The authors wish to acknowledge the VISUALIA visual therapy clinic, which partially supported the development of the computer-based test, under agreement with the University of Oviedo (FUO-EM-104-12).

The study design was approved by the Basque Country Ethics Committee and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained either from participants enrolled in the study or their legal guardians. Figure 1 represents the protocol steps.
1. Participant Recruitment
<ol>
	<li>Recruit patients with the following characteristics: between 7-14 years of age with refractive amblyopia and/or successfully treated strabismic amblyopia (strabismus children are only eligible to participate if misalignment of the visual axes has been successfully corrected with glasses, visual therapy, and/or strabismus surgery); with monocular best corrected distance visual acuity of &#8805;0.1 logMAR21; and stereoacuity measured in the 800&#34;-200&#34; range (coarse-moderate stereo-deficiency)22 according to a random-dot stereogram test23.
	<ol>
		<li>To determine &#34;refractive amblyopia&#34;, consider the interocular refractive difference error by means of autorefraction under cycloplegia. 
		NOTE: Anisometropic amblyopia is defined when a patient presents an interocular refractive difference error of &#8805;1 spherical dioptre (D), (spherical equivalent). Isometropic amblyopia is defined when the cycloplegic refractive error in each eye of &#8805;4.00 D hypermetropia or myopia and/or &#8805;2.00 D astigmatism and the interocular refractive difference error is &#60;1 D.</li>
		<li>To determine successfully treated strabismic amblyopia, check for the absence of strabismus using a monocular cover-uncover test and stimulating accommodation with a letter acuity score of 20/30 to ensure that the subject looks at the letter with the fovea when performing the fixing movement24.</li>
	</ol>
	</li>
	<li>Exclude patients with the following characteristics: strabismus; hyperphoria (upward deviation) of 2 prism diopters or more; nystagmus; hypermetropic anisometropia, in which the patient presents a spherical equivalent difference between the eyes of 3 diopters (or more) if corrected with glasses (to prevent aniseikonia); any ocular pathology; attention-deficit/hyperactivity disorder; and any cognitive disorder. Exclude patients without access to a computer or without internet connection at home.</li>
</ol>
2. Visual Evaluations
<ol>
	<li>Perform the Baseline Optometric Evaluation prior to beginning this section for collection of baseline data and ensure all appropriate inclusion/exclusion criteria.
	<ol>
		<li>Measure best corrected distance visual acuity (BCVA) with Early Treatment Diabetic Retinopathy Study (ETDRS visual acuity chart).</li>
		<li>Check for the absence of strabismus as explained in step 1.1.2.</li>
		<li>Measure stereoacuity at a constant distance of 40 cm, under light illumination of 120 cd/m2. 
		NOTE: Illumination control guarantees contrast consistency between dots and background in the test pattern. Patient head-to-test distance is a critical variable because it is included in the equation that sets stereoacuity result.
		<ol>
			<li>Measure global stereoacuity with a random-dot stereogram (to avoid monocular cues) conducted according to the test manufacturer&#39;s instructions.</li>
			<li>Measure local stereoacuity using a contour test despite the presence of monocular cues with a Wirt Circles test conducted according to the test manufacturer&#39;s instructions.</li>
		</ol>
		</li>
		<li>Measure the refractive error by cycloplegic refraction (1% cyclopentolate) following Pediatric Eye Disease Investigator Group (PEDIG) guidelines25,26.</li>
		<li>Rule out any ocular pathology with an in-depth study of the anterior (slit lamp) and posterior (indirect ophthalmoscope) poles.</li>
	</ol>
	</li>
	<li>Conducting the optometric center first visit with patients.
	<ol>
		<li>Create a patient profile in the game service application.
		<ol>
			<li>Set the patient&#39;s user identifier and password.</li>
			<li>Set the patient&#39;s interpupillary distance.</li>
		</ol>
		</li>
		<li>Install the computerized stereoscopic game on the patient&#39;s computer.</li>
		<li>Configure the game by clicking on the configuration panel.
		<ol>
			<li>Set the physical dimensions of the patient&#39;s computer screen.</li>
			<li>Ensure that the user is wearing the anaglyph glasses correctly (with the red filter over the left eye).</li>
		</ol>
		</li>
		<li>Measure the patient&#39;s stereopsis basal acuity using the computerized stereoscopic game.
		<ol>
			<li>Ask the patient to play the game under supervision, as explained in section 3.4.</li>
			<li>Consult the result stored in the cloud using the game service application.</li>
		</ol>
		</li>
