The study receives funding from the Federal Ministry of Education (BMBF) via the grant CSB (01 EO 0801). The Center for Stroke Research Berlin (CSB) is an integrated research and treatment centre. We thank the Stiftung Felgenhauer for financial support.
We thank Richard A. Dargie for corrections to the English text.

1. Preparation of Study Position
<ol>
	<li>Let the patient take a seat, with distance of 2 m in front of the screen, (203 x 152 cm covering 58.15 degree of the visual angle on the horizontal axis and 43.61 degree of the visual angle on the vertical axis, resolution: 1400 x 1050), in a fixed base simulation car seat imitating a real car seat. Help the patient to adjust the seat distance to the pedal with the handle underneath. Help to adjust the backrest.</li>
	<li>Instruct the patient how to use the simulation car gadgets (brakes, turn signal, steering wheel).</li>
	<li>Instruct the patient in the task: Drive as you would do in a real non-simulated driving situation. The road is a one way single lane road with curves (smallest radius 500 m, biggest radius 1,200 m) and without traffic. Be vigilant to street signs and break-down cars emerging on both sides of the road. React to the notion of potentially hazardous events such as wild boar or balls approaching the road as soon as possible by either pressing the brake or using the turn-signal or both, what seems to be appropriate in the respective driving situation. While pressing the pedal, the car speeds up to a constant speed of 70 km/h unless the brake is used1. The drive takes about 10 min.</li>
	<li>Inform the patient about simulation sickness. In case malaise, nausea or sweating occurs, interrupt testing session.</li>
	<li>A test drive with less task density is conducted to get used to the simulation situation and to prevent simulation sickness by allowing enough time to adjust2.</li>
</ol>
2. Calibration of Eye Tracker
<ol>
	<li>In the second testing session, after the patient is properly seated and has received enough time for practice, place the Eye-Tracker on the patient's head and adjust it by pulling the flexible straps. The head camera laser should be pointing to the middle of the screen. Adjust the head camera's focusing on the pupil.</li>
	<li>Instruct the patient to look at the five dots according to the lead of the mouse arrow for calibration.</li>
	<li>Start the simulation.</li>
	<li>Complete calibration with adding the horizontal calibration: Patient fixates overlay picture (of an eye) on screen on the left, then follows the eye moving across the screen and fixates it again on the right side.</li>
	<li>Test the calibration by asking the patient to fixate on specific objects on the screen, and matching it with the overlay eye picture, which indicates the gaze position calculated by the software. The calibration is successful, if gaze and overlay picture meet at the same spot on screen. A vertical drift of the eye tracker acuity may occur during the drive. Evaluate the amount of drift by visual inspection at the beginning and at the end of the drive, checking the need for retesting.</li>
	<li>If calibration was successful, turn off overlay pictures. If not repeat process of calibration until it is successful.</li>
	<li>For quick assessment of compensatory behavior of gaze-movements turn on overlay eye pictures.</li>
</ol>
3. Simulation
<ol>
	<li>Proceed with simulation by asking the patient to start driving.</li>
	<li>Let the patient drive diverse routes (each a 6,500 m in rural areas and approximately 10 min duration) with different task difficulty due to level of distraction by surrounding environment. Each patient drives three routes.</li>
	<li>Immediate assessment of oculomotor behavior: Turn on the overlay eye pictures and visualize the gaze behavior of a patient during testing session: The eye-tracker continuously sends coordinates of the actual gaze position to the simulation software SILAB. In return SILAB projects the overlay eye picture, which is an image of an eye, on the screen exactly on the spot where the patient looks at. This can not only be used to prove quality of calibration, but also to make gaze behavior immediately visible not only to you but also to the patient.</li>
</ol>
4. Analysis
<ol>
	<li>For data recording use SILAB Software at a sample rate of 100 Hz. Use SILAB Software also to record speed, reaction times (use of turn signal, brake).</li>
	<li>Perform statistical analysis of head- and eye-movement parameters with Matlab (MathWorks Company, Natick, USA). Use the following criteria:
	<ol>
		<li>Define saccades as sections of the gaze trajectory where gaze velocity exceeds 30&deg; /s and gaze amplitude is larger than 1&deg;(as eye movements below 1&deg; belong to microsaccades). Cluster saccades occurring within 80 ms. Define sections between saccades as fixations. Define head-movements as movements exceeding 6&deg; /sec 11 and an amplitude of more than 3&deg;. Exclude simultaneous head-and eye-movements with directory in the opposite direction as they represent no gain in gaze amplitude.</li>
		<li>Define object fixations as fixation on an object with gaze position maximal 1, 24&deg; apart from the object on the x-axis and 1, 66&deg; on the y-axis. Objects are not triggered according to the patients gaze position, but consider eccentricity of object to gaze position by calculating it when the object appears3.</li>
		<li>Calculate the average length of the participants' fixations (mean fixation durations) and the spread of search in the horizontal and vertical meridians (the variance of the fixation locations).</li>
	</ol>
	</li>
	<li>Measure reaction times in two ways: As a first mode (first detection) measure reaction time as first detection by either fixation or manual detection: If the patient fixates the object first and responds manually afterward (in the majority of cases), then chose the fixation time as reaction time as first detection. If the patient uses the turn signal or brake pedal first as an indicator without first fixating the object, then chose manual reaction time as first detection. As a second mode (manual reaction), measure reaction time by manual reaction (brake or turn signal) only.</li>
</ol>
5. Representative Results
We recruited 6 patients of different ages (35-71 years of age) with incomplete hemianopia after ischemic PCA infarction (4 on the right and 2 on the left hemisphere) and 85 healthy controls of various ages (20-75 years of age, equally distributed) to determine age related changes in eye- and head-movements as well as driving performance as a reference group. They did not report cognitive deficits, neurological or psychiatric deficits or diseases and visual acuity was higher than 0.5. The medical history was taken and experiences with virtual media explored. The study was conducted in conformity with the declaration of Helsinki and was approved by the local ethics committee. Written informed consent was obtained from all participants. All subjects were unaware of the purpose of the experiments.
