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Department of Applied Neurobiology
What We Do - Products and Projects
Silent Brain Seizures
Patients with acute brain injury often suffer neurologic deterioration and worsening of their injury within a few days after emergency care. Several known processes can contribute to this worsening, but the mechanisms are still largely unknown. The Neurophysiology and EEG Laboratory focuses on patterns of abnormal brain activity that may cause expansion of an injury focus or otherwise worsen recovery from the primary injury. “Silent brain seizures” are firestorms of uncontrolled brain activity that may damage brain tissue by releasing toxic neurochemicals and depleting the brain’s energy reserves. Importantly, these non-convulsive seizures (NCS) occur without any outward manifestation in physical convulsions and can therefore only be detected with brain wave recordings, or EEG. Increased clinical awareness of the frequency of NCS has led several leading clinical neurophysiologists to argue for the use of continuous EEG monitoring in brain-injured patients. [NCS White Paper] In our laboratory, we employ this continuous EEG approach to monitor rats for several days following traumatic or ischemic brain injury. With this technique, we have characterized a rat model of NCS and other clinically defined EEG abnormalities and are able to study their pharmacology and pathogenic effects. Top
Cortical Spreading Depression
Another type of ‘silent brain seizure’ that occurs spontaneously following acute, focal brain injury is known as cortical spreading depression (CSD). CSD is a wave of neuronal/glial depolarization that propagates from the injury focus into adjacent brain tissue that may be at risk for further deterioration or death. Although CSD has been studied in animals for over 60 years, only recently in 2002 has it been shown to occur commonly in the human brain after spontaneous or traumatic brain injury. We participate in the Co-Operative Study on Brain Injury Depolarizations (www.cosbid.org) to investigate CSD and its potential role a secondary insult adversely affecting recovery from brain injury in humans. In the Neurophysiology Laboratory, we study CSD in animal models of ischemic and traumatic brain injury with acute and chronic recording preparations. Click here for an explanation of CSD in non-scientific language. Top
Delayed Neuronal Cell Death
Our Department employs a variety of in vitro (i.e. cell culture) and in vivo models to investigate the role of caspase-dependent and caspase-independent apoptotic neuronal cell death. We are currently studying this process in neuronal culture models of hypoxia/hypoglycemia, oxidative stress, ionic imbalance, and EAA-mediated toxicity as well as in experimental rat models of brain trauma. Several experimental drugs are being used to study the role of the caspase-3, calpain, mitochondrial cytochrome c and MAPKinases in the process of delayed neuronal cell death in neuroprotection protocols. Techniques currently being used include COMET Assay Analysis, TUNEL staining, acid fuchsin staining, caspase-3 activity and pulse-field electrophoresis analysis of DNA strand breaks. Top
Genomics
Our Department is currently studying the genomic response to brain injury in the rat using DNA microarray techniques to map the injury-induced changes in over 1,000 CNS related functional genes. We also use smaller, pathway-specific microarrays to study the mechanism of neuroprotection offered by advanced development drugs. Using this global approach to study CNS gene expression allows us ultimately select and target specific genes and/or gene families of interest for subsequent quantification of mRNA levels using real time QRT/PCR and regional localization of mRNA expression using in situ hybridization. Top
Proteomics and Biomarkers
The study of altered gene regulation after brain injury provides information on the molecular biology underlying the brain's responses to such injury. However, many cellular responses to injury do not involve changes in the levels of gene transcription. For this reason, our lab is also analyzing the changes in protein and peptide expression that result from brain injury, to further elucidate the post-translational mechanisms underlying the response to trauma. We have been using traditional proteomic techniques (2D-gel electrophoresis followed by MS analysis to identify the proteins), degradomic applications and PowerBlot analysis to identify alterations in the proteins following various types of experimental brain injury. The aim is to define specific biomarkers of brain injury that will permit more precise post-injury identification of the anatomical location and cellular mechanisms of injury, as well as provide a quantitative index of injury severity while offering a real-time, longitudinal information platform defining prognosis directing therapeutic decisions. Top
Neuroprotection
