Olfactory ensheathing cells promote collateral axonal branching in the injured adult rat spinal cord

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Experimental Neurology 185 (2004) 15–25

Olfactory ensheathing cells promote collateral axonal branching

in the injured adult rat spinal cord

M.I. Chuah,* D. Choi-Lundberg, S. Weston, A.J. Vincent, R.S. Chung,J.C. Vickers, and A.K. West

NeuroRepair Group, School of Medicine, University of Tasmania, Hobart, Tasmania 7001, Australia

Received 13 February 2003; revised 20 August 2003; accepted 16 September 2003

Abstract

In recent years, injection of olfactory ensheathing cells (ECs) into the spinal cord has been used as an experimental strategy to promote

regeneration of injured axons. In this study, we have compared the effects of transplanting encapsulated ECs with those injected directly into

the spinal cord. The dorsal columns of adult rats were cut at T8–9 and rats in experimental groups received either EC-filled porous polymer

capsules or culture medium (CM)-filled capsules with ECs injected at the injury site. Control rats were in three groups: (1) uninjured, (2)

lesion with transplantation of CM-filled capsules and (3) lesion with transplantation of CM-filled capsules and injections of CM. Three weeks

after injury, Fluororuby was injected into the hindlimb motor and somatosensory cortex to label corticospinal neurons. Observations indicated

that there were a few regenerating fibres, up to 10, in the EC-treated groups. In rats that received encapsulated ECs, regenerating fibres were

present in close association with the capsule. Rats that received EC injections demonstrated a significant increase in the number of collateral

branches from the intact ventral corticospinal tract (vCST) compared with the corresponding control, CM-injected group (P = 0.003), while a

trend for increased collateral branches was observed in rats that received encapsulated ECs (P = 0.07).

D 2003 Elsevier Inc. All rights reserved.

Keywords: Olfactory ensheathing cells; Spinal cord; Regeneration; Corticospinal tract; Collateral sprouting

Introduction

Traumatic injury to the spinal cord triggers a series of

cellular reactions that often result in wide ranging functional

deficits (e.g., reviews by Fawcett and Asher, 1999; Schwab

and Bartholdi, 1996). Within the first few days of injury,

there is massive neuronal and glial cell death (Emery et al.,

1998). Axons distal to the injury site degenerate while

reactive astrocytes divide to form scar tissue, which inhibits

subsequent axonal regrowth by a combination of mechan-

ical and trophic mechanisms (Lindsay, 1986). The injury

site is a complex environment containing reactive astro-

cytes, fibroblasts, immune cells and a mix of growth factors,

inhibitory factors, cytokines and other agents, some soluble

and some associated with the surface of specific cells

(Schwab and Bartholdi, 1996).

0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.expneurol.2003.09.008

* Corresponding author. Discipline of Anatomy and Physiology,

School of Medicine, University of Tasmania, Private Bag 24, Medical

Sciences Building, College Road, Hobart, Tasmania 7001, Australia. Fax:

+61-3-62262679.

E-mail address: [email protected] (M.I. Chuah).

In light of the restrictive nature of the injured central

nervous system, various experimental strategies have been

developed to alter this environment so that axonal repair can

take place. One of these is the use of olfactory ensheathing

cells (ECs). Indeed, ECs injected into the injured spinal cord

have resulted in a remarkable degree of axonal regeneration

and functional recovery (e.g., Imaizumi et al., 1998; Nash et

al., 2002; Ramon-Cueto et al., 1998), including remyelina-

tion of demyelinated axons (Li et al., 1997; Imaizumi et al.,

1998). Perhaps most remarkable is the restoration of climb-

ing ability of rats whose spinal cords have undergone

complete transection (Ramon-Cueto et al., 2000). However,

studies have yet to investigate the mechanisms through

which ECs operate in the novel environment of the injured

spinal cord.

Results of in vitro studies have demonstrated that ECs

express cell surface molecules such as N-CAM, N-cadherin

and laminin (Chuah and Au, 1994; Chuah et al., 1991;

Treloar et al., 1996). They also produce many growth

factors including several forms of neuregulins and some

neurotrophins (Chuah and West, 2002; Chuah et al., 2000;

Woodhall et al., 2001). It is not known whether the

M.I. Chuah et al. / Experimental Neurology 185 (2004) 15–2516

reparative properties of ECs in the spinal cord can be

attributed to these soluble growth factors or whether direct

contact between ECs and host tissue is crucial for spinal

cord repair. In the present study, we have compared the

effect of encapsulated ECs with that of ECs injected directly

into the injured spinal cord. The dorsal corticospinal tract

(dCST) was cut at T8–9 and the extent of repair was

assessed qualitatively and quantitatively by examining seg-

ments of the spinal cord rostral and caudal to the lesion. We

have also investigated the response of the uninjured ventral

corticospinal tract (vCST) to ECs that are encapsulated or

implanted directly into the spinal cord. To our knowledge,

this is the first study to examine the influence of ECs on

collateral sprouting in the spinal cord.

Materials and methods

All procedures involving animals were approved by the

Animal Experimentation Ethics Committee of the Univer-

sity of Tasmania and are consistent with the Australian Code

of Practice for the Care and Use of Animals for Scientific

Purposes.

Ensheathing cell purification and encapsulation

Ensheathing cells were purified and cultured on the basis

of the method of Chuah and Teague (1999) with modifica-

tions (Vincent et al., 2003) to increase the yield of ECs while

maintaining a high level of purity. The olfactory nerve layer

(ONL) and mucosa (OM) were isolated from 3-day-old

hooded Wistar rats euthanised in ice. The ONL was peeled

away from the glomerular and deeper layers of the olfactory

bulb and the OM was scraped from both sides of the nasal

septum after cutting away the surrounding respiratory tissue.

