Behavioral recovery from spinal cord injury following delayed application of polyethylene glycol
Center for Paralysis Research, Institute for Applied Neurology, Department of Basic Medical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1244, USA
*e-mail: cpr{at}vet.purdue.edu
Accepted 16 October 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: spinal injury, neurotrauma, spinal cord, nerve injury, central nervous system injury, fusogen, polyethylene glycol, guinea pig.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During the acute phase, a delayed, progressive and self-propagating wave of cell and tissue destruction is initiated, with the injury leading to cell death, the formation of a cicatrix and the irreversible loss of distal segments of axons through Wallerian degeneration (Griffin et al., 1995). This latter dynamic is often referred to as secondary injury (Honmou and Young, 1995
), and it is the loss of white matter, which cannot be replaced, that frames the more permanent behavioral loss accompanying the long-term, or chronic, phase of the injury.
Techniques aimed at regenerating new and functional connections by promoting nerve regeneration from intact proximal segments show clinical promise in cases of SCI. Inhibition of endogenous inhibitors of central nervous system (CNS) regeneration, application of novel growth factors to the lesioned spinal cord, neurotransplantation of both fetal nervous tissue and activated peripheral nervous system macrophages into the spinal lesion and the application of direct current electrical fields are all thought to facilitate regrowth of spinal cord white matter and/or to facilitate new functional synaptic connections (Schwab et al., 1993; Bregman et al., 1996
; Benowitz et al., 1999
; Lazarov-Spiegler et al., 1996
; Borgens et al., 1999
). The last three techniques mentioned have now moved into human clinical study in cases of severe SCI. Another approach, which is aimed at recovering functional deficits irrespective of the time since the original spinal injury, is to restore physiological conduction through intact but non-functional white matter by K+ channel blockade. This technique is also at the stage of human clinical testing (Shi and Blight, 1997
; Hansebout et al., 1993
; Hayes et al., 1993
).
It is a matter of debate whether greater success in reducing long-term injury has been achieved in recent years by attacking the initial phase of the insult. One such example, adopted as a means of standard management of acute spinal cord injury, has been the administration of large doses of the steroid methylprednisilone within hours of the injury (Bracken et al., 1990). This approach, often referred to as neuroprotection, claims to ameliorate behavioral loss by reducing the extent of secondary injury, although the efficacy and safety of this therapy are now under question (Short et al., 2000
; Pointillart et al., 1999
).
We suggest another approach to treating the acute injury. In this method, the application of a hydrophilic surfactant physically repairs damaged membranes, leading to a rapid (minutes to hours) recovery of cellular structure and function. This treatment is designed to permit immediate recovery of nerve impulse conduction in injured fibers, to reverse the permeabilization of the plasmalemma and immediately to seal breaches in it that would probably progress to dissolution and axotomy.
We have reported that a brief administration of the fusogen polyethylene glycol (PEG) to completely transected, but reapposed, adult guinea pig spinal axons can induce anatomical reconnection of the severed proximal and distal segments and immediate recoveries of compound action potential conduction through fused axons (Shi et al., 1999). We have also shown that a similar application of PEG to severe compression injuries also leads to an immediate recovery of compound action potential conduction through the lesion (Shi and Borgens, 1999
). These procedures were repeated in vivo in experiments in which an aqueous solution of PEG was applied for 2 min to a standardized compression injury to adult guinea pig thoracic spinal cord. A swift recovery of both spinal cord conduction, measured by somatosensory evoked potentials (SSEPs), and behavioral function, measured by the recovery of the cutaneus trunci muscle (CTM) reflex (Borgens et al., 1987
; Blight et al., 1990
), occurred in PEG-treated animals (Borgens and Shi, 2000
).
