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The Journal of Neuroscience, August 15, 1998, 18(16):6176-6185
Transplanted Olfactory Ensheathing Cells Remyelinate and Enhance
Axonal Conduction in the Demyelinated Dorsal Columns of the Rat Spinal
Cord
Toshio
Imaizumi1, 2,
Karen L.
Lankford1, 2,
Stephen G.
Waxman1, 2,
Charles A.
Greer3, and
Jeffery D.
Kocsis1, 2
1 Department of Neurology, Yale University School of
Medicine, New Haven, Connecticut 06510, 2 PVA/EPVA
Neuroscience Research Center, Veterans Affairs Medical Center, West
Haven, Connecticut 06516, and 3 Department of Neurosurgery,
Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
Olfactory ensheathing cells (OECs), which have properties of both
astrocytes and Schwann cells, can remyelinate axons with a Schwann
cell-like pattern of myelin. In this study the pattern and extent of
remyelination and the electrophysiological properties of dorsal column
axons were characterized after transplantation of OECs into a
demyelinated rat spinal cord lesion. Dorsal columns of adult rat spinal
cords were demyelinated by x-ray irradiation and focal injections of
ethidium bromide. Cell suspensions of acutely dissociated OECs from
neonatal rats were injected into the lesion 6 d after x-ray
irradiation. At 21-25 d after transplantation of OECs, the spinal
cords were maintained in an in vitro recording chamber
to study the conduction properties of the axons. The remyelinated axons
displayed improved conduction velocity and frequency-response properties, and action potentials were conducted a greater distance into the lesion, suggesting that conduction block was overcome. Quantitative histological analysis revealed remyelinated axons near and
remote from the cell injection site, indicating extensive migration of
OECs within the lesion. These data support the conclusion that
transplantation of neonatal OECs results in quantitatively extensive
and functional remyelination of demyelinated dorsal column axons.
Key words:
olfactory ensheathing cell; transplantation; demyelination; remyelination; dorsal column; spinal cord
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INTRODUCTION |
Olfactory ensheathing cells (OECs)
exhibit a number of unique properties that make them attractive
candidates for cell therapies designed to remyelinate demyelinated CNS
axons. Although OECs normally do not produce myelin, studies
have shown that OECs can myelinate axons both in vitro
(Devon and Doucette, 1992 ) and in vivo (Franklin et al.,
1996). OECs are pluripotent, exhibiting properties of both Schwann
cells and astrocytes (for review, see Ramon-Cueto and Valverde, 1995).
Previous studies have shown that, although transplantation of Schwann
cells alone results in myelination of demyelinated or amyelinated
spinal cord axons, transplantation of Schwann cells, together with
astrocytes, produces much more extensive areas of remyelination
(Blakemore and Crang, 1985; Honmou et al., 1996) as well as
improvements in the conduction properties of the remyelinated axons
(Honmou et al., 1996). Because OECs exhibit both astrocyte-like and
Schwann cell-like properties, we reasoned that they might be able to
restore axonal conduction properties without cotransplantation of
astrocytes. Recent evidence indicating that OECs can promote
regeneration of dorsal root ganglion (DRG) axons into the dorsal horn
(Ramon-Cueto and Nieto-Sampedro, 1994) and of corticospinal axons
across a transection site in the spinal cord (Li et al., 1997) further
suggests the possibility that remyelinating cell therapies by using
OECs also might facilitate nerve regeneration after injuries involving
both axonal demyelination and transection. This may be significant in
light of recent results that indicate some axonal transection in
demyelinating lesions of multiple sclerosis (MS) patients (Trapp et
al., 1998) (for review, see Waxman, 1998).