Here, we demonstrate representative test results of two patients tested 7- 9 months after stroke incident with incomplete hemianopia (Figure 1) on the right side with and without compensatory behavior as well as a healthy subject as a control. The healthy control was chosen due to similar age, driving and computer game experience.
Patient A exhibited compensatory saccadic movement to the side where the visual defect is located resulting in normal performance in driving simulation compared to a healthy control with successful detection of and reaction to possible hazards in a rural driving situation. However, patient B did not show compensatory saccadic movement and revealed poor performance in driving simulation with missing out on peripheral objects in the blind field causing prolonged reaction times or collisions. Nevertheless, throughout the drives, patient B adopted compensatory behavior causing fewer collisions, without being instructed to do so. The testing was performed head-unrestrained allowing realistic conditions and to detect the possible influence of head movements in compensatory behavior.
The patients were asked to drive the course as they would do in a real non-simulated driving situation. Compared to the healthy subject patient A performed saccades 1.7 times more frequently, which predominantly covered the side of the screen where the visual defect was located (63%). The amplitudes of the saccades in Patient A and the control were similar (mean amplitude: 5.5 degree in the healthy subject versus 5.3 degree in the patient). The fixation duration of patient A was shorter compared to the healthy control (mean fixation duration of 381 ms in patient A versus 483 ms in the control).
In contrast patient B and the control explored equally frequent both sides of the screen. Figure 2 illustrates the distribution of fixations on the screen during the first drive of patient A, the healthy subject and patient B. Patient B performed 3.4 less saccadic movements compared to patient A covering half the size of the amplitude of patient A (mean amplitude: 5.5 patient A versus 2.9 degree in patient B). Patient B showed longer fixation durations compared to both healthy control and patient A (mean fixation duration 1049 ms).
Patient A and patient B performed almost no head-movements (1 to 2) while the healthy control executed a few (5 to 10) head-movements per driving session contributing to gaze amplitude.
Figure 3 demonstrates the influence of eccentricity of object position with respect to gaze position on the reaction time, demonstrated separately for the left and right side of the visual field. The figure illustrates the increase of reaction time due to eccentricity in the two subjects illustrated separately for both sides of the visual field. Some reaction times at very small eccentricities are less than 50 ms. These are not realistic reaction times but rather due to scanning of possible hazardous locations along the road, or objects appearing at the point of fixation of the patient. We did not filter those events because this also represents a certain driving behavior of interest: acknowledging and yielding to potential hazardous locations. (The graphic also shows that there were less reaction times noted for patient B due to missed objects in his blind field.)
In patient A and the healthy control all objects were detected and no collisions occurred. In patient B though, reaction times differed clearly between right (blind) and left (sighted) field: Patient B detected objects occurring in the blind field 1.6 times slower compared to the sighted field and collided 4 times with objects occurring in the blind field (median reaction times: right (blind) field: 4411.66 ms versus left (sighted) field: 2810 ms).
Hence, patient A compensated his loss of right eccentric vision well by an increased number of saccadic movements reaching the side of the visual field defect. It is still unclear though if this compensatory strategy becomes insufficient with higher workload. Evidence for this is suggested in the graph for the left visual field: While the patient managed to react equally fast on the right side due to lateralization of saccadic movements, he showed longer reaction times at greater eccentricities on the left side, suggesting a possible cost of the strategy with regard to performance. However, the healthy control also shows slight differences of reaction times comparing both sides, which might also be due to the fact that the healthy control performed one drive less than the patients. To test whether this represents a stable effect, more trials would be needed.
In contrast to patient A, patient B presented a representative outcome of a patient lacking compensatory behavior and its effect on driving performance: the lack of compensatory saccadic movements into the blind field resulted in collision with objects appearing in the blind field and prolonged reaction times. Nevertheless, throughout the drives, the patient spontaneously started to perform more saccades into the right visual field with greater amplitude, resulting in smaller incidence of collisions.
<img alt="Figure 1A" src="/files/ftp_upload/4427/4427fig1A.jpg" /> 
Figure 1A. Patient A, automated 30 &deg; threshold perimetry.
<img alt="Figure 1B" src="/files/ftp_upload/4427/4427fig1B.jpg" /> 
Figure 1B. Patient B, automated 30 &deg; threshold perimetry.
<img alt="Figure 2" src="/files/ftp_upload/4427/4427fig2.jpg" /> 
Figure 2. Distribution of fixations on screen for patient A, patient B, and healthy control.
<img alt="Figure 3" src="/files/ftp_upload/4427/4427fig3.jpg" /> 
Figure 3. Reaction times to objects appearing at various eccentricities in the visual field, for patient A, patient B, and healthy control.
1This tempomat was implemented to assure comparability of reaction times between age groups as it is known that older drivers reduce speed as a possible compensatory mechanism 7.
2Simulation sickness is described as nausea, sweating or dizziness persisting during a driving session. There is different data on frequency of occurrence ranging from 9 % to 37 % depending on age as it occurs more likely in the elderly 8, 9, 10. Thorough preparation with practice drives long enough for each individual for proper adjustment reduce the chance of simulation sickness.
3Per drive there are 4 wild boar and 4 balls programmed to approach from each side of the road at two different eccentricities, at straight parts of the course and at different intervals of the course to prevent a testing habit. Appearance of objects is triggered by the subject passing flow points on the road.

