Brain-Derived Neurotrophic Factor Stimulates Hindlimb Steppingand Sprouting of Cholinergic Fibers after Spinal Cord Injury

Lyn B. Jakeman, Ping Wei, Zhen Guan, and Bradford T. StokesDepartment of Physiology and Spinal Cord Injury Research Center, The Ohio State University, 1645 Neil Avenue, Columbus, Ohio 43210

Received February 27, 1998; accepted July 31, 1998

Neurotrophic factors have been proposed as a thera-peutic treatment for traumatic brain and spinal cordinjury. The present study determined whether exog-enous administration of one such factor, brain-derivedneurotrophic factor (BDNF), could effect behavioralrecovery and/or histopathological changes after spinalcord injury. Adult rats received a mild or moderatecontusion injury or complete transection of the mid-thoracic spinal cord. Immediately thereafter, they wereinfused intrathecally with vehicle or BDNF for 28 days.Behavioral recovery was evaluated for 6 weeks afterinjury, at which time the rats were sacrificed and thespinal cord tissue was examined histologically. Theinfusion of BDNF resulted in acute stimulation ofhindlimb activity. These effects included activation ofalternating airstepping in injured rats when the hind-limbs were unloaded as well as slight improvements inthe rate of recovery in open field locomotion score.BDNF infusion was also associated with enhancedgrowth of cholinergic fibers at the injury epicenter, butdid not affect white matter sparing or density ofserotonergic axons at or below the injury site. Basedon immunohistochemical detection of BDNF proteindistribution, these described effects are likely to bemediated by the activation of cells and axons withinthe central injury region and the along the peripheralrim of the spinal cord. Together, these findings demon-strate that the exogenous infusion of BDNF after spi-nal trauma can influence postinjury outcome throughmechanisms that include acute stimulation of hind-limb activity and neuritogenesis at the injury site.r 1998 Academic Press

Key Words: neurotrophin; BDNF; trauma; spinal pat-tern generator; regeneration; functional recovery.

INTRODUCTION

Cases of traumatic spinal cord injury (SCI) are oftenclassified as anatomically and/or functionally incom-plete. Pathological specimens examined at autopsyfrequently reveal a central region of necrosis andloosely packed cells, with a rim of spared white matter

at the injury site and preservation of tissue above andbelow the injury (33, 34). This spared tissue provides apromising target for treatments to promote functionalplasticity and provoke improved recovery (57). Possibleintervention strategies include neuroprotective thera-pies to prevent or reduce secondary injury (43, 72),neuritogenic approaches to promote functional regen-eration either across or around the lesion site (32, 55,56, 70), and/or rehabilitation strategies to encouragetraining of spared neural circuits relevant to usefulfunction (17).

Exogenous administration of brain-derived neuro-trophic factor (BDNF) is one promising strategy forpromoting recovery, as BDNF has been shown to affectall three facets of repair described above. First, thereare several examples of neuroprotective actions ofBDNF on spinal cord neurons. In newborn animals,BDNF application can rescue axotomized neurons inthe red nucleus, dorsal column of Clarke, facial nucleus,and the spinal cord ventral horn (14, 15, 64, 71). Inadult rats, BDNF infusion can restore cholinergic mark-ers or prevent death of motoneurons after ventral rootaxotomy (14, 16, 24, 37, 49, 66, 72). Second, BDNF alsoenhances axonal sprouting in vivo. For example, chronicadministration of BDNF induces sprouting of retinalganglion cells (53) and serotonergic axons (44) in thebrain. BDNF can also enhance axonal elongation into aperipheral nerve graft or into implants of Schwanncells or fibroblasts in the injured spinal cord (29, 46,69, 74), and intrathecal (i.t.) application of BDNF canpromote extensive axonal growth from motoneuronsafter root avulsion (37, 49). Finally, recent studiesprovide evidence that BDNF plays an important role insynaptogenesis and plasticity through direct modula-tion of neuronal activity and regulation of synapticfunction (35, 38, 39, 65). To date, however, there havebeen no reports of such activation of synaptic activityfollowing BDNF administration to the spinal cord.

Despite the potential importance of neuronal rescueand regeneration after injury, there is also very littleexperimental evidence of histological effects of exog-enous neurotrophin administration following head orspinal cord trauma (42, 45, 47). In cerebral ischemia

EXPERIMENTAL NEUROLOGY 154, 170–184 (1998)ARTICLE NO. EN986924

1700014-4886/98 $25.00Copyright r 1998 by Academic PressAll rights of reproduction in any form reserved.

models, intraventricular BDNF infusion can reduce theextent of cell death if administered prior to the onset ofischemia (6, 54). A related study suggests that BDNFtreatment may reduce posttraumatic necrosis surround-ing a midline needlestick wound of the lumbar spinalcord (50). Therefore, in the present study, we wished todetermine if BDNF administration would act as aneuroprotective, neuritogenic, or plasticity agent whenadministered following traumatic injury to the spinalcord. We have evaluated behavioral and histopathologi-cal changes after SCI in rats receiving pharmacologicaldoses of BDNF through an indwelling intrathecal cath-eter. In the first 2 to 3 weeks after injury, BDNFtreatment stimulated alternating stepping and en-hanced the activity of the hindlimbs in open fieldlocomotion. In addition, BDNF enhanced sprouting ofcholinergic fibers into the lesion site, where penetrationof the neurotrophin was most complete. These resultsdemonstrate that BDNF can effect both acute andchronic changes in the injured spinal cord and suggestthat this factor may serve as a tool for the futuretreatment of traumatic SCI.

