Quantifying acute changes in neurometabolism following blast-induced traumatic brain injury.

Brain health is largely dependent on the metabolic regulation of amino acids. Brain injuries, diseases, and disorders can be detected through alterations in free amino acid (FAA) concentrations; and thus, mapping the changes has high diagnostic potential. Common methods focus on optimizing neurotransmitter quantification; however, recent focus has expanded to investigate the roles of molecular precursors in brain metabolism. An isocratic method using high performance liquid chromatography with electrochemical cell detection was developed to quantify a wide range of molecular precursors and neurotransmitters: alanine, arginine, aspartate, serine, taurine, threonine, tyrosine, glycine, glutamate, glutamine, and γ-Aminobutyric acid (GABA) following traumatic brain injury. First, baseline concentrations were determined in the serum, cerebrospinal fluid, hippocampus, cortex, and cerebellum of naïve male Sprague Dawley rats. A subsequent study was performed investigating acute changes in FAA concentrations following blast-induced traumatic brain injury (bTBI). Molecular precursor associated FAAs decreased in concentration at 4 h after injury in both the cortex and hippocampus while those serving as neurotransmitters remained unchanged. In particular, the influence of oxidative stress on the observed changes within alanine and arginine pathways following bTBI should be further investigated to elucidate the full therapeutic potential of these molecular precursors at acute time points.

Spatial Intracranial Pressure Fields Driven by Blast Overpressure in Rats.

Free-field blast exposure imparts a complex, dynamic response within brain tissue that can trigger a cascade of lasting neurological deficits. Full body mechanical and physiological factors are known to influence the body's adaptation to this seemingly instantaneous insult, making it difficult to accurately pinpoint the brain injury mechanisms. This study examined the intracranial pressure (ICP) profile characteristics in a rat model as a function of blast overpressure magnitude and brain location. Metrics such as peak rate of change of pressure, peak pressure, rise time, and ICP frequency response were found to vary spatially throughout the brain, independent of blast magnitude, emphasizing unique spatial pressure fields as a primary biomechanical component to blast injury. This work discusses the ICP characteristics and considerations for finite element models, in vitro models, and translational in vivo models to improve understanding of biomechanics during primary blast exposure.

Impaired orthotopic glioma growth and vascularization in transgenic mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia among the aging population and is characterized pathologically by the progressive intracerebral accumulation of beta-amyloid (Abeta) peptides and neurofibrillary tangles. The level of proangiogenic growth factors and inflammatory mediators with proangiogenic activity is known to be elevated in AD brains which has led to the supposition that the cerebrovasculature of AD patients is in a proangiogenic state. However, angiogenesis depends on the balance between proangiogenic and antiangiogenic factors and the brains of AD patients also show an accumulation of endostatin and Abeta peptides which have been shown to be antiangiogenic. To determine whether angiogenesis is compromised in the brains of two transgenic mouse models of AD overproducing Abeta peptides (Tg APPsw and Tg PS1/APPsw mice), we assessed the growth and vascularization of orthotopically implanted murine gliomas since they require a high degree of angiogenesis to sustain their growth. Our data reveal that intracranial tumor growth and angiogenesis is significantly reduced in Tg APPsw and Tg PS1/APPsw mice compared with their wild-type littermates. In addition, we show that Abeta inhibits the angiogenesis stimulated by glioma cells when cocultured with human brain microvascular cells on a Matrigel layer. Altogether our data suggest that the brain of transgenic mouse models of AD does not constitute a favorable environment to support neoangiogenesis and may explain why vascular insults synergistically precipitate the cognitive presentation of AD.

Chronic Anxiety- and Depression-Like Behaviors Are Associated With Glial-Driven Pathology Following Repeated Blast Induced Neurotrauma.

Long-term neuropsychiatric impairments have become a growing concern following blast-related traumatic brain injury (bTBI) in active military personnel and Veterans. Neuropsychiatric impairments such as anxiety and depression are common comorbidities that Veterans report months, even years following injury. To understand these chronic behavioral outcomes following blast injury, there is a need to study the link between anxiety, depression, and neuropathology. The hippocampus and motor cortex (MC) have been regions of interest when studying cognitive deficits following blast exposure, but clinical studies of mood disorders such as major depressive disorder (MDD) report that these two regions also play a role in the manifestation of anxiety and depression. With anxiety and depression being common long-term outcomes following bTBI, it is imperative to study how chronic pathological changes within the hippocampus and/or MC due to blast contribute to the development of these psychiatric impairments. In this study, we exposed male rats to a repeated blast overpressure (~17 psi) and evaluated the chronic behavioral and pathological effects on the hippocampus and MC. Results demonstrated that the repeated blast exposure led to depression-like behaviors 36 weeks following injury, and anxiety-like behaviors 2-, and 52-weeks following injury. These behaviors were also correlated with astrocyte pathology (glial-fibrillary acid protein, GFAP) and dendritic alterations (Microtubule-Associated Proteins, MAP2) within the hippocampus and MC regions at 52 weeks. Overall, these findings support the premise that chronic glial pathological changes within the brain contribute to neuropsychiatric impairments following blast exposure.

Glial Activation in the Thalamus Contributes to Vestibulomotor Deficits Following Blast-Induced Neurotrauma.

Vestibular impairment has become a frequent consequence following blast-related traumatic brain injury (bTBI) in military personnel and Veterans. Behavioral outcomes such as depression, fear and anxiety are also common comorbidities of bTBI. To accelerate pre-clinical research and therapy developments, there is a need to study the link between behavioral patterns and neuropathology. The transmission of neurosensory information often involves a pathway from the cerebral cortex to the thalamus, and the thalamus serves crucial integrative functions within vestibular processing. Pathways from the thalamus also connect with the amygdala, suggesting thalamic and amygdalar contributions to anxiolytic behavior. Here we used behavioral assays and immunohistochemistry to determine the sub-acute and early chronic effects of repeated blast exposure on the thalamic and amygdala nuclei. Behavioral results indicated vestibulomotor deficits at 1 and 3 weeks following repeated blast events. Anxiety-like behavior assessments depicted trending increases in the blast group. Astrogliosis and microglia activation were observed upon post-mortem pathological examination in the thalamic region, along with a limited glia response in the amygdala at 4 weeks. These findings are consistent with a diffuse glia response associated with bTBI and support the premise that dysfunction within the thalamic nuclei following repeated blast exposures contribute to vestibulomotor impairment.

Connexin 43 Inhibition Sensitizes Chemoresistant Glioblastoma Cells to Temozolomide.

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ-blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM, and perhaps other TMZ-resistant cancers.

A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications.

The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.

A diphtheria toxin negative selection in RNA interference screening.

RNA interference (RNAi) screening is a powerful technique for understanding the molecular biology of cancer and searching drug targets. Genes and their upstream activators that are essential for the survival of cancer cells often dictate cancer formation/progression. Hence, they are preferable therapeutic targets. Identifying these genes using RNAi is, however, problematic because knocking them down leads to cell death. Here we describe a diphtheria toxin (DT) negative selection method to circumvent the problem of cell death in RNAi screening. DT fails to kill mouse cells due to the lack of functional DT receptor (DTR). Thus, we first prepare a construct encoding a human functional DTR driven by the promoter of mouse Atf5, a gene essential for the survival of malignant glioma. Then a DT-sensitive mouse malignant glioma cell line is established by over-expressing this DTR. Finally, an RNAi screen is performed in this cell line and genes that activate Atf5 expression are identified. The negative selection approach described here allows RNAi screening to be used for identifying genes controlling cell survival in cancers or perhaps other human diseases with potential in therapeutic intervention.