformaldehyde, postfixed overnight, and thentransferred to a 30% sucrose solution for 3 days. Spinalcords were sectioned in the sagittal plane at 35-�m intervalsin blocks of 15 mm length at, and distal to, the lesion site.To examine lesion extent and graft survival, Nissl stainswere performed in series of 1-in-7 sections in all animals

(Fig. 1). Lesions were considered adequate on Nissl stain ifthey extended through all dorsal white matter in midlinesections of the spinal cord. The observer was blinded togroup identity. To examine growth responses of host corti-cospinal axons, series of 1-in-5 sections were processedusing the avidin–biotin–peroxidase method, as previouslydescribed (Figs. 3 and 4; Weidner et al., 2001). Growth ofthe lesioned corticospinal tract was determined by quanti-fying the number of BDA-labeled axons crossing a verticalline drawn through 35-�m-thick parasagittal sections of thespinal cord at the rostral end of the lesion site, and 5, 10, 15,and 20 mm caudal to the lesion site, as previously described(Schnell et al., 1994). The number of crossing axons wasquantified separately in gray matter, dorsal white matter,and ventral matter in each of 1-in-5 sections labeled forBDA reaction product. The total number of BDA-labeledaxons in each region per animal was summed and meanvalues among groups were compared using two-tailed Stu-dent’s t test.

To determine whether experimental manipulations influ-enced lesion size, the volume of spinal cord at the lesion sitewas measured using the fractionator method and NeuroLu-cida software (Gunderson, 1987). Briefly, the area of spinalcord underlying the lesion site was digitally acquired from

Table 1Modified 21-point BBB scale

Points Criteria

0 No observable hindlimb (HL) movement1 Slight movement of one or two joints, usually the hip and/or knee2 Extensive movement of one joint or extensive movement of one joint and slight movement of one other joint3 Extensive movement of two joints4 Slight movement of all three joints of the HL5 Slight movement of two joints and extensive movement of the third6 Extensive movement of two joints and slight movement of the third7 Extensive movement of all three joints of the HL8 Sweeping with no weight support or plantar placement of the paw with no weight support9 Plantar placement of the paw with weight support in stance only (i.e., when stationary) or occasional, frequent, or consistent weight-supported

dorsal stepping and no plantar stepping10 Occasional weight-supported plantar steps, no forelimb (FL)–HL coordination11 Frequent to consistent weight-supported plantar steps and no FL–HL coordination12 Frequent to consistent weight-supported plantar steps and occasional FL–HL coordination13 Frequent to consistent weight-supported plantar steps and frequent FL–HL coordination14 Consistent weight-supported plantar steps, consistent FL–HL coordination, and predominant paw position during locomotion is rotated

(internally or externally) when it makes initial contact with the surface as well as just before it is lifted off at the end of stance, or frequentplantar stepping, consistent FL–HL coordination, and occasional dorsal stepping

15 Consistent plantar stepping and consistent FL–HL coordination and no toe clearance or occasional toe clearance during forward limbadvancement

16 Consistent plantar stepping and consistent FL–HL coordination during gait and toe clearance occuring frequently during forward limbadvancement

17 Consistent plantar stepping and consistent FL–HL coordination during gait and toe clearance occuring consistently during forward limbadvancement

18 Consistent plantar stepping and consistent FL–HL coordination during gait and toe clearance occuring consistently during forward limbadvancement; predominant paw position parallel at initial contact and lift off; tail up most of the time

19 Consistent plantar stepping and consistent coordinated gait; consistent toe clearance; predominant paw position parallel at initial contact andlift off; tail consistently up; trunk instability

20 Consistent plantar stepping and coordinated gait, consistent toe clearance, predominant paw position parallel throughout stance, consistenttrunk stability, tail consistently up

21 Normal gait

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a Sony camera attached to an Olympus BH-2 microscope at12.5� magnification from each Nissl-stained section of thespinal cord. The areas were summed and multiplied by theintersample interval (175 �m) to obtain a volume estimateof spared spinal cord through the lesion site.

Results

Recipients of NT-3-secreting cells exhibit modest butsignificant differences in functional outcomes

Six months after the initial injury, or 3 months aftergrafting of NT-3-secreting or �-gal-producing cells, loco-motion was assessed on the 21-point modified Basso-Beat-tie-Bresnehan (BBB) (Basso et al., 1995). Over 5 days ofsequential testing, recipients of NT-3-secreting fibroblastsexhibited a mean score of 12.1 � 0.48 (�SEM) on the BBBscale, and control-lesioned subjects exhibited a mean scoreof 10.6 � 0.59 (P � 0.05, repeated measures ANOVA; Fig.2). Thus, NT-3-grafted subjects exhibited a modest butsignificant improvement in locomotor outcomes comparedto lesioned control subjects. A score of 10 or 11 on the21-point BBB scale reflects occasional to consistent weight-supported plantar steps that lack forelimb–hindlimb coor-dination, whereas a score of 12 on the BBB scale indicatesfrequent to consistent weight-supported plantar steps to-gether with occasional forelimb–hindlimb coordination.Thus, functionally, the mean 1.5-point improvement on theBBB scale in NT-3-grafted subjects reflected more consis-tent hindlimb weight support together with occasional hind-limb–forelimb gait coordination.

