1. The Measurement of Suppression
<ol>
	<li>Before beginning the measurement of suppression, give the patient detailed instructions (see 1.8) and show the visual stimuli relating to steps 1.2-1.8 below on the laptop screen. All patients should wear full refractive correction (glasses/contact lenses) throughout the procedure, suitably adjusted for the working distance.</li>
	<li>The goggles (eMagin 8700) should be fitted to the patient (over the top of spectacles/contact lenses) and properly adjusted. Ensure that the screens sit parallel to the ocular surface. The screens can be adjusted to account for each patient's interpupillary distance. Instructions for connecting and calibrating the goggles can be found here: <a href="http://www.3dvisor.com/wpcontent/uploads/2010/08/UserGuide10.7.pdf" target="_blank">http://www.3dvisor.com/wpcontent/uploads/2010/08/UserGuide10.7.pdf</a>.</li>
	<li>The stimuli consist of two populations of moving dots, one population moving in a common direction (the 'signal' dots) and the other moving in random directions (the 'noise' dots). The observer's task is to report the signal direction. Task difficulty is manipulated by varying the relative proportion of signal to noise dots in the stimulus.</li>
	<li>The custom Matlab programs which control these measurements utilize a 3-down 1-up staircase procedure. There are two separate measurement stages. The first provides a binocular threshold, that is, the threshold number of signal dots required when both signal and noise dots are presented to both eyes at high contrast. The second program measures the contrast imbalance required to achieve the same threshold when the dot populations are presented to separate eyes.</li>
	<li>Different images are displayed on the goggles screens through the use of a Matrox DualHead2Go, (Montreal, Canada) device.</li>
	<li>To begin the suppression assessment, first measure a binocular threshold. Here the same visual stimuli are shown to both eyes and the minimum number of signal dots required to accurately judge the direction of the dot movement is measured.</li>
	<li>To align the eyes, the screens will display a black half cross in each eye against a white screen. Using the keyboard, the patient is asked to move the half of the cross seen by the amblyopic eye until it lines up with the half seen by the fellow eye. It is important that the patient is advised that the image seen by the amblyopic eye may be blurry, but it is the alignment that is important. If the moving image disappears from view ask the patient to blink more frequently as this may improve the visibility of the image. Exact ocular alignment (pixel values) can be extracted following each measurement to ascertain the repeatability of this task.</li>
	<li>Once the patient reports that the half crosses are aligned, the measurement procedure can begin. Each measurement takes about three minutes to complete. Five short staircase measurements are completed for each of the two parts of the measurement. The following instructions should be given before the measurement begins:</li>
	<li>Instructions for the observer: 
	"During this test you will be asked to make a judgment about the direction of moving images." 
	"You will see a group of white/grey dots against a grey background. After each presentation I will ask you to decide if the dots are mostly moving to the left or right using the left and right arrow keys on the keyboard in front of you." 
	"There are two groups of dots, some move smoothly to the left or right (the signal dots), the other group appear to move in random directions." 
	"Please try to decide which direction the signal dots are moving in (right or left) amongst the confusion of the random dots." "As you continue to make decisions the number of random dots will increase, making it more difficult to see the signal dots, when it gets so difficult you cannot tell the direction, please just take your best guess".</li>
	<li>The first measurement can now start. A good 'last minute' instruction is to remind the participant to blink regularly ("a good time to blink is when you press the button").</li>
	<li>Five measurements using the binocular staircase paradigm are now made. Ocular alignment is repeated before each measurement is started.</li>
	<li>The result obtained from each measurement is the threshold number of signal dots (out of 100 dots total) required to correctly judge the direction when both eyes see the same stimuli. The program also reports the range of responses as a standard error of the measurement.</li>
	<li>For the next measurement, the contrast staircase, the five results from the binocular staircases are averaged and the resulting threshold defines the number of signal dots presented to the amblyopic eye. The remaining noise dots are presented to the non-amblyopic eye at varying contrasts to assess suppression.</li>
	<li>For this measurement the amblyopic eye always sees high contrast dots whereas the contrast of the noise dots shown to the non-amblyopic eye is varied using a staircase procedure. At the start of the staircase a fixed number of signal dots (obtained from step 1.11) are shown to the amblyopic eye at 100% contrast and the remaining number of noise dots are shown to the fellow eye at 0% contrast (i.e. no noise dots are visible resulting in minimal suppression). Correct identification of the signal dot direction results in an increase in the contrast of the noise dots shown to the fellow eye according to the 3-down 1-up staircase algorithm. Throughout the measurement, the contrast of the noise dots shown to the fellow eye is varied by the staircase until task performance converges on 79% correct. This indicates that the signal and noise dots are being combined between the two eyes to produce the same level of task performance that was observed under binocular viewing conditions (step 1.11). The ratio of the contrast of the signal dots presented to the amblyopic eye (always 100%) relative to the contrast of the noise dots shown to the fellow eye at threshold is a measure of the balance point. Increasing the contrast of the noise dots shown to the fellow eye above this point would result in suppression of the signal dots shown to the amblyopic eye and impairment in task performance.</li>
	<li>Five measurements are made of the contrast imbalance and an average result is calculated. The result informs the examiner about the level of contrast imbalance that is necessary to overcome suppression and allow the amblyopic eye and fellow eye to see the dots simultaneously.</li>
</ol>
2. Training Using this Method
<ol>
	<li>The contrast imbalance found in step 1.14 above can be used as a starting point for a training regime. The training involves integrating the use of dichoptic images with the contrast imbalance into a video game format ("tetris"). The video game can be implemented on either the virtual reality goggles or on an ipod touch device.</li>
	<li>The video game tetris involves a series of falling blocks which are fitted together to form complete lines. The amblyopic eye sees full contrast blocks, and the fellow eye sees reduced contrast images. Information presented to each eye must be perceived simultaneously for successful play.</li>
	<li>Over time as the training regime continues the contrast imbalance is reduced by increasing the contrast to the fellow eye, making it more difficult for the visual system to overcome the suppression caused by more similar dichoptic images.</li>
	<li>Training duration should be 1-2 hr a day and should be continued until no further improvement in contrast imbalance (increase in contrast presented to the fellow eye) is observed. During the training regime, checks of monocular and binocular function should be made regularly (determined by age of patient and underlying condition). Tests of monocular visual acuity, stereopsis and standard tests of suppression are particularly important for reporting progress.</li>
</ol>

