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The Journal of Neuroscience, December 15, 1998, 18(24):10514-10524
Regeneration of Adult Rat Corticospinal Axons Induced by
Transplanted Olfactory Ensheathing Cells
Ying
Li,
Pauline M.
Field, and
Geoffrey
Raisman
The Norman and Sadie Lee Research Centre, Division of Neurobiology,
National Institute for Medical Research, Medical Research
Council, London NW7 1AA, United Kingdom
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ABSTRACT |
Precisely localized focal stereotaxic electrolytic lesions were
made in the corticospinal tract at the level of the first to second
cervical segments in the adult rat. This consistently destroyed all
central nervous tissue elements (axons, astrocytes, oligodendrocytes,
microglia, and microvessels) in a highly circumscribed area.
In a group of these rats immediately after lesioning, a
suspension of cultured adult olfactory ensheathing cells was
transplanted into the lesion site. Within the first week after
transplantation, the cut corticospinal axons (identified by anterograde
transport of biotin dextran) extended caudally along the axis of the
corticospinal tract as single, fine, minimally branched sprouts that
ended in a simple tip, often preceded by a small varicosity. By 3 weeks, the regenerating axons, ensheathed by P0-positive
peripheral myelin had accumulated into parallel bundles, which now
extended across the full length of the lesioned area and reentered the
caudal part of the host corticospinal tract.
The transplants contained two main types of cells: (1)
p75-expressing S cells, which later formed typical peripheral
one-to-one myelin sheaths around individual ensheathed axons, and (2)
fibronectin-expressing A cells, which aggregated into tubular sheaths
enclosing bundles of myelinated axons. The point of reentry of the
axons into the central nervous territory of the caudal host
corticospinal tract was marked by the resumption of oligodendrocytic
myelination. Thus the effect of the transplant was to form a
"patch" of peripheral-type tissue across which the cut central
axons regenerated and then continued to grow along their original
central pathway.
Key words:
regeneration; olfactory ensheathing cells; corticospinal tract; white matter; adult spinal cord repair; axon
growth; myelinated tracts; transplantation
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INTRODUCTION |
Transplants of peripheral nerve into
the visual system have established that cut axons can reestablish
functional connections in the adult CNS (Vidal-Sanz et al., 1987 ). The
transplants recruited axons from the retinal ganglion cells, bypassed
their normal retinofugal pathways through the optic nerve, and
delivered the regenerating fibers directly to the deafferented terminal
field in the tectum. In practice, however, most injuries involve
multiple fiber systems. In these situations, repair would require that
the regenerating axons reenter their original tracts to be distributed
to their correct terminal sites.
Myelinated fiber tracts of the adult CNS have a complex and regular
arrangement of three types of glial cells (Suzuki and Raisman, 1992).
When a tract is damaged, the cut axons produce local sprouts at the
site of injury (Ramon y Cajal, 1928; Li and Raisman, 1995), but even
with minimal disturbance to the tract glial framework (Davies et al.,
1996), the sprouts did not reenter the distal part of the tract.
To make the damaged tracts favorable for the regeneration of cut adult
axons, we (Li and Raisman, 1994, 1997) and others (Xu et al., 1997)
have studied transplantation of cultured Schwann cells. Schwann cells
integrate into the host tract glial structure (Brook et al., 1993; Li
and Raisman, 1997). They greatly increase axon sprouting in lesions of
the corticospinal tract (CST), but few sprouts reenter the distal tract
(Li and Raisman, 1994). The reluctance of axon sprouts to leave the
Schwann cell environment of the transplant and reenter the glial
environment of the distal CST resembles the inability of regenerating
cut dorsal root fibers to leave the peripheral nerve/Schwann cell
environment of the dorsal roots and reenter the glial environment of
the dorsal spinal cord (Bignami et al., 1984).
To identify a source of cells that might enable the regenerating axons
to reenter the host CST, we therefore sought a situation in which adult
axons are normally able to enter the CNS. In the olfactory system, the
sensory neurons are replaced throughout adult life, and the newly
formed axons continually reenter the CNS (Moulton, 1974; Barber and
Raisman, 1978a,b; Graziadei and Montigraziadei, 1979, 1980; Wilson and
Raisman, 1981). The entry point of the olfactory axons into the
olfactory bulb is associated with special glial cells, known as
olfactory ensheathing cells (OECs) (Blanes, 1898; Raisman, 1985;
Valverde and Lopez-Mascaraque, 1991; Ramón-Cueto and
Nieto-Sampedro, 1992; Barnett et al., 1993). Ramón-Cueto and
collaborators (Ramón-Cueto and Nieto-Sampedro, 1994) reported
that transplants of cultured OECs mediate the reentry of regenerating
dorsal root axons into the dorsal spinal gray matter and that
injections of OECs increased axon growth into Schwann cell-filled
guidance channels transplanted into the spinal cord (Ramón-Cueto
et al., 1998).
In the present study we injected cultured OECs into focal lesions in
the rat CST. This experimental paradigm optimizes the opportunity for
repair by ensuring that (1) damage is largely restricted to a single
tract, (2) the cut axon sprouts come at once into direct contact with
the transplanted cells, (3) the distance to be crossed by the
regenerating axons is minimized, and (4) the advancing sprouts find
themselves in direct contact and alignment with the distal part of
their own tract.
