4-Aminopyridine derivatives enhance impulse conduction in guinea-pig spinal cord following traumatic injury.

4-Aminopyridine (4-AP), a potassium channel blocker, is capable of restoring conduction in the injured spinal cord. However, the maximal tolerated level of 4-AP in humans is 100 times lower than the optimal dose in in vitro animal studies due to its substantially negative side effects. As an initial step toward the goal of identifying alternative potassium channel blockers with a similar ability of enhancing conduction and with fewer side effects, we have synthesized structurally distinct pyridine-based blockers. Using isolated guinea-pig spinal cord white matter and a double sucrose gap recording device, we have found three pyridine derivatives, N-(4-pyridyl)-methyl carbamate (100 microM), N-(4-pyridyl)-ethyl carbamate (100 microM), and N-(4-pyridyl)-tertbutyl (10 microM) can significantly enhance conduction in spinal cord white matter following stretch. Similar to 4-AP, the derivatives did not preferentially enhance conduction based on axonal caliber. Unlike 4-AP, the derivatives did not change the overall electrical responsiveness of axons to multiple stimuli, indicating the axons recruited by the derivatives conducted in a manner similar to healthy axons. These results demonstrate the ability of novel constructs to serve as an alternative to 4-AP for the purpose of reversing conduction deficits.

A comparative study of the quantitative accuracy of three-dimensional reconstructions of spinal cord from serial histological sections.

We evaluated the accuracy of estimating the volume of biological soft tissues from their three-dimensional (3D) computer wireframe models, reconstructed from histological data sets obtained from guinea-pig spinal cords. We compared quantification from two methods of three-dimensional surface reconstruction to standard quantitative techniques, Cavalieri method employing planimetry and point counting and Geometric Best-Fitting. This involved measuring a group of spinal cord segments and test objects to evaluate the accuracy of our novel quantification approaches. Once a quantitative methodology was standardized there was no statistical difference in volume measurement of spinal segments between quantification methods. We found that our 3D surface reconstructions' ability to model precisely actual soft tissues provided an accurate volume quantification of complex anatomical structures as standard approaches of Cavalieri estimation and Geometric Best-Fitting. Additionally, 3D reconstruction quantitatively interrogates and three-dimensionally images spinal cord segments and obscured internal pathological features with approximately the same effort required for standard quantification alone.

Rapid recovery from spinal cord injury after subcutaneously administered polyethylene glycol.

Arguably a seminal event in most trauma and disease is the breakdown of the cell membrane. In most cells, this is first observed as a collapse of the axolemmas barrier properties allowing a derangement of ions to occur, leading to a progressive dissolution of the cell or its process. We have shown that an artificial sealing of mechanically damaged membranes by topical application of hydrophilic polymers such as polyethylene glycol (PEG) immediately restores variable levels of nerve impulse conduction through the lesion. This was documented by a rapid recovery of somatosensory evoked potential (SSEP) conduction, and by recovery of the cutaneous trunchi muscle (CTM) reflex in PEG-treated animals. The CTM reflex is a sensorimotor behavior dependent on an intact (and identified) white matter tract within the ventrolateral funiculus of the spinal cord, and is thus an excellent index of white matter integrity. We show that PEG can be safely introduced into the bloodstream by several routes of administration. Using a fluorescein decorated PEG, we demonstrate that the polymer specifically targets the hemorrhagic contusion of the adult guinea pig spinal cord when administered through the vasculature, but not intact regions of the spinal cord. A single subcutaneous injection (30% weight by weight in sterile saline) made 6 hr after a standardized spinal cord contusion in adult guinea pigs was sufficient to produce a rapid recovery of SSEP propagation through the lesion in only PEG-treated animals, accompanied by a statistically significant recovery of the CTM reflex. These data suggest that parenterally administered PEG may be a novel treatment for not only spinal injury, but head injury and stroke as well.

An oscillating extracellular voltage gradient reduces the density and influences the orientation of astrocytes in injured mammalian spinal cord.

