Tracing neuronal connections with lipophilic carbocyanine dyes has revolutionized neuroanatomical tract tracing and is an essential feature to understand development of brain connections in both control and mutant mice in particular since multiple colors of dyes can be used. More recently, lipophilic dyes have also gained ground in labeling blood vessels by directly staining the endothelial cell membranes upon contact. A persistent problem that blocks even wider use of these extremely successful dyes is their limited combination with other procedures such as immunostaining or detailed histology that requires dehydration for embedding, since the dye molecules are not permanently bound to the membranes and can either leak out or be easily washed away by lipophilic solvents or detergents. Attempts to overcome these problems have thus far been at best partially successful. Therefore, to broaden even further the use of carbocyanine lipophilic dyes we propose to develop fixable carbocyanine dyes that can be bonded to lysine groups in membrane bound proteins thereby retaining the dyes in the membranes even after the lipid bilayers has been removed with detergents or organic solvents. We plan to optimize the use of these dyes by developing a protocol that allows combination of multiple distinct fluorophores to maximize the information gained from a given model organism. Specifically, our Specific Aims are: (1) Synthesize four spectrally distinct fixable lipophilic dyes, three for nerve tract tracing and one or blood vessel labeling, that are compatible with standard fixation techniques used in tissue processing and immunocytochemistry protocols. Dyes synthesized will feature an aromatic N-hydroxysuccinimide ester group to provide covalent anchoring to membrane proteins. (2) Evaluate fixable dyes in standardized test systems in fixed tissue using processing techniques needed for high resolution histology at the light microscope level. In this aim we will characteriz how long these dyes are retained in tissue after treatment with organic solvents and detergents and how washing out progresses over time. We will also characterize the conditions under which these dyes can be best combined with each other and with immunochemistry and/or staining for dying cells. (3) Create test products to market each dye alone or in combination to the research community. We will develop test products to be sent to 5-10 collaborators in the neuroscience community along with the published protocol developed in SA2. Combined, these three aims will provide novel reagents useful for studies related to breaches in the blood-brain barrier such as tumors, trauma and neurodegenerative diseases as well as normal development.

Public Health Relevance

Studying the normal and diseased brain with fluorescence imaging techniques requires probes with improved performance. We will generate high performance fluorescent dyes for membrane labeling of nerve tracts and blood vessels compatible with long-term tissue preservation techniques and other known fluorescent cell markers. The possible multicolor labeling will provide detailed knowledge of the developing brain and disease models such as brain tumors, fetal alcohol syndrome, neurodegenerative diseases, trauma or other disruptions of the blood-brain barrier.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1-IMST-L (11))
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Flicker, Paula F
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Molecular Targeting Technologies, Inc.
West Chester
United States
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Fritzsch, Bernd; Duncan, Jeremy S; Kersigo, Jennifer et al. (2016) Neuroanatomical Tracing Techniques in the Ear: History, State of the Art, and Future Developments. Methods Mol Biol 1427:243-62
Hewitt, Stephen M; Baskin, Denis G; Frevert, Charles W et al. (2014) Controls for immunohistochemistry: the Histochemical Society's standards of practice for validation of immunohistochemical assays. J Histochem Cytochem 62:693-7