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.

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).

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.

Transected dorsal column axons within the guinea pig spinal cord regenerate in the presence of an applied electric field.

Using an implanted battery and electrodes, we have imposed a weak, steady electrical field across partially severed guinea pig spinal cords. We have analyzed regeneration of dorsal column axons in experimental animals and sham-treated controls at 50-60 days postinjury by anterograde filling of these axons with the intracellular marker horseradish peroxidase and by employing a marking device to identify precisely the original plane of transection (J. Comp. Neurol. 250: 157-167, '86). In response to electric field applications, axons grew into the glial scar, as far as the plane of transection in most experimental animals. In a few animals axons could be traced around the margins of the lesion (but never through it). Moreover, these fibers returned to their approximate positions within the rostral spinal cord before turning toward the brain. In sham-treated controls, ascending axons were found to terminate caudal to the glial scar, and rarely were any fibers found within the scar itself. Axons were never observed to cross into the rostral cord segment. These findings suggest that an imposed electrical field promotes growth of axons within the partially severed mammalian spinal cord, that a steady voltage gradient may be an environmental component necessary for axonal development and regeneration, and that some component(s) of the scar impede or deflect axonal growth and projection.