Under these highly defined circumstances we observed that OECs induced
rapid, aligned growth of cut CST axon sprouts across the lesion and
into the caudal CST. In the present paper we describe the unique
morphology of these regenerating axons and the cellular arrangements by
which the OECs form a bridge conveying them across the lesion and
mediating their reentry. In a companion study we have shown that this
regeneration can restore lost functions (Li et al., 1997).
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MATERIALS AND METHODS |
Cell cultures. Syngeneic cells from the outer
(nerve fiber and glomerular) layers of adult female AS rat
olfactory bulbs were plated out, as in the method of Ramón-Cueto
and Nieto-Sampedro (1992), but without purification. Before
transplantation, the cells were cultured for 14-17 d in DMEM/F12
Nutrient Mix + 10% fetal calf serum (Life Technologies, Gaithersburg,
MD). Immunostaining (Fig. 1) confirmed
that after 10-20 d, the cultured cells had segregated into clusters of
the two major cell types described by Doucette and Devon (1994),
Barnett et al. (1993), and Ramón-Cueto and Nieto-Sampedro (1992):
(1) p75LOW AFFINITY NEUROTROPHIN
RECEPTOR +, S100+ S cells (named after their
resemblance to Schwann cells), and (2) vimentin+ fibronectin (FN)+ A
cells (named after a resemblance to astrocytes; but see comment
in Discussion). A proportion of both cell types stained with GFAP. At
higher power and lower cell density, individual S cells can be seen as
elongated, fine spindles, and A cells as more flattened.
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Figure 1.
Clusters of p75-immunoreactive S cells
(brown) and fibronectin-immunoreactive A cells
(purple). Fifteen days in culture. Scale bar, 100 µm.
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Surgical procedures. Details of the procedures are given in
Li and Raisman (1994). Briefly, the corticospinal tract was destroyed in 129 adult female rats (200-240 gm body weight) of a locally bred AS
strain by a current of 10 µA passed for 8-10 min through a stainless
steel electrode inserted stereotaxically on one side between the first
and second cervical segments. In 86 rats after lesioning, the electrode
was withdrawn, a glass micropipette was inserted into the same
position, and 3-5 µl of a suspension containing ~100,000 cultured
OECs was injected into the lesion site. In 73 rats (22 with lesions
alone, 51 with lesions and transplanted OECs), the corticospinal axons
were anterogradely labeled, at 6-10 d before rats were killed,
by injection of biotin dextran (BD) either into the contralateral
medullary pyramids or spanning the entire contralateral sensorimotor cortex.
Perfusion and preparation of material. After survivals
of 6 d to 3 months (n = 10 at 6 d, 19 at
10 d, 7 at 2 weeks, 21 at 3 weeks, 51 at 4 weeks, 4 at 6 weeks, 3 at 7 weeks, 11 at 9 weeks, 3 at 3 months), (1) 26 operated rats (11 with lesions alone, 15 with lesions and transplanted OECs) and 5 normal
rats were perfused with PBS, and immunohistochemical analysis
was performed on 10 µm cryostat sections; (2) 73 rats (22 with lesion
alone, 51 with lesions and transplanted OECs) were fixed by perfusion
with a mixture of 4% paraformaldehyde and 0.15% glutaraldehyde and
0.4% picric acid in 0.1 M phosphate buffer (PB), and
100-µm-thick vibratome sections were used for light microscopic
visualization of BD (n = 52), for confocal analysis of
the whole transplant region (n = 15), or for electron
microscope immunohistochemistry (n = 6); and (3) for
electron microscopy, 30 rats (10 with lesions alone, 20 with lesions
and transplanted OECs) were fixed with a mixture of 1%
paraformaldehyde and 1% glutaraldehyde in PB, and 200-µm-thick vibratome sections taken through the CST were osmicated, dehydrated, and flat-embedded in resin, semithin (1-2 µm) sections were stained with 1% methylene blue and Azur II, and ultrathin sections were stained with uranyl acetate and lead citrate.
Histology. For single antibody application, 10 µm cryostat
sections were fixed in acid alcohol or 4% paraformaldehyde in PB and
incubated with the primary antibody as in Table
1. The secondary antibody (anti-mouse or
anti-rabbit as appropriate) was either directly conjugated to HRP
(1:100) or biotinylated (1:500) and developed in ABC (1:300; Vector,
Burlingame, CA). For simultaneous visualization of p75 and FN or P0 and
MOG, the fixed cultures or cryostat sections were incubated with
the first primary and then the appropriate secondary antibodies (as in
Table 1), visualized by diaminobenzidine (DAB) (brown) or by
nickel-glucose oxidase (Ni-GOD) (black) (Li and Raisman, 1995),
followed by incubation with the second primary antibody, and the
appropriate secondary antibody was visualized with VIP (Vector)
(purple).
For light microscopy, BD was visualized with Ni-GOD. For electron
microscopy of BD, the sections were frozen and thawed (Henry et al.,
1994) before ABC incubation. For confocal microscopy, the sections were
incubated with p75 (1:100) and ABC overnight, washed in PBS, and
incubated in biotinylated secondary antibody (1:200) for 1 hr. The
BD-labeled axons were visualized with avidin-fluorescein (green), and
the transplanted OECs were visualized with avidin-rhodamine (red).