<ol>
	<li>Bouwmeester, L., Heutink, J., Lucas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+visual+training+for+patients+with+visual+field+defects+due+to+brain+damage:+a+systematic+review.">The effect of visual training for patients with visual field defects due to brain damage: a systematic review.</a> Journal of Neurology, Neurosurgery, and Psychiatry. 78, 555-564 (2007).</li><li>Roth, T., Sokolov, A. N., Messias, A., Roth, P., Weller, M., Trauzettel-Klosinski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+explorative+saccade+and+flicker+training+in+hemianopia.+A+randomized+controlled+study.">Comparing explorative saccade and flicker training in hemianopia. A randomized controlled study.</a> Neurology. 72, 324-331 (2009).</li><li>Freedman, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+the+eyes+and+head+during+visual+orienting.">Coordination of the eyes and head during visual orienting.</a> Experimental brain research. 190, 369-387 (2008).</li><li>Martin, T., Riley, M. E., Kelly, K. N., Hayhoe, M., Huxlin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually-guided+behavior+of+homonymous+hemianopes+in+a+naturalistic+task.">Visually-guided behavior of homonymous hemianopes in a naturalistic task.</a> Vision Research. 47, 3434-3446 (2007).</li><li>Hayhoe, M. M., Ballard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+movements+in+natural+behavior.">Eye movements in natural behavior.</a> Trends in Cognitive Sciences. 9, 188-194 (2005).</li><li>Wood, J. M., McGwin, G., Elgin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemianopic+and+quadrantanopic+field+loss,+eye+and+head+movements,+and+driving.">Hemianopic and quadrantanopic field loss, eye and head movements, and driving.</a> Investigative ophthalmology &#38; visual science. 52, 1220-1225 (2011).</li><li>Cantin, V., Lavalli re, M., Simoneau, M., Teasdale, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mental+workload+when+driving+in+a+simulator:+effects+of+age+and+driving+complexity.">Mental workload when driving in a simulator: effects of age and driving complexity.</a> Accident; analysis and prevention. 41, 763-771 (2009).</li><li>Brooks, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulator+sickness+during+driving+simulation+studies.">Simulator sickness during driving simulation studies.</a> Accident; analysis and prevention. 42, 788-796 (2010).</li><li>Allen, R. W., Park, G. D., Fiorentino, D., Rosenthal, T. J., Cook, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+simulator+sickness+as+a+function+of+age+and+gender.">Analysis of simulator sickness as a function of age and gender.</a> , (2006).</li><li>Liu, L., Watson, B., Miyazaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VR+for+the+Elderly:+Quantitative+and+Qualitative+Differences+in+Performance+with+a+Driving+Simulator.">VR for the Elderly: Quantitative and Qualitative Differences in Performance with a Driving Simulator.</a> Cyberpsychol. Behav. 2, 567-577 (1999).</li><li>Einh&#228;user, W., Moeller, G. U., Schumann, F., Conradt, J., Vockeroth, J., Bartl, K., Schneider, E., K&#246;nig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye-Head+Coordination+during+Free+Exploration+in+Human+and+Cat.+Basic+and+Clinical+Aspects+of+Vertigo+and+Dizziness.">Eye-Head Coordination during Free Exploration in Human and Cat. Basic and Clinical Aspects of Vertigo and Dizziness.</a> Ann. N.Y. Acad. Sci. 1164, 353-366 (2009).</li><li>Bowers, A. R., Mandel, A. J., Goldstein, R. B., Peli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Driving+with+Hemianopia,+I:+Detection+Performance+in+a+Driving+Simulator.">Driving with Hemianopia, I: Detection Performance in a Driving Simulator.</a> Investigative Ophthalmology and Visual Science. 50, 5137-5147 (2009).</li><li>Cockelbergh, T. R. M., Brouwer, W. H., Cornelissen, F. W., van Wolffelaar, P., Kooijman, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Visual+Field+Defects+on+Driving+Performance.">The Effect of Visual Field Defects on Driving Performance.</a> Archives of Ophthalmology. 120, 1509-1516 (2002).</li></ol>

