April 2, 2010
Tissue Engineering, Imaging Neuronal Circuits Featured In Cold Spring Harbor Protocols
The goal of tissue engineering is to recapitulate healthy human organs and tissue structures in culture, and then transplant them into patients, where they are fully integrated. This is a complicated process, and the use of high-throughput imaging systems that allow researchers to directly monitor transplanted tissues in live animals over time is important for improving the culturing and implantation techniques, as well as the design of artificial tissue scaffolds. By using transgenic animals with cell-specific fluorescent reporters, parameters such as tissue perfusion, donor cell survival, and donor-host cell interaction/integration can be observed. In the April issue of Cold Spring Harbor Protocols (http://www.cshprotocols.org/TOCs/toc4_10.dtl), Mary Dickinson and colleagues from the Baylor College of Medicine (http://www.bcm.edu/star/?PMID=3157) present a protocol for the use of "The Mouse Cornea as a Transplantation Site for Live Imaging of Engineered Tissue Constructs." This is a modified version of the classical corneal micropocket angiogenesis assay, which employs it as a live imaging "window" to monitor angiogenic hydrogel tissue constructs. As one of April's featured articles, it is freely available on the journal's website (http://cshprotocols.cshlp.org/cgi/content/full/2010/4/pdb.prot5416).
Neurons are organized into anatomical and functional groups called "circuits". The activity of these circuits is traditionally monitored using conventional electrophysiological techniques. But some cells, such as the submandibular ganglia, are difficult to impale for intracellular recordings. Instead, viral vectors can be used to deliver fluorescent calcium sensors for detecting activity in a living animal. "Calcium Imaging of Neuronal Circuits In Vivo Using a Circuit-Tracing Pseudorabies Virus," from Lynn Enquist and colleagues at Princeton University (http://www.molbio1.princeton.edu/labs/enquist/), provides detailed instructions for the use of the pseudorabies virus (PRV) as a vector for imaging connectivity and activity of neuronal circuits. PRV has a broad host range but does not infect higher-order primates, and it travels along chains of synaptically connected neurons. The PRV strain used in this procedure encodes G-CaMP2, a sensitive fluorescent calcium sensor protein. Available in the April issue of Cold Spring Harbor Protocols (http://www.cshprotocols.org/TOCs/toc4_10.dtl), the method allows for reliable detection of endogenous circuit activity at single-cell resolution. As one of April's featured articles, it is freely available on the journal's website (http://cshprotocols.cshlp.org/cgi/content/full/2010/4/pdb.prot5410).
About Cold Spring Harbor Protocols: Cold Spring Harbor Protocols (www.cshprotocols.org) is a monthly peer-reviewed journal of methods used in a wide range of biology laboratories. It is structured to be highly interactive, with each protocol cross-linked to related methods, descriptive information panels, and illustrative material to maximize the total information available to investigators. Each protocol is clearly presented and designed for easy use at the bench"”complete with reagents, equipment, and recipe lists. Life science researchers can access the entire collection via institutional site licenses, and can add their suggestions and comments to further refine the techniques.