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P.E.D.S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+amblyopia+and+strabismus+in+African+American+and+Hispanic+children+ages+6+to+72+months+the+multi-ethnic+pediatric+eye+disease+study.">Prevalence of amblyopia and strabismus in African American and Hispanic children ages 6 to 72 months the multi-ethnic pediatric eye disease study.</a> Ophthalmology. 115, 1229-1236 (2008).</li><li>Hess, R. F., Li, X., Lu, G., Thompson, B., Hansen, B. 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+contrast+dependence+of+the+cortical+fMRI+deficit+in+amblyopia;+a+selective+loss+at+higher+contrasts.">The contrast dependence of the cortical fMRI deficit in amblyopia; a selective loss at higher contrasts.</a> Hum. Brain Mapp. 31, 1233-1248 (2010).</li><li>Li, X., Dumoulin, S. O., Mansouri, B., Hess, R. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+masking+in+strabismic+amblyopia:+attenuation,+noise,+interocular+suppression+and+binocular+summation.">Contrast masking in strabismic amblyopia: attenuation, noise, interocular suppression and binocular summation.</a> Vision research. 48, 1625-1640 (2008).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+suppression+in+amblyopia.">The role of suppression in amblyopia.</a> Invest. Ophthalmol. Vis. Sci. 52, 4169-4176 (2011).</li><li>Rahi, J., Logan, S., Timms, C., Russell-Eggitt, I., Taylor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk,+causes,+and+outcomes+of+visual+impairment+after+loss+of+vision+in+the+non-amblyopic+eye:+a+population-based+study.">Risk, causes, and outcomes of visual impairment after loss of vision in the non-amblyopic eye: a population-based study.</a> Lancet. 360, 597-602 (2002).</li><li>Black, J. M., Thompson, B., Maehara, G., Hess, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+compact+clinical+instrument+for+quantifying+suppression.">A compact clinical instrument for quantifying suppression.</a> Optom. Vis. Sci. 88, 334-343 (2011).</li><li>Hess, R. F., Mansouri, B., Thompson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+binocular+approach+to+treating+amblyopia:+antisuppression+therapy.">A binocular approach to treating amblyopia: antisuppression therapy.</a> Optom. Vis. Sci. 87, 697-704 (2010).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+sensory+eye+dominance+in+the+normal+visual+system:+a+new+technique+and+insights+into+variation+across+traditional+tests.">Quantifying sensory eye dominance in the normal visual system: a new technique and insights into variation across traditional tests.</a> Invest. Ophthalmol. Vis. Sci. 51, 6875-6881 (2010).</li><li>Zhang, P., Bobier, W., Thompson, B., Hess, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binocular+Balance+in+Normal+Vision+and+Its+Modulation+by+Mean+Luminance.">Binocular Balance in Normal Vision and Its Modulation by Mean Luminance.</a> Optom. Vis. Sci. , (2011).</li><li>To, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+game+platform+for+treatment+of+amblyopia.">A game platform for treatment of amblyopia.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 19, 280-289 (2011).</li><li>Hess, R. F., Mansouri, B., Thompson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+binocular+approach+to+the+treatment+of+amblyopia+in+adults+well+beyond+the+critical+period+of+visual+development.">A new binocular approach to the treatment of amblyopia in adults well beyond the critical period of visual development.</a> Restor. Neurol. Neurosci. 28, 793-802 (2010).</li><li>Goodman, L. K., Black, J. M., Phillips, G., Hess, R. F., Thompson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+binocular+interactions+in+two+cases+of+alternating+strabismus.">Excitatory binocular interactions in two cases of alternating strabismus.</a> J. Aapos. 15, 345-349 (2011).</li><li>Holmes, J. M., Clarke, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amblyopia.">Amblyopia.</a> Lancet. 367, 1343-1351 (2006).</li><li>Brainard, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Psychophysics+Toolbox.">The Psychophysics Toolbox.</a> Spat. Vis. 10, 433-436 (1997).</li><li>Pelli, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VideoToolbox+software+for+visual+psychophysics:+transforming+numbers+into+movies.">The VideoToolbox software for visual psychophysics: transforming numbers into movies.</a> Spat. Vis. 10, 437-442 (1997).</li></ol>