No conflicts of interest declared.

The new established method enables the examination of visual exploratory behavior of patients with visual field defects caused by a stroke. The test design also offers an immediate approach to evaluate compensatory gaze behavior: By turning on the overlay eye pictures the examiner can visualize the gaze behavior of a patient during testing session. Hence, it allows a very quick and immediate assessment as to whether the patient has adopted a compensatory gaze behavior. It also allows patients to become aware of it by visualizing gaze movements by an overlay eye picture moving across the screen as a gaze indicating feedback tool. The role of head-movements in compensatory gaze behavior is still unclear. In our control group head-movements were more common among the elderly. The healthy control performed more head-movements than the patients. Head-movements may play a bigger role when the field of view tested is wider than in our set-up. Hence we could not identify head-movements as part of compensatory gaze behavior in our patient. However more patients need to be examined to clarify the role of head-movements in compensatory behavior.
Limitations of the study are the following: Retesting becomes necessary in some individuals due to vertical drift of the eye tracker throughout the drive. Objects appear naturally along the road and not at a fixed eccentricity triggered by the gaze position. Nevertheless current gaze position in relation to the object is considered when interpreting reaction times.
Patients with visual field defects have been tested before in simulated and real driving settings: Bowers et al. 12 and Cockelbergh et al. 13 carried out studies in a driving simulator and demonstrated poorer driving performance in patients compared to healthy controls. However they did not record eye- and head-movements and individual differences could not be related to visual exploratory behavior. Wood et al. 6 tested in a real life situation and established an evaluation of driving performance of patients with visual field defects. Head- and eye-movements were analyzed via video and post-test scoring by two independent researchers, thus dealing with inter-rater-reliability. Nevertheless they did not provide a quantitative analysis of fixation durations, saccades, and head movements and evaluation depended on a certified driving rehabilitation specialist. The advantage of our set up with simulated driving is the easy and fast assessment within a clinical setting, recording of well-defined parameter of eye- and head- movements as well as reaction times. It is possible to control the level of distraction and expose each driver to a similar driving situation with standardized routes and conditions allowing comparability. Roth 2 has shown that SCT improves search behavior on the blind sight in natural search tasks. By adjusting the level of distraction in the driving courses, it will be possible to prove if, and at what level, the compensatory behavior fails with higher workload. Comparing simulated to real driving studies, it seems appropriate to teach compensatory behavior in a simulated environment and expose the patient to a real driving situation as a second step. Especially since the latter enables to evaluate safety of driving.
In the future we intend to include characterization of different levels of compensatory behavior by analyzing saccades, amplitudes and distribution. This could help to offer more individual rehabilitation plans adjusted to the patient's current level of compensatory behavior. Secondly, as patient B reveals spontaneous adoption of a compensatory strategy, we like to test the design as a possible tool for rehabilitation purposes: Driving simulation not just as a diagnostic test design but also for specific training, instructing the patient to perform compensatory saccade behavior. Combined with immediate visualization of gaze behavior by the gaze indicating eye overlay pictures this might provide a feedback mechanism to arise the attention to a compensatory strategy.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>Driving Simulator Software SILAB</td>
			<td>Wuerzburg Institute for Traffic Sciences GmbH (WIVW)</td>
			<td>&nbsp;</td>
			<td><a href="http://www.wivw.de/index.php.en" target="_blank">http://www.wivw.de/index.php.en</a></td>
		</tr>
		<tr>
			<td>EyeSeeCam</td>
			<td>University of Munich Hospital 
			Clinical Neurosciences</td>
			<td>&nbsp;</td>
			<td><a href="http://eyeseecam.com" target="_blank">http://eyeseecam.com</a></td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Estimated costs and time for establishment 20,000 Euro, 3 months.</td>
		</tr>
	</tbody>
</table>