It is well established that transplantation of CNS glial cells can
result in anatomical (Blakemore and Crang, 1985; Gumpel et al., 1987;
Duncan et al., 1988; Rosenbluth et al., 1990; Franklin et al., 1995) as
well as electrophysiological (Utzschneider et al., 1994) repair of
demyelinated or amyelinated CNS axons. Furthermore, transplantation of
purified O2-A progenitor cells alone has been observed to produce
significant remyelination of demyelinated spinal cord lesions (Groves
et al., 1993), demonstrating that replacement of oligodendrocytes is
sufficient to produce some repair of demyelinated CNS lesions. Despite
the appeal of oligodendrocyte replacement, immunological considerations
in MS patients argue in favor of developing a peripheral source of
cells. Peripherally derived Schwann cells or OECs are more accessible
than CNS glia and could, at least in principle, be obtained from the
host's nerves and thereby avoid the risk of cell rejection or the need for immunosuppression. Furthermore, because MS lesions result from
autoimmune attacks on oligodendrocytes, transplanted oligodendrocytes potentially could be at risk for secondary immunological attack, whereas Schwann cells and OECs, expressing different surface antigens, may not.
Although Schwann cells represent a readily accessible source of
myelin-forming cells, the apparent requirement for cotransplantation of
astrocytes to produce extensive areas of Schwann cell myelination in
the CNS indicates a potential limitation to the usefulness of Schwann
cells for remyelinating cell therapies (Franklin et al., 1992).
Therefore, in this study we have asked whether OECs, which exhibit both
astrocyte-like and Schwann cell-like properties, can remyelinate
extensive areas of a demyelinated spinal cord lesion and restore
electrophysiological properties of the remyelinated axons.
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MATERIALS AND METHODS |
Experiments were performed on 20 (6 unoperated controls; 7 EB-X-lesioned, and 7 EB-X-lesioned with transplants) female Wistar rats; one EB-X animal subsequently was eliminated from analysis because
of inadequate lesion size.
Induction of demyelination. The induction of the
lesion was a modification of the method developed by Blakemore and
Patterson (1978) and is described in detail in a previous paper (Honmou et al., 1996). A 40 Gy surface dose of x-ray irradiation was delivered through a 2 × 4 cm opening in a lead shield (4 mm thick) to the spinal cord caudal to T10, using a Siemens Stabilipan radiotherapy machine (250 kV, 15 mA, 0.5 mm Cu; 1 mm Al filters, SDD 28 cm, dose
rate 220.9 cGy/min; Siemens AG, Erlangen, Germany). At 3 d after
x-ray irradiation, a laminectomy was performed at T11, and 0.5 µl of
0.3 mg/ml ethidium bromide (EB) in saline was injected unilaterally
into the dorsal column at depths of 0.7 and 0.5 mm at three
longitudinal sites 2 mm apart, for a total of six injections (Felts and
Smith, 1992; Honmou et al., 1996).
Cell preparation and transplantation. OECs were separated
from neonatal rats (2 or 3 d old), as described previously (Chuah and Au, 1993). After the removal of meningeal membranes, olfactory nerve layers were dissected from the olfactory bulb and were cut into
pieces and dipped in cold DMEM (4-6°C) in sterile condition. The tissue blocks of the ONL were triturated through a flame-narrowed glass pipette, filtered through gauze, and then retriturated until single-cell suspensions were obtained. OECs were concentrated at
2.4-3.0/µl in DMEM. The demyelinated rats were anesthetized with
ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). A dorsal
laminectomy was performed at the T11 vertebral level under sterile
conditions. A bolus of 0.5 µl of the OEC suspension was injected via
a glass micropipette into the midpoint of the EB-X lesion 0.7 and 0.5 mm in depth over a period of 90 sec; the micropipette was left in
position for 30 sec before slowly being withdrawn. The transplanted
site was marked with sterile charcoal.
Electrophysiological recording. Rats were killed under
sodium pentobarbital anesthesia (75 mg/kg, i.p.) for physiological experiments 21-25 d after cell injection. The vertebral column between
T8 and L2 was removed and placed in cold (4-6°C) dissecting solution
[containing (in mM) 135 choline chloride, 20 choline bicarbonate, 1.0 KCl, 1.2 KH2PO4, and 90 dextrose]. The spinal cord region between vertebral regions T9 and L1
was removed and was pinned in a recording chamber constantly perfused
with zero-Ca2+ modified Krebs' solution
[containing (in mM) 124 NaCl, 3.0 KCl, 2.0 MgCl2, 26 NaH2CO3,
1.3 NaHPO4, and 10 dextrose] bubbled with 95%
O2/5% CO2 at a drip rate of 4.0-5.5
ml/min at room temperature for 30 min. Then CaCl2 (2.0 mM) was added to the solution. At 80 min after the addition
of CaCl2, the electrophysiological recordings were
obtained at 26°C. The Krebs' solution in the chamber gradually was
warmed to 36°C and maintained at this temperature with a TC-102 temperature controller (Medical Systems, Greenvale, NY ). After a 90 min incubation at 36°C, additional recordings were obtained.
