Andrew R. Blight - US grants

The structure and electrophysiological characteristics of central spinal axons will be determined during successive stages of the inflammatory response that follows spinal cord contusion injury, in cats. The time-course and mechanism of dysfunction in surviving axons will be investigated, using intracellular microelectrode recording and current-injection in an in vitro preparation of spinal cord. The quantitative extent of axonal survival and myelination will be examined with light- and electron-microscopy, for comparison with physiological recordings in the same tissue. The myelination and electrophysiology of selected individual axons will be correlated by electrophoretic injection of horseradish peroxidase at the time of recording, and subsequent processing for microscopy. The time-course and extent of phagocytic infiltration of the tissue, as a possible contributive factor in axonal pathology, will also be examined. The experiments relate to the problem of defining the extent to which functional loss in traumatic paraplegia and paraparesis is be due to factors other than direct destruction of axons, particularly their demyelination and incomplete remyelination. This will address the utility of attempting to develop therapy for these currently untreatable conditions through improvement of remyelination or treatment of demyelination pathophysiology. The study will provide information on the role of the myelin sheath in action potential conduction, and the physiological effects of demyelination and remyelination in the central nervous systems. This basic knowledge will also be of significance outside the particular context of spinal trauma.

biomedical equipment purchase;

The goal of this project is to examine the relation between partial remyelination in the injured nervous system and the clinically significant problem of action potential conduction block. These studies will be performed in a guinea pig model of chronic spinal cord injury and will involve intracellular recording from myelinated nerve fibers within strips of spinal cord white matter isolated in artificial media. The physiological responses and conduction properties of these fibers will be recorded and related to the characteristics of their myelin sheaths, as determined by intracellular injection of the marker biocytin and subsequent morphologic reconstruction from serial sections. This information will be used to explore the relation between morphological and physiological changes using computer based mathematical models of action potential conduction. A broader survey of the range of myelination deficits in these white matter tracts will also be performed by conventional means of osmium tetroxide fixation and teasing apart of single fibers. A particular goal of this study will be to identify differences between axons that respond and those that do not respond to the application of 4- aminopyridine (4-AP) with a change in the temperature of conduction block or of frequency-response characteristics. Previous experiments have shown that 4-AP, a potassium channel blocking drug, can restore conduction of impulses in some axons of chronically injured spinal cord that otherwise fail to conduct at physiological temperature. The same drug has also been shown to improve neurologic function in animals and human subjects with chronic spinal cord injury, and may prove to be a useful symptomatic treatment, particularly in some cases of incomplete spinal cord injury. It is important to understand the cell physiological basis of these responses, in order to optimize this approach to reducing functional deficits following injury to the nervous system. The experiments will address the potential role of a number of factors in the relation between changes in the myelination of central axons and their conduction characteristics. These include the dimensions of node and internode, with attendant effects on impedance matching; changes in the resting potential of the nerve fiber and their influence on excitability; alterations in the accumulation of extracellular potassium as a result of action potential transmission; and direct involvement of internodal ion channels exposed to large electrical potential transients as a result of dramatic reductions of myelin sheath thickness. A mathematical model of the myelinated axon will be used to explore the possible interaction between these factors and the blockade of voltage-dependent potassium channels, equivalent to the effects of CAP and other drugs.

The goal of this project is to conduct pre-clinical tests of components of the extracellular matrix molecule tenascin-C that offer potential as therapeutics for enhancing nerve fiber regeneration. This may lead to the development of a treatment for spinal cord injury, a devastating condition for which there are no reparative treatments available. The study is based on a new understanding of how the fhA-D component of tenascinC regulates neurite outgrowth. The project will examine effects of delivering fnA-D recombinant protein, or its active peptide sequence (VFDNFVLK ) to the site of an experimental spinal cord injury. Hemisection of the dorsal thoracic (T9) spinal cord of rats will be used to examine the response of corticospinal and primary afferent nerve fibers to transection and treatment with the protein and peptide. Groups of animals will be assigned to treatment with fnA-D, VFDNFVLK, or buffer delivered intrathecally for two weeks from an implanted osmotic pump. Hind limb function will be monitored with a grid-walking test. The primary measure of treatment effect will be immunohistological analysis of axonal regeneration, using Cholera toxin injected either into the motor cortex or into the sciatic nerves. Effects on regeneration of axons will be examined qualitatively and measured quantitatively. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE