THE ANATOMICAL RECORD 294:847–857 (2011)

Olfactory Ensheathing CellTransplantation Into Spinal CordProlongs the Survival of MutantSOD1G93A ALS Rats Through

Neuroprotection and RemyelinationYING LI,1,2 JIANLING BAO,1,3 NIKAN H. KHATIBI,4 LIN CHEN,1,2,5

HONGMEI WANG,1 YAOKUI DUAN,2 HONGYUN HUANG,1,2,5*

AND CHANGMAN ZHOU6*1Beijing Hongtianji Neuroscience Academy, Beijing, China

2Institute of Neuroscience, Taishan Medical University, Beijing, China3Medical Laboratory Center, First Affiliated Hospital of Xin Jiang Medical University

Urumqi, Xin Jiang, China4Department of Anesthesiology, Loma Linda University Medical Center,

Loma Linda, California5Department of Neurosurgery, Beijing Rehabilitation Center, Beijing, China

6Department of Anatomy and Embryology, Peking University Health Science Center,Beijing, China

ABSTRACTAmyotrophic lateral sclerosis (ALS) is a progressively fatal, incurable,

neurodegenerative disorder. In this study, we investigated whether olfac-tory ensheathing cells (OEC) transplantation could provide protection tomotor neurons and enable remyelination in mutant SOD1G93A transgenicrats with ALS. Seventy-two rats were divided into four groups: SOD1G93A

rats (n ¼ 20); mediumþSOD1G93A rats (n ¼ 20); OECsþSOD1G93A rats (n¼ 24); and another eight wild-type rats were used as controls. About 5 lL(1 � 105) OECs in DF12 medium was injected into the dorsal funiculus ofthe thoracic spinal cord at a predetermined depth. Survival analysisrevealed a significant increase in the survival time in OECþSOD1G93A

rats. Body weight records and inclined board test showed a significant dif-ference between OECþSOD1G93A and SOD1G93A from the onset at 7 daysto 11 days (P < 0.05). Four weeks following transplantation, motor neu-ron counts in the ventral horn of the spinal cord noted a significant motorneuron loss in SOD1G93A rats when compared with wild-type rats (P <0.001), and much less neuronal loss and collapse was noted inOECþSOD1G93A rats when compared with SOD1G93A rats(P < 0.001);immunohistochemistry and Western blot analysis of choline acetyltrans-ferase supported the motor neuron count. Images of confocal microscopeindicated that the transplanted OECs had survived for more than 4weeks and migrated 4.2 mm through the spinal cord. Evidence of remyeli-nation of transplanted OEC was captured with triple fluorescence label-ing of green fluorescent protein, neurofilament, and myelin basic protein

Grant sponsor: National Natural Science Foundation ofChina; Grant number: 30971527.

The first two authors contributed equally to this article.

*Correspondence to: Hongyun Huang, MD, PhD, Beijing Hon-gtianji Neuroscience Academy, Xi-Xia-Zhuang, Ba-Da-Chu, Shi-Jing-Shan District, Beijing 100144, China. E-mail: hongyunh@gmail.com (or) Changman Zhou, MD, PhD, Department of Anatomy

and Embryology, Peking University Health Science Center, 38Xueyuan Rd, Beijing 100191, China. E-mail: changmanzhou@hotmail.com

Received 24 August 2010; Accepted 22 January 2011

DOI 10.1002/ar.21362Published online 17 March 2011 in Wiley Online Library(wileyonlinelibrary.com).

VVC 2011 WILEY-LISS, INC.

and was further confirmed by Western blot analysis of MPB. In conclu-sion, the transplanted OECs could serve as a source of neuroprotectionand remyelination to modify the ALS microenvironment. Anat Rec,294:847–857, 2011. VVC 2011 Wiley-Liss, Inc.

Keywords: amyotrophic lateral sclerosis; olfactory ensheathingcells; SOD1G93A rats; neuroprotection; remyelination

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a progressivelyfatal, incurable, neurodegenerative disease characterizedby motor neuron cell death within the central nervoussystem (Qureshi et al., 2009; Schmidt et al., 2009). ForALS victims, this translates clinically into moderate tosevere skeletal muscle wasting, paralysis, and even ashortened lifespan from failed respiratory efforts (Cleve-land and Rothstein, 2001).