The surface of the demyelinated lesion was identified by its
translucence and dorsomedial position; the site of OEC injection was
identified by the charcoal spot. A bipolar silver wire stimulating electrode was placed on the caudal end of the normal region of the
dorsal column. Stimulus amplitude was set 20% above the strength that
elicited a maximal compound action potential (CAP). Stimuli (50 µsec
duration) were delivered via a constant current stimulus isolation unit
(Master-8, AMPI). The electrical responses were recorded with glass
microelectrodes (10 µm tip diameter, 3-10 M resistance) filled
with 150 mM NaCl and positioned within the dorsal columns
2.0-5.0 mm from the stimulating electrode. CAPs were recorded with a
preamplifier (Axoprobe-1, Axon Instruments, Foster City, CA) and
displayed on a digital oscilloscope (4094C, Nicolet Instruments,
Madison, WI). CAP amplitudes were measured from peak to peak and were
compared with the control CAP amplitude. All data were expressed as
mean ± SD.
Histological examination. After electrophysiological
recordings were made, spinal cord tissue was fixed for 24 hr in 2%
paraformaldehyde plus 2% glutaraldehyde (w/v in 0.14 M
Sorensen's buffer). Tissue was washed three times, stored overnight in
buffer, and cut into 2 mm segments. Then spinal cord segments were
post-fixed with 1% osmium tetroxide (Polysciences, Warrington, PA) for
4 hr and embedded in Epox-812 (Ernest Fullam, Latham, NY). Tissue was
serially sectioned on an ultramicrotome, and 1 µm sections were
collected every 0.25 mm. Finally, the sections were counterstained with 0.5% methylene blue/0.5% azure II in 0.5% borax.
To assess the degree of remyelination produced by transplanted OEC
cells, we first measured cross-sectional areas of the medial aspect of the dorsal column within each section, the demyelinated region of the dorsal column, and any "myelin-rich" areas with a
Nikon Microphot microscope with a 4× lens coupled to a MCI CCD2 video
camera and interfaced to an Image 1 processing system. Regions showing
different degrees of myelination are readily apparent at this
magnification by their staining density (see Fig.
1). Then the percentage of the target
region that was demyelinated and the sizes of the regions within the
lesion showing relatively high and low percentages of myelination were
calculated. The numbers of myelinated axons in 3-10 representative
fields in the "myelin-rich" (i.e., remyelinated) and
"myelin-poor" (i.e., demyelinated) regions of the lesion were
counted at 100× magnification. Small nonmyelinated axons were more
difficult to identify with this type of preparation, and their numbers
were not included in this analysis. The degree of remyelination at each
level of the spinal cord was calculated by using the formula:
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where Nx = estimated number of myelinated
axons at position X, MR = average
density of myelinated axons in the myelin-rich region of the lesion,
MP = average density of myelinated axons in the
myelin-poor region of the lesion, AR = area of
the myelin-rich region of the lesion, and AP = area of myelin-poor region of the lesion.
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Figure 1.
Illustration of regions of the dorsal column
identified for subsequent analysis of myelinated axon density and size.
A, Cross section of an EB-X-lesioned dorsal column with
transplanted OECs. Areas A, B, and
C indicate the dorsomedial, centromedial, and
ventromedial regions of the dorsal column, respectively; within these
areas the sizes (cross-sectional area) of myelinated axons were
measured for one representative video field every millimeter along the
length of the spinal cord segment that was studied. B,
Photomicrograph shown in A, with the target (central
region used for analysis) dorsal column areas, lesioned area, and
"myelin-rich" and "myelin-poor" areas outlined.
