No Evidence for Chronic Demyelination in Spared Axons after

Development/Plasticity/Repair

No Evidence for Chronic Demyelination in Spared Axonsafter Spinal Cord Injury in a Mouse

Jurate Lasiene,1,2 Larry Shupe,3 Steve Perlmutter,3 and Philip Horner1

Departments of 1Neurological Surgery, 2Psychology, and 3Physiology and Biophysics, University of Washington, Seattle, Washington 98195

The pattern of remyelination after traumatic spinal cord injury remains elusive, with animal and human studies reporting partial tocomplete demyelination followed by incomplete remyelination. In the present study, we found that spared rubrospinal tract (RST) axonsof passage traced with actively transported dextrans and examined caudally to the lesion 12 weeks after mouse spinal cord contusioninjury were fully remyelinated. Spared axons exhibited a marginally reduced myelin thickness and significantly shorter internodes.CASPR (contactin-associated protein) and Kv1.2 channels were used to identify internodes and paranodal protein distribution propertieswere used as an index of myelin integrity. This is the first time the CNS myelin internode length was measured in a mouse. To betterunderstand the significance of shortened internodes and thinner myelin in spared axons, we modeled conduction properties usingMcIntyre’s et al. model of myelinated axons. Mathematical modeling predicted a 21% decrease in the conduction velocity of remyelinatedRST axons attributable to shortened internodes. To determine whether demyelination could be present on axons exhibiting a patholog-ical transport system, we used the retroviral reporter system. Virally delivered green fluorescent protein unveiled a small population ofdystrophic RST axons that persist chronically with evident demyelination or abnormal remyelination. Collectively, these data show thatlasting demyelination in spared axons is rare and that remyelination of axons of passage occurs in the chronically injured mouse spinalcord.

Key words: myelin; remyelination; CASPR; trauma; mouse; conduction; internode; spinal cord injury; axons

IntroductionExperimental strategies such as cell transplantation designed topreserve myelin or to induce remyelination have lead to success-ful improvements in function after spinal cord injury (SCI) ifadministered in acute or subacute phases after SCI (Li et al., 1997;Akiyama et al., 2002; Cao et al., 2005; Cummings et al., 2005;Keirstead et al., 2005; Lepore and Fischer, 2005). However, remy-elination strategies fails if transplants are delivered chronicallyafter SCI (Keirstead et al., 2005; Karimi-Abdolrezaee et al., 2006).The primary data regarding myelin status in chronic injury isincomplete and a poor correlation between rodent and humanstudies exists. The majority of human cases do not show progres-sive chronic demyelination, whereas only a few cases report per-sistent demyelination (Kakulas, 1999; Norenberg et al., 2004;Guest et al., 2005). Because previous studies did not correlatefiber integrity with myelin condition, the key question remainsunanswered: what is the chronic myelin status of spared or intact

white matter? Because myelin sheaths not only ensure successfulpropagation of action potentials, but also participate in axonalmaturation, transport and survival (Brady et al., 1999; Edgar etal., 2004) it is important to understand the pattern of remyelina-tion within individual axons to develop the most appropriatemyelin repair strategies.

Rodent models of SCI reveal electrophysiological and mor-phological signs of demyelinated axons (Gledhill et al., 1973;Blakemore, 1974; Gledhill and McDonald, 1977; Blight, 1985;Gensert and Goldman, 1997; Cao et al., 2005). Initial demyelina-tion is thought to be caused by necrosis of oligodendrocytes;however, it is not exactly clear what causes prolonged oligoden-drocyte death weeks and months after injury (Blight, 1985;Crowe et al., 1997). In accordance with morphological studies ofmyelin disintegration (Balentine, 1978; Griffiths and McCulloch,1983), proteolipid protein (PLP) and myelin basic protein (MBP)expression is significantly reduced after injury (Wrathall et al.,1998). In addition, immunohistochemistry studies demonstratethat sodium and potassium channels as well as contactin-associated protein (CASPR) distribution lose their compact andrestricted staining pattern in demyelinated fibers (Karimi-Abdolrezaee et al., 2004). Myelin damage results in decreasedaction potential conduction velocity or a conduction block andtranscranial magnetic motor-evoked potentials show longer la-tency responses (Fehlings and Nashmi, 1995). Weeks after SCI,demyelination is evident mostly in the medial area rostral andcaudal to the lesion site; however, thin profiles of new myelin areobserved and their number increases with time (Griffiths and

Received Dec. 29, 2006; revised Feb. 25, 2008; accepted Feb. 25, 2008.This work was supported by National Institutes of Health Grants NSO46724 and NS040867, the International

Foundation for Research on Paraplegia, and a Royalty Research Grant (University of Washington, Seattle, WA). Wethank J. Trimmer (University of California, San Diego, CA) for providing polyclonal CASPR antibody, F. Galimi (Uni-versity of Sassari, Sassari, Italy) for a gift of viral plasmids, D. Possin and N. Gill (University of Washington, Seattle,WA) for technical help with electron microscopy, and J. Petruska (State University of New York, Stonybrook, NY) forcomments on this manuscript. P.H. is a member of the University of Washington Institute for Stem Cell and Regen-erative Medicine and the Center on Human Development and Disability.

Correspondence should be addressed to Philip Horner, Department of Neurological Surgery, University of Wash-ington, Seattle, WA 98195. E-mail: [email protected].

