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Bridging Lesions in Cord
Volume 7, Number 4, 2001THE NEUROSCIENTIST
REVIEW
Bridging Areas of Injury in the Spinal Cord
MARY BARTLETT BUNGE
The Miami Project to Cure Paralysis
University of Miami School of Medicine
There is a devastating loss of function when substantial numbers of axons are interrupted by injury to the
spinal cord. This loss may be eventually reversed by providing bridging prostheses that will enable axons to
regrow across the injury site and enter the spinal cord beyond. This review addresses the bridging strategies that are being developed in a number of spinal cord lesion models: complete and partial transection
and cavities arising from contusion. Bridges containing peripheral nerve, Schwann cells, olfactory
ensheathing glia, fetal tissue, stem cells/neuronal precursor cells, and macrophages are being evaluated as
is the administration of neurotrophic factors, administered by infusion or secreted by genetically engineered
cells. Biomaterials may be an important factor in developing successful strategies. Due to the complexity of
the sequelae following spinal cord injury, no one strategy will be effective. The compelling question today is:
What combinations of the strategies discussed, or new ones, along with an initial neuroprotective treatment,
will substantially improve outcome after spinal cord injury? NEUROSCIENTIST 7(4):325–339, 2001
KEY WORDS Cellular bridges, CNS regeneration, CNS injury, Neurotrophins, Transplantation
Laceration injuries or those that lead to cavitation in the
spinal cord interrupt descending and ascending axons.
Whereas loss of gray matter may be small, it is the interruption of fiber tracts that leads to devastating
sequelae for the injured person. How can we bridge
these interrupted areas with permissive cells or materials to enable severed axons to once again extend to appropriate areas on either side of the lesion? A number
of investigators have conducted experiments to test cellular and noncellular components to achieve this goal of
eliciting growth across the lesion. Of course, this is but
the first step. Strategies to save as much tissue as possible directly following injury and to support growth beyond the bridge to reach areas for reconnection will be
different and additive. Thus, it will be important to combine neuroprotective agents and guidance techniques
with bridging to accomplish the long-term goal of improving outcome after injury. This overview will discuss
only bridging strategies in the adult spinal cord. Additional strategies utilizing cell/polymer scaffolds to
Work in the Bunge laboratory has been supported by NINDS grant
NS09923, The Miami Project to Cure Paralysis, The Christopher
Reeve Paralysis Foundation, and the Heumann, Hollfelder, and Rudin
Foundations. I thank Dr. N. Kleitman for helpful comments on the
manuscript and C. Rowlette, S. Knockee, and J. Cox for word processing. I also appreciate the contributions of illustrations by Drs. J.
Guest (The Miami Project; Fig. 5), A. Ramón-Cueto (Centro de
Biologia Molecular “Severo Ochoa,” Universidad Autónoma, Madrid;
Fig. 9), and X. M. Xu (color renditions of figures in Xu, Chen, and
others 1997; Xu and others 1999; Figs. 2 and 11). Dr. C. W.
Christman (Richmond, VA) finalized the drawings in Fig. 1.
Address correspondence to: Mary Bartlett Bunge, Ph.D., The Miami
Project to Cure Paralysis, University of Miami School of Medicine,
P.O. Box 016960 (R-48), Miami, FL 33101 (e-mail: [email protected]).
bridge CNS injuries are described in a review by
Harvey (2000) (see also Plant and others 2000). Also,
the review by Houweling and others (1998) contains
supplementary information about bridging experiments.
The Complete Transection Model
Peripheral Nerve Transplantation
A landmark article published in 1980 (Richardson and
others 1980) that importantly influenced subsequent spinal cord injury (SCI) research involved bridging a complete transection gap in the spinal cord with peripheral
nerve. Why complete transection and why peripheral
nerve? Complete transection lesions yield unambiguous
results about axonal regeneration compared with sprouting from noninjured axons and sparing of fibers. Peripheral nerve was tested because the rationale was to place
an environment known to support axonal regeneration
into a milieu in which adequate regeneration did not occur. This concept had been embraced by Spanish workers in the early 1900s (Ramón y Cajal 1928). The
growth of fibers into peripheral nerve implanted in the
central nervous system (CNS) prompted Ramón y Cajal
to speculate that if the environment is suitable, axons
from central neurons are able to respond and regrow. In
fact, that most perspicacious of investigators further
hinted at a combination strategy by hypothesizing that
“nutritive” and “orienting” substances will be required
to achieve axonal regeneration in the adult. By the time
the Aguayo team (Richardson and others 1980) investigated the efficacy of peripheral nerve transplantation
into the spinal cord, tracing techniques had become
available, allowing clear demonstration of the source of
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Box 1: Spinal Cord Injury Models:
Transection
Complete transection
Over hemisection
Dorsal hemisection
Lateral hemisection
Tract lesion
regenerated fibers in this and related studies (rev. in
Aguayo 1985).
Peripheral nerve was placed into a complete transection
gap of 5 or 10 mm (Richardson and others 1980). After
3 to 4 months, tracing demonstrated that fibers grow into
and across the implant from both stumps. A mean of
5850 myelinated axons are found in the graft if the nearby
dorsal roots are avulsed. Regenerated fibers do not leave
the graft, and corticospinal tract (CST) fibers do not enter
these grafts (Richardson and others 1982). Of course,
another possibility is to skirt the lesion area completely.
Other work by the Aguayo team (rev. in Aguayo 1985)
showed that ends of a piece of peripheral nerve inserted
into the medulla and the spinal cord results in fiber
ingrowth as much as 30 mm along the graft.
Efforts to achieve functional improvement after
peripheral nerve grafting have been made by the Olson
team (Cheng and others 1996). Their strategy combined
compressive wiring of the spinal column, transplantation of 18 thin peripheral nerves oriented to extend from
white matter to gray matter, and the use of fibrin glue to
which fibroblast growth factor-1 was added. If one of
these steps is omitted, the improvement in locomotion
and evidence of growth of axons from corticospinal and
a variety of brainstem neurons into the grafts and
beyond are not found. The investigators reported movements in the three joints of the hind limbs, partial support of body weight, and contact placing of the hind
limbs, suggestive of CST involvement. Reports confirming these studies have not yet appeared in the literature.
Possible reasons for this are the surgical expertise (causing minimal damage) and care with which this study
was executed and the way in which the nerves were
inserted into the spinal cord. Further discussion may be
found in another review (Bunge 2000).
Schwann Cell/Ensheathing Glia Transplantation
Because Schwann cells (SCs) are considered to be responsible for the supportive milieu provided by peripheral nerve, they have been substituted for nerve in
bridges (Fig. 1). SCs possess many attributes such as
extracellular matrix production, surface adhesion and
integrin molecules, and growth factor secretion that are
favorable for axonal growth (Plant and others 2000).
Purified populations can be prepared in numbers adequate to construct bridges. An attractive concept is that
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Box 2: Bridges across Transection Gap
Peripheral nerve
Rat Schwann cells (SCs)
Human SCs
Olfactory ensheathing glia
Peripheral nerve-activated macrophages
SCs from peripheral nerve of a spinal cord–injured person could be placed in culture for expansion. Not only
rat but also human SCs can be rapidly expanded, nearly
10,000 to 100,000 times, with a final purity of 95% to
98% (Bonamichi and others 1997; G. Casella and P. Wood,
unpublished observations). From a 1-cm piece of human
nerve, a graft 30 m long and 2.6 mm in diameter can be
prepared in 6 weeks. This, then, could be transplanted
into a spinal cord lesion in the same person. Human SCs
have been found to be as effective as rat SCs in bridges;
they support axonal regrowth and ensheathment and
myelination of regenerated central nerve fibers (Guest
and others 1997). Finally, the SCs can be genetically
modified during their culture period.
Bridges of purified SCs (4–5 mm long) unite
transected adult rat spinal cord stumps (Xu, Chen, and
others 1997) (Fig. 2); fibers grow into both ends of the
graft, as in the peripheral nerve work cited above. Much
of the SC bridge work tested a cable of SCs in Matrigel
(a commercial preparation of basal lamina components)
inside a PAN/PVC polymer channel (Fig. 3), with the
stumps inserted 1 mm into each end of the channel to
be in apposition to the cable. A channel is advantageous
in some ways. In the nude rat transection/human SC
transplantation paradigm, the effectiveness of an
SC/Matrigel cable with or without the surrounding
channel has been compared (Guest and others 1997).
The SCs, once compacted into a cable inside the channel, can be removed for transplantation without the
channel (Fig. 4). Whereas the spinal cord stumps deteriorate less and there tend to be more regenerated fibers
in the graft without a channel, in the presence of a channel, nerve fibers are in more parallel array (Fig. 5) and
there is less invasion of scar tissue into the graft-cord
interfaces. But no substantial difference in neuroanatomic
or functional outcome is observed.
When polymer-enclosed SCs are transplanted into
adult Fischer rats, spinal neurons as far away as levels
C3 and S4 extend ascending and descending fibers into
the graft, where myelinated axons and 8 times as many
unmyelinated but ensheathed axons are found (Xu,
Chen, and others 1997) (Table 1) (Figs. 6, 7). It is only
when SCs are present in the graft that considerable
n u m b e r s o f a x o n s a r e f o u n d w e e k s l a t e r.
Immunoreactive serotonergic and noradrenergic axons
grow for a short distance into the rostral end of the
graft, indicating a modest brainstem response; CST
regeneration into the graft and tracer-labeled fiber
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Fig. 2. A Schwann cell (SC) graft inside a polymer channel
placed between completely transected stumps of adult Fischer
rat thoracic spinal cord 1 month before perfusion. The channel
has been cut open to reveal the SC bridge. The ends of the
bridge are fused well to the stumps. Bar, 1 mm. (Courtesy of Dr.
X. M. Xu, color rendition of Fig. 3 in Xu, Chen, and others 1997.)
Fig. 1. Cartoons of experimental strategies in adult rat thoracic
spinal cord. A, A Schwann cell (SC) bridge implanted in a complete transection gap. SCs and stumps of transected spinal cord
are enclosed within a polymer channel (Xu, Chen, and others
1997). B, Injection of olfactory ensheathing glia (OEG) (dark
blue) into the spinal cord stumps near the ends of the polymer-enclosed SC bridge. Tracer (orange) was delivered 6
weeks later at level C7. Tracer-labeled axons are present
beyond the distal interface, and lumbar cord nerve cell bodies
are labeled, suggesting that their severed axons regrow across
both graft-cord interfaces (Ramón-Cueto and others 1998). C,
Injection of OEG (dark blue) into the spinal cord stumps after
complete transection (Ramón-Cueto and others 2000). D, Infusion of neurotrophins (light blue) into the distal end of a polymerenclosed SC cable (Xu, Guénard, Kleitman, Aebischer, and
Bunge 1995). E, Injection of SCs into the distal spinal cord to
create a trail 5 mm long beyond the transection site and also
into the transection site itself. Tracer (orange) was injected into
the distal end of the trail later to identify nerve cells that had
extended axons the length of the trail (Menei and others 1998).
F, Insertion of an SC-containing hemi-channel into the right side
of the spinal cord (Xu and others 1999). All cartoons except F
are sagittal section representations; F depicts a horizontal view.
(Drawings finalized by Dr. C. W. Christman.)
egress from the SC graft into the distal spinal cord are
not found.