The pooled tissues were digested in MEM HEPES modifi-

cation (MEMH; Sigma, NSW, Australia) containing 0.25%

trypsin (Gibco, NY) and 0.03% collagenase (Sigma) for 15

min at 37jC. This incubation was repeated twice with fresh

solution, then replaced with culture medium (CM) composed

of MEM D-valine modification (Sigma) supplemented with

10% dialysed fetal calf serum (10000 MW cut-off; Sigma)

and penicillin–streptomycin–amphotericin B (Gibco) (Tisay

and Key, 1999). The digested tissue was triturated, filtered

through a sterile nylon gauze (80 Am pore size) and centri-

fuged for 10 min at 500 � g. Resuspended cells were plated

in CM. After 24 h, the culture was treated with 10� 4 M

cytosine-h-D-arabinofuranoside (AraC; Sigma) for 48 h to

minimise the population of fibroblasts. Purified ECs were

enriched in 125 Ag/ml bovine pituitary extract (Sigma) for 2

days before passage.

For encapsulation, ECs were scraped from 25-cm2 flasks

(Sarstedt, NC), centrifuged at 500 � g and resuspended in

CM. By means of a Hamilton syringe, they were introduced

into a polyvinylidene fluoride (PVDF) hollow fibre of 1.0-

mm diameter with a MW cut-off of 300000 Da (Spectrum,

CA). After loading, each end of the fibre was heat sealed to

form a capsule measuring 6–8 mm long and containing

about 105 ECs. Control capsules contained CM alone with

no ECs. All capsules were placed in CM overnight at 37jCin 5% carbon dioxide before transplanting to the spinal cord

the following day.

Hoechst blue-labeling of ensheathing cells

Ensheathing cells that were used for direct injections into

the spinal cord were prelabeled with Hoechst 33342 (Sig-

ma). One day before injection, ECs that were growing in a

25-cm2 flask were incubated in 10 ng/ml Hoechst 33342 in

CM and diluted from a stock of 10 mg/ml in dimethyl

sulfoxide. The cells were incubated for 40 min at 37jC,washed four times in MEMH and returned to fresh CM. A

small sample of cells was scraped off the flask and viewed

under ultraviolet filter to check that the staining had been

successful. Before implantation, the CM was collected to

test whether any unincorporated Hoechst dye had remained

in the solution. A fresh flask of unlabeled cells was

incubated overnight with this medium and checked under

ultraviolet filter. Only labeling of a barely detectable level

was observed. This procedure was done as a precautionary

measure to ascertain that host tissues were not exposed to

excess dye in solution during the process of injecting ECs

into the spinal cord.

Thirty minutes before injection, ECs were scraped from

half the flask, centrifuged at 500 � g and resuspended at a

concentration of about 104 cells/Al CM.

Spinal lesion surgeries

Adult rats weighing 270–380 g were anaesthetised with

3% isoflurane with oxygen, and maintained at 2–2.5%

isoflurane with oxygen at 0.5 l/min throughout the surgery.

Rats were injected subcutaneously with 0.1 ml/kg Rimadyl

(carprofen 50 mg/ml, a nonsteroidal anti-inflammatory and

analgesic; Pfizer Animal Health, UK), intramuscularly with

0.1 ml/kg Norocillin L.A. (procaine penicillin G 150 mg/ml

and benzathine penicillin G 112.5 mg/ml; Norbrook Labo-

ratories, UK) and intraperitoneally with 40 ml/kg Hart-

mann’s saline or 0.9% saline (Baxter Healthcare, NSW,

Australia). Hair from the back was removed with clippers

and skin swabbed with 70% ethanol and povidone-iodine. A

sterile surgical drape was placed over the rat and the skin

over spinous processes of thoracic vertebrae 6–11 (T6–

T11) was incised and retracted. Attachments of muscles to

T7–T10 spinous processes were cut and the muscles

retracted. The spinous processes of T8 and T9 were re-

moved with rongeurs and a laminectomy performed using a

dental drill and rongeurs to expose the dorsal spinal cord.

The dura was incised with microscissors. The dorsal col-

umns, which include the dCST, were cut by lowering

microscissors attached to a stereotaxic arm to a depth of

1.5 mm below the dorsal surface of the spinal cord and

M.I. Chuah et al. / Experimental Neurology 185 (2004) 15–25 17

cutting twice. The width of the cut was 1.5–2.0 mm, centred

on the midline. Experimental trials were first done to

ascertain that this surgical procedure produced a consistent

total transection of the dCST on both sides.

Two groups of rats received injections of ECs (n = 6) or

CM (n = 5) followed by placement of a CM-containing

PVDF capsule during the same lesion surgery. ECs sus-

pended in CM or CM alone were loaded into a 10-Al syringewith 26-s gauge bevelled needle (Hamilton, Reno, NV)

using the ‘‘fast up’’ function of an sp310i syringe pump

(World Precision Instruments, Victoria, Australia) mounted

on a stereotaxic frame. One microlitre of ECs or CM was

injected with the pump set at an injection rate of 1 Al/min at

each of four sites, 0.3 mm to the right and left of the midline

and 2–3 mm anterior and posterior to the injury site. All

injections were 1.0 mm deep to the dorsal surface of the

spinal cord. These coordinates were selected to place the

injected material just lateral to the dCST. Additionally, 4

Al of ECs or CM was injected at 1 Al/min at the injury site

on the midline. After each of the five injections, the needle

was left in place for 1 min. Following the injections, a

PVDF fibre capsule filled with CM was inserted into the

dorsal median sulcus of the spinal cord, centred anteropos-

teriorly on the injury site. Two additional groups of rats

received a PVDF fibre capsule loaded with ECs (n = 5) or

CM (n = 6), but no injections of ECs or CM, during the

same lesion surgery. The capsule was inserted into the

dorsal median sulcus of the spinal cord, centred anteropos-

teriorly on the injury site.

