JOURNAL OF NEUROTRAUMA Volume 9, Number 2, 1992 Mary Ann Liebert, Inc., Publishers

Problematic Issues in

Spinal Cord Injury

EDWARD L. HOGAN

developing an approach to understanding and designing effective for spinal cord injury include consideration of the effect of physical injury on circulation, the roles of relevant mediators, including calcium, peptides, and eicosanoids, the limited value of electrophysiologic studies for functional monitoring, and the pharmacokinetics and mechanisms of action of potentially

Thetherapy

numerous problems related to

protective drugs.

For the past several years, we have been seeking the efficient application of preclinical drug trials. Our approach has been much influenced by current concepts of the pathology and pathogenesis of the traumatic lesion, taking into consideration the evolution of tissue damage and necrosis as a result of a cascade of chemical reactions, effects on circulation, differential effects on axons and glia, infiltration of neutrophils, and consideration of specific mediators, particularly calcium, calcium-activated phospholipases generating eicosanoids, including leukotrienes, calcium-activated neutral proteinase, free radicals, kininogens, and

kinins. We use lesion size as a key end point measure in trials of agents that are putatively protective. Vascular permeability in the lesion is one such measure because of the key role that reduced barrier function plays in trauma pathogenesis. In the past, we employed radioactive human serum albumin, labeled with l25I. More recently, we have developed a technique that uses fluorescein isothiocyanate dextran (FITC-D). The method is simple and improves assessment of permeability at the trauma lesion site. The kinetics are linear, with fluorescence proportional to the concentration administered. The peak fluorescence occurs 2 h after trauma (with FITC-D administration 2 h before injury). Fluorescence in the lesion is proportional to the impact injury in a standard rat model. Methylprednisolone in a very high dose and the kappa opiate agonist U50488H have been found effective in this screening. The end points employed have included the vascular injury index (VII) determined by the FITC-D method. Additional measures were gravimetric determination of edema and measurement of neutrophil infiltration by myeloperoxidase (MPO) activity. We now examine these drugs in more detail by study of several drug doses and impact forces. Of the methods used, the vascular injury index is simplest, and we consider it to be preferred for use in animal (preclinical) screening. Another end point measure with which we are working appears promising after preliminary use. The principle of this method is examination of retrograde axonal transport across the lesion using Fluorogold. This approach has the great advantage of assessing integrity of fiber tracts, which is the major determinant of the functional disability consequent on spinal cord injury. It is an application of axonal transport methodology resembling the work of Tator's group, who examined transport of horseradish peroxidase across a lesion caused by clip compression. The advantages of Fluorgold include: it does not fade rapidly, does not diffuse into neighboring fibers, is compatible with other anatomic techniques, and can be quantified. Its disadvantage is that it is laborious. Video imaging techniques and biochemical assay of transported fluorescence are being examined as we attempt to reduce the necessary effort. Preliminary data indicate that fluorescence in the red

Department of Neurology, Medical University of South Carolina, Charleston, South Carolina. 161

HOGAN

peaks at 72 h after application of the marker and at 96 h in the cerebral motor cortex. The extent of Fluorogold labeling of cells in the red nucleus of the rat subjected to spinal cord impact injury was reduced markedly in comparison with the sham-operated control, and the reduction was proportional to the trauma force. Initial experiments suggest that high-dose methylprednisolone protected neurons, (i.e., a relative preservation of marker transport) in the motor cortex. It is important to stress that a pharmacologie approach to screening must encompass a large number of drugs and animals and requires a cooperative effort of many laboratories. nucleus

REFERENCES BANIK, N.L., HOGAN, E.G., and HSU, C.Y. (1987). The multimolecular cascade of spinal cord injury: Studies on prostanoids, calcium and proteinases. Neurochem. Pathol. 7, 57-77. HOGAN, E.L., HSU, C.Y., and BANIK, N.L. (1986). Calcium-activated mediators of secondary injury in spinal cord. CNS Trauma 3, 175-179. HSU, C.Y., HALUSHKA, P.V., SPICER, K.M., et al. (1988). Temporal profile of thromboxane-prostacyclin imbalance in experimental spinal cord injury. J. Neurol. Sei. 83, 55-62. MIDHA, R., FEHLINGS, M.G., TATOR, C.H., et al. (1987). Assessment of spinal cord injury by counting corticospinal and rubrospinal neurons. Brain Res. 410, 299-308. QU, Z.X., XU, J., PEROT, P.L., and HOGAN, E.L. (1991). A sensitive fluorometric method for measurement of vascular permeability in spinal cord injury. J. Neurotrauma 8, 149-156.

