Temporal and Spatial Dynamics of Peroxynitrite-Induced Oxidative Damage after Spinal Cord Contusion Injury

Abstract

The reactive nitrogen species peroxynitrite (PN) has been suggested to be an important mediator of the secondary oxidative damage that occurs following acute spinal cord injury (SCI). The PN decomposition products nitrogen dioxide (*NO(2)), hydroxyl radical (*OH), and carbonate radical (*CO(3)) are highly reactive with cellular lipids and proteins. In this immunohistochemical study, we examined the temporal (3, 24, and 72 h, and 1 and 2 weeks) and spatial relationships of PN-mediated oxidative damage in the contusion-injured rat thoracic spinal cord (IH device, 200 kdyn, T10) using 3-nitrotyrosine (3-NT), a marker for protein nitration by PN-derived *NO(2) and 4-hydroxynonenal (4-HNE), an indicator of lipid peroxidation (LP) initiated by any of the PN radicals. Minimal 3-NT or 4-HNE immunostaining was seen in sham, non-injured spinal cords. In contrast, both markers showed a substantial increase at 3 h post-injury at the epicenter, that extended throughout the gray matter and into the surrounding white matter. At 24 and 72 h, the oxidative damage expanded circumferentially to involve all but a small rim of white matter tissue at the injury site, and longitudinally as much as 6-9 mm in the rostral and caudal directions. The staining was observed in neuronal soma, axons, and microvessels. At all time points except 3 h, there was no significant difference in the mean rostral or caudal extent of 3-NT and 4-HNE staining. By 1, and more so at 2 weeks, the longitudinal extent of the oxidative damage staining was greatly decreased. The spatial and temporal overlap of 3-NT and 4-HNE staining supports the concept that PN is involved in both damage produced by lipid peroxidation and protein nitration, and that antioxidant agents that target PN or PN-derived radicals should be effective neuroprotectants for acute SCI if administered during the first post-injury hours.

Antioxidant Therapies for Acute Spinal Cord Injury

“…Interestingly, the time course of PN-mediated 3-NT after rat contusion SCI mirrors the timing of LP-related 4-HNE levels in terms of early onset, peak at 24 h, and persistence for at least 7 days, although by day 14 the 3-NT immunostaining has waned much more than that for 4-HNE [22]. A re-examination of FIG.…”

“…A more recent and more extended immunoblotting/immunohistochemical time course study in the rat contusion model has confirmed that the increase in 4-HNE occurs as early as 1 h, peaks at 24 h, and remains significantly elevated for at least 7 days [23]. However, 4-HNE immunohistochemical staining at 14 days after SCI reveals a persistent elevation [22]. The species or type of SCI model does not seem to make a difference, because in a guinea pig compression injury model the 4-HNE levels in injured spinal tissue also peaked at 24 h and persisted for at least 7 days [24].…”

“…An increase in LP that appears in the injured cord within the first hour has also been evidenced in terms of a depletion of endogenous antioxidants, including ascorbic acid [16], alpha-tocopherol (also known as vitamin E) [17], glutathione [18], and ubiquinol-9 and ubiqunol-10 [19]. More contemporary immunohistochemical and immunoblotting methods have been recently used, which reveals an impressive increase in the levels of the LP-derived aldehydic breakdown products 4-HNE [20][21][22][23] and acrolein [24,25].…”

A Calpain Inhibitor Enhances the Survival of Schwann CellsIn Vitroand after Transplantation into the Injured Spinal Cord

“…These include elevated levels of reactive oxygen species, reactive nitrogen species, and nitric oxide (NO; Carrico et al, 2009;Hamada et al, 1996;Liu et al, 1999Liu et al, , 2003Nakahara et al, 2002;Vaziri et al, 2004;Xiong and Hall, 2007). Blocking factors that induce cell death (for example, formation of reactive oxygen species and lipid peroxidation, or inducible nitric oxide synthase [iNOS] activity) can prevent necrosis and increase cell survival, both in vitro and after transplantation into the injured brain (Blasig et al, 2002;Grasbon-Frodl et al, 1996;Matsuda et al, 2005;Nakao et al, 1994).…”

Acute Molecular Perturbation of Inducible Nitric Oxide Synthase with an Antisense Approach Enhances Neuronal Preservation and Functional Recovery after Contusive Spinal Cord Injury

“…Uncontrolled NO production can lead to tissue injury and damage to cellular constituents through the generation of reactive nitrogen species, such as peroxynitrite, which is involved in the nitration of proteins and lipids that prevents their normal functioning (Liu et al, 2001;Rubbo and Radi, 2008;Xiong et al, 2007). Peroxynitrite may also be a major trigger of lipid peroxidation (Radi et al, 1991); after CNS insults studies have shown a similar spatial and temporal localization of 3-nitrotyrosine (3-NT), a marker for protein nitration, and 4-hydroxynonenal (4-HNE), which is used to identify lipid peroxidation following both traumatic brain injury (Hall et al, 2004) and SCI (Carrico et al, 2009). Peroxynitrite has additionally been shown to inhibit mitochondrial respiration (Bolañ os et al, 1995), as well as produce DNA strand breakage, which can trigger cell death through the activation of poly(ADP-ribose) polymerase (PARP; Szabó , 2003).…”