Physical injury to the spinal cord gives rise to a variety of environmental changes including ischemia, free radical formation, massive ion shifts, and changes in osmotic equilibria. Many believe that in terms of neuronal death, spread of necrosis and neurological deficits these """"""""secondary"""""""" insults do far more damage than the physical trauma. Some of the therapies developed to address these conditions include low temperature, hyperbaric oxygen, corticosteroids, free radical scavengers, hyperosmotic agents, and dimethyl sulfoxide (DMSO). However, the efficacies of many of these treatments are still a matter of controversy and the isolated effects of such therapies have not been investigated quantitatively in terms of neuronal survival. The previous grant period established baseline data on reactions of single neurons in culture to physical trauma (dendrite amputation and shock wave trauma) by defining the probability of cell survival under normal culture conditions as a function of lesion parameters. The observed response categories (rapid cell death within 1 to 2 hours, or survival with slow recovery over more than 24 hrs) elucidated, on the cellular level, two major clinical problems: (1) there is little time for intervention to save acutely traumatized neurons, and (2) those neurons which are recovering from physical trauma may be especially vulnerable to secondary insults for at least one and possibly several days. We propose to address directly these vital problems in the highly simplified and well controlled environment of neuronal monolayer cell culture. Using unique laser cell surgery techniques, routine biochemical methods, and novel multielectrode analyses of network spike activity, this research will focus on four areas of investigation: (1) identification of those factors of the secondary insult which pose the greatest threat to injured neurons during the recovery period. (2) Assessment of the effects of various commonly applied therapies on the continued survival of recovering neurons under normal culture conditions and in conjunction with secondary insults. (3) Investigation of early (15 min to 1 h) physical, chemical, or pharmacological interventions that may slow down the rapid cell deterioration or death and """"""""buy time"""""""" until established therapies may be attempted. In addition (4), we plan to investigate the ionic basis of the damage gradients that we have shown to develop in transected processes. Our observation that neurons continue to die in calcium-free medium has focused attention on endogenous Ca++ and has led to the formation of the sodium/endogenous calcium hypothesis of cell death. We wish to be given the opportunity to test this hypothesis thoroughly.