Here, for the first time, we evaluate fully the behavioral character of the recovered CTM reflex produced by a delayed application of PEG and confirm our observations of the physiological recovery of conduction in 100 % of these spinally injured animals.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Application of PEG
An aqueous solution of PEG (Mr 1800, 50 % w/v in distilled water) was applied with a Pasteur pipette directly onto the exposed injury (dura removed) for 2 min and then removed by aspiration. This procedure was performed approximately 7 h after the lesioning procedure (see above). The region was immediately washed with isotonic Krebs solution (NaCl, 124 mmol l1; KCl, 2 mmol l1; KH2PO4, 1.24 mmol l1; MgSO4, 1.3 mmol l1; CaCl2, 1.2 mmol l1; dextrose, 10 mmol l1; NaHCO3, 26 mmol l1; sodium ascorbate, 10 mmol l1), which was also aspirated to remove excess PEG. In sham-treated animals, the injury site was re-exposed surgically at approximately 7 h post-lesion, a control application of water (vehicle) was applied for 2 min, followed by a lavage with Krebs solution subsequently removed by aspiration. The wounds were closed, and the animals were kept warm with heat lamps until awaking. Guinea pigs were housed individually and fed ad libitum.
Behavioral analysis
The CTM reflex (Fig. 1) is observed as a rippling of the skin on the back of the animal following light tactile stimulation. These contractions can be measured by tattooing a matrix of dots on the animals shaved back. When the skin contracts towards the point of tactile stimulation, the dots move in this direction. Practically, a receptive field is determined prior to injury by stimulating the back skin of the sedated animal by lightly touching it with a monofilament probe. When skin outside the receptive field is stimulated, there is no response. By exploring the entire back with the probe, the boundary of the receptive field can be established. This boundary is drawn directly onto the shaved back of the guinea pig using a marker (Fig. 2). Stimulation inside the boundary produces CTM contractions, but stimulation outside it does not. The entire procedure was videotaped by a camera mounted approximately 0.92 m above the examination table. The same procedure was used to determine the region of CTM loss (areflexia) and subsequent recovery, when it occurred. These behavioral tests (and the physiological test described below) were performed approximately 24 h, 3 days, 2 weeks and 1 month post-treatment.
|
|
The overall pattern of skin movement can be quite complex in response to focal stimulation. Thus, we chose to restrict our quantitative evaluation of back skin movement to the peak contractions in response to stimulation, i.e. to the region of skin where markers were displaced by the greatest distance. When the region of peak skin contraction had been determined, the videotape was reversed to a time just prior to stimulation and skin movement. The videotape was then advanced at intervals of 1/24th of a second so that a time point prior to, and just at the peak of skin contraction, could be captured to the computer. These frames were superimposed over images of the animals, and the distance of peak contraction was divided by the time required to produce it. This provided a measure of the velocity of skin contraction (Fig. 2). The character of skin movement following tactile stimulation was determined at the pre-injury evaluation for all but four animals, and for all animals at 1 day, 3 days, 2 weeks and 1 month post-treatment. When the peak contraction was determined for any one animal, a protractor was used to measure the angle at which the skin was pulled towards the monofilament probe relative to an imaginary line perpendicular to the long axis of the animal at the midline. The peak contraction of the skin was recorded as a positive angle when the skin pulled towards the probe and as a negative angle in the infrequent cases in which the skin pulled away from the probe. We also recorded whether this peak response occurred on the same side of the midline as the point of stimulation or on the other side (a contralateral response).
Physiological recordings of somatosensory evoked potentials
Subdermal electrodes stimulated nerve impulses from the tibial nerve of the hindleg (stimuli trains in sets of 200 at 3 Hz; stimulus amplitude 3 mA square wave, 200 µs in duration) (Fig. 3). Evoked potentials, more properly termed somatosensory evoked potentials (SSEPs), were conducted through the spinal cord to the sensory cortex of the brain. To record SSEPs, a pair of subdermal electrodes, located above the level of the contralateral cortex, was used with a reference electrode usually located in the ipsilateral pinna of the ear. The stimulation and computer management of evoked potential recordings utilized a Nihon Kohden Neuropak 4 stimulator/recorder and PowerMac G3 computer. In all animals, failure to record an SSEP was confirmed to be due to the absence of evoked potential conduction through the lesion by a control test carried out at the same time. The medial nerve of the forelimb was stimulated, initiating evoked potentials in a neural circuit above the level of the crush injury. To perform this test, recording electrodes were left in place while stimulating electrodes were relocated to stimulate the median nerve of the foreleg.
|
Statistical analyses
The MannWhitney two-tailed test was used to compare the means of the data derived from experimental and sham-treated groups. To compare the proportions between groups, Fishers exact test was used. All tests were performed using Instat software.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As will be discussed below, since none of the control animals recovered an SSEP, 13 of the 15 experimental animals with the best electrical recordings were evaluated quantitatively. Similarly, a full evaluation of CTM functioning by stop-frame videographic analysis was carried out on the three controls that recovered the reflex and on 13 of the 15 recovered PEG-treated animals for comparison.