The failure of axons to leave SC grafts is of obvious
concern. A gauntlet of chondroitin sulfate proteoglycan
(CSPG) at the distal SC graft-host cord may be, in part,
responsible. CSPGs, up-regulated in CNS injury, have
been implicated in failure of neurite growth in many in
vitro and in vivo studies (reviewed in Fawcett and Asher
1999 and Fitch and Silver 1999). In the complete
transection/SC transplantation paradigm, the
proteoglycans, CS-56 antigen, neurocan, phosphacan,
and NG2, are expressed near the proximal and distal
interfaces (Plant and others 2001). Moreover, the CS-56
antigen and phosphacan are more heavily expressed at
the distal than the proximal interface only when SCs are
present in the channel. We do not yet understand in
what ways SCs interact differently with the distal than
the proximal stump. Proximal stumps may differ from
distal ones in tissue viability, types of degenerating and
regenerating axons, blood-brain barrier deficiency, number of macrophages/microglia, and blood circulation, as
examples. New initiatives have begun to reduce CSPG
in areas of CNS injury to potentially improve axonal
regeneration (Lemons and others 1999; Moon and others 1999; Yick and others 2000).
There are three strategies that lead to at least modest
growth from the SC graft into the distal spinal cord.
Human SCs can be grafted into nude rats, which accept
cells from other species. The regenerative response is
improved compared with the Fischer rat model in some
ways, but this reflects the use of the nude rat, not the
human SCs (J. Guest, unpublished observations). When
grafting is done in a similar way, a comparable number
of myelinated axons is found in the human SC graft
(Guest and others 1997) (Table 1). Despite the distant
location of the nerve cell bodies in the brain stem,
serotonergic and noradrenergic fibers are detected
within the grafts. A small number of propriospinal and
sensory fibers extend throughout the graft and into the
distal spinal cord for a maximum distance of 2.6 mm.
Inclined plane and BBB open field locomotion evaluation (Basso and others 1995) has revealed that in both
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Fig. 3. Scanning electron micrographs of a PAN/PVC channel and the contained Schwann cell (SC) graft. After maintenance in culture
overnight, the SC-Matrigel mix that initially fills the channel contracts to yield a solid cable of SCs, smaller in diameter than that of the
channel (left). The inner diameter of the channel is 2.6 mm, the width of the thoracic spinal cord of a 170 g Fischer rat. The SCs
become aligned during the syneresis process (right). Bar, right panel, 20 µ m. (From Guénard and others 1992.)
Box 3: Axonal Growth from Graft into
Cord
Peripheral nerve bridge + additional strategies
Human Schwann cell (SC) bridge in nude rat
Rat SC bridge + ensheathing glia
Neurotrophin-engineered SCs
Rat SC hemibridge
Olfactory ensheathing glia
Peripheral nerve-activated macrophages
Fig. 4. A 3 to 4 mm bridge of human Schwann cells placed in a
complete transection gap of an adult nude rat thoracic spinal
cord 6 weeks earlier (upper panel ). This graft was inserted
without an enclosing channel. In the higher magnification (lower
panel ), a ventral view (dura intact) is illustrated. The union of
the bridge and stumps appears seamless (arrows), and scarring and cavitation are not obvious. Bar, 5 mm. (From Guest
and others 1997.)
cases there is a modest but statistically significant
improvement in behavioral scores when human SCgrafted animals are compared with animals transplanted
with similar cables but capped at the distal end to prevent outgrowth into the distal spinal cord (Guest and
others 1997); also, during the open field testing, animals
with open-ended grafts exhibit more frequent and longer
episodes of alternating stepping than those with capped
grafts. In a few animals with bridging grafts, there is
evidence of some form of contact-placing response, but,
because the animals lacked retrograde tracing evidence
of CST regeneration, Guest and colleagues (1997)
328
assumed that these responses represent propriospinal
placing mediated by local neuronal circuits.
A treadmill test, developed at The Miami Project
(Broton, Nikolic, and others 1996), enables kinematic
analysis of limb positions. When transected-only
(Broton, Xu, and others 1996) and human SC-transplanted rats are compared in this way, the former exhibit
rhythmic alternating stepping 20% of the time and the
latter exhibit stepping 60% of the time they are on the
treadmill (J. Guest and J. Broton, unpublished observations). An interpretation is that the graft increases excitability in the distal stump, possibly by influencing the
activity of the lumbar-stepping pattern-generating neurons. There is no evidence of fore- to hind-limb
coordination.
A second strategy in which some outgrowth of regenerated fibers from the SC graft into the spinal cord is
observed is the combination with methylprednisolone
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Box 4: Brainstem Axonal Growth into
Distant Thoracic Implant
Peripheral nerve bridge + additional strategies
Human Schwann cell (SC) bridge in nude rat
Rat SC bridge + neuroprotection
Rat SC bridge + neurotrophins
Rat SC hemibridge
Neurotrophin-engineered fibroblasts or SCs
Olfactory ensheathing glia
Fetal CNS + neurotrophins
Fig. 5. The same section of an interface between host nude rat
spinal cord and a human Schwann cell graft, immunostained for
neurofilaments (upper panel ) and glial fibrillary acidic protein
(lower panel ). The convex surface of the host spinal cord is well
demarcated by the staining for astrocytic filaments. The upper
panel shows that the nerve fibers leaving the host spinal cord
become disorganized at the interface but assume a longitudinal
course once in the graft. Bar, 1 mm. (From Zwimpfer and Guest
1999.)
administration at the time of transection and Fischer rat
SC transplantation (Chen and others 1996). Methylprednisolone is now used routinely in the United States
within 8 h after human SCI to reduce secondary injury.
With methylprednisolone, the SC bridge contains more
myelinated axons and more spinal neurons extend axons
into the graft (Table 1) (Fig. 8, top). Also, brainstem
neurons respond (mean = 57); serotonergic and
noradrenergic axons are found 2.0 to 2.5 mm into the
graft. This is a significant finding because when peripheral nerve is implanted at the same level as the SC graft
a response of brainstem neurons is not seen (Richardson
and others 1984; Houle and others 1994). Thus, the
administration of methylprednisolone overcomes, to
some degree, the distance between graft and brainstem.