Myelinated, demyelinated, and remyelinated areas were identified at low
magnification by the staining density. C-F, Diagrams
showing the cross-sectional areas of the target dorsal column area,
demyelinated area, and myelin-rich and myelin-poor areas within the
lesion, respectively. These areas were used to calculate the percentage
of the target areas lesioned as well as the estimated numbers of
remyelinated axons at leach level of the cord.
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Calculated numbers of myelinated axons at each level then were
integrated across distance to establish the total amount of axon length
that was remyelinated and expressed in axon millimeters. Estimates of
the percentage of axons remyelinated were obtained by dividing the
numbers of axon millimeters remyelinated by the product of the area of
the lesion and the average myelinated axon density in unlesioned dorsal
columns. The total sizes of the lesioned areas also were calculated by
integrating the size of the lesion across distance. Lengths of the
lesioned and remyelinated areas were determined from the distance
between the first and last sections that showed detectable areas of
complete demyelination and the distance between sections with at least
200 remyelinated axons.
To assess the types of fibers remyelinated by OECs, we measured the
cross-sectional areas of myelinated and unmyelinated axons in one video
field each for three different anatomical regions of the dorsal column.
Cross-sectional areas of all myelinated axons were measured in one
video field each in the dorsomedial, centromedial, and ventromedial or
corticospinal tract region of the dorsal column at 1 mm intervals along
the dorsal column (see Fig. 1A). Myelinated axon size
data for each region were pooled for all animals within each treatment
condition. The numbers of axons in each 1 µm2 size
(area) category were determined for control and remyelinated axons, and
the size distribution of myelinated axons was compared by using a
 2 test.
For some experiments, ultrathin sections also were collected from
regions of the spinal cord showing extensive remyelination. Sections
were stained with uranyl acetate and lead citrate and examined with a
Zeiss EM902A electron microscope (Oberkochen, Germany) operating at 80 kV. Electron micrographs of remyelinated areas were taken at 7000 and
50,000× magnification.
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RESULTS |
Remyelination of the EB-X lesion after OEC transplantation
Histological examination of spinal cords confirmed that the EB
injection and x-ray irradiation of the spinal column resulted in
extensive demyelination of the central region of the dorsal columns,
with an average of 90 ± 3% (n = 4 animals) of
the target area demyelinated at the widest part of the lesion. To avoid
the possibility that large numbers of spared axons could confound interpretation of the conduction properties of demyelinated axons, we
did not include animals in any subsequent analysis if  75% of the
target dorsal column area was lesioned at the widest point in the
lesion. EB-X lesions resulted in demyelinating lesions typically 5-7
mm in total length, with an average distance of 6 ± 0.25 mm
(n = 8 animals). Total volumes of the lesioned areas averaged 0.9 ± 0.3 mm3 (n = 4 animals) for lesioned areas without transplanted cells and 1.0 ± 0.2 mm3 (n = 4 animals) for lesioned
areas with transplanted OECs, or an estimated average loss of
120,000 ± 20,000 mm (n = 4 animals) of myelinated
axon length for each lesion. In control lesions without cell
transplants, demyelination of axons within the lesioned area was
essentially complete, with <2% of axons within the lesioned area
either being spared demyelination or spontaneously remyelinated.
Transplantation of OECs into previously demyelinated regions of the
dorsal column resulted in remyelination of large areas of the EB-X
lesion, near the site of cell transplantation at the approximate center
of the lesion, and for 2-3 mm rostral and caudal to the midpoint.
Large myelin-rich areas were detected readily in lesions containing
transplanted OECs (Fig. 2C),
but not in lesioned dorsal columns into which only culture medium was
injected (Fig. 2B). Migration of myelin-forming cells
was extensive, with at least 200 remyelinated axons being detected
across distances of 4-6 mm along the spinal cord (average 5.0 ± 0.5 mm; n = 4 animals).
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Figure 2.
A-C, Low-magnification light
micrograph of cross sections through dorsal columns from unlesioned
control rats (A), EB-X-lesioned rats
(B), and animals with EB-X lesions, followed by
the transplantation of 30,000 OECs (C).