Following the lesion, injections (in two groups) and

capsule placement (in all four groups), the retracted muscles

were sutured with 5–0 silk and the skin closed with Michel

wound clips. The skin was swabbed with povidone-iodine

and the rat returned to its individual home cage under a heat

lamp for 1 h. Rats were injected daily for 3 days following

surgery subcutaneously with 0.1 ml/kg Rimadyl (carprofen

50 mg/ml).

Cortical injections of Fluororuby

To anterogradely label the corticospinal tract, rats were

injected with Fluororuby (Molecular Probes, Eugene, OR)

into the hindlimb motor and somatosensory cortex, which

contains most corticospinal neurons projecting to the lum-

bosacral enlargement of the spinal cord (Li et al., 1990).

Fluororuby injections were performed 3 weeks after spinal

lesion surgeries and also on a group of uninjured rats

weighing 340–365 g (n = 4). Rats were anaesthetised and

injected with Rimadyl, Norocillin L.A. and saline as for the

spinal surgeries. They were placed in a stereotaxic frame

with gas anaesthesia mask (David Kopf Instruments,

Tujunga, CA). Scalp hair was removed with clippers, the

scalp swabbed with 70% ethanol and povidone-iodine, and

incised. Scalp flaps were retracted and fascia cleared from

the skull with cotton tip applicators. The bregma and

lambda were levelled in the horizontal plane by adjusting

the bite bar. Burr holes were drilled in the skull above the

injection sites at the following coordinates relative to the

bregma: 0.9 mm posterior, 2.0 mm lateral; 1.9 mm posterior,

2.4 mm lateral; and 2.9 mm posterior, 2.6 mm lateral, on

both the right and left (coordinates on the basis location of

hindlimb somatosensory cortex by Paxinos and Watson,

1986). Half a microlitre of Fluororuby solution (10%

rhodamine–dextran, MW 10000 in water with 0.5% Triton

X100) was injected at each of the six sites 1.8 mm below the

dura at a rate of 0.5 Al/min using a 10 Al Hamilton syringe

with 33 gauge needle controlled by an sp310i syringe pump.

The needle was left in place for 1 min after each injection.

Burr holes were filled with bone wax and the scalp closed

with suture clips. The skin was swabbed with povidone-

iodine and the rat returned to its individual home cage under

a heat lamp for 30 min. Rats were injected daily for 2 days

following the surgery subcutaneously with 0.1 ml/kg

Rimadyl (carprofen 50 mg/ml).

Histology

Rats were anaesthetised with 0.4 ml Nembutal (200 mg/

kg; Boehringer Ingelheim, NSW, Australia) 2 weeks after

injection of Fluororuby. They were perfused transcardially

with cold phosphate buffered saline (PBS) followed by 4%

paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After

perfusion, the vertebral column and attached brain were

dissected and placed in cold 4% buffered paraformaldehyde

overnight. The following day, the spinal cord was removed

en bloc from the rest of the vertebral column and cut into

segments at levels indicated in Fig. 1A. The rostral ends of

spinal cord segments were marked with a solution of 15%

Alcian Blue in 50% glycerol to facilitate orientation of

specimens on the cryostat (Jung Frigocut 2800E, Leica).

All segments were stored in buffered 4% paraformaldehyde

at 4jC until ready for sectioning, at which time they were

embedded in Tissue-TekR O.C.T. compound (ProSciTech,

QLD, Australia). The approximately 2.8-cm length of cord

containing the capsule was sectioned (50 Am thickness) in

the sagittal plane while all other sections were made in the

transverse plane at the cervical, upper thoracic and mid-

thoracic level. Sections were floated on water bath to

remove O.C.T. compound and then mounted on slides

coated with 2% 3-aminopropyltriethoxysilane in acetone

(Sigma) (Henderson, 1989). It should be noted that when

the sections were floated, the capsule often became dis-

lodged from the rest of the spinal cord tissue, leaving an

oval-shaped space in the spinal cord section. Sections were

allowed to dry for 1 h in the dark and coverslipped using

DAKO fluorescent mounting medium. To prevent evapora-

tion and drying of sections, nail polish was used to seal the

edges of coverslips.

Histological examinations were performed with a fluo-

rescence attachment mounted on an Olympus BX50 micro-

scope and images captured using an Olympus DP50 digital

camera with the aid of Viewfinder Lite and Studio Lite 1.0

Fig. 1. (A) Dissected brain and spinal cord of rat. Arrows indicate the levels

at which the spinal cord is cut into segments. Transverse sections (50-Amthickness) were made at the cervical (C), upper thoracic (UT), mid-thoracic

(MT) and upper lumbar level (immediately caudal to the bracketed

segment). The bracketed segment contains the lesion and was longitudinally

serially sectioned. Scale bar = 1.5 cm. (B) Transverse section of the spinal

cord at the mid-thoracic level showing the Fluororuby-labeled dorsal

corticospinal tract. Scale bar = 0.8 mm. Inset shows a magnified image of

the fibres of the vCST. Scale bar = 200 Am.


software (Pixera Corporation). To count the number of

dCST fibres in transverse sections, images were enlarged

to A4 size and printed. A plastic transparency was placed

over the fluorescent tract and individual axons were marked

off with a pen as the counting proceeded. Images of vCST at

the lower thoracic level were similarly captured and counted

at a magnification of 450�.

In sagittal sections, an 8-mm length of the spinal cord

caudal to the capsule was used for counting the number of

axonal branches from the vCST fibres. All serial sagittal

sections of each spinal cord segment were examined at

400� magnification, and the number of fibres branching

directly from vCST fibres (as exemplified by branching

shown in Fig. 4H) was counted. Similarly, the number of

terminal branches of axons present in the grey matter was

also counted. Terminal branches appeared characteristically

as an arborisation extending from a single axon and whose

area of innervation was restricted to the grey matter. In the

uninjured animals, counts were conducted at the cor-

responding similar level. All counts were performed by a

person blinded to the treatment group. Statistical evaluation

of counts and comparison among groups were performed

with SigmaStat 2.03 (Jandel Scientific Software Corpora-

tion, San Rafael, CA) and the results presented in Figs. 6A

and B. A one-way ANOVA was applied to detect differ-

ences among groups, followed by multiple pair wise com-

parison (Tukey test). A P value of < 0.05 was considered

statistically significant and data were presented as mean FSEM.

