Mild traumatic brain injury (mTBI), particularly mild "blast type" injuries resulting from improvised exploding devices and many sport-caused injuries to the brain, result in long-term impairment of cognition and behavior. Our central hypothesis is that there are inflammatory consequences to mTBI that persist over time and, in part, are responsible for resultant pathogenesis and clinical outcomes. We used an adaptation (1 atmosphere pressure) of a well-characterized moderate-to-severe brain lateral fluid percussion (LFP) brain injury rat model. Our mild LFP injury resulted in acute increases in interleukin-1α/β and tumor necrosis factor alpha levels, macrophage/microglial and astrocytic activation, evidence of heightened cellular stress, and blood-brain barrier (BBB) dysfunction that were evident as early as 3-6 h postinjury. Both glial activation and BBB dysfunction persisted for 18 days postinjury.

Perinatal hypoxia/ischemia (HI) is a common cause of neurological deficits in children. Interleukin-1 (IL-1) activity has been implicated in HI-induced brain damage. However, the mechanisms underlying its action in HI have not been characterized. We used a 7-day-old rat model to elucidate the role of nuclear factor-jB (NF-jB) activation in HI stimulation of IL-1 signaling. HI was induced by permanent ligation of the left carotid artery followed by 90 min of hypoxia (7.8% O 2 ). Using ELISA assays, we observed increased cell death and caspase 3 activity in hippocampus and cortex 3, 6, 12, 24 and 48 h post-HI. IL-1b protein expression increased, beginning at 3 h after HI and lasting until 24 h post-HI in hippocampus and 12 h post-HI in cortex. Intracerebroventricular injection of 2lg IL-1 receptor antagonist (IL-1Ra) 2 h after HI significantly reduced cell death and caspase 3 activity. Electrophoretic mobility shift assay analyses of hippocampus and cortex after HI for NF-jB activity showed increased p65/p50 DNA-binding activity at 24 h post-HI. Western blot analyses showed significant nuclear translocation of p65. Protein expression levels of two known inflammatory agents, inducible nitric oxide synthase and cycloxygenase 2, known to be transcriptionally regulated by NF-jB, also increased at 24 h after HI. All these HI-induced changes were reversed by IL-1Ra blockade of IL-1 signaling, consistent with IL-1 triggering of inflammatory apoptotic outcomes via NF-jB transcriptional activation. The observed increase in cytoplasmic phosphorylated inhibitor jBa (IjBa) and nuclear translocation of Bcl-3 24 h after HI was also significantly attenuated by IL-1Ra blockade, suggesting that HI-induced IL-1 activation of NF-jB is via both the degradation of IjBa and the nuclear translocation of Bcl-3. Keywords: Bcl-3, hypoxia/ischemia, inhibitor jBa, interleukin-1, nuclear factor-jB. Perinatal hypoxia/ischemia (HI) during gestation/delivery or the accidental asphyxia of infants is a common cause of neurological deficits and delayed cognitive and behavioral deficits (Kaltschmidt et al. 1994). A rapidly expanding body of evidence indicates that inflammatory mediators, including inflammatory cytokines, contribute substantially to the pathogenesis associated with perinatal HI brain injury (Hagberg et al. 1996;Bona et al. 1999;Hedtjarn et al. 2002). The best-characterized early response inflammatory cytokine is interleukin-1 (IL-1) (Szaflarski et al. 1995). The IL-1 family includes the agonists IL-1a and IL-1b, the endogenous receptor antagonist IL-1 receptor antagonist (IL-1Ra), and a newly discovered member, IL-18/IL-1c (Shapiro et al. 1998). IL-1b has consistently been detected in central nervous system after injury to the brain or spinal cord (Rothwell et al. 1997;Nesic et al. 2001). There is also evidence showing a correlation between increased IL-1b levels and subsequent neurodegeneration. Thus, administration of exogenous IL-1b markedly exacerbates neuronal/glial damage in rodents exposed to focal cerebral ischemia or...

Cysteinyl-S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins due to physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia reperfusion model, and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique’s power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.

Perinatal hypoxia/ischemia (HI) is a common cause of neurological deficits in children. Our goal was to elucidate the underlying mechanisms that contribute to the neurological sequelae of HI-induced brain injury. HI was induced by permanent ligation of the left carotid artery followed by 90 min of hypoxia (7.8% O2) in female P7 rats. A two-dimensional differential proteome analysis was used to assess changes in protein expression in cortex 2 h after HI. In total, 17 proteins reflecting a 2-fold or higher perturbation of expression after HI as compared to sham-treated pups were identified by mass spectrometry. Of the altered proteins, 14-3-3epsilon and TUC-2, both playing an important role in the development of the central nervous system, decrease after HI, consistent with an early disturbance of cortical development. Also affected, DARPP-32 and alpha-synuclein, two proteins important for dopamine neurotransmission, increased more than 2-fold 2 h after HI injury. The differential expression of these proteins was validated by individual Western blot assays. The expression of several metabolic enzymes and translational factors was also perturbed early after HI brain injury. These findings provide initial insights into the mechanisms underlying neurodegenerative events after HI and may allow for the rational design of therapeutic strategies that enhance neuronal adaptation and compensation after HI.