driving simulation in the clinic: testing visual exploratory behavior in daily life activities in patients with visual field defects

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily life, despite similar visual deficits. We assume this could be due to variable development of compensatory strategies such as altered visual scanning behavior. Scanning compensatory therapy (SCT) is studied as part of the visual training after infarction next to vision restoration therapy. SCT consists of learning to make larger eye movements into the blind field enlarging the visual field of search, which has been proven to be the most useful strategy1, not only in natural search tasks but also in mastering daily life activities2. Nevertheless, in clinical routine it is difficult to identify individual levels and training effects of compensatory behavior, since it requires measurement of eye movements in a head unrestrained condition. Studies demonstrated that unrestrained head movements alter the visual exploratory behavior compared to a head-restrained laboratory condition3. Martin et al.4 and Hayhoe et al.5 showed that behavior demonstrated in a laboratory setting cannot be assigned easily to a natural condition. Hence, our goal was to develop a study set-up which uncovers different compensatory oculomotor strategies quickly in a realistic testing situation: Patients are tested in the clinical environment in a driving simulator. SILAB software (Wuerzburg Institute for Traffic Sciences GmbH (WIVW)) was used to program driving scenarios of varying complexity and recording the driver's performance. The software was combined with a head mounted infrared video pupil tracker, recording head- and eye-movements (EyeSeeCam, University of Munich Hospital, Clinical Neurosciences).
The positioning of the patient in the driving simulator and the positioning, adjustment and calibration of the camera is demonstrated. Typical performances of a patient with and without compensatory strategy and a healthy control are illustrated in this pilot study. Different oculomotor behaviors (frequency and amplitude of eye- and head-movements) are evaluated very quickly during the drive itself by dynamic overlay pictures indicating where the subjects gaze is located on the screen, and by analyzing the data. Compensatory gaze behavior in a patient leads to a driving performance comparable to a healthy control, while the performance of a patient without compensatory behavior is significantly worse. The data of eye- and head-movement-behavior as well as driving performance are discussed with respect to different oculomotor strategies and in a broader context with respect to possible training effects throughout the testing session and implications on rehabilitation potential.

Watch this Scientific Journal Video about Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects at JoVE.com

Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects

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.

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily ...

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17.2K Views.  Universitätsmedizin Charité. 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.

Video: Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Integration of Brain Tissue Saturation Monitoring in Cardiopulmonary Exercise Testing in Patients with Heart Failure

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction (HF). However, in clinical cardiopulmonary exercise testing (CPET), cerebral hemodynamics is not assessed. NIRS is used to measure cerebral tissue oxygen saturation (SctO2) in the frontal lobe. This method is reliable and valid and has been utilized in several studies. SctO2 is lower during both rest and peak exercise in patients with HF than in healthy controls (66.3 &#177; 13.3% and 63.4 &#177; 13.8% vs. 73.1 &#177; 2.8% and 72 &#177; 3.2%). SctO2 at rest is significantly linearly correlated with peak VO2 (r = 0.602), oxygen uptake efficiency slope (r = 0.501), and brain natriuretic peptide (r = -0.492), all of which are recognized prognostic and disease severity markers, indicating its potential prognostic value. SctO2 is determined mainly by end-tidal CO2 pressure, mean arterial pressure, and hemoglobin in the HF population. This article demonstrates a protocol that integrates SctO2 using NIRS into incremental CPET on a calibrated bicycle ergometer.

This protocol integrated near-infrared spectroscopy into conventional cardiopulmonary exercise testing to identify the involvement of the cerebral hemodynamic response in exercise intolerance in patients with heart failure.

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction ...