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Research Report

Cyclosporin-A enhances non-functional axonal growing aftercomplete spinal cord transection

Antonio Ibarraa,b,⁎,1, Edson Hernández a,b,1, Joel Lomelic,2, Dante Pinedab,1,Maribel Buenrostrob,1, Susana Martiñóna,b,1, Elisa Garciaa,b,1, Nayeli Floresa,b,1,Gabriel Guizar-Sahaguna,b,1, Dolores Corread, Ignacio Madrazob,1

aUnidad de Investigación Médica en Enfermedades Neurológicas, HE, CMN Siglo XXI, IMSS,Av. Cuauhtemoc No. 330, Col. Doctores, C.P. 06720, México City, MexicobProyecto CAMINA A.C. Tlalpan No. 4430 Col. Toriello Guerra, C.P. 14050, México City, MexicocEscuela Superior de Medicina del IPN. Plan de San Luis y Díaz Mirón CP 11340 México D.F., MexicodLaboratorio de Inmunología Experimental, Subdirección de Medicina Experimental, Instituto Nacional de Pediatría,Torre 8vo, Piso. Av. Insurgentes Sur 3700-C, Col. Insurgentes Cuicuilco, CP 04530 México DF, Mexico

A R T I C L E I N F O

⁎ Corresponding author. Fax: +52 55 55735545E-mail address: iantonio65@yahoo.com (AAbbreviations: BBB, Basso–Beattie–Bresnah

tract; CsA, cyclosporin-A; SC, spinal cord; GA1 Fax: +52 55 55735545.2 Fax: +55 57296000x62608.

0006-8993/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainres.2007.02.056

A B S T R A C T

Article history:Accepted 21 February 2007Available online 1 March 2007

Therapeutic approaches that promote both neuroprotection and neuroregeneration wouldbe valuable for spinal cord (SC) injury therapies. Cyclosporin-A (CsA) is animmunosuppressant that, due to its mechanism of action, could both protect andregenerate the neural tissue after injury. Previous studies have already demonstrated thatintraperitoneal administration of CsA at a dose of 2.5 mg/kg/12 h during the first 2 days afterSC contusion, followed by 5 mg/kg/12 h orally, diminishes tissue damage and improvesmotor recovery. In order to evaluate the effect of this CsA dosing regimen on axonal growth,we assessed motor recovery, presence of axons establishing functional connections andexpression of GAP-43 in rats subjected to a complete SC transection. The Basso–Beattie–Bresnahan rating scale did not show difference in motor recovery of CsA or vehicle-treatedrats. Moreover, somato-sensorial evoked potentials demonstrated no functionalconnections in the SC of these animals. Nevertheless, histological studies showed that: i)a significant number of CsA-treated rats presented growing axons, although they deviatedperpendicularly at the edge of the stumps, surrounding them, ii) the expression of GAP-43 inanimals treated with CsA was higher than that observed in the control group. Finally,anterograde tracing of the corticospinal tract of rats subjected to an incomplete SCtransection showed no axonal fibers reaching the caudal stump. In summary, CsAadministered at the dosing-regimen that promotes neuroprotection in SC contused ratsinduces both GAP-43 expression and axonal growth; however, it failed to generatefunctional connections in SC transected animals.

© 2007 Elsevier B.V. All rights reserved.

Keywords:GAP-43Motor recoveryNeuroregenerationSomatosensory evoked potentialSpinal cord injuryTherapy

.. Ibarra)an; CNS, central nervous system; CDPs, cord dorsal evoked potentials; CST, corticospinaP-43, growth-associated protein-43

er B.V. All rights reserved.

l

Fig. 1 – Motor recovery of rats subjected to a SC-transectionand treated with cyclosporin-A or vehicle. BBB rating scoreshowed no significant difference between the studied groups(p>0.05, two factor ANOVA for repeated measures). Eachpoint represents the mean±S.E.M. of 10 rats.