		<li>Using patient stereopsis basal acuity, set the patient&#39;s basal level in the game service application.
		<ol>
			<li>The computerized stereoscopic game defines three stimulation categories, each associated with a stereoscopic acuity interval value: poor (840&#34;-300&#34;), coarse (480&#34; -210&#34;) and moderate-fine stereopsis (300&#34; -30&#34;). Patients begin with the finest level at which they can identify the stereopsis stimulus. For example, in a patient with a stereo threshold of 720&#34;, assign the poor stereopsis level on the patient&#39;s profile.</li>
		</ol>
		</li>
		<li>Explain to participants how to perform the exercises at home, as explained in section 3.</li>
	</ol>
	</li>
	<li>Perform check-up visits on completion of every 15 therapy sessions with the computerized stereoscopic game during the training period.
	<ol>
		<li>Open the game service application, open the patient profile and check both compliance and stereopsis results data.
		<ol>
			<li>Remind parents and participants about the importance of respecting the working distance from the screen.</li>
			<li>Remind parents and participants about the importance of compliance.</li>
		</ol>
		</li>
		<li>Evaluate the logMAR BCVA at distance with an ETRDS test to check for any deterioration from the starting values.</li>
		<li>Measure the patient&#39;s stereopsis acuity using the computerized stereoscopic game (step 2.2.4)</li>
		<li>Set the patient&#39;s basal level in the game service application (step 2.2.5).</li>
	</ol>
	</li>
	<li>Perform the final optometric evaluation on completion of 60 therapy sessions with the computerized stereoscopic game (end of treatment) to collect outcome data. 
	NOTE: This evaluation reproduces the baseline optometric evaluation, stressing the measurement of global and local stereopsis.</li>
	<li>Perform the follow-up optometric evaluation 6 months after completion to ensure stability of the results. 
	NOTE: This evaluation reproduces the baseline optometric evaluation, stressing the measurement of global and local stereopsis.</li>
</ol>
3. Treatment Exercises Performed at Home
<ol>
	<li>Explain to participants that they must follow a course of training using the computerized stereoscopic game at home for sixty 8 min sessions.</li>
	<li>Explain that each session must be performed on a different day and that five sessions per week must be completed. Explain to patients that the research team will have access to their compliance and results data in the cloud.</li>
	<li>Instruct patients to visit the optometry center for a check-up visit after every 15 sessions, which should be completed within 3 weeks to consider compliance as 100%, and in all cases, within a maximum of 6 weeks.</li>
	<li>Explaining how to use the computerized stereoscopic game
	<ol>
		<li>Ask the participant to sit at a distance of 80 cm from the computer screen. Make it clear that patients should not try to cheat the program by moving closer to the screen.</li>
		<li>Ensure that the room is dimly lit, avoiding any reflections on the computer screen.</li>
		<li>Explain that the program takes the form of a videogame, in which a random-dot image conceals a hidden silhouette. The silhouette can only be seen in three dimensions while wearing anaglyph glasses.</li>
		<li>Give the patient a pair of anaglyph glasses and explain how to wear them, focusing on which filter goes to which eye (with the red filter over the left eye).</li>
		<li>Provide the patient with an identifier and password to log in to the game (step 2.2.1).</li>
		<li>Instruct the patient to use the mouse to select which silhouette appears, selecting one figure from the four options shown at the bottom of the screen (Figure 2).</li>
		<li>Explain what happens after the selection of a silhouette.
		<ol>
			<li>If the answer is correct, the software emits a high-pitched sound, and the correct image appears in the form of a full-color picture as a reward.</li>
			<li>If the answer is incorrect, the software emits a deep sound. The player still has two more attempts to find the right answer.</li>
			<li>If there are three consecutive wrong answers, the software will show the correct answer.</li>
		</ol>
		</li>
		<li>Explain to the patient that after each trial, the software generates a new screen with a random-dot image concealing a new hidden silhouette.</li>
		<li>Explain to the patient that the hidden silhouette becomes more difficult to find as the session progresses and that this is normal.</li>
		<li>Explain to the patient that, once the session is complete, the software stores the outcomes in a cloud server, allowing the optometrist to track compliance and stereoacuity evolution remotely. 
		NOTE: Stress the importance of a stable Wi-Fi connection and of closing the computerized stereoscopic game properly.</li>
	</ol>
	</li>
</ol>

