Division of Neurosurgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Shi, Riyi,
Tomoko Asano,
Neil C. Vining, and
Andrew R. Blight.
Control of Membrane Sealing in Injured Mammalian Spinal Cord
Axons.
J. Neurophysiol. 84: 1763-1769, 2000.
The process of sealing of damaged axons was
examined in isolated strips of white matter from guinea pig spinal cord
by recording the "compound membrane potential," using a sucrose-gap
technique, and by examining uptake of horseradish peroxidase (HRP).
Following axonal transection, exponential recovery of membrane
potential occurred with a time constant of 20 ± 5 min, at 37°C,
and extracellular calcium activity
([Ca2+]o) of 2 mM. Most
axons excluded HRP by 30 min following transection. The rate of sealing
was reduced by lowering calcium and was effectively blocked at
[Ca2+]o 0.5 mM, under
which condition most axons continued to take up HRP for more than
1 h. Sealing at higher
[Ca2+]o was blocked by
calpain inhibitors (calpeptin and calpain inhibitor-1) indicating a
requirement for type II (mM) calpain in the sealing process. Following
compression injury, the amplitude of the maximal compound action
potential conducted through the injury site was reduced. The extent of
amplitude reduction was increased when the tract was superfused with
calcium-free Krebs' solution (Ca2+ replaced by
Mg2+). These results suggest that the fall in
[Ca2+]o seen following
injury in vivo is sufficient to prevent membrane sealing and may
paradoxically contribute to axonal dieback, retrograde cell death, and
"secondary" axonal disruption.
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INTRODUCTION |
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Influx of calcium ions is
recognized as an essential mediator of intracellular damage and cell
death in a range of pathological conditions, including mechanical
trauma (Schanne et al. 1979; Schlaepfer and Bunge
1973
). Repair of damage to nerve membranes has also been shown
to be a calcium-dependent process (Xie and Barrett 1991
;
Yawo and Kuno 1985
), but this observation has received less attention, and the potential conflict between these two phenomena in injury has not been examined. The possibility of opposing effects is
particularly significant in the context of CNS trauma, where extracellular calcium activity
([Ca2+]o) at the injury
site falls immediately by 1-2 orders of magnitude and can remain
depressed for hours (Stokes et al. 1983
; Young et
al. 1982
). This prolonged depression of calcium, which may be
common to a number of other pathological conditions, has been viewed as
potentially beneficial, by reducing the driving force for cellular
calcium influx. However, the sealing of severed and partially damaged
axons may be compromised by such conditions, which would affect the
positive interpretation of these ionic changes.
The present study was designed to examine the process of
membrane sealing in adult mammalian spinal cord axons isolated in vitro, and the dependence of sealing on extracellular calcium activity.
Sucrose gap recordings were used to monitor membrane potential changes
and to record axonal conduction in strips of white matter isolated from
guinea pig spinal cord, by techniques that have been described
previously (Shi and Blight 1996). Uptake of horseradish
peroxidase (HRP) by the injured axons correlated with
electrophysiological findings and provided additional information on
the distribution of axonal damage and the time course of resealing (to
large molecules) of the cut membrane. The response of isolated spinal
cord axons to blunt compression was also examined, using an
electrically controlled manipulator (Shi and Blight
1996
). These experiments aimed to determine the effects of
altered sealing on the recovery of action potential conduction in nerve
fiber tracts subjected to more clinically relevant injuries.
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METHODS |
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All experiments were performed in vitro on strips of spinal cord white matter, isolated from 64 adult, female, Hartley strain guinea pigs. Histological studies were performed on the same tissues that were used for physiological analysis.
Isolation of spinal cord
The technique for isolation of the cord was described previously
(Shi and Blight 1996). Briefly, guinea pigs were
anesthetized deeply with ketamine (80 mg/kg), xylazine (12 mg/kg), and
acepromazine (0.8 mg/kg) and were perfused through the heart with 500 ml oxygenated, cold Krebs' solution to remove blood and lower core
temperature. The vertebral column was excised, and the spinal cord was
removed, immersed in cold Krebs' solution, and immediately subdivided, first along the sagittal midline, and then each half of the cord was
cut radially, to produce ventral, lateral, and dorsal strips of white
matter. The composition of the Krebs' solution was as follows (in mM):
124 NaCl, 2 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2 CaCl2, 20 dextrose, 10 sodium ascorbate, and 26 NaHCO3,
equilibrated by bubbling with 95% O2-5%
CO2 to produce a pH of 7.2-7.4. In calcium-free
Krebs' solution, the calcium chloride was replaced by equimolar
magnesium chloride.
