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Introduction
Historically, the challenge of spinal cord injury (SCI) was seemingly
insurmountable until experiments by Aguayo and David 1 challenged the myth that
neurons of the central nervous system (CNS) were not capable of regeneration as
were neurons of the peripheral nervous system (PNS). In recent years, research has
bolstered the idea that the central nervous system (CNS) can regenerate and has
made clinical therapy of CNS damage not an unreasonable goal for the next 20
years. A cursory review of the CNS regeneration literature identified two general
themes in current research. First, successful CNS regeneration requires the
promotion of pro-regeneration factors and inhibition of anti-regeneration factors.
Second, practical therapies will depend on a multi-step process to restore CNS
function. Philip Horner and Fred Gage identified some of the crucial steps needed
to repair damaged axons and restore function: 1) damaged neurons must be viable
when treatment is given, 2) axonal growth and extension must be encouraged, 3)
repaired axons must enervate the appropriate target effector cells and 4) synaptic
structure and function must be repaired5. Strategies for CNS regeneration are just
beginning to explore combinations of the steps that will reverse the damaged CNS.
Spinal cord injury (SCI) injury can be classified into two modes: acute
injury in which blunt trauma compresses or severs the spinal cord and gradual
injury in which disease or injury initiates a complex biochemical pathway that ends
in CNS impairment. Girardi et al. called these modes of CNS injury primary and
secondary injury cascades7. In fact, Horner and Gage recognized that secondary
injury would be more difficult to treat because of
“chronic pathological
sequelae.”5 Accordingly, CNS damage that results in loss of specific cell
populations without chronic pathology has been the most commonly used model
for CNS damage in animals. These damaged neuronal cells (and accessory cells)
must be enticed to repair themselves otherwise the damaged cells will undergo cell
death and result in a net loss in the total number of cells. Experimentally, several
types of cells have been used to encourage neuronal cell survival in animal models.
They include fetal tissue grafts, embryonic stem cells, multi-potent stem cells
(from adults), Schwann cells (that are normally found in the peripheral nervous
system but not the CNS), and olfactory ensheathing cells.
Another important area of research that must be applied to CNS regeneration
is the study of the normal biochemical signaling that occurs in adult neurons to
maintain cell number and integrity. There is strong data that suggested
neurotrophic factors (NTFs) provide pro-survival signals to neurons and that
deprivation of these signals results in cell death, primarily through apoptosis. For
example, deprivation of neurotrophic factors (NTFs) resulted in apoptosis, but,
over-expression of Bcl-2, an anti-apoptotic protein, rescued these neurons from
cell death2. NTFs that stimulate axonal growth have been used in many different
models to facilitate CNS regeneration. One of the major problems using NTFs is
simply the number neurotrophic protein families and the number of molecules in
these families. Horner and Gage listed ten families of proteins, each family with
several members, that have been studied as potential candidates for nerve
regeneration. In addition, it is known that different glial cells secrete different
NTFs suggesting differential utilization of these factors by different cell
subpopulations. This complexity means that the identities, the combinations, and
the concentrations of these factors need to be studied and may not be the same for
all types of CNS pathology. Unfortunately, no complete picture exists of which
NTFs are required and which are dispensable, what combinations of NTFs will be
most efficacious, what concentrations of NTFs are necessary, and what temporal
requirements exist for the NTFs. Obviously, the fewer the factors needed, the more
practical and more cost effective the regeneration strategy will be, but, it is not at
all certain that such a simple solution is possible.
Review of Papers
In two papers3,4, Xu et al. introduced and then expanded on a model of unilateral
CNS repair using an acrylonitrile/vinyl chloride mini-channel that was seeded with
Schwann cells from the sciatic nerve in a rat model. The authors created a hemisected model of CNS damage by cutting the right half of the spinal cord at the
eighth thoracic vertebra. The therapeutic approach included restoration of the dura,
a thin membrane that surrounds the CNS, as one feature that distinguished this
approach from others. The authors argued that this restored the physiological
environment of the CNS and may contribute to the success of the graft by restoring
cerebrospinal fluid circulation. Because 50-60% of CNS injuries in humans occur
in the cervical region7, mini-channels are a viable treatment strategy, compared to
larger channels, because of the limited space available in the cervical region of the
spinal cord. In the first paper, Xu at al. demonstrated that the Schwann cell-seeded
mini-channel system (and the factors secreted by the transplanted Schwann cells)
allowed axons from transected neurons to grow into and through the mini-channel,
emerge on the other side of the mini-channel, and integrate into the caudal segment
of the rat CNS. Unfortunately, the number of axons capable of performing these
steps was too few to expect significant restoration of motor and/or sensory function
lost due to the hemi-section. Therefore, the authors hypothesized that the addition
of NTFs, which have demonstrated the ability to induce axonal growth and
regeneration in various models, would improve the model by increasing the
number of axons in the mini-channel and the host spinal cord. In the first paper, a
non-degradable mini-channel plus extracellular matrix or the mini-channel plus
extracellular matrix plus transplanted Schwann cells were the two experimental
groups. In the second paper, combinations of NTFs were delivered to the caudal
side of the mini-channel graft using mini-osmotic pumps, in addition to the seeded
mini-channels. This review will focus on the data from the second paper but refer
to data from the first paper when it contributes to the discussion.
Five experimental groups were included in the second paper:
Experimental Group
Mini-
Matrigel Schwann
channel
BDNF$
NT-3&
PBS Number of
cells
MG@ + BDNF/NT-3


