We will develop a suite of analytical methods to determine the activity of ectopeptidases in brain tissue. Ectopeptidases are membrane-bound peptidases facing the extracellular space. They are widely understood to act as a clearance mechanism for peptides. However, recent research points to other, more subtle and important roles for these enzymes. For example, some peptides are activated, and others have their activity altered by ectopeptidases. In stroke, neurons may be damaged. Certain peptides can protect neurons in this and other neurodegenerative conditions. We hypothesize that when neurons are damaged, ectopeptidase activity is changed in order to support the neuroprotective functions of particular neuropeptides (galanin, dynorphins and enkephalins). However, the tools that can test this idea do not exist. The methods that we will create largely focus on obtaining samples. Our experimental model is the organotypic culture of the hippocampal formation. We have learned how to withdraw fluid from the extracellular space in these preparations using electroosmotic flow. Electroosmotic flow results from the interaction of an externally applied electric field and the natural charges on the surfaces of cells. By using this approach, we can pull peptide solutions through small, few hundred

Public Health Relevance

Methods developed in this project will be widely applicable in- and outside of neuroscience for detailed investigations of the concentrations and changes in concentrations of substances in the extracellular space of tissues. The health focus in this project is particularly stroke, however findings will apply to other neurodegenerative diseases, such as Alzheimer's, Parkinson's, and ALS. These new methods will, in this project, be used to determine how certain peptides act to protect neurons from damage in stroke. The project will establish that the role of ectopeptidases, enzymes that can abolish, augment, or alter the effect of neuropeptides, in protecting neurons is more important than is currently appreciated. A plausible mechanism is that neuroprotective peptides exert their protective effect by reducing the inflammatory response of microglia. There are currently no drugs used clinically that reduce neuroinflammation effectively.

National Institute of Health (NIH)
Research Project (R01)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Edmonds, Charles G
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University of Pittsburgh
Schools of Arts and Sciences
United States
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Ou, Yangguang; Wu, Juanfang; Sandberg, Mats et al. (2014) Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures. Anal Bioanal Chem 406:6455-68
Sandberg, Mats; Patil, Jaspal; D'Angelo, Barbara et al. (2014) NRF2-regulation in brain health and disease: implication of cerebral inflammation. Neuropharmacology 79:298-306
Groskreutz, Stephen R; Weber, Stephen G (2014) Temperature-assisted on-column solute focusing: a general method to reduce pre-column dispersion in capillary high performance liquid chromatography. J Chromatogr A 1354:65-74
D'Angelo, Barbara; Ek, C Joakim; Sandberg, Mats et al. (2013) Expression of the Nrf2-system at the blood-CSF barrier is modulated by neonatal inflammation and hypoxia-ischemia. J Inherit Metab Dis 36:479-90
Correa, Fernando; Ljunggren, Elin; Patil, Jaspal et al. (2013) Time-dependent effects of systemic lipopolysaccharide injection on regulators of antioxidant defence Nrf2 and PGC-1* in the neonatal rat brain. Neuroimmunomodulation 20:185-93
Rupert, Amy E; Ou, Y; Sandberg, M et al. (2013) Electroosmotic push-pull perfusion: description and application to qualitative analysis of the hydrolysis of exogenous galanin in organotypic hippocampal slice cultures. ACS Chem Neurosci 4:838-48
Rupert, Amy E; Ou, Y; Sandberg, M et al. (2013) Assessment of tissue viability following electroosmotic push-pull perfusion from organotypic hippocampal slice cultures. ACS Chem Neurosci 4:849-57
Guy, Yifat; Faraji, Amir H; Gavigan, Colleen A et al. (2012) Iontophoresis from a micropipet into a porous medium depends on the ýý-potential of the medium. Anal Chem 84:2179-87
Faraji, Amir H; Cui, Jonathan J; Guy, Yifat et al. (2011) Synthesis and characterization of a hydrogel with controllable electroosmosis: a potential brain tissue surrogate for electrokinetic transport. Langmuir 27:13635-42
Guy, Yifat; Rupert, Amy E; Sandberg, Mats et al. (2011) A simple method for measuring organotypic tissue slice culture thickness. J Neurosci Methods 199:78-81

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