DOI:10.1523/JNEUROSCI.4756-07.2008Copyright © 2008 Society for Neuroscience 0270-6474/08/283887-10$15.00/0

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McCulloch, 1983). Electron microscopy of remyelinated nodesshows normal morphology, with myelin lamellae terminating incytoplasm-filled loops on each side of the node (Gledhill et al.,1973). Collectively, these studies demonstrate dynamic changesin myelin status including significant demyelination but also my-elin regeneration.

In the present study, we used multiple methods to discrimi-nate spared axons (that pass through the lesion) versus damagedaxons simultaneously evaluating myelin health within individualnerve segments in the chronically injured mouse spinal cord. Weselectively studied the RST because it is a heavily myelinated tractand important for rodent locomotion (Waldron and Gwyn,1969; Holstege, 1987). We quantified the amount of demyelina-tion, myelin thickness, and internodal lengths in spared versussevered axons. To our knowledge, this is the first study to com-bine functional axonal labeling and the simultaneous examina-tion of myelin indices. The results establish a tight correlationbetween myelin status and axon integrity.

Materials and MethodsFemale C57BL/6 mice (8 weeks of age) were used in this study. All sur-gical procedures and experimental manipulations were in accordancewith a protocol approved by the Animal Care and Use Committee of theUniversity of Washington.Surgical procedures. Mice, anesthetized with intraperitoneal injections(0.5 ml/20 g) of avertin (tribromoethnol in tert-amyl alcohol), under-went midthoracic (T9) laminectomy after which an impact probe waslowered onto the dura and the cord was rapidly displaced 0.5 mm (mod-erate SCI) using the Ohio State University contusion device. After con-tusion injury, muscle and skin were closed in layers, and then animalswere kept on a heating plate (38°C) until fully awake. Subcutaneoussaline, gentomicin (APP, Schaumburg, IL) and intramuscular buprenor-phine (Reckitt Benckiser Healthcare, Hull, UK) were administered for 7and 2 d, respectively. Bladder expression was performed two times dailyuntil voluntary control returned.

Tracing of the rubrospinal tracts. To examine myelin characteristicsaround rubrospinal fibers, the Rubrospinal tracts were anterogradelytraced immediately after SCI or 8 weeks after contusion injury or inuninjured controls. Animals received bilateral pressure injections total-ing 2 �l per animal of either a 10% solution of biotinylated dextran amine[BDA 10,000 molecular weight (MW); Invitrogen, Carlsbad, CA] or rho-damine dextran amine (Fluoro-Ruby 10,000; Invitrogen) into the rednuclei (coordinates: 3.4 –3.6 mm posterior to bregma, 0.5–1 mm lateralto midline, 3.6 mm depth) delivered by Hamilton 5 �l syringe fitted witha 33 gauge Hamilton needle. The needle was left in place for 2 min toensure proper dye distribution. Sixteen to 18 d after tracer injections,animals were perfused with appropriate fixatives.

Lentivirus production. We produced vector stocks by transient trans-fection of 293T cells grown in DMEM (Invitrogen), 10% FBS (HyClone,Logan, UT), 2 mM glutamine (Invitrogen), and 1% of penicillin-streptomycin (Invitrogen) with the pCMV-eGFP expression vector, en-velope plasmid pMDG, packaging plasmid pMDLg/p RRE, and transfervector plasmid pRSV.Rev (a gift from F. Galimi, University of Sassari,Sassari, Italy) by calcium phosphate DNA precipitation as described pre-viously (Dull et al., 1998). Supernatants were collected 48 and 72 h aftertransfection, filtered, and concentrated by two successive ultracentrifu-gations. p24 antigen was assayed by ELISA (PerkinElmer, Wellesley,MA).

Viral transfection of the red nuclei. To label RST axons we used a retro-viral reporter system that does not rely on an active axonal transport, butrather on passive diffusion. pCMV-GFP lentivirus (titer of 10 9-10 10,total volume 5 �l per animal) was pressure injected bilaterally into thered nuclei as described above. Animals were perfused 30 d after viralinjections with 4% paraformaldehyde.

Immunoelectromicroscopy. After an overdose with beupanasia-D(Schering-Plough, Union, NJ), animals were killed by intracardiac per-fusion with 0.5% glutaraldehyde (Ted Pella, Redding, CA) and 3% para-

formaldehyde (Ted Pella) in 0.1 M phosphate buffer, pH 7.4. Spinal cordswere removed and sectioned at 60 �m on a Vibratome. The BDA wasdetected by the following protocol: washed three times for 10 min in 0.1M PB, then washed three times for 10 min a 0.1 M Tris buffer, 3 h incu-bation in an avidin-peroxidase complex (ABC elite; Vector Laboratories,Burlingame, CA) according to the manufacturers suggestion, washedthree times for 10 min in Tris-based buffer (TB), first preincubation for10 min in 0.4% ammonium nickel sulfate, second preincubation for 10min in 0.4% ammonium nickel sulfate and 0.7 mg/ml diaminobenzidine,and reaction in 0.4% ammonium nickel sulfate and 0.7 mg/ml diamino-benzidine with 0.004% hydrogen peroxide in TB. The reaction wasstopped by washing extensively in TB. The sections were then washedthree times for 10 min in 0.1 M PB buffer. After osmication in 1% OsO4for 1h, sections were washed again in 0.1 M PB buffer, dehydratedthrough an ascending alcohol series ending in 2� 10 min in propyleneoxide, and subsequently embedded using the Eponate 12 kit (Ted Pella).Then, semithin sections were cut at 1 �m, mounted on glass slides andstained with toluidine blue or Richardon’s stain. Ultrathin sections werecut at 60 nm on a Reichert Ultracut S microtome by Leica (Nussloch,Germany), mounted on nickel grids, uranyl acetate and lead citratestained, and viewed under a Philips (Aachen, Germany) TEM/CM 10electron microscope.

