September 2012: This Month in JoVE

Summary

This September in JoVE, researchers from the School of Medicine at the Free University of Berlin demonstrate a novel method for studying how stroke patients compensate for visual field defects. To do this, our authors make use of a driving simulator complete with brakes, a steering wheel, and turn signals. Using driving simulation software and sophisticated eye tracking, researchers can compare the gaze behavior of stroke patients as they navigate through virtual driving courses with varying degrees of complexity. Though posterior cerebral artery infarction can lead to similar visual deficits in patients, some are able to navigate through the driving courses by developing compensatory eye movements, while others crash into dangerous obstacles, like wild boars. Through the analysis of compensatory gaze behavior employed by patients, our authors see great potential for using driving simulation as a tool to rehabilitate stroke patients trying to overcome the blind spots in their visual fields.

Protocol

Doppler Optical Coherence Tomography of Retinal Circulation

Ou Tan¹, Yimin Wang¹, Ranjith K. Konduru², Xinbo Zhang¹, SriniVas R. Sadda², David Huang^1
1Department of Ophthalmology, Oregon Health and Science University , ²Department of Ophthalmology, University of Southern California

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Micropipette Aspiration of substrate-attached cells to estimate cell stiffness

Myung-Jin Oh¹, Frank Kuhr¹, Fitzroy Byfield², Irena Levitan^1
1Section of Respiratory, Critical Care and Sleep Medicine, Department of Medicine, University of Illinois, ²Institute for Medicine and Engineering, University of Pennsylvania

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Time-lapse imaging of neuroblast migration in the mouse forebrain

Jivan Khlghatyan, Armen Saghatelyan
The Cellular Neurobiology Unit, Centre de Recherche Université Laval Robert-Giffard

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Vladimir A. Timoshevskiy, Atashi Sharma, Igor V. Sharakhov, Maria V. Sharakhova
Department of Entomology, Virginia Tech

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Jason M. Beckta^{1, 2}, Scott C. Henderson³, Kristoffer Valerie^{1, 2, 4
1}Department of Radiation Oncology, Virginia Commonwealth University, ²Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, ³Department of Anatomy & Neurobiology, Virginia Commonwealth University, ⁴Massey Cancer Center, Virginia Commonwealth University

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Cell tracking using photoconvertible proteins during zebrafish development

Verónica A. Lombardo, Anje Sporbert, Salim Abdelilah-Seyfried
Max Delbrück Center for Molecular Medicine

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Driving Simulation in the Clinic: Testing visual exploratory behavior in daily life activities in patients with visual field defects

Johanna Hamel^{1, 2}, Antje Kraft¹, Sven Ohl³, Sophie De Beukelaer¹, Heinrich J. Audebert^{1, 2}, Stephan A. Brandt^1
1Department of Neurology, Universitätsmedizin Charité, ²Center for Stroke Research Berlin (CSB), Universitätsmedizin Charité, ³Berlin School of Mind and Brain, Humboldt Universität zu Berlin

Patients with visual deficits after stroke report about different constraints in daily life most likely due to variable compensatory strategies, which are difficult to differentiate in clinical routine. We present a clinical set-up which allows measurement of different compensatory head- and eye-movement-strategies and evaluating their effects on driving performance.