The cutaneus trunci muscle reflex
Application of PEG produced a very rapid recovery of CTM function in 73 % of treated animals within the first 24 h compared with a complete lack of spontaneous recovery in sham-treated animals at this time (Fig. 4). Some spontaneous recovery of the CTM reflex in controls began to appear on day 3, resulting in three recoveries out of a total of 13 animals (23 %) by 1 month (Table 1). In marked contrast, 11 of 15 PEG-treated animals recovered the reflex activity within the region of areflexia during the first day post-treatment (Table 1) (Fig. 4), and another three animals by 1 month (total number of animals showing 93 %; P0.0003; Fishers exact test, two-tailed) (Fig. 4). The area of recovered areflexic back skin was 27.6±8.6 % in the 15 PEG-treated animals and 18.3±3.4 % (means ± S.E.M.) in the three controls. Thus, the total area of PEG-mediated recovery was not statistically different from that occurring spontaneously, although infrequently (P=0.28; MannWhitney, two-tailed test).
|
|
|
In the three spontaneous recoveries in control animals, the peak distance of contraction (1 mm) and its velocity (25 mm s1) were identical in two of them and the reflex was unchanged after recovery. In the third case, a reduction in the velocity of CTM contraction was measured, while the angle of contraction and the peak distance of contraction remained unchanged (Table 2).
Recovery of conduction through the injured spinal cord
As in previous studies, SSEPs usually segregated into two peaks following tibial stimulation in the uninjured animal: an early-arriving peak (approximately 2035 ms) and a late-arriving peak (4050 ms). Table 1 shows the proportion of animals recovering an SSEP in the experimental population, which was 87 % for the first day after PEG treatment and by week 4 had reached 100 %. Not one sham-treated animal recovered conduction over the same period (Table 1). Fig. 5A shows a typical example of an SSEP recorded from a sham-treated animal, as well as a median nerve control procedure. Such control procedures were undertaken for any measurement that failed to demonstrate a repeatable SSEP and, in every case, demonstrated that the absence of evoked potentials was due to a failure to conduct them through the lesion. In Fig. 5B, a typical process of PEG-mediated recovery is shown. Note that the latency of the recovering evoked potentials was greater than normal in the early stages of recovery, but gradually declined with time (depicted for the early-arriving peaks). Fig. 6 shows that normal latency was not reached during the 1 month of observation and also plots the magnitude of the early-arriving evoked potentials, which recover to more than 50 % of their pre-injury values.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The spinal injury
The means of injury we chose was a constant-displacement injury in which each spinal cord received a severe lateral compression for approximately the same distance and for the same duration. This is a different approach from previous studies of impact injuries (usually dropping a weight, usually 20 g from 20 cm, onto the dorsal aspect of the exposed spinal cord). These different approaches have been discussed and described elsewhere (Blight, 1988, 1991
; Collins and Kauer, 1979
). We looked for a method that would produce a repeatable injury among animals, at least with respect to the anatomical consequences of the technique. In our hands, the constant-displacement injury method produces lesions that are not statistically different among animals, as determined by three-dimensional computer reconstruction of the vertebral segment containing the injury (Moriarty et al., 1998
; Duerstock et al., 2000
). The resulting hemorrhagic lesion is fairly typical of clinical injuries in that it spreads in severity from the center of the spinal cord (destroying most gray matter) to the margins, leaving a subpial rim of spared white matter over a longitudinal distance of approximately one vertebral segment (Moriarty et al., 1998
) (see also Duerstock and Borgens, 2002
). Moreover, the technique does not require special laboratory equipment, such as stereotaxic frames and impactors, and is simple to carry out. Nevertheless, all attempts at standardizing an injury to the spinal cord are still associated with some variation in functional recovery among animals. This is believed to be due to biomechanical differences between the cords, to the variable level of endogenous sealing of mechanically damaged axons (Krause et al., 1994
; Shi et al., 2000
) and to other factors that cannot be controlled (Blight, 1991
). This is evident in this report from the modest number of control animals showing CTM recovery. A more precise anatomical and behavioral outcome can be obtained by specific transection techniques; however, severing tracts, or the spinal cord itself, is generally not a clinically meaningful injury model (Borgens, 1992
).