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The computer-based test was developed at the University of Oviedo by S.M.-G., who is a co-author of this manuscript. Subsequent to completion of this study, VISIONARY TOOL, S.L. (found at &#60;www.visionarytool.com&#62;), a private entity, approached S.M.-G. and J.A.P.-C. with an opportunity to take part in the development of a computerised visual training tool, which includes several games and tests, one of which is the random-dot hidden silhouette game used in this article.

Presented here is a protocol for the direct stimulation of stereoacuity, in which random-dot stereo images are used to enhance stereoscopic acuity in stereo-deficient subjects. Four preceding studies have evaluated the results of direct stimulation16,17,18,19. This latest protocol contributes additional features to the abovementioned interventional models.
The model of intervention proposed is intended for patients with a history of strabismic or anisometropic amblyopia, who have already received treatment (i.e., optical correction, occlusion, strabismus surgery, vision therapy) and achieved a best corrected visual acuity of at least 0.1 logMAR, but whose stereoacuity remains low (between 200&#34;-800&#34;). The goal of the protocol is to improve stereoacuity in cases like these.
Direct stimulation of stereopsis has already been shown to be effective in enhancing stereoacuity in stereo-deficient subjects16,17,18,19. However, for a stimulation system to be feasible, therapy must be performed in the patient&#39;s home to reach the 3,000-20,000 trials needed for learning to occur.
In the previously published study that validated this procedure and is summarized above, 11 subjects improved their stereoacuity20. However, five of the subjects experienced no increase in stereoacuity (Figure 3). This may be attributable to the presence of small-angle strabismus undetectable in a cover test. Read inferred that, since images from the left and right eyes should be located within Panum&#39;s area of fusion, normal stereoacuity should require alignment within 0.6 prism diopters28. Panum&#39;s fusional area is &#177;5-20 min of arc (0.1-0.6 prism diopter in the fovea), and it may be that alignment within this window is needed to support high-grade stereoscopic acuity29. A study conducted by Holmes et al. showed that a cover test failed to detect deviations below &#177;3 prism diopters; therefore, the presence of undetectable strabismus could compromise a patient&#39;s ability to acquire fine stereoacuity24.
Gamification has been used to enhance patient motivation and compliance. Moreover, the program stores data in the cloud after each session, making it possible for the practitioner to track a patient&#39;s activity remotely on a daily basis. Thanks to this feature, compliance results are excellent (88.36%) and comparable to those recorded in two earlier studies, in which amblyopic subjects received dichoptic stimulation treatment using an iPad at home10,11. They are also much better than the reported results of a PEDIG study under similar conditions, in which only 22.5% of the sample managed to complete over 75% of the treatment prescribed13. The compliance demonstrated here also exceeds that reported by studies that evaluated the effectiveness of occlusion treatment in amblyopia (70% compliance when 6 h of occlusion are prescribed, and 50% when 12 h are prescribed)30. A web application has the added advantage that parents are not required to keep a record of their child&#39;s compliance13. The optometrist&#39;s only duty is to access the server and check the data collected for each patient at the end of each session using the computerised stereoscopic game program.
During the training period, patients visit the optometry center (check-up visits), allowing the optometrist to stress the importance of user-to-screen distance. Optometrists also set the stimulation category (poor, coarse, moderate-fine) during these check-up visits. Perceptual learning theories predict that improvements are less likely if the patient does not work at his or her threshold (e.g., if the patient moves closer to the screen or works in an easier stimulation category). These findings were corroborated in the study carried out to validate this protocol20. User-to-screen distance is out of the software&#39;s control and is therefore the responsibility of the patient or patient&#39;s parents.
The decision to use a random-dot approach for the design of the computerized stereoscopic game may be critical. Stimulation through random-dot stereoscopic images is never inconsequential: even patients working below their threshold experience improvements. In a process of perceptual learning, repeated exposure to a random-dot stimulus alone will enhance binocular vision. The patient&#39;s task, and one that is particularly difficult for patients with a history of strabismus31, is to fuse the correlated random dots perceived by each eye12 without suppression. This enhances their ability to distinguish the correlated dots (signal) from those unable to be fused (noise). Training of this type may have improved the disparity detector response, given that the perceptual learning would have improved the fusional response and improved the patient&#39;s ability to detach the signal from noise32.
One of the risks of the perceptual learning approach is selectiveness. This method has demonstrated that random-dot stereogram training is not selective, because learning is transferred to medial lateral stereoacuity measured with a Wirt Circles test. Another finding that demonstrates the effectiveness of this treatment method is the stability of the results achieved. Different studies have examined whether improvements achieved in subjects with amblyopia as a result of perceptual learning training are stable16,17,19,33. This model has demonstrated the stability of the improvements measured with a random-dot stereoacuity test at a 6 month follow-up visit.
Several limitations have been detected. The software design requires the stimulation category to be set manually, when this process should ideally be automatic according to the patient&#39;s evolution. The pass level condition implemented could be improved by considering the possibility of moving the patient back to a coarse stereoacuity setting if the patient fails to pass a level on several consecutive occasions. In any case, a staircase procedure is discarded, because one of the goals of gamification is to improve patient motivation through game mechanics. The patient should experience the sensation of progress and success, regardless of whether their clinical condition is improving or deteriorating. This is achieved by concealing easier trials within the game flow (though not with a standard staircase procedure, whose goal is to quickly and accurately determine the threshold limit, at which performance is 50%). Another improvement is to monitor the patient&#39;s distance from the screen automatically. However, we are not aware of a solution that does not involve the use of special hardware, though it may be worth testing custom-built webcam head-tracking software.
Other limitations are due to the study design and include the following: (1) the majority of subjects had a history of strabismus (the sample of subjects with a history of anisometropic amblyopia was too small); (2) the age range was restricted to 7-14 years; and (3) the stereoacuity range was between 800&#34;-200&#34;. In future studies, it would be interesting to verify the therapeutic effect on anisometropic amblyopia and coarser stereoacuity and in older subjects.