Immunohistochemistry. Animals were killed with an overdose withbeupanasia-D (Schering-Plough) followed by intracardiac perfusionwith 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Spinalcords were removed, rubrospinal tracts were dissected rostral and caudalto the lesion and incubated overnight with primary monoclonal or poly-clonal CASPR (1:100, a generous gift from J. S. Trimmer, University ofCalifornia, Davis, CA), and monoclonal Kv1.2 (1:200; NeuroMab, Davis,CA) antibodies. Then, tissue was incubated with secondary antibodiesagainst mouse and rabbit Alexa 488 (1:250, Invitrogen) and Cy5 (1:250;Jackson ImmunoResearch, West Grove, PA). Some caudal parts of therubrospinal tracts were transferred to slides with a small pool of 0.1 M PBwhere single positively labeled rubrospinal fibers were mechanicallyteased for single fiber analysis. Single fibers were stained by the samemethod as whole rubrospinal tracts. Some of the lesion sites were cryo-protected in 30% sucrose in 1� PBS overnight and flash frozen in OCT,and then 16-�m-thick horizontal sections were cut on a cryostat. Thetissue was incubated overnight with primary guinea pig GFAP antibody(1:500). Stained rubrospinal tracts or single fibers were viewed underNikon (Tokyo, Japan) Eclipse TE200 confocal microscope fitted withBio-Rad (Hercules, CA) Laser Sharp 2000. Later, fibers were recon-structed with Volocity software (Improvision Lexington, MA).

Quantitative PCR. Spinal cords were removed from killed animals thathad undergone moderate contusion spinal cord injury (as describedabove) 24 h (n � 2), 5 d (n � 3), or 2 weeks (n � 3) earlier, and RNA wasextracted with the Invitrogen PureLink Micro-to-Midi RNA kit. RNAwas transcribed to DNA with Qiagen (Hilden, Germany) reverse tran-scription kit. DNA samples were analyzed in triplicate by using the ABIPRISM 7900 sequence detection system (Applied Biosystems, FosterCity, CA). The sequence of MBP sense primer was 5�-ATGGCATCACAGAAGAGACC-3� and antisense primer was 5�-CATGGGAGATCCAGAGCGGC-3�, for �-actin, the sense primer was5�-CCACTGCCGCATCCTCTTCC-3� and antisense primer was 5�-CTCGTTGCCAATAGTGATGACCTG-3�. Each 50 �l quantitative PCR(qPCR) consisted of 1 �l of sample DNA, 1.5 �l of 10 �M sense andantisense primers, and SYBR green PCR mix (Qiagen). Reactions wereperformed in optical 384-well reaction plates (ISC BioExpress, Kaysville,UT). qPCR assays were run according to the manufacturer’s directions,and results were analyzed with Sequence Detection System software (Ap-plied Biosystems). Injured spinal cord tissue samples were comparedwith control samples taken from the same level of the spinal cord (i.e.,thoracic level 8 –10). �-actin served as an internal control for total RNAlevels. Fold changes were calculated as follows: �Ct � average Ct of targetgene (MBP) � average Ct of endogenous control gene (�-Actin), then wecalculated ��Ct � �Ct treated � �Ct untreated, and finally the foldchange � 2ˆ-[average ��Ct].

Quantification methods. G-ratios (axon diameter/total fiber diameter)were determined for all BDA-labeled axons. We measured G-ratios for all

3888 • J. Neurosci., April 9, 2008 • 28(15):3887–3896 Lasiene et al. • Spared Axons Are Remyelinated

BDA-labeled axons in randomly chosen sections within a 3 mm zonestarting 200 �m caudal to the lesion. To remove bias we analyzed allpositively labeled axons in a section (n � 80 axons from control mice;n � 92 axons from injured). Control measurements were performed atthe T10 –T11 levels, which correspond to the same location in the injuredanimals. Photos were taken of positively BDA-labeled axons at the fol-lowing magnifications: 2950 –3900� for overview, and 8900 –52000�for closer examinations. Later, photographs were digitized by scanningwith Epson (Long Beach, CA) Perfection 4870 Photo. Measurements ofmyelin sheath thickness and axon diameter were made by using NIHImage-J. A calculation of tissue shrinkage/correction factor was not madeand we assumed that tissue processing effected control and injured tissuesimilarly. Internode lengths were measured by tracing axons from oneparanode identified by CASPR staining to another using Image-J. Mul-tiple internodes were analyzed from a total of 99 and 100 BDA-labeledaxons from control and injured tissue, respectively. To ensure thatCASPR and Kv1.2 labels were colocalized on Fluoro-Ruby-positive axo-lemma, we used Volocity software to view three-dimensional reconstruc-tions of the tissue from confocal microscope z sections.

Mathematical modeling. McIntyre et al.’s (2001) model of a mamma-lian myelinated axon, adapted from the database archive of the NEU-RON simulation environment (http://senselab.med.yale.edu/senselab/modeldb/), was implemented to estimate the effects of the observedchanges in internode length, axon diameter, and myelin thickness onaxonal conductance. Briefly, the axon is modeled as a multicompartmen-tal double cable, with separate representations of the axolemma and themyelin sheath. Nodes of Ranvier, paranodal myelin attachment seg-ments, paranodal main segments, and internodal segments are includedas separate compartments with different geometry and electrical proper-ties. Action potentials are generated at the nodes with modifiedHodgkin–Huxley equations that incorporate nonlinear fast Na �, persis-tent Na �, slow K � conductance, a linear leak conductance, and mem-brane capacitance. The internodes have no active conductance. The axo-lemma and myelin sheath in paranodal and internodal segments eachhave a passive linear conductance in parallel with membrane capaci-tance. Myelin capacitance is represented as a function of capacitance perunit area and myelin thickness.