Micrographs B and C show areas near the
centers of the lesioned areas. D-F, High magnification
of images of the centers of the dorsal column shown in the panels
directly above these. Few myelinated axons can be
detected in the center of the lesioned dorsal column without
transplanted OECs, but many can be observed in the lesion with
transplanted OECs. Calibration in A refers to
A-C and that in D to
D-F.
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Examination of dorsal column axons at the electron microscopic level
revealed myelination patterns consistent with interpretations at the
light microscopic level of myelin-rich and myelin-poor areas within the
transplanted spinal cords. At the ultrastructural level, peripheral
areas of OEC transplanted lesions that were categorized as myelin-poor
contained primarily densely packed nonmyelinated axons, with small
numbers of scattered myelinated axons and cell bodies of undetermined
type (Fig. 3A). Little
extracellular space was observed between axons in these areas. In the
more central portions of the lesions, however, the majority of axons
either exhibited the characteristic features of myelination, with
multiple layers of closely apposed membrane surrounding the axons (Fig. 3B,C), or appeared to be in earlier stages of myelin
formation (Fig. 3C). The relative amounts of extracellular
space also tended to be larger in regions of apparent remyelination.
Although we occasionally observed structures consistent with astrocytic
processes in transplanted regions, astrocyte-like cells were not
prominent features of lesioned dorsal columns.
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Figure 3.
Electron micrographs of representative regions of
EB-X-lesioned area of the OEC transplanted dorsal column shown in
Figure 2C. A, Peripheral edge of EB-X
lesion. Note primarily nonmyelinated axons. B,
C, Central areas of the lesion showing examples of
remyelinated axons. Intercellular spacing is increased, and most axons
appear either to be myelinated (B) or to be in
some stage of myelin formation (C).
Inset in C, High magnification of
remyelinated axon showing multilayered membrane structure consistent
with that of myelin surrounding the axon. A-C, 12,000×
magnification; inset, 170,000× magnification.
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The total amount of remyelination observed in lesions with transplanted
OECs was significantly greater than that observed in control lesions
without transplanted cells (Fig. 4).
Transplantation of 24,000-30,000 OECs resulted in the remyelination of
~22,000 ± 3000 mm (n = 4 animals) of total axon
length as compared with 2000 ± 1000 axon mm (n = 4 animals) either spared or spontaneously remyelinated in control
lesions (p < 0.001, unpaired Student's t test). Using the density of myelinated axons in unlesioned
spinal cords as a reference, these results indicate that
transplantation of OECs results in remyelination of ~17 ± 2%
of demyelinated axons, compared with 1.8 ± 0.3% of axons spared
or remyelinated in control lesions without transplanted cells. Because
dorsal column areas tend to decrease in EB-X-lesioned dorsal columns
and increase in lesions with transplanted OECs, these figures probably
represent an underestimate of the difference between control lesions
and lesions with transplanted OECs.
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Figure 4.
Graphs showing areas of dorsal columns and
EB-X-lesioned areas as well as the estimated numbers of remyelinated
axons at successive points along the length of the spinal cord for a
representative EB-X-lesioned dorsal column (A)
and a lesioned dorsal column after 30,000 transplanted OECs
(B). The data shown here are for the same lesions
shown in Figure 2B, C.
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OECs appeared to be capable of remyelinating axons in all areas of the
medial aspect of the dorsal column, although individual experiments
often showed that remyelination was localized to a given portion of the
dorsal columns. The overall shapes of the myelin-rich areas within a
given lesion also tended to be similar in sections separated by a 1 mm
or more, implying that OECs can migrate more readily along the parallel
rather than the perpendicular axonal axis and that small differences in
positioning of the injection site could be responsible for differences
in the subsequent patterns of remyelination. Although some axons in all
size categories were remyelinated in lesions with OEC transplants, size
distributions of remyelinated axons in the OEC transplant condition
appeared to be skewed toward larger diameter axons when compared with
myelinated axon sizes for similar regions of control dorsal columns
(p < 0.005, 2 test) (Fig.