We have studied the cellular basis for recovery from acute spinal cord injury induced by applied electric fields. We have emphasized this recovery is due to the regeneration of spinal axons around and through the lesion, and have begun to evaluate the contribution of other cells to the recovery process. We have imposed a voltage gradient of about 320 microV/mm across puncture wounds to the adult rat spinal cord in order to study the accumulation and orientation of GFAP+ astrocytes within and adjacent to the lesion. This electric field was imposed by a miniaturized electronic implant designed to alternate the polarity of the field every 15 minutes. Astrocytes are known to undergo hyperplastic transformation within injured mammalian cords forming a major component of the scar that forms in response to injury. We have made three observations using a new computer based morphometry technique: First, we note a slight shift in the orientation of astrocytes parallel to the long axis of the spinal cord towards an imaginary reference perpendicular to this axis by approximately 10 degrees--but only in undamaged white matter near the lesion. Second, the relative number of astrocytes was markedly, and statistically significantly, reduced within electrically--treated spinal cords, particularly in the lesion. Third, the imposed voltage gradient statistically reduced the numbers of astrocytes possessing oriented cell processes within the injury site compared to adjacent undamaged regions of spinal cord.

Endogenous electric current is associated with normal development of the vertebrate limb.

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.

Cellular engineering: molecular repair of membranes to rescue cells of the damaged nervous system.

PURPOSE: The acute administration of hydrophilic polymers (polyethylene glycol) can immediately seal nerve membranes, preventing their continuing dissolution and secondary axotomy. Polymer application can even be used to reconnect, or fuse, the proximal and distal segments of severed axons in completely transected adult mammalian spinal cord. CONCEPT: The sealing or fusion of damaged nerve membranes leads to a very rapid (minutes or hours) recovery of excitability in severely damaged nerve fibers, observed as a rapid return of nerve impulse conduction in vitro, as well as an in vivo recovery of spinal cord conduction and behavioral loss in spinal cord-injured adult guinea pigs. RATIONALE: Surfactant application produces a rapid repair of membrane breaches through mechanisms of interaction between the polymers and the aqueous phase of damaged membranes, and their ability to insert into, or seal, the hydrophobic core of the axolemma exposed by mechanical damage. DISCUSSION: This new technology applied to severe neurotrauma offers a clinically safe and practical means to rescue significant populations of spinal cord nerve fibers within 8 hours after damage--preventing their continued dissolution and secondary axotomy by secondary injury mechanisms. Application of this novel technology to other injuries to the peripheral and central nervous system is discussed, as well as a general application to soft tissue trauma.

Giant multinucleated macrophages occur in acute spinal cord injury.

Using a cell-isolation and -culture procedure specific for macrophages, we report the existence of giant (more than 50 microm diameter), multinucleated macrophages within an acute, 5-day-old adult rat spinal cord injury. The size and multinuclearity of these isolated giant cells was confirmed using transmission electron microscopy. Giant macrophages are markers for long-term infection, disease, and chronic injury in other soft tissues and are unexpected in the acute inflammatory stage of central nervous system injury. To our knowledge, this descriptive report is the first confirming the existence of giant macrophages in any injured nervous tissue, with additional data suggesting some of these cells to be multinucleated.

Advances in three-dimensional reconstruction of the experimental spinal cord injury.

Three-dimensional (3D) computer reconstruction is an ideal tool for evaluating the centralized pathology of mammalian spinal cord injury (SCI) where multiple anatomical features are embedded within each other. Here, we evaluate three different reconstruction algorithms to three-dimensionally visualize SCIs. We also show for the first time, that determination of the volume and surface area of pathological features is possible using the reconstructed 3D images themselves. We compare these measurements to those calculated by older morphometric approaches. Finally, we demonstrate dynamic navigation into a 3D spinal cord reconstruction.

The macrophage in acute neural injury: changes in cell numbers over time and levels of cytokine production in mammalian central and peripheral nervous systems.

We evaluated the timing and density of ED-1-positive macrophage accumulation (ED 1 is the primary antibody for the macrophage) and measured cytokine production by macrophages in standardized compression injuries to the spinal cord and sciatic nerves of individual rats 3, 5, 10 and 21 days post-injury. The actual site of mechanical damage to the nervous tissue, and a more distant site where Wallerian degeneration had occurred, were evaluated in both the peripheral nervous system (PNS) and the central nervous system (CNS) at these time points. The initial accumulation of activated macrophages was similar at both the central and peripheral sites of damage. Subsequently, macrophage densities at all locations studied were statistically significantly higher in the spinal cord than in the sciatic nerve at every time point but one. The peak concentrations of three cytokines, tumor necrosis factor &agr; (TNF &agr; ), interleukin-1 (IL-1) and interleukin-6 (IL-6), appeared earlier and were statistically significantly higher in injured spinal cord than in injured sciatic nerve. We discuss the meaning of these data relative to the known differences in the reparative responses of the PNS and CNS to injury.

Immediate recovery from spinal cord injury through molecular repair of nerve membranes with polyethylene glycol.