SC# + PBS



SC + BDNF



SC + NT-3



SC + BDNF/NT-3



Animals


4



4
4

3

8
(@=Matrigel, #=Schwann cell, $=brain derived neurotrophic factor, &=neurotrophin-3)
Photographs of the critical, sequential steps in the surgical procedure were shown
in Figure 2. The spinal cord was exposed after careful layer-by-layer dissection of
the area between the seventh and the ninth thoracic vertebrae. The spinal cord was
exposed (after the dura was carefully preserved for suturing at the end of the
operation) and the spinal cord was transected which resulted in a 2.5-2.8 mm
lesion. The lesion was located at the eighth thoracic vertebra. The mini-channel
graft was transplanted into the lesion site and the two stumps of the spinal cord
were placed into the open ends of the 3 mm mini-channel. Next, an Alset miniosmotic pump cannula was placed 2.5 mm from the caudal end of the minichannel. The Alset mini-osmotic pumps were designed to release 5 ug of
factor(s)/day for a total of 28 days (0.83 ug/ul of human brain derived neurotrophic
factor [BDNF] and neurotrophin-3 [NT-3]). Although human BDNF and NT-3
were administered to the rats, sequence comparison, using the BLAST algorithm,
of BDNF (gi 6978569 for rat and gi 4502393 for human) and NT-3 (gi 205774 for
rat and gi 189303 for human) amino acid sequences confirmed 95% sequence
identity. Finally, all the dissected layers were closed with 10-0 silk suture.
Thirty days after mini-channel transplantation, histology of transverse
sections from the middle of the mini-channels demonstrated that new tissue had
grown into the channel, creating what the authors called a tissue cable. This cable
represented Schwann cells transplanted with the mini-channel but also in-growth
of new cells and cellular structures. Normally, an individual nerve fiber (one single
neuron) is located in a bundle with many other nerve fibers. (A nerve is a actually a
bundle of nerve fibers.) A nerve fascicle is the next level of organization and
consists of a bundle of nerve fibers surrounded by the perineurium. The
perineurium consists of concentric layers of flattened cells that alternate with
layers of fine, collagenous, longitudinal fibers. The fascicle is easily identified
histologically. The next level of organization is the epineurium that consists of
multiple nerve fascicles or a nerve trunk encapsulated by connective tissue, blood
vessels, and lymphatics6. Gross histology in Figure 3 A showed that the tissue
cable in the channel was well vascularized and recapitulated an epineurium-like
structure,
including
multiple
fascicles,
blood
vessels,
and
lymphatics.
Magnification of the transverse section in Figure 3 B showed individual
myelinated axons surrounded by connective tissue. In the first paper, the authors
included a photomicrograph of normal epineurium that was more organized than
the tissue cables in the mini-channels. So even though many of the features of the
epineurium were present, there was a visible difference in the degree of
organization between the tissues. The electron micrograph in Figure 5 A confirmed
the presence of myelinated axons and unmyelinated axons in the mini-channel
tissue cable. In the first paper, pre-labeling of transplanted Schwann cells
demonstrated that the Schwann cells located in the mini-channel were from donor
cells not host cells. So, presumably, the Schwann cells that surround the axons that
have grown into the mini-channel, depicted in Figure 5 C, were from transplanted
Schwann cells rather than host cells, although the data is not shown.
In the next experiment, the numbers of axons that grew into the minichannels from the various experimental groups were quantified by simply counting
the number of axons. The results from the first and second paper are summarized
in the table below:
Paper
Experimental
Group
Avg. # of
myelinated axons
SEM
Number of
Animals
First
MG
185
72.3
4
SC
1004