Recipients of NT-3-secreting cells exhibit a significantimprovement in the number and distance of corticospinalaxons extending distal to a chronic injury site

All animals exhibited complete dorsal hemisection le-sions on Nissl stain (Fig. 1). In 11 subjects (n � 6 NT-3-grafted and n � 5 �-gal-grafted), lesions inadvertentlytransected the spinal cord completely. When the behavioralanalysis was repeated to exclude subjects with completelesions, findings of partial functional recovery in NT-3-grafted subjects persisted (mean BBB score in NT-3-grafted, 12.6 � 0.4; �-gal-grafted, 11.2 � 0.6, P � 0.05).The volume of spinal cord lying ventral to the lesion sitewas also quantified in all subjects to determine whetherNT-3 gene delivery altered lesion size compared to recipi-ents of �-gal-producing grafts. The 1-in-7 parasagittal sec-tions containing ventral spinal cord within 1 mm of thelesion site were scanned into NeuroLucida software andtotal spared spinal cord volume was reconstructed and mea-sured; mean volumes did not differ between NT-3-graftedand �-gal-grafted groups (NT-3, 3.3 � 1.2 mm3; �-gal, 2.9� 1.5 �m3; P � 0.61). Thus, lesion size among the twoexperimental groups did not differ significantly, and there-fore the superior level of function in NT-3 graft recipientswas not attributable to a reduction in lesion size after thesecond surgery. All subjects exhibited surviving grafts inthe lesion cavity and surviving suspension grafts 6 mmcaudal to the lesion (Figs. 1 and 3).

The number of BDA-labeled corticospinal axons cross-ing a vertical line drawn through the rostral aspect of thelesion site, and 5, 10, 15, and 20 mm distal to the lesion site,was quantified in BDA-labeled sections, as previously de-scribed (Schnell et al., 1994). BDA-labeled corticospinalaxons were separately measured in gray matter, dorsal whitematter, and ventral white matter at each distance from thelesion site (Fig. 3). NT-3-grafted subjects exhibited a sig-nificant increase in the number of BDA-labeled corticospi-nal axons in host gray matter when measured 0, 5, 10, and15 mm caudal to the lesion site compared to �-gal-graftedsubjects (P � 0.05; Figs. 3 and 4). A significant differencein axonal number between groups was no longer detectablein gray matter 20 mm caudal to the lesion site. The numberof corticospinal axons was greatest adjacent to the lesionsite and was steadily reduced in number more caudally. Noaxons were detected in dorsal white matter caudal to thelesion site, and no difference in the number of ventral CSTaxons was detected between the two groups (P � 0.67). Inno case were corticospinal axons observed to penetrateNT-3-secreting grafts in the lesion site, consistent withobservations in acute lesion studies (Grill et al., 1997b).Notably, corticospinal axon density was greatest in graymatter adjacent to the caudal aspect of the lesioned dorsalcorticospinal tract (Fig. 3). The latter observation, takentogether with the lack of difference in number of ventralCST axons between NT-3 and control groups, indicates thatgrowth of lesioned dorsal axons rather than the spared

Fig. 2. Functional performance on the BBB scale. Five days of repeatedtesting on the BBB scale performed 6 months after the initial spinal cordinjury demonstrates modest yet significant functional superiority in NT-3-grafted subjects compared to �-gal-grafted animals (P � 0.05, repeatedmeasures ANOVA). Subjects received grafts 3 months after the initialspinal cord injury.

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Fig. 3. BDA label of corticospinal tract. (a) Composite photomicrograph demonstrating the labeled corticospinal tract (CST) approaching a site of spinal cord injury (dashedlines) containing an NT-3-secreting cell graft (G). GM, gray matter; VWM, ventral white matter. Scale bar, 0.50 mm. (b) Camera lucida drawing of BDA-labeled corticospinalaxons in an NT-3-grafted subject compared to (c) a �-gal-grafted animal. A greater number of axons are evident in the NT-3-grafted subject, confirmed by quantification(see below and text). Scale bar, 1 mm. (d) BDA-labeled CST approaching the lesion site in an NT-3-grafted and (g) a �-gal-grafted subject. Extent of labeling of theCST is similar in the various groups rostral to the lesion site. Scale bar, 150 �m. (e) At the lesion site, labeling of the CST abruptly discontinues in NT-3-graftedand (h) �-gal-grafted subjects. Scale bar, 50 �m. (f) In the host gray matter underlying the caudal aspect of the lesion site, bundles of BDA-labeled axonswere often evident coursing distally in NT-3-grafted subjects, whereas fewer such axons were observed in (i) �-gal-grafted subjects. Scale bar, 10 �m.