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1. Introduction

Any therapy applied after spinal cord (SC) injury should bothprotect the remaining healthy cells from secondary damage,and restore the hurt neural tissue. Regarding the first, manystrategies have been shown useful, which enhance motorrecovery in experimental models (Boran et al., 2005; Ibarra etal., 2003, 2004; Sharma et al., 2006a,b). For the second purpose,several strategies have been evaluated, but no effectivetherapy is yet available. Cyclosporin-A (CsA) is an immuno-suppressant agent which has been successfully proven as aneuroprotective drug (Borlongan et al., 2003; Diaz-Ruiz et al.,1999, 2000, 2004, 2005; Ibarra et al., 2003; Okonkwo et al., 1999;Sullivan et al., 2005). It is one of the few drugs assessed underan established dosing schedule in models of SC injury (Diaz-Ruiz et al., 1999, 2000; Ibarra et al., 1996, 2003). By inhibitingcalcineurin, a calcium-dependent phosphoserine–phospho-threonine protein phosphatase, CsA induces expression of thegrowth-associated protein-43 (GAP-43), involved in neuronalprocess extension (Alonso et al., 1995; Curtis et al., 1993;Strittmatter et al., 1992). Also, it inhibits romatase activity (acalcineurin-independent enzyme), and thus promotes neu-roregeneration (Palladini et al., 1996; Sosa et al., 2005;Sugawara et al., 1999; Wei et al., 2004).

Currently, there are no data available on the neuroregen-erative properties of CsA at the dosing scheme that promotesneuroprotection in models of SC injury (Ibarra et al., 1996).Thus, a unique therapy with CsA that protects and restoresneural tissue was evaluated herein.

2. Results

2.1. Long-term effects of CsA on rats with completeSC-transection

2.1.1. Functional outcomeAs can be seen in Fig. 1, SC-transected animals presented verypoor motor recovery, as assessed by the BBB-rating scale,without significant difference between CsA and vehicle-treated rats. At the end of the study, average BBB score was3.0±0.82 (mean±S.E.M.) for CsA-treated and 2.9±0.68 forvehicle-treated rats (p>0.05, two factor ANOVA for repeatedmeasures). Both laminectomized groups presented a score of21, which was maintained throughout the study (not shown).

2.1.2. Electrophysiological resultsAfter electric stimulation of the sciatic nerve, spinal corddorsum evoked potentials (CDPs) were evaluated at lumbarand thoracic levels. The reliability of the technique waspreviously assessed in an independent set of non-treatedrats (3 sham-operated and 3 transected animals). A triphasicwave was recorded at T7 level before surgery; however, 7 daysafterwards only sham operated animals presented thiscomponent (see Fig. 2), indicating total disruption of axonalfibers in the transected rats. As expected, all rats (sham ortransected) presented a triphasic wave from lumbar nervesindicating transmission of the electric stimulus (Fig. 2). Onceconfirmed the reliability of the technique, the CDPs of treated

rats were evaluated. One hundred and twenty days aftersurgical procedure, sham-operated rats presented a triphasicwave recorded at T7 and L6 SC levels. The comparison oflatency or amplitude of the different components of thewavesshowed no significant differences between treated anduntreated rats (p>0.05); this finding ruled out a direct effectof CsA on electrophysiological response (see Figs. 2 and 3).Injured animals only presented the signal recorded at L6 (seeFig. 2).

2.1.3. Morphological findingsAnalysis of the site of injury using silver stain showed that itwas complete in all cases. The end of the proximal and distalstumps presented signs of degeneration, characterized bysubstitution of the normal spinal cord tissue by large cysts(Fig. 4). In many cases, some axons surrounding the stumpswere identified, although their relative amount varied accord-ing to treatment: 70% of CsA-treated rats showed moderate toabundant growing axons (Figs. 4A and B); this was onlyobserved in 10% of the vehicle-treated rats (p=0.025, Fisherexact probability test; Figs. 4C and D). No growing axonstraversing the injury site were observed; in all cases theyturned next to the surface of the proximal stump (Fig. 4B).Most of them emerged from the spinal cord, although somearose from the neighboring peripheral roots (Fig. 4B).