MATERIALS AND METHODS

Design. These experiments were designed to evalu-ate the effects of BDNF following injury of threedifferent severity levels to the midthoracic spinal cord(vertebral level T8). The growth factor was adminis-tered, beginning immediately after injury, with anosmotic minipump attached to a preimplanted intrathe-cal cannula. The tip of the cannula was placed justcaudal to the level of injury (vertebral T9). In Study I,30 rats received a mild contusion injury (0.9 mmdisplacement) at the level of the T8 vertebra using theOSU spinal cord injury electromechanical injury device(7, 61). Three doses of BDNF (50, 100, or 150 µg/day)were compared with vehicle control and injury onlyanimals (n 5 6/group). In Study II, 44 rats received amoderate injury (1.15 mm displacement) and wereinfused with vehicle or BDNF in the same doses asabove (n 5 11/group). Four of these rats were sacrificedat 3 weeks postinjury to determine the distribution ofBDNF in the neuropil of the spinal cord during theperiod of infusion. In Study III, 8 rats received acomplete transection and were infused with eithervehicle (n 5 4) or 100 µg BDNF/day (n 5 4). All infu-sions continued for a total of 28 days with the pumpreplaced at 14 days postinjury under metathane anes-thesia. Behavioral recovery was evaluated at 1, 3, 8, 15,22, 35, and 42 days after injury. The rats were sacrificedafter the last test session and spinal cord tissue wasprepared for histological analysis. Data were analyzedonly from animals in which the cannula was attachedto the pump and patent at the time of replacement and

sacrifice and the tip was within 5 mm of the injury site(n 5 3–10/group).

Cannula placement and injury. All surgery wasperformed under aseptic conditions. Adult femaleSprague–Dawley rats (Harlan, 225–275 g) were anes-thetized (ip) with ketamine HCl (80 mg/kg) and xyla-zine (10 mg/kg). A partial laminectomy was performedat the T12–T13 vertebral junction. A small incision wasmade in the lateral meninges, and a 1.5 cm length ofstretched PE60 tubing (0.2–0.4 mm final outer diam-eter, prefilled with sterile Dulbecco’s phosphate-buff-ered saline) was threaded below the arachnoid, placingthe tip directly under the T9 vertebra. The free end ofthe cannula was sealed and secured to the overlyingmusculature with 4-0 suture. The wound was closed inlayers, topical antibiotic and fluids were administered,and the rats were allowed to recover in warmed cages.For injuries, rats were anesthetized as above andspinal cord contusion injury was performed at the T8vertebral level as described previously (10, 62). Fortransections at the same level, the underlying spinalcord was cut in two sites, approximately 3 mm apart,and the intervening tissue was removed by gentleaspiration around the full circumference of the spinalcord. The transection site was packed with sterileGelfoam (Upjohn and Pharmacia) and the wound wasclosed in layers. Immediately after contusion or transec-tion injury, the preimplanted cannula was flushed withthe appropriate infusate and an ALZET 2002 osmoticminipump (Alza, Inc., infusion rate 0.5 µl/h) filled withBDNF (recombinant human BDNF, Amgen, Inc., di-luted to 4.15, 8.3, or 12.5 mg/ml) or vehicle (Dulbecco’sphosphate-buffered saline (PBS), containing 1 mg/mlrat serum) was attached to the end of the catheter. Theskin wound was closed and the animals were allowed torecover as described above. After injury, bladders wereexpressed two or three times daily until reflexes re-turned (within 2 weeks). Gentamycin (Gentocin, Scher-ing-Plough, 1 mg/kg) was administered from immedi-ately preop until the return of bladder function andagain at any sign of bladder infection. A vitamin Csupplement (5 mg/day) was given to all rats throughoutthe experiment to maintain low urinary pH. The ratswere weighed twice weekly and supplemented with ahigh caloric nutrient paste (Nutrical, Evsco Pharmaceu-ticals) while the postoperative weight was #90% ofpreinjury weight (usually 1–2 weeks postinjury).

Behavioral testing. All behavioral testing and analy-sis was performed by observers blinded to treatmentgroup. Prior to surgery, rats were acclimated to theopen field testing area and trained to criterion (,0.2footfalls/s) on a 2 3 2-cm2 wire mesh grid as describedpreviously (63). Overground locomotion was evaluatedin all animals using the Basso, Beattie, Bresnahan(BBB) open field locomotion scale (4, 5). Rats weretested for performance in open field locomotion and grid

171BDNF INFUSION AFTER SPINAL CORD INJURY

walking 3 days after cannula placement surgery; onlythose with normal locomotion and meeting preinjurycriteria were entered into the study. The rats in StudyII and Study III were also trained to tolerate hand-heldthoracic support for quantitative analysis of airstep-ping. The support training criteria required threesuccessive periods of $15 s of relaxed posture whilebeing suspended below the forelimbs between an inves-tigator’s thumb and fingers, thus providing the rat withfirm, but not confining, support to the upper thoraciccage. For quantitative analysis of airstepping, the ratswere videotaped for 1 min of thoracic support eachweek using a standard video camera at 1/60–1/250 sshutter speed. Tapes were viewed and analyzed usingApple VideoPro software. Analysis of airstepping wasadapted from Van Hartesveldt et al. (67). Each segmentof the 1-min recording in which the rat was relaxed astrained for $15 s was timed and analyzed. Hindlimbairstepping was defined as coordinated, rhythmic, andalternating hindlimb activity, with most movementsdirected along the midline. Stepping occurred in ‘‘bouts’’of activity separated by brief periods of flexor relax-ation. Total step cycles, the number of stepping bouts,and the duration and frequency of each stepping boutwithin the 151-s segments were determined.