The techniques described in this manuscript form the basis of two patents held by BT and RFH, one of which is also held by JC and LT.

The suppression measurement and treatment techniques described in this paper critically depend on manipulation of the contrast of the images seen by each eye. Specifically, the relative interocular contrast imbalance at which binocular combination occurs, i.e. the "balance point contrast", provides a measure of suppression6,11,17 . In addition, by repeatedly exposing patients with amblyopia and suppression to "balanced" visual stimuli, it is possible to reduce suppression and potentially improve both monocular and binocular visual function12,15,16 . These techniques represent an advance in suppression measurement and a new approach to amblyopia treatment respectively. Currently available clinical tests for assessing suppression such as the Worth 4 lights test and the Bagolini striated lenses assess whether suppression is present or absent, and other tests such as the sbisa bar quantify the level of suppression by the use of neutral density filters. Our technique provides additional clinical information by allowing for the strength of suppression to be precisely quantified. Amblyopia, which is often associated with suppression, is generally treated by occluding the non-amblyopic eye or degrading the image in the non-amblyopic eye in order to encourage use of the amblyopic eye18. While this technique is effective in improving amblyopic eye function18, it does not directly address the binocular deficits associated with amblyopia. Our technique directly targets binocular visual function and we have found that this approach can improve both monocular and binocular function in adult patients with amblyopia12,15,16. The measurement and treatment approaches described are suitable for use in adults and children, although the games may need to be adapted for use in younger pediatric populations. We are currently developing additional games for this purpose.
Our techniques do have limitations. Some patients with a strabismus and strong suppression find it challenging to align the images shown to each eye at the start of the suppression measurement protocol. While it is possible for such patients to align the images17, this can be time consuming. With regards to treatment it is important to work alongside a suitably qualified clinician throughout the training regime, and to consider the risk of diplopia (double vision) if suppression is reduced. Our prior work on this treatment approach has focused on patients with anisometropia or small-angle strabismus and no patients have developed diplopia, however the risk of diplopia should be carefully considered by a suitably qualified clinician on a patient by patient basis before the treatment is administered.
When setting up the techniques for the first time it is important to ensure that all hardware connections are secure and that all relevant drivers are installed and updated on the host computer. It is also important to use a computer equipped with a graphics card that is compatible with the Matrox graphics board described in the protocol and that supports a screen resolution of 1600x600 (once the Matrox drivers are installed) to ensure that each goggle screen has a distinct image. We have found that most problems encountered when setting up the system occur due to incorrect screen resolution settings and the absence of up to date drivers for the necessary hardware. It is also important to ensure that both goggle screens have equal luminance prior to measuring suppression to ensure accurate and stable measurements.
The techniques described provide a foundation for further development of suppression measurement and treatment approaches. In particular, the development of more engaging video games based on the core principle of varying contrast between the eyes could make this approach more efficient and more appealing to younger patients. Aside from their clinical use, these techniques can also be used in a research setting to explore the neural mechanisms underlying suppression and the role that inhibitory interactions between the eyes play in the normal visual system13.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment for the measurement of suppression</td>
 </tr>
 <tr>
 <td>Computer equipped with a graphics card compatible with a Matrox Duel Head to Go device (see below) and Psychtoolbox19,20 </td>
 <td>Any Suitable Manufacturer</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Matlab software of a version compatible with Psychtoolbox</td>
 <td>Mathworks</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Psychtoolbox and custom software for stimulus generation</td>
 <td>Psychtoolbox</td>
 <td></td>
 <td>Psychtoolbox is available for download from <a href="http://psychtoolbox.org" target="_blank">psychtoolbox.org</a> and the additional custom software is available from the corresponding author upon request. </td>
 </tr>
 <tr>
 <td>Matrox Duel Head to Go</td>