2.2. Effect of CsA on GAP-43 expression

As GAP-43 is involved in axonal growth, it was looked for in anew set of experiments, in the lesion site by immunofluores-cence 10 days after a complete SC-transection. Fig. 5A showsthat CsA-treated animals presented a higher amount (902±57)(mean±S.E.M. of integrated density) of this molecule ascompared to vehicle-treated animals (571±121) (p=0.03), butwith a similar distribution, i.e. limited to the gray matter. Theimmunoreactivity was predominantly observed in the areasurrounding the central canal (area X) and lamina VII (see Figs.5B, C and D).

Fig. 2 – Spinal cord dorsum evoked potentials (CDPs) recorded at L6 and T7 spinal cord segments. After stimuli, all studiedanimals presented the electrophysiological response at L6 segment; however, only sham operated rats (laminectomy)presented the CDPs recorded at thoracic level. CsA, cyclosporin-A.

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2.3. Effect of CsA on the corticospinal tract axonal growth

The corticospinal tract (CST) plays a central role in move-ment control, through its terminals in the intermediate grayand ventral horn, including motoneurons (Liang et al., 1991).Thus, we proceeded to evaluate these fibers 2 months afteran incomplete SC-transection. In no case, the CST reachedthe lesion epicenter. All CsA-treated rats presented con-voluted and tortuous fluorescent fibers extending in bundlesalong the ventral part of the dorsal columns (see Figs. 6Aand B). However, 1–2 mm before the center of the lesionthey disappeared and none was observed reaching orpenetrating the caudal stump. No anterograde tracing wasobserved in vehicle-treated rats suggesting a strong degen-erative process (Figs. 6C and D). It is worth mentioning that,in most cases, CsA treated rats presented axons with amorphology of regenerated fibers: a tortuous curse and thepresence of a growth-cone-like tip (Figs. 7A and B) (Stewardet al., 2003).

3. Discussion

Currently, the cure of neurodegenerative diseases representsone of the most important challenges for scientists. Thelimited ability of neural cells to replicate and restore damagedtissue constitutes one of themain problems to face. Numerousstudies have reported interesting approaches to solve it;however, none of them has proven to be satisfactory (Byrneset al., 2005; GrandPre et al., 2002; Hashimoto et al., 2005;Mansilla et al., 2005; Nash et al., 2002); despite some have been

able to induce axonal growing, almost all have failed to inducefunctional connections.

In the present work, using the CsA-dosing regimen thatpromotes neuroprotection in incomplete SC injury models(Diaz-Ruiz et al., 1999; Ibarra et al., 2003), we provide evidence ofthe positive action of this drug upon axonal growth: asignificant percentage of CsA-treated rats presented growingaxons; however, functional recovery was not demonstrated.Furthermore, a higher expression of GAP-43 was observed.Nevertheless, the growing fibers were unable to cross thesection gap or to make effective connections yielding neurolo-gical recovery. CsA-treated rats presented poor motor outcomeand absence of CDPs at T7. It isworth tomention that a possibledirect action of CsA on electrical transmission or a technicalfailure in electrical stimulation were ruled out as cause of CDPsabsence, since it did not induce an alteration of the compo-nents of the wave in sham-injured rats, and all animalspresented the triphasic wave obtained from L6 SC segment.Thus, the absence of the T7 wave in SCI rats, suggests the lackof axonal connections. The histological findings furtherdemonstrated that those axons extending from the cranialstump were deviated transversely and did not cross the site ofinjury. Currently, it is well known that after adult nervoussystem trauma, a healing process occurs, producing a thickwound scar, which constitutes the major impediment for axonregeneration (Hermanns et al., 2001; Stichel et al., 1999b). Infact, numerous studies have demonstrated that inhibition ofscar formation promotes massive axon elongation across thelesion site (Stichel et al., 1999a,b). Thus, it is plausible to suggestthat themain cause of axonal deviationwas the presence of thescar, which acted as a physical/chemical barrier for the growing

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cones (Stichel et al., 1999a). Another possible cause of axonaldeviation is the action of repulsive factors. Previous studieshave demonstrated a marked up-regulation of repulsive