Recording chamber
The construction of the recording chamber is illustrated in Fig. 1. A strip of isolated spinal cord white matter, approximately 35 mm in length, was supported in the central compartment and continuously superfused with oxygenated Krebs' solution (~2 ml/min). The ends of the tissue were carried through the sucrose gap channels to side compartments filled with isotonic (120 mM) potassium chloride (Fig. 1A). The white matter strip was sealed on either side of the sucrose gap channels, using fragments of plastic cover-slip and a small amount of silicone grease to attach the cover slip to the walls of the channel and seal around the tissue. Isotonic sucrose solution was continuously run through the gap channels at a rate of 1 ml/min. The temperature of the chamber was maintained with a thermostatically controlled Peltier unit in the base (Cambion Instruments). The axons were stimulated and compound action potentials recorded at opposite ends of the strip of white matter by silver/silver chloride wire electrodes positioned within the side chambers and the central bath. The central bath was similarly connected to instrument ground.
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Stimuli, in the form of constant-current unipolar pulses of 0.1 ms duration, were controlled with a pulse generator connected through a WP Instruments isolation unit. Recordings were made using a bridge amplifier and Neurocorder (both from Neurodata Instruments) for digital data storage on videotape. Subsequent analysis was performed using custom Labview software (National Instruments) on a Macintosh Power PC computer.
Transection
To study the response of the nerve fibers to transection, the tissue strip was cut at the face of the recording sucrose gap, using micro-scissors. The scissors were also used to cut through the tissue at the center of the chamber, so that the isolated tract could be transferred to HRP solution at different times after injury for evaluation of sealing at the cut ends (Fig. 1B).
Compression
A flat, raised surface was provided at the center of the recording chamber, against which the isolated white matter strip could be compressed, using a rod attached to a motorized micromanipulator (Fig. 1C). The end of the rod provided a compression surface of 2.5 mm along the length of the tissue, with a transverse width of 7 mm, such that it was wider than the tissue, even under compression. The compression rod was positioned perpendicularly to the tissue and was brought to a point of contact with its surface. After baseline measurements of conduction were obtained, the rod was advanced by means of the manipulator motor at a speed of 24 µm/s. The compound action potential and the displacement of the rod were monitored during the compression, and the compression was stopped when the potential reached a set, target amplitude. The rod was then removed rapidly upward, to relieve pressure on the tissue, and the recovery of the compound potential was monitored.
HRP histochemistry
To examine disruption of axons, segments of white matter strips were transferred at different times after injury (the times and the number of tissue samples depending on the physiological recordings required) to oxygenated Krebs' solution containing 0.015% HRP (Sigma type VI). After incubation for 1 h at room temperature, the tissue was fixed by immersion in 2.5% glutaraldehyde in phosphate buffer. Transverse sections of the tissue were cut at 30 µm on a Vibratome and stained with diaminobenzidine reaction to reveal the extent of HRP uptake into damaged axons. Sections at approximately 1 mm from the plane of transection and at 5 mm distance were compared, to control for the presence of damaged fibers that might be unrelated to the deliberate injury. Sections were examined and photographed with a Nikon Optiphot microscope.
Initial experiments were performed to determine whether quantitative morphometry could be performed, using 1-µm plastic sections. The density of staining with HRP was insufficient to provide a clear distinction between lightly stained and unstained fibers in the semi-thin sections. The presence or absence of stain was relatively unequivocal in the thicker Vibratome sections, but these sections did not allow accurate quantitative analysis of axon numbers, particularly for the smaller diameter nerve fibers. Observations were therefore restricted to major shifts in axonal uptake of HRP under different conditions.