In addition, a striking finding is that more spinal cord
tissue inserted into each end of the polymer channel
survives; the inserted tissue largely deteriorates without
this treatment. The egress of fibers from the graft may
be related to the reduction in secondary spinal cord
tissue loss adjacent to the graft. In more recent work,
Oudega and colleagues (1999) showed that die-back of
one of the tracts, the vestibulospinal tract, is significantly diminished when methylprednisolone is administered after completely transecting the spinal cord (with
no SC graft).
A third strategy, combining transection/Fischer rat SC
transplantation with injection of olfactory ensheathing
glia (OEG) into the stumps beside the graft (Fig. 1),
leads to substantial growth of regenerated fibers from
the graft into the distal spinal cord. Why ensheathing
glia? They are found in that part of the olfactory system
where nerve fiber growth continues throughout adulthood (reviewed in Ramón-Cueto and Avila 1998;
Kleitman and Bunge 2000). This was the rationale for
testing them earlier to see if the dorsal root entry zone,
usually a barrier to regenerating sensory neurites, would
allow axons to enter the spinal cord after cutting and
reapposing dorsal roots to the thoracic spinal cord. After
injecting OEG beside the entry zone, regrowing axons
are not only able to enter the spinal cord but also extend
within the dorsal horn to appropriate laminae (RamónCueto and Nieto-Sampedro 1994). These axons are
accompanied by OEG, highly migratory cells. Stimulusevoked conduction of signals through the regenerated
sensory fibers is restored to some extent, an indicator
that functional synapses are formed (Navarro and others
1999). Ramón-Cueto and Nieto-Sampedro’s 1994 work
prompted Ramón-Cueto and others (1998) to determine
if the presence of OEG promotes growth across SC
graft-cord interfaces. Preparations of 200,000 OEG
from adult rats, purified on the basis of their expression
of p75, were injected into each stump adjacent to the
SC bridge. This strategy does, indeed, improve the ability of regenerating fibers to cross both interfaces and
grow into the spinal cord, more than 2.5 cm for ascending fibers (Figs. 1, 9, 10). The Hoechst-labeled OEG are
found consistently in areas where regeneration is seen.
Serotonergic fibers, detected by immunostaining, regenerate not through the channel but in a connective tissueOEG milieu on the outside of the channel and reenter
the distal spinal cord where they extend for at least 1.5 cm.
Following this work, OEG were injected on each side
of a transection without an implanted SC bridge
(Ramón-Cueto and others 2000) (Fig. 1); they migrated
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Table 1. Numbers of myelinated axons in the SC graft and neurons in the spinal cord that extend axons into the graft in differing transplantation procedures
Myelinated Axons
Strategy
Capped
Channel
Open
Channel
Capped
Channel
Open
Channel
Rat SC/Matrigel
(Matrigel only)
501 ± 83
(71 ± 25)
1990 ± 594
(3 ± 0.9)
306 ± 69
1064 ± 145
a
Reference
Xu, Chen, and others
(1997); Xu, Guénard,
Kleitman, and Bunge
(1995)
Guest and others (1997)
Chen and others
(1996); Bunge and
Kleitman (1999)
Xu, Guénard, Kleitman,
Aebischer, and Bunge
(1995)
Oudega and others
(1997)
Xu and others (1999)
Responding Cord Neurons
Human SC/Matrigel
(Matrigel only)
Rat SC/Matrigel +
methylprednisolone
(SC/Matrigel +
vehicle)
Rat SC/Matrigel +
BDNF, NT-3
(SC/Matrigel +
vehicle)
Rat SC/Matrigel +
IGF-1, PDGF
(Rat SC/Matrigel
only)
Rat SC/Matrigel in
hemi-channel
(Matrigel only)
a
1442 ± 514
1159 ± 308
3237 ± 2478
1116 ± 113
2083 ± 321
(335 ±108)
(1324 ± 342)
(284 ± 88)
(1064 ± 145)
1523 ± 292
967 ± 104
(882 ± 287)
316 ± 42
(504 ± 78)
1004 ± 126
(185 ± 72.3)
550 ± 89
(126 ± 18)
The numbers are means ± SEM. The information in parentheses pertains to control animals. SC = Schwann cells.
a. Initial experiments were conducted with distally capped channels to focus on responses of descending tracts.
from both stumps into the transection site. The rats so
treated showed improved voluntary hind-limb movement, plantar hind paw placement, body weight support,
and proprioception and light touch by 3 to 7 months.
Serotonergic, noradrenergic, and CST fibers crossed the
lesion, entered the distal spinal cord, and extended for
up to 3 cm (to L6); the animals showing the best behavioral recovery exhibited fibers that extended for greater
distances. Whereas serotonergic and noradrenergic
fibers grew in spinal cord tissue and invaded regions
they normally innervate, CST fibers extended along the
pia mater and reentered the spinal cord after lengthy
growth. In general, CST growth is not seen in complete
transection models. They consistently regenerate only in
surviving spinal cord tissue (Schwab and Bartholdi
1996; Grill and others 1997). They do not respond to
the peripheral nerve or SC environment, with the one
exception of the complex peripheral nerve bridging
strategy (Cheng and others 1996).