In the past, scientific research has alluded to a num-ber of potential physiological mechanisms as explana-tions for the disease state. This includes the formationof protein aggregates (Watanabe et al., 2001; Ross andPoirier, 2004), axonal transport defects (LaMonte et al.,2002; Roy et al., 2005), oxidative damage (Cleveland andLiu, 2000; Weishaupt et al., 2006), mitochondrial defects(Gajewski et al., 2003), glutamate toxicity (Ludolphet al., 2000), viral infections (Berger et al., 2000), auto-immune attacks (Alexianu, 1995; Staines, 2008), andalterations in calcium homeostasis (Guatteo et al., 2007;von Lewinski et al., 2008). Despite these efforts, theexact etiology behind ALS formation is still unknownand an effective therapeutic modality does not exist tilldate.

One of the challenges faced with treating ALS victimshas been the difficulty in dealing with the diffuse natureof motor neuron cell death. Recently, however, scientificresearch has suggested that transplantation of neuralstem/precursor cells may be a promising therapeuticstrategy for combating the effects of ALS (Julien, 2001;Lepore and Maragakis, 2007; Ferrero et al., 2008; Maz-zini et al., 2008; Morita et al., 2008; Garbuzova-Davisand Sanberg, 2009). Preliminary pilot studies in humansubjects have suggested that transplantation of fetal ol-factory ensheathing cells (OECs) in patients with ALSappears to slow the rate of clinical progression in thefirst 4 months following transplantation (Huang et al.,2008). OECs, a specific glial cell type residing in theolfactory system, assist axonal extension of olfactory sen-sory neurons from the peripheral to the central nervoussystem (Ramon-Cueto and Avila, 1998). In experimentsusing animal models of spinal cord injury, OECs pro-moted the regeneration of injured spinal pathways andenhanced motor recovery (Ramon-Cueto and Nieto-Sam-pedro, 1994; Li et al., 1997; Ramon-Cueto and Avila,1998). Additionally, human clinical trials in China(Huang et al., 2003, 2006a,b) have suggested that func-tional and sensory recovery can occur in patients withspinal cord lesions who receive OECs grafts from fetalolfactory bulbs. However, despite these promising efforts,no study has cross-examined the neurophysiologicalmechanism of transplanted OECs in prolonging the sur-vival time in ALS victims.

As a result, in this study, we investigated the potentialtherapeutic effects of transplanted OECs in rat modelsof ALS. As previous experiments have indicated thatOEC transplantation in mice models of ALS (via thefourth cerebral ventricle) revealed no adverse effects andno significant differences between mice with OEC trans-plantation and mice without transplantation (Moritaet al., 2008), we hypothesized that transplanted OECscould protect motor neurons and enable remyelination inmutant SOD1G93A transgenic rats with ALS.

MATERIALS AND METHODS

All of the studies were carried out in accordance withthe Guidelines for Animal Experimentation at BeijingHongtianji Neuroscience Academy. An overview of theexperimental design is given in Fig. 1.

Animals

Seventy-two Sprague–Dawley SOD1G93A male ratswere used in this experiment. Of these rats, 64 were di-vided into three groups, the SOD1G93A rats (n ¼ 20),mediumþSOD1G93A rats (n ¼ 20), and OECþSOD1G93A

rats (n ¼ 24). Another eight wild-type rats were used asnormal control.

As previously described, Sprague–Dawley rats carry-ing the SOD1G93A mutation (Taconic Company, German-town, New York) were used as an ALS animal model(Herbik et al., 2006). The colony was subsequently main-tained by breeding SOD1G93A male rats with femalewild-type Sprague–Dawley rats with �50% of the result-ing offspring carrying the SOD1 mutation. Wild-type lit-termates were used for controls.

Identification of Mutant SOD1G93A

Transgenic Rats

Sprague–Dawley pups were genotyped between 4 and5 weeks of age. A small piece of tail was cut and boiledin 300 lL of 0.02 M Ethylenediaminetetraacetates(EDTA) and 1 mM NaOH for 1 hr. The samples werethen vortexed and centrifuged for 5 min. Polymerasechain reaction (PCR) was then performed by using 4 lLof this solution. For each sample, a master mix was usedthat contained 2.5 lL 10� PCR buffer, 5 lL 5� Q solu-tion, 0.2 lL Taq DNA Polymerase (Qiagen, Valencia,CA), 2.5 lL 2.5 mM dNTP Mix (Invitrogen, Carlsbad,CA), 0.05 lL human/mouse primer (50 CAG CAG TCACAT TGC CCA GGT CTC CAA CAT G 30), 1 lL humanSOD1 primer (50 CCA AGA TGC TTA ACT CTT GTAATC AAT GGC 30), and 9.75 lL ddH2O. Positive andnegative controls were used in all PCR reactions (Lladoet al., 2006).