The main focus of our neuroprotection research is to facilitate preclinical development of neuroprotective drugs which would mitigate acute brain injuries. In doing so, we “cherry-pick” lead drug candidates initially developed by pharmaceutical companies and test them in our well established in vitro (hypoxia and neural toxin) and in vivo (ischemia and trauma) rat models of acute brain injury under Collaborative Research and Development Agreements (CRDAs). Our past and present CRADA partners include Roche Biosciences, Millennium Pharmaceuticals, Guilford Pharmaceuticals, Cognetix Inc., Panacea Pharmaceuticals, and Neuren Pharmaceuticals. In the past decade we have evaluated over a dozen neuroprotective drug candidates representing a wide spectrum of therapeutic targets. These developmental drugs are tested using rigid, comprehensive pharmacological criteria to capture efficacy, potency, toxicity and, critically, evaluations of therapeutic window. Our short (24-72 h) and long (7-21 days) term outcome measures of neuroprotection include quantitative histopathology, immunohistochemistry, EEG analysis, physiological monitoring, and functional/cognitive assessments. A non-pharmacological approach to neuroprotection, i.e. brain specific hypothermia, is also being explored as a method to induce rapid and safe induction of brain cooling to selectively reduce cerebral metabolic rate while maintaining the normal systemic physiology after brain injury. Top
Inflammation and brain injury
Acute injury to the brain often involves a robust neuro-inflammatory response and associated recruitment of inflammatory leukocytes to the site of injury. Expression of inflammatory molecules such as chemokines and endothelial adhesion molecules by the injured brain can promote the diapedesis of white blood cells through capillary walls and into the brain parenchyma. The infiltrating leukocytes are known to be primary sources of pro-inflammatory cytokines, which may be detrimental to the injured brain. In response, recent neuroprotection approaches have focused on modulation of transcriptional factors, such as NF- kB, that control the expression of multiple pro-inflammatory genes. In particular, the use of proteasome inhibitors has been shown to effectively blunt NF- kB mediated gene expression and have indicated efficacy in the treatment of several neuro-inflammatory disorders. Our department is currently investigating time course changes in inflammatory genes regulated by NF- kB and targeting this response with novel therapeutics such as proteasome inhibitors (provided by our collaborative partners in the pharmaceutical industry) with our overall aim to improve current strategies for limiting secondary brain damage due to inflammation. Top
Antimalarial drug development
Malaria poses a threat across several continents: Eurasia (Asia and parts of Eastern Europe), Africa, Central and South America. Bradley (1991) estimates human exposure at 2,073,000,000 with infection rates at 270,000,000, illnesses at 110,000,000, and deaths at 1,000,000. Significant mortality rates are attributed to infection by the parasite Plasmodium falciparum, with an estimated 90% among African children. A worldwide effort is ongoing to chemically and pharmacologically characterize a class of artemisinin compounds which might be promising antimalarial drugs. The U.S. Army is studying the efficacy and toxicity of several artemisinin semi-synthetic compounds: arteether, artemether, atelinate, and atesunate. The World Health Organization and the U.S. Army selected arteether for drug development and possible use in the emergency therapy of acute, severe malaria.
Together, with Division of Experimental Therapeutics (WRAIR & AFRIMS) colleagues, we have investigated the neurotoxic effects of artemisinin compounds (arteether, artemether, DQHS, artelinate and artesunate) in non-human primate and other animal models. Animals were given compounds intravenously and the following were evaluated pre- and post-exposure: i) neurology; ii) pharmacokinetics; iii) CBC; iv) urine analysis; v) smooth and skeletal muscle and viscera samples. The cytopathology and systems neuropathology was studied. The resulting findings were included in a WRAIR NDA application submitted to the FDA for the use of intravenous artesunate. Clinical trials are ongoing. Top
Chemical Defense Research
Soman (GD), Sarin (GB), and VX are recognized chemical warfare nerve agents (CWNA). Current understanding of the physiological and pathological effects of these agents at low-levels of exposure is insufficient to provide adequate prediction, protection, or diagnosis of adverse health effects. The data generated by ongoing studies evaluates: i) neurological signs and impairments; ii) physiological consequences following exposure; and iii) behavioral deficits that may ensue. Research in the neuropathology of low-dose exposure examines: i) which subacute low-dose exposure paradigms induce brain pathology; ii) the first appearance of lesions; iii) the progress of the injury; iv) the distribution of the injury and the functional systems that are injured; and v) the possibility of exacerbated health risks in subjects living for long periods following low-dose exposure to CWNA. The resulting data can provide guidance and focus for further neurological, neuropsychiatric, physiological, and neurobehavioral testing. The short-term and long-term human health risks may be extrapolation from the animal data.
Pharmacological or hormonal compounds that may serve to protect the brain from nerve agent exposure are being investigated with WRAIR and USAMRICD colleagues. The neuropathology of CWNA exposures is characterized and then compared with subjects given neuroprotective compounds using different protective paradigms. Top