QU, Z.X., XU, J., PEROT, P. L., and HOGAN, E.L. (1991). U-50488H reduces vascular permeability and edema in rat spinal cord injury. Abstr. Soc. Neurosci. 17, 421. XU, J., COX, R., LI, Z-H., et al. (1992). Evaluation of acute spinal cord injury by counting neurons labeled by Fluoro-Gold, a new retrograde transport tracer. FASEB J. 6:A 1062. Address reprint requests to: Edward L. Hogan, M.D.

Medical

Department of Neurology University of South Carolina 171 Ashley Avenue Charleston, SC 29425

DISCUSSION Dr. Tator: The use of tracer techniques for outcome measures is a great example of the use of a powerful tool from the basic neuroscientists. There have not been enough such approaches in injury work, especially as an outcome measure after weeks or months of injury. Dr. Hogan: The interesting thing has been the discrepancy between HRP and Fluorogold. Fluorogold is related to transport mechanisms and reflects continuity of the axon, but the metabolic parameters are unknown. Dr. Michel: Dr. Hogan tested many compounds with three different outcome parameters, and only one compound (methylprednisolone) appears effective. What does that mean? Dr. Hall: Those negatives are not truly negatives until you extensively explore different doses and dosing regimens. There is no easy way out for examining potential agents for acute traumatic injury. All those results tell you is that a given dose, a certain drug does not work. Without dose-response studies, you really cannot be sure that you have adequately tested a compound. Dr. Faden: When one does screening, one has to pay extremely careful attention to dose-response curves and the pharmacokinetics of such compounds. Dr. Wrathall: One outcome measure is designed to look for vascular mechanisms, and a drug or a dose-response curve might be different for that mechanism than for another mechanism. A positive aspect of 162

END POINT MEASURES: VASCULAR

using multiple models would lie in their sensitivity to different mechanisms. We know that there are many ways in which the injury could be helped. The particular dose-response curve that we are interested in finally is going to be the dose that will be useful in people in clinical trials, and that is going to have to be based on some long-term functional measures. Dr. Hall: This is why I said there is no easy way out of our problem. I have been at this for a long time and devoted my last 10 years to two drugs. There has not been any other way to do it. Dr. Faden: What should be the accepted criteria of a test or outcome measure to forcefully discriminate severity of injury? Two specific questions would be: What kind of correlation coefficient would we really like to see for acceptance? What kind of sensitivity does the test have as a discriminator in a drug testing situation? One would have to look in rather a definitive way at a very large number of animals and also correlate those with accepted measures, whether it be inclined plane or whatever. Dr. Young: Testing must be done in stages. There are several drugs for which at least one positive report is available. From those drugs, you must cull out the most promising and do systematic dose-response studies to ascertain precisely the parameters within which the drug is effective. We have reached the point at which this testing cannot be done in any single laboratory. It must involve multiple laboratories agreeing on a specific set of parameters to test. One issue that everybody here has agreed on is that before taking a drug to clinical trial, there must be a functional end point as the outcome measure. Biochemical measures are not enough, electrophysiologic measures are not enough, anatomic measures are not enough. Ultimately, you must show some functional effect. Many investigators have done very specialized experiments looking at specific mechanisms and have identified promising drugs. We are at another stage of investigation with drugs that we think are promising. We need to get the data to convince people to start clinical trials, and this requires a big investment. Relevant to selecting a drug for a clinical trial are: Is the drug broad spectrum? Does the drug have a long time window of treatment? Is it more potent than another agent? The more data you can gather to address those questions, the more likely is it that the drug will become acceptable to the parties involved. What worries me a great deal is that we are talking about volumes of animal experiments that are beyond any single laboratory. There is no possibility that we in a single laboratory, within a span of 5 years, can put together a convincing story that will bring a drug to clinical trial.

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Problematic issues in spinal cord injury.

JOURNAL OF NEUROTRAUMA Volume 9, Number 2, 1992 Mary Ann Liebert, Inc., Publishers Problematic Issues in Spinal Cord Injury EDWARD L. HOGAN develo...
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