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RESULTS |
The corticospinal axons were identified by BD anterograde labeling
and by selective immunostaining for  calcium/calmodulin-dependent protein kinase II (CaMII) ((Terashima, 1995) (Fig.
2A,B). They form a compact, well delineated tract of
~0.5 mm diameter, located in the ventromedial part of the dorsal
columns, ventral to the gracile and cuneate fasciculi. The CST consists
of ~50,000 axons on each side; the large majority are myelinated. In
semithin or ultrathin sections or with neurofilament (NF)
immunohistochemistry (Fig. 2C), they are distinguished from
the larger myelinated ascending sensory axons of the gracile and
cuneate fasciculi by their rather uniform diameter (~1 µm) and by
their much higher glial density.
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Figure 2.
The normal rat corticospinal tract
(cs) in horizontal section (A) and
cross section (B, C, at the level of the lesions and
transplants; arrows in A). A,
B, CaMII; C, NF. cu, Cuneate
fasciculi; gr, gracile fasciculi; x,
fibers leaving the pyramidal decussation. Scale bars, 500 µm.
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Lesions
The lesions were largely confined to the CST (Fig.
3), and they destroyed an ovoid area of
tissue ~0.5 mm in width and 0.5-1.0 mm in rostrocaudal length. The
area of total destruction of the host spinal tissue was clearly
demarcated from the myelinated fiber bundles of the adjacent intact
spinal tracts with their associated cellular framework of astrocytes,
oligodendrocytes, and microglia. Within the lesioned areas, all CNS
components were completely eliminated. This was shown by light
microscopic immunohistochemistry of CAMII and NF for axons, CC1
for oligodendrocytes (Shuman et al., 1997), OX42 for ramified
microglia, and GFAP for astrocytes. Semithin and ultrathin sections
confirmed these conclusions and showed that the lesioned area contained
only debris and amoeboid macrophages.
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Figure 3.
Horizontal sections through a lesion
(les) placed in the CST (cs) on one side
to show the complete destruction of axons and glia within the lesioned
area. A, CAMII; B, NF; C,
GFAP. Arrow indicates midline. Survival times:
A, 3 weeks, B, 1 week, C,
6 weeks. Scale bar: A, 500 µm; B, C,
200 µm.
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As described in a previous publication (Li and Raisman, 1995), the cut
ends of the CST axons arborized in the rostral part of the lesioned
area (Fig. 4), but in none of the 69 animals with lesions alone (i.e., with no transplanted OECs) in this or
the previous series were there any situations in which we have observed such axons to cross the lesioned area.
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Figure 4.
A, B, Terminal sprouting of cut,
BD-labeled axons in lesions of the CST. Survival: 3 weeks. Scale bar,
20 µm.
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General morphology of OEC transplants
The transplanted OECs formed a conspicuous, densely hypercellular
mass, enclosed within the ovoid, smoothly outlined lesioned area, and
elongated along the axis of the host CST. Immunostaining for p75,
fibronectin, GFAP, L1, and laminin (Fig.
5) clearly shows the position, size, and
shape of the mass of transplanted cells. Compared with lesions alone,
where a dense astrocytic scar develops in the host CST (Li and Raisman,
1995), there is only a slight upregulation of GFAP in the immediately
adjacent host tract astrocytic processes. As in the case of transplants
of embryonic central neural tissue (e.g., Lawrence et al., 1984) or
cultured peripheral nerve Schwann cells (Brook et al., 1993; Li and
Raisman, 1997), the transplanted OECs are highly angiogenic, and from
the earliest times they induce a dense plexus of microvessels (clearly
shown by laminin immunostaining) (Fig. 5E,F), which
contrasts conspicuously with the low vascularity characteristic of the
surrounding white matter.
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Figure 5.
Immunohistochemical characterization of
transplanted OECs. A, p75; B, fibronectin
(FN); C, GFAP; D,
L1; E, F, laminin (LN).
gm, Gray matter; wm, white matter.
Arrows indicate blood vessels with associated OECs
radiating out from the transplant. Survival times: 10-14 d. Scale
bars: A-E, 500 µm; F, 50 µm.
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Rostral and caudal to the transplants, there is considerable
disorganization of the proximal and distal host CST caused by retrograde and orthograde axonal degeneration, fragmentation of myelin,
and major invasion of macrophages, and phagocytic activity by
microglia, macrophages, and astrocytes. From the earliest times observed, the individual transplanted OECs in the lesioned area become
elongated along the tract axis and migrate both rostrally and caudally
into the host CST. Later (see Fig. 11) the caudally directed migration
becomes very prominent.
Axon response
From the earliest times observed (6 d), the BD-labeled
regenerating cut corticospinal axons in contact with the transplanted OECs adopt a unique morphology (Fig. 6).
The regenerating axons are slightly expanded in diameter as they enter
the transplanted area, and they have a moderate number of varicosities.