Fig. 3 – Analysis of spinal cord dorsumpotential tracings obtainedvehicle. The latency of the first (A and B) and second (C and D) columbar (A and C) or thoracic (B and D) SC-segments of those ratsamplitude of the wave (E and F) was similar in all groups. The trrepresent mean±S.E.M. of 8 rats.

molecules (i.e. semaphorin and Eph-B3), which are involvedin axonal regeneration inhibition after SC injury (Miranda et al.,1999; Niclou et al., 2006).

from thoracic or lumbar segments of rats treatedwith CsA ormponents of the triphasic wave was very similar, either atin which the evoked electrical stimuli was detected. Theiphasic wave was abolished in SC-transected rats. Bars

Fig. 4 – Representative photomicrographs of silver stained spinal cords at the site of injury. Longitudinal sections (4×) ofCsA (A and B) or vehicle (C and D) treated rats. Figures B and D show amplified (10×) details of A and C, respectively. A and Bshow moderated amount of growing axons surrounding the cystic area at the end of the parent stumps; most growingaxons emerge from preserved spinal cord tissue, although some axons apparently emerge from a nerve root (r) in B.Scarce axons which follow a similar pattern to the one described in A and B, were observed in C and D. No axons crossfrom one stump to the other. p, proximal stump; d, distal stump.

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The failure of CsA to favor functional regeneration was alsoobserved when the CSTwas evaluated. None of vehicle-treatedrats presented labeled fibers along the analyzed area (1 cmcranial or caudal to the site of transection). It has beendescribedthat axotomized corticospinal fibers undergo progressive andcontinuing retrograde degeneration so they completely disap-pear (Pallini et al., 1988). This phenomenon is thought to berelated to secondary ischemia or to deficiency of growth factors(Sayer et al., 2002), and it could be the reason for the absence ofCST fibers in vehicle treated rats. Noteworthy, this was not thecase for animals subjected to CsA; despite none of the axonscrossed the siteof injuryall presentedCST-labelled fibers. Eitherby calcineurin-dependent or independent mechanisms, CsA iscapable to diminish several harmful events developed aftercentral nervous system (CNS) damage, i.e. lipid peroxidation,apoptosis, disturbance of mitochondrial function and inflam-mation (Diaz-Ruiz et al., 2000, 2004, 2005; Domanska-Janik et al.,2004; Khaspekov et al., 1999; Ruiz et al., 2000; Terada et al., 2003).Thus, CsA could be providing a favorable microenvironment toprevent lesion-induced axonal die-back. Previous studies havedemonstrated the protective role of CsA inmodels of CNS injury(Alessandri et al., 2002; Diaz-Ruiz et al., 1999; Ibarra et al., 2003;

Sullivan et al., 2005). Neuroregenerative properties of CsA couldalso explain the presence of CST fibers in treated animals. As itwas already shown, in most CsA-treated rats the axonspresented morphology suggestive of development in progress(Steward et al., 2003). This, in turns, would be favoring thepresence of fibers in the injured area. Some studies havesuggested the neuroregenerative properties of CsA (Avramutand Achim, 2003; Gold, 1997; Palladini et al., 1996; Sugawaraet al., 1999); one of them, reported electrophysiological andneurological reestablishment as a result of regenerative pro-cesses (Palladini et al., 1996). Despite our results suggested apositive effect of CsA on axonal growing, no functionalconnections or motor recovery could be observed. It is difficultto explain the cause of disparity in results; however, somedifferences in the study design and data presented couldexplain it. Firstly, different rat strains were used, which couldhave distinct microenvironments for positive and negativefactors participating in axonal regeneration; for instance,scarring process or expression of repulsive molecules could beuniquely modulated in each strain. Secondly, no axons tippedwith a growth coneor branching patterns (i.e. truly regeneratingaxons and not only spared fibers) were shown in the work cited

Fig. 5 – Expression of GAP-43 in the transected spinal cord of CsA or vehicle treated rats. CsA-treated animals presented ahigher amount of this molecule as compared to vehicle-treated ones (A) (p=0.03, Student t-test). New born brain (NB) was usedas positive control. The expression of GAP-43 presented a similar distribution in vehicle (C) and CsA-treated rats (D) and waslimited to the graymatter. Picture (B) represents a negative control using an irrelevant primary antibody. * Different from vehicletreated rats (p=0.03, Student t-test), ** Different from the other groups (p=0.0002, ANOVA followed by the Tukey's multiplecomparison test). Bars represent mean±S.E.M. of 4 rats.