5). Differences in the diameter of
myelinated axons were particularly apparent in the corticospinal tract
region of the dorsal column, where the average size (area) of
myelinated axons was increased from 1.72 ± 0.09 µm2 in control spinal cords to 2.55 ± 0.06 µm2 in the OEC remyelinated condition.
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Figure 5.
Graphs illustrating the size (area) distribution
of myelinated axons in the dorsomedial (A),
centromedial (B), and ventromedial
(C) of the dorsal columns (designated as areas
A, B, and C in Fig.
1B). Each graph shows the percentage of axons in
each size category for unoperated controls (white bars)
and OEC remyelinated dorsal columns (black bars).
Representative fields were analyzed every millimeter along the dorsal
column. A minimum of 250 control axons and 1200 OEC remyelinated axons
were measured from at least three different experiments to produce each
graph. The population distribution for each region is shifted toward
larger axon sizes in the OEC remyelinated condition, but the effects
were most marked in the ventromedial or corticospinal tract region of
the dorsal column.
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Enhanced axonal conduction after remyelination by OECs
Consistent with histological evidence for extensive demyelination
of EB-X-lesioned dorsal column and substantial remyelination by
transplanted OECs, field potential recordings revealed significant reductions in conduction velocity and frequency-response properties in
EB-X-lesioned dorsal columns, whereas conduction velocities and
frequency-response properties in lesioned dorsal columns with transplanted OECs were more similar to control unlesioned dorsal columns. Figure 6A
shows four superimposed CAPs recorded at 1 mm increments with respect
to the site of stimulation for a control dorsal column. Similar
recordings from the demyelinated and remyelinated dorsal columns are
shown in Figure 6, B and C, respectively.
Conduction velocity was reduced significantly in the demyelinated
region of the EB-X lesion at both 26°C (0.85 ± 0.27 m/sec,
n = 7) and 36°C (1.21 ± 0.37 m/sec,
n = 7) as compared with the normally myelinated regions
of the dorsal columns (8.35 ± 2.22 m/sec, at 26°C,
n = 6; 12.17 ± 2.47 m/sec, at 36°C,
n = 6). After OEC transplantation-induced remyelination
the conduction velocity was increased at both 26°C (4.52 ± 2.32 m/sec, n = 6; p < 0.006, t
test) and 36°C (7.40 ± 3.78 m/sec, n = 6;
p < 0.006) as compared with demyelinated axons. A
summary of these results is shown graphically in Figure
7.
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Figure 6.
Field potential recordings from control
(A), demyelinated (B), and
transplanted (C) dorsal columns. Compound action
potentials were recorded at four points at 1 mm increments
(arrows) along the dorsal columns. Note the increase in latency
for recordings obtained from the demyelinated axons and the decrease in
latency for those obtained from the dorsal columns with OEC
transplantation.
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Figure 7.
Summary data of conduction velocity for normal
(n = 6), demyelinated (n = 7),
and remyelinated (n = 6) dorsal column axons at
26°C (left) and 36°C (right).
Conduction velocity of the demyelinated axons was reduced as compared
with control at both temperatures (p < 0.00001, Student's t test). Conduction velocity of the
transplant group was increased as compared with the demyelinated axons
but decreased as compared with the normal axons. Significance levels
are indicated by asterisks (*p < 0.05; **p < 0.01; shown as comparisons of normal
or demyelinated groups to the transplant group).
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In normal dorsal columns the amplitude of the CAP decreased with
increasing conduction distance. The CAPs from the demyelinated dorsal
columns showed larger increases in latency and larger amplitude decrements with increased conduction distance as compared with control
dorsal column recordings, indicating extensive conduction block (Fig.
8). The responses from the region
remyelinated by OECs displayed less amplitude decrement with increasing
conduction distance (Fig. 8, triangles); unlike the
recordings from the demyelinated areas in which virtually no response
was present at 4 mm from the recording site, the remyelinated axons
were able to conduct at least 5 mm into the lesion, suggesting a
reduction of conduction block.
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Figure 8.
Amplitude decrement with conduction distance for
normal (n = 6), demyelinated (n = 7), and transplanted (n = 6) dorsal columns at
26°C (A) and 36°C (B).