Other conventional experimental methods have also been vitiated, or found to be unnecessary, by the extremely rapid recovery produced by PEG in prior published experiments and unpublished pilot trials. Thus, blinding of evaluators to the treatment used was not performed in this study since, within a matter of a hours, the experimentally treated animals could be identified with greater than 90 % accuracy in CTM studies or with 100 % accuracy when evaluating SSEP recovery. The most important precaution in these series of experiments was the determination that the measures of damage are statistically similar between animals after injury and before PEG treatment.
The CTM reflex as a functional indicator of spinal cord behavioral recovery
The skin movement is dependent on sensory afferents projecting as a long tract of axons in each ventral funiculus of the spinal cord (just lateral to the spinothalamic tract) to nuclei of CTM motor neurons located at the cervical/thoracic junction left and right of the midline (Blight et al., 1990; Thierault and Diamond, 1998
) (Fig. 1). There are some contralateral afferent projections within the cord, but these appear to play only a very marginal role in the normal expression of the reflex. This mainly local response to stimulation is further represented by the fact that there are no contralateral projections between the nuclei of CTM motor neurons (Blight et al., 1990
). The reflex is bilaterally organized into segmentally arranged receptive fields, displays little supraspinal control and is lost following spinal injury, producing a region of areflexia below the level of the lesion (Borgens et al., 1990
; Blight et al., 1990
; Thierault and Diamond, 1988
) (Fig. 1). Following transection, recovery of the CTM reflex within this region of areflexia is not usually observed for the rest of the life of the animal and is infrequent (
20 %) following severe crush lesions to guinea pig spinal cord (Blight et al., 1990
; Borgens and Shi, 2000
; Borgens and Bohnert, 2001
).
Although these injuries to the spinal cord also produce locomotor deficits in the animals, we have ignored these deficits and their apparent changes over time. This is because standing, stepping and overall locomotion in small animal models of SCI are locally controlled and generated within the spinal cord and so have little dependence on supraspinal control and organization (for a review, see Borgens, 1992). We also note that the recovery of the CTM and the recovery of SSEP conduction should be considered as independent outcomes. The long tracts associated with the CTM are bilateral and located in the ventrolateral funiculus, while SSEP conduction (initiated by stimulation of the tibial nerve) is more a measure of dorsal column activity (see below).
Behavioral recovery
We have not previously evaluated the character of the CTM behavioral recovery by comparing its characteristics with those of the pre-injury reflex. Here, we have shown that the recovered reflex activity is statistically similar in terms of the direction, distance and velocity of CTM contractions when compared with the normal reflex. The entire receptive field lost after injury was not restored, however. The largest area of back showing recovery after PEG treatment approached 50 % of the original area of areflexia. Given that we evaluated only one specific sensorimotor reflex, which is dependent on an identified white matter tract, this amount of appropriately organized recovery was surprising. The recovery of the receptive fields on the back was also variable in location and was sometimes revealed as islands of CTM-receptive flank that stood out within a still-areflexic region of back skin. This variability in the way in which recovery was re-established is believed to result from the variable re-recruitment of afferent axons into conduction through the lesion (see also Blight et al., 1990; Borgens et al., 1987
, 1990
). In summary, direct application of this hydrophilic polymer to the site of a spinal cord injury can rapidly reverse behavioral loss, restoring an appropriately organized behavioral response as well as nerve impulse conduction through the lesion within a clinically useful time frame.
