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.

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.

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.