Note that axons in the transplant group show less amplitude decrement
with increasing conduction distance than axons in the EB-X lesion
condition; p value notation and n values
are the same as described in Figure 7.
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Paired stimulus pulses applied at various intervals were used to study
the recovery properties of the axons; interstimulus intervals ranged
from 1 to 100 msec at 26°C and from 0.5 to 100 msec at 36°C. CAP
amplitude recovery was reduced and delayed in demyelinated regions but
improved after the OEC transplantation at both temperatures (Fig.
9). The ability of dorsal column axons to
transmit trains of action potentials was examined via
frequency-response analysis, using 25-200 Hz stimulus trains (0.5 sec
duration) at 26°C and 25-800 Hz at 36°C. Control dorsal column
axons displayed a progressive amplitude decrement to 10% at 26°C
(200 Hz) and 30% of initial amplitude at 36°C (800 Hz) (Fig.
10). At both temperatures the
demyelinated axons showed nearly complete conduction block at high
frequencies. This defect in conduction was reversed partially, however,
by OEC transplantation, and the OEC-remyelinated axons showed
considerable restoration in their ability to follow
high-frequency stimulation (Fig. 10).
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Figure 9.
Amplitude recovery for the second of two stimuli
at varying interstimulus intervals for normal, demyelinated, and
transplanted dorsal columns at 26°C (A) and
36°C (B). There is less amplitude decrement for
the remyelinated axons than for the demyelinated axons.
p value notation and n values are the
same as described in Figure 7 and indicate the comparison between the
normal or demyelinated condition to the transplant group.
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Figure 10.
Frequency-response properties of normal,
demyelinated, and transplanted dorsal columns at 26°C
(A) and 36°C (B). The
demyelinated axons display considerable reduction in their ability to
follow high-frequency stimulation, but the remyelinated axons are able
to follow higher frequencies of stimulation. Comparisons of responses
between normal or demyelinated groups are shown related to the
transplant group. p value notation and n
values are the same as described in Figure 7.
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DISCUSSION |
In the present study we demonstrate that transplantation of OECs
into EB-X demyelinated adult rat spinal cords results in relatively
extensive remyelination of demyelinated axons and recovery of
electrophysiological function. Histological, ultrastructural, and
electrophysiological data all support the idea that transplanted OECs
can produce substantial repair of demyelinated spinal cord axons. The
pattern of remyelination produced by transplanted OECs was very similar
to that produced by cotransplantation of neonatal Schwann cells and
astrocytes (Honmou et al., 1996), i.e., relatively thick myelin with
large cytoplasmic and nuclear regions surrounding remyelinated axons,
suggesting that a single OEC remyelinates a single axonal segment. A
notable difference between remyelination by Schwann cells and OECs was
that transplantation of neonatal Schwann cells without astrocyte
cotransplantation resulted in only small restricted areas of
remyelination, whereas OECs were capable of migrating and remyelinating
axons across distances of several millimeters without the need for
transplantation of additional cell types. This observation suggests
that astrocytes may produce a signal that induces the migration of
Schwann cells, as suggested by Honmou et al. (1996). Indeed,
preliminary studies in our laboratory suggest that injections of
Schwann cells cultured in conditioned astrocyte medium display more
extensive myelination than Schwann cells alone.
OECs have a number of unique properties that could account for their
ability to remyelinate axons in the CNS. As summarized by Doucette and
colleagues (Doucette, 1991, 1993; Doucette and Devon, 1993, 1994) and
recently reviewed by Ramon-Cueto and Valverde (1995), these cells share
properties of both Schwann cells and astrocytes. They express
p7SNGFR, L1, PSA-NCAM, laminin, central-GFAP,
myelin basic protein, neuropeptide Y, and glial nexin and may
contribute to the secretion of NGF. In olfactory nerve they typically
do not express a myelinating phenotype and appear morphologically
similar to astrocytes (Doucette and Devon, 1993). However, the
addition of OECs to cultures of DRG neurons results in their
development of a well defined myelinating phenotype similar to that
observed by Schwann cells (Devon and Doucette, 1992; Ramon-Cueto and
Nieto-Sampedro, 1993). Moreover, Franklin et al. (1996) demonstrated
that the injection of OECs into the demyelinated rat spinal cord
results in remyelination. Although one possibility is that
transplantation of OECs may result in in vivo
differentiation of both astrocyte-like and Schwann cell-like cells that
interact to provide conditions appropriate for relatively extensive
migration and myelination, we did not see extensive evidence for the
presence of astrocytes in the lesioned area. Consequently, we tend to
favor the possibility that the OECs intrinsically possess greater
myelinating and migratory potential than Schwann cells.
