Temporal relationship of peroxynitrite-induced oxidative damage, calpain-mediated cytoskeletal degradation and neurodegeneration after traumatic brain injury

Abstract

We assessed the temporal and spatial characteristics of PN-induced oxidative damage and its relationship to calpain-mediated cytoskeletal degradation and neurodegeneration in a severe unilateral controlled cortical impact (CCI) traumatic brain injury (TBI) model. Quantitative temporal time course studies were performed to measure two oxidative damage markers: 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) at 30 min, 1, 3, 6, 12, 24, 48, 72 h and 7 days after injury in ipsilateral cortex of young adult male CF-1 mice. Secondly, the time course of Ca⁺⁺-activated, calpain-mediated proteolysis was also analyzed using quantitative western-blot measurement of breakdown products of the cytoskeletal protein α-spectrin. Finally, the time course of neurodegeneration was examined using de Olmos silver staining. Both oxidative damage markers increased in cortical tissue immediately after injury (30 min) and elevated for the first 3–6 h before returning to baseline. In the immunostaining study, the PN-selective marker, 3NT, and the lipid peroxidation marker, 4HNE, were intense and overlapping in the injured cortical tissue. α-Spectrin breakdown products, which were used as biomarker for calpain-mediated cytoskeletal degradation, were also increased after injury, but the time course lagged behind the peak of oxidative damage and did not reach its maximum until 24 h post-injury. In turn, cytoskeletal degradation preceded the peak of neurodegeneration which occurred at 48 h post-injury. These studies have led us to the hypothesis that PN-mediated oxidative damage is an early event that contributes to a compromise of Ca⁺⁺ homeostatic mechanisms which causes a massive Ca⁺⁺ overload and calpain activation which is a final common pathway that results in post-traumatic neurodegeneration.

Introduction

There is compelling evidence supporting the role of oxidative damage in the delayed secondary neuronal cell death which is initiated by the primary traumatic brain injury (TBI). The first work in this regard, conducted by Kontos and his colleagues, demonstrated the formation of superoxide radicals in cerebral microvasculature in a cat fluid percussion TBI models (Kontos and Povlishock, 1986, Kontos and Wei, 1986). Using the salicylate trapping method, Hall and coworkers detected a rapid, but transient rise in brain hydroxyl radical (OH) in experimental head injury models (Hall et al., 1993, Hall et al., 1994, Smith et al., 1994). This was elegantly confirmed by others using brain microdialysis techniques to monitor salicylate-trapped OH over time in the same animals (Globus et al., 1995). The increase of OH is followed by an increase in lipid peroxidation products (Smith et al., 1994). The strongest support for a role of oxidative damage in the acute pathophysiology of TBI is derived from the fact that several antioxidant compounds have been shown to be neuroprotective in TBI models (Hall et al., 1994, Awasthi et al., 1997, Mori et al., 1998, Marklund et al., 2001).

The reactive oxygen species (ROS) peroxynitrite (PN), which can produce highly reactive and cytotoxic free radicals, has been suggested to be a key player in post-traumatic secondary brain oxidative damage (Hall et al., 1999, Hall et al., 2004). Peroxynitrite is formed by the chemical reaction of nitric oxide (NO), produced by nitric oxide synthase (NOS), with superoxide radical (O₂⁻) whenever the two are produced in close proximity (Saran et al., 1990). All three NOS isoforms (eNOS, iNOS, and nNOS), as a key element in production of PN, have been shown to be up-regulated in brain tissue following TBI (Cobbs et al., 1997, Wada et al., 1998, Gahm et al., 2000, Orihara et al., 2001). In addition, a novel isoform of Ca⁺⁺-sensitive NOS (mtNOS), discovered within mitochondria (Bates et al., 1995, Lopez-Figueroa et al., 2000, Giulivi, 2003), has been shown to contribute to mitochondrial production of NO and PN (Radi et al., 2002). The PN-derived free radicals (NO₂, OH, CO₃⁻) can induce extensive oxidative damage to cellular membranes, proteins and DNA (Beckman, 1996, Murphy et al., 1998, Radi, 1998). Each of the PN-derived free radicals can initiate lipid peroxidation (LP), or cause protein carbonylation by reaction with susceptible amino acids (e.g. lysine, cysteine, arginine). Moreover, aldehydic LP products (e.g. 4-hydroxynonenal) can bind to cellular proteins compromising their structural and functional integrity (Neely et al., 1999). Additionally, NO₂ can nitrate 3 positions of tyrosine residues in proteins. As a result, 3-nitrotyrosine (3NT) is used as a specific footprint of PN-induced cellular damage (Hall et al., 1997).

In recent work we investigated the role of PN in a mouse model of diffuse closed head injury which demonstrated the spatial and temporal coincidence of PN-induced protein nitration and lipid peroxidation and that this oxidative damage precedes, and therefore may have a causal role in post-traumatic neurodegeneration (Hall et al., 2004). However, TBI is a complex disorder that is impossible to fully reproduce in a single model. For example, we have found significant differences between the time courses of neurodegeneration in mouse models of diffuse (Kupina et al., 2003) and focal (Hall et al., 2005a, Hall et al., 2005b) TBI. Hence, the magnitude and timing of post-traumatic secondary injury mechanisms probably vary across head injury models. Most importantly, if we are to accurately test the efficacy of novel neuroprotective pharmacological treatments, it is important to first understand the time course of the relevant secondary injury mechanisms in models of focal as well as diffuse TBI.

Thus, the present study was conducted to examine the temporal and spatial characteristics of PN-induced cortical oxidative damage in a model of severe controlled cortical impact (CCI) focal TBI, and its relationship to calpain-mediated cytoskeletal degradation and neurodegeneration. Markers for PN-induced protein nitration (3-nitrotyrosine, 3NT) and lipid peroxidation (4-hydroxynonenal, 4HNE) were measured using immuno-slotblotting and immuno-histochemistry. Secondly, we employed western-blotting methods to look at the time course of calpain-mediated cytoskeletal degradation in order to assess the temporal relationship of oxidative damage to Ca⁺⁺−mediated proteolytic degradation. Finally, we used de Olmos silver staining to measure the evolution of post-traumatic neurodegeneration. The results demonstrate that the PN-mediated oxidative damage is an early event that probably contributes to an exacerbation of neuronal intracellular Ca⁺⁺ overload, massive calpain activation and cytoskeletal degradation and neurodegeneration.