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RESULTS |
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Spinal cord strips placed in the recording chamber showed a period
of stabilization of the resting membrane potential, requiring 30-60
min. During this time, the "compound resting membrane potential" recorded across the sucrose gap became more negative, and the amplitude
of the maximal evoked compound action-potential increased. The form and
quantitative characteristics of the compound action potential and of
the "gap potential" or "compound resting membrane potential"
have been described previously (Shi and Blight 1996). The action potential corresponds closely to the action potential recorded in single large mammalian spinal cord axons using
intracellular microelectrodes (Blight and Someya 1985
).
The amplitude of the gap potential was determined to be 16 ± 3 (SD) mV, or approximately three times the amplitude of the peak of the
compound action potential. To provide a "normalized value" of gap
depolarization for different white matter strips, the measured gap
potential (in mV) at any given time following injury was related to the
initial peak of gap depolarization seen immediately following
transection (which was normalized to a value of 1). This served to
overcome the variability in measured absolute potential. The amplitude
of the potential recorded from a given tissue strip is arbitrary in
itself, with a value based on the particular configuration of the
tissue in the sucrose gap, and its passive electrical characteristics.
Response to transection
When the white matter strip was cut near the central face of the sucrose gap, the gap potential was reduced within seconds toward bath potential (0 mV) and then began to repolarize slowly (Fig. 2A). Full recovery of the initial resting potential required approximately 45-60 min at 37°C and 2 mM [Ca2+]o. The form of the recovery curve was approximately exponential, with a time constant of 20 min. At 25°C, the rate of recovery of resting potential was slower, with a time constant of approximately 40 min, but was still exponential to the same baseline (Fig. 2B).
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Staining cut axons wih HRP
HRP histochemistry showed a comparable rate of sealing of the axonal membrane to entry of the enzyme molecule. At 37°C and 2 mM [Ca2+]o, almost all axons were stained when transferred to HRP solution between 1 and 5 min after transection (Fig. 3A), but very few fibers were stained when transferred at 30 min or 1 h post transection (Fig. 3, B and C).
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Effects of low calcium activity
The rate of recovery of the gap potential decreased with
decreasing calcium concentration in the medium, from 2 to 0.5 mM (Fig.
4A). Further reduction in
calcium concentration had little effect on the rate of recovery, and,
at all concentrations 0.5 mM, there was no approach to the original
baseline polarization (Fig. 4B). This change in the rate of
recovery of compound membrane potential was reflected in the maintained
uptake of HRP. Most axons in spinal cord strips maintained in Krebs'
solutions with 0.5 mM
[Ca2+]o continued to take
up HRP for more than 1 h after transection (Fig. 3E).
Spinal cord strips maintained in Krebs' solution with 1 mM
[Ca2+]o showed an
intermediate rate of sealing to HRP, the number of axons staining at
1 h (Fig. 3D) being greater than the number at 30 min
in solutions with 2 mM
[Ca2+]o (Fig.
3B).
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Calpain inhibition
The dependence of sealing on relatively normal concentrations of calcium raised the possibility that the process of repair was dependent on millimolar or type II calpain. This possibility was examined by incubating spinal cord strips in Krebs' solution containing 2 mM Ca2+ and either calpeptin or calpain inhibitor-I. Both blockers of calpain activity produced a similar suppression of gap potential recovery (Fig. 5) and maintenance of HRP uptake (Fig. 3F) to those seen with calcium concentration below 0.5 mM (Figs. 3E and 4A).
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Response to compression
The loss and recovery of action potential conduction in the
isolated strips exposed to focal compression injury was similar to that
reported previously (Shi and Blight 1996). Briefly, the normal compound action potential recorded at one end of the strip in
response to electrical stimulation at the other was reduced in
amplitude by focal compression in the middle of the strip. On removal
of compression, there was a partial recovery of amplitude within 5-6
min, which then became stable for the succeeding hour of incubation.
The amplitude of this partial recovery was compared in two groups of spinal cord strips, one incubated throughout in 2 mM Ca2+ Krebs' solution, and the other in which the superfusion medium was changed to 0 mM Ca2+ Krebs' at the time of compression. The amplitude of the recovered compound action potential was significantly greater in the group of spinal cord tracts maintained in Krebs' solution with 2 mM [Ca2+]o (Fig. 6).