The trajectory of the individual BD-labeled axons is constrained within
the longitudinal rostrocaudal axis of the CST. The axons appear almost
entirely unbranched, except for occasional spine-like protrusions, and they taper to a diameter of ~0.1-0.2 µm. Through-focus
examination of the full thickness of the 100-µm-thick vibratome
sections clearly shows that at earlier survivals (6-10 d), the axons
have minute, simple tips (Fig. 6, asterisks), which can be
clearly seen to end freely, within the thickness of the block of tissue
included in the sections. From as early as 10 d, some regenerating
axons have completely traversed the central part of the transplants, and their free tips have already left the caudal end of the mass of
transplanted OECs and continued in a straight line, to end at a
distance of  1 mm into the distal part of the host CST.
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Figure 6.
Morphology of regenerating axon sprouts
labeled by anterograde transport of BD. A, Bundle of
aligned, unbranched regenerating cut CST axons advancing through an OEC
transplant. B, C, High-power photographs showing the
freely ending tips (*) of regenerating CST axons preceded by small
varicosities. Survival time: 10 d. Scale bars: A,
200 µm; B, C, 20 µm.
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Electron microscopy of transplanted cell types
S cells
We identify the S cells by their resemblance to the
transplanted Schwann cells seen in our previous studies (Li and
Raisman, 1994). The S cells (Fig.
7A, indicated by S)
are solitary and have overall rounded outlines with dark cytoplasm and
smooth, rounded dark nuclei with dark areas of heterochromatin. The
cell surfaces emit microvillous processes, and from the earliest times observed, S cell processes make direct membrane contact with the axons,
forming very thin, single layers, intimately investing the
individual axons and their varicosities, and extending all the way to
their tips (Fig. 8). Cross sections (Fig.
9) show that the single S cell processes
can ensheathe multiple, small-diameter axon sprouts (as in developing
or unmyelinated peripheral nerve). From as early as 10 d, the
abaxonal surfaces of the S cells are covered by a basal lamina (Fig. 9,
arrowheads) facing a collagen-containing extracellular
space.
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Figure 7.
Electron micrographs of longitudinal sections.
A, S and A cells; B, fascicle of
parallel, longitudinal A cell processes. C,
Collagen-containing extracellular space; d, cytoplasmic
lipid droplets in A cell processes; f, filopodial
extensions of A cells; m, S cell microvillus. Survival:
10 d. Scale bars: A, 5 µm; B, 2 µm.
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Figure 8.
A, Preterminal varicosities of two
regenerating axons with central cores of neurofilaments
(f) and wrapping by very fine S cells
processes (S, drawn enlarged in B).
Survival: 10 d. Scale bar, 5 µm.
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Figure 9.
Electron micrograph of a cross section
through a bundle of fine regenerating axons (x)
enwrapped by S cell processes (S), whose abaxonal
surfaces are covered with a basal lamina (arrowheads),
in a collagen-containing extracellular space (C),
with an outer sheath of slender, curving sheets of loosely apposed A
cell processes (a). Survival: 10 d. Scale
bar, 1 µm.
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A cells
Compared with the S cells, the A cells (Fig. 7A,
indicated by A) are larger and more elongated, with paler
cytoplasm, containing lipid droplets, and larger, paler nuclei with
more irregularly shaped, somewhat rectangular outlines. They lie in the
collagen-containing extracellular space and do not contact axons. The A
cells aggregate in close contact with each other, with membrane
thickenings over much of the contact area. In longitudinal sections,
the clusters of A cells and their processes (Fig. 7B) extend
in parallel arrays along the rostrocaudal axis of the CST. At the
leading edge of the cluster, the A cells are prolonged into two or
three long, thin, filopodia-like streamers (Fig. 7A,
indicated by f). In cross section, the sheet-like,
curving A cell processes (Fig. 9, indicated by a) can be
seen to form layered shells, enclosing groups of axons and their
associated S cell wrapping.
Longer-term axon and olfactory-ensheathing cell configuration
In 15 rats at 3 weeks to 3 months survival, we prepared confocal
pictures of the whole transplant area in consecutive 1 µm steps
throughout the entire thickness in each of the five to six members of a
continuous series of longitudinal 100-µm-thick vibratome sections
through the 10-15 mm longitudinal block of spinal cord containing the
transplant (i.e., an aggregate of 500-600 scans from each transplant).
We used BD to identify the regenerating corticospinal axons and
concomitant p75 to identify the transplanted OECs. This provided a
complete visualization of the whole cross-sectional area of each
100-µm-thick section of the transplant region (Fig. 10). The regenerating corticospinal
axons form parallel bundles confined to the transplant area. They were
almost entirely unbranched, in direct alignment with the rostrocaudal
axis of the CST, and passed uninterruptedly into the distal host CST
caudal to the transplant.
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Figure 10.
A compilation of 100 sequential 1 µm confocal
scans showing the regenerating, BD-labeled corticospinal axons
(green fluorescence) passing uninterruptedly,
without branching through a transplant of p75-immunoreactive
(red fluorescence) OECs in the lesioned CST and
reentering the caudal host tract. *, Region enlarged in
B. Survival time: 3 weeks. Scale bar, 200 µm.
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From 3 weeks onward, the regenerating axons become ensheathed by
P0-immunoreactive myelin, which is characteristic of peripheral myelin
[as in the dorsal roots (Fig.