Amblyopia is a developmental disorder of spatial vision frequently associated with the existence of strabismus, anisometropia, or form deprivation at a young age1. The incidence of amblyopia among the general population ranges from 1.3%-3.6%2. Conventional amblyopia therapy starts with the correction of any refractive error, followed by occlusion of the good eye with a patch or atropine penalization3. Although treatment outcomes of conventional treatment are initially good, with 73%-90% of patients experiencing improved visual acuity, this improvement does not equate to normal visual acuity in about 50% of patients. Moreover, children frequently experience deterioration even after successful treatment2. A previous study showed that, with respect to binocular vision, as few as 28% of anisometropic patients with amblyopia recorded two or more levels of improvement as a result of treatment4. There has been little research aimed at evaluating strategies for the improvement of stereopsis in cases of amblyopia, despite the fact that stereopsis is essential to visual perception in humans5. Conventional amblyopia therapy, which produces good results for visual acuity but poor outcomes in terms of binocular vision, can benefit from the development of an interventional model aimed at improving stereoacuity in patients with a history of amblyopia2.
Over the last 10 years, some researchers have proposed an alternative approach to understanding the course of amblyopia6,7. This understanding has motivated the proposal of a interventional model focused on anti-suppression dichoptic training for the recovery of binocular vision8,9. Amblyopia therapy involving targeted gamified activities for patient use at home has been reported successful in a number of cases10,11.
Notwithstanding, this dichoptic training is ineffective for improving stereoscopic acuity12. Two contemporary clinical trials using the anti-suppression dichoptic training model reported no stereoacuity improvement11,13. The most recent research indicates, however, that dichoptic stimulation is capable of reducing suppression depth and extent and improving binocular vision (restoring simultaneous binocular perception). In some cases, this coincides with improved stereoacuity14,15.
Some studies have proposed a different intervention approach that is focused on the direct stimulation of stereopsis through perceptual learning activities16,17. These studies are confined to a succession of cases treated under laboratory conditions. In Astle et al.&#39;s study, stereopsis was stimulated in two adult anisometropic amblyopes16 over the course of nine laboratory sessions. Treatment included binocular stimulation using a mirror stereoscope with stereogram pairs based on random-dot images. Ding and Levi stimulated stereopsis in a course of treatment conducted entirely under laboratory conditions, using a mirror stereoscope and stereograms based on Gabor patches18. Their subjects were five adults, four of which having no stereopsis and one being stereo-deficient. Subjects were required to perform between 3,000-20,000 perceptual learning training trials.
Furthermore, Xi et al. studied anisometropic amblyopes over the course of 10 -13 perceptual learning training sessions, during which 3-D anaglyph textures were used to stimulate stereopsis19. Finally, in Vedamurthy et al.&#39;s study, 11 stereo-deficient adults were engaged in visuo-motor task training (a &#34;squash-the-bug&#34; game) in a virtual reality environment17. These subjects performed 12,600 trials in 35 sessions over the course of 8-11 weeks.
Direct stimulation of stereopsis has been performed in laboratory studies, but this therapy model is time-consuming and difficult to apply in a daily clinical practice, especially with children. Thus, a feasible therapy model has been devised for which a successful proof of concept has been previously presented20. This protocol incorporates the results of a prospective, randomized, double-blind, parallel-group study based on perceptual learning treatment using random-dot stimuli in a video game format to improve stereoacuity. An in-depth explanation of the protocol followed in this study is presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autorrefractometer, model TRK 1P</td>
			<td>Topcon, Japan</td>
			<td></td>
			<td>Refractive error measurements by autorrefraction</td>
		</tr>
		<tr>
			<td>Computerized Stereoscopic Game</td>
			<td>University of Oviedo, Spain</td>
			<td></td>
			<td>The computer-based test itself was developed at the University of Oviedo by SM-G, coauthor of this manuscript. After finishing this study, a private company named VISIONARY TOOL (www.visionarytool.com) has contacted both SM-G and JAP-C to participate in the development of a computerized visual training tool. This tool includes several games and tests. The one used in this article, based on random dot hidden silhouettes, is one of them.</td>
		</tr>
		<tr>
			<td>Randot Preschool Stereoacuity Test</td>
			<td>Stereo Optical Company Inc, USA</td>
			<td></td>
			<td>Global stereoacuity test</td>
		</tr>
		<tr>
			<td>Screen model</td>
			<td>SIFIMAV, Italy</td>
			<td></td>
			<td>Logarithmic visual acuity chart ETDRS format</td>
		</tr>
		<tr>
			<td>Wirt Circles Test</td>
			<td>Stereo Optical Company Inc, USA</td>
			<td></td>
			<td>Local stereoacuity test</td>
		</tr>
	</tbody>
</table>