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Preparation of OECs

OECs were collected from the olfactory bulb of neona-tal ‘‘green’’ rats (Lewis-EGFP F463.5, Stable-singleinsertion site, single-chromosome 5) purchased from theRat Resource Research Center, which ubiquitouslyexpressed green fluorescent protein (GFP) (Sasaki et al.,2004). The tissues were incubated for 40 min at 37�C inDispase II (Roche, Mannheim, Germany) and thentransferred into a 0.25% collagenase type IA (Sigma) inDulbecco’s modified Eagle medium/Ham’s F12 (DMEM/F12) and incubated for 30 min at 37�C. After centrifuga-tion, the cell pellet was resuspended in the DMEM/F12with 10% fetal bovine serum and plated on poly-L-lysine(PLL)-coated dishes. At 80–90% confluence, the cellswere harvested and passed with DMEM/F12 containing1% insulin-transferrin-selenium supplement (ITS-X,Gibco) and 50 ng/mL neurotrophin 3 (NT3, PeproTechEC) onto a PLL-coated dish. Cells were maintained inthis medium and passed five times until OECs werepurified to more than 95% (Bianco et al., 2004).

To characterize the GFP positive OECs, a double fluo-rescence labeling was performed at 7 days of culture.The cell culture plate was fixed with 4% paraformalde-hyde in phosphate buffer and then treated with antibodyp75 (1:500 Santa Cruz) and an appropriate secondaryantibody conjugated to rhodamine isothiocyanate (RITC)(1:200 Santa Cruz) for fluorescence immunohistochemis-try staining; in this way, the GFP positive cells can beidentified to be the OECs.

OES Transplantation

OECs were transplanted into the SOD1G93A transgenicrats at 100 days old. The OECs with GFP (GFP-OECs)were transplanted into the thoracic region of the spinalcord as previously described (Sasaki et al., 2004, 2006)with some modifications. Rats were first anesthetizedwith 4% (0.12 mL/kg) chloral hydrate, and then a lami-nectomy was performed between vertebrae T6 and T8.

About 5 lL (1� 105) OECs in DF12 medium wereinjected into the dorsal funiculus of thoracic spinal cord(T7) through a glass micropipette at depths of 0.7 mmand 0.4 mm.

Controls and Animal Processing

The mediumþSOD1G93A rats were established as vehi-cle delivery controls. These animals were injected withthe same volume (5 lL) of culture medium (DF12) with-out OECs. The other group of SOD1 rats and the wild-type rats were established as additional controls andwere observed. Eight rats in each group were sacrificedat 4 weeks following OEC transplantation (four ratsfor histology and immunohistochemistry, another fourrats for Western blot analysis). In addition, fourOECþSOD1G93A rats were sacrificed at 2 weeks (tworats) and 4 weeks (two rats) only for fluorescence-marked OEC trance. Animals were deeply anesthetizedwith sodium pentobarbital (50 mg/kg i.p.) before sacrificeand processed separately for immunohistochemistry orWestern blot analysis.

Clinical Assessment and Inclined Board Test

The animal behavior test was performed in 12SOD1G93A rats in each group, that is, SOD1, Medium,and OECs group. Disease onset, survival time, and bodyweight were measured each day, whereas the inclinedboard test was performed at every 2-day intervalsthroughout the study. Disease onset was determinedwhen tremor/weakness was identified during suspensionby the tail and/or during voluntary walking. SOD1G93A

rats were assessed once daily for morbidity and mortal-ity from OECs transplantation (age ¼ 100 days) to death(age ¼ 147 days). Body weights were recorded every day.An inclined board test was performed at 2-day intervals.This test involved placing rats on a wooden board, whichwas gradually elevated by five degree increments. Themaximum angle (slope of the curve) at which the ani-mals could maintain position on the inclined board for30 sec was recorded.

Histology

Four rats in each group were histologically evaluatedat 4 weeks (age ¼ 128 days, 1 day after disease onset)after OEC transplantation. All rats were anesthetizedwith 4% chloral hydrate and perfused transcardiallywith 4% paraformaldehyde in phosphate buffer.

A thoracic spinal cord segment (up to 1 cm craniallyand 1 cm caudally from the T7 injection point) wasremoved and postfixed in the same fixative and cryopro-tected in a series of sucrose solutions: 10%, 15%, and20% sucrose in Phosphate-Buffered Saline (PBS) at 4�Cfor 2 days. The tissue was then embedded in Tissue-Tec(Sakura, Japan) and frozen in liquid nitrogen-cooled iso-pentane. Twenty-micrometer-thick sections were cuttransversely on a cryostat (Leica CM3050 S).