Histological and immunocytochemical procedures.The rats were anesthetized as above and exsangui-nated with 0.1 M PBS and perfused with 4% paraformal-dehyde in PBS. The site of the cannula tip was markedand the spinal cords were removed and cryoprotected in30% sucrose. Cryostat sections (20 µm) were takenfrom the epicenter, 3–6 and 6–9 mm rostral and caudalto the epicenter of all specimens with contusion injury.Every fifth section was stained with luxol fast blue andcresyl violet as described previously (7, 10). Interveningsections were used for immunohistochemical analysis(see below). Spared white matter was outlined manu-ally on digital images while viewing sections at 1003and percentage of sparing was calculated as the amountof spared white matter/total cross sectional area of thesection. For those animals with complete transections,a 1-cm segment surrounding the injury site was post-fixed 24 h and embedded in paraffin for serial section-ing to verify the completeness of the transection.

For immunocytochemical staining, sections werewashed in 0.1 M PBS and incubated in 0.5% BSA, 1%normal serum, and 0.1% Triton X-100. Sections fromdifferent treatment groups were always mounted andincubated on the same slide. Primary antibodies raisedagainst BDNF (rabbit anti-BDNF;Amgen, Inc.; 1:1000),choline acetyltransferase (goat anti-ChAT; Chemicon;1:1000), serotonin (rabbit anti-5-HT; Eugene-Tech;1:3000), or neurofilament proteins (mouse RT-97; Boeh-ringer Mannheim; 1:50) were applied to the tissuesections for 18–72 h at 4°C. Sections were incubatedwith the appropriate biotinylated secondary antibody

(1:400, Vector Laboratories, Burlingame, CA) for 1–3 hat room temperature. Labeled sites were visualizedusing Vectastain ABC immunoperoxidase and 3,38-diaminobenzidine–H2O2. Quantification of propor-tional area (neurofilament and 5-HT) was performedusing the MCID image analysis system (Imaging Re-search, St. Catherine’s, Ontario, Canada) by manuallysetting the relative optical density threshold to identifypositively stained profiles within a defined target area.Fiber length per section (ChAT) was determined byimaging the entire cavity region of each of four sections/specimen at high magnification (403 objective) andtracing the length of all positively stained fibers. Speci-ficity of BDNF-, ChAT-, and 5-HT-like immunoreactiv-ity (abbreviated as BDNF-IR, ChAT-IR, or 5-HT-IR)was determined in representative sections by preabsorp-tion of the primary antibody with purified BDNF(Amgen, Inc., 15 mg/ml), 5-HT (Sigma, 20 mg/ml), orChAT (Sigma, ,3.7 mg/ml), respectively.

Statistics. Behavioral measures were compared us-ing two-way ANOVA with time as a repeated measure(SAS Statistical Systems; Proc GLM; Error forGroup 5 subject (GROUP)). When ANOVA interactioneffects (group 3 time) were significant (P , 0.05), posthoc pairwise comparisons were performed using Tukey’sHSD to compare groups at each day (67). Comparison ofeffects following complete transections (vehicle vs 100µg/day) were determined by repeated measures t testfollowed by post hoc means corrected t tests at eachtime point (Graphpad Prism2). All histological outcomemeasures were analyzed using one-way ANOVA fol-lowed by Tukey’s HSD test if main effects were signifi-cant. Contingency data (animals/group reaching a scoreor criteria) across treatment groups are presented aspercentages only. Significance for all analyses wasdefined at P , 0.05.

RESULTS

Behavioral Effects of BDNF: Airstepping. Blindedobservations of rat behavior in Study I (0.9 mm injury)revealed a striking effect of BDNF treatment on hind-limb activity. The vehicle-treated or injury-only ani-mals uniformly exhibited either flaccid paralysis oroccasional uncoordinated hindlimb spasms during thefirst 3 weeks after injury, if they were suspended bygrasping under the forelimbs. However, when thoserats with the highest doses of BDNF were transportedfrom cage to cage or lowered into the open field area fortesting, many of them exhibited vigorous alternatingstepping of the hindlimbs in the absence of intentionalor obvious external stimulation (‘‘airstepping’’). In or-der to evaluate these effects quantitatively, rats inStudy II and Study III were trained to rest in a relaxedposture during hand-held thoracic support. Analysis ofairstepping frequency in the 1.15-mm contusion groups

172 JAKEMAN ET AL.

revealed a dose-dependent trend in the number of steps(Fig. 1a) and increased stepping bouts (Fig. 1b) duringperiods of 15 or more s of relaxed torso posture. Thestep frequency within bouts did not change with BDNFdose (Fig. 1c), suggesting that a BDNF stimulated afixed pattern of neural activity.