Results

Histology and axonal labeling

Previous studies have shown that corticospinal neurons

are present not only in the motor cortex but also in some

other cortical areas including the supplementary motor area,

prefrontal cortex and somatosensory cortex (Li et al., 1990;

Miller, 1987). On the basis these findings and several

experimental trials, we have found that bilateral injection

of Fluororuby into the hindlimb motor and somatosensory

cortex consistently produced intense labeling of the cortico-

spinal tracts (Fig. 1B). In the uninjured rat, the number of

fibres in the bilateral dCST averaged 339 F 18, 348 F 11

and 284 F 28 at the cervical, upper and middle thoracic

levels, respectively (Fig. 2A). The fibres were present in

compact arrangement as two well-defined tracts. The

smaller component of vCST averaged 55 F 4 fibres bilat-

erally at the mid-thoracic level and fibres were distributed

diffusely over a wide area of the ventral funiculus. As

reported in previous studies (Liang et al., 1991; Weidner

et al., 2001), we also found occasionally a few corticospinal

fibres, usually numbering fewer than six on each side, being

present in the lateral funiculi, just ventral to the dorsal horns.

Axonal counts rostral to the injury site

The number of Fluororuby-labeled dCST axons was

counted in all groups at the cervical, upper and mid-thoracic

levels (Fig. 2A). At the cervical, upper and mid-thoracic

level, there was no statistically significant difference in the

number of dCST fibres in all treatment groups, although a

higher number of fibres in the groups that received ECs

(encapsulated or injected) were present at the mid-thoracic

level. The control group that received CM-containing cap-

sules together with CM injections had the least number of

fibres with 86 F 20 fibres at the mid-thoracic level. This

value translates to 27 F 5% of the number of fibres present

at the cervical level (Fig. 2B). Compared to the other control

group which received only a CM-containing capsule, this

Fig. 2. (A) Graphical presentation of the number of labeled dorsal

corticospinal fibres at the cervical, upper and mid-thoracic level in all

groups. The rats which received EC or CM injections also had culture

medium (CM)-containing capsules placed over the lesion. There was no

statistically significant difference among the treatment groups. (B) The

number of labeled dorsal corticospinal fibres at the upper and mid-thoracic

levels expressed as a percentage of fibres at the cervical level within each

group. Except for the uninjured group, all treatment groups showed a

reduction in the number of dorsal corticospinal fibres from the cervical to

the mid-thoracic level. The data also indicate that many of the corticospinal

neurons still survive 5 weeks post-lesion. Data are presented as mean FSEM.

Fig. 3. Tracing of a typical sagittal section of the spinal cord showing the

cavity which marks the location of the capsule (*) and its spatial orientation

with the dorsal corticospinal tract (dCST). The grey matter constitutes the

middle one-third of the section. The labeled arrows indicate the

approximate locations from which Fig. 4 is obtained. R = rostral, C =

caudal. Scale bar = 2.5 mm.


group sustained additional acute injury from multiple CM

injections, hence the comparatively fewer fibres that were

labeled. The persistence of Fluororuby-labeled fibres at 5

weeks post-axotomy even in the absence of any cellular

transplants suggests that a considerable proportion of dCST

neurons still survive. This was somewhat surprising given

that it is widely accepted that neurons undergo chromatol-

ysis and eventual cell death following traumatic injury (e.g.,

Schwab and Bartholdi, 1996).

Corticospinal tract fibres near the lesion site

Dissection of the spinal cord at 5 weeks post-surgery

showed that during this time, a layer of fibrous connective

tissue had grown over the capsule, marked by a slight

protrusion on the dorsal surface (Chuah and West, 2002).

Longitudinal sections revealed that the capsule had shifted

ventrally in the spinal cord during the recovery period (Fig.

3) and in some animals had caused some compression of the

vCST. The cut edge of the dCST was visible as a bright red

fluorescent bundle with the axotomised edge directly ap-

posed to the rostral tip of the capsule (Figs. 3 and 4A).

In both control and EC-treated rats, small numbers of

nerve sprouts were observed emanating from the cut stump

and sides of the dCST (Fig. 4B). Small numbers of regener-

ating fibres, up to 10, were present in EC-treated rats. In the

EC-encapsulated rats, the regenerating fibres were observed

in close association with the capsule, along its sides and

caudally as well (Figs. 4C, D and E). These regenerating

fibres were characterised by axonal varicosities and theywere

often closely apposed, running in a parallel direction, typical

of dCST fibres. In rats that received encapsulated ECs, some

nerve fibres were observed to be curving closely around the

caudal edge of the capsule (Fig. 4E), a location and direc-

tional growth that are not associated with the normal trajec-

tory of the dCST. In contrast, in rats that had ECs injected

directly into the spinal cord, fine nerve fibres projected

caudally away from the caudal tip of the CM-containing

capsule (Fig. 4F). The longest of these caudally projecting

fibres extended as far as 10mmdistal to the caudal edge of the

capsule. In summary, the observations indicate that there

were a few regenerating fibres from the sectioned dCST of

rats which had received ECs (encapsulated or injected).

However, regeneration was probably hampered by large areas

of necrosis, macrophages and tissue scarring in both control

and EC-treated rats. It was observed that some of the

regenerating sprouts terminated when confronted by areas

of brightly fluorescent cells, presumably macrophages (Fig.