11A)], but which is
completely absent from the normal CST or other spinal tracts. The P0
immunostaining provides a striking overall picture of the regenerating
axons passing through the transplanted region and their reentry into the distal host CST. Concomitant with the expression of P0, the expression of p75, which was a conspicuous marker of S cells at earlier
times, becomes reduced throughout most of the transplant (although not
as much as in the case of Schwann cells when they form myelin in
developing or regenerating peripheral nerve (Taniuchi et al., 1988) or
after transplantation into the CST (Li and Raisman, 1997). L1 was also
greatly downregulated, although still present at low levels throughout
the transplants. Fibronectin immunostaining remained as dense as at the
earlier times.
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Figure 11.
A, Longitudinal section of a
transplant of OECs extending from the injection site
(IS) for >5 mm caudally (open arrows) in
the denervated host CST. Immunohistochemistry for P0
(black) demonstrates the peripherally myelinated
internodal segments of the regenerating host CST axons passing through
the transplant. Small, single arrows indicate P0
immunostaining of host Schwann cell peripherally myelinated axons in
the dorsal roots. B, The caudal end of the cell
migration where the columns of transplanted OECs interdigitate with the
glial cells rows of the distal host CST and their associated P0
myelinated axons reenter the oligodendrocytic territory of the distal
host CST. C, Enlargement (from A) of the
migrating OEC column with P0 axons. Blue, Thionin
counterstain of cells. D, Electron micrograph of a
BD-labeled corticospinal axon with peripheral-type myelin taken from
the center of the transplant. Survival: 3 months
(A-C), 4 weeks (D). Scale
bars: A, 500 µm; B, 200 µm:
C, 100 µm: D, 2 µm.
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Longitudinal semithin and ultrathin sections (Fig.
12) show that the axons are traveling
through regions that have a structure that closely resembles peripheral
nerve and remains devoid of any central glial cell types (astrocytes,
oligodendrocytes, or microglia). The S cells no longer enwrap multiple
axons but now consistently express the characteristic peripheral-type,
one-to-one myelinating relationship with the axons, in which the S cell
outer cytoplasmic tongues and all their abaxonal surfaces are closely and uniformly clothed by a basal lamina. Apart from their contact with
their individual axons, the S cells do not make contact with other S
cells or any other cell type. The S cell myelin is thicker (~20
turns) and (with our fixative procedure) better-preserved than the
central, oligodendrocytic myelin in the adjacent host spinal white
matter tracts, and the periodicity of the peripheral myelin is 10%
greater than that of the central myelin (Peters et al., 1976; Li and
Raisman, 1997). There are frequent nodes and Schmidt-Lantermann clefts.
Electron microscopy of the anterogradely transported BD label clearly
confirms the corticospinal identity of the peripherally myelinated
regenerating axons (Fig. 11D).
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Figure 12.
Electron micrograph of a longitudinal section
through a group of S cells forming typical peripheral myelin around
regenerating axons (x). The processes
(a) of the A cells form a perineurial-like
sheath. C, Collagen-containing extracellular space;
d, cytoplasmic lipid droplets; arrows,
regions of intercellular A cell junctions. Survival: 30 d. Scale
bar, 5 µm.
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As the S cells form myelin, the A cells become compacted into tubular
sheaths (Fig. 12) that span the length of the transplants. The sheaths
consist of reduplicated, closely apposed thin sheets of cytoplasmic A
cell processes (Fig. 12, indicated by a) bound by extensive,
complex intercellular junctions (Fig. 12, arrows). As in the
case of the perineurial sheaths of peripheral nerve, the A cell sheaths
lie in a collagen-containing extracellular space and enclose a
territory of ~5-10 µm in diameter, containing ~3-10 S cells and
their associated axons.
Reentry of the regenerating axons into the CNS
P0 immunostaining shows that the distal part of the host CST
becomes selectively infiltrated by a dense mass of transplanted OECs
(Fig. 11), extending caudally within the boundaries of the host CST for
distances of ~5 mm at 3-4 weeks and >10 mm at 3 months. At the
caudal boundary, there is no appreciable disruption of the alignment of
the host glial cells, and the streams of OECs and P0 myelinated axons
interdigitate smoothly with the longitudinal interfascicular glial rows
(Suzuki and Raisman, 1992) of the distal host CST. After crossing the
transplants, the peripherally myelinated regenerating corticospinal
axons continue uninterruptedly into the distal part of the host CST,
coextensive with the mass of caudally migrating OECs. Combined P0 and
MOG immunostaining (Fig. 13A,B) shows the transition,
at a single internode, from the peripheral myelin of the transplant
region (black = P0) to the oligodendrocytic territory
of the distal host CST (purple = MOG). In
electron micrographs this transition was represented by "mixed"
nodes with peripheral myelin rostrally and central myelin caudally
(Fig. 13C).
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Figure 13.
A, B, The junction
(arrows) where the regenerating CST axons leave the S
cell peripherally myelinated environment (p;
black = P0) of the transplant to reenter the distal
host CST, where they become centrally myelinated (c;
purple = MOG) by the host oligodendrocytes.