Schwann Cell/Neurotrophin Transplantation
That neurotrophins are salutary in the SC grafting paradigm was discovered by Xu, Guénard, Kleitman,
Aebischer, and Bunge (1995), who studied the regeneration response to SC grafts and neurotrophins delivered
into the polymer channel, not in this case a bridge because the distal end was blocked to concentrate on de330
scending fibers (Fig. 1). BDNF and NT-3 were delivered together for 14 days after transplantation; the animals were maintained for an additional 14 days. A
month later, myelinated axon number in the graft and
retrogradely labeled neurons in the rostral spinal cord
were both increased with neurotrophin administration
(Table 1, Fig. 8). Also, in SC/neurotrophin but not SC/
vehicle grafts, some nerve fibers were immunoreactive
for serotonin in the graft at least 5 mm from the rostral
cord-graft interface. A mean of 92 neurons in the
brainstem extended axons at least 5 mm into the bridge;
this is in contrast to SC bridges without neurotrophins
in which there was essentially no response from the
brainstem because of the distance involved. Thus, regeneration of some neuronal populations distant from
the spinal cord transection and implant can be elicited
by a combination of trophic factors and a favorable cellular substrate. But not all growth factors have the same
effect in this paradigm. The inclusion of neurotrophic
factors, IGF-1 and PDGF, in the SC transplant does not
promote axonal regeneration into the bridge; axonal
regrowth into the transplant is diminished up to 63% although SC myelination of ingrowing axons is promoted
(Oudega and others 1997) (Table 1).
In another complete transection study (Menei and
others 1998), SCs were infected with a retroviral vector
carrying the human prepro BDNF/cDNA and then transplanted into the spinal cord. The engineered SCs were
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Fig. 6. To assess the response of neurons to a Schwann cell
graft positioned between completely transected stumps of adult
Fischer rat spinal cord, Fast Blue is injected into the middle of
the 4 to 5 mm long graft. Axons from neurons A and D pick up
tracer because they grow to or beyond (B, E) the injection site;
neurons such as C do not regenerate far enough to become
labeled. Neurons grow into the graft from both stumps. More
neurons are labeled close to the graft, and labeling is seen primarily in laminae VII and VIII where most propriospinal nerve
cells are located. The brackets contain numbers of labeled neurons found at the spinal cord level indicated in one of the traced
animals studied. (From Xu, Chen, and others 1997.)
deposited in a 5 mm long trail extending into the distal
spinal cord from the transection site and into the
transection site itself (Fig. 1). No polymer channel was
used in these experiments. The trails were largely intact
for at least a month, as determined by examining SCs
prelabeled with Hoechst dye. Cues for this strategy
were taken from two studies, one by Brook and others
(1994), who transplanted SCs in columns extending
through the thalamus and across the choroid fissure into
the hippocampus where they acted as bridges that
enable directed axonal growth across boundary membranes of the brain. The second study by Oudega and
Hagg (1996) will be mentioned later. When animals
with engineered SCs were compared with those receiving untreated SCs, more serotonergic and some
adrenergic axons were seen in the trail beyond the
transection site (Menei and others 1998). After injecting
a retrograde tracer, Fast Blue, at the distal end of the SC
trail, more retrogradely traced neurons were found in
the brainstem, mostly in reticular and raphe nuclei
Fig. 7. This survey electron micrograph portrays the content of
a Schwann cell (SC) graft placed in a complete transection gap.
Scattered SC-myelinated axons occur throughout. Numerous
nonmyelinated axons are present as well but are not readily visible at this low magnification. Well-developed fascicles of
SC-myelinated and -ensheathed axons (surrounded by differentiated perineurium in some cases) are present in other areas of
the graft as illustrated earlier in Xu, Chen, and others (1997).
Bar, 5 µ m. (From the study by Xu, Chen, and others 1997.)
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Fig. 8. These panels illustrate the injection sites (*) of Fast Blue, into a Schwann cell (SC) graft about 5 mm from the rostral spinal
cord stump, and the nearest labeled nerve cell bodies in the Fischer rat spinal cord. The host-graft interfaces are indicated by arrowheads. The rostral end of the channels is designated by large arrows. The injected Fast Blue has not reached the interfaces; the Fast
Blue that is deposited along the epineurial-like covering of the SC graft does not enter the graft or host spinal cord. The nearest
labeled cell bodies of the spinal cord are within the channel in the animal given methylprednisolone at the time of SC transplantation
(top), indicating improved survival of the spinal cord stump inserted into the channel compared with animals not receiving this compound. In contrast, despite improved regeneration following the administration of BDNF and NT-3, increased tissue survival is not
seen, because the nearest labeled cell bodies are rostral to the channel (bottom). The insets show labeled neuronal somata at higher
magnification. Sagittal sections; bars, 300 µ m (upper panel ) and 200 µ m (lower panel ). (From Chen and others 1996 and Xu, Guénard,
Kleitman, Aebischer, and Bunge 1995.)
(means, 135 vs. 22, mostly in vestibular nuclei for untreated
SC transplants). When the animals did not receive SCs,
no serotonergic or noradrenergic axons were seen beyond
the transection, and no labeled neurons were found rostral
to the transection. Thus, transplantation of SCs secreting human BDNF improves the regenerative response
across the transection site and into the thoracic spinal
cord. Behavioral testing was not done in this study.
Other Transplantation Strategies
When bundles of 10,000 carbon filaments were implanted into a complete thoracic transection lesion, fi332
bers labeled following intracortical injections of tracer
were visualized among the filaments and up to 5.6 mm
distal to the implanted fibers (Khan and others 1991). In
a lumbar model, Houle and Ziegler (1994) employed
NGF-treated nitrocellulose strips as bridges 5 weeks after complete transection. Fetal spinal cord tissue was
positioned between the strips. Six weeks later, this combination led to growth of numerous sensory fibers
rostralward along the strips and into the rostral spinal
cord; in the absence of NGF, fewer fibers extended beyond the caudal host-graft interface and along the strips.
Polylactic/polyglycolic acid has been fashioned into
channels to implant SCs into complete spinal cord gaps
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Fig. 9. This photomicrograph illustrates the numerous fibers that regenerate into a Schwann cell graft from the rostral Fischer rat spinal cord stump in the presence of injected olfactory ensheathing glia. The interface between the spinal cord (left) and the graft (right)
lies between the arrowheads. This section was stained with GAP-43 antibody to enable visualization of axons. (Courtesy of Dr. A.