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In addition, experiments were performed, changing the perfusion of the injured spinal cord strip from calcium-free to 2 mM calcium Krebs' solution at 30 min following compression injury (Fig. 7A). Even after this delay, the amplitude of the compound potential was increased significantly within a few minutes of restoring extracellular calcium (Fig. 7B).
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DISCUSSION |
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Neurons are able to recover from mechanical injury, including loss
of dendritic or axonal processes, because they have the ability to seal
breaks in the plasmalemma, and do so surprisingly rapidly. This sealing
of cut processes and the consequences for cell survival have been
examined previously in large, isolated, invertebrate axons (Yawo
and Kuno 1985), in cultured vertebrate cells (Lucas et
al. 1985
; Shi et al. 1989
; Xie
and Barrett 1991
), and, more recently, in reptilian peripheral
axons (David et al. 1997
). While influx of sodium is
clearly implicated in the cytotoxic effects of mechanical damage to
cells (Rosenberg and Lucas 1996
), the process of sealing
itself is also clearly calcium dependent (Gallant 1988
;
Xie and Barrett 1991
) and appears to involve an essential role of calcium-activated phospholipase-A2 (Yawo and Kuno 1985
). The precise dependence of resealing on
calcium-mediated mechanisms has not been established, nor has the
phenomenon been examined in adult mammalian axons of the CNS, where
repair is of key clinical relevance. The potential significance of
these mechanisms is highlighted by the finding that extracellular
calcium activity in the CNS is profoundly depressed by mechanical
trauma (Stokes et al. 1983
; Young et al.
1982
). The present experiments were designed to use a dynamic
measure of sealing in mammalian spinal axons to determine whether the
extracellular calcium changes seen in spinal cord trauma might play a
role in neurological recovery, as has been suggested, based on
observations in vivo (Young 1992
).
Gap potential as a measure of sealing
It may appear counterintuitive to study injury mechanisms in isolated tissues of this kind because the tissue is injured several times, once by transection when the spinal cord is removed from the animal, a second time when the ventral white matter strip is cut from the length of isolated cord, and a third when the strip is finally cut or compressed. However, following the initial trauma of isolation, a minimum of 1 h at 20°C and 1 h in 37°C was given to allow the cord to recover, both in terms of metabolism and membrane sealing. Furthermore, the experimental injuries were made at least 10 mm (several length-constants) from the end of the strip, where the isolating transverse cuts were made. Thus the experimental injury was spatially as well as temporally separated from the injuries created during extraction.
A simple experiment confirmed that the injury of extraction was unlikely to affect the characteristics of sealing in the experimental transection. Spinal tracts were transected on one side of the chamber for an initial recording in 2 mM Ca2+, then transected again on the other side of the chamber an hour later (switching the recording amplifier to the opposite end of the chamber). The second injury resulted in recovery of compound resting membrane potential with the same characteristics as recovery from the first transection. Therefore it seems unlikely that the response to experimental injury in vitro is significantly conditioned by the injury involved in tissue isolation.
Nerve fibers in isolated spinal cord strips completely sealed to HRP
1 h after transection in Krebs' solution containing 2 mM
Ca2+. However, the axons were practically
completely accessible to HRP one hour following transection in
solutions containing 0.5 mM Ca2+ or a combination
of 2 mM Ca2+ and 30 µm calpain inhibitor-I. The
recovery of membrane integrity for the large molecule correlated well
with recovery to baseline of the measured "compound resting membrane
potential" (Leppanen and Stys 1997a,b
), supporting the
use of this potential as an indicator of the recovery of membrane integrity.
The compound resting potential would be expected to relate to
ionic permeability changes, and it may therefore provide a
measure of membrane permeability changes that are more subtle than the more extensive membrane disruption required for access of HRP. In
addition, the initial phase of potential recovery occurred consistently, even in the absence of sealing to HRP or of subsequent return to the original baseline resting membrane potential. The initial
phase of recovery of the gap potential therefore seems to be based not
on sealing of a proportion of the nerve fibers in the tract, but
perhaps on a decrease in the core conductance near the cut end of the
fibers. This would be consistent with the kind of constriction that has
been seen in morphological studies of giant axons (Krause et al.