A brief application of the hydrophilic polymer polyethylene glycol (PEG) swiftly repairs nerve membrane damage associated with severe spinal cord injury in adult guinea pigs. A 2 min application of PEG to a standardized compression injury to the cord immediately reversed the loss of nerve impulse conduction through the injury in all treated animals while nerve impulse conduction remained absent in all sham-treated guinea pigs. Physiological recovery was associated with a significant recovery of a quantifiable spinal cord dependent behavior in only PEG-treated animals. The application of PEG could be delayed for approximately 8 h without adversely affecting physiological and behavioral recovery which continued to improve for up to 1 month after PEG treatment.

Anatomical repair of nerve membranes in crushed mammalian spinal cord with polyethylene glycol.

Acute damage to axons is manifested as a breach in their membranes, ion exchange across the compromised region, local depolarization, and sometimes conduction block. This condition can worsen leading to axotomy. Using a novel recording chamber, we demonstrate immediate arrest of this process by application of polyethylene glycol (PEG) to a severe compression of guinea pig spinal cord. Variable magnitudes of compound actions potentials (CAPs) were rapidly restored in 100% of the PEG-treated spinal cords. Using a dye exclusion test, in which horseradish peroxidase is imbibed by damaged axons, we have shown that the physiological recovery produced by polyethylene glycol was associated with sealing of compromised axolemmas. Injured axons readily imbibe horseradish peroxidase-but not following sealing of their membranes. The density of nerve fibers taking up the marker is significantly reduced following polyethylene glycol treatment compared to a control group. We further show that all axons-independent of their caliber-are equally susceptible to the compression injury and equally susceptible to polyethylene glycol mediated repair. Thus, polyethylene glycol-induced reversal of permeabilization by rapid membrane sealing is likely the basis for physiological recovery in crushed spinal cords. We discuss the clinical importance of these findings.

Functional reconnection of severed mammalian spinal cord axons with polyethylene glycol.

We describe a technique using the water-soluble polymer polyethylene glycol (PEG) to reconnect the two segments of completely transected mammalian spinal axons within minutes. This was accomplished by fusing completely severed strips of isolated guinea pig thoracic white matter maintained in vitro in a double sucrose gap recording chamber. The faces of the severed segments were pressed together, and PEG (MW 1,400-3,500 d; approximately 50% by weight in distilled water) was applied directly to this region through a micropipette and removed by aspiration within 2 min. Successful fusion was documented by the immediate restored conduction of compound action potentials through the original transection and by the variable numbers of fused axons in which anatomical continuity was shown to be restored by high-resolution light microscopy and by the diffusion of intracellular fluorescent dyes through fused axons. These data support the conclusion that some severed and subsequently PEG-fused spinal axons both demonstrate restored anatomical continuity and also are physiologically competent to conduct action potentials. This work adds to our previous demonstration that PEG application can immediately repair severely crushed, rather than cut, spinal cord white matter, and may lead to novel treatments for acute trauma to the central and peripheral nervous systems.

An imposed oscillating electrical field improves the recovery of function in neurologically complete paraplegic dogs.

We show that an applied electric field in which the polarity is reversed every 15 minutes can improve the outcome from severe, acute spinal cord injury in dogs. This study utilized naturally injured, neurologically complete paraplegic dogs as a model for human spinal cord injury. The recovery of paraplegic dogs treated with oscillating electric field stimulation (OFS) (approximately 500 to 600 microV/mm; n = 20) was compared with that of sham-treated animals (n = 14). Active and sham stimulators were fabricated in West Lafayette, Indiana. They were coded, randomized, sterilized, and packaged in Warsaw, Indiana, and returned to Purdue University for blinded surgical implantation. The stimulators were of a previously unpublished design and meet the requirements for phase I human clinical testing. All dogs were treated within 18 days of the onset of paraplegia. During the experimental applications, all received the highest standard of conventional management, including surgical decompression, spinal stabilization (if required), and acute administration of methylprednisolone sodium succinate. A radiologic and neurologic examination was performed on every dog entering the study, the latter consisting of standard reflex testing, urologic tests, urodynamic testing, tests for deep and superficial pain appreciation, proprioceptive placing of the hind limbs, ambulation, and evoked potential testing. Dogs were evaluated before and after surgery and at 6 weeks and 6 months after surgery. A greater proportion of experimentally treated dogs than of sham-treated animals showed improvement in every category of functional evaluation at both the 6-week and 6-month recheck, with no reverse trend. Statistical significance was not reached in comparisons of some individual categories of functional evaluation between sham-treated and OFS-treated dogs (ambulation, proprioceptive placing); an early trend towards significance was shown in others (deep pain), and significance was reached in evaluations of superficial pain appreciation. An average of all individual scores for all categories of blinded behavioral evaluation (combined neurologic score) was used to compare group outcomes. At the 6-month recheck period, the combined neurologic score of OFS-treated dogs was significantly better than that of control dogs (p = 0.047; Mann-Whitney, two-tailed).