Serial transverse sections of the thoracic spinal cordwere then stained in 0.1% cresyl violet solution for 3–10 min. This was followed by a quick rinse in distilledwater, and later differentiation in 95% ethyl alcohol for2–30 min before being dehydrated in 100% alcohol 2 �5 min. Finally, the sections were cleared in xylene 2 �

Fig. 1. Experimental protocol of OECs transplantation was per-formed at the age of 100 days, disease onset at the age of 127 days,and the outcomes (i.e., survival, body weight, and incline board test)were recorded from 100 days to death (146 days). The OECs tracewas performed at 2 weeks (age ¼ 114 days) and 4 weeks (age ¼ 128days) after OECs transplantation, and the Nissl staining, immunohisto-chemistry, and Western blot analyses were performed at 4 weeks (age¼ 128) following transplantation.

THERAPEUTIC EFFECTS OF TRANSPLANTED OECs IN ALS RATS 849

5 min and mounted with permanent mounting mediumto allow for counting of the motor neurons. The positivecells in the ventral horn of gray matter were counted inevery fifth section, and the results of at least 10 sectionsfrom four rats were averaged (Llado et al., 2006; Turneret al., 2009).

Another series of sections from each group was con-ducted using immunohistochemistry of choline acetyl-transferase (ChAT) and neurofilament (NF) antiserum.The spinal cord sections were incubated with 3% H2O2

to quench the endogenous peroxidase and were followedby 30-min incubation with normal serum at roomtemperature (Yan et al., 2006; Chen et al., 2007). Thesections were incubated overnight at 4�C with rabbitanti-ChAT (AB5042, 1:400; Chemicon).

Trace of transplanted OEC. To evaluate themigration of transplanted OECs, EGFP fluorescence-marked OECs groups were observed under confocalmicroscopy. Another four rats in the OECs transplantedgroup were anesthetized and perfused with 4% parafor-maldehyde at 2 weeks (two rats) and 4 weeks (two rats).The thoracic spinal cord segments were cut verticallyand observed under confocal microscope. The GFP wasexcited by an argon laser at 488 nm and was emitted at530 nm.

Double and triple fluorescence labeling. Toobserve the migration and remyelination of transplantedOECs using double staining, vertical sections were incu-bated with goat anti-NF200 (Sigma, 1:200) and treatedwith secondary antibody (donkey anti-goat IgG conju-gated with Texas Red). Using triple labeling, sectionswere first incubated with goat anti-NF200 antibodies(Sigma, 1:200) and rabbit anti-myelin basic protein(MBP, 1;200; Abcam). Sections were then treated withsecond antibody conjugated with fluorescent dye (donkeyanti-goat IgG conjugated with Texas Red; Santa Cruz);goat anti-rabbit IgG-AMCA (blue aminomethylcoumarinacetate, 1:200; Jackson Immuno Research, Pennsylvania,PA) in the dark for 2 h at room temperature. Sectionswere cover-slipped with 30% glycerin and observedunder confocal microscopy. The Texas Red was excitedby a dye laser at 595–605 nm and was emitted at620 nm. The GFP was excited by an argon laser at488 nm and was emitted at 530 nm. The absorption ofAMCA blue flourescence was at 350 nm, and the emis-sion peak at 450 nm.

Western Blot Analysis

To quantify and evaluate the effect of transplantedOECs on the degeneration of motor neurons and remye-lination of transplanted OEC into the spinal cord, West-ern blot analysis of ChAT, NF, and MBP wereperformed. Rats were anesthetized and sacrificed withan overdose of pentobarbital (150 mg/kg) and then per-fused with ice-cold PBS from the left ventricle. The spi-nal cord was removed, frozen, and stored at �80�C untilprotein extraction took place. Tissues were homogenizedin ice-cold lysis buffer (0.32 mol/L sucrose, 1 mmol/Lethylenediaminetetraacetate, 5 mmol/L Tris-HCl, pH 7.4,0.1 mmol/L phenylmethylsulfonyl fluoride, 10 lmol/Leupepsin, 10 lmol/L pepstatin A, and 1 mmol/L b-mer-captoethanol). The protein content was determined by

Bio-Rad protein assay. Equal amounts of protein perlane (50 lg) were loaded onto an 8% polyacrylamide geland separated by electrophoresis at 90 V for 30 min andthen 120 V for 1.5–2.5 hr. The proteins were then trans-ferred to nitrocellulose at 200 V for 2 hr, and the mem-brane was blocked with 5% nonfat dry milk/0.5% Tween-20 in Tris-buffered saline for 2–2.5 hr. The nitrocellulosewas then incubated with different antibodies overnightat 4�C: rabbit anti-ChAT (AB5042, 1:800; Chemicon),goat anti-NF200 (1:800; Sigma), and rabbit anti-MBP(1:800; Abcam). The membrane was treated with horse-radish peroxidase-conjugated secondary antibody for60 min at 37�C and then exposed to X-ray film. The X-ray films were scanned, and the optical density wasdetermined by Bio-Rad Image analysis. As an internalcontrol, the same nitrocellulose was incubated with anantibody specifically for b-actin (1:1,000; Santa Cruz)after being stripped.

