Jen Hill Lucas

Recording of spontaneous activity with photoetched microelectrode surfaces from mouse spinal neurons in culture

A matrix of photoetched gold conductors integrated into the floor of a tissue culture chamber has been used to record from mammalian spinal cord neurons grown on the insulation layer of the multielectrode plate. Spontaneous activity has been monitored from tissue microfragments less than 150 micrometers in diameter and from thin sheets of spinal cell aggregates. Maximum spike amplitudes of 360 microV with signal-to-noise ratios of 8:1 have so far been achieved and the spontaneous activity maintained for several days. Recording electrode impedances measured between 4 and 7 M omega at 1 kHz. Conductor tips were deinsulated with laser pulses that formed shallow craters 2 micrometers deep and 12 micrometers in diameter. Addition of colloidal gold or platimum black was not necessary to achieve satisfactory recordings.

Effect of Glutathione Augmentation on Lipid Peroxidation after Spinal Cord Injury

Lipid peroxidation (LPO) is considered a major factor in damage spread after spinal cord injury (SCI). Therapies that limit LPO after SCI have demonstrated some utility in clinical trials, but more effective treatments are needed. In the present study the effects of augmenting SC levels of the endogenous antioxidant glutathione (GSH) on LPO after SCI were studied in a rat contusion injury model. A significant decrease in GSH occurred 1h after SCI which was paralleled by increases of 123% in malondialdehyde (MDA) and >500% in the 4-hydroxyalkenals (4-HAs), two LPO products. SC irrigation with gamma-glutamylcysteine (GC) preserved GSH and reduced 4-HAs below naive levels but had no effect on MDA. By 24 h after SCI, MDA returned to naive levels but 4-HAs were still elevated. Once again, GC treatment reduced 4-HAs. 4-HAs are much more reactive than MDA and are considered among the most toxic LPO products. These results suggest that (1) conditions after SCI may favor particular branches of the LPO pathway leading to differential LPO product levels, (2) MDA measurement is not by itself an adequate test for the presence or magnitude of LPO after SCI, (3) binding of GSH to 4-HAs may be an important mechanism by which the GSH system confers protection against LPO after SCI, and (4) SC GSH can be augmented after trauma by local irrigation with GC. These results also suggest that GSH augmentation may be an effective strategy for curtailment of LPO-mediated damage in acute phase SCI.

Adhesion of cultured mammalian central nervous system neurons to flame-modified hydrophobic surfaces

SummarySurface wettability is an excellent indicator of the ability of cells to adhere to a culture substrate. We have determined that brief exposure of a hydrophobic culture surface to a propane flame may increase wettability more than 1200% via the deposition of ionic combustion products. Previously nonadherent mouse spinal cord cells will adhere to and differentiate morphologically on a hydrophobic surface after flaming. Central nervous system cells remain adhered to flamed surfaces for periods of 2 mo. or longer and demonstrate spontaneous electrical activity during that time. Secondary modification of a flamed surface with polylysine further enhances the strength of single cell adhesion, thereby retarding mobility and promoting neurite extension. Flaming also enhances the wettability of common culture materials such as glass and polystyrene, as well as metal. Flaming of hydrophobic substrates through masks permits creation of discrete adhesion islands and patterns which may be used for a variety of investigations requiring maintenance of different cell types in separate regions of a culture surface.

REVIEW ■ : Physical Injury of Neurons: Important Roles for Sodium and Chloride Ions:

There is growing evidence that ions other than Ca2+ play important roles in the deterioration of neuronal elements in both gray and white matter after physical injury. This review features information gathered with a tissue culture model of dendrite transection regarding the contributions of Na+ and CI- to ultrastructural damage and neuronal death. This information and the results of other in vitro investigations of physical and ischemic/excitotoxic injuries indicate that elevation of internal Na+ is an early event that may contribute significantly to neuronal injury through effects on Na+-driven transport mechanisms. Proposed deleterious consequences include cytoplasmic acidification, reduced mitochondrial energy production, and elevation of intracellular Ca2+ and extracellular excitatory amino acids to toxic levels. Prevention of Na+ entry into neurons after injury has been found to limit ultrastructural damage, prevent death, and preserve electrophysiological function. Although the role of CI- in neuronal injury is less well defined, there is also evidence that elevation of intracellular CI- contributes to structural damage, particularly to the smooth endoplasmic reticulum. In terventions that limit Na+- and CI--mediated damage to injured neurons may have utility in neurosurgery and as acute phase treatments for nervous system trauma and other pathological states. NEURO SCIENTIST 3:89-101, 1997

In vitro Simulation of Neural Trauma by Laser

A serious lack of knowledge about central nervous system trauma is encountered on the cellular level where the inability to create precise experimental lesions of known magnitude has limited our understanding of the reactions of single cells to injury. We used a laser cell surgery technique developed in this laboratory to manipulate neurons in a controlled environment, in order to observe pathologic reactions during and immediately after the injury. This technique is especially suited for axonal and dendritic amputations close to the perikaryon. The laser provided three different physical modes of injury to neurites: direct vaporization of cytoplasm, pressure wave damage from external vaporization of substrate material, and photobiologically-induced localized cytoskeletal destruction leading to the slow pinching of processes followed by transection. Our data indicated a great similarity between laser impact damage and the cellular damage produced by physical trauma to the central nervous system.