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(Steward et al., 2003); neither there was histological evidence ofaxons reaching the caudal stump (Palladini et al., 1996).

Finally, it is worth to mention that, although the doseregimenused in thiswork is useful to promote neuroprotection,it is not necessarily adequate to induce functional connections,especially in long-lasting studies. Future studies should testother dose-regimens with the aim of promoting functionalrecovery. Besides, a combined therapy of CsA, fibroglial scarinhibitors and repulsive molecule-neutralizers could be tested.

4. Experimental procedures

4.1. Study design

Three sets of experiments were performed. Firstly, in orderto study the long-term effects of the neuroprotective CsA-

regimen on the axonal growth, two groups of rats (n=10per group) were subjected to a complete SC-transection.Once injured, the animals were treated with CsA or vehiclefor a period of 120 days. Rats subjected to laminectomyonly, and treated (n=8) or not (n=8) with CsA were used ascontrols. In a second set of experiments, two groups of rats(n=8 per group) were subjected to complete SC-transection,and then treated with CsA or vehicle until the end of thefollow-up. Ten days after injury, the amount (n=4 pergroup) and distribution (n=4 per group) of GAP-43 wasevaluated.

In a third set of experiments, the effect of the CsAneuroprotective-dosing scheme on corticospinal fibers axonalgrowthwas analyzed. For this purpose, two groups of rats (n=5per group) were subjected to incomplete transection of dorsalSC at T9, including the CST. Once injured, the animals wereimmediately treated either with CsA or vehicle, and after

Fig. 6 – Anterograde labelling of the corticospinal tract of SC-transected animals treated with CsA (A) or vehicle (C). Boxes inpictures (A) and (C) are shown amplified in (B) and (D) respectively. Only CsA-treated rats presented labeled fibers; however, innone of these animals the fibers reached the site of lesion or the caudal stump.

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2months of therapy, the CST of all animals was anterogradelylabeled.

4.2. Experimental animals

Adult female Sprague–Dawley (SPD) rats (13–14 weeks old,200–220 g) were supplied by the Animal Breeding Center ofCamina Research Project. The rats were age-matched andhoused under light- and temperature-controlled room. Effortswere made to minimize the number of animals used and theirsuffering. All procedures were in accordancewith the NationalInstitutes of Health (US) Guide for the Care and Use ofLaboratory Animals and the Mexican Official Norm onPrinciples of Laboratory Animal Care.

4.3. Spinal cord injury and animal care

Rats were anesthetized by intramuscular injection of keta-mine (77.5 mg/kg, Probiomed, Mexico City, Mexico) andxylazine (12.5 mg/kg; Fort Dodge Laboratories, Fort Dodge,Iowa). One hour after induction of anesthesia, their spinalcords were exposed by laminectomy at T9 level, and the dura-mater was penetrated with a 30-gauge needle to perform acomplete or incomplete SC transection. Complete transectionwas performed by sliding a straight-edged scalpel bladethrough the spinal cord. Accuracy of the lesion was verifiedvisually and by passing a micro-hook through the entireinternal contour of the dura. For incomplete transection

approximately 75% of the cord (including the corticospinaltract) was transversely cut with iridectomy scissors. Afterinjury, the aponeurotic plane and the skin were separatelysutured with nylon thread.

Sterile beds and filtered water were replaced daily. Bladderexpression was assisted by massage at least twice a day, untilnormal function was attained. All rats were carefully mon-itored for evidence of urinary tract infection or any other signof systemic disease. During the first 10 days after lesion, and incase of hematuria after this period, they received a course ofenrofloxacine (Marvel, México) in the drinking water at a doseof 64 mg/kg/day.

