The temporal sequence combined with the recently demonstrated role of CL hydroperoxides (CL-OOH) in in vitro models of apoptosis suggest that CL-OOH may be both a key in vivo trigger of apoptotic cell death and a therapeutic target in experimental traumatic brain injury.

Manganese superoxide dismutase (MnSOD) provides the first line of defense against superoxide generated in mitochondria. SOD competes with nitric oxide for reaction with superoxide and prevents generation of peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. Thus, sufficient amounts of catalytically competent MnSOD are required to prevent mitochondrial damage. Increased nitrotyrosine immunoreactivity has been reported after traumatic brain injury (TBI); however, the specific protein targets containing modified tyrosine residues and functional consequence of this modification have not been identified. In this study, we show that MnSOD is a target of tyrosine nitration that is associated with a decrease in its enzymatic activity after TBI in mice. Similar findings were obtained in temporal lobe cortical samples obtained from TBI cases versus control patients who died of causes not related to CNS trauma. Increased nitrotyrosine immunoreactivity was detected at 2 h and 24 h versus 72 h after experimental TBI and co-localized with the neuronal marker NeuN. Inhibition and/or genetic deficiency of neuronal nitric oxide synthase (nNOS) but not endothelial nitric oxide synthase (eNOS) attenuated MnSOD nitration after TBI. At 24 h after TBI, there was predominantly polymorphonuclear leukocytes accumulation in mouse brain whereas macrophages were the predominant inflammatory cell type at 72 h after injury. However, a selective inhibitor or genetic deficiency of inducible nitric oxide synthase (iNOS) failed to affect MnSOD nitration. Nitration of MnSOD is a likely consequence of peroxynitrite within the intracellular milieu of neurons after TBI. Nitration and inactivation of MnSOD could lead to selfamplification of oxidative stress in the brain progressively enhancing peroxynitrite production and secondary damage.

Traumatic brain injury (TBI) from blast injury is often complicated by hemorrhagic shock (HS) in victims of terrorist attacks. Most studies of HS after experimental TBI have focused on intracranial pressure; few have explored the effect of HS on neuronal death after TBI and none have been done in mice. We hypothesized that neuronal death in CA1 hippocampus would be exacerbated by HS after experimental TBI. C57BL6J male mice were anesthetized with isoflurane, mean arterial blood pressure (MAP) was monitored, and controlled cortical impact (CCI) delivered to the left parietal cortex followed by 1) continued anesthesia (CCI only), or either 60 or 90 min of volume controlled HS. Parallel 60 or 90 min HS only groups were also studied. After HS (± CCI), 6% hetastarch was used targeting MAP ≥50 mmHg during a 30 min Pre-Hospital resuscitation phase. Then, shed blood was re-infused and hetastarch given targeting MAP ≥60 mmHg during a 30 min Definitive Care phase. Neurological injury was evaluated at 24h (fluorojade C) or 7 days (CA1 and CA3 hippocampal neuron counts). HS reduced MAP to 30−40 mmHg in all groups, p<0.05 vs CCI only. Ipsilateral CA1 neuron counts in the 90 min CCI+HS group were reduced at 16.5±14.1 vs 30.8±6.8, 32.3±7.6, 30.6±2.2, 28.1±2.2 neurons/100 Im in CCI only, 60 min HS only, 90 min HS only, and 60 min CCI+HS, respectively, all p<0.05. CA3 neuron counts did not differ between groups. Fluorojade C staining confirmed neurodegeneration in CA1 in the 90 min CCI+HS group. Our data suggest a critical time window for exacerbation of neuronal death by HS after CCI and may have implications for blast injury victims in austere environments where definitive management is delayed.

We reported that adenosine A(1) receptor (A(1)AR) knockout (KO) mice develop lethal status epilepticus after experimental traumatic brain injury (TBI), which is not seen in wild-type (WT) mice. Studies in epilepsy, multiple sclerosis, and neuro-oncology suggest enhanced neuro-inflammation and/or neuronal death in A(1)AR KO. We hypothesized that A(1)AR deficiency exacerbates the microglial response and neuronal damage after TBI. A(1)AR KO and WT littermates were subjected to mild controlled cortical impact (3 m/sec; 0.5 mm depth) to left parietal cortex, an injury level below the acute seizure threshold in the KO. At 24 h or 7 days, mice were sacrificed and serial sections prepared. Iba-1 immunostaining was used to quantify microglia at 7 days. To assess neuronal injury, sections were stained with Fluoro-Jade C (FJC) at 24 h to evaluate neuronal death in the hippocampus and cresyl violet staining at 7 days to analyze cortical lesion volumes. We also studied the effects of adenosine receptor agonists and antagonists on (3)H-thymidine uptake (proliferation index) by BV-2 cells (immortalized mouse microglial). There was no neuronal death in CA1 or CA3 quantified by FJC. A(1)AR KO mice exhibited enhanced microglial response; specifically, Iba-1 + microglia were increased 20-50% more in A(1)AR KO versus WT in ipsilateral cortex, CA3, and thalamus, and contralateral cortex, CA1, and thalamus (p < 0.05). However, contusion and cortical volumes did not differ between KO and WT. Pharmacological studies in cultured BV-2 cells indicated that A(1)AR activation inhibits microglial proliferation. A(1)AR activation is an endogenous inhibitor of the microglial response to TBI, likely via inhibition of proliferation, and this may represent a therapeutic avenue to modulate microglia after TBI.