Following complete transection, the rats infused withvehicle never showed spontaneous airstepping behav-ior. In contrast, two/four of the spinal transected ratsinfused with BDNF showed airstepping, one at 2, 3, 4,and 5 weeks postinjury and one at 3 weeks postinjuryonly. In all of these animals, the transection wasconfirmed as complete at sacrifice.

Behavioral recovery: Open field locomotion. Boththe time course and the final extent of recovery afterSCI in rats is dependent on the severity of the initialinjury (4, 5, 7). In addition to effects on airstepping, weexamined and found subtle differences in the rate offunctional recovery (treatment 3 time interaction ef-fect; P , 0.05) with all three injury severities in ani-mals treated with BDNF. After 0.9-mm displacementcontusion injury, post hoc analysis revealed differencesin BBB score between groups on day 15 postinjury(100 5 150 µg/day . 50 µg/day 5 vehicle; Tukey’s HSD;P , 0.05) (Fig. 2a). Those rats with BDNF infusionshowed greater likelihood of consistent forelimb–hindlimb coordination at 2 weeks postinjury than ve-hicle or control animals (BBB score $ 15: 0/4 injuryonly, 2/5 vehicle, 1/5 50 µg/day, 3/4 100 µg/day, and 4/5150 µg/day; Fig. 2b).

Following 1.15-mm displacement injury, all rats wereparalyzed at 1 day postinjury. Those infused withvehicle declined further by 3 days postinjury and thenshowed modest recovery over the next 6 weeks (Fig. 3a;(5)). In the presence of BDNF, the initial paralysis(3 days postinjury) was attenuated, thus affecting aslightly accelerated recovery to the same plateau. Inaddition to the effect at 3 days, a higher percentage ofrats in the high-dose groups was able to step with fullweight support on each hindlimb (BBB score $ 10.0) at8 days postinjury (1/10 vehicle-treated animals, 1/10 50µg/day, 8/10 100 µg/day, and 6/10 150 µg/day; Fig. 3b).No differences were seen in this group after 8 dayspostinjury.

Greater effects of BDNF on locomotor recovery wereobserved following spinal cord transection (Study III;Fig. 4). After a complete transection, adult rats nor-mally regain only slight movements of the joints of thehindlimbs over the course of recovery (5, 13). In con-trast, the rats receiving the infusion of BDNF hadincreased hindlimb use, reflected in higher BBB scoresdue to extensive flexion of two or more joints of eachhindlimb at all times examined. Differences betweenvehicle and BDNF-treated rats were significant at 8,15, and 22 days postinjury. In addition, three of the fourBDNF-treated rats achieved a BBB score of 8 (plantar

placement) and two had a score of 9 (weight support instance) during the period of BDNF infusion, while thehighest score achieved by any of the vehicle-treatedrats was 4.5.

Histological analysis. Histological analysis of spi-nal cord tissue after contusion injury was performedwith three principle objectives. The first was to esti-mate the distribution of BDNF penetration, both dur-ing and after pump infusion. The second was to deter-mine if the presence of BDNF led to an improvement intissue preservation at the injury site. The last goal wasto determine if the infusion stimulated growth ofknown fiber populations, particularly with regard tochanges in cholinergic and serotonergic axons.

BDNF immunohistochemistry. The fixation proce-dures used for these specimens favored a conservativeestimate of penetration, while minimizing staining ofendogenous BDNF (1). To evaluate BDNF distributionduring the period of pump infusion, one rat per groupwas sacrificed at 3 weeks postinjury. As shown in Figs.5a and 5c, the strongest BDNF-IR was present at theepicenter of the injury, where the protein was distrib-uted throughout the remaining white matter and withinthe scar tissue and loose cellular debris. Intense immu-noreactivity was also associated with the meningessurrounding the spinal cord and staining was evidentwithin the dorsal roots. In contrast, no immunostainingwas detected in the animals infused with vehicle or 50µg BDNF/day (Fig. 5b). Just caudal to the injury site,BDNF-like activity was present in the higher doseanimals, where it extended into the superficial dorsalhorn and approximately 250–300 µm along the periph-eral white matter rim (Fig. 5d). Staining was specificfor BDNF, as all immunoreactivity was blocked whenthe antibody was preabsorbed with 15 mg/ml BDNF(Figs. 5e and 5f).

The extent of BDNF penetration during the infusionwas examined at other levels of the spinal cord as well.Slight BDNF-IR was detected in the peripheral whitematter and the superficial dorsal horn at the cervicalenlargement (Fig. 6a) and in upper lumbar segments(Fig. 6b) of rats receiving 100 or 150 µg/day BDNF. Nostain was present in vehicle controls (Figs. 6c and 6d).To determine the residual BDNF distribution aftercessation of the infusion, sections from rats sacrificed at6 weeks postinjury were also incubated with BDNFantibodies as described above. In those specimens withBDNF infusion, a thick rim of BDNF-IR was foundalong the meninges surrounding the spinal cord(Fig. 6e). In contrast with those specimens obtainedduring the infusion, however, BDNF-IR was not de-tected within the dorsal horn or peripheral whitematter of the spinal cord in these specimens.