4G). The small number of regenerating fibres is consistent

with the finding that there were not statistically significant

differences in the number of dCST fibres rostral to the lesion.

In contrast to the dense, compact arrangement of fibres in

the dCST, the brightly labeled vCST is composed of fibres

that are distributed over a wide area in the ventral half of the

spinal cord. In a few animals that received encapsulated or

injected ECs, branches from the vCST caudal to the lesion

extended to the dorsal columns and grew distally in the

space originally occupied by the dCST (Fig. 4H). These

fibres were new collateral sprouts because they did not

exhibit any axonal branches or terminations as they coursed

through the grey matter. In addition, in some EC-treated

(injected or encapsulated) rats, distally projecting fibres in

Fig. 4. Longitudinal sections of spinal cord segment containing lesion and capsule, 2 weeks after injection of Fluororuby. ‘‘capsule’’ marks the cavity which

previously housed a capsule containing ECs. (A) During post-lesion recovery, the capsule has been displaced ventrally in the spinal cord and appears to be in

the path of the sectioned dorsal corticospinal tract. Nerve sprouts projecting from the sides of the tract (asterisk) are visible at higher magnification. Scale bar =

800 Am. (B) Arrow indicates a fibre sprouting from the dorsal corticospinal tract (dCST) in the area marked by asterisk in A. The fibre ends in an arborisation

of fine sprouts. Scale bar = 300 Am. (C) Arrows indicate two fine nerve fibres present in the tissue overlying the EC-containing capsule. Scale bar = 250 Am.

(D) Arrows indicate a nerve fibre running close to the ventral edge of the capsule (C) containing ECs. Scale bar = 700 Am. (E) Regenerating nerve fibres

(arrows) running close to the EC-containing capsule, curving around its caudal edge and sandwiched between the capsule and clusters of brightly fluorescent

cells, presumably macrophages. Scale bar = 180 Am. (F) Section from spinal cord bearing lesion injected with ECs and transplanted with capsule containing the

culture medium. A nerve fibre (arrows) is observed coursing away from the caudal edge of the capsule. Scale bar = 400 Am. (G) Arborising fine nerve sprouts

(arrow) appear to terminate when confronted with aggregates of fluorescent cells, presumably macrophages. Scale bar = 150 Am. (H) Section from a region of

spinal cord caudal to the lesion and which received encapsulated ECs. A collateral branch (arrows) from the ventral corticospinal tract in the ventral funiculus

extends across the grey matter and then projects dorsally in the region where the original dorsal corticospinal tract was located. Scale bar = 600 Am. (I) Similar

section caudal to the lesion from a different rat which received encapsulated ECs. Fibres of the vCST (arrowheads) are clearly observed in the ventral funiculus.

A caudally projecting fibre (arrows) in the dorsal funiculus originates from the fibre network present in the grey matter. Scale bar = 800 Am.


Fig. 4 (continued).


the dorsal columns, caudal to the lesion, were observed to

originate from a network in the grey matter (Fig. 4I). This

unusual axonal route as described in Figs. 4H and I was not

observed in animals from both control groups.

Although ECs injected directly into the spinal cord were

prelabeled fluorescently with Hoescht blue (Fig. 5A), there

was difficulty in localising the cells 5 weeks posttransplan-

tation. In some animals, ECs remained near the injection site

(Fig. 5B), while in others, very few ECs were present (Fig.

5C). Viable ECs could be distinguished from the yellowish

crystalloid autofluorescence of macrophages (Bunn et al.,

2002; Fig. 6 in Takami et al., 2002) by their blue fluores-

cence under UV stimulation (Fig. 5B, inset). In small areas

where the two types of fluorescence overlapped, it is possible

that the fluorescence could be a consequence of macro-

phages having phagocytosed dead ECs.

Axonal branching from the ventral corticospinal tract

In addition to the number of labeled vCST fibres, axonal

fibres observed to be clearly branching directly from the

vCST (as shown in Fig. 4H) were also counted. Initial

overview showed that there was variability in the proportion

of labeled fibres in the vCST. This is due in part to the fact that

the total number of fibres in this tract is considerably less than

that of the dCST, about 19% and probably also because the

motor neurons are dispersed over a wider cortical region. To

normalize the data, the number of axonal branches and

terminations was expressed as a ratio of the number of labeled

vCST fibres (Figs. 6A and B). ANOVA analysis of the data

showed that there was a statistically significant difference

among treatment groups (P = 0.01). The two control groups

(lesion with transplantation of CM-filled capsule; lesion with

Fig. 5. (A) Olfactory ensheathing cells labeled with Hoescht blue before

implantation. Scale bar = 40 Am. (B) Injection site (asterisk) in spinal cord

surrounded by aggregates of ECs prelabeled with Hoescht blue. Ensheath-

ing cells can be distinguished by their blue fluorescence (arrow, inset) while

the autofluorescence of macrophages (arrowhead, inset) is yellowish

crystalloid in appearance. In this specimen, no fibres are present near the

injection site or ECs. Scale bar = 250 Am. Scale bar in inset = 125 Am. (C)

Only a few ECs (arrow) are present near this injection site (asterisk). Scale

bar = 180 Am.

Fig. 6. (A) Graphical presentation of the ratio of axonal collateral branches

relative to the number of ventral corticospinal fibres. The group which

received EC injections had significantly more collateral branches than the

group which received only culture medium (CM) injections ( P = 0.003). In

these two groups, a capsule containing CM was also inserted in the spinal

cord. In comparison, the group that received encapsulated ECs showed a

similar trend of increased collateral branching although the difference with

the corresponding control was not statistically significant ( P = 0.07). There

was no statistical difference between the group that received EC injections

with the one that received encapsulated ECs ( P = 1.00). (B) Graphical

presentation of the ratio of fibre terminations in the grey matter relative to

the number of ventral corticospinal fibres. The two groups which received

either EC injections or encapsulated ECs showed a trend of increased ratio

of terminating fibres in the grey matter caudal to the lesion. Data are

presented as mean F SEM.


transplantation of CM-filled capsule, and injections of CM)

had markedly lower ratios of branches to fibres compared

with the unlesioned group. This could be attributed to the fact

that the ventrally displaced capsule damaged some of the

branches of the vCST. Also, the control group that received

injections of CM sustained additional acute injury.