Ramón-Cueto, from the investigation reported in Ramón-Cueto and others 1998.)
(Oudega and others 2001). The interest in testing this
material is that it is biodegradable and would, over time,
avoid the continued irritation of the spinal cord and possible disruption of the host-graft junction by remaining
channels (Guest and others 1997). This material is well
tolerated by the spinal cord (Gautier and others 1998).
Rapalino and others (1998) transplanted autologous
macrophages, first exposed ex vivo to peripheral nerve
segments, into completely transected thoracic rat spinal
cord. The rats were evaluated with a number of techniques: BBB open field testing, cortically evoked
hind-limb muscle responses, and immunochemistry and
tracing to detect growth of fibers across the transection
site. Behavioral recovery was seen in the form of
increased movement of the hind limbs, plantar placement of the hind paws, and weight support by 8 weeks.
Retransection led to complete loss of recovered locomotor activity, including the improved evoked potentials.
Nerve fibers were present across the transection site in
recovered animals only.
The Partial Transection Model
Partial transection injuries consist of dorsal or lateral
hemisections, highly focal damage, or “over-hemisections”
in which more than half the spinal cord is removed. Advantages are that the animals are not as paralyzed and
there is remaining tissue to serve as a bridge. In comprehensive studies of neutralization of myelin-related
neurite growth inhibitory substances by the antibody,
IN-1, CST fibers unfailingly extend through the remaining ventral spinal cord tissue (reviewed in Schwab and
Bartholdi 1996). In fact, in a study of CST fiber regeneration in dorsally hemisected thoracic spinal cord, they
consistently avoided transplants of embryonic spinal
cord, newborn pons, collagen, amnion extracellular
matrix, gelfoam, laminin-coated nitrocellulose filters,
carbon filaments, and glass fibers (Schnell and Schwab
1993). In this study, no fibers entered any of the implanted tissue or materials. In a recent study investigating OEG in which there was no remaining tissue bridge
(Ramón-Cueto and others 2000), some CST fibers
chose to regenerate along the pia for some distance below the complete transection before entering the gray
matter.
Ensheathing Glia/Schwann Cell Transplantation
The efficacy of OEG in axonal regeneration was tested
in very localized lesions of the CST between levels C1
and C2 by Raisman’s group (Li and others 1997, 1998).
Suspensions containing OEG injected into these lesions
aligned with the degenerating CST and migrated beyond
the area of damage. Axons extended into and 2 to 3 mm
beyond the injury site and were myelinated with
Schwann-like myelin until they reentered the CNS
where myelin was formed by oligodendrocytes. A thin
layer of cytoplasm abutting on regenerating axons suggested that the glia escorted regenerating fibers across
the lesion. In four animals whose forelimbs functioned
normally following complete lesioning, transplanted
cells were present along the entire extent of the lesion;
in three animals in which forelimb function did not return to normal, OEG did not bridge the entire lesion
rostrocaudally. Thus, improved function correlated with
the presence of a continuous bridge of glia across the
lesion. Imaizumi and others (2000) reported both improved conduction and sensory axonal regeneration foll ow i n g OE G ( a n d S C , b o t h f r e s h l y i s o l a t e d )
transplantation on either side of severed T11 dorsal col-
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Box 5: Improvement in Locomotion with
Transplantation
Peripheral nerve graft + additional strategies
Neurotrophin-engineered fibroblasts
Human Schwann cell bridge in nude rat
Olfactory ensheathing glia
Peripheral nerve-activated macrophages
Embryonic stem cells
Collagen bridge + neurotrophin
Fetal CNS
Fig. 10. By presenting tracer to fibers in the cervical spinal
cord region, the extension of these fibers into the Schwann cell
(SC) graft and beyond into the caudal spinal cord stump may
be detected. This is shown in the top panel; traced fibers extend
through the distal interface (arrows) between the graft (left) and
the spinal cord (right). This is seen when olfactory ensheathing
glia (OEG) are injected into the spinal cord near the ends of the
SC bridge. When OEG are not injected, there is no evidence of
fibers leaving the SC graft (bottom panel, distal interface indicated by arrows). Horizontal sections; the bar represents 160 µ m
(top) or 50 µ m (bottom). (From Ramón-Cueto and others 1998.)
umns. The transplants apparently induced a new rapidly
conducting pathway across the transection site.
Polymer (PAN/PVC) channels enclosing rat SCs but
only half the diameter of the spinal cord (Fig. 1) lead to
improved regeneration (Xu and others 1999) compared
with SC-filled channels into which the entire spinal cord
stumps are inserted (described above; Xu, Chen, and
others 1997). The model consists of placement of the
channel on the right side of the spinal cord at T8 (Fig.
11). A mean of 1000 myelinated axons and 9 times
more unmyelinated axons was found at the graft midpoint, as in the Xu, Chen, and others (1997) work. But,
in addition to propriospinal and sensory axons, neurons
(mean = 125) from a number of brainstem regions
extended fibers into the graft without additional treatment. Moreover, some regenerating axons exited the
graft to extend into the distal spinal cord as far as 3.5
mm. These axons grew toward the gray matter where
they formed bouton-like structures. The response of
brainstem neurons and growth of fibers into the distal
spinal cord may result, in part, from restoration of
334
cerebrospinal fluid circulation and relatively more stable
cord-graft interfaces due to the more limited laminectomy. As in the larger channel paradigm (Xu, Guénard,
Kleitman, Aebischer, and Bunge 1995), infusion of
BDNF and NT-3 improves regeneration; a larger number of axons leave the graft to enter the spinal cord and
grow as much as 6 mm beyond (Xu, Bamber, and others
1997). Montgomery and others (1996) placed SC-filled
polycarbonate channels into the dorsal half of the spinal
cord. Typical SC/myelinated and ensheathed axon fascicles were found in the interior of these tubes. Axons
grew in from both rostral and caudal locations. No
quantitation was performed, and whether the regenerated axons leave the graft was not tested.