126
11
10
6
4
SC + PBS
734
139
4
SC + BDNF
907
121
4
SC+ NT-3
861
254
4
SC + BDNF/NT-3
935
323
3
Second MG + BDNF/NT-3
The MG + BDNF/NT-3 group only had an average of 10 axons in the mini-channel
which was much smaller than the findings in the first paper. No explanation for
this observation was given, although the two groups are not strictly identical. The
Schwann cells were critical for attracting the damaged axons into the mini-channel,
even though these cells are not native to the CNS. However, the addition of either
BDNF (907) or NT-3 (861) did not significantly increase the average number of
axons in the mini-channel compared to the SC + PBS control (734). Also, the
combination of the BDNF and NT-3 (935) did not function in an additive manner
much less a synergistic manner, on the average number of axons in the minichannel. Although the NTFs were added to the caudal side of the mini-channel
with the hope of increasing the number axons in the mini-channel, the NTFs, alone
or in combination, did not significantly increase the number of axons located in the
mini-channel.
Next, anterograde transport was used to measure the extent of axon
integration into the host spinal cord on the caudal side of the graft. Two
compounds
were
used
for
anterograde
transport,
Phasellus
vulgaris-
leucoagglutinin (PHA-L) and biotinylated-dextran amine (BDA). The compounds
were injected 3 mm from the rostral end of the mini-channel and traveled in the
anterograde direction, down the axon from the cell body of the neuron towards the
synapse. A partial summary of the anterograde transport experiments from the first
and second papers is in the table below:
Paper
Experimental Group
Max. Distance
(mm)
Numbers of
Animals
First
MG
~0
4 of 4
SC
~3
4 of 101
MG + BDNF/NT-3
~0
N.I.C.
SC + PBS
Limited
N.I.C.
SC + BDNF
~6
2 of 4
SC + NT-3
~4.5
2 of 4
SC + BDNF/NT-3
~6
4 of 5
Second
N.I.C.=not indicated clearly
1: This represents two different groups, each group receiving PHA-L at different times.
During the anterograde transport studies, the left side of the spinal cord was
transected prior to administration of PHA-L or BDA to prevent the compounds
from traveling around the mini-channel rather that through the mini-channel.
Histology demonstrated the absence of stained axons on the left hemi-section of
the spinal cord. In the first paper, the maximum distance of axon integration,
measured from the caudal end of the channel, was ~3 mm. In the second paper,
with the addition of NTFs, alone or in combination, the average distance was
increased approximately two-fold. In the SC + BDNF/NT-3 group, there was a
steep drop-off in the number of axons as the distance from the caudal end of the
mini-channel increased. However, Figure 9 also clearly showed the substantial
increase in the maximum distance traveled. Furthermore, the axons actually
extended beyond the location of the osmotic pump cannula (2.5 mm from the
caudal end of the mini-channel) by approximately 3.5 mm, suggesting that the
distance may be increased further if the NTF gradient can be provided over longer
distances.
In addition to distance measurements, some observations about the axonal
structures were made. In the SC + NT-3 group (Figure 6), anterograde-transport
staining showed axonal arborization or branching of the terminal ends of the axons.
The staining also identified bouton-like bodies consistent with synapse formation.
In the SC + BDNF group (Figure 7), anterograde-transport staining also identified
bouton-like structures. The authors also noted fascicle-like structures in this group
using
toluidine
blue
staining.
Unfortunately,
no
staining
for
known
neurotransmitters was included in the paper. This relatively simple experiment
would have reinforced the argument that the bouton-like structures were consistent
with synapse formation. Also, Figure 7 H identified a particularly long axon