White matter sparing and axonal elongation at theepicenter. To determine the effects of BDNF on protec-tion of the myelinated fiber pathways of the thoracic

173BDNF INFUSION AFTER SPINAL CORD INJURY

spinal cord, we used conventional luxol fast blue stain-ing to examine white matter sparing at the epicenter.Following a 0.9-mm displacement injury, spared whitematter represented 33.2–50.3% of the total epicenterarea, corresponding to previous reports describing amild injury with this device (mean 5 37.2; s 5 5.7).Spared white matter represented 1.6–36.2% of the totalepicenter area after 1.15 mm displacement as describedpreviously for a moderate injury level (mean 5 16.9;

FIG. 2. Effects of BDNF infusion on overground locomotionfollowing mild (0.9 mm displacement) contusion injury. Rats wereinfused intrathecally with BDNF beginning immediately after injury(n 5 4–6/group). (a) Time course of locomotor recovery. Values repre-sent means 6 SEM of BBB score, averaged across the two hindlimbs.Repeated measures ANOVA showed significant interaction ofgroup 3 time (F 5 1.71; P 5 0.0273). Post hoc Tukey’s HSD revealeddifferences between the two highest doses of BDNF and vehicle or 50µg BDNF/day (P , 0.05). (b) A higher percentage of these animalsdemonstrated consistent coordination between forelimbs and hind-limbs at 15 days postinjury).

FIG. 1. BDNF stimulates alternating airstepping when the hind-limbs are unloaded: All data plotted from 3 weeks postinjury (n 5 5/group). (a) Stepping cycles were counted for each videotape segmentof $15 s in which the rats exhibited a relaxed posture as trained(minimal head, forelimb, or torso lateral movements). Values repre-sent means 6 SEM of complete cycles counted/segment time.Rats with vehicle infusions had flaccid paralysis of the hind-limbs. One-way ANOVA revealed a dose-dependent upward trendthat did not reach significance (P 5 0.0591). (b) The number ofstepping bouts/second of relaxed suspension were counted. Step-ping bouts differed as a function of dose (P 5 0.0458). (c) Cyclefrequency was determined by counting complete step cycles within anactive stepping bout (i.e., constant stepping periods). Only thoseanimals with stepping bouts were used (vehicle, 0/5; 50, 1/5; 100, 3/5;150, 4/5).

174 JAKEMAN ET AL.

s 5 9.8). There were no differences in the cross-sectional area or the proportional area of spared whitematter at the injury epicenter as a function of treat-ment. In all transected animals, the gelfoam filled thelesion cavity, and there was no evidence of myelinatedfibers traversing the transection site.

ChAT-IR. Choline acetyltransferase is present inmotoneurons and their processes in the spinal cord. Inuninjured controls, ChAT-IR was distributed through-out the gray matter. Most staining was associated withmotoneurons and proximal dendrites within the ven-tral horn and intermediolateral cell column (Fig. 7a). Inaddition, dense staining was seen in small longitudinalsegments from motor axons in the normal ventral andlateral white matter (Fig. 7b). At the lesion epicenter ofvehicle controls, punctate staining was found in smallprofiles in the residual rim of white matter, and a lightbackground cellular staining was observed in the cavity

region (Fig. 7c). When viewed at high magnification,small, darkly staining fibers were occasionally foundwithin the cellular matrix. Such profiles were infre-quent in vehicle-treated animals (Fig. 7d) but weremore common among the animals treated with higherdoses of BDNF (Figs. 7e and 7f). The staining in thesefibers was blocked when the antibody was preincubatedin the presence of excess purified ChAT (Figs. 7gand 7h).

The total length of ChAT-IR fibers/section rangedfrom 50 µm to over 2 mm within the cavity region. Inone-third of the specimens, total fiber length exceeded500 µm. Differences were found between doses, withthe 100 µg/day group containing significantly greaterChAT-IR fiber growth than 50 µg/day or vehicle con-trols (Fig. 8a). However, there were no differences inthe number of ChAT-IR neurons or proportional area inspinal cord sections from 3 mm rostral or 3 mm caudalto the epicenter (data not shown), suggesting thatBDNF did not affect survival or ChAT activity ofsurrounding motoneurons in this model. To determineif the effect on fiber growth into the epicenter wasspecific for cholinergic axons, an additional set ofsections was stained with a mouse antibody directedagainst the heavy (200-kDa) neurofilament protein(RT-97) to identify spared or regenerating axons withinthe injury site. In the cavity region of the epicenter,immunoreactive fibers traversed the epicenter inbundles that were often associated with connectivetissue septae and blood vessels. The proportional areaof the lesion cavity that had RT-97-positive fiber stain-ing ranged from 3.8 to 28%, but there were no differ-

FIG. 3. Effects of BDNF infusion on overground locomotionfollowing moderate (1.15 mm displacement) contusion injury (n 5 10/group). (a) The effects of BDNF infusion on the open field locomotionscore were seen as early as 3 days postinjury. Repeated measuresANOVA revealed significant interaction of group 3 time (F 5 32.95;P , 0.001). Post hoc Tukey’s HSD showed differences between vehicleand all other groups at 3 days postinjury (dpi) (*P , 0.05). (b) Agreater percentage of rats with BDNF infusion had weight supportedsteps with both hindlimbs at 8 days postinjury (n 5 10/group).