Physiological and anatomical recovery
The most striking effect of PEG application in all SCI studies in this series, in vitro and in vivo, has been the uniform recovery of conduction through the lesion in response both to transection (and reattachment) and to compression injury (Shi and Borgens, 1999; Shi et al., 1999
; Borgens and Shi, 2000
; Shi and Borgens, 2000
; Borgens and Bohnert, 2001
). This recovery of conduction, documented by the recovery of either compound action potential in vitro or of SSEP conduction in vivo, occurred in 100 % of the spinal cords treated with PEG. It is very clear that the fusogenic action of PEG (Nakajima and Ikada, 1994
; Davidson et al., 1976
) alters the dynamics of axolemma injury to permit a very rapid re-establishment of excitability in the region of conduction block. The magnitude of recovered compound action potentials in spinal cord strips in isolation can be nearly doubled by the addition of the fast K+ channel blocker 4-aminopyridine, suggesting that the repaired region of membrane is still somewhat leaky to K+ and not yet a perfect seal (Shi and Borgens, 1999
).
We have also shown that the PEG-restored membrane is repaired sufficiently to exclude the uptake of a large-molecular-mass intracellular tracer, horseradish peroxidase (HRP) (Shi and Borgens, 2000). This label is normally taken up into breaches of the nerve membrane, a common method of loading neurons or their processes with this intracellular dye (Borgens et al., 1986
; Malmgren and Olsson, 1977
). Compression-injured spinal cords showed a dramatic uptake of HRP into white matter axons at the epicenter of the injury when evaluated only 15 min post-injury. This uptake was greatly reduced by PEG treatment, and the effect was independent of axon diameter. Thus, the dye-exclusion test (Asano et al., 1995
) proved that the polymeric seal is indeed just that and is sufficient to interfere with the ability of even large-molecular-mass compounds to cross the membrane after damage (Shi and Borgens, 2001
). Physical reconnection of axons within completely severed strips of guinea pig spinal cord ventral white matter also demonstrated the capability of a brief PEG treatment to reunite membranes sufficiently to seal in small-molecular-mass fluorescent intracellular markers (rhodamine- and fluorescein-decorated dextrans; Mr 8000) (Shi et al., 1999
).
Finally, the companion paper (Duerstock and Borgens, 2002) demonstrates that this anatomical sealing of single cells can also be represented at the level of the whole tissue. The histopathological character of the in vivo spinal lesion is also reduced by PEG treatment.
The mechanisms of PEG-mediated repair
This report is one of a series that has explored the ability of a cell fusogen, PEG, to reconnect severed mammalian spinal cord axons and to seal the axolemma of severely compressed/crushed spinal axons. We have previously discussed what are believed to be the mechanisms of action of PEG, in particular, as well as the mechanisms that may be shared with non-ionic triblock polymers such as the poloxamines and poloxamers discussed below (for a review, see Borgens, 2001) (see also Borgens and Shi, 2000
; Lee and Lentz, 1997
; Lentz, 1994
; Marks et al., 2001
). Briefly, sealing of membrane breaches by high-molecular-mass molecules such as PEG may involve a dehydration of the plasmalemma where closely apposed regions of the bilayer resolve into each other, i.e. the structural components of the plasmalemma are no longer partitioned by the polar forces associated with the aqueous phase. The lipid core of the membrane, exposed across a breach in it, would be expected to flow together in the absence of an aqueous barrier (following dehydration of the membrane). Subsequent to the removal of the polymer and rehydration, the now-continuous phase undergoes spontaneous reassembly of its structural elements, including protein components and complex lipid components. This reorganization of cellular water is believed to result from the strongly hydrophilic structure of PEG.
Poloxamers are complex polymers of a PEGpropylene glycolPEG structure. They too can seal membrane breaches (see below), but perhaps through a slightly different mechanism. It is proposed the hydrophobic head group inserts itself into the membrane breach, sealing it by plugging it (Marks et al., 2001). We have preliminary evidence that Poloxamer 188 can also induce axolemma sealing and induce recovery from adult guinea pig SCI in a manner similar to PEG (D. Bohnert and R. B. Borgens, unpublished observations). It is also possible that these non-ionic detergents, like PEG, may seal membrane breaches initially as a surfactant film, although this does not explain the permanent nature of polymer-mediated seals, even after the polymers have been removed.
