Center for Paralysis Research, Department of Basic Medical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907
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ABSTRACT |
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Shi, Riyi and
Richard B. Borgens.
Acute repair of crushed guinea pig spinal cord by polyethylene glycol.
We have studied the responses of adult guinea pig spinal cord
white matter to a standardized compression within a sucrose gap
recording chamber. This injury eliminated compound action potential
(CAP) conduction through the lesion, followed by little or no recovery
of conduction by 1 h postinjury. We tested the ability of
polyethylene glycol (PEG) to repair the injured axons and restore
physiological function. Local application of PEG (1,800 MW, 50% by
weight in water) for ~2 min restored CAP conduction through the
injury as early as 1 min post PEG application. The recovery of the CAP
1 h was significantly greater in treated compared with control spinal
cords (controls = 3.6% of the preinjury amplitude; PEG
treated = 19%; P < 0.0001, unpaired Student's
t-test). Stimulus-response analysis indicated that the
susceptibility for recovery was similar for all calibers of axons after
PEG application. The enhanced recovery of conduction after PEG
treatment was associated with an early alteration in conduction
properties relative to control spinal cords. This included increased
refractoriness and sensitivity to potassium channel blockade using
4-aminopyridine (4-AP). Normally 4-AP enhanced the amplitude of the
recovering CAPs by ~40% in control spinal cords; however this effect
was nearly doubled to ~72% in PEG treated spinal cords. Because
severe clinical injuries to the spinal cord (and some peripheral
nerves) are both resistant to medical treatment and usually produced by compression, we discuss the possible clinical benefits of PEG application.
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INTRODUCTION |
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The water-soluble polymer polyethylene glycol
(PEG) has the unique ability to fuse cell membranes. This class of
compounds first was evaluated as a means to couple several individual
cells into one as a means to study various aspects of cell biology and to introduce genetic material from one cell into another (Ahkong et al. 1987; Davidson et al. 1976
;
Nakajima and Ikada 1994
; O'Lague and Huttner
1980
). Single giant axons of crayfish and earthworm also have
been fused using PEG (Bittner et al. 1986
; Krause
and Bittner 1990
; Krause et al. 1991
). The
ability of these types of polymers to join cell membranes also has been
exploited to instantaneously seal cell membranes, reversing
the permeabilization of the membrane produced by damage or disease.
This has obvious medical application where such permeabilization leads
to cellular death or tissue atrophy (Lee et al. 1992
,
1993
).
When the membranes of axons are crushed or compressed, various
alterations in their intracellular and extracellular ionic domains
immediately occurs. This is due to a compromised electrical and ionic
barrier. These changes also can block the transmission of action
potentials. For example, conduction block can occur due to a local
collapse of the membrane potential in concert with a higher
permeability to K+ ions. Natural resealing of the
compromised membrane eventually may lead to a restoration of ionic
equilibrium and conduction (Blight 1993; Shi and
Blight 1996
). However, if the membrane lesion is severe, the
outcome will be a progressive localized dissolution of the membrane and
myelin, resulting in permanent conduction block to action potential
propagation (Shi and Blight 1996
). In the most severe
injuries, this process of "secondary injury" will lead to
continued permeabilization of the axolemma and its complete collapse,
sometimes causing the death of the neuron. This is particularly true if
the insult to the nerve process occurs close to the neuron's soma
(Lucas et al. 1985
; Shi et al. 1989
). In
surviving neurons, such progressive degeneration eventually will lead
to the separation of the axon and Wallerian degeneration of the distal
segment (Fawcett and Keynes 1990
; Ochs
1980
). An artificial means to immediately seal and repair nerve
membrane lesions might intervene in these processes, rescuing the
distal segment of the axon from eventual dissolution, restoring
variable levels of conduction, and to some unknown extent, producing a
recovery of lost behaviors.
Here we evaluate the ability of PEG to repair crushed nerve fibers of the CNS. We test the capability for immediate repair of nerve membranes after a standardized and severe compression injury to isolated adult guinea pig white matter.