 <td>Matrox</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Z800 3D dual pro-HMD video goggles </td>
 <td>eMagin Corporation</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment for the treatment of suppression</td>
 </tr>
 <tr>
 <td>iPod Touch</td>
 <td>Apple</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Screen overlay to allow for dichopic viewing of the iPod</td>
 <td>Spatial View</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Custom software for the tetris treatment game</td>
 <td></td>
 <td></td>
 <td>The software was built using Spatial View's proprietary SDK, full details have been published previously15</td>
 </tr>
 </tbody>
</table>

the measurement and treatment of suppression in amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

The level of suppression found in the described method is dependent on the underlying cause of the amblyopia, previous treatment, use of refractive correction and visual acuity. Because each patient has a very unique history, it is difficult to define 'normal' values of suppression or make comparisons between people. Generally we expect those with worse visual acuity to have a deeper level of suppression9. During training, as we reduce the contrast imbalance between the eyes, the depth of suppression typically reduces and this improves a range of binocular and monocular functions. This change in suppression has been demonstrated in several patient populations (see Figures 3 and 4 in reference12, Table 3 and Figure 9 in reference 15 and Figure 2 in reference 16). It is important to note that detailed measurements of suppression using this technique have the potential to enhance our understanding of the amblyopia syndrome.
<img alt="Figure 1" src="/files/ftp_upload/3927/3927fig1.jpg" /> 
 Figure 1. The equipment used for measuring suppression which includes 1) laptop 2) goggles 3) signal splitter.
<img alt="Figure 2" src="/files/ftp_upload/3927/3927fig2.jpg" /> 
 Figure 2. The goggles fitted correctly on a study participant.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/3927/3927fig3highres.jpg" src="/files/ftp_upload/3927/3927fig3.jpg" /> 
 Figure 3. An overview of the random dot kinetogram stimuli (Step 1.2/1.3). The top panel demonstrates the alignment phase where a half cross is displayed to each eye monocularly as a cue for binocular alignment. The middle panel demonstrates the signal to noise paradigm where signal dots are presented to the amblyopic eye only and the noise dots are presented to the fellow eye, under binocular conditions the signal and noise are combined to give a motion coherence threshold which is then utilized in the contrast varying stage of the measurement. The bottom panel demonstrates the contrast threshold, this procedure uses the motion coherence threshold at a fixed level of signal to noise, and displays varying contrast levels between the two eyes. The point at which the two eyes see a balanced binocular input is the contrast threshold or "balance point".
<img alt="Figure 4" src="/files/ftp_upload/3927/3927fig4.jpg" /> 
 Figure 4. (Step 1.7) Alignment screens as seen through the goggles, horizontal and vertical arrow keys are used to align targets until an entire cross is seen.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/3927/3927fig5highres.jpg" src="/files/ftp_upload/3927/3927fig5.jpg" /> 
 Figure 5. An overview of the training protocol.
<img alt="Figure 6" src="/files/ftp_upload/3927/3927fig6.jpg" /> 
 Figure 6. Example of clinical and psychophysical data obtained from one adult participant with strabismic amblyopia who completed the training procedure (steps 2.1 to 2.4). Figure 6A demonstrates the change in visual acuity (measured with a logarithmic style chart in logMAR units over four weeks of training for both the amblyopic eye (AME) and fellow eye (FFE)). Smaller LogMAR values indicate better visual acuity. The cumulative number of hours of game play is shown in brackets on the x-axis. Figure 6B demonstrates the change in contrast imbalance over four weeks of training. The y-axis shows the contrast that could be tolerated in the fellow eye, therefore larger values indicate less suppression. At the start of training only 30% contrast could be tolerated in the fellow eye before the amblyopic eye was suppressed. However as training progressed, more contrast could be tolerated indicating a reduction in suppression. Figure 6C shows the improvement in stereoacuity over the training period (measured with the Randot stereo test). Zero on the y-axis indicates no stereo vision and increasing values indicate improving stereo sensitivity (units are the reciprocal of the stereo threshold in seconds of arc).
<img alt="Figure 7" src="/files/ftp_upload/3927/3927fig7.jpg" /> 
 Figure 7. Standard clinical tests used to assess binocular vision status before and after training including the Worth 4 dot test (top left), Bagollini lenses (top right), Randot Stereopsis test (bottom left) and TNO stereopsis test (bottom right).