C, Electron micrograph of a node (arrows)
between an internode (top), which is peripherally
myelinated (p) by transplanted S cells, and the
adjacent caudal internode (bottom), which is centrally
myelinated (c) by an oligodendrocyte of the
distal host CST. Survival times: 5 weeks (A, B); 4 weeks
(C). Scale bars: A, B, 5 µm;
C, 2 µm.
Figure 14.
Summary of the cellular changes associated
with the advance of the regenerating cut CST axons
(black) across lesions (gray)
repaired with transplants of OECs (red and
green) and the reentry of the regenerating axons into
the oligodendrocytic territory (blue) of the distal host
CST. A, At shorter survivals, the axons advance,
intimately clothed by thin sheets of S cell cytoplasm
(red), and flanked at a distance by elongated A cells
(green) aligned along the rostrocaudal tract
axis. B, At longer survivals, the S cells myelinate the
regenerating axons with peripheral myelin (red) and are
clothed by a basal lamina (dashed line). The A cells
form tubular, perineurial-like outer sheaths aligned along the tract
axis and bridging the lesion area. Thus, where they leave the rostral
host CST (above the transplant), and where they reenter the caudal host
CST (below the transplant) the regenerating axons are myelinated by
central myelin (blue) formed by the host tract
oligodendroglia.
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DISCUSSION |
The axonal response
The tip of a growing axon acts as a sensory structure, exploring
the environment and changing shape (Mason and Wang, 1997) in response
to the molecular signals that determine advance, collapse, or turning
(Fan et al., 1993; Tessier-Lavigne and Goodman, 1996; Mason and Wang,
1997).
The regenerating CST axons induced by OEC transplants have three
characteristic morphological features. (1) At shorter survivals, they
have simple, unbranched tips, ending freely in the transplants. They
resemble the simple tips of developing axons in the optic stalk (Chan
et al., 1998, their Fig. 3). We do not see the expanded, hand-like
growth cones with filopodia found in tissue culture (Argiro et al.,
1984) or at choice points in developing systems (Bovolenta and Mason,
1987). (2) The regenerating axons form parallel fascicles aligned with
the long axis of the CST. This is in contrast to the highly tortuous,
varicose, branching neuromatous masses and pseudoterminal arborizations
found after transplantation of Schwann cells (Li and Raisman, 1994).
(3) At longer survivals, the regenerating axons traverse the entire
rostrocaudal axis of the transplants and continue uninterruptedly into
the denervated host CST.
We believe these to be regenerating axons that have been cut (rather
than surviving uncut axons that have been demyelinated and remyelinated
by the transplanted OECs) because (1) the lesioning procedure results
in a circumscribed area of macrophage-infiltrated debris, totally
devoid of central glia and axons (Fig. 3), indicating that all
components of CNS tissue have been totally destroyed. In 69 rats (from
this and the previous studies) with lesions alone and BD labeling of
the CST, the swollen ends of the cut axons produced branching sprays of
short sprouts in the rostral edge of the lesions (Fig. 4) (Li and
Raisman, 1994, their Fig. 3; Li and Raisman, 1995, their Figs. 3, 4),
but we have never seen axon sprouts traversing a lesion without
transplanted OECs. (2) At increasing survival times after
transplantation of OECs into the lesioned area, we see the free tips of
the regenerating cut axons extending progressively into and through the
transplants (Figs. 6, 8).
Reentry of the regenerating CST axons into the host CST
Because of the caudal migration of the transplanted OECs, the
regenerating axons reenter the distal host CST up to 10 mm caudal to
the injection site. After approximately 3 weeks, these axons are
myelinated, for their course through the transplants, by peripheral myelin formed by the transplanted S-type OECs. From the internode at
the point where they reenter the distal part of the CST, however, they
become myelinated by oligodendrocytes. For individual axons the point
of reentry is indicated by the presence of a mixed
peripheral-to-central myelin node, which can be identified by P0/MOG
combined immunohistochemistry or in electron micrographs (Fig. 13). The
reformation of central myelin indicates that the axons have left the
peripheral environment of the transplant and reentered the
oligodendrocytic CNS environment of the host CST.
At present we have not established how far the regenerating CST axons
grow or the nature of their terminal distribution. However, it seems
likely that they do form effective contacts, because in a functional
study we found that OEC-induced regeneration of cut CST fibers across a
complete unilateral CST lesion restores a specific directed forepaw
reaching function (Li et al., 1997).
The composition and behavior of the transplanted OECs
There are two distinct types of transplanted cells,
differing in their phenotype, structure, and behavior. One of the most striking features of the OEC transplants is the "cooperation" between these two cell types. At the earlier stages the A cells form
clusters with pioneering filopodia, advancing around the S
cell-ensheathed axons. At later stages the S cells myelinate the axons
and the A cell clusters coalesce into tubular, perineurial-like structures traversing the whole rostrocaudal length of the transplants (Fig. 14). A cells were named after a
supposed resemblance to astrocytes (based on the expression of GFAP)
(Barber and Lindsay, 1982; Franceschini and Barnett, 1996). However,
both the expression of fibronectin in culture (Fig. 1) and their tissue
arrangement after transplantation (Fig. 12), indicate a strong
resemblance to fibroblasts.