Statistical Analysis

Kolmogorov–Smirnov test of normality was insignifi-cant for all variables. Measures were compared everyday (body weight) or every 2 days (inclined board test)with respect to the baseline condition, using an ANOVAfor repeated measures. Comparisons of the mean scoresof the two groups were made by using the unpairedTukey test. Survival analysis was performed by theKaplan–Meier curve from SPSS 11.5J for Windows(SPSS). The null hypothesis was rejected when P � 0.05for all tests. Statistical analyses were performed usingthe Graph-pad Prism software (version 5.0).

RESULTSIdentification of OECs Before andAfter Transplantation

Prior to transplantation, cells were identified by sim-ple morphology—small nucleus, thin cytoplasm, and fineprocesses were detected at 3 days (Fig. 2A). After seed-ing onto PLL-coated flasks, the cultured OECs wereobserved after 7 days of culture with GFP green fluores-cence marker (Fig. 2B) and p75 red florescence marker(Fig. 2C). To identify the characterization of OEC, highermagnification was used, which showed parts of the greenfluorescence cells to have purer populations of p75 posi-tive OECs (Fig. 2D–F). An enrichment of OEC culture ofup to five generations could be achieved depending onthe proportion of p75 in the starting population of cells.

At both 2 (age ¼ 114 days) and 4 weeks (age ¼ 128days) after OEC transplantation in the thoracic spinalcord (T7) of SOD1G93A rats, the vertical sections revealeda large number of GFP-expressing cells broadly scat-tered within both gray and white matter areas extend-ing several millimeters in both rostral and caudal sidesfrom the injection site (Fig. 2G,H). Two weeks followingtransplantation, OECs showed a restricted pattern ofcell dispersion (Fig. 2G) comparable with that of theintact spinal cord; this suggested that there was a dif-fuse distribution of GFP-expressing OECs in the spinalcord of SOD1G93A rats. Additionally, 4 weeks followingtransplantation, there was a more distal dispersion ofGFP-expressing OECs, indicating that there was a netcell migration rather than pressure-induced spreading ofcells (Fig. 2H).

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Clinical Assessment and Behavioral Test

To investigate whether OEC transplantation hadtherapeutic effects on rats with ALS, the disease onset,survival time, disease progression (Fig. 3), and bothbody weight and behavioral tests were performed andrecorded (Fig. 4).

Disease onset. Animals were observed each day fortremors/weaknesses, which were identified during tailsuspension and/or during voluntary walking; this indi-cated the onset of the disease. There was no significantdifference between OECsþSOD1G93A rats and SOD1G93A

rats (or MediumþSOD1G93A rats) at 127 days, indicatingthat the disease onset was not affected by OEC trans-plantation (Fig. 3A).

Survival time. On average, SOD1G93A rats survivedfor 140.1 days (F 3.7, n ¼ 12), whereas the OECþSOD1G93A

rats survived for 146.9 days (F 8.9, n ¼ 12, P < 0.05,unpaired t test). MediumþSOD1G93A rats survived for140.33 days (F 7, n ¼ 12, P > 0.001 versus OECþSOD1G93A

rats, unpaired Tukey test). Kaplan–Meier survival analysisrevealed a significant increase (more than 6 days) in sur-vival of OECþSOD1G93A rats versus SOD1G93A rats orMediumþSOD1G93A rats (P< 0.01; Fig. 3A).

Disease duration. The analysis of disease onset andanimal survival time was shown in Fig. 3B. OECþSOD1G93A

rats had a significantly prolonged survival time for morethan 7 days when compared with SOD1G93A rats andMediumþSOD1G93A rats, respectively (P < 0.01). There was

Fig. 2. Morphological identification of OECs before transplantationand the trace at 2 and 4 weeks after transplantation. (A) OEC observa-tion under discrepancy microscopy at 3 days of culture. (B) The expres-sion of GFP by transplanted EGFP-OEC at 7 days of culture. (C) Theexpression of P75 NTR by transplanted EGFP-OEC at 7 days of culture.(D–F) High magnifications of OECs double staining by GFP and P75

under confocal microscopy. Image (D) shows the GFP positive cells,whereas image (E) shows the P75 positive cells; image (F) is the mergerof both (D) and (E). Some of the GFP positive cells were colocalized withP75 positive OECs. (G, H) The EGFP-OEC was traced at 2 weeks and 4weeks after transplantation. Migration as long as 4.2 mm in both sidestogether (H). Bar¼ 100 lm (A–C), 5 lm (D–F), 100 lm (G), and 1 mm (H).