Watch this Scientific Journal Video about The Measurement and Treatment of Suppression in Amblyopia at JoVE.com

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

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49.5K Views.  University of Auckland. Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Video: The Measurement and Treatment of Suppression in Amblyopia

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

The Use of Mixed Reality in Custom-Made Revision Hip Arthroplasty: A First Case Report

The technology of 3D printing and visualization of anatomical structures is rapidly growing in various fields of medicine. A custom-made implant and mixed reality were used to perform complex revision hip arthroplasty in January 2019. The use of mixed reality allowed for a very good visualization of the structures and resulted in precise implant fixation. According to the authors' knowledge, this is the first described case report of the combined use of these two innovations. The diagnosis preceding the qualification for the procedure was the loosening of the left hip's acetabular component. Mixed reality headset and holograms prepared by engineers were used during the surgery. The operation was successful, and it was followed by early verticalization and patient rehabilitation. The team sees opportunities for technology development in joint arthroplasty, trauma, and orthopedic oncology.

A complex revision hip arthroplasty was performed using a custom-made implant and mixed reality technology. According to the authors&#39; knowledge, this is the first report of such procedure described in the literature.

The technology of 3D printing and visualization of anatomical structures is rapidly growing in various fields of medicine. A custom-made implant and mixed ...

Surgical Treatment of an Endolymphatic Sac Tumor

Endolymphatic sac tumors (ELST) are low-grade papillary adenocarcinoma originating from the endolymphatic sac. Usually slow-growing, with local aggressiveness and a low risk of distant metastases, ELST can be sporadic but also frequently associated with von Hippel Lindau disease. The current treatment of ELST is primarily surgical resection. A 55-year-old woman accessed our otologic tertiary level referral center for a sudden worsening of hearing loss in her left ear and vertigo. A magnetic resonance (MRI) and computer tomography scan study subsequently showed a mass in the petrous bone; hence, the presence of an ELST was hypothesized. After embolization of the mass, the patient underwent surgical removal of the lesion. The resection of the mass was done through a translabirinthine approach, with an uneventful procedure. No residual disease remained after surgery. After 24 months of radiologic follow up with MRI, there are no signs of recurrence disease. This paper reports the management of this sporadic ELST, as well as the follow up results, providing clinicians this protocol for the handling of such a challenging otologic skull base surgery and rare disease.

Here, we discuss clinical and radiological features of endolymphatic sac tumors and report the methodology and results of a surgical removal in a case in care at our otologic referral center.