Richard B. Borgens

Polyethylene glycol immediately repairs neuronal membranes and inhibits free radical production after acute spinal cord injury.

Membrane disruption and the production of reactive oxygen species (ROS) are important factors causing immediate functional loss, progressive degeneration, and death in neurons and their processes after traumatic spinal cord injury. Using an in vitro guinea pig spinal cord injury model, we have shown that polyethylene glycol (PEG), a hydrophilic polymer, can significantly accelerate and enhance the membrane resealing process to restore membrane integrity following controlled compression. As a result of PEG treatment, injury‐induced ROS elevation and lipid peroxidation (LPO) levels were significantly suppressed. We further show that PEG is not an effective free radical scavenger nor does it have the ability to suppress xanthine oxidase, a key enzyme in generating superoxide. These observations suggest that it is the PEG‐mediated membrane repair that leads to ROS and LPO inhibition. Furthermore, our data also imply an important causal effect of membrane disruption in generating ROS in spinal cord injury, suggesting membrane repair to be an effective target in reducing ROS genesis.

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 ~8 h without adversely affecting physiological and behavioral recovery which continued to improve for up to 1 month after PEG treatment.—Borgens, R. B., Shi, R. Immediate recovery from spinal cord injury through molecular repair of nerve membranes with polyethylene glycol. FASEB J. 14, 27–35(2000)

Understanding Secondary Injury

Secondary injury is a term applied to the destructive and self-propagating biological changes in cells and tissues that lead to their dysfunction or death over hours to weeks after the initial insult (the “primary injury”). In most contexts, the initial injury is usually mechanical. The more destructive phase of secondary injury is, however, more responsible for cell death and functional deficits. This subject is described and reviewed differently in the literature. To biomedical researchers, systemic and tissue-level changes such as hemorrhage, edema, and ischemia usually define this subject. To cell and molecular biologists, “secondary injury” refers to a series of predominately molecular events and an increasingly restricted set of aberrant biochemical pathways and products. These biochemical and ionic changes are seen to lead to death of the initially compromised cells and “healthy” cells nearby through necrosis or apoptosis. This latter process is called “bystander damage.” These viewpoints have largely dominated the recent literature, especially in studies of the central nervous system (CNS), often without attempts to place the molecular events in the context of progressive systemic and tissue-level changes. Here we provide a more comprehensive and inclusive discussion of this topic.

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.

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.

Well-Ordered Porous Conductive Polypyrrole as a New Platform for Neural Interfaces

We present the preparation of electrically conductive, porous polypyrrole surfaces and demonstrate their use as an interactive substrate for neuronal growth. Nerve growth factor (NGF)-loaded porous conducting polymers were initially prepared by electrochemical deposition of a mixture of pyrrole monomers and NGF into two- or three-dimensional particle arrays followed by subsequent removal of a sacrificial template. Morphological observation by scanning electron microscopy (SEM) revealed these to possess high regularity and porosity with well-defined topographical features. A four-point probe study demonstrated remarkable electrical activities despite the presence of voids. In addition, we investigated the effects of these surfaces on cellular behaviors using PC 12 cells in the presence and absence of electrical stimulation. Our results suggest that the surface topography as well as an applied electrical field can play a crucial role in determining further cell responses. Indeed, surface-induced preferential regulation leads to enhanced cellular viability and neurite extension. Establishing the underlying cellular mechanisms in response to various external stimuli is essential in that one can elicit positive neuronal guidance and modulate their activities by engineering a series of electrical, chemical, and topographical cues.

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. J Neurosci. Res. 66:1179–1186, 2001.

Acrolein-Mediated Mechanisms of Neuronal Death

It is well known that traumatic injury in the central nervous system can be viewed as a primary injury and a secondary injury. Increases in oxidative stress lead to breakdown of membrane lipids (lipid peroxidation) during secondary injury. Acrolein, an alpha,beta‐unsaturated aldehyde, together with other aldehydes, increases as a result of self‐propagating lipid peroxidation. Historically, most research on the pathology of secondary injury has focused on reactive oxygen species (ROS) rather than lipid peroxidation products. Little is known about the toxicology and cell death mediated by these aldehydes. In this study, we investigated and characterized certain features of cell death induced by acrolein on PC12 cells as well as cells from dorsal root ganglion (DRG) and sympathetic ganglion in vitro. In the companion paper, we evaluated a possible means to interfere with this toxicity by application of a compound that can bind to and inactivate acrolein. Here we use both light and atomic force microscopy to study cell morphology after exposure to acrolein. Administration of 100 μM acrolein caused a dramatic change in cell morphology as early as 4 hr. Cytoskeletal structures significantly deteriorated after exposure to 100 μM acrolein as demonstrated by fluorescence microscopy, whereas calpain activity increased significantly at this concentration. Cell viability assays indicated significant cell death with 100 μM acrolein by 4 hr. Caspase 3 activity and DNA fragmentation assays were performed and supported the notion that 100 μM acrolein induced PC12 cell death by the mechanism of necrosis, not apoptosis.

Polyethylene Glycol Improves Function and Reduces Oxidative Stress in Synaptosomal Preparations following Spinal Cord Injury

Spinal cord injury (SCI) results in rapid and significant oxidative stress. We have previously demonstrated that administration of polyethylene glycol (PEG) inhibits oxidative stress using an in vitro model of SCI. In this study we tested the effects of PEG in vivo, to elucidate the mechanism of PEG-mediated neuroprotection. We show that a compression injury at T10-11 induced diffusive oxidative stress in crude synaptosomal preparations, correlated with synaptosomal dysfunction and increased intrasynaptosomal calcium. Administration of PEG immediately post-injury produced a marked decrease in synaptosomal oxidative stress and calcium, associated with an increase in synaptosomal function. Confocal microscopy using fluorescein conjugated PEG revealed that PEG entered the cells of the injured spinal cord, placing the polymer in a position to directly interact with cellular organelles. PEG attenuates calcium-induced functional compromise of normal spinal cord synaptosomes and mitochondria in vitro. These results indicate that PEG may exert its neuroprotective effect through direct interaction with mitochondria, besides its known ability to rescue neurons and their axons by repairing the plasma membranes. We submit that PEG is likely to interfere with the cascade of secondary injury by several mechanisms of action that in concert reduce oxidative stress.

Effective repair of traumatically injured spinal cord by nanoscale block copolymer micelles

Spinal cord injury (SCI) results in immediate disruption of neuronal membranes followed by extensive secondary neurodegenerative processes. A key approach for repair of SCI is sealing the damaged membranes early. Here we show that axonal membranes injured by compression can be effectively repaired by using self-assembled monomethoxy poly(ethylene glycol)-poly(D,L-lactic acid) di-block copolymer micelles (60 nm diameter). Injured spinal tissue incubated with micelles showed rapid restoration of compound action potential and reduced calcium influx into axons. Much lower micelle concentration is required for treatment than the positive control, polyethylene glycol. Intravenously injected micelles effectively recovered the locomotor function and reduced the volume and inflammatory response of the lesion in SCI rats. The micelles showed no adverse effects after systemic administration to live rats. Our results suggest that copolymer micelles can interrupt the spread of primary SCI damage with minimal toxicity.