Free radicals are highly reactive molecules implicated in the pathology of traumatic brain injury and cerebral ischemia, through a mechanism known as oxidative stress. After brain injury, reactive oxygen and reactive nitrogen species may be generated through several different cellular pathways, including calcium activation of phospholipases, nitric oxide synthase, xanthine oxidase, the Fenton and Haber-Weiss reactions, by inflammatory cells. If cellular defense systems are weakened, increased production of free radicals will lead to oxidation of lipids, proteins, and nucleic acids, which may alter cellular function in a critical way. The study of each of these pathways may be complex and laborious since free radicals are extremely short-lived. Recently, genetic manipulation of wild-type animals has yielded species that over- or under-express genes such as, copper-zinc superoxide dismutase, manganese superoxide dismutase, nitric oxide synthase, and the Bcl-2 protein. The introduction of the species has improved the understanding of oxidative stress. We conclude here that substantial experimental data links oxidative stress with other pathogenic mechanisms such as excitotoxicity, calcium overload, mitochondrial cytochrome c release, caspase activation, and apoptosis in central nervous system (CNS) trauma and ischemia, and that utilization of genetically manipulated animals offers a unique possibility to elucidate the role of free radicals in CNS injury in a molecular fashion.

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.

Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.

The hippocampal CA1 neurons are selectively vulnerable to global ischemia, and neuronal death occurs in a delayed manner. The threshold of global ischemia duration that induces neuronal death has been studied, but the relationship between ischemia duration and glial death in the hippocampal CA1 area has not been fully studied. We examined neuronal/glial viability and morphological changes in the CA1 subregion after different durations of global ischemia. Global ischemia was induced in Sprague-Dawley rats by 10, 5, and 3 min of bilateral common carotid artery occlusion and hypotension. At 1-56 days after ischemia, the morphological reactions of neurons, astrocytes, oligodendrocytes, and microglia were immunohistochemically evaluated. Most of the hippocampal CA1 pyramidal neurons underwent delayed death at 3 days after 10/5 min of ischemia, but not after 3 min of ischemia. The number of astrocytes gradually declined after 10/5 min of ischemia, and viable astrocytes showed characteristic staged morphological reactions. Oligodendrocytes also showed morphological changes in their processes after 10/5 min of ischemia. Microglia transformed into a reactive form at 5 days only after 10/5 min of ischemia. These data suggest that some morphological changes in glial cells were not dependent on neuronal cell death, but their own reactions to the different severity of ischemia.

Cerebral gene expressions change in response to traumatic brain injury (TBI), and future trauma treatment may improve with increased knowledge about these regulations. We subjected C57BL/6J mice to injury by controlled cortical impact (CCI). At various time points post-injury, mRNA from neocortex and hippocampus was isolated, and transcriptional alterations studied using quantitative real-time polymerase chain reaction (PCR) and gene array analysis. Spatial distribution of enhanced expression was characterized by in situ hybridization. Products of the upregulated transcripts serve functions in a range of cellular mechanisms, including stress, inflammation and immune responses, and tissue remodeling. We also identified increased transcript levels characterizing reactive astrocytes, oligodendrocytes, and microglia, and furthermore, we demonstrated a novel pattern of scattered cell clusters expressing the chemokine Cxcl10. Notably, a sustained increase in integrin alpha X (Itgax), characterizing antigen-presenting dendritic cells, was found with the transcript located to similar cell clusters. In contrast, T-cell receptor alpha transcript showed only a modest increase. The induced P-selectin (Selp) expression level in endothelial cells, and chemokines from microglia, may guide perivascular accumulation of extravasating inflammatory monocytes differentiating into dendritic cells. In conclusion, our study shows that following TBI, secondary injury chiefly involves inflammatory processes and chemokine signaling, which comprise putative targets for pharmaceutical neuroprotection.

The authors have developed a method for routine monitoring of disturbances in brain energy metabolism and extracellular levels of excitatory amino acids using intracerebral microdialysis in 10 patients with subarachnoid hemorrhage. Microdialysis was conducted for periods ranging from 6 to 11 days after ictus. Altogether, 16,054 chemical analyses from 1647 dialysate samples were performed. Concentrations of the energy-related substances lactate, pyruvate, glucose, and hypoxanthine were measured, and the lactate/pyruvate ratio was calculated. The excitatory amino acids glutamate and aspartate were measured. The microdialysis data were matched with computerized tomography findings, clinical course, and outcome. The results support the concepts that microdialysis is a promising tool for chemical monitoring of the human brain and that extracellular fluid levels of lactate, lactate/pyruvate ratio, glucose, hypoxanthine, and glutamate are useful markers of disturbances in brain energy metabolism in neurointensive care patients. These results have generated a working hypothesis that the pattern of these extracellular markers may help differentiate between various causes of energy perturbations, such as hypoxia and different degrees of ischemia. The correlation between the dialysate levels of excitatory amino acids and outcome supports the concept of glutamate receptor overactivation in acute human brain injury.

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Defective Cu,Zn-superoxide dismutase (SOD1) is responsible for some types of amyotrophic lateral sclerosis, and ventral horn motor neurons (VMN) have been shown to die through a mitochondria-dependent apoptotic pathway after chronic exposure to high levels of reactive oxygen species (ROS). VMN are also selectively vulnerable to mild spinal cord injury (SCI); however, the involvement of SOD1, ROS, and apoptosis in their death has not been clarified. Mild compression SCI was induced in SOD1-overexpressing transgenic rats and wild-type littermates. Superoxide production, mitochondrial release of cytochrome c, and activation of caspase-9 were examined, and apoptotic DNA injury was also characterized. In the wild-type animals, increased superoxide production, mitochondrial release of cytochrome c, and cleaved caspase-9 were observed exclusively in VMN after SCI. Subsequently, a majority of VMN (75%) selectively underwent delayed apoptotic cell death. Transgenic animals showed less superoxide production, mitochondrial cytochrome c release, and caspase-9 activation, resulting in death of only 45% of the VMN. These results suggest that the ROS-initiated mitochondrial signaling pathway possibly plays a pivotal role in apoptotic VMN death after SCI and that increased levels of SOD1 in VMN reduce oxidative stress, thereby attenuating the activation of the pathway and delayed cell death.