In all studies, we have used an application of an aqueous solution of PEG (50 % w/v in distilled water) for 2 min. We have detected no difference in response using PEG solutions prepared with polymers with a relative molecular mass of 400 to approximately 3000 (D. Bohnert and R. B. Borgens, unpublished observations), but believe that the viscosity of the solution may be more important to PEG-mediated repair than the molecular mass of the polymer. Using fluorescently decorated PEG, we have traced the distribution of PEG after various routes of administration in guinea spinal cord injury. PEG clearly targets the hemorragic lesion and is only faintly detected in uninjured spinal cord following a direct application to the injury site (Borgens and Bohnert, 2001). More importantly, subcutaneous or intravenous application labels the lesion just as well. This may be more important to an eventual clinical use where intravascular injection of PEG may be beneficial during emergency care and later applied directly to the lesion during surgical management of the injury. At present, we are testing the application of hydrophilic polymers administered both topically and through the blood supply in veterinary cases of severe, acute paraplegia in dogs secondary to disc herniation and fracture dislocation (see also Blight et al., 1991
; Borgens et al., 1999
).
Membrane repair in other types of injury
As mentioned above, non-ionic detergents, so-called triblock polymers, are mainly composed of PEG and may share mechanisms of action in reversing cell permeabilization. Their structure usually incorporates a high-molecular-mass central hydrophobic core with hydrophilic PEG side chains. Poloxamer 188 (P188) has been shown to reverse muscle cell death subsequent to high-voltage insult (Lee et al., 1992). Isolated rat skeletal muscle cells were labeled with an intracellular fluorescent dye that leaked out of the cells after high-voltage trauma. This insult was sufficient to disrupt muscle membranes, allowing the leakage of the marker in 100 % of the control preparations. Treatment of skeletal muscle cells in vitro with P188 reduced or even eliminated leakage of dye following the injury. Further in vivo tests extended these results: an intravenous injection of P188 produced physiological and anatomical recovery of rat muscle following electric shock (Lee et al., 1992
). This approach has also been tested for its ability to reverse cell death in a testicular reperfusion injury model in rats (Palmer et al., 1998
). P188 can also seal heat-shocked muscle cells in vitro, as shown by an inhibition of calcein dye leakage from cells induced by elevated temperature (Padanlam et al., 1994
). P188 rescues fibroblasts from lethal heat shock (Merchant et al., 1998
). Another biocompatible detergent (Poloxamer 1107; administered intravenously) was used in an in vivo testicular ischemia/reperfusion injury model in rats as well as for inhibiting the leakage of hemoglobin from irradiated erythrocytes (Palmer et al., 1998
; Hannig et al., 1999
). These studies demonstrate that non-ionic biocompatible detergents and large hydrophilic molecules can reverse the permeabilization of cell membranes and also that they can be administered through the vascular system to reach damaged target cells.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asano, T., Shi, R. and Blight, A. R. (1995). Horseradish peroxidase used to examine the distribution of axonal damage in spinal cord compression injury in vitro. J. Neurotrauma 12, 993.
Benowitz, L. J., Goldberg, D. E., Madsen, J. R., Soni, D. and Irwin, N. (1999). Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury. Proc. Natl. Acad. Sci. USA 96, 1348613490.
Blight, A. R. (1988). Mechanical factors in experimental spinal cord injury. J. Am. Paraplegia Soc. 11, 2634.[Medline]
Blight, A. R. (1991). Morphometric analysis of a model of spinal cord injury in guinea pigs, with behavioral evidence of delayed secondary pathology. J. Neurol. Sci. 103, 156171.[Medline]
Blight, A. R., Toombs, J. P., Bauer, M. S. and Widmer, W. R. (1991). The effects of 4-aminopyridine on neurological deficits in chronic cases of traumatic spinal cord injury in dogs: a phase I clinical trial. J. Neurotrauma 8, 103119.[Medline]
Blight, A. R., McGinnis, M. E. and Borgens, R. B. (1990). Cutaneus trunci muscle reflex of the guinea pig. J. Comp. Neurol. 296, 614633.[Medline]
Borgens, R. B. (1992). Applied voltages in spinal cord reconstruction: History, strategies and behavioral models. In Spinal Cord Dysfunction, vol. 3 (ed. L. S. Illis), pp. 110145. Oxford: Oxford Medical Publications.
Borgens, R. B. (2001). Cellular engineering: Molecular repair of membranes to rescue cells of the damaged nervous system. J. Neurosurg. (in press).