Approximately 500 control and 500 injured model axons were createdusing the axon diameter, internode length, and myelin thickness dataobtained in this study. The diameter of each Fluoro-Ruby-stained axonwas used to create 8 model axons with 121 nodes and 120 internodes of

variable length. The 120 lengths were takenfrom a uniform distribution (one SD about themean) calculated from the measured internodelengths of axons with diameters within 0.5 �m.The myelin thickness of each of the eight axonswas taken as the myelin thickness of one of theeight BDA-stained axons with the most similardiameters. Thus, data from all labeled axonsfrom all animals were used to create the simu-lated axons. The electrical and other geometricparameters were taken from McIntyre et al. forsimulated axons with diameters �3 �m, andextrapolated for smaller axons using the mea-surements of Berthold et al. (1983).

The model axons propagated an action po-tential in response to a suprathreshold, depo-larizing current step delivered to the node atone end. Conduction velocity was measured asthe distance between the 10th and 100th nodes,divided by the action potential conduction timebetween those nodes. All simulations were runin NEURON (Hines and Carnevale, 1997).

Simulations of conduction were also run us-ing the earlier model of Brill et al. (1977) forcomparison. This simpler model, availablefrom the NEURON database, represents theaxon and myelin sheath as a single conductor.Membrane dynamics in the internodes are afunction of myelin conductance and capaci-

tance, which is estimated from the relationship between the capacitanceand conductance of a coaxial cable (Goldman and Albus, 1968).

Statistics. All anatomical data were analyzed using two-tailed t testbecause we were comparing means of two independent randomly se-lected populations with normal distributions. The significance level wasp � 0.05.

ResultsMouse contusion leads to early, widespread myelin pathologyTo establish the commonality of contusion-induced myelin pa-thology in our mouse model with that of rat and larger species, weconducted the following experiments. First, we established thepresence of demyelination at early time points after injury. TheRST was traced with bilateral injections of rhodamine-dextran-amine (Fluoro-Ruby) bilaterally in the red nuclei (Fig. 1A) at thesame day that the mice underwent moderate contusion injury atT9. Two and a half weeks later, we observed 9 –14% of extensivelylabeled axons with CASPR spreading caudal to the lesion (Fig.2Aa’). We used the staining properties of CASPR concentrated inthe paranodal regions of the axon (Poliak and Peles, 2003) andvoltage-gated Kv1.2 channels concentrated in the juxtaparanodalregions as an index of myelin integrity (Karimi-Abdolrezaee etal., 2004), because myelin stains themselves do not provide satis-fying direct investigation (Fig. 3H). Furthermore, sectionsstained with toluidine blue taken 2 weeks after injury showedevidence of some demyelinated axons immediately caudal (100 –300 �m) to the lesion (Fig. 2B) compared with control animals(Fig. 2C). Additional evidence of early demyelination is the pres-ence of remyelinated dorsal column axons 12 weeks after injury(Fig. 2D). Finally, quantitative PCR analysis revealed a significantdecrease in MBP expression levels both at the lesion site and 1mm caudal at 24 h, 5 d and 2 weeks after injury (Fig. 2E). Thesedata show that the mouse contusion lesion creates myelin pathol-ogy and decreased myelin synthesis throughout the rostrocaudalaxis of the lesion similar to what has been reported in rats.

To establish whether cut axons persist with chronically demy-elinated segments, we performed the following experiments. We

Figure 1. Injections into the red nuclei resulted in extensive bilateral labeling of the rubrospinal tracts. A, Bilateral injections ofFluoro-Ruby resulted in dye filling the red nuclei. White arrows indicate the red nuclei in a coronal section. B, Fluoro-Rubyextensively labeled the rubrospinal tract in control animals (arrows). C, Labeled rubrospinal tract (black arrows), visible in a wholecord under dissecting fluorescent microscope, extends beyond the lesion (white arrow) in injured animals. D, Coronal section ofinjured spinal cord caudal to the lesion epicenter shows axons (some indicated with arrows) filled with Fluoro-Ruby (red) next toGFAP (green) labeled reactive tissue. E, Note that Fluoro-Ruby axons (red, white arrows) traverse the reactive astrocyte arealabeled with GFAP (green) in a horizontal section. Scale bars: A, 500 �m; B, C, 2 mm; D, 100 �m; E, 200 �m. R, Rostral; C, caudal;D, dorsal.

Lasiene et al. • Spared Axons Are Remyelinated J. Neurosci., April 9, 2008 • 28(15):3887–3896 • 3889

examined myelin indices on bilaterallyFluoro-Ruby traced RST axons 12 weeksafter moderate contusion injury(Schmued et al., 1990) (Fig. 1). Fluoro-Ruby extensively labeled RSTs in controlmice (Figs. 1C, 3C,D) and revealed mor-phologically intact fibers of passage ex-tending caudally to the lesion in the in-jured animals (Figs. 1B,D,E, 3B,E–G).Dextrans have been shown to rely on intactaxonal transport (Glover et al., 1986; Ter-asaki et al., 1995); hence, we classifiedFluoro-Ruby-labeled axons as activelytransporting. As described previously,many axons severed rostral to the injurysite ended in bulbs that were filled withFluoro-Ruby (Li and Raisman, 1995) (Fig.3A, inset). Such axons exhibited a variablepattern of CASPR and Kv1.2 protein stain-ing (Fig. 3A). As described previously, in-ternodal proteins were found to be dif-fused along the axolemma and in additionwe observed CASPR restricted to abnor-mally narrow clusters. Irregular CASPRand Kv1.2 protein distribution indicativeof aberrant myelination (Arroyo et al.,2002; Karimi-Abdolrezaee et al., 2004) al-ternated with normal-looking CASPR-labeled paranodal sites (Fig. 3A).