1994) and that has been shown to be unaffected by replacement
of extracellular Ca2+ with
Mg2+ (Gallant 1988
).
HRP uptake as a measure of sealing
Changes in intra-axonal staining with HRP were interpreted as measuring axolemmal sealing (sufficient to exclude diffusion of the HRP molecule into the axon). It seems possible that uptake or transport of HRP along the axon could be affected by higher calcium; however, the control for this was to observe HRP transport in white matter exposed to high calcium and HRP immediately after transection. There was no profound reduction in HRP transport when the axons were exposed to HRP before membrane sealing could occur (Fig. 3A). There was also no reduction in the intensity of staining in individual axons exposed at intermediate timepoints, only an increase in the number of axons showing no staining at all (Fig. 3B).
Calcium dependence of sealing following transection
Resealing of axons in this preparation, recorded by either
electrophysiological or histological means, depends on extracellular calcium ion activity in the millimolar range. In solutions with calcium
activity below 0.5 mM, sealing does not take place within the duration
of the experiment, up to several hours. This observation, combined with
the blocking effect of calpain inhibitors on the resealing process,
indicates that the sealing of myelinated nerve fibers in the mammalian
CNS depends on the activity of millimolar calpain. This is consistent
with the selective presence of this form of the enzyme within axons of
the CNS (Hamakubo et al. 1986). The mechanism of the
role of millimolar calpain is not clear in this context, or in axon
sealing in other contexts. It may be that some degree of
calpain-dependent rapid axoskeletal degradation is necessary to begin
the axolemmal membrane sealing process. It could be imagined that this
might involve the need to free the axonal membrane from the rigidity of
the axoskeleton to allow sufficient spatial reorganization to bring the
cut ends together. The present study did not examine the role of
phospholipase-A2 in the sealing process (Yawo and Kuno
1985
). Unfortunately, the available inhibitors of
phospholipase-A2 are themselves toxic to normal axons (Xie and
Barrett 1991
).
Perhaps most importantly, from the aspect of understanding the pathology of human CNS injury, the recovery of action potential conduction in white matter tracts that have been injured by blunt compression is also calcium dependent in this range. Conduction block in some axons injured in low extracellular calcium concentrations appears to be restored when normal calcium activity is returned. This indicates that resealing of critically damaged, but nontransected axons may play a significant role in recovery from blunt contusion injury in the CNS. It seems likely that the membrane damage produced by stretch or compression injury may simply represent on a smaller and more distributed scale the same kind of membrane disruption represented by the more dramatic case of axonal transection.
Membrane sealing is required to halt the progressive cycle of
depolarization, calcium and sodium entry, consequent metabolic disruption, and cytoskeletal disassembly that produces "dieback" of
cut axons. An important additional role is the repair of more subtle
membrane damage, which may produce an increase in nonspecific membrane
permeability without immediately breaking axonal continuity. Such
critical changes in membrane permeability, induced by stretch or blunt
impact, may be responsible for the kind of progressive, axonal
breakdown that has been described in "diffuse axonal injury" following brain trauma (e.g., Buki et al. 2000;
Povlishock 1992
). Similar processes may occur in the
injured spinal cord, given the similarity of the mechanical disruption,
although no studies have addressed the specific presence of this kind
of pathology either in human studies or animal models. Such
"occult" injury may be accessible to therapeutic interventions
aimed at accelerating membrane sealing and halting the ionic disruption
that accompanies the loss of membrane integrity at the site of injury.
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ACKNOWLEDGMENTS |
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We thank T. M. Kelly, J. Gadzia, and B. Houston for invaluable assistance.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-33687, a grant from the Canadian Spinal Research Organization, and a fellowship from the Ishikawa Prefecture to T. Asano.
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FOOTNOTES |
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Present address and address for reprint requests: R. Shi, Dept. of Basic Medical Sciences/CPR, Purdue University, West Lafayette, IN 47907-1244 (E-mail: riyi{at}vet.purdue.edu).
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 23 August 1999; accepted in final form 13 June 2000.
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REFERENCES |
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