stained in the host spinal cord. The tissue is a three-dimensional object but the
histological sections only represent two dimensions. Transport studies using a dye
or compound monitored using three dimensional radiological equipment would be
an exciting way to track the length and continuity of the axons that re-integrate into
the host spinal cord. This experiment would be more informative but may be
impractical. Finally in the SC + BDNF/NT-3 group (Figure 8), staining identified
bundles longitudinal axons in a fascicle-like structure. Normally, neurons or nerve
fibers are bundled together in a fascicle, so Figure 8 D demonstrated the
recapitulation of this level of CNS structure. (See Figure 10 for a surprisingly
simple but informative summary of the data.)
Conclusions
The progression of any research area is composed of small incremental changes
published in the scientific literature, until a critical mass is reached when a more
complete understanding is achieved. Although the critical mass in CNS
regeneration research has not been reached, Xu et al. have created an exciting
model of CNS regeneration that provided a few new steps in the process. The
Schwann cell-seeded mini-channels with NTFs provided survival signals to the
transected neurons and promoted axonal repair and growth. Although the addition
of NTFs did not increase the number of axons in the mini-channel, it did increase
the distance the axons integrated into the host spinal cord. Some questions still
need to be addressed. For example, the authors noted that only anterograde
transport was used in the second paper because the majority of axons in the minichannel were derived from neurons on the rostral side of the mini-channel. Can
improvements be made to increase the numbers of axons from neurons on the
caudal side of the mini-channel? Were the concentration and duration of NTFs
physiologically relevant? How long do the re-integrated axons survive in the host
spinal cord? Do the bouton-like bodies secrete neurotransmitters? How can
function be assessed in this experimental model? What other NTFs could be used
in this system? Since Schwann cells were required to attract damaged axons into
the mini-channel, would concatenation of the seeded mini-channels attract the
axons exiting the caudal end of one mini-channel into the adjacent mini-channel?
These and other questions need answers if Horner and Gages’ prediction of clinical
trials in ten years is to come true.
End Notes
1. Axonal Elongation into Peripheral Nervous System “Bridges” after Central
Nervous System Injury in Adult Rats. Science. 214: 931-933, 1981.
2. The Relationship Between Neuronal Survival and Regeneration. Annual Review
of Neuroscience. 23:579-612, 2000.
3. Regrowth of Axons into the Distal Spinal Cord through a Schwann cell-seeded
Mini-channel Implanted into Hemisected Adult Rat Spinal Cord. European Journal
of Neuroscience. 11: 1723-1740, 1999.
4. Neurotrophins BDNF and NT-3 Promote Axonal Re-entry into the Distal Host
Spinal Cord through Schwann Cell-seeded Mini-channels. European Journal of
Neuroscience. 13:257-268, 2001.
5. Regenerating the Damaged Central Nervous System. Nature. 407: 963-970,
2000.
6. Stedman’s Medical Dictionary. 25th Ed. 1990. Williams and Wilkins, Baltimore,
MD.
7. Advances and Strategies for Spinal Cord Regeneration. Tissue Engineering in
Orthopedic Surgery. 31: 465-471, 2000.
Progress in a Schwann cell-seeded Mini-channel
Model of CNS Regeneration
Eugene H. Kang
BE553
Dr. Keith Gooch
Friday 27 April 2001
Included articles:
Regrowth of Axons into the Distal Spinal Cord through a Schwann cell-seeded
Mini-channel Implanted into Hemisected Adult Rat Spinal Cord. European Journal
of Neuroscience. 11: 1723-1740, 1999.
Neurotrophins BDNF and NT-3 Promote Axonal Re-entry into the Distal Host
Spinal Cord through Schwann Cell-seeded Mini-channels. European Journal of
Neuroscience. 13:257-268, 2001.