eprintid: 16225 rev_number: 34 userid: 1458 dir: disk0/00/01/62/25 datestamp: 2013-01-30 14:51:19 lastmod: 2016-11-15 14:06:37 status_changed: 2013-01-30 14:51:19 type: thesis_degree metadata_visibility: show contact_email: amy.elisabeth.rupert@gmail.com item_issues_count: 0 eprint_status: archive creators_name: Rupert, Amy Elisabeth creators_email: aeh37@pitt.edu creators_id: AEH37 title: Development of Electroosmotic Sampling for the Investigation of Galanin-Degrading Ectopeptidase Activity in the Hippocampus ispublished: unpub divisions: sch_as_chemistry full_text_status: public keywords: method development, hippocampus, sampling, electroosmosis, galanin, ectopeptidase, ischemia abstract: There are currently no methods that have been successfully used to sample the extracellular component of brain slice preparations with the precision developed for in vivo sampling methods. We developed a slice-compatible method that removes fluid by electroosmosis. Two designs were investigated: one utilizing a single capillary where flow originates below the tissue and fluid pulled upwards and one using two capillaries where flow is between the two capillary lumens. The two-capillary approach is analogous to push-pull perfusion wherein a source ‘push’ capillary with a tapered tip is inserted into the tissue and a collection ‘pull’ capillary is positioned at the surface in close proximity to the source capillary. Voltage is applied across proximal capillary ends, which invoked fluid flow from source capillary into the tissue then to the collection capillary. Damage studies addressed minimization of perturbation of tissue by electric fields in both single and push-pull models. Flow rates were quantified for the two-capillary model using HPLC analysis of collected fluid. Numerical simulations aided understanding of electric field distribution and fluid flow within the tissue. We then investigated the hydrolysis of exogenous galanin in the extracellular space after ischemic pre-conditioning, a method in which mild, short ischemia creates resistance in the brain against a longer duration of ischemia 24-48 hours later. The efficacy of many neuropeptides, including galanin, is controlled by hydrolysis of active peptide into smaller active and inactive fragments by ectopeptidases. We used push-pull electroosmotic sampling and MALDI mass spectrometry to identify hydrolysis products created after passing exogenous galanin through tissue. We then quantified the hydrolysis of galanin after ischemic pre-conditioning. We showed that pre-conditioned cultures have decreased galanin hydrolysis in the CA1 and DG areas, but not the CA3. Finally, we treated cultures with inhibitors for metallo- and aminopeptidases and quantified the resulting relative changes in galanin hydrolysis. Results indicate that metallopeptidases, particularly those that use zinc, are likely responsible for galanin degradation in the CA1 region of the hippocampus. Neuron specific aminopeptidase may also be hydrolyzing galanin. The distribution of hydrolysis products determined with MALDI-MS also indicate aminopeptidase activity. date: 2013-01-30 date_type: published pages: 131 institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Weber, Stephen etdcommittee_name: Robinson, Rena etdcommittee_name: Michael, Adrian etdcommittee_name: Sandberg, Mats etdcommittee_email: sweber@pitt.edu etdcommittee_email: rena@pitt.edu etdcommittee_email: amichael@pitt.edu etdcommittee_email: mats.sandberg@anatcell.gu.se etdcommittee_id: SWEBER etdcommittee_id: RENA etdcommittee_id: AMICHAEL etdcommittee_id: etd_defense_date: 2012-10-05 etd_approval_date: 2013-01-30 etd_submission_date: 2012-10-31 etd_release_date: 2013-01-30 etd_access_restriction: immediate etd_patent_pending: FALSE thesis_type: dissertation degree: PhD citation: Rupert, Amy Elisabeth (2013) Development of Electroosmotic Sampling for the Investigation of Galanin-Degrading Ectopeptidase Activity in the Hippocampus. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/16225/1/AmyRupertDissertation2012-2.pdf