In the present study we have examined the electrophysiological
properties of the OEC-induced remyelinated axons. The results indicate
that the conduction velocity of the remyelinated axons is restored
toward normal values and that the axons are capable of following
relatively high-frequency stimulation trains. Moreover, impulses
conduct a greater distance along their longitudinal axis in the dorsal
columns of the remyelinated axons than in the demyelinated axons,
suggesting that conduction block is overcome in the axons remyelinated
by OEC transplantation. These data suggest that a sufficient nodal
Na+ channel density is present in the OEC
remyelinated axons to establish relatively normal conduction.
Examination of the morphology of the remyelinated spinal cord indicates
substantial structural differences as compared with normal spinal cord.
The density of axons is lower in the remyelinated dorsal columns; this
results from the increased space occupied by the cell bodies and nuclei
of the OECs as compared with oligodendrocytes. In this regard, the
general morphological appearance of the remyelinated axon regions in
the dorsal columns is reminiscent of peripheral nerve. Extracellular
microelectrode recordings from sciatic nerve are of relatively low
amplitude as compared with recordings in CNS white matter. The larger
extra-axonal space of both peripheral nerve and the reconstructed
spinal cord white matter could provide a greater extra-axonal current
pathway that would lower the extracellular current density recorded by
the extracellular microelectrode tip, resulting in a reduced amplitude
of the response. The reduced amplitude of the CAP of the remyelinated
axons also likely reflects a reduction in the number of axons
conducting action potentials.
Recently, Li et al. (1997) reported that injecting a suspension of OECs
at the site of a lesion in the corticospinal tract resulted in the
extension of transected corticospinal axons through the lesion. The
OECs cells adopted a myelinating phenotype with individual axons that
persisted through the lesion site. Those animals with successful axon
extension through the lesion also exhibited a restoration of directed
forepaw reaching. Moreover, recent studies indicate that some axonal
transection can occur in MS patients (Trapp et al., 1998) (for review,
see Waxman, 1998). Given that in contusive spinal cord injury and
perhaps in some MS patients both axonal transection and demyelination
can occur, the OEC may offer a unique opportunity to address both types
of pathologies by a monocellular approach.
Concluding remarks
Our current data indicate that OECs may be good candidates for
remyelinating cell therapies. OECs migrate extensively and remyelinate
a significant fraction of demyelinated axons without the need for
cotransplanting other cell types. Although we cannot rule out the
possibility of unusually long internodal distances for OEC remyelinated
axons, the amount of remyelinated axon length relative to the numbers
of transplanted OECs also suggests that at least some OECs divide after
transplantation. Ultrastructural evidence of ongoing myelination at 4 weeks after transplantation implies that even greater repair may be
possible with longer recovery times, although in this model it was
necessary to terminate the experiment after a relatively short time
period to minimize any confounding effects of spontaneous
remyelination. Notable, in addition to histological evidence for
remyelination by OECs, the transplantation of OECs into demyelinated
lesions also results in improved conduction properties.
| |
FOOTNOTES |
Received Feb. 23, 1998; revised May 28, 1998; accepted June 4, 1998.
This work was supported in part by the National Multiple Sclerosis
Society, National Institutes of Health (Grant NS10174), and the Medical
Research Service, Department of Veterans Affairs.
Correspondence should be addressed to Dr. Jeffery D. Kocsis, Department
of Neurology, Yale University School of Medicine Neuroscience Research
Center, Veterans Affairs Medical Center, Building 34, West Haven, CT
06516.
| |
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Top
Abstract
Introduction
