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Peripheral blood neutrophil activity profiles in dogs with spinal cord injuries: a longitudinal analysis

A challenge in treating secondary spinal cord injury (SCI) has been developing agents that can penetrate the central nervous system. Thus, there is an emphasis on targeting peripheral immune responses post‐injury, as it is well recognized that a majority of inflammatory cells within the injured cord originate from the periphery. Limited data in rodents and human SCI shows an increase in circulating neutrophils, enhanced neutrophil oxidative burst activity, and an increase in circulating monocytes. This study was carried out in dogs with naturally‐occurring SCI resulting from intervertebral disk herniation (IVDH) to longitudinally characterize the peripheral post‐injury immune cell populations. These studies are feasible since we can obtain serial blood draws, and can measure clinical outcomes. Texas A&M see approximately 120 cases of canine IVDH associated SCI each year, which are primarily dachshunds breed. This data from dogs can be extremely useful since canine SCI parallels human SCI with respect to identifiable contusion with sustained compression; treatment modalities including decompression and physical rehabilitation; and histopathic, molecular and magnetic resonance lesion phenotype. The objective of our laboratory is to provide a large animal model of naturally‐occurring SCI to recapitulate translational rodent research, where blood and cerebrospinal fluid volumes are too low to analyze longitudinally. This project focuses on identifying and enumerating resting and activated neutrophils in dogs with SCI, peripheral injury (long bone fracture), and in healthy controls recruited from Texas A&M University. Blood draws of 10 mL were collected on the day of admission and 3, 7, 30 and 90 days post‐SCI, and on single day from dogs with long bone fractures and healthy controls. The modified Frankel score (MFS) was used as a coarse measure of injury severity to stratify patients into severe vs. mild‐moderate injury. Isolated neutrophils from peripheral blood were evaluated using commercially available canine and mouse antibodies: CADO48A (canine specific neutrophil marker), CD11b, CD45, and CD62L (L‐ selectin). Neutrophil oxidative burst activity was determined by observing the conversion of non‐fluorescent dihydrorhodamine123 (DHR) to fluorescent rhodamine123 by cellular oxidation. To our knowledge, this is the first time longitudinal analysis of peripheral neutrophil activation has been performed in dogs. Preliminary results from a small number of animals show that there are differences in L‐selectin expression and mean fluorescent intensities of DHR conversion detected after SCI or peripheral injury compared to healthy controls. The increased numbers of CADO48Ahigh/CD62Lhigh cells in SCI dogs compared to peripheral injured dogs could demonstrate these cells as targets for immune therapy, and provide a window for therapeutic intervention. This study demonstrates changes in the peripheral immune cells in an animal model that closely resembles human pathogenesis of neuroinflammation after spinal cord injury.