THERAPEUTIC EFFECTS OF TRANSPLANTED OECs IN ALS RATS 851

no significant difference between the SOD1G93A rat andMediumþSOD1G93A rat (P < 0.01).

Body weight. The daily recorded body weightsshowed that there was no significant difference withregards to body weight onset between the groups(Fig. 4A). A significant difference was found betweenOECþSOD1G93A and SOD1G93A (or MediumþSOD1G93A)at 7–10 days (age ¼ 134–137 days) after the diseaseonset (P > 0.05). At 11–13 days (age ¼ 138–140 days),there was no significant difference between the groups(P < 0.05). However, the body weight of OECþSOD1G93A

continued to decline until death was reached at about 20days (age ¼ 147 days) after disease onset (Fig. 4C), incontrast to the SOD1G93A and MediumþSOD1G93A ratgroups that reached death at 13 days (age ¼ 140 days).

Inclined board test. There was no significant differ-ence between the groups (Fig. 4B) before the diseaseonset (age ¼ 127 days). A significant difference wasfound between the OECþSOD1G93A and SOD1G93A

groups (MediumþSOD1G93A) at 7 and 9 days (age ¼ 134and 136 days), respectively, after the disease onset (P >0.05). Because the inclined board test was performed at2-day intervals following the disease onset, there was nosignificant difference at 1, 3, 5 (age ¼ 128, 130, 132days) and 11 days (age ¼ 138 days) after disease onset(P < 0.05).

Motor Neuron Cell Quantification

Both Nissl staining (Fig. 5) and motor neuron cellcount (Fig. 6A) showed a significant loss in motor neuroncells in SOD1G93A and MediumþSOD1G93A rats whencompared with wild-type rats (P < 0.001). SOD1G93A

rats that received OEC transplantation showed a muchsmaller neuronal loss area and collapse than SOD1G93A

rats (Fig. 4A1–D2). At 4 weeks, Nissl staining of OECtransplants showed that nearly all the motor neuronssuffered from some degree of cell loss (neuronal degener-ation) in the SOD1G93A and MediumþSOD1G93A rats(Fig. 5B1,B2,C1,C2). However, motor neuron cells weremore clearly recognized in the figure, and the number ofmotor neuron cells was significantly much more than inthe SOD1G93A or MediumþSOD1G93A rats (Fig. 6A).

Immunohistochemistry staining of ChAT was similarto what was found with Nissl staining (Fig. 5A3–D3). Toevaluate the ChAT protein levels in the spinal cordbetween the OEC transplanted and nontransplantedSOD1G93A rats, Western blot quantification was con-ducted. The results showed that the protein levels ofChAT in both SOD1G93A and MediumþSOD1G93A weresignificantly decreased (versus wild-type rats, P < 0.05);however, the ChAT protein levels in the OECs trans-plantation rats was significantly higher than that inboth SOD1G93A and MediumþSOD1G93A (P < 0.05;Fig. 6B). Additionally, to evaluate the regeneration ofnerve fibers, the NF protein levels were analyzed byWestern blot analysis (Fig. 6C). The results showed thatthe protein levels of NF in OECs transplantation weresignificantly increased than that in both SOD1G93A andMediumþSOD1G93A (P < 0.05).

Migration of Transplanted OECs

The transplanted OECs were traced on a vertical sec-tion of the SOD1G93A rat spinal cord after 4 weeks oftransplantation to identify and characterize the survivaland migration. At low magnification, the GFP-markedcells were viable and migrated nearly 4.2 mm in boththe rostral and caudal directions (Fig. 7A). At high mag-nification (Fig. 7B,C), double staining for GFP (trans-planted OECs) and NF (neuronal fibers) indicated thatthe transplanted OECs marked by GFP with its proc-esses (green fluorescence marked) migrated along theneuronal fibers (red fluorescence marked) both at close(Fig. 7B) and distal (Fig. 7C) points from the injectingsite.