Spared RSTs are remyelinated withsignificantly shorter internodesAfter confirming the aberrant myelinationstatus of fibers cut rostrally to the lesion,we sought to determine the myelin statusof spared, actively transporting fibers ofpassage caudal to the lesion. To do this weexamined internodal proteins in isolatedwhole tracts or single mechanically teasedfibers that extended 0.5 mm caudally andbeyond the lesion epicenter (Figs. 1B,3B,E). In addition, CASPR and Kv1.2 pro-tein distribution on Fluoro-Ruby-labeledaxons had a normal compact spatial distri-bution indicating the presence of healthymyelin (Fig. 3F,H). We did not observediffuse or dispersed protein distributionon the labeled fibers. Importantly, intactaxons of passage had significantly shorterinternodes (mean, 217.6 �m; n � 98; p � 0.0001, two-tailed ttest) (Fig. 3B,E,F) compared with control animals (mean, 389.2�m; n � 99) (Fig. 3C,D,G), suggesting that this population hadbeen remyelinated (Blakemore, 1974; Gledhill and McDonald,1977) (Fig. 4). Because mean axon diameter decreased by 18%, asdescribed previously (Blight, 1983), and was not statistically dif-ferent from controls ( p � 0.06, two-tailed t test), we also com-pared axons with small/medium (�3 �m) and large (3– 4 �m)diameters in control and injury groups to control for diameterchanges. We confirmed that myelin internodes are significantlyshorter within both groups. The short internodes (�100 �m)were either randomly located on the axon interspersed with av-erage length internodes or were found to be next to each other. In

addition, we observed short internodes (�100 �m) located 2.5cm away from the lesion epicenter.

Spared RST axons exhibit thinner to normal myelin thicknessIn addition to examining internode length in longitudinal sec-tions, we examined the quality and thickness of myelin in spared,actively transporting axons. Bilateral injections of BDA into thered nuclei were made 8 weeks after moderate contusion injury inadult mice (Reiner et al., 2000). Three weeks after injection, spi-nal cords were processed for immunoelectromicroscopy and themyelin G-ratio (axon diameter/total fiber diameter) was mea-sured for axons immunopositive for BDA (see Materials andMethods) (Fig. 5). We did not observe organelles or neurofila-ment compaction in positively labeled fibers normally indicative

Figure 2. Demyelination is evident in mice at early time points after moderate spinal contusion injury. A, Flouro-Ruby-labeledRST axons demonstrated visible CASPR spreading (arrows and a’) caudal to the injury after 2.5 weeks after injury, indicative ofdemyelination. Some neighboring axons exhibited compacted CASPR labeling (arrowheads). B, A light micrograph of a toluidineblue stained section taken immediately caudal to the injury (2 weeks after moderate contusion injury at T9). In lateral whitematter adjacent to the dorsal horn (D), there are degenerating axons (arrows and b’) and evident microcavities (mc) interspersedwith normally myelinated axons (white arrowheads). C, A light micrograph of toluidine blue stained section of control cord takenventral to the dorsal horn demonstrates normally myelinated axons. D, A light micrograph of a section, stained with Richardson’sstain, taken at the lesion sight of dorsal columns after 12 weeks after injury. There are many axons that after being demyelinatedhad been remyelinated by Schwann cells, indicated by thick myelin sheaths as opposed to thinner myelin sheaths produced byoligodendrocytes. E, MBP expression was measured with qPCR at different time points after moderate contusion injury comparedwith control spinal cord. MBP expression was significantly decreased at the lesion sight and 1 mm caudal to the lesion epicenter at24 h and continued to decrease at 5 d and 2 weeks after injury.


of axon degeneration (Balentine, 1978). Axons positive for BDAin injured animals (Fig. 5B) contained no evidence of demyeli-nated segments, but had an increased g-ratio (mean, 0.71; n �91) compared with uninjured controls (mean, 0.68; n � 79; p �0.06, two-tailed t test) (Fig. 5E). Because there was a trend towardsmaller axon diameter in injured animals, axons were sorted intosmall/medium (�2 �m in diameter) and large (�2 �m in diam-eter) caliber groups to further characterize the changes in G-ratio.Only small/medium axons had a significantly increased G-ratio( p � 0.025, two-tailed t test), indicating thinner myelin. Largeaxons did not have significantly thinner myelin as evidenced bystatistically normal G-ratios ( p � 0.243, two-tailed t test). Over-all, although there was a trend for thinner myelin in injured micecompared with uninjured controls, the increase in G-ratio wasbelow that reported previously for nontraced axons (Blight, 1993;

Totoiu and Keirstead, 2005). In addition, G-ratio increase waspositively related to internode length increase, which could indi-cate that thinner myelin tended to be of a longer internode (Fig.5F). The distinct difference between the present analysis and theexisting literature is the use of prospective axon labeling to delin-eate spared axons.