Like transplanted Schwann cells (Li and Raisman, 1997), transplanted
OECs suppress scar formation by the host astrocytes. There was no
long-term astrocytic hypertrophy or reorganization to form the thick,
reduplicated astrocytic scars that are typically found after comparably
sized CST lesions without transplants (Li and Raisman, 1995). At the
caudal end of the columns of migrating OECs, the longitudinal glial
cell alignment of the distal host CST was preserved.
The ability of transplanted OECs to myelinate central axons is all the
more striking because the axons with which they are normally
associated, in the olfactory system, are totally unmyelinated (Doucette, 1991). Devon and Doucette (1992) demonstrated that OECs
myelinate the neurites of dorsal root ganglion cells in culture, and
transplanted OECs (Imaizumi et al., 1998) or an OEC cell line (Franklin
et al., 1996) is able to remyelinate axons and enhance conduction in a
gliotoxic lesion of the spinal cord. In both of these situations, as in
the present study, they produce peripheral-type myelin.
In contrast to transplanted Schwann cells (Li and Raisman, 1994, 1997),
the greater caudal migration of the OECs (of up to 10 mm) (also see
Imaizumi et al., 1998) may be important for maintaining the alignment
of the CST axons and presenting the regenerating axon tips to the
caudal part of the host tract.
Anton and collaborators have shown the importance of p75 for Schwann
cell migration in vitro (Anton et al., 1994). The expression of p75 on transplanted OECs and Schwann cells (Li and Raisman, 1997)
may similarly be involved in their migration in vivo. In our
previous experiments (Li and Raisman, 1997), p75 had disappeared from
the transplanted Schwann cells by 2 months. In the present material a
proportion of the transplanted OECs were still expressing p75 at 3 months. This prolonged maintenance of p75 expression may contribute to
the enhanced migration of OECs compared with Schwann cells. The
transplanted OECs also express high levels of molecules such as laminin
(Liesi, 1985) and L1, which may contribute to both the cell migration
and the induction of axon growth (Burden-Gulley et al., 1997; Lahrtz et
al., 1997).
Relationship to central myelin
There have been previous reports of regeneration of corticospinal
axons after intracerebral transplantation of hybridoma cells secreting
an antibody against a central myelin-associated molecule (Schnell and
Schwab, 1990; Schnell et al., 1994; Bregman et al., 1995). These axons
were reported as branched, and their course was deflected by extensive
cyst formation at the lesion site, so that they did not reenter the
distal CST but descended in abnormal locations in the spinal gray
matter. Further work will be needed to elucidate the relationship to
the present observations, where we found cyst formation to be minimal
and the regenerating axons were unbranched, maintained their original
position and alignment, and directly reentered the white matter of the
caudal CST, where they became remyelinated by oligodendrocytes.
In view of the evidence that myelin-associated proteins are inhibitory
to axon growth (Bandtlow et al., 1990; DeBellard et al., 1996), our
observation that the regenerating axons ultimately become myelinated by
oligodendrocytes does not necessarily mean that at the time when the
regenerating sprouts are reentering the host CST they have made contact
with central myelin. As shown by the present electron microscopic
study, the newly growing axon sprouts traverse the lesion enwrapped in
the cytoplasm of S cells and flanked by migrating A cells and are not
in contact with central myelin. On their reentry into the caudal host
CST, they may continue to be isolated from any residual myelin by the
phagocytic cells, which engulf the degenerating myelin associated with
the degenerating distal parts of the cut corticospinal axons and by the
membranes of the array of numerous, longitudinal astrocytic processes
present in the host tract.
Conclusion and forward look
The conditions of the present experiment were designed to optimize
the possibility for repair. The corticospinal fibers form one of the
most circumscribed tracts in the spinal cord. The lesion was small, and
the reparative cells were injected into it immediately after axotomy.
The observed regeneration distance of 10 mm is sufficient for the CST
axons to reach the level of the motoneuron pools supplying the forelimb
muscles. In a preliminary series (unpublished data) we have obtained
functionally effective repair when transplantation was delayed for 5 weeks after the lesion. However, to obtain a comparable result in the
larger, more disorganized spinal cord lesions, including much longer
term lesions typically encountered in clinical situations in which
several different tracts are involved, further interventions will
probably be needed to enable the regenerating axons to bridge the
greater distances and correctly realign with the appropriate distal
tracts (Cheng et al., 1996; Xu et al., 1997).
| |
FOOTNOTES |
Received Aug. 28, 1998; revised Oct. 1, 1998; accepted Oct. 7, 1998.
This project was supported by the British Neurological Research Trust,
the International Spinal Research Trust, the Barnwood House Trust, and
Smith's Charity. Dr. Daqing Li provided invaluable consultation and
collaboration. We are grateful to Yewande Ajayi, Bernice Watt, and
Tammaryn Johnson for their excellent and innovative technical support.
Correspondence should be addressed to Dr. Geoffrey Raisman, Division of
Neurobiology, National Institute for Medical Research, The Ridgeway,
Mill Hill, London NW7 1AA, UK.
| |
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9428 - 9434.