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METHODS |
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Animal use and care
Adult female guinea pigs of 350-500 g body wt were used for
these studies. The spinal cord was isolated from deeply anesthetized animals (60 mg/kg ketamine hydrochloride, 0.6 mg/kg acepromazine maleate, and 10 mg/kg xylazine, im). After anesthesia, the animal was
perfused transcardially with cold (15°C) Krebs solution [which contained (in mM) 124 NaCl, 2 KCl, 1.2 KH2PO4,
1.3 MgSO4, 1.2 CaCl2, 10 dextrose, 26 NaHCO3, and 10 sodium ascorbate, equilibrated with 95%
O2-5% CO2]. The vertebral column was removed
rapidly using bone forceps and scissors by previously described
techniques (Shi and Blight 1996, 1997
). The spinal cord
was divided into four longitudinal strips, first by midline sagittal
division, then by separating the dorsal and ventral halves with a
scalpel blade against a plastic block. Only the ventral white matter
was used for this study. These 35- to 38-mm-long strips of spinal cord
white matter usually will be referred to below as "cords" or
"spinal cords" for ease of description. Spinal cords were
maintained in continuously oxygenated Krebs solution for 1 h
before mounting them within the recording chamber. This was to ensure
their recovery from dissection before experiments were begun.
Double sucrose gap recording technique
The double sucrose gap recording chamber and in vitro injury
model already have been described in previous publications, and we
refer the interested reader to these reports (Shi and Blight 1996, 1997
). The construction of the recording chamber and the placement of the spinal cord within it is illustrated in Fig. 1, A and
B.
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A standardized compression injury was produced with a
stepper-motor-controlled rod, which compressed the spinal cord while suspended inside the recording chamber (Shi and Blight
1996) (Fig. 1B). The basic recovery profile after
such standardized compression in normal Krebs solution has been
characterized previously and published (Shi and Blight
1996
).
Every electrophysiological test was digitized in real time and captured to the computer for subsequent quantitative evaluation. All records also were recorded on VHS magnetic tape as a means of back up documentation. All solutions used in the PEG repair process were made on the day of their use.
PEG repair procedure
First, typical physiological functioning of the isolated white matter strip removed to the recording chamber required ~0.5-1 h of incubation time while immersed in oxygenated Krebs to stabilize. In initial experiments, once the CAP propagation had stabilized, the Krebs solution was replaced with Ca2+ free Krebs (Ca2+ replaced with equimolar Mg2+).
Second, the spinal cord strip then was crushed by the techniques described in the preceding section, while simultaneous stimulation and recording continued.
Third, a solution of PEG in distilled water (50% by weight) was applied by a pressure injection through a micropipette. A vital dye was added to the PEG solution to monitor its continuous application to the lesion site in a stream ~0.5 mm wide for ~1-2 min. The PEG was applied to one side of the lesion, washed over it, and immediately removed by constant aspiration on the other side using a second pipette.
Fourth, immediately after the PEG application, the bathing media in the central chamber was replaced with a continuous stream of oxygenated normal Krebs solution. The physiological properties of the PEG treated spinal cord were monitored continuously for 1 h. Usually, a weak recovering CAP was evident within 6-15 min of the PEG application.
The preceding technique should be considered as a basic one, from which testing of several variations described in the following text were performed.
For example, we tested the response of "recovering" axons to the additional application of the fast potassium channel blocker, 4-aminopyridine (4-AP). In this trial, five separate cords were treated with an application of PEG as described earlier and compared with five control cords. One hour after compression, 100 µM 4-AP (in Krebs solution) was applied for 15 min and then washed free with normal Krebs solution as described earlier.
Fifth, in a final series of experiments, the requirement for the injury to be carried out in Ca2+-free media was tested. In these experiments, the cord was compressed while it was immersed in normal Krebs' solution.
Statistical treatment
Before and after the application of 4-AP, we used Student's t-tests to compare recovering action potential amplitude between the control and PEG treated group. Comparisons of action potential amplitude also were made between the two PEG-treated groups.
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RESULTS |
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PEG-mediated repair of crushed spinal cord strips
Approximately 0.5 h after the equilibration of the spinal
cord strip in the recording chamber, the Krebs solution in the central compartment was replaced with a Ca2+-free Krebs and the
spinal cord was crushed by previously described techniques. In every
spinal cord tested in this group of 20 (10 control and 10 experimental), this procedure resulted in the immediate and total loss
of CAP propagation from the point of stimulation to the point of
recording. Figure 2 shows an individual
record of one typical control experiment and a PEG-treated experimental spinal cord strip. Note the immediate and complete loss of the CAP in
both preparations, and the initial recovery of the CAP in the PEG
treated spinal cord by 5 min posttreatment (Fig. 2B). The
earliest recorded recoveries of a CAP occurred within 1-2 min after
PEG treatment. In control preparations, three cords never regained
conduction during the 1 h of continuous observation. In contrast,
not one PEG-treated spinal cord providing the data summarized in Fig.