Remyelination of Transplanted OECs

To evaluate the remyelination of transplanted OECs,triple immunofluorescence labeling of GFP from GFP-transgenic animal, NF, and MBP immunohistochemistrywas performed on vertical sections of spinal cordSOD1G93A rats after 4 week of transplantation; thesewere compared with SOD1G93A rats without transplanta-tion (Fig. 8). The results showed that SOD1G93A ratswithout cell transplantation, GFP was negative (Fig.8A1). This is in contrast to the transplanted OECs withgreen fluorescence of GFP, which were clearly shown inthe thoracic spinal cords of OECþSOD1G93A rats(Fig. 8B1). The neuronal fibers with red fluorescence ofNF-200 and the myelin with blue fluorescence of MBPstaining were positive in neuronal fibers of OEC rats

Fig. 3. Survival and disease progression (A). Kaplan–Meier survivalanalysis reveals a significant increase in the survival ofOECþSOD1G93A rats versus SOD1G93A (P < 0.01). (B) The disease du-ration in OEC transplantation rats is significantly longer than SOD1G93A

(P < 0.01). Survival analysis was performed by the Kaplan–Meiercurve from SPSS 11.5J for Windows (SPSS). Comparisons of themean scores of the two groups were made by using the unpairedTukey test as posthoc test.

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Fig. 4. Body weight and behavior (A, B). Analysis of the bodyweights and behavior from the age of 100 days (OEC transplantation)to 147 days (the day of rat death) showed no difference before onset.(C) OEC transplantation significantly prevented weight loss inOECþSOD1G93A versus SOD1G93A from 7 to 10 days (age ¼ 134–137days) after disease onset (P < 0.01). (D) The angles (slope of the

curve) in the inclined board test were significantly different betweenOECþSOD1G93A and SOD1G93A at 7 (age ¼ 134 days) and 9 days(age ¼ 136 days), respectively, after disease onset (P > 0.05). ANOVAwas used for repeated measures. Comparisons of the mean scores ofthe two groups were made by using the unpaired Tukey test.

Fig. 5. Histological evidence of motor neuronal protection inOECþSOD1G93A rats (A1–D1) using Nissl staining. The location of(A1)–(D1) was shown in the window of (A1). (A2–D2) High magnifica-tion of motor neurons. The motor neurons nearly collapse in SOD1G93A

(B2) and MediumþSODG93A rats (B3). (A3–D3) The expression of ChATimmunohistochemistry in the ventral horn of various groups. Bar ¼50 lm (A1–D1 and A3–D3) and 5 lm (A2–D2).

THERAPEUTIC EFFECTS OF TRANSPLANTED OECs IN ALS RATS 853

(8B2,B3); however, fluoresces were quite weak in theSOD1G93A rats without cell transplantation (Fig.8A2,A3).

The structure of neuronal fibers with the myelinsheath was in good order (Fig. 8B2,B3), which was incontrast to the disorderly nature seen in the SOD1G93A

rats without cell transplantation (Fig. 8A2,A3). After themerger of the two (Fig. 8B2,B3), it was shown (Fig. 8B4)in the OECs transplanted spinal cord that the GFP-expressing OECs with long processes distributed longitu-dinally along the axon fibers—these were stained withNF (red) and MBP (blue). Using higher magnification,the GFP-expressing OECs were associated with remyeli-nated fibers, containing both MBP-positive myelin andNF. The quantification of MBP Western blotting showedthat the content of MBP in OECs transplanted spinalcord was significantly higher than that in the spinalcord of SOD1G93A without cell transplantation andMediumþSOD1G93A rats (P > 0.05).

DISCUSSION

ALS is a progressively fatal, incurable, neurodegenera-tive disorder victimizing an estimated one to three indi-viduals per 100,000 worldwide. Over the last fewdecades, extensive research on OECs has suggested thepotential of these cells to act as neuroregeneration-pro-moting molecules (Franssen et al., 2007). As a result, inthis study, we investigated whether OEC transplantationcould provide protection to motor neurons and enableremyelination in mutant SOD1(G93A) transgenic rats withALS. We found that OEC transplantation could in factprolong survival and improve the overall function oftransgenic SOD1G93A rats with ALS, suggesting that thistype of treatment may be a feasible option in the future.

Transplanted OECs Protect MotorNeuron Cells From Death

Our results demonstrated that OECs transplantedALS rats had a significantly higher number of motorneurons and ChAT protein levels than nontransplantedrats (Figs. 5 and 6). This can be partly explained by thesupportive environment OECs provide. In essence, themigration of transplanted OECs to the sites of neurondegeneration allow for the development of a nourishingenvironment where the remaining motor neurons cansurvive and function through secretion of growth/sur-vival factors such as Nerve Growth Factor (NGF), Glial-Derived Neurotrophic Factor (GDNF), Neurotrophin-3(NT-3), Neurotrophin-4/5 (NT-4/5) (Saarma and Sariola,1999; Woodhall et al., 2001; Lipson et al., 2003). This issimilar to a recent data that has implicated the microen-vironment of the motor neuron rather than the motorneuron itself as a primary target of the pathophysiology(Borchelt, 2006). Moreover, in addition to the findingthat the transplanted OECs could migrate at long dis-tances (4.2 mm) along the spinal cord, we also foundthat they could rescue motor neurons within the ventralhorn of the spinal cord (Fig. 6).