Genetically Modified Cell Transplantation
A very important pioneering effort has been made by
the Gage-Tuszynski team to genetically modify
fibroblasts to provide neurotrophic factors to spinal cord
(reviewed in Tuszynski and others 1999). The cells embedded in a collagen gel are positioned in thoracic dorsal hemisection lesion cavities. There is robust growth
of sensory and coerulospinal axons into the graft if the
fibroblasts secrete NGF, for example. These grafts could
potentially serve as bridges for certain fiber populations,
but sensory and coerulospinal axons have not yet been
observed to exit the graft. In one of the most recent
studies, Grill and others (1997) severed the CST bilaterally or the entire dorsal half of the mid-thoracic spinal
cord to remove additional tracts. The more extensive
lesioning led to functional deficits lasting for 2 months,
as observed with a locomotion grid task that requires
sensory motor integration, thus partially reflecting the
function of supraspinal motor projections to the spinal
cord. When fibroblasts genetically modified to secrete
NT-3 were grafted into the more extensive lesion, significant sensorimotor functional improvement occurred
and a significant increase in CST axon growth at and up
to 8 mm distal to the injury site was observed. In no
case did CST axons enter grafts, confirming that CST
fibers regenerate only into surviving ventral gray matter.
SCs have also been genetically modified to produce
NGF and tranplanted into uninjured midthoracic adult
rat spinal cord for 2 weeks to 1 year (Tuszynski and
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Fig. 11. Implantation of a Schwann cell (SC) graft-containing polymer hemi-channel that is one-half the diameter of the spinal cord.
Panel A illustrates a dorsal view of an adult Fischer rat spinal cord following this procedure. The bridge between the two laterally
hemisected stumps inside the channel is shown in panel B (arrows). Panel C presents a ventral view. Panel D is a toluidine
blue-stained plastic cross section of the intact spinal cord (left) and implanted channel (arrows) and SC bridge (F ). The dorsal (DH )
and ventral (VH ) horns are visible in the intact left hemi-cord. The contents of boxes labeled E and F are not included in this assemblage. Bars, A through C, 2.0 mm and D, 100 µ m. (Courtesy of Dr. X. M. Xu, color rendition of Fig. 4 in Xu and others 1999.)
others 1998). In vivo expression of the human NGF
transgene lasts for at least 6 months. At 3 months and
later, the NGF-secreting grafts slowly increase in size in
contrast to nontransduced SC grafts. After 2 weeks, the
transduced transplants contain numerous sensory axons
and, after 4 months, dopaminergic and noradrenergic
axons, which continue to increase to 6 months. Control
grafts contain far fewer axons. No graft, transduced or
not, contains serotonergic or CST axons. Again, genetic
manipulation of SCs to increase their production of a
neurotrophin improves their ability to support axonal
regeneration in the adult rat spinal cord.
Another team (Liu and others 1999) transplanted
fibroblasts genetically engineered to produce BDNF
into partial cervical hemisection cavities created to
interrupt the rubrospinal tract on one side. One to two
months later, 7% of rubrospinal tract neurons possessed
axons that were present three to four segments caudal to
the transplants. These axons, in and around the transplants and in white matter caudal to the transplant, terminated in appropriate gray matter regions. These fibers
were not seen when unmodified fibroblasts were transplanted. Behavioral testing showed that recipients of
BDNF-producing fibroblasts exhibited significant recovery of forelimb function, which was abolished by a second lesion.
Neurotrophin Administration
Cervical axotomy leads to atrophy of rubrospinal neurons in the adult rat. This can be prevented if BDNF is
infused near the cell bodies 7 to 14 days later; in addition, expression of “regeneration genes,” GAP-43 and
T∝ 1-tubulin, is stimulated, receptor expression for
BDNF is maintained, and the number of axons that regenerate into a peripheral nerve graft placed into the lateral hemisection cavity is increased (Kobayashi and
others 1997). The stimulation of these genes is correlated with the increase in rubrospinal axon regeneration
into the graft. The graft was not tested as a bridge because only the proximal end of the nerve was in apposition to the spinal cord. Whereas peripheral nerve grafts
also have not been tested as bridges by Houle and colleagues (Ye and Houle 1997), valuable characterization
of neurotrophic factor influence on axonal regeneration
into these grafts has been accomplished.
The neurotrophin NGF has been tested as a supplement to peripheral nerve grafts in dorsal columns. Infusion of NGF into peripheral nerve grafts inserted into
adult rat dorsal columns causes a greater number of spinal cord neurons to regenerate axons into the graft, and
some of these neurons are farther from the graft site
than when NGF is not present (Fernandez and others
1990). Oudega and Hagg (1996) found that infusion of
NGF into the spinal cord beyond a peripheral nerve
graft in the dorsal columns leads to increased numbers
of regenerating sensory fibers that exit the graft into the
spinal cord. This work is important in suggesting ways
in which regenerated fibers can be lured from bridges
into the spinal cord. Neurotrophic factors, possibly in
gradients, may be a key to overcoming barriers to
axonal growth at the SC graft-distal cord interface. It is
not yet known whether the use of IN-1 antibody, efficacious in neutralizing myelin-related growth inhibitory
molecules (Schnell and Schwab 1990), will enhance
growth through such an interface as well as along the
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Box 6: Spinal Cord Injury Models:
Cavitation
Impaction instrument
Weight-drop device
Inflatable balloon
Photochemical technique (Bunge and others 1994)
Compression clip (Fehlings and Tator 1995)
(Oudega and others 1999; Bunge and others 2000). The
animals were maintained for 2 to 12 weeks. In control
rats, not injected with cells, a large cavity was evident.
