June 2012: This Month in JoVE

Summary

Back in 1905, in what is now the Czech Republic, Eduard Zirm performed the first corneal transplantation surgery (keratoplasty), which restored vision to a patient blinded by corneal injury. Today, eye banks all over the world prepare, store, and distribute donated corneas to hospitals so that thousands of sight-saving keratoplasties can be performed every year. In June 2012, JoVE has its eye on two research groups, one from Italy and the other from Michigan, who demonstrate two distinct methods for corneal graft preparation prior to transplantation.

Protocol

Hybridization in situ of salivary glands, ovaries and embryos of vector mosquitoes

Jennifer Juhn¹, Anthony A. James^{2, 1
1}Department of Molecular Biology and Biochemistry, University of California, Irvine , ²Department of Microbiology and Molecular Genetics, University of California, Irvine

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Maureen Sinclair¹, Jun-Yong Huang^{2, 1
1}Institute for Cell and Molecular Biosciences, University of Newcastle, United Kingdom, ²Institute for Cell and Molecular Biosciences, University of Newcastle

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

A simplified technique for In situ excision of cornea and evisceration of retinal tissue from human ocular globe

Mohit Parekh¹, Stefano Ferrari¹, Enzo Di Iorio¹, Vanessa Barbaro¹, Davide Camposampiero¹, Marianthi Karali², Diego Ponzin¹, Gianni Salvalaio^{3, 1
1}Fondazione Banca Degli Occhi del Veneto O.N.L.U.S. , ²Telethon Institute for Genetics & Medicine (T.I.G.E.M.), ³Fondazione Banca Degli Occhi del Veneto, O.N.L.U.S.

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Efficient differentiation of mouse embryonic stem cells into motor neurons

Chia-Yen Wu¹, Dosh Whye^{2, 1}, Robert W. Mason¹, Wenlan Wang^1
1Nemours Biomedical Research, Alfred I. duPont Hospital for Children, ²Department of Pediatrics, Columbia University Medical Center

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Maria A. Woodward¹, Michael Titus², Kyle Mavin², Roni M. Shtein^1
1Department of Ophthalmology, University of Michigan , ²MidWest Eye Banks

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Endothelialized microfluidics for studying microvascular interactions in hematologic diseases

David R. Myers^{1, 2, 3, 4,*}, Yumiko Sakurai^{1, 2, 3, 4,*}, Reginald Tran^{1, 2, 3, 4}, Byungwook Ahn^{1, 2, 3, 4}, Elaissa Trybus Hardy^{1, 2, 3, 4}, Robert Mannino^{1, 2, 3, 4}, Ashley Kita^{1, 2, 3, 4}, Michelle Tsai^{1, 2, 3, 4}, Wilbur A. Lam^{1, 2, 3, 4
1}Department of Pediatrics, Emory University School of Medicine, ²Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, ³Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta , ⁴Winship Cancer Institute of Emory University
^* These authors contributed equally

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (<30 μm) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

N. Jeremy Hill¹, Disha Gupta^{2, 1}, Peter Brunner^{2, 1}, Aysegul Gunduz^{2, 1}, Matthew A. Adamo³, Anthony Ritaccio², Gerwin Schalk^{1, 2, 4, 5, 6, 7
1}Wadsworth Center, New York State Department of Health, ²Department of Neurology, Albany Medical College, ³Department of Neurosurgery, Albany Medical College, ⁴Department of Neurosurgery, Washington University, ⁵Department of Biomed. Eng., Rensselaer Polytechnic Institute, ⁶Department of Biomed. Sci., STATE UNIVERSITY OF NEW YORK AT ALBANY, ⁷Department of Elec. and Comp. Eng., University of Texas at El Paso

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.