stereoacuity improvement using random-dot video games

Conventional amblyopia therapy involves occlusion or penalization of the dominant eye, though these methods enhance stereoscopic visual acuity in fewer than 30% of cases. To improve these results, we propose a treatment in the form of a video game, using random-dot stimuli and perceptual learning techniques to stimulate stereoacuity. The protocol is defined for stereo-deficient patients between 7-14 years of age who have already received treatment for amblyopia and have a monocular best corrected distance visual acuity of at least 0.1 logMAR. Patients are required to complete a perceptual learning program at home using the video game. While compliance is stored automatically in the cloud, periodic optometry center visits are used to track patient evolution and adjust the game&#39;s stereoscopic demand until the smallest detectable disparity is achieved. The protocol has proved to be successful, and effectiveness is gauged in terms of a two-level gain on a random stereoacuity test (global stereoacuity or cyclopean stereoacuity reference test). Moreover, the random-dot stimuli learning transfers to medial lateral stereoscopic acuity according to a Wirt Circles test, in which success criteria is a final stereoacuity of over 140&#34;, and the attained enhancement corresponds to no less than two levels of stereoscopic acuity. Six months later, a random-dot stereoacuity test recorded no reduction in the stereoacuity that was achieved.

As a representative example of the results that can be achieved following this protocol, we summarize the results of a recent study carried out by Portela et al.20. Figure 3 and Figure 4 show the outcomes that were obtained.
Sixteen stereo-deficient subjects aged between 7-14 years of age were included in this study, four of whom had a history of refractive amblyopia (2 anisometropic and 2 isometropic). Twelve of the subjects had a history of successfully treated strabismic amblyopia, and four of these had a history of both strabismic and anisometropic amblyopia. Eleven of the 12 subjects with a history of strabismic amblyopia presented esotropia, and one presented exotropia. All participants had previously received amblyopia therapy and achieved good levels of visual acuity but did not attain a fine level of stereoacuity (less than or equal to 200&#34;). All but one of the subjects were able to complete the 60 assigned training sessions that were 8 min each (8 h in total). Compliance was considered to be 100% when patients completed the training in less than 12 weeks and 0% when the training took more than 24 weeks. On average, subjects took 79 days to complete the 60 sessions (IQR = 66-102 days); therefore, they surpassed the minimum recommended compliance of five sessions per week. Compliance outcomes were excellent (88.36%).
Visual acuity among the subjects remained stable during and post-therapy. Stereoacuity, however, improved in a significant number of subjects (see Figure 3). The means, medians, and minimum and maximum values are presented in Table 1. When these were analyzed using the Mann-Whitney U test, stereoacuity improved significantly after treatment (Random-Dot Stereoacuity test, p = 0.019; Wirt Circles test, p = 0.014). For a better understanding, Figure 4 shows a graphic presentation of the improvement in stereoacuity between the start and end of therapy.
