June 2012: This Month in JoVE

1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Editorial
 

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.

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Chao, W., Kolski-Andreaco, A. June 2012: This Month in JoVE. J. Vis. Exp. (64), e4467, (2012).

Abstract

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.

Our authors from Italy show us their technique for excising the cornea from the ocular globe, which involves first dunking it in a series of sterilizing solutions preparing it for sterile handling, creating an incision on the scleral surface, and ultimately separating the corneal-scleral rim away from the globe. Once the cornea is removed, the endothelial cell density and viability is checked and it is prepared for long-term storage, during which time the media is periodically tested in high-throughput fashion with other grafts deposited at the eye bank.

In corneal transplantation surgeries, full-thickness grafts are used in a procedure called penetrating keratoplasty; however, JoVE learns that in some diseased corneas, only the endothelial layer is affected. Our authors from the University of Michigan & Midwest Eye Bank demonstrate further processing of donor corneas with Descemet's stripping automated endothelial keratoplasty (DSAEK), a procedure that involves grafting only the corneal endothelial layer. This relatively recent procedure is made possible via the use of a microkeratome, a device that can precisely cut through the cornea so that the endothelial layer can be separated for transplantation. The uniformity and quality of the donor tissue is verified using slit lamp and specular microscopy, and the graft is stored for corneal transplantation surgery.

Together, two groups of authors, from different continents, have documented the steps for removing the entire cornea from the eye and isolating the posterior layer for endothelial keratoplasty - a process that decreases the risk of infection or graft rejection and improves a patient's ability to see.

In JoVE Neuroscience, a multidisciplinary team of clinicians and scientists introduce new research applications of the electrocorticography (ECoG). The ECoG is an invasive procedure that uses strip and grid electrodes placed directly on the brain to localize seizure foci in epilepsy patients. This procedure can also be used to map cortical regions that need to be spared during resective surgery. Cortical mapping is accomplished by monitoring whether stimulation of a given electrode results in the disruption of movement or speech. Patients are typically subjected to intracranial monitoring to locate seizure foci for about a week, and this duration provides a unique window of opportunity for researchers to study the human brain in action with the ECoG, which has better signal-to-noise properties and less susceptibility to recording artifacts than the noninvasive electroencephalography (EEG).

After determining baseline activity at rest, our authors record brain activity in the high gamma frequency range, during simple cognitive, or motor tasks. Then, these investigators demonstrate how to use the SIGFRIED software system to perform rapid, real-time functional mapping based on ECoG signals, which are further analyzed to provide information regarding the brain regions associated with particular tasks.

In Bioengineering, JoVE encounters a team of scientists who show that the microvasculature can be approximately recreated on a chip. This "chip" is actually a device with microfluidic channels that are coated with endothelial cells. SU-8 photolithography is used to etch the channel pattern onto a silicon wafer, which acts as a mold for PDMS. Once the device has been fabricated, it is seeded with endothelial cells, which are cultured in the channels. Thanks to the transparency of PDMS, the cells can be imaged via microscopy. This in vitro model of the microvasculature provides a controlled microenvironment that can be used to study normal hemodynamic processes as well as in hematologic disease. This short summary is a mere glimpse at some of JoVE's content for the month of June. Further investigation might lead one to methods for in situ hybridization of adult mosquito tissue and embryo, visualizing mitosis in drosophila, and differentiating embryonic stems cells into motor neurons. Stay tuned.

Protocol

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

Jennifer Juhn1, Anthony A. James2, 1
1Department 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 Huang2, 1
1Institute 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 Salvalaio3, 1
1Fondazione 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 Whye2, 1, Robert W. Mason1, Wenlan Wang1
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. Shtein1
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. Myers1, 2, 3, 4,*, Yumiko Sakurai1, 2, 3, 4,*, Reginald Tran1, 2, 3, 4, Byungwook Ahn1, 2, 3, 4, Elaissa Trybus Hardy1, 2, 3, 4, Robert Mannino1, 2, 3, 4, Ashley Kita1, 2, 3, 4, Michelle Tsai1, 2, 3, 4, Wilbur A. Lam1, 2, 3, 4
1Department 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 Gupta2, 1, Peter Brunner2, 1, Aysegul Gunduz2, 1, Matthew A. Adamo3, Anthony Ritaccio2, Gerwin Schalk1, 2, 4, 5, 6, 7
1Wadsworth 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.

Disclosures

No conflicts of interest declared.

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