3 failed to recover CAP conduction after
PEG treatment. In four more control spinal cords, the recovery of the
CAP was not observed for ~20 min. Figure 3 provides a summary graph
of the 10 control and the 10 experimental spinal cords treated and
monitored identically, save the experimental application of PEG to the
lesion site. PEG treatment always provided a striking increase in the
amplitude of recorded CAPs, averaging 19% of the original
pretransection amplitude, and always facilitated the CAP recovery in
100% of the cases tested. At every time point testedincluding the
10-min postinjury period
recovered CAP amplitudes were statistically
significantly greater than control preparations (Fig. 3). Figure 3 also
shows that the injury need not be carried out in Ca2+-free
media to produce functional repair as claimed by Bittner for
invertebrate axons (Krause and Bittner 1990
) (refer to
DISCUSSION).
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Electrophysiological properties of the repaired spinal cords
The PEG repaired spinal cords showed typical conduction properties (as observed in recovering untreated cords); however, some differences in their electrophysiological properties were revealed by further evaluation.
Figure 4A shows the effect of injury on the normal recovery of CAP amplitudes. Typically, the recovered CAP was dampened in amplitude across all threshold intensities of excitation. We also evaluated if this reduced magnitude of the CAP occurred across all caliber spectra of injured axons within the spinal cord strip or was manifest in only large or small diameter axons. Figure 4B shows the actual amplitudes of control compound potentials at 1 h postinjury, plotted against the preinjury amplitude at the same stimulus intensity. A least-squares linear regression was not significantly different from 1:1 linearity, suggesting that there was no difference between the susceptibility to damage of axons of different stimulus thresholds.
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In Fig. 4C, we plotted two hypothetical lines representing outcomes after PEG treatment. Note that if larger axons of a lowered stimulus threshold were more susceptible to PEG, the data would be shifted as in the gray line (a). In the opposite situation, the hatched line (c) shows a shift in the opposite direction should small caliber axons with a higher stimulus threshold be repaired. In Fig. 4D, the actual data taken from the PEG-treated population is plotted in the same manner as in Fig. 4B. The near unity slope of the relation of amplitude response before and after injury indicated no consistent selectivity of PEG-mediated improvement of conduction in fibers of lower or higher threshold.
Although PEG appeared to be able to repair axons of a wide range of calibers similar to the natural recovery process observed in control cords, the electrophysiological properties of PEG-mediated recoveries was not the same as controls. Figure 5A shows the classical relationship between the timing of paired stimuli and the amplitude of the two elicited CAPs. Paired stimuli in which the interstimulus interval was between 0.6 to 15.0 ms demonstrated typical dampening of the CAP amplitude soon after the absolute refractory period. When the interval between the paired stimuli was longer than this, a plateau was reached where the first and second CAPs were of an identical magnitude-marking the extent of the relative refractory period.
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Figure 5B shows control data derived from four separate experiments. The abscissa shows the magnitude of the second CAP of the pair as a percent of the magnitude of the first elicited CAP. The ordinate shows the log of the interstimulus interval ranging from 0.6 to 15 ms. This sigmoidal plot is typical, beginning with stimuli that do not elicit a second AP during the absolute refractory period and ending at the termination of the relative refractory period.
Furthermore Fig. 5B shows that this relationship was not disturbed by the injury, as pre- and postinjury data points were not significantly different along this sigmoidal curve. This did not hold true, however, for PEG-treated spinal cords. The early and robust recovery of CAPs produced by PEG demonstrated a typical period of absolute refractory as before the injury and experimental treatment. Moreover the relative refractory period also appeared to terminate when a similar stimulus interval to control preparations was achieved. During the refractory period of PEG-treated cords, the amplitude of the second CAP was slightly reduced when compared with that before the crush and PEG treatment (Fig. 5C). However, this latter relationship was not statistically significant.
Potassium channel blockade as an adjunct to PEG-mediated recovery of conduction
It is a common feature of injured cells to loose intracellular potassium to the extracellular milieu through compromised membrane. In axons, this may be sufficient to suppress action potential conduction. Thus we attempted to determine if blockage of fast potassium channels with 4-AP would affect the properties of conduction immediately after PEG repair.
Figure 6A shows the enhancement of the CAP in crushed (but untreated with PEG) spinal cord by 4-AP. In this individual record, the initial recovered CAP at 1 h postinjury is shown, and the enhanced CAP after 100 µM 4-AP treatment is superimposed on it. After documentation of the 4-AP enhanced CAP, the blocker was washed out, and the media in the central compartment was replaced with normal Krebs solution. The CAP fell to pretreatment levels by 15 min and was indistinguishable from the original record. This final waveform is superimposed on the other two CAPs in Fig. 6A but cannot be discriminated from the pretreatment electrical record. In this single test, 4-AP reversibly enhanced the recovered CAP by ~40%.
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Figure 6B shows an identical test performed on a PEG-treated spinal cord, in which 4-AP was administered at 1 h post-PEG application. In this individual test, the second CAP was enhanced reversibly by ~70%. After the near doubling of the CAP, 4-AP was washed out as described, and the CAP fell to pretreatment levels as in controls (Fig. 6A). Figure 6C shows the group data, five spinal cords in each group. The percent enhancement of the PEG-mediated recovery for the group data mirrors that discussed above for the individual experiments (~70% enhancement in the experimental group; ~40% in the control group). This experimental enhancement was statistically significantly greater than that observed in the controls. (P < 0.05, unpaired Student's t-test)
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DISCUSSION |
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After severe compression/contusion of the guinea pig spinal cord,
a complete conduction block occurs in response. For reasons that are
understood, such as the initiation of endogeneous repair mechanisms
(Shi and Blight 1996; Xia and Barrett
1991
; Yawo and Kuno 1985
), and for reasons that
are not, a small population of axons seal their cellular lesions and
are capable of propagating action potentials through the damaged area
once again. In our standardized injury model, compound action potential
propagation through the damaged area never recovered before 10 min
postinjury, whereas some cords never recovered at all during the hour
of measurement and evaluation. Furthermore, this natural recovery
process never resulted in CAPs greater than ~7% of the
pretransection amplitude.
Within a few minutes after the application of the water-soluble polymer PEG, an immediate recovery of CAP propagation through the lesion occurred. The recovered CAP amplitude slowly increased with time to a peak of ~20% of the initial CAP amplitude. Moreover, this level of recovery was always statistically significantly higher than control amplitudes, observed at every time point tested, and occurred in 100% of the experimentally treated spinal cords. It is clear that a topical application of PEG can immediately repair severe compression injury to the mammalian spinal cord leading to significant increases in functional recovery as defined by the enhanced capacity to propagate nerve impulses through the lesion. This report is the first to demonstrate PEG-mediated repair of crushed mammalian nervous tissue.
We have employed a concentration of PEG (50% by weight in water) and
MW (1,800 Da) that commonly has been used by many to fuse both
nonneuronal cells and neuron-like cells (PC 12) in vitro (Davidson et al. 1976; O'Lague and Huttner
1980
). Bittner has used similar PEG MWs and concentrations to
fuse or repair single axons in crayfish and earthworms, while
suggesting that additional use of a Ca2+-free hypotonic
bathing medium was required for successful treatment (Bittner et
al. 1986
; Krause and Bittner 1990
; Krause
et al. 1991
). We have shown that a physiological, balanced
media and the aforementioned PEG solution is all that is required to
produce functionally significant repair in mammalian spinal cords (see
following text). Moreover, in other experiments where completely
transected guinea pig spinal cords were fused with PEG, we have learned
there was no specific PEG MW critical to the process
having tested PEG
solutions using 400, 1,400, 1,800, 2,000, and 3,700 Da (unpublished observations).
Mammalian spinal cord in isolation
The ability to resolve even small increases in a recovering CAP
was permitted by the use of the double sucrose gap chamber of the
design described here and elsewhere (Shi and Blight 1996, 1997
). Briefly, the ends of the isolated strip of spinal cord white matter are bathed in isotonic KCL and are approximately at
intracellular potential during the recording session, while the central
compartment contains physiological media at extracellular potential.