Stereoacuity improved by at least one level in 11 subjects when stereoacuity was measured with the random stereoacuity test. Where stereoacuity was evaluated with the Wirt Circles test, improvement of at least one level was also observed in 11 subjects. Clinically speaking, an improvement in stereoacuity measured with a random stereoacuity test is significant when the improvement reaches at least two levels (Adam&#39;s criteria)27, and this was achieved in seven subjects. Where the Wirt Circles test was used, an improvement of at least two levels and a stereoacuity equal or better than 140&#34; is considered significant (Levi&#39;s criteria)12, and this was achieved in 10 subjects. After 6 months, the outcomes were stable according to the random-dot stereoacuity test. This is the reference test to measure stereoacuity, with its main feature being its excellent test-retest reliability23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60236/60236fig1a.jpg" />
Figure 1: Protocol steps. <a href="https://www.jove.com/files/ftp_upload/60236/60236fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60236/60236fig2a.jpg" />
Figure 2: Logical process of the game. The subject must indicate which figure appears, selecting one from those shown at the bottom of the screen (left image). If the answer is correct, the software emits a high-pitched sound, and the same image appears in picture form (right image). If the subject provides three consecutive correct answers, the software generates a new screen with a random-dot image representing a finer stereopsis. If the subject provides a wrong answer, the software emits a deep sound and the random-dot image remains the same (left image). Finally, if the subject provides three consecutive wrong answers, the software will show the correct answer (right image). This figure is adapted from Portela et al.20 with permission from Optometry and Vision Science. <a href="https://www.jove.com/files/ftp_upload/60236/60236fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60236/60236fig3a.jpg" />
Figure 3: Measurements of basal and post-treatment levels of stereoacuity. Random-dot stereoacuity (RDS) and Wirt Circles tests were used to measure stereoacuity. Measurements are in log seconds of arc. This figure is adapted from Portela et al.20 with permission from Optometry and Vision Science. <a href="https://www.jove.com/files/ftp_upload/60236/60236fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60236/60236fig4.jpg" />
Figure 4: Medians of stereoacuity data before and immediately post treatment for each stereoacuity test. (A) Random-dot stereoacuity (RDS) test and (B) Wirt Circles test. Boxes indicate 25% and 75% quartiles. Measurements are in log seconds of arc. This figure is adapted from Portela et al.20 with permission from Optometry and Vision Science. <a href="https://www.jove.com/files/ftp_upload/60236/60236fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Baseline Stereo</td>
			<td colspan="2">Post-Treatment Stereo</td>
		</tr>
		<tr>
			<td></td>
			<td>Wirt</td>
			<td>RST</td>
			<td>Wirt</td>
			<td>RST</td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>293.13&#177;271.17</td>
			<td>475.00&#177;240.84</td>
			<td>107.50&#177;51.60</td>
			<td>305.63&#177;306.50</td>
		</tr>
		<tr>
			<td>Median</td>
			<td>200 [95 to 400] 95%</td>
			<td>400 [250 to 800] 95%</td>
			<td>100 [60 to 140] 95%</td>
			<td>150 [100 to 700] 95%</td>
		</tr>
		<tr>
			<td>Min</td>
			<td>50</td>
			<td>200</td>
			<td>40</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Max</td>
			<td>800</td>
			<td>800</td>
			<td>200</td>
			<td>800</td>
		</tr>
	</tbody>
</table>
Table 1: Means, standard deviation, medians, interquartile ranges, and maximum and minimum stereoacuity values. The left columns show baseline stereoacuity data, and the right columns show post-treatment stereoacuity outcomes. Stereoacuity was measured using random-dot stereoacuity (RDS) and Wirt Circles tests. Measurements are in seconds of arc. This figure is adapted from Portela et al.20 with permission from Optometry and Vision Science.

