Evidence that infiltrating neutrophils do not release reactive oxygen species in the site of spinal cord injury

Abstract

The release of reactive oxygen species (ROS) by neutrophils, which infiltrate the region of damage following spinal cord injury (SCI), was investigated to determine if such release is significant following spinal cord injury. The relationship of extracellular levels of hydroxyl radicals and hydrogen peroxide obtained by microdialysis sampling and oxidized protein levels in tissue to neutrophil infiltration following spinal cord injury was examined. Neither of the reactive oxygen species were elevated in the site of spinal cord injury relative to their concentrations in normal tissue at a time (24 h) when the numbers of neutrophils were maximum in the site of injury. Surprisingly, ablation with a neutrophil antiserum actually increased the level of oxidized proteins in Western blots. Thus, our findings are (1) that neutrophils, which infiltrate the site of damage following a spinal cord injury, do not release detectable quantities of reactive oxygen species; and (2) that the presence of neutrophils reduces the concentrations of oxidized proteins in the site of spinal cord injury. Therefore, release of reactive oxygen species by neutrophils does not contribute significantly to secondary damage following spinal cord injury. Reduced levels of oxidized proteins in the presence of neutrophils may reflect removal of damaged tissue by neutrophils.

Introduction

Severe and permanent crippling caused by spinal cord injury (SCI) is caused in part by processes secondary to the initial insult. Because they are potential targets for clinical intervention, it is important to understand these secondary processes. Possible agents of secondary damage include reactive oxygen species (ROS) (Anderson et al., 1985, Demopoulos et al., 1982, Hall and Braughler, 1993, Liu et al., 1991, Liu, 1993) and infiltrating immune cells (McPhail and Harvath, 1993, Taoka and Okajima, 1998). This study explores whether the latter generate the former following SCI.

Polymorphonuclear leukocytes, which are mostly neutrophils, infiltrate the injured human spinal cord in large numbers (Tator and Koyanagi, 1997). There is evidence that infiltrating immune cells contribute to secondary damage following experimental SCI (Hamada et al., 1996, Taoka et al., 1997a, Taoka et al., 1997b), but there is also evidence that they do not (Dusart and Schwab, 1994, Holtz et al., 1989). Neutrophils may help recovery by removing damaged tissue, but they may simultaneously contribute to secondary damage by attacking viable, functional neurons. Neutrophils may also influence recovery from CNS trauma by summoning macrophages into the damaged tissue. The latter likely aid in the regeneration of peripheral axons by removing damaged tissue (Blight, 1985, Blight, 1992, Stadtman, 1993) and by storing cholesterol from the myelin they ingest and then restore during remyelination of regenerating axons (Boyles et al., 1989). Like neutrophils, macrophages may also worsen damage by attacking healthy neurons (Blight, 1992).

In the respiratory burst, neutrophils convert large quantities of oxygen to the superoxide anion O₂⁻, which they release (Jones, 1993, McCord and Fridovich, 1969, McNeil et al., 1989, Noble et al., 2002). O₂⁻ is a precursor to the much more reactive hydroxyl radical. Products of the respiratory burst in neutrophils oxidize phospholipids in vitro (Zimmerman et al., 1997), so this process may damage viable tissue (Fridovich, 1978). ROS release by neutrophils may participate in the destruction of infectious organisms (Klebanoff, 1974, Tonai et al., 2001) and/or removal of damaged tissue (McNeil et al., 1989, McPhail and Harvath, 1993). Considerable superoxide is generated following head trauma (Kontos and Wei, 1986), cerebral inflammation (Kontos et al., 1992), and SCI (Liu et al., 1998). Administration of various superoxide dismutases, enzymes that convert O₂⁻ to H₂O₂, lowers mortality from compression SCI in rats (Taoka et al., 1995), and recovery from ischemic or traumatic brain injury improves in patients treated with superoxide dismutase conjugated to polyethylene glycol (Muizelaar et al., 1993). In summary, there is considerable evidence that both neutrophil aggregation in the site of trauma and the generation of ROS are harmful following CNS trauma, suggesting that following SCI, neutrophils generate damaging ROS through their respiratory burst.

The only established treatment effective for treating SCI in humans is administration of the glucocorticoid methylprednisolone; this must begin within 8 h after injury (Bracken and Holford, 1993), and the benefits thereof are modest. However, treatment of SCI often cannot commence within 8 h of trauma; and even when it does, adding a second type of treatment effective at longer times might further improve ultimate recovery. In this vein, Taoka and Okajima (1998) have proposed that methylprednisolone treatment might act synergistically with blockers of the actions of neutrophils because methylprednisolone does not influence the activation of neutrophils in their SCI model. Damaging actions of neutrophils could provide a target for delayed treatment of SCI, as neutrophil concentrations are elevated for about 4–48 h after a contusion injury to the rat spinal cord (Carlson et al., 1998, Xu et al., 1991), the type of injury inflicted in the present work. To help develop therapeutic approaches to treating SCI, it is important to characterize better the generation of ROS by neutrophils and their actions following SCI. To this end, we investigated whether ROS generation and protein oxidation correlate with neutrophil infiltration following spinal cord injury. We compared the production of ROS in injured versus normal spinal cord tissue when maximum numbers of neutrophils were present in the site of injury. In addition, we compared the oxidation of proteins following SCI in normal rats to such oxidation in sites of injury in which infiltration of neutrophils was prevented by their removal from the circulation. Whether neutrophils cause secondary damage by generating ROS following CNS trauma is hitherto unaddressed by in vivo experiments.