The compartments are separated by a viscous sucrose gap, which can be
thought of as analogous to a limiting membrane. Although this
electrical seal is not perfect, this recording arrangement can produce
electrical records of superior resolution
approaching the
signal-to-noise ratio of intracellular recordings. In our other studies
where PEG has been used to fuse completely transected strips of spinal
cord white matter in isolation, CAPs of only 1-2% of the original
magnitude could be revealed clearly against noise (to appear
elsewhere). In this study, the demand for high-resolution discrimination between preinjury and recovered CAP propagation was less
a factor given the immediate reoccurrence of PEG-mediated CAPs at a
time when control spinal cords could not conduct action potentials at
all, and the substantial amplitudes of the recovered CAPs at times >10
min postinjury relative to their preinjury levels. We have outlined
other advantages when studying the physiology of spinal cord injury
using this technique and direct the interested reader to these
discussions (Shi and Blight 1996
, 1997
).
Fusigens and membrane repair
PEG has been used for >30 yr as a means to fuse cells. This
allows the production of giant cells from many small ones facilitating electrophysiological study or manipulation of membranes. The fusion of
cells also facilitated the exchange of genetic material between cells
and the formation of hybridomas during the production of monoclonal
antibodies, as well as serving as a model for the vesicular fusions
that normally occur during the biology of cells (Lee and Lentz
1997; Lentz 1994
). Even so, the actual molecular
mechanisms of action permitting membrane fusion by PEG is still under
investigation (Lee and Lentz 1997
). It is clear that
cellular fusion occurs in a stepwise manner when adjacent membranes
touch in the presence of PEG, membrane fusion occurring before the
fusion of the cells and the mixing of cytoplasms (Ahkong et al.
1987
). At the level of the membrane, acute dehydration of the
fusing plasmalemmas permits the intermingling of glyco/protein/lipidic
structures, which resolve into each other first at the outer membrane
leaflet and, subsequently, the inner membrane leaflet (Lee and
Lentz 1997
). Rehydration apparently leads to a spontaneous form
of structural self-assembly within the aqueous plane of the membrane.
PEG-mediated axonal recovery
Severe and local compression to spinal cord white matter produces
a "lesion" in axons that in the least may be characterized by a
compromised electrical seal and a collapse in the local resting potential of the axon at this site. This usually leads to three possible outcomes: 1) the breach in the membrane at the site
of damage seals itself, which may preserve the axon's anatomic
integrity and capacity to propagate APs. 2) In some cases,
this natural recovery process may preserve the anatomic integrity of
the axonbut may not restore its ability to conduct action potentials
in some types of axons. This is especially true if axons within the
local area of injury becomes denuded of myelin. And 3)
failure in the natural process of axonal sealing may lead to physical
separation of the axon and the death of the distal segment in
mammals
or of the neuron itself if the injury to the nerve process is
close to the soma (Blight 1993
; Blight and
Decrescito 1986
; Lucas et al. 1985
; Shi
et al. 1989
).
We believe the chemical properties of PEG that allow membranes to
resolve into each other permitting the complete fusion of two or more
closely opposed cells provides a very rapid mechanism of repair of the
acute membrane breach. We are using a horseradish peroxidase (HRP) dye
exclusion test (Asano et al. 1995), together with a new
method of computer-managed morphometry (Moriarty et al.
1998
) to anatomically evaluate the difference in axonal
membrane repair by PEG relative to control spinal cords. This test was based on the hypothesis that a PEG-induced "seal" or membrane "repair" will exclude the intracellular uptake of HRP by damaged cells after injury. Our preliminary evidence is that PEG-treated spinal
cord strips are indeed better sealed than control cords. These studies
will be reported elsewhere.
In this physiological study, we have determined similarities and
differences between the natural mechanisms of axonal repair and those
mediated by PEG. First, a least-squares linear regression analysis of
pre- and postinjury CAP amplitudes suggests that PEG-mediated repair
can occur across all levels of stimulus thresholds, reflecting axon
diameters, as does the natural recovery process in untreated spinal
cord strips. In other words, all spinal axons regardless of their
caliber are equally susceptible to PEG-mediated repair (see Shi
and Blight 1996 for a similar analysis of axonal recovery from
compression injury). The differences between natural repair and that
produced by PEG application are more striking. First, this injury is
very severe; 30% of control spinal cords never recovered any capacity
to conduct CAPs during the 1-h period of evaluation after injury. On
the other hand, there was no instance where PEG did not initiate a
measurable physiological recovery. On a more subtle level, there
appears to be a slightly reduced CAP amplitude during the period of
relative refractory in only PEG-mediated CAPs relative to control
cords. One explanation for this observation may be that in control
cords a severely compromised and dysfunctional population of axons may
become completely nonfunctional, revealing more normal conduction
properties in that population that survive the injury. PEG may rescue a
portion of such severely compromised axons, recruiting them into the
CAP and perhaps accounting for its slightly different conduction properties.