Borgens, R. B., Blight, A. R. and McGinnis, M. E. (1990). Functional recovery after spinal cord hemisection in guinea pigs: The effects of applied electric fields. J. Comp. Neurol. 296, 634653.[Medline]
Borgens, R. B., Blight, A. R. and Murphey, D. J. (1986). Axonal regeneration in spinal cord injury: A perspective and new technique. J. Comp. Neurol. 250, 168180.[Medline]
Borgens, R. B. and Bohnert, D. (2001). Rapid recovery from spinal cord injury following subcutaneously administered polyethylene glycol. J. Neurosci. Res. (in press).
Borgens, R. B., Blight, A. R. and McGinnis, M. E. (1987). Behavioral recovery induced by applied electric fields after spinal cord hemisection in guinea pig. Science 238, 366369.[Medline]
Borgens, R. B. and Shi, R. (2000). Immediate recovery from spinal cord injury through molecular repair of nerve membranes with polyethylene glycol. FASEB J. 14, 2735.
Borgens, R. B., Toombs, J. P., Breur, G., Widmer, W. R., Water, D., Harbath, A. M., March, P. and Adams, L. G. (1999). An imposed oscillating electrical field improves the recovery of function in neurologically complete paraplegic dogs. J. Neurotrauma 16, 639657.[Medline]
Bracken, M. B., Shepard, M. J., Collins, W. F., Holford, T. R., Young, W., Baskin, D. S., Eisenberg, H. M., Flamm, E., Leo-Summers, L. and Maroon, J. (1990). A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. New Engl. J. Med. 322, 14051461.[Abstract]
Bregman, B. S., Dai, H. N., Lin, T., James, C. V., Schwab, M. E. and Schnell, L. (1996). Transplants, neurotrophic factors and myelin-associated neurite growth inhibitors: effects on recovery of locomotor function after spinal cord injury in rats. Soc. Neurosci. Abstr. 22, 764.
Collins, W. F. and Kauer, J. S. (1979). The past and future of animals models used for spinal cord trauma. In Neural Trauma (ed. J. J. Popp, R. S. Bourke, L. R. Nelson and H. K. Kimelberg), pp. 273279. New York: Raven Press.
Davidson, R. L., OMalley, K. and Wheeler, T. B. (1976). Induction of mammalian somatic cell hybridization by polyethylene glycol. Somat. Cell Genet. 2, 271280.[Medline]
Duerstock, B. S., Bajaj, C. L., Pascucci, V., Schikore, D., Lin, K.-N. and Borgens, R. B. (2000). Advances in three-dimensional reconstructions of the experimental spinal cord injury. Comput. Med. Im. Grap. 24, 389406.
Duerstock, B. S. and Borgens, R. B. (2002). Three-dimensional morphometry of spinal cord injury following polyethylene glycol treatment. J. Exp. Biol. 205, 1324.
Griffin, J. W., George, E. B., Hsieh, S. T. T. and Glass, J. D. (1995). Axonal degeneration and disorders of the axonal cytoskeleton. In The Axon (ed. S. G. Waxman, J. Kocsis and P. Stys), pp. 375390. New York: Oxford University Press.
Hannig, J., Yu, J., Beckett, M., Weichselbaum, R. and Lee, R. C. (1999). Poloxamine 1107 sealing of radiopermeabilized erythrocyte membranes. Int. J. Radiat. Biol. 75, 379385.[Medline]
Hansebout, R. R., Blight, A. R., Fawcett, S. and Reddy, K. (1993). 4-Aminopyridine in chronic spinal cord injury: A controlled, double-blind, crossover study in eight patients. J. Neurotrauma 10, 118.[Medline]
Hayes, K. C., Blight, A. R. and Potter, P. J. (1993). Preclinical trial of 4-aminopyridine in patients with chronic spinal cord injury. Paraplegia 31, 216224.[Medline]
Honmou, O. and Young, W. (1995). Traumatic injury to the spinal axons. In The Axon (ed. S. G. Waxman, J. D. Kocsis and P. K. Stys), pp. 480503. New York: Oxford University Press.
Krause, T. L., Fishman, H. M., Ballinger, M. L. and Bittner, G. D. (1994). Extent and mechanism of sealing in transected giant axons of squid and earthworms. J. Neurosci. 14, 66386651.[Abstract]
Lazarov-Spiegler, O., Solomon, A. S., Zeev-Brann, A. B., Hirschberg, D. L., Lavie, W. and Schwartz, M. (1996). Transplantation of activated macrophages overcomes central nervous system regrowth failure. FASEB J. 10, 12961302.
