Elsevier

Experimental Neurology

Volume 215, Issue 2, February 2009, Pages 308-316
Experimental Neurology

Increased oxidative activity in human blood neutrophils and monocytes after spinal cord injury

https://doi.org/10.1016/j.expneurol.2008.10.022Get rights and content

Abstract

Traumatic injury can cause a systemic inflammatory response, increasing oxidative activity of circulating leukocytes and potentially exacerbating the original injury, as well as causing damage to initially unaffected organs. Although the importance of intraspinal inflammation after human spinal cord injury is appreciated, the role of the systemic inflammatory response to this injury is not widely recognised. We investigated oxidative activity of blood leukocytes from nine cord-injured subjects and six trauma controls (bone fractures without CNS injury) at 6 h–2 weeks after injury, comparing values to those of ten uninjured subjects. Neutrophil and monocyte free radical production, evaluated by flow cytometry, increased significantly more in cord injury subjects than in trauma controls (6-fold vs 50% increases). In leukocyte homogenates, the concentration of free radicals increased significantly more in cord injury subjects (2-fold) than in the trauma controls (1.6-fold) as did activity of myeloperoxidase (2.3-fold vs. 1.7-fold). Moreover, in homogenates and blood smears, expression of the NADPH oxidase subunit gp91phox and of the oxidative enzyme, inducible nitric oxide synthetase was 20–25% greater in cord injury subjects than in trauma controls. Expression of the pro-inflammatory transcription factor NF-κB and of cyclooxygenase-2 increased similarly after both injuries. Finally, aldehyde products of tissue-damaging lipid peroxidation also increased significantly more in the plasma of spinal cord injury subjects than in trauma controls (2.6 fold vs. 1.9-fold). Spinal cord injury causes a particularly intense systemic inflammatory response. Limiting this response briefly after cord injury should protect the spinal cord and tissues/organs outside the CNS from secondary damage.

Introduction

Primary spinal cord injury (SCI) is followed by secondary damage at the injury site, in which inflammation and a large intraspinal influx of leukocytes play an important role (Blight, 1992, Fleming et al., 2006, Saville et al., 2004, Taoka and Okajima, 1998, Tator and Fehlings, 1991). In response to SCI, the release of leukocytes from marginal pool stores and the bone marrow into the circulation increases the number of cells available to cause damage (Furlan et al., 2006). The state of these leukocytes will impact on their actions upon entering the injured cord. Leukocytosis is part of the well-known systemic inflammatory response to physical trauma; leukocyte activation consists of increased oxidative activity, phagocytic behaviour and migration (Bhatia et al., 2005, Shih et al., 1999, Tanaka et al., 1991). This inflammatory response can exacerbate the primary cord injury and also cause bystander damage to organs and tissues unaffected by the original injury (Baskaran et al., 2000, Bhatia et al., 2005, Utagawa et al., 2008). Animal studies have shown that CNS injury causes a more intense systemic inflammatory response than general trauma, with ensuing damage to organs such as the lungs and liver (Campbell et al., 2003, Gris et al., 2008). Although inflammation is a recognized source of secondary damage to the injured cord (Fleming et al., 2006), the intensity and character of a systemic inflammatory response to human SCI have not been studied.

What are the destructive characteristics of activated circulating inflammatory cells? They may upregulate expression of oxidative enzymes, proteases and other potentially damaging molecules such as pro-inflammatory cytokines (Wang et al., 1997). Indeed, rat studies have confirmed that SCI causes a neutrophilia and changes the character of neutrophils, priming them for greater oxidative burst activity (Gris et al., 2008). An increased capacity for oxidative burst in neutrophils, within hours of SCI in rats, can be induced by circulating pro-inflammatory cytokines and chemokines (Campbell et al., 2005, Wang et al., 1997), as well as by molecules such as platelet activating factor (Botha et al., 1996). Upon entry into the injured cord, these leukocytes would be primed to produce an oxidative burst, releasing proteolytic enzymes and inducing or extending the damage.

Although the time-course and other characteristics of the inflammatory leukocyte infiltration into the injured human spinal cord have been studied (Fleming et al., 2006), the impact of SCI on these cells while still in the circulation is less well understood. Using flow cytometry, western blotting, biochemical analyses and immunohistochemical staining, we assessed the presence of reactive oxygen species, expression of a pro-inflammatory transcription factor, expression/activity of oxidative enzymes, and the concentration of products of cell membrane damage (lipid peroxidation) in blood samples obtained from SCI patients. Results were compared to those from a control group who suffered orthopaedic trauma with no CNS injury and from uninjured able-bodied subjects.

Section snippets

Patient enrollment

These studies were approved by the University of Western Ontario Research Ethics Board for the Review of Health Sciences Research Involving Human Subjects. Venous blood samples were obtained from twenty-five subjects after consent was obtained according to the Declaration of Helsinki. Exclusion criteria were polytrauma, a personal or family history of peripheral neuropathy or autoimmune disease, significant cognitive limitations, malignant cancer within five years of the study, chronic liver

Leukocyte counts and oxidative burst after SCI

The total leukocyte counts at 3.5 ± 1 h after injury in SCI (17.1 ± 1.4 × 109 cells/L) or trauma control (14.7 ± 1.2 × 109 cells/L) subjects were greater than normal (4–10 × 109 cells/L). This increase was caused by a neutrophilia as neutrophil counts at this time after SCI were 14.8 ± 1.3 × 109 cells/L and in trauma controls they were 12.6 ± 0.9 × 109 cells/L, compared to normal (2–7.5 × 109 cells/L). In contrast, the monocyte counts in SCI and trauma controls (0.8 ± 0.1 × 109 and 0.7 ± 0.1 × 109 cells/L, respectively)

Discussion

Following traumatic SCI, neutrophils and then monocyte/macrophages infiltrate the lesion from the circulation (Fleming et al., 2006). Our study reveals that insult to the spinal cord caused a particularly intense induction of oxidative burst and associated enzymes in the circulating leukocytes, significantly greater than that in the trauma control subjects. The highly activated neutrophils and monocytes after SCI could play a significant role in the secondary damage that occurs within and

Acknowledgments

We especially thank all of the volunteers and patients for contributing to this study. We appreciate that the SCI and trauma subjects made the decision to contribute to this research, and permitted the blood samples to be drawn, under circumstances when their own personal needs were very great. We also thank all of the fellows, nurses and staff at the University Health Sciences Centre Victoria Hospital Campus and Robarts Research Institute who facilitated our work by coordinating the timely

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