[Abstract]
[Full Text]
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Y. Li, Y. Sauve, D. Li, R. D. Lund, and G. Raisman
Transplanted Olfactory Ensheathing Cells Promote Regeneration of Cut Adult Rat Optic Nerve Axons
J. Neurosci.,
August 27, 2003;
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7783 - 7788.
[Abstract]
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M. J. Ruitenberg, G. W. Plant, F. P. T. Hamers, J. Wortel, B. Blits, P. A. Dijkhuizen, W. H. Gispen, G. J. Boer, and J. Verhaagen
Ex Vivo Adenoviral Vector-Mediated Neurotrophin Gene Transfer to Olfactory Ensheathing Glia: Effects on Rubrospinal Tract Regeneration, Lesion Size, and Functional Recovery after Implantation in the Injured Rat Spinal Cord
J. Neurosci.,
August 6, 2003;
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[Abstract]
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G. Raisman
A promising therapeutic approach to spinal cord repair
J R Soc Med,
June 1, 2003;
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A. Lakatos, P. M. Smith, S. C. Barnett, and R. J. M. Franklin
Meningeal cells enhance limited CNS remyelination by transplanted olfactory ensheathing cells
Brain,
March 1, 2003;
126(3):
598 - 609.
[Abstract]
[Full Text]
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Y. Li, P. Decherchi, and G. Raisman
Transplantation of Olfactory Ensheathing Cells into Spinal Cord Lesions Restores Breathing and Climbing
J. Neurosci.,
February 1, 2003;
23(3):
727 - 731.
[Abstract]
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H. H. Nash, R. C. Borke, and J. J. Anders
Ensheathing Cells and Methylprednisolone Promote Axonal Regeneration and Functional Recovery in the Lesioned Adult Rat Spinal Cord
J. Neurosci.,
August 15, 2002;
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7111 - 7120.
[Abstract]
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O. Guntinas-Lichius, K. Wewetzer, T. L. Tomov, N. Azzolin, S. Kazemi, M. Streppel, W. F. Neiss, and D. N. Angelov
Transplantation of Olfactory Mucosa Minimizes Axonal Branching and Promotes the Recovery of Vibrissae Motor Performance after Facial Nerve Repair in Rats
J. Neurosci.,
August 15, 2002;
22(16):
7121 - 7131.
[Abstract]
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T. Takami, M. Oudega, M. L. Bates, P. M. Wood, N. Kleitman, and M. B. Bunge
Schwann Cell But Not Olfactory Ensheathing Glia Transplants Improve Hindlimb Locomotor Performance in the Moderately Contused Adult Rat Thoracic Spinal Cord
J. Neurosci.,
August 1, 2002;
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6670 - 6681.
[Abstract]
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G. W. Plant, P. F. Currier, E. P. Cuervo, M. L. Bates, Y. Pressman, M. B. Bunge, and P. M. Wood
Purified Adult Ensheathing Glia Fail to Myelinate Axons under Culture Conditions that Enable Schwann Cells to Form Myelin
J. Neurosci.,
July 15, 2002;
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[Abstract]
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J. Lu, F. Feron, A. Mackay-Sim, and P. M. E. Waite
Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord
Brain,
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[Abstract]
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J. V. Coumans, T. T.-S. Lin, H. N. Dai, L. MacArthur, M. McAtee, C. Nash, and B. S. Bregman
Axonal Regeneration and Functional Recovery after Complete Spinal Cord Transection in Rats by Delayed Treatment with Transplants and Neurotrophins
J. Neurosci.,
December 1, 2001;
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[Abstract]
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M. B. Bunge
Book Review: Bridging Areas of Injury in the Spinal Cord
Neuroscientist,
August 1, 2001;
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325 - 339.
[Abstract]
[PDF]
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M. Murray and I. Fischer
Transplantation and Gene Therapy: Combined Approaches for Repair of Spinal Cord Injury
Neuroscientist,
February 1, 2001;
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28 - 41.
[Abstract]
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H. B. Treloar, J. C. Bartolomei, B. W. Lipscomb, and C. A. Greer
Mechanisms of Axonal Plasticity: Lessons from the Olfactory Pathway
Neuroscientist,
February 1, 2001;
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[Abstract]
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T W Ford, C W Vaughan, and P A Kirkwood
Changes in the distribution of synaptic potentials from bulbospinal neurones following axotomy in cat thoracic spinal cord
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[Abstract]
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K. T. Tisay and B. Key
The Extracellular Matrix Modulates Olfactory Neurite Outgrowth on Ensheathing Cells
J. Neurosci.,
November 15, 1999;
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[Abstract]
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L. I. Benowitz, D. E. Goldberg, J. R. Madsen, D. Soni, and N. Irwin
Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury
PNAS,
November 9, 1999;
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[Abstract]
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Y. Liu, D. Kim, B. T. Himes, S. Y. Chow, T. Schallert, M. Murray, A. Tessler, and I. Fischer
Transplants of Fibroblasts Genetically Modified to Express BDNF Promote Regeneration of Adult Rat Rubrospinal Axons and Recovery of Forelimb Function
J. Neurosci.,
June 1, 1999;
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[Abstract]
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Top
Abstract
Introduction