Fig. 4. BDA-labeled corticospinal axons distal to the chronic lesion site. (a–c) Examples of complex arbors of BDA-labeled axons located 10 mm distal tothe chronic spinal cord lesion site in three different NT-3-grafted subjects. Note the complexity and extensive branching of individual neurites compared tothe less extensive branching pattern observed in �-gal-grafted subjects in (d–f). �-Gal-grafted subjects contained fewer of these regions of branching spinalcord axons and less complex branching in regions that contained axons. Scale bar, 10 �m in a–d, 5 �m in e and f. (g) Quantification of total meanBDA-labeled axonal crossings of the spinal cord caudal to the lesion site (see Methods for details) reveal a significant increase in axonal crossings inNT-3-grafted subjects, up to 15 mm caudal to the lesion site (P � 0.05, Student’s t test).

ventral axons likely accounted for the increase in axondensity in NT-3-grafted animals.

Discussion

Findings of this study indicate for the first time thatgrowth of chronically injured corticospinal tract axons canbe promoted by cellular delivery of NT-3 to the lesion site.Further, NT-3 delivery also slightly but significantly ame-liorates functional deficits on a standard locomotor taskcompared to control grafted subjects that do not receivegrowth factors after chronic spinal cord injury. The superi-ority of function in NT-3-grafted subjects is not attributablesimply to gross sparing of spinal cord parenchyma afterlesions, since the volume of the ventral spinal cord at thelesion site did not differ significantly when comparing NT-3-grafted and �-gal-grafted groups.

The experimental paradigm of this study subjected ani-mals to an initial dorsal hemisection injury and then exam-ined the ability of NT-3-secreting cell grafts to promoteaxonal growth and to influence function 3 months later, or6 months after the initial injury. Animals were not function-ally tested before the grafting procedure at the 3-month timepoint; however, dorsal hemisection lesions typically resultin mean BBB scores of 15.0 � 0.3 (Jones and Tuszynski,unpublished observations), a number consistent with previ-ous reports using similar lesions (Merkler et al., 2001). YetBBB scores of control-lesioned subjects measured 6 monthsafter the initial injury (3 months after the grafting proce-dure) in this experiment averaged 10.6, indicating that thesecond surgical procedure, in which scar was removed fromthe chronic lesion site, may have caused further damage tothe spinal cord. This supposition is supported by the factthat 8% of animals exhibited complete loss of ventral spinalcord parenchyma on histological examination. However,overall volumes of ventral spinal cord did not differ amongthe remaining subjects in �-gal-grafted and NT-3-graftedgroups, indicating that the lesions were consistent in extentbetween groups and that functional benefits in NT-3 sub-jects were not simply due to neuroprotection. Nonetheless,the greater BBB scores of NT-3-grafted subjects comparedto �-gal-grafted subjects at 6 months may have representedan amelioration of further functional decline following thesecond surgical procedure. It is clear that cellular NT-3delivery enhanced the growth of chronically injured corti-cospinal axons, but whether the functional superiority ofNT-3-grafted subjects resulted from this corticospinalgrowth has not been proven. Axonal systems in the spinalcord that exhibit NT-3 sensitivity include corticospinal ax-ons, dorsal column sensory axons, and motor neurons thatinnervate the muscle spindle (Altar et al., 1993; Ernfors etal., 1994; Farinas et al., 1994; Hory-Lee et al., 1993; Kleinet al., 1994; Kucera et al., 1995; Maisonpierre et al., 1990;Tessarollo et al., 1994). Of these NT-3-sensitive systems, itis most likely that enhanced locomotor outcomes would

result from effects on corticospinal motor axons, rather thandorsal column sensory axons or ventral muscle spindleneuronal somata.

Cellular delivery of NT-3 to chronically injured cortico-spinal axons promoted their growth for distances of up to 15mm distal to the chronic injury site. This extent of growthexceeds the 8-mm distance that we previously reported 3months following grafts of NT-3-secreting fibroblasts incollagen to acute lesions to the corticospinal tract (Grill etal., 1997b). However, unlike the present study, the experi-ment of Grill et al. did not utilize distal or “downstream”NT-3-secreting suspension grafts. Thus, it is possible thatthe presence of a distal trophic factor source amplified thedistance over which corticospinal axons extended after in-jury. Other factors might also have contributed to this im-provement of axon growth in chronically injured, NT-3-grafted animals. For example, the chronic state of injurymight enhance the sensitivity of corticospinal axons toNT-3, although this seems unlikely given the downregula-tion of regeneration-associated genes, such as �-tubulin andGAP-43, that occurs in the absence of regeneration 2 weeksafter injury (Tetzlaff et al., 1991; Theriault et al., 1992).Future experiments will determine whether grafts of NT-3-secreting cells placed only caudal to injury sites will resultin similar distances of axonal growth and, conversely,whether grafts placed in the lesion site only will supportgrowth over 15-mm distances after chronic injury.