Pair-wise comparison showed that spinal cords that had

ECs injected into the vicinity of the lesion had significantly

more branches from intact vCST fibres compared with the

corresponding control animals (P = 0.003). Interestingly,

there was a trend for the spinal cords that had encapsulated

ECs to have more axonal branches than those which

received CM containing capsules (P = 0.07). No significant

difference in axonal branching was obtained in a compar-

ison between the EC-injected and EC-encapsulated groups

(P = 1.00). The ratio of fibre terminations in the grey matter

to vCST fibres did not differ among treatment groups (Fig.

6B). However, it should be noted that generally there were

more fibre terminations in the two groups that received ECs.

In particular, the group that had ECs injected into the

vicinity of the lesion had a mean value of 9.2 F 2.7

terminations per fibre compared with its control which

had only 3.1 F 0.7 (P = 0.075).


Discussion

We have compared the effect of encapsulated versus

injected ECs on collateral axonal sprouting and regeneration

in the injured spinal cord. We have shown for the first time

that collateral axonal branching from the intact vCST is

significantly increased in the presence of ECs. A small

number of regenerating fibres were present in the two

groups of rats which received ECs (encapsulated and

injected), consistent with results that have been reported

previously by other research groups (Ramon-Cueto et al.,

2000; Takami et al., 2002).

Response of vCST to injury in the dorsal columns

In this study, ECs transplanted into the spinal cord

following injury to dCST resulted in increased collateral

branches from the vCST. These new branches either termi-

nated in the grey matter or extended dorsally and caudally

along the original path of the dCST. The response of vCST

to injury in the dCST has generally been overlooked in EC-

transplantation studies, but this observation of collateral

sprouting is particularly important in the accurate interpre-

tation of results. Unless nerve fibres are traced to their

origin, their mere presence in the dorsal column caudal to a

dCST lesion may not be solely attributed to regenerating

fibres. It is possible that previous studies which reported the

presence of regenerating dCST fibres following EC trans-

plantation could have included axons from vCST in their

observations (Li et al., 1998; Nash et al., 2002; Takami et

al., 2002). Li et al. (1998) labeled CST axons by injecting

biotin dextran either into the medullary pyramid or the entire

contralateral sensorimotor cortex. Such an extensive injec-

tion would most likely have labeled the vCST. In a recent

study in which the thoracic spinal cord was contused, a

quantitative analysis was done on the number of fibres in the

dorsal column rostral to and near the lesion (Takami et al.,

2002). Although the authors inferred that the presence of the

fibres near the lesion is indicative of regeneration, they also

conceded that some could be originating from the spared

vCST. Unless a total transection of the spinal cord is

performed, the neuroanatomical contribution of collateral

sprouting to fibres caudal to the lesion cannot be entirely

discounted.

Similarly, the contribution of collateral sprouting from

descending motor tracts to functional recovery should not be

underestimated (Murray and Goldberger, 1974). An early

report has suggested limited sprouting from mid-brain

derived tracts following thoracic spinal cord sectioning

(Prendergast and Misantone, 1980). More recently, the

sparing of a few fibres in the ventral funiculus of the rat

spinal cord was sufficient to significantly improve stepping

abilities in locomotion (Schucht et al., 2002). A study on

adult rats reported that spontaneous sprouting from the

vCST following lesion of the dCST correlated with the rats’

ability to retrieve food pellets. If the vCSTwas subsequently

injured, functional recovery was abolished (Weidner et al.,

2001).

Influence of ECs on the response of the vCST

Although a statistically significant increase in collateral

sprouting was only observed in rats that had ECs injected

directly into the injury site, those that received encapsulated

ECs demonstrated a trend for increased branching (P =

0.07). When the group that received EC injections was

compared with the one that received encapsulated ECs,

there was no clear statistically significant difference between

them (P = 1.00). The promotion of collateral sprouting by

diffusible growth factors is supported by evidence from

numerous studies on different types of neurons (Cohen-Cory

and Fraser, 1995; Gallo and Letourneau, 1998; He et al.,

1992; Schnell et al., 1994). The peculiar course of a few

regenerating fibres closely enveloping the caudal end of a

capsule containing ECs, as shown in Fig. 4E, is suggestive

of an affinity between the nerve fibres and capsule. Whether

diffusible factors are involved in inducing this behaviour

will need to be confirmed in future experiments.

Other controlling mechanisms could account for our

observations. The fact that only the group that received

EC-injections demonstrated a statistically significant in-

crease in the ratio of branches when compared with their

corresponding control group suggests that injected ECs

could have altered the inflammatory response at the

injury site. One possibility is by suppressing the expres-

sion of inhibitory molecules such as chondroitin sulphate

proteoglycan.

Developmental studies have shown that an increase in

the polysialic acid level of the neural cell adhesion molecule

(NCAM) on CST axons coincides with the initiation of

collateral branching (Daston et al., 1996; Joosten et al.,

1996). The cell adhesion molecule NCAM is expressed by

ECs both in vivo and in vitro (Chuah and Au, 1993;

Miragall et al., 1988). It is possible that in our study,

NCAM expressed by encapsulated ECs was isolated within

the capsule, and hence lesioned fibres were subjected to

diminished stimulation, thus accounting for the less exuber-

ant collateral branching. Preliminary scanning electron mi-

croscopy of encapsulated ECs retrieved from the spinal cord

shows that ECs appear to be embedded within a mucoid

material, suggesting that secreted extracellular matrix may

be trapped within the capsule and cannot be accessed by

regenerating fibres.