FIG. 4. Effects of BDNF infusion on hindlimb movements in theopen field following complete transection. Rats were infused withBDNF for 28 days beginning immediately after injury (n 5 3–4/group). t test with repeated measures identified differences betweenscores from the two groups across time ( P 5 0.001). Differences onday 8, 15, and 22 were determined by post hoc means corrected t tests(*P , 0.05).

175BDNF INFUSION AFTER SPINAL CORD INJURY

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FIG. 6. Distribution of BDNF-IR during and after infusion. (a) Transverse section through cervical enlargement of a rat infused with 100µg BDNF/day and sacrificed at 3 weeks postinjury. Note penetration of BDNF-IR into the peripheral white matter (arrows) and superficialdorsal horn (small arrowhead). (b) Transverse section through upper lumbar spinal cord of the same rat shown in (a). (c,d) Transverse sectionsthrough cervical enlargement and upper lumbar spinal cord of rat infused with vehicle. (e) Transverse section through the injury epicenter of arat sacrificed 6 weeks after injury, i.e., after cessation of BDNF infusion. BDNF-IR is restricted to the peripheral meninges and within smallcells in the white matter, but no diffuse staining was seen in the dorsal horn or peripheral white matter. (f) Corresponding section from a ratinfused for 28 days with vehicle and sacrificed at 6 weeks postinjury. No residual BDNF-IR was detected. Bars, 500 µm.

177BDNF INFUSION AFTER SPINAL CORD INJURY

178 JAKEMAN ET AL.

ences in axonal fiber area between the treatmentgroups (Fig. 8b).

Serotonin immunoreactivity. After injury, serotoner-gic fibers were dramatically depleted caudal to theinjury and the density of fibers rostral to the injury sitewas increased. At the epicenter, 5-HT-specific immuno-reactive fibers were primarily restricted to the periph-eral rim of spared tissue. Occasionally, punctate axonsextended for 100–200 µm into the lesion center, butthese were very rare. Immunoreactive fiber densitywas expressed as a proportion of the entire epicentercross-sectional area and as a proportion of the area ofthe remaining white matter rim. Proportional area of5-HT-IR ranged from 0.3 to 7.3% of the entire cross-sectional area and 1.2–25.9% of area the remainingwhite matter rim, but there was no effect of treatmenton either measure (Fig. 8c).

DISCUSSION

The use of neurotrophic factors as a therapeuticstrategy after SCI has been proposed for nearly adecade (41, 47, 57), yet there is very little known aboutthe consequences of exogenous administration in mod-els of SCI or trauma. One possible reason in the case ofBDNF is that this factor shows limited diffusion intothe parenchyma following intracerebroventricular (icv)or intrathecal administration (1, 16, 48, 73). The re-stricted penetration of BDNF is primarily due to thepresence of noncatalytic, high-affinity trkB receptorsfound on the ependyma of the ventricles and themeninges of the spinal cord (1, 73). Trauma (25),axotomy (19), and neurotoxicity (8) may all induceprolonged up-regulation of these truncated trkB recep-tors after injury. Nevertheless, there is evidence fromicv infusion studies that high doses of BDNF can diffusebeyond the ependymal layers, presumably by supersatu-rating those receptors that line the ventricles (48).Based on those data, the present study employedpharmacological doses of BDNF (50–150 µg/day) toimprove diffusion into the neuropil of the injured spinalcord. Our results demonstrate that, during the infu-sion, BDNF can penetrate into the peripheral rim of thespinal cord, extending from cervical to lumbar levels.Upon cessation of treatment, residual BDNF is primar-ily restricted to the peripheral meninges.

The most striking effects of BDNF on behavior were

observed when trained rats with spinal cord contusionor transection were suspended from just below theforelimbs. Under these conditions, vehicle-treated ratsdid not exhibit alternating stepping during the first 3weeks postinjury. In contrast, some spontaneous airstep-ping was seen with all doses of BDNF. Various forms ofalternating hindlimb stepping or fictive locomotor activ-ity have been reported previously from data obtained inrats, cats, and humans following partial or completeSCI (18, 27, 52). This highly coordinated activity isbelieved to be organized within the lumbar spinal cordby the presence of interneuronal networks collectivelyreferred to as central pattern generators (CPGs) (27).In most cases, however, activation of CPG activitysufficient to induce hindlimb stepping requires exter-nal stimulation of afferent or descending pathways. Forinstance, stepping can be induced by a moving tread-mill, peritoneal or tail stimulation, bladder distention,or electrical stimulation (2, 21, 26, 30). In newborn rats,stepping can also be elicited after SCI by administra-tion of clonidine or L-dopa (31, 67). However, thesepharmacological agents will not induce spontaneousstepping in adult spinal animals (3, 21, 52). In thepresent study, the number of bouts of airstepping wasincreased with increasing BDNF dose, but there waslittle variation in the cycle frequency. Therefore, it islikely that the primary effect of BDNF was to reducethe threshold for activation of existing CPGs.