4.4. CsA or vehicle administration

To reach therapeutic non toxic serum levels of CsA(Sandimmune; Novartis Pharma AG, Basel, Switzerland), weused the scheme previously standardized for SC injurymodels (Ibarra et al., 1996) and that has been reported tobe neuroprotective (Diaz-Ruiz et al., 1999; Ibarra et al., 2003).Briefly, 6 h after SC injury, rats were given 2.5 mg/kg/12 hintraperitoneally (i.p.) followed by 5 mg/kg/12 h orally, untilthe end of the study. Oral administration was given byinserting a water-lubricated curved blunt stainless steelcatheter into the esophagus, twice daily. Physiologic salinesolution (i.p.) or olive oil (oral) only, was administered tovehicle-treated rats according to the same regimen describedabove.

Fig. 7 – Photomicrograph of a representative growth conefound in the corticospinal tract of rats subjected toSC-transection and CsA treatment (A). Figure in B is anamplified detail of A. One to two millimeters away from theend of the proximal stump, the presence of a tip with agrowth-cone-like specialization was found in mostCsA-treated rats. GC, growth cone; A, axon.

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4.5. Motor recovery assessment

Behavioral recovery was assessed by the use of the Basso–Beattie–Bresnahan (BBB) open-field test of locomotor ability(Basso et al., 1995). Recovery was scored on a scale of 0(complete paralysis) to 21 (complete mobility) by two obser-vers blinded to the treatment given to the rat being observed.

4.6. Electrophysiological recordings

Two laminectomies, one at the thoracic (T8) and other at thelumbar (L6) level were performed in the anesthetized rat. Afterlaminectomy and under deep anesthesia, two chlorinatedsilver electrode balls were placed on the dorsum of the spinalcord. Once in position, the spinal cord dorsum potentials(CDPs) were obtained as follows: current pulses using needleelectrodes were applied on the left hind limb.With the stimuliapplied, CDPs were evoked at the lumbar level. The electrodeswere placed at the best location to obtain greater amplitudesof CDPs. The presence of the CDPs was a test of the function-ality of the afferent tracts, and a positive result at the thoraciclevel was a test of afferents regeneration. The CDPs wereamplified and averaged by a Nicolet Compact Four EMG

device. The impedance between electrodes was automaticallyfixed at 2000 to 5000 kΩ. Every recordingwas the average of 300sweeps. These values were digitized and stored for subse-quent analysis and illustration. The threshold of the CDPs wasselected according to the lowest intensity of stimuli necessaryto obtain a CDP. In order to explore the possibility ofdifferential regeneration according to the kind of afferents,we applied stimuli at 2, 3, 4, 6 and 12 times the threshold (×T).Only the latter are shown in this work.

4.7. Histological analysis

To analyze axons at the site of injury, rats were anaesthetized120 days after lesion, and perfused via the ascending aorta,with 50 ml physiological saline, followed by 500 ml 10%formaldehyde, using a peristaltic pump at 30 ml/min. A 12-mm-long fragment of SC containing the injured zone in thecenter was resected and placed in the same fixing solution for1 week. Tissues were embedded in paraffin. Longitudinal 10-μm-thick sections were obtained sequentially every 650 μm.Sections mounted on slides were impregnated with silvernitrate using the Sevier–Munger's technique as previouslydescribed (Guizar-Sahagun et al., 2004). The relative amount,location and course of axons were evaluated by a treatment-blinded expert. Rats with absence or scarce number of axonsat the end of the stumps, were considered “rats with non-growing axons”, while animals showing moderate to abun-dant axons at both stumps were classified as “rats withgrowing axons”.