Survival of ECs in the spinal cord

Although the exact extent of EC survival in the injured

spinal cord has yet to be quantified, several studies (Nash et

al., 2002; Ramon-Cueto et al., 1998, 2000) have shown that

ECs are able to survive in the spinal cord over a long period,

up to 8 months after implantation (Ramon-Cueto et al.,

2000). In this study, it is expected that some ECs will have


died in the spinal cord by 5 weeks post-implantation.

However, it is unlikely that most of them had been phago-

cytosed by macrophages. In our study, autofluorescence of

macrophages could be clearly distinguished from Hoechst-

labeled ECs, and for most instances these two types of

fluorescence did not overlap. Most recently, DeLucia et al.

(2003) showed that a cell line of Hoescht-labeled ECs

remained viable and was not phagocytosed by macrophages

or microglia 2 weeks following transplantation.

In recent experiments on encapsulated ECs in the spinal

cord, we have found that the ECs survive in the capsules

and continue to express growth-promoting molecules such

as neuregulins (Woodhall et al., 2003). Real time quantita-

tive RT-PCR performed on encapsulated ECs retrieved from

the spinal cord showed that the expression profile of the

various isoforms of neuregulins changed in response to the

novel CNS environment.

One possible reason why it was difficult to localize ECs

injected into the spinal cord may be that ECs continued to

proliferate, and hence the fluorescent label had been pro-

gressively diluted. Preliminary experiments in our laborato-

ry show that ECs undergo increased proliferation when

exposed to spinal cord tissue. Another reason is that the

conditions influencing migration of ECs in the spinal cord

are not clearly understood. It has been reported that ECs

tended to migrate preferentially to the vicinity of the lesion,

suggesting that they may be responding to signals from

injured neurons (Boruch et al., 2002). However, in a study

in which the medial longitudinal fasciculus was transected,

ECs migrated preferentially away from the lesion (Gudino-

Cabrera et al., 1999). The resolution of the fate and

migratory behaviour of ECs in the spinal cord is particularly

important if they are to be developed into a clinically

relevant therapeutic agent.

Acknowledgments

This work was funded by the Motor Accident and

Insurance Board (MAIB) of Tasmania, International In-

stitute for Research in Paraplegia and the National Health

and Medical Research Council. We thank Dr Jim Stanko-

vich for advice on statistical analysis.

References

Boruch, A.V., Conners, J.J., Pipitone, M., Deadwyler, G., Storer, P.D.,

Devries, G.H., Jones, K.J., 2002. Neurotrophic and migratory properties

of an olfactory ensheathing cell line. Glia 33, 225–229.

Bunn, H.J., Woltmann, G., Grigg, J., 2002. Applicability of laser scanning

cytometry to study paediatric alveolar macrophages. Eur. Respir. J. 20,

1437–1443.

Chuah, M.I., Au, C., 1993. Cultures of ensheathing cells from neonatal rat

olfactory bulbs. Brain Res. 601, 213–220.

Chuah, M.I., Au, C., 1994. Olfactory cell cultures on ensheathing cell

monolayers. Chem. Senses 19, 25–34.

Chuah, M.I., Teague, R., 1999. Basic fibroblast growth factor in the pri-

mary olfactory pathway: mitogenic effect on ensheathing cells. Neuro-

science 88, 1043–1050.

Chuah, M.I., West, A.K., 2002. Cellular and molecular biology of en-

sheathing cells. Microsc. Res. Tech. 58, 216–227.

Chuah, M.I., David, S., Blaschuk, O., 1991. Differentiation and survival of

rat olfactory epithelial neurons in dissociated cell culture. Dev. Brain

Res. 60, 123–132.

Chuah, M.I., Cossins, J.-M., Woodhall, E., Tennant, R., Nash, G., West,

A.K., 2000. Glial Growth Factor 2 induces proliferation and structural

changes in ensheathing cells. Brain Res. 857, 265–274.

Cohen-Cory, S., Fraser, S.E., 1995. Effects of brain-derived neurotrophic

factor on optic axon branching and remodelling in vivo. Nature 378,

192–196.

Daston, M.M., Bastmeyer, M., Rutishauser, U., O’Leary, D.D.M., 1996.

Spatially restricted increase in polysialic acid enhances corticospinal

axon branching related to target recognition and innervation. J. Neuro-

sci. 16, 5497–5499.

DeLucia, T.A., Conners, J.J., Brown, T.J., Cronin, C.M., Khan, T., Jones,

K.J., 2003. Use of a cell line to investigate olfactory ensheathing cell-

enhanced axonal regeneration. Anat. Rec. 271B, 61–70.

Emery, E., Aldana, P., Bunge, M.B., Puckett, W., Srinivasan, A., Keane,

R.W., Bethea, J., Levi, A.D., 1998. Apoptosis after traumatic human

spinal cord injury. J. Neurosurg. 89, 911–920.

Fawcett, J.W., Asher, R.A., 1999. The glial scar and central nervous system

repair. Brain Res. Bull. 49, 377–391.

Gallo, G., Letourneau, P.C., 1998. Localised sources of neurotrophins ini-

tiate axon collateral sprouting. J. Neurosci. 18, 5403–5414.

Gudino-Cabrera, G., Pastor, A.M., de la Cruz, R.R., Delgado-Garcia, J.M.,

Nieto-Sampedro, M., 1999. Limits to the capacity of transplants of

olfactory glia to promote axonal regrowth in the CNS. NeuroReport

11, 467–471.

He, Y., Yao, Z., Gu, Y., Kuang, G., Chen, Y., 1992. Nerve growth factor

promotes collateral sprouting of cholinergic fibres in the septohippo-

campal cholinergic system of aged rats with fimbria transection. Brain

Res. 586, 27–35.