Electrically mediated regeneration and guidance of adult mammalian spinal axons into polymeric channels.

An extracellular electric field has been shown to influence the regeneration of nerve fibers within the adult mammalian spinal cord. However, in these studies, few axons were labeled by local application of intracellular markers relative to the number of axons transected. This has limited an evaluation of the robustness of the response, and the direction of growth of regenerating axons that might be influenced by the orientation of the applied voltage gradient. In this study, a hollow silicone rubber tube (c. 6 mm x 1 mm outside diameter) containing a cathodal (negative) electrode was inserted longitudinally into the dorsal half of the adult guinea-pig spinal cord. The electric field ( approximately 100 microV/mm) was imposed within the damaged spinal cord with an implanted d.c. stimulator for about three weeks. Based on previous studies, this orientation of the electric field would be expected to both initiate axonal regeneration and guide growing axons to, and into, the silicone guidance channel. In experimental animals (n = 20), a robust regeneration of axons into the tube was observed in more than half the cases. These axons were traced from surrounding white and gray matter by anterograde and retrograde labeling using a tetramethylrhodamine-conjugated dextran as an intracellular marker. Control animals (n = 16) received tubes with inactive electrodes. It was rare to find any axons within control guidance channels, since adult mammalian central nervous system axons do not regenerate. This report provides evidence for not only the facilitated regeneration of adult mammalian central axons, but also their guidance, by an imposed electric field.

Acute repair of crushed guinea pig spinal cord by polyethylene glycol.

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 approximately 2 min restored CAP conduction through the injury as early as 1 min post PEG application. The recovery of the CAP

Two- and three-dimensional computer graphic evaluation of the subacute spinal cord injury.

We have evaluated three-week-old compression lesions of the rat spinal cord using two-dimensional and three-dimensional morphometry, reconstruction, and visualization techniques. We offer a new computer assisted method to determine the number and density of macrophages within the spinal lesion using the macrophage specific monoclonal label ED1. We also provide quantitative information on pathological cyst formation and cavitation. This technique does not require: (1) subjective identification of the cell type, (2) human interaction with the data during the phase of quantification, and (3) can be applied to any sampling paradigm based on immunocytochemical labeling. Using novel algorithms based on solutions to 'correspondence' and 'branching' problems inherent in cross-sectional histological data, we provide three-dimensional reconstructions and visualizations of the macrophagic lesions and cysts imbedded within it. Our three-dimensional surface reconstructions can be interrogated to determine volumes and surface areas of structures within the data set. Using these methods we have learned that macrophage numbers approach the maximum density possible for such isodiametric cells (approximately 12 microm diameter) in the central lesion ranging from 4000-7000 cells per mm2 of lesion. At the time point studied, macrophage numbers would have peaked following the initial insult, and would not be expected to decline for several months. While the density of macrophages is highest in the region of most tissue damage, we show that the central regions of cavitated and cystic spinal parenchyma is not. We discuss how this density of cells may effect the secondary pathological responses of the spinal cord to injury.

The responses of mammalian spinal axons to an applied DC voltage gradient.

We have imposed a steady, rostrally negative, weak (ca 0.4 mV/mm) voltage gradient across transections of ascending white matter tracts in the adult guinea pig using an implanted stimulator and electrodes for about 1 month. We have evaluated the projections of these axons relative to the transection approximately 2 months postinjury by anterograde transport of injected tetramethylrhodamine-conjugated dextran and the use of an indwelling marker device which locates the plane of the original transection. Tract tracing was accomplished with conventional epifluorescence microscopy and confocal laser microscopy. Sham-treated control spinal cords contained well-filled lateral and dorsal column ascending tracts terminating caudal to the lesion which formed at the level of the hemisection. Electric field-treated spinal cords contained similarly labeled columns of axons that penetrated the lesion within the caudal segment of the spinal cord, branched within it, and in some cases such branches projected across the plane of transection. Ascending axons also passed around the lesion through undamaged parenchyma, branched repeatedly at the plane of the hemisection, and passed into the rostral segment of the spinal cord. Spear-shaped endings typical of growth cones were found at the terminals of these processes which often branched again within the rostral segment. Centrally projecting fibers, their processes, and the overall level of branching in these projections was not observed in our previous studies using high molecular weight horseradish peroxidase tracers.

