Inflammation plays a critical role in secondary damage after spinal cord injury (SCI). Currently, there is no widely accepted, FDA approved, therapeutic for mitigating inflammation following SCI. L-selectin is an adhesion receptor that facilitates recruitment of leukocytes into sites of inflammation. Preliminary data in the Noble-Haeusslein lab show improved sparing and long-term recovery after SCI in L-selectin knockout or wild- type mice treated with diclofenac acid (DFA), a non-steroidal anti-inflammatory drug (NSAID) that induces L- selectin shedding via cleavage at the membrane proximal domain. DFA was effective when administered immediately and at 3 hours, but not at 8 hours, post-SCI. L-selectin, therefore, represents a potential therapeutic target to reduce secondary damage in the acutely injured spinal cord. However, the effect of L- selectin shedding on the recruitment of specific leukocyte subsets remains undefined. The hypothesis of this proposal is that L-selectin shedding, through cleavage at the membrane proximal domain, reduces the recruitment of pro-inflammatory subsets of leukocytes following SCI. The objectives are to determine the effect of L-selectin shedding on recruitment of specific leukocyte subsets, confirm that DFA achieves it beneficial effects via L-selectin shedding, and identify a new candidate therapeutic for future studies.
Specific Aim 1 will test the hypothesis that L-selectin shedding reduces infiltration of specific subsets of leukocytes into the acutely injured spinal cord. Flow cytometry will be performed up to 72 hours post-SCI in wild-type (WT) and L-selectin knockout (KO) mice treated with DFA or a vehicle control at 3 hours post-injury. In vivo imaging will be utilized to observe the behavior of immunolabeled leukocyte populations in vessels in the acutely injured spinal cord.
Specific Aim 2 will test the hypothesis that benefit of DFA is specific to shedding of L-selectin at the membrane proximal domain. Leukocyte infiltration will be quantified by flow cytometry up to 72 hours post-SCI in L(E)-Same mice that lack the cleavage site in the membrane proximal domain of L-selectin, rendering leukocytes resistant to L-selectin shedding. Long-term neurological recovery will be measured using the Basso Mouse Scale (BMS) and grid walk tests to determine if the effect of DFA is abolished in L(E)-Same mice.
Specific Aim 3 will test the hypothesis that N-phenylanthranalic acid, an NSAID with an improved safety profile compared to DFA, induces L-selectin shedding and improves long-term recovery after SCI. WT mice will be treated with N-phenylanthranalic acid at 3 hours post-SCI. L-selectin shedding will be quantified by flow cytometry and ELISA up to 72 hours post-injury. Leukocyte infiltration will be assessed using flow cytometry and long-term neurological recovery will be measured based on the BMS. The collective results will help uncover the role of L-selectin in recruitment of specific leukocyte populations after SCI and validate L- selectin shedding as a therapeutic strategy in the acutely injured spinal cord. The findings from this proposal may be applicable to other central nervous system disorders marked by damaging inflammation.
Spinal cord injury is a severely debilitating event with limited effective treatment options for the patient beyond rehabilitation. The proposed research will examine the contribution of the adhesion receptor, L-selectin, to recruitment of leukocytes into the acutely injured spinal cord and subsequent long-term recovery. This work will define mechanisms underlying L-selectin-dependent leukocyte recruitment and validate a novel therapeutic strategy based on disrupting L-selectin function.