Henderson, C., 1989. Aminoalkylsilane: an inexpensive simple preparation

for slide adhesion. J. Histotechnol. 12, 123–124.

Imaizumi, T., Lankford, K.L., Waxman, S.G., Greer, C.A., Kocsis, J.D.,

1998. Transplanted olfactory ensheathing cells remyelinate and enhance

axonal conduction in the demyelinated dorsal columns of the rat spinal

cord. J. Neurosci. 18, 6176–6185.

Joosten, E.A.J., Reshilov, L.N., Gispen, W.H., Bar, P.R., 1996. Embryonic

form of N-CAM and development of the rat corticospinal tract: immu-

no-electron microscopical localization during spinal white matter in-

growth. Dev. Brain Res. 94, 99–105.

Li, X.G., Florence, S.L., Kaas, J.H., 1990. Areal distributions of cortical

neurons projecting to different levels of the caudal brain stem and spinal

cord in rats. Somatosens. Motor Res. 7, 315–335.

Li, Y., Field, P.M., Raisman, G., 1997. Repair of adult rat corticospinal tract

by transplants of olfactory ensheathing cells. Science 277, 2000–2002.

Li, Y.L., Field, P.M., Raisman, G., 1998. Regeneration of adult rat cortico-

spinal axons induced by transplanted olfactory ensheathing cells. J.

Neurosci. 18, 10514–10524.

Liang, L., Moret, V., Wiesendanger, M., Rouiller, E., 1991. Corticomoto-

neuronal connections in the rat: evidence from double-labeling of mo-

toneurons and corticospinal axon arborizations. J. Comp. Neurol. 311,

356–366.

Lindsay, R.M., 1986. Reactive gliosis. In: Fedoroff, S., Vernadakis, A.

(Eds.), Astrocytes: Cell Biology and Pathology of Astrocytes. Academ-

ic Press, Orlando, pp. 231–262.

Miller, M.W., 1987. The origin of corticospinal projection neurons in rat.

Exp. Brain Res. 67, 339–351.

Miragall, F., Kadmon, G., Husmann, M., Schachner, M., 1988. Expres-

sion of cell adhesion molecules in the olfactory system of the adult

mouse: presence of the embryonic form of N-CAM. Dev. Biol. 129,

516–531.

Murray, M., Goldberger, M.E., 1974. Restitution of function and collateral


sprouting in the cat spinal cord: the partially hemisected animal. J.

Comp. Neurol. 158, 19–36.

Nash, H.H., Borke, R.C., Anders, J.J., 2002. Ensheathing cells and methyl-

prednisolone promote axonal regeneration and functional recovery in

the lesioned adult rat spinal cord. J. Neurosci. 22, 7111–7120.

Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates,

second ed. Academic Press, Sydney.

Prendergast, J., Misantone, L.J., 1980. Sprouting by tracts descending from

the midbrain to the spinal cord: the result of thoracic funiculotomy in

the newborn, 21-day-old, and adult rat. Exp. Neurol. 69, 458–480.

Ramon-Cueto, A., Plant, G.W., Avila, J., Bunge, M.B., 1998. Long-distance

axonal regeneration in the transected adult rat spinal cord is promoted by

olfactory ensheathing glia transplants. J. Neurosci. 18, 3803–3815.

Ramon-Cueto, A., Cordero, M.I., Santos-Benito, F.F., Avila, J., 2000.

Functional recovery of paraplegic rats and motor axon regeneration in

their spinal cords by olfactory ensheathing glia. Neuron 25, 425–435.

Schnell, L., Schneider, R., Kolbeck, R., Barde, Y., Schwab, M.E., 1994.

Neurotrophin-3 enhances sprouting of corticospinal tract during devel-

opment and after adult spinal cord lesion. Nature 367, 170–173.

Schucht, P., Raineteau, O., Schwab, M.E., Fouad, K., 2002. Anatomical

correlates of locomotor recovery following dorsal and ventral lesions of

the rat spinal cord. Exp. Neurol. 176, 143–153.

Schwab, M.E., Bartholdi, D., 1996. Degeneration and regeneration of ax-

ons in the lesioned spinal cord. Physiol. Rev. 76, 319–370.

Takami, T., Oudega, M., Bates, M.L., Wood, P.M., Kleitman, N.,

Bunge, M.B., 2002. Schwann cell but not olfactory ensheathing glia

transplants improve hindlimb locomotor performance in the moder-

ately contused adult rat thoracic spinal cord. J. Neurosci. 22,

6670–6681.

Tisay, K.T., Key, B., 1999. The extracellular matrix modulates olfactory

neurite growth on ensheathing cells. J. Neurosci. 19, 9890–9899.

Treloar, H.B., Nurcombe, V., Key, B., 1996. Expression of extracellular

matrix molecules in the embryonic rat olfactory pathway. J. Neurobiol.

31, 41–55.

Vincent, A.J., West, A.K., Chuah, M.I., 2003. Morphological plasticity of

ensheathing cells is regulated by cAMP and endothelin-1. Glia 41,

393–403.

Weidner, N., Ner, A., Salimi, N., Tuszynski, M.H., 2001. Spontane-

ous corticospinal axonal plasticity and functional recovery after

adult central nervous system injury. Proc. Natl. Acad. Sci. 98,

3513–3518.

Woodhall, E., West, A.K., Chuah, M.I., 2001. Cultured olfactory ensheath-

ing cells express Nerve Growth Factor, Brain-derived Neurotrophic

Factor, Glia Cell Line-derived Neurotrophic Factor, and their receptors.

Mol. Brain Res. 88, 203–213.

Woodhall, E., West, A.K., Vickers, J.C., Chuah, M.I., 2003. Olfactory

ensheathing cell phenotype following implantation in the lesioned spi-

nal cord. Cell. Mol. Life Sci. 60, 2241–2253.

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