Modest changes in the recovery profile in overgroundlocomotion were observed in all three experimentalparadigms, although the activity was manifestedslightly differently in each experiment, depending uponthe severity of injury. Following contusion injury, ratswith or without BDNF regained the equivalent degreeof locomotor ability, but those receiving BDNF reachedrelative milestones of hindlimb function sooner afterinjury. Rats with mild injuries were able to demon-strate consistent forelimb–hindlimb coordination sooner,and those with moderate injuries were able to place andsupport weight in on their hindlimbs sooner. Similareffects on the time course of recovery have been re-ported following transplantation of neurotrophin-secreting fibroblasts into a contusion injury site inadult rats (37). Following complete transection, how-ever, the differences were more profound. Those ani-mals with no BDNF treatment regained only slightmovement of the joints of the hindlimbs over 6 weeks

FIG. 7. BDNF effect on ChAT expression in the spinal cord. (a) ChAT-IR in normal ventral horn of the midthoracic spinal cord (vh).Staining is limited to the ventral horn motoneurons and processes, many of which extend into the lateral (lwm) and ventral white matter. (b)Higher magnification photomicrograph of normal cholinergic axons sectioned longitudinally within the ventral white matter. (c) Transversesection through the center of the injury site in a vehicle-treated animal. Staining includes dark profiles in the residual rim of white matter(open arrows) and diffuse cellular labeling in the center of the cavity. Within the central cavity, darkly stained ChAT fibers can be seen(arrowheads). When present, these ChAT-IR fibers in the lesion cavity were clearly identified at higher magnification. (d) Enlargement of thecentral cavity region from rat which was infused with vehicle for 28 days and sacrificed at 6 weeks postinjury. An occasional ChAT-IR fiber ispresent. (e) Numerous ChAT-IR fibers are found in the center of the lesion in another rat that was infused with 100 µg BDNF/day. (f) Densebundle of ChAT-IR fibers from rat infused with 150 µg BDNF/day. (g) High-power magnification of isolated ChAT-IR fibers in the center regionof an injury site. (h) Immunostaining of the same region of an adjacent section to that in (g), following preincubation of the polyclonal antibodywith 3.7 mg/ml purified ChAT protein. Bars: a, d, 100 µm; b, e, f, g, h, 50 µm; c, 500 µm.

179

(see also Ref. 5). BDNF treatment stimulated extensivehindlimb movements in all animals (defined as .50%of the maximal range of the joints) and facilitatedmovements of two of the animals sufficient to permitsuccessful plantar placement and, in one case, fullweight support on both hindlimbs. While modest, thisdegree of improvement is similar in magnitude to thatobtained with recent, highly invasive experimentaltreatments performed in completely transected ani-mals (13, 51). The present study shows, however, thatthis degree of functional recovery can be attained aftercomplete transection in the absence of axonal regenera-tion across the site of injury. In this case, all transec-tions were filled with gelfoam and the absence ofmyelinated axons verified in serial paraffin sections.This suggests that BDNF was able to effect thesehindlimb movements by stimulating neurons below thelevel of the transection and the activating local cir-cuitry. Interestingly, a similar enhancement of hind-limb movements has also been seen in rats that re-ceived a complete transection 8 weeks following asevere contusion injury, with no other treatment (5).Thus, the lumbar spinal cord is capable of local synap-tic plasticity sufficient to mediate plantar placementand occasional weight support, and BDNF treatmentmay act by either enhancing local synaptic activity orpromoting similar plastic changes in local circuitry.

Recent evidence has demonstrated that BDNF exertsprofound effects to enhance synaptic transmission,both in vitro and in vivo (60, 65). Several underlyingmechanisms may contribute to increased synaptic activ-ity, including an increase in neuronal firing rate (38,58), an increase in neurotransmitter synthesis or turn-over (58), or an increase in postsynaptic efficacy (38,39). After SCI, any of these acute actions on synapticactivity could enhance hindlimb activity during locomo-tion or airstepping. Unfortunately, it is not possible todetermine the mechanism of action of BDNF on behav-ioral changes in this model without additional pharma-cological and pharmacokinetic studies. However, basedon the immunocytochemical detection of exogenously

FIG. 8. Quantitative analysis of fiber growth into the centralcavity area. (a) ChAT-IR fiber length/section was determined bysummating the length of all positively stained profiles within thecavity. Values represent means 6 SEM of the averaged length fromthree to four sections/specimen (n 5 7–8/group). One-way ANOVAP 5 0.0253; post hoc analysis using Tukey’s HSD comparison, 100µg/day . vehicle; 100 µg/day . 50 µg/day (*P , 0.05). (b) The relativeaxonal ingrowth from all fiber types within the lesion center wasestimated by proportional area measurements of positive stainingwith the monoclonal neurofilament antibody, RT-97. The area of thecavity containing neurofilament-like immunoreactivity was ex-pressed as a proportion of the cavity area at the injury center. Valuesrepresent means 6 SEM of proportional area values for two sections/specimen (n 5 6/group). (c) The proportional area of 5-HT-like immu-noreactive fibers within the peripheral white matter rim at theepicenter was not different between groups (n 5 6/group; P 5 0.3598).

180 JAKEMAN ET AL.

administered BDNF in our model, it is most likely thatthese effects are mediated by changes in the activity ofthe primary afferent fibers in the dorsal root or syn-apses upon second order neurons found in the superfi-cial dorsal horn. By reducing the threshold for activa-tion of these fibers, BDNF may contribute to increasedreflex activity, which would be manifest in increasedhindlimb movements after complete transection.

