Targeting L-Selectin to Improve Neurologic and Urologic Function After Spinal Cord Injury

Abstract

We are evaluating the efficacy of diclofenac (DFA), an anti-inflammatory agent with L-Selectin sheddase activity, in a murine model of spinal cord injury. Scope: These studies have focused on the efficacy of DFA in the context of dose, optimal therapeutic window, and dependency on injury severity, using clinically relevant outcome measures that include neurologic assessments and assays of bladder function. Major findings:-Mice with spinal cord injury (SCI) were administered a range of doses of DFA (60mg/kg, 40mg/kg, 20mg/kg, 10mg/kg, 5mg/kg, 1mg/kg, or vehicle) and L-selectin sheddase activity was monitored at 8 hours, 1 day, 3 days, and 7 days using ELISA. 40mg/kg and 60mg/kg DFA were the minimally effective doses to induce L-selectin sheddase activity, which was detected at 8 and 24 hours. Mice were immediately administered vehicle, 40mg/kg DFA, or 60mg/kg DFA after a mild SCI. All mice showed similar marked neurological improvement over time, based upon traditional assessments of neurological function (coordination and kinematics), consistent with the natural recovery that accompanies mild forms of SCI. Importantly, neurologic recovery was improved in mice receiving 40mg/kg, based upon assessment of the percentage of mice frequently/consistently plantar stepping. These encouraging findings not only highlight efficacy of DFA at 40mg/kg after SCI, but reveal a sensitive assay for recovery of function after mild injury that is critical to the spinal cord injured patient; namely the ability to plantar step. Subsequent studies will utilize this assay as well as standard assays of neurological function to assess efficacy in paradigms of moderate and severe injury.. Significance: We can reliably monitor L-selectin sheddase activity in response to DFA administration. This will allow for the robust detection of L-selectin sheddase activity during Task 4a of Specific Aim 4. We identified the minimal effective dose of DFA, 40mg/kg, which induces L-selectin shedding. Utilizing this dose, we demonstrated neurologic recovery after immediate administration to mice receiving mild SCI, using an assay that critically assesses the ability to step. This finding, together with earlier results showing efficacy of 40mg/kg DFO after a moderate SCI, reinforce the candidacy of DFA as a therapeutic for the spinal cord injured patient. Given these successes, we are optimally positioned to proceed with Specific Aim 2, where we will evaluate the window and duration of efficacy after a moderate SCI. Finally, we have established protocols for assessing urological function that will be utilized for Specific Aim 3.