Shortened internodes lead to decreased conduction velocityAlthough the changes in myelin indices are surprisingly minimal,there is a possibility that decreased internodal length and myelinthickness could combine to significantly alter the conductionproperties of spared axons. To better understand the predictedrelationship between shortened internodes and thinner myelin inspared axons, action potential conduction in control and injuredaxons was simulated using the double cable axon model of

Figure 3. Actively transported Fluoro-Ruby tracer revealed morphologically intact rubrospinal axons caudal to the lesion with compact CASPR and Kv1.2 paranodal protein labeling. A, Paranodalprotein CASPR (white arrows) staining on Fluoro-Ruby filled axons that were cut rostrally to the injury indicates nodes that do not seem to be fully formed and are abnormally close in proximity toeach other. Inset shows axons ending in bulbs filled with Fluoro-Ruby. B, A reconstruction of RTS tract portion (arrows) caudal to the injury. C, A reconstruction of RTS tract (arrows) portion in a controlanimal thoracic cord. D, Internodes were measured by using a digital overlay tracing the distance between one CASPR labeled paranode to another. Arrows point at nodes of Ranvier in a controlanimal (mean, 389.2 �m). E, Internodes measured in injured animals were significantly shorter (mean, 217.6 �m; p � 0.0001) than in control animals. F, CASPR and Kv1.2 protein staining ininjured animals was compact and spatially restricted on Fluoro-Ruby filled axons similarly to control animals. Arrows indicate nodes of Ranvier, arrowheads show Kv1.2 channel labeling. G, Arrowsindicate nodes of Ranvier on a single axon teased from a control animal thoracic cord. H, Myelin stain receptor-interacting protein label (arrowheads) shows how a myelin tube envelops a labeledaxon, which correlates with CASPR stain. Scale bars: A, D, E, H, 20 �m; B, C, G, 100 �m; F, 10 �m.


McIntyre et al. (2002). Control and injured axons were con-structed using axon diameter, internode length and myelin thick-ness measurements from Fluoro-Ruby and BDA-stained axons.Simulated conduction velocities ranged from 6 to 48 m/s foraxons with diameters from 1.2 to 7.9 �m.

The simulations showed a decrease in conduction velocity forthe injured axons, but no conduction failures or changes in actionpotential waveform. The average conduction velocity of the ax-ons constructed with data from the spinal injured animals was32% slower than the velocity of the control group (15.3 vs 22.4m/s, p � 0.01). The change in the distribution of axon diametersafter injury contributes significantly to this effect (Fig. 6B). How-ever, the majority of Fluoro-Ruby-stained axons were between2.0 and 4.0 �m, with similar means for both control and injuredgroups which allowed us to control for a diameter change. Sim-ulations of axons constructed only with data from axons with 2– 4�m diameter still showed a 21% decrease in average conductionvelocity for the injured group (15.9 vs 20.2 m/s, p � 0.01) (Fig.6A). To see the contribution of myelin thickness change to con-duction velocity (CV), we repeated the simulation using the samemyelin thicknesses for both the control and injury groups (i.e.,only internode length differed between the control and injuryaxons), which produced the same result. Thus, decreased axondiameter and internode length, but not myelin thickness, arelikely to contribute to significant slowing of rubrospinal axonsbelow the level of a contusion injury.

Simulations were also performed using the single cable modelof Brill et al. (1977). The decrease in conduction velocity of thesimulated injured axons was not as great as with the double cable

model: 14% slower than the control axons. There was no conduc-tion failure for any simulated axons.

Passive axon labeling reveals a pathologic population withmyelin deficitsAs stated above, spared axons with intact axonal transport (dex-tran labeled) were myelinated but occasionally we observed de-myelinated unlabeled axons below the lesion. To determinewhether demyelination could be present on axons exhibiting apathological transport system we used a lentiviral reporter(pCMV-GFP) to infect rubrospinal neurons 8 weeks after injury.Viral transfection resulted in axons extensively filled with greenfluorescent protein (GFP) (Fig. 7A). The majority of axons(96.5%) exhibited healthy morphology whereas a small sub-population of axons (3.5%) showed dystrophic morphology withelaborate bulbous protrusions along their axolemma (Fig.7A,B,D) different from the controls (Fig. 7C). Axons that exhib-ited normal morphology also showed the same pattern of CASPRand Kv1.2 staining as Fluoro-Ruby-labeled axons (Figs. 3B–H,7B). CASPR and Kv1.2 labeling was spatially compact and re-stricted to the paranodal regions; we did not observe spreading ordiffusion of the paranodal proteins along the GFP filled axonswith normal morphology (Fig. 7B). In dystrophic, GFP-labeledaxons that demonstrated globular protrusions, CASPR and Kv1.2proteins were not always present in a predictably periodic andcompact manner. Surprisingly, we observed some regular paran-odal protein staining interspersed with diffused or no stainingalong the axolemma (Fig. 7E). In both cases where dystrophicGFP-labeled processes could be followed longitudinally along thecord, they terminated in dystrophic end-bulbs indicating thatthey had retracted from their target. At this time, we cannot drawany conclusions whether healthy axons instigate remyelinationor whether myelin preserves spared axons.

DiscussionIn this study, we describe the remyelination pattern at subacuteand chronic survival periods of spared mouse RST axons thatextend caudally past a moderate contusion lesion. We used acombination of classical anatomical techniques and new molec-ular markers to examine myelin indices around actively trans-porting axons and passively filled tracts. Our data confirms thatmouse contusion injury results in myelin pathology includingdemyelinated axons during the subacute postinjury period. Im-portantly, our data show that by 12 weeks after injury, RST axonsof passage with active axonal transport are fully remyelinated asevidenced by the presence of myelin indices similar to control butsignificantly shortened internodes. These observations establish atight correlation between axon sparing and the occurrence ofremyelination in the chronic period.