Anterograde labeling with rhodamine dextran amine (RDA,Fluoro-ruby; Molecular Probes, Eugene, OR) was used toidentify axons from the corticospinal tract (CST) at the siteof injury. Two months after incomplete SC transection, fiveanimals from each group (CsA or vehicle-treated) were re-anesthetized, and the dye (10% in PBS; 5 μl per cortex) wasbilaterally injected into the hindlimb area of the cortex (depthof 2.6 mm from the dura; 3.3 mm posterior and 2.8 mm lateralto bregma). Two weeks after RDA injection, the animals wereperfused with PBS, followed by 4% paraformaldehyde andthen the spinal cord was collected and cryoprotected in 30%sucrose for at least 3 days. A 20-mm block of the spinal cord,with the injured site at the center, was excised and embeddedin Tissue-Tek (Sakura Finetek, CA, USA). Serial longitudinalcryosections 20-μm-thick, were cut and collected onto gelatin-coated slides. Theywere thenmountedwith antifading oil andcoverslips and examined under a Zeiss laser-scanning con-focal microscope (LSM-510) or a Zeiss axioplane 100 fluores-cence light microscope. All sections were inspected andanalyzed by an observer who was blinded to treatment.

4.8. GAP-43 extraction, electrophoresis and Westernblotting

Immediately after euthanization, a 10-mm block of the spinalcord, with the injured site at the center, was excised andprocessed as was previously described (Eastwood and Harri-son, 2001). Briefly, tissue samples were weighted and homo-genized in 10 vol. (10 μl/μg) of cold suspension buffer (0.1 MNaCl, 0.01 M Tris–HCl, 0.001 M EDTA, 1 μg/ml aprotinin, 100μg/ml phenylmethylsulphonyl fluoride) and centrifuged at

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13,000×g for 5 min. The remaining pellet was resuspended in500 μl of suspension buffer containing 1% sodium dodecylsulphate, boiled for 10 min, and centrifuged at 13,000×g for5min. The protein content of the supernatant was determinedusing the Bradford assay (Bio-Rad Lab. Inc. CA, USA) (Bradford,1976). Samples were processed in a dual mini vertical 12%polyacrylamide gel electrophoresis system (Owl, NH, USA).Protein was electrotransferred from gels to nitrocellulosemembranes at 100 V for 1 h. Non-specific binding sites onthe membranes were blocked with 5% non fat dry milk inPBS-Tween, overnight at 4 °C. The nitrocellulose sheets werethen incubated with an anti-GAP-43 monoclonal antibody(Clone 7B10, Zymed Lab. Inc. CA USA) diluted 1:1000 inblocking buffer, for 2 h at room temperature (RT). After 3washes with blocking buffer, blots were incubated with anti-mouse antibody conjugated to horseradish peroxidase,diluted 1:200 in blocking buffer for 1.30 h at RT and visualizedusing the enhanced chemiluminescence detection kit (Amer-sham, Pharmacia Biotech, UK). Quantitative integrated opti-cal density measurements for each band were carried outusing computerized videodensitometry (Total Lab ControlCenter 1.0).

4.9. Immunohistochemistry

Ten days after SCI, each rat was intracardially perfused andprepared for immunohistochemical studies as describedpreviously (Ibarra et al., 2004). The spinal cords were removed,postfixed overnight, and transferred to sucrose 30% forcryoprotection for at least 3 days. A 10-mm block of the spinalcord, including the injured site, was excised and divided in thecranial and caudal segments. The cranial segment wasembedded in Tissue-Tek and then, frozen axial blocks wereserially sectioned (20 μm) from the epicenter. After havingdischarged 1000 μm of tissue, sections were taken up forstaining. Tissue samples were then incubated for 1 h with themonoclonal antibody anti-GAP-43 (2.5 μg/ml; Clone 7B10,Zymed Lab. Inc. CA). After rinsing, sections were incubatedwith the secondary antibody, FITC-conjugated goat anti-mouse IgG (1:100; Zymed Lab. Inc. CA), for 1 h at RT. Theywere then prepared for examination under a Zeiss laser-scanning confocal microscope (LSM-510) or a Zeiss Axioplane100 fluorescence light microscope. The results were analyzedby an observer who was blinded to the treatment received bythe rats.

4.10. Statistical analysis

Data were analyzed by the GraphPad Prism 3.0 software.Numeric variables were analyzed by ANOVA or Student t-test.Statistical significance was considered relevant when p≤0.05.

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