All three types of cellular implants were well integrated
with the spinal cord, reduced injury-induced tissue loss,
improved axonal sparing/regeneration of propriospinal
projections (by 3 times) and of brainstem axons (by 1.4
times), and decreased CST die-back (by 40%).
Potential Bridging Strategies
spinal cord. NT-3, a neurotrophin to which CST fibers
respond, improves growth of these fibers into a collagen
matrix transplanted in a lesion of the CST (Houweling
and others 1998). No CST fibers leave the collagen
implant despite functional recovery (as assessed by a
grid walk test) in rats receiving the NT-3-containing collagen implants. Joosten and others (1995) found earlier
that whether the collagen is introduced in a fluid or gel
state makes a difference in promoting CST growth.
Only when collagen is added in the fluid state are
tracer-labeled CST axons present in the implant, perhaps because apposition to the spinal cord is better or
because an astroglial scaffolding structure forms only in
the initially fluid collagen.
Transplantation into Models of Cavitation
Because studying cysts that develop from contusion is
highly clinically relevant (Bunge and others 1997),
transplantation into the spinal cord to bridge these cavities has been initiated. In 1991 and 1996, Martin and
others published accounts of injecting SC suspensions
into a spinal cord cavity created by an inflatable balloon. SC survival is better when transplantation is done
immediately or at 10 days than at 3 days. Also, the
gliotic reaction is less with immediate injection. The
grafts contain numerous axons, mainly from nearby dorsal roots; neither CST nor monoaminergic fibers are detectable. Paino and others (Paino and Bunge 1991;
Paino and others 1994) prepared SCs, either in bands of
Bungner on the original collagen substratum or added
as suspensions later to collagen, and transplanted them
into photochemical lesion-induced dorsal column cavities (Bunge and others 1994) after rolling the cell-carrying collagen into jelly rolls. Profuse growth of axons
into the implants occurred but their source was not
known. Injection of fibroblasts, genetically modified to
produce NGF or BDNF, into an acute contusion injury,
accelerates locomotor recovery (BBB testing; Basso and
others 1995) and leads to larger cross-sectional areas of
the spinal cord at the epicenter than when nontransduced cells are implanted (Kim and others 1996). More
axons, enhanced myelinogenesis, and new oligodendrocytes are found in transplants of BDNF- or NT-3-producing fibroblasts at 10 weeks after contusion of adult
rat spinal cord (McTigue and others 1998). More recently,
SCs and/or OEG have been injected into thoracic cavities 1 week after contusion by the NYU impactor
336
Transplantation of fetal CNS tissue into adult spinal
cord has been studied extensively (reviewed in Reier
and others 1992 and Bregman 1994). Fetal tissue survives well, differentiates, forms synapses, becomes
vascularized, fills the lesion cavity, becomes integrated
with the spinal cord, reduces gliosis around the lesion,
and may provide trophic factors. At present, this type
of transplant may be more appropriate to provide immature neuronal populations that become relay stations
(Bregman and others 1993) rather than bridges in the
adult. Host axons grow into this type of transplant, and
neurons in the transplanted tissue extend axons into the
host tissue, but generally fibers have not been traced all
the way across. Host CST and brainstem-spinal axons
project into fetal transplants, but their distribution is restricted to within 200 µm of the host-transplant border.
CNS tissue transplants enable some degree of functional recovery, however (Bregman 1994). When transplants of E14 fetal spinal cord tissue are placed into
cervical or thoracic spinal cord hemisection lesions in
adult rats in combination with the administration of
BDNF, NT-3, or NT-4/5 at the site of injury, there is an
increase in the extent of CST, serotonergic, and
noradrenergic growth into the transplants after 1 to 2
months (Bregman and others 1997). In this case,
neurotrophins promote more extensive axonal growth
into the fetal transplants.
The application of stem cells or neural progenitor
cells (e.g., Shihabuddin and others 1999) to devise therapeutic strategies for the CNS by replenishing neurons
and/or glia may lead to bridging SCI lesions. In a new
article testing the efficacy of neural differentiated
embryonic stem cells transplanted into a 9-day contusion cavity in the rat spinal cord, the cells survived and
differentiated into astrocytes (19% ± 4%), oligodendrocytes (43% ± 6%), and neurons (8% ± 5%) (McDonald and others 1999). The cells migrated as far as 8 mm
away from the rostral or caudal lesion edge. Hind-limb
weight support and coordination (as assessed by BBB
open field testing; Basso and others 1995) partially
improved by 2 weeks with the transplantation of these
cells. Gelfoam impregnated with cultured microglia
placed into a thoracic dorsolateral cavity led to the presence of neurites (along with blood vessels and SCs) in
the implant (Rabchevsky and Streit 1997). Injection of
embryonic serotonergic neurons near a complete thoracic transection did not serve as a bridge. But the dense
meshwork of graft-derived 5HT+ fibers in the ventral
horn enabled recovery of bilateral alternating, rhythmic
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locomotor-like activity, possibly resulting from activation of the central pattern generator (Feraboli-Lohnherr
and others 1997).
Prospectus
The many new advances in basic neuroscience research
are providing more and more opportunities to devise
promising therapeutic strategies to improve outcome after SCI. The numerous causes of restricted axonal regeneration after injury in the CNS will require a
multifaceted approach to advance successful treatment.
Much more needs to be learned about mechanisms that
protect spinal cord tissue after injury; that govern appropriate presentation, dosage, transport, and signaling of
neurotrophic molecules; that diminish the influence of
inhibitory molecules; that differentiate between destructive and beneficial effects of macrophages/microglia;
that reveal effective ways of genetically modifying cells
for transplantation; that improve the efficacy of
biomaterials; that instruct in the use of guidance molecules; and that indicate the most appropriate choice for
cellular bridges. It will be a challenging but exciting
journey for the scientist and an inspiring and eventually
successful one for those in wheelchairs.
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