Reduction of the current of injury leaving the amputation inhibits limb regeneration in the red spotted newt.

Immediately following amputation of the limb in salamanders, a strong, steady, and polarized flow of ionic current is produced by the injury. Current flows in a proximodistal direction within the limb stump and is associated with a fall in electrical potential of about 50 mV/mm near the stump's end. This current is electrogenically driven by the Na(+)-dependent, internally positive transcutaneous voltage of the intact skin of the limb stump. Reduction of this EMF, the skin's battery, by topical application of Na+ blocking agents leads to inhibition or disruption of normal limb regeneration. This suggests electrical factors are a critical control of limb regeneration. Here we test another means to reduce the injury current and its associated electrical field within the forelimb stump of red spotted newts. A fine (40 gauge), insulated, multistrand wire was inserted beneath the skin of the animal's back, with the uninsulated portion terminating either at the shoulder region or at the base of the tail. When this cathodal (negative) electrode is connected to a regulated current source, sufficient current was pulled into the stump end from an external anode (placed in the water the animal was immersed in) to markedly reduce or null the endogenous current for the first 8 days following amputation. The extent of limb regeneration in sham-treated and experimentally treated animals was determined 1 month following amputation at the elbow. Sham-treated animals regenerated normally, with most producing digits within this time. Limb regeneration was completely arrested, or caused to be strikingly hypomorphic, in half of the experimentally treated animals. This effect was independent of where the subcutaneous electrode was placed and suggests that electrical (physiological) factors are indeed a critical control of limb regeneration in urodeles.

Uncoupling histogenesis from morphogenesis in the vertebrate embryo by collapse of the transneural tube potential.

We have shown that unidirectional pumping of Na+ out of the neural tube's luminal fluids in amphibian embryos produces a large potential difference (40-90 mV, lumen negative to the abluminal surface). This transneural tube potential (TNTP) is analogous to the Na+ dependent transepithelial potential (TEP) that exists across surface ectoderm. This TEP is retained in ectoderm after it is internalized when the neural folds fuse to form the neural tube. The TNTP can be markedly reduced for several hours by injection of the Na+ channel blockers amiloride or benzamil into the lumen by iontophoresis through microelectrodes. Here we describe the effect of TNTP modification on developmental anatomy. Axolotl embryos possessing a fused and closed neural tube (stage 21-23) were injected with either amiloride or benzamil and allowed to continue development for 36-52 hr. These were compared to control embryos injected with vehicle alone, or to embryos in which amiloride or benzamil was iontophoresed just beneath surface ectoderm. All embryos in which the TNTP was reduced were grossly defective. These were characterized by a disaggregation of the cells comprising the structures that had already begun to form (otic primordia, brain, spinal cord, notochord) as well as a failure in the development of new structures. Remarkably, some of these embryos displayed continuing development of external form in the complete absence of concomitant internal histogenesis. We discuss the ways in which a large endogenous voltage gradient associated with an epithelial potential difference (the TNTP) may be required both for the structural integrity of the early neuroepithelium, and a prerequisite for normal morphogenesis.

Three-dimensional gradients of voltage during development of the nervous system as invisible coordinates for the establishment of embryonic pattern.

We are interested in the generation of endogenous electric fields associated with ionic currents driven through the vertebrate embryo by the transepithelial potential of its surface ectoderm. Using a non-invasive vibrating electrode for the measurement of ionic current, we have provided measurements of currents traversing amphibian embryos, and a preliminary report of the internal, extracellular voltage gradient under the neural plate which polarizes the embryo in the rostral/caudal axis (Metcalf et al. [1994] J. Exp. Zool. 268:307-322). Here we complete a description of this gradient in electrical potential (ca. 10 mV/mm, caudally negative), describe a simultaneous gradient organized in the medial/lateral axis (ca. 5-18 mV/mm, negative at the margins of the neural folds), and describe their appearance and disappearance during ontogeny of the axolotl embryo. Both voltage gradients are not expressed until neurulation, and disappear at its climax. This appearance and disappearance correlates with the shunting of current out of the lateral margins of the neural folds in rostral regions of the embryo beginning at stage 15, and is not associated with a more substantial current leak from the blastopore which appears at gastrulation. A steady blastopore current is still present after neural tube formation when intra-embryonic electric fields have been extinguished. We discuss the direct experimental tests supporting the hypothesis that these extracellular electric fields both polarize the early vertebrate embryo and serve as cues for morphogenesis and pattern.