Ionic controls of axonal injury and membrane repair
In other work, we have evaluated the way that ionic changes
produced by injury impact the response of axons to insult
(Borgens 1988; Borgens et al. 1980
;
Shi and Blight 1997
). It is known that injury to cell
membranes leads to an increase in K+ permeability that
contributes to AP conduction block (Blight 1989
;
Shi and Blight 1996
, 1997
). In myelinated axons, even
acute damage to the myelin sheath leads to K+ current
shunting and depressed or inhibited conduction
both events can be
reversed variably by K+ channel blockade using 4-AP
(Blight 1989
; Pratt et al. 1995
; Shi et al. 1997
). This fast potassium channel blocker
both enhances the safety factor for conduction as well as extending the
length constant for AP propagation across regions of demyelination
(Blight 1989
; Shi and Blight 1996
, 1997
).
In the studies reported here, we expected an enhanced amplitude of the
CAP in even control cords based on our prior studies and these
mechanisms of action. We were surprised, however, by the nearly
doubling of CAP amplitudes in PEG-treated cords, suggesting that PEG
repaired membrane itself is particularly leaky to K+. We
propose a mechanism of synergistic action of 4-AP with PEG repair in
Fig. 7. In any event, 4-AP that has been
used to restore conduction and behavioral recovery in clinical spinal
cord injury should be viewed as a possible adjunct to the development
of any clinical use of PEG in nerve repair (see next section).
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Clinical implications
It is likely that this procedure can provide a novel means of
treating severe, acute neurotrauma. In addition to immediate improvements in conduction, repair of crushed axons in peripheral nerves leading to a rescue of their distal segments would provide the
added benefit of reducing atrophy or degeneration of target cells or so
called "end organs." Many target tissues require innervation for
sustenance and/or survival. Currently trauma to peripheral nerves close
to the trunk of the body are still problematic in that slowly
regenerating axons (~1 mm/d) may not reach downstream targets before
their irreversible atrophy or degeneration. This is in spite of the
fact that fasicular alignment and grafting can provide the enhanced
possibility for functional reconnection (Fawcett and Keynes
1990; Ketchum 1982
; Thomas 1988
).
It is also possible that PEG-mediated fusion of even transected axons
could become a component of microsurgical grafting techniques because the conventional resection of peripheral nerve trunks before fasicular grafting exposes the severed tips of proximal and distal axonal segments
making them available for fusion.
In the spinal cord, transection of white matter is rarely a clinical
occurrenceclinical spinal cord injury usually involves severe
compression/contusion of the spinal cord followed by centrally occurring hemorrhagic necrosis of gray and white matter. As discussed earlier, "secondary injury" leads to progressive loss of white matter due to a local deterioration of cell membranes at the site of
the lesion. In such a clinical scenario, PEG-mediated repair of crushed
white matter
as shown to be possible in this report
could take on
real clinical significance. With continued development of these
techniques for the clinic, one might reasonably expect more immediate
recovery of conduction (and variable function) as well as the rescue of
variable amounts of injured white matter. To these ends, we already
have moved this treatment approach to experimental tests in vivo using
both spinal cord and peripheral nerve injury models in adult guinea pigs.
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ACKNOWLEDGMENTS |
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We acknowledge the expert technical assistance of D. Bohnert, L. Moriarty, and J. Pryor during the conduct of these experiments and C. Cornell and J. Sanson for manuscript preparation.
Financial assistance was provided by the United States Army Medical Research and Materials Command (DAMD17-94-J-4242) and National Science Foundation Grant BES9631560 to R. B. Borgens.
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FOOTNOTES |
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Address reprint requests to R. B. Borgens.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 August 1998; accepted in final form 6 January 1999.
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REFERENCES |
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