The concept of chronically demyelinated axons is based onanatomical, pharmacological and cell based studies. Demyelina-tion has been widely reported after chronic spinal cord injury inanimals (Gledhill et al., 1973; Blakemore, 1974; Gledhill andMcDonald, 1977; Blight, 1983) and limited demyelination is ob-served in humans (Gensert and Goldman, 1997; Kakulas, 1999;Guest et al., 2005). Previous studies suggest that remyelination inexperimental injury models is mostly incomplete and that newmyelin is abnormally thin and contributes to conduction failure(Griffiths and McCulloch, 1983; Scolding and Lassmann, 1996;Nashmi and Fehlings, 2001). Abnormally thin myelin as well asshortened internodes are thought to be indicators of new myelin(Gledhill and McDonald, 1977; Blight, 1993). Combined withevidence that mRNA levels for myelin proteins MBP and PLP

Figure 4. Internodal distances in injured and control animals were statistically different. A,Internodes measured in injured animals (mean, 217.6 �m; gray bars) were significantly shorterthan in control animals (mean, 389.2 �m; p � 0.0001; black bars). B, There is a correlationbetween increased axon diameter and increased internode length in injured (empty triangles,R 2 � 0.22) and control (black squares, R 2 � 0.12) conditions.


(Wrathall et al., 1998) are reduced in the injured spinal cord,these studies suggest myelin may be unstable or insufficientlythick to insulate spared axons. The implications are that chronicdemyelination in the absence of axonal loss can contribute todysfunction of the injured spinal cord.

Previous work on 4-aminopyridine (4-AP), a potassiumchannel blocker, demonstrated some neurological improvementafter SCI in animal models (Bostock et al., 1981; Blight, 1989).One theory is that improvement results from blockage of potas-sium channels exposed on demyelinated, intact axons. However,4-AP also potentiates synaptic transmission and skeletal muscletwitch tension (Smith et al., 2000) and human clinical trials of4-AP in chronic SCI have been inconclusive so far where somereport modest and temporary improvement on motor and sen-sory functions especially reduction in spasticity, whereas othersdo not find significant beneficial effects (Potter et al., 1998; vander Bruggen et al., 2001; Wolfe et al., 2001; DeForge et al., 2004).

In addition to potassium channel blockers, cell transplanta-tion with the purpose of remyelinating fibers has been one of themain therapeutic approaches to improve function after SCI (Li et

al., 1997; Akiyama et al., 2002; Cao et al.,2005; Cummings et al., 2005; Keirstead etal., 2005; Lepore and Fischer, 2005). Re-myelination by transplanted cells of differ-ent kinds is readily observable, but only ifsuch transplants are delivered early,namely before 3 weeks after SCI (Keirsteadet al., 2005; Karimi-Abdolrezaee et al.,2006). It could be that transplanted cellsoutcompete endogenous oligodendrocyteprogenitors that are slower to divide anddifferentiate than primed transplantedcells. Early remyelination could be benefi-cial to stabilize and preserve surviving ax-ons because myelin provides tropic andstructural support and chronically demy-elinated axons have been noted to develop

severe pathologies in PLP, myelin-associated glycoprotein mu-tants, and multiple sclerosis lesions (Griffiths et al., 1998; Yin etal., 1998; Blakemore et al., 2000; Bjartmar and Trapp, 2003).

To date, anatomical studies of demyelination have been basedon correlative analyses, which do not take in to account the statusof the axon (spared or severed) or its site of origin. Our findingsindicate that myelin indices immediately caudal to the injury siteare near control values and establish a correlation between axonalsparing and myelin status. Although myelin thickness was re-duced, the G-ratios measured were within the 0.6 – 0.8 range,which is considered a theoretically optimal range for axon con-duction (Waxman, 1980). Our results correlate with studies thatobserved complete remyelination after myelin was experimen-tally removed but the axon remained intact (Blakemore andMurray, 1981; Jeffery and Blakemore, 1995). Important caveatsto the current approach include the use of the murine modelwhere anatomical distances are small compared with larger spe-cies. For example, it may be that demyelination on spared axonsis strictly limited to the injury epicenter. In the mouse, this would

Figure 5. Actively transporting axons labeled with BDA and examined with the electron microscope do not show extensive demyelination. A, A coronal cut through the lesion that illustrates theappearance of the spared rim of white matter. B–D, Asterisks indicate BDA filled axons that have mature looking myelin caudal to the injury (B, arrow) that is similar to that of control spinal cord (C,D). E, Axons in injured animals had a tendency for bigger G-ratios (mean, 0.71; R 2 � 0.2) compared with controls (mean, 0.68; p � 0.06; R 2 � 0.4). F, G-ratio correlates within internode lengthin control (R 2 � 0.96) and injured (R 2 � 0.81) animals. Scale bars: A, C, 300 �m; B, D, 2 �m. D, Dorsal.

Figure 6. CV as a function of internode length and axon diameter. A, Histograms of the conduction velocities of simulatedcontrol (open bars) and injured (filled bars) axons with diameters between 2.0 and 4.0 �m. B, Histograms of the conductionvelocities of simulated control (open bars) and injured (filled bars) axons of all diameters. CV was calculated to decrease by22–30% in injured animals.


be a region of 0.4 – 0.8 mm in size, an areawe were unable to examine because of theinterference of intense lesion-relatedautofluorescence.