Lee, J. and Lentz, B. R. (1997). Evolution of lipid structures during model membrane fusion and the relation of this process to cell membrane fusion. Biochemistry 36, 62516259.[Medline]
Lee, R., River, L. P., Pan, F. S. and Wollmann, L. (1992). Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo. Proc. Natl. Acad. Sci. USA 89, 45244528.[Abstract]
Lentz, B. R. (1994). Induced membrane fusion; potential mechanism and relation to cell fusion events. Chem. Physiol. Lipids 73, 91106.[Medline]
Malmgren, L. and Olsson, L. (1977). A sensitive histochemical method for light and electron microscopic demonstration of horseradish peroxidase. J. Histochem. Cytochem. 25, 12801283.[Medline]
Marks, J. M., Pan, C.-Y., Bushell, T., Cromie, W. and Lee, R. C. (2001). Amphiphilic, tri-block copolymers provide potent, membrane-targeted neuroprotection. FASEB J. (in press).
Merchant, F. A., Holmes, H. A., Capelli-Schellpfeffer, M., Lee, R. C. and Toner, M. (1988). Poloxamer 188 enhances functional recovery of lethally heat-shocked fibroblasts. J. Surg. Res. 74, 131140.
Moriarty, L. J., Duerstock, B. S., Bajaj, C. L., Lin, K. and Borgens, R. B. (1998). Two- and three-dimensional computer graphic evaluation of the subacute spinal cord injury. J. Neurol. Sci. 155, 121137.[Medline]
Nakajima, N. and Ikada, Y. (1994). Fusogenic activity of various water-soluble polymers. J. Biomater. Sci. Polym. Ed. 6, 751759.[Medline]
Padanlam, J. T., Bischof, J. C., Cravalho, E. G., Tompkins, R. G., Yarmush, M. L. and Toner, M. (1994). Effectiveness of poloxamer 188 in arresting calcein leakage from thermally damaged isolated skeletal muscle cells. Ann. N.Y. Acad. Sci. 92, 111123.
Palmer, J. S., Cromie, W. L. and Lee, R. C. (1998). Surfactant administration reduces testicular ischemiareperfusion injury. J. Urol. 159, 21362139.[Medline]
Pointillart, V., Petitjean, M. E., Wiart, L., Vital, J. M., Lassie, P., Thicoipe, M. and Dabadie, P. (2000). Pharmacological therapy of spinal cord injury during the acute phase. Spinal Cord 38, 7176.[Medline]
Schwab, M. E., Kapfhammer, J. P. and Bandtlow, C. E. (1993). Inhibitors of neurite growth. Annu. Rev. Neurosci. 16, 565595.[Medline]
Shi, R., Asano, T., Wining, N. C. and Blight, A. R. (2000). Control of membrane sealing in injured mammalian spinal cord axons. J. Neurophysiol. 84, 17631769.
Shi, R. and Blight, A. R. (1997). The differential effects of low and high concentrations of 4-aminopyridine on axonal conduction in normal and injured spinal cords. Neuroscience 77, 553562.[Medline]
Shi, R. and Borgens, R. B. (1999). Acute repair of crushed guinea pig spinal cord by polyethylene glycol. J. Neurophysiol. 81, 24062414.
Shi, R. and Borgens, R. B. (2000). Anatomic repair of nerve membrane in crushed mammalian spinal cord with polyethylene glycol. J. Neurocytol. 29, 633644.[Medline]
Shi, R., Borgens, R. B. and Blight, A. R. (1999). Functional reconnection of severed mammalian spinal cord axons with polyethylene glycol. J. Neurotrauma 16, 727738.[Medline]
Short, D. J., El Marsy, W. S. and Hones, P. W. (2000). High dose methylprednisolone in the management of acute spinal cord injury a systematic review from a clinical perspective. Spinal Cord 38, 273286.[Medline]
Thierault, E. and Diamond, J. (1988). Noccieptive cutaneous stimuli evoke localized contractions in a skeletal muscle. J. Neurophysiol. 60, 446447.