The behavioral effects of BDNF infusion were nolonger present when the infusion pump was removed.In addition, there was a general decrease in all of theseeffects after 2 weeks. This decrease may be due to lossof activity of the growth factor, decreased responsive-ness of the receptors, or changes due to plasticity ofspinal cord circuits. Recent reports have shown that thebiological efficacy of BDNF may be diminished by asmuch as 60% over 2 to 3 weeks when stored at bodytemperature (16). In the present study, however, be-cause the pumps were replaced with fresh neuro-trophin at 14 days postinjury, it is unlikely that decayaccounts for all of the observed decrease in hindlimbstimulation. Alternatively, the prolonged infusion ofBDNF can cause a decrease in the binding of BDNF tofull-length trkB receptors and decreased responsive-ness to the acute biological effect of BDNF in vitro or invivo (22). However, in the case of acute analgesic effectsof BDNF infusion into the brainstem, the down-regulation of trkB protein does not necessarily corre-spond to a decrease in behavioral effects (23). There-fore, additional work must be done to evaluate the timecourse of behavioral changes in the context of trkBreceptor internalization in this model.

A final contribution to the decrease in behavioraleffects of BDNF infusion might be the result of thecontinuing plasticity of the injured spinal cord. Forexample, the protracted recovery of spared descendingpathways may help to modulate the acute effects ofBDNF infusion by restoring inhibitory regulation of thelocal interneuronal circuitry. Conversely, BDNF itselfmay contribute to functional plasticity and synapsedevelopment. For example, the role of BDNF in modu-lating synapse formation in development is well known(11, 12, 60). Therefore, it is possible that exogenousBDNF treatment could be combined with carefullydesigned training paradigms to enhance developmentof useful function after injury (17).

In addition to acute synaptic effects, BDNF has beenshown to promote axonal sprouting in a variety ofmodels of spinal cord injury. For example, BDNFstimulates growth of descending axons into peripheralnerve grafts (74) or Schwann cell-filled channels (69)after partial spinal cord lesions. Genetically engineeredfibroblasts that secrete BDNF stimulate increased axo-nal ingrowth (29, 46), and BDNF application in combi-nation with fetal spinal cord transplants can enhanceregeneration of descending axons (9). Intrathecal admin-

istration of BDNF also stimulates extensive sproutingof motor axons following ventral root avulsion (37, 49).In the current study, ChAT-immunoreactive fibers atthe injury epicenter showed a morphology which closelyresembled that of motoneuron axons cut in a longitudi-nal axis. These fibers may represent direct axonaloutgrowth from injured motoneurons, intermediolat-eral cell column neurons, or from proximal dendritesnear the site of severe trauma (40). Growth in thecenter of the lesion cavity showed some selectivity forcholinergic neurons, because we found no difference inthe total axon density in the epicenter as defined withantibodies raised against the heavy neurofilament sub-unit. The effect of BDNF on cholinergic fiber growthwas also in contrast with the lack of effect on sproutingof serotonergic fibers. These results indicate that sero-tonergic fibers may require a more substantive sub-strate to support growth at the injury site. In thisregard, the infusion of BDNF may provide the greatestpromise as an adjunct to transplantation of nonneuralcells such as fibroblasts or Schwann cells or fetal spinalcord tissue. Thus, we have demonstrated that BDNFinfusion can induce sprouting of at least one type offiber into the epicenter region, where the penetration ofthe protein was greatest. Because the white matter andscar tissue surrounding the injury epicenter are sourcesof cell surface molecules that are inhibitory to axonalgrowth (20, 57) even greater effects of BDNF on sprout-ing may be possible when additional approaches pro-vide ways to enhance penetration into target regions ofthe gray matter.

In conclusion, we have demonstrated that the chronicinfusion of BDNF following SCI exerts acute behavioraleffects and stimulates sprouting of cholinergic fibers atthe injury site. The mechanisms of these effects arelikely to be multifaceted. The behavioral actions in-clude acute stimulation of local synaptic activity, includ-ing increased activation of CPGs and local reflexes.This is potentially useful in light of recent clinical datawhich have demonstrated that a combination of activa-tion and control of existing spinal CPG activity throughaggressive physical therapy can improve voluntarylocomotion in spinal cord injury patients (17, 28). Thechronic effects of BDNF on cholinergic fibers suggeststhat exogenous BDNF infusion may also contribute toregrowth of damaged neurons. Strategies to improvethe penetration of BDNF into the spinal cord paren-chyma will be important to extend these investigations.Together, these results support the long-held proposalthat BDNF infusion may be a promising component ofadjunct therapy to promote functional recovery andregeneration after injury.

ACKNOWLEDGMENTS

Supported by NS-10124/2 (BTS), NS-37321 (BTS/LBJ), and theBremer Foundation (L.B.J.). The initial dose ranging studies were

181BDNF INFUSION AFTER SPINAL CORD INJURY

supported in part by Amgen, Inc. Full-length hrBDNF and rabbitpolyclonal anti-BDNF antibody were provided by Amgen/RegeneronPartners. Osmotic minipumps supplied in part through agreementwith Alza, Inc. Additional technical support was provided by Ms. PatWalters, Dr. YiFei Chen, Mr. Ryan Veverka, and Mr. Michael Wilson.Statistical analysis was performed with consultation from Dr. M.Moeschberger. The authors thank Drs. L. Williams, S. Wiegand,D. M. Basso, D. M. McTigue, and P. Popovich for helpful discussionsduring execution and analysis of these experiments and usefulcriticisms during preparation of the manuscript.

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