The presence of clusters of varicosities along BDA-labeled axons distal to the lesion site suggests the formationof axonal terminals in the denervated spinal cord. Thisraises the possibility that axonal growth may actually belimited by the presence of potential neuronal targets forextending corticospinal axons. As mentioned above, thepresence of a “downstream” intraparenchymal graft of NT-3-secreting cells may have extended this distance of distalaxonal growth. Interestingly, in separate experiments wehave placed grafts of fetal spinal cord tissue into the dorsalhemisection lesion site, together with implants of gelfoamsoaked in BDNF and NT-3; in contrast to a previous report(Coumans et al., 2001), we observed corticospinal axonpenetration into the graft but nearly immediate arrest ofaxonal growth upon encountering presumably attractive fe-tal neuronal targets (Lu and Tuszynski, unpublished obser-vations). Thus, the optimal bridge in a spinal cord lesion sitemay be one that is free of neuronal elements that couldresult in premature abortion of axonal regeneration.

Future studies will address whether debridement of thechronic injury site is necessary to elicit axonal growth inresponse to a neurotrophic factor source. Traditionally, theglial “scar” has been considered a barrier to axonal growth(Fawcett and Asher, 1999; Fitch and Silver, 1999; Jones andTuszynski, 2002; Jones et al., 2002) that becomes particu-larly impenetrable in chronic stages of injury. However,debridement of the scar in the region of injury can lead tofurther spinal cord damage, as occurred in the present study.Yet despite the presence of a marked upregulation of several

54 M.H. Tuszynski et al. / Experimental Neurology 181 (2003) 47–56

inhibitory extracellular matrix molecules around sites ofacute SCI, many axons pass through these putatively inhib-itory boundaries if presented with a positive growth stimu-lus such as growth factors (Jones and Tuszynski, unpub-lished observations). Clearly, the safety profile of growthfactor delivery after chronic injury would be improved iflesion debridement was not required, and this possibility isbeing examined in ongoing studies.

Growth of BDA-labeled axons distal to the lesion sitecould hypothetically have resulted either from regenerationof transected dorsal corticospinal tract axons or from sprout-ing of uninjured ventral corticospinal axons. Clearly, a pro-portion of dorsally lesioned axons did regenerate becauseaxons were observed to directly emerge from the lesioneddorsal projection and to project caudally for extensive dis-tances through remaining gray matter underlying the lesion(e.g., Fig. 3f). Further, if NT-3 gene delivery had inducedsprouting of ventral corticospinal axons, one would predictthat these axons, if branching from the ventral white matter,would have increased the density of axons in the ventralcorticospinal tract. Yet the density of BDA-labeled axons inthe ventral corticospinal tract did not differ between NT-3and control-grafted subjects (see Results). Alternatively,sprouting of ventral corticospinal axons might have oc-curred only from their terminals in gray matter, and acontribution of axonal growth from this source cannot beexcluded. Although unlikely, another possibility that mustbe considered is that corticospinal growth distal to the injurysite originated from the fews axons that might have beeninjured for the first time by resection of the chronic scar.

Previously, we have documented that growth factor produc-tion from genetically modified cells is sustained for at least the3-month time period of this study, based on ELISA, Westernblot, and RT–PCR measures of genetically modified primaryfibroblasts (Blesch et al., 1999; Grill et al., 1997b; Tuszynski etal., 1994). Thus, it is highly likely that growth factor produc-tion was sustained throughout this study.

Thus, axons in a chronically injured state retain neuro-trophin sensitivity. Recent studies indicate that long-pro-jecting spinal systems, such as the corticospinal tract, re-main in close proximity to a lesion site for extended timeperiods after injury (Houle and Jin, 2001), including obser-vations derived from primate spinal cords 3 months afterinjury (Tuszynski et al., 2002). Thus, the opportunity toelicit renewed growth from chronically injured axons maypersist for extended time periods. Since the majority ofhumans with spinal cord injury were injured several yearsearlier, these findings may be of relevance to the develop-ment of clinically relevant neural repair strategies.

Acknowledgments

This study was supported by the Hollfelder Foundation, theCal-Diego PVA, the Veterans Administration, and the NIH.

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