Because intact axons were remyeli-nated, we sought to label RST axons,which may be severed or lack functionaltransport by using a retroviral reporter(CMV-GFP) as a passive labeling tech-nique. The purpose of this approach was tolabel demyelinated or thinly myelinatedaxons described previously to persist afterspinal cord injury (Waxman, 1989; Totoiuand Keirstead, 2005). In 3.5% of axonstraced in this manner caudal to the lesion,we observed some demyelination or ab-normal patterns of remyelination. Thissubpopulation of axons exhibited a verysimilar pattern of demyelination or abnor-mal remyelination to fibers cut rostral tothe lesion. The data from the passivelyGFP-labeled axons and axons severed ros-tral to the lesion demonstrate that abnor-mal myelin or demyelination may be con-fined to severed or degenerating axons.These data highlight three important find-ings: (1) demyelination is primarilypresent in severed axons in the chronic pe-riod after spinal cord injury, (2) dystro-phic axons can exhibit variable regions offull and incomplete remyelination alongseveral millimeters, and (3) complex swell-ings are periodic along demyelinated pro-cesses and persist in a small population forat least 12 weeks. It is important to con-sider that axonal degeneration may be acontinuum after injury and it cannot bedetermined from the present data whetherfunctionally transporting axons promoteremyelination or whether remyelinationleads to the return/maintenance of axonaltransport.

Some virally and dextran-labeled axonsin our studies resembled dystrophic axonsdescribed in head and spinal cord injurymodels (Li and Raisman, 1995; Povlishocket al., 1997). Previous work suggests theseare dynamic structures and can be foundclose to the site of the lesion up to 1 weekafter injury, but it is not yet possible todetermine if they are in a state of degener-ation or persistent abortive growth (Tomet al., 2004). Future research should bedone to determine whether passively la-beled dystrophic axons are degenerating slowly and demyelinat-ing or if they are stable, yet dynamic structures.

To further evaluate the implications of our myelin analyses inspared axons we adapted a mathematical model of single axonalconduction. The rationale for this was twofold. First, evidence inhumans and rodents with chronic spinal cord injury suggest re-duced conduction velocity, longer refractory period and reducedexcitability (Waxman, 1980; Blight, 1983; Nashmi and Fehlings,2001). Second, we theorized that a combination of shortened

internodes with a marginally reduced myelin thickness couldcause an increase in conduction failure or significant decrease inconduction velocity. McIntyre’s et al.’s (2002) theoretical modelof a single myelinated axon simulations predicted a 21% decreasein conduction velocity caused by changes in myelin for sparedremyelinated RST axons with the physical parameters measuredin our studies. This is a moderate reduction in conduction veloc-ity and the effect on locomotion is likely moderate or negligible inmice. However, a 21% CV decrease might be important in larger

Figure 7. pCMV-GFP lentivirus infection resulted in passively GFP-filled rubrospinal axons. A, Of labeled axons, 96.5% were oflarge to small caliber and had an appearance similar to that of control uninjured axons. However, 3.5% of labeled axons had adystrophic and bulbous appearance (arrowheads, inset shows high magnification of a globular protrusion). B, Higher magnifica-tion of the rubrospinal tract caudal to the lesion. The arrowhead indicates an atrophic axon. Note that this axonal profile containsregular swellings, abnormal thin sections and no apparent internodes. In myelinated axons, internodes contain readily visiblenodes of Ranvier (arrows). C, Control axons were of large to small caliber with spatially compact CASPR labeling; arrows indicatesome nodes of Ranvier. D, Dystrophic axon exhibiting diffuse CASPR labeling (arrows). Scale bars: A, 100 �m; inset, 20 �m; B–D,40 �m.


mammals and the actual effect on nerve function will only bedetermined by physical measurement of CVs in the future.

In experiments where conduction velocity and compound ac-tion potential (CAP) were measured chronically after a contusionlesion similar to the injury severity modeled here, Van de Meentet al. (1996) showed a similar (12.5%) drop in conduction veloc-ity to that predicted by our calculations. Importantly, these stud-ies show that changes in latency are not a strong predictor offunctional outcome, whereas a decrease in the CAP amplitudetightly correlates with recovery in locomotion. These data indi-cate that the myelin status of spared long-tract axons in thechronically contused spinal cord is less likely to contribute tofunctional deficits than is axonal loss.

Nonetheless, the functional conclusions that can be drawnfrom our simulations are limited. Our model examines only theconduction of a single action potential, but does not take intoaccount what the effect might be when trains of action potentialsare conducted along the axon (which would be more physiolog-ically applicable). This may be particularly relevant in light offindings that remyelinated axons fatigue more readily than con-trols (Nashmi and Fehlings, 2001). Fatigue in combination with adrop in velocity might contribute in a significant way to loss oflocomotor function. In addition, our simulations assume thatmembrane conductances and other biophysical properties of theaxon are unchanged after remyelination. There is no quantitativedata available for these parameters after spinal cord injury, andconsequently there was no basis to model such pathophysiology.Direct electrophysiological studies of descending axons, includ-ing an analysis of frequency-dependent conduction, will be re-quired to elucidate definitively the physiology of remyelinatedaxons after spinal cord injury.

ConclusionsThe clinical implications of our findings are potentially impor-tant. Our data suggest that, given time, endogenous remyelina-tion of spared axons of passage is close to that of uninjured axons.This new myelin likely contributes to the spontaneous functionalrecovery observed after injury (Blight, 1993). In the future, thedynamism of demyelination and remyelination needs to be stud-ied with a careful consideration of whether an axon is intact orsevered.

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No Evidence for Chronic Demyelination in Spared Axons after