Migration of Transplanted OECs

It has been hypothesized that implanted OECs areable to acquire certain properties enabling them tomigrate from the site of injection and crossover to thelesion site (Franssen et al., 2007). One study in particu-lar (Su et al., 2009) reported that astrocytes in the glialscar release Tumor necrosis factor-a (TNF-a), which canattract OECs to the site and possibly explain the exten-sive migration. However, various studies over the yearshave attempted to quantify the distance OECs couldmigrate. Markers such as a Hoechst stain have beenused by some researchers who have recorded migratingdistances as far as 1.5 cm (Franssen et al., 2007). Otherssuch as Lankford et al. (2008) found that OECs couldmigrate extensively (about 5 mm) in an irradiated spinalcord, but very little in an intact cord (Franssen et al.,2007). In our study, we found that OECs from GFP-transgenic rats could migrate close to 4.2 mm; the GFPpositive cells migrate approximately 2 mm in 2 weeksand 4 mm in 4 weeks, implying that the migration isongoing in ALS rats. The reason for the longer distanceof OEC migration may be attributed to the characteristicpathology of ALS and spinal cord injury. ALS is a

Fig. 6. Motor neuron quantification and the expression of proteinlevels of ChAT, NF, and MBP in the thoracic spinal cord. (A) The num-ber of motor neurons in hemi section. There was a significant differ-ence between OECþSOD1G93A rats and nontransplanted SOD1G93A

rats or MediumþSOD1G93A rats (P < 0.01). (B, C) The protein levels ofChAT, NF, and MBP were significantly decreased when compared withwild type (P < 0.01); however, it was significantly different in theOECþSOD1G93A rats versus nontransplanted SOD1G93A rats orMediumþSOD1G93A rats (P < 0.01).

854 LI ET AL.

Fig. 7. Confocal imaging of transplanted OECs migration in the spi-nal cord. (A) Migration as long as 4.2 mm in both directions. Thesquares (B) and (C) show the location of high magnification of (B1)–(B3) and (C1)–(C3). The images (B1) and (C1) show the transplanted

EGFP-OEC; (B2) and (C2) show the expression of NF immunofluores-cence; (B3) and (C3) show the relationship between transplanted OECand fibers. The arrows point the processes of OECs.

Fig. 8. The triple fluorescence labeling of remyelination observedunder confocal microscopy. (A1–A4) SOD1G93A rat; (B1–B6)OECþSOD1G93A rats. The exogenous OEC marked by GFP with longprocesses is distributed longitudinally along the axon fibers in the spi-nal cord of OECþSOD1G93A rats (B1), which was negative in theSOD1G93A rats without OEC (A1). The myelin of axon fibers werestained separately by NF and MBP immunohistochemistry by red (B2)

and blue colors (B3) in OECþSOD1G93A rats. It was a very weak stain-ing in SOD1G93A rats without OEC (A2, A3). Images (A4) and (B4) werethe merger of (A1–A3) and (B1–B3). To demonstrate the remyelinationof transplanted OECs, a high magnification showed the GFP-express-ing OEC associated with remyelinated fibers, containing MBP positivemyelin (B5, B6). Bar ¼ 50 lm (A1–B4), 20 lm (B5), and 5 lm (B6).

neurodegenerative disease that specifically targets motorneurons in spinal cord injury, and damage to the axonaltract is the primary target that results in permanentloss of functional connections.

Remyelination of Transplanted OECs

Although evidence has recently been provided indicat-ing that OECs do have the capacity to remyelinate(Franssen et al., 2007), it remains unclear whetherOECs are directly or indirectly responsible for this pro-cess. Previous studies have suggested endogenous stemcells invading the lesion site myelinated axons ratherthan the implanted OECs. However, in our study, wefound that the positive GFP expression indicated thatthe remyelination took place from exogenous sourcesand not endogenously as suggested. Specifically, the tri-ple fluorescence labeling under confocal microscopy andthe quantification of MBP Western blotting demonstratea clear proof of remyelination of transplanted OEC inthe spinal cord of our ALS rats. This could potentiallyexplain why there was such an improvement seen in ourmeasured outcomes following transplantation.

In conclusion, this study suggests that the transplan-tation of OECs in the spinal cord could prolong the sur-vival of mutant SOD1 (G93A) ALS rats. In fact, thetransplanted OECs could serve as a source of neuropro-tection and remyelination, adjusting the ALS microen-vironment. Further studies are warranted to assess thepotential of these cells in ALS disease.

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