We propose that L-selectin, which is expressed on all leukocytes in the blood, may have a pathophysiologic role in demyelination after spinal cord injury. Axonal demyelination is a significant pathologic feature of spinal cord injury and contributes to functional impairment. Myelin loss in fiber tracts is not restricted to initially damaged axons but rather occurs in intact axonal tracts. These fiber tracts are often dysfunctional because of this progressive demyelination. Furthermore, this demyelination is not accompanied by extensive remyelination. We hypothesize that L-selectin, a leukocyte cell adhesion receptor, modulates demyelination after spinal cord injury by directly targeting infiltrating leukocytes to myelinated fiber tracts. Leukocyte/myelin interactions generate focal oxidative stress/injury contributing to demyelination. Collectively, these events limit the extent of functional recovery.
Three aims are proposed to test this hypothesis. In Specific Aim 1, we will compare leukocyte infiltration in the injured cord of L-selectin null and wild-type mice. Flow cytometry will be used to identify and quantify specific leukocyte populations, including neutrophils and macrophages, that have infiltrated into the injured cord. We predict equivalent levels of infiltration in the two types of mice but significantly reduced demyelination in the null mice. If this result is obtained, we will attempt to determine which leukocyte population is involved in demyelination through adoptive transfer experiments. Isolated leukocytes from L-selectin wild-type animals will be transferred into null mice just before spinal cord injury.
In Specific Aim 2, we will compare demyelination and evaluate locomotor recovery after spinal cord injury in L-selectin null and wild-type mice. Quantitative light and ultrastructural studies will be used to evaluate the extent of demyelination and axonal pathology. Functional recovery will be assessed over a period of 6 weeks post injury using complementary approaches that examine performance on a rotorod and an inclined grid and coordinated movement in an open field.
In Specific Aim 3, we will compare oxidative stress stress in white matter regions undergoing demyelination in L-selectin null and wild-type mice. Homogenates of spinal cord will be prepared for analysis of either lipid peroxidation (4-hydroxyalkenals), glutamine synthetase, and protein carbonyls. Double immunolabeling will be used to study the regional relationship between 4-hydroxynonenal/protein complex, a major aldehydic product formed by peroxidation of unsaturated fatty acids, protein carbonyls, and the presence of infiltrating leukocytes.
Together, these studies offer a focused effort to better understand the direct role that L-selectin plays in demyelination after spinal cord injury. The long term objective of these studies is to develop improved therapies for the treatment of the spinal cord injured patient. The proposed studies represent an important first step toward accomplishing this objective.