Watch this Scientific Journal Video about Stereoacuity Improvement using Random-Dot Video Games at JoVE.com

Stereoacuity Improvement using Random-Dot Video Games

Presented here is a protocol to improve stereoacuity using gamified perceptual learning software based on random-dot stimuli. Patients are stereo-deficient subjects without strabismus. The protocol combines optometry center visits with home exercises using software. Compliance and stereoacuity evolution are stored in the cloud.

Conventional amblyopia therapy involves occlusion or penalization of the dominant eye, though these methods enhance stereoscopic visual acuity in fewer ...

stereoacuity-improvement-using-random-dot-video-games

Research

JoVE Journal

Behavior

14.0K Views.  University of Oviedo. Presented here is a protocol to improve stereoacuity using gamified perceptual learning software based on random-dot stimuli. Patients are stereo-deficient subjects without strabismus. The protocol combines optometry center visits with home exercises using software. Compliance and stereoacuity evolution are stored in the cloud.

Video: Stereoacuity Improvement using Random-Dot Video Games

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Holistic Facial Composite Creation and Subsequent Video Line-up Eyewitness Identification Paradigm

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim to a crime may create from memory a holistic facial composite of the culprit with the assistance of a police operator. The aim is that the composite is recognized by someone who believes that they know the culprit. For this paradigm, participants view a culprit actor on video and following a delay, participant-witnesses construct a holistic system facial composite. Controls do not construct a composite. From a series of arrays of computer-generated, but realistic faces, the holistic system construction method primarily requires participant-witnesses to select the facial images most closely meeting their memory of the culprit. Variation between faces in successive arrays is reduced until ideally the final image possesses a close likeness to the culprit. Participant-witness directed tools can also alter facial features, configurations between features and holistic properties (e.g., age, distinctiveness, skin tone), all within a whole face context. The procedure is designed to closely match the holistic manner by which humans&#8217; process faces. On completion, based on their memory of the culprit, ratings of composite-culprit similarity are collected from the participant-witnesses. Similar ratings are collected from culprit-acquaintance assessors, as a marker of composite recognition likelihood. Following a further delay, all participants &#8212; including the controls&#160;&#8212; attempt to identify the culprit in either a culprit-present or culprit-absent video line-up, to replicate circumstances in which the police have located the correct culprit, or an innocent suspect. Data of control and participant-witness line-up outcomes are presented, demonstrating the positive influence of holistic composite construction on identification accuracy. Correlational analyses are conducted to measure the relationship between assessor and participant-witness composite-culprit similarity ratings, delay, identification accuracy, and confidence to examine which factors influence video line-up outcomes.

This applied experimental paradigm replicates circumstances by which an eyewitness to a real crime may create a holistic facial composite of the culprit from memory, and then attempt to identify the culprit from a video line-up containing the culprit or one in which he or she is not present.

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim ...

Reducing State Anxiety Using Working Memory Maintenance

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock. Anxiety is probed throughout the safe and threat blocks using the acoustic startle reflex, which is potentiated under threat (Anxiety-Potentiated Startle (APS)). By conducting the Sternberg WM paradigm during the threat of shock and probing the startle response during either the WM maintenance interval or the intertrial interval, it is possible to determine the effect of WM maintenance on APS.

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working ...

Frame-by-Frame Video Analysis of Idiosyncratic Reach-to-Grasp Movements in Humans

Prehension, the act of reaching to grasp an object, is central to the human experience. We use it to feed ourselves, groom ourselves, and manipulate objects and tools in our environment. Such behaviors are impaired by many sensorimotor disorders, yet our current understanding of their neural control is far from complete. Current technologies for investigating human reach-to-grasp movements often utilize motion tracking systems that can be expensive, require the attachment of markers or sensors to the hands, impede natural movement and sensory feedback, and provide kinematic output that can be difficult to interpret. While generally effective for studying the stereotypical reach-to-grasp movements of healthy sighted adults, many of these technologies face additional limitations when attempting to study the unpredictable and idiosyncratic reach-to-grasp movements of young infants, unsighted adults, and patients with neurological disorders. Thus, we present a novel, inexpensive, and highly reliable yet flexible protocol for quantifying the temporal and kinematic structure of idiosyncratic reach-to-grasp movements in humans. High speed video cameras capture multiple views of the reach-to-grasp movement. Frame-by-frame video analysis is then used to document the timing and magnitude of pre-defined behavioral events such as movement start, collection, maximum height, peak aperture, first contact, and final grasp. The temporal structure of the movement is reconstructed by documenting the relative frame number of each event while the kinematic structure of the hand is quantified using the ruler or measure function in photo editing software to calibrate 2 dimensional linear distances between two body parts or between a body part and the target. Frame-by-frame video analysis can provide a quantitative and comprehensive description of idiosyncratic reach-to-grasp movements and will enable researchers to expand their area of investigation to include a greater range of naturalistic prehensile behaviors, guided by a wider variety of sensory modalities, in both healthy and clinical populations.

This protocol describes how to use frame-by-frame video analysis to quantify idiosyncratic reach-to-grasp movements in humans. A comparative analysis of reaching in sighted versus unsighted healthy adults is used to demonstrate the technique, but the method can also be applied to the study of developmental and clinical populations.

Prehension, the act of reaching to grasp an object, is central to the human experience. We use it to feed ourselves, groom ourselves, and manipulate ...

Use of a Video Scoring Anchor for Rapid Serial Assessment of Social Communication in Toddlers

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency&#8212;which is foundational for both social learning and social competency&#8212;is hypothesized to be disrupted in autism spectrum disorder (ASD). The ability to quantify the full range of RSB during the toddler period, when core symptoms of ASD often arise, is pivotal for evaluating early risk for ASD, characterizing social development, and tracking response to early interventions. However, important parameters of variation in RSB&#8212;especially prior to the development of verbal language&#8212;can be nuanced and difficult to characterize using questionnaire-based methods. To address this challenge, we developed a system for measuring quantitative variation in RSB in toddlers (ages 18 - 30 months) that incorporated not only standard questionnaire data from caregivers but also a novel set of video-referenced items, through which a respondent compares the behavior of a subject to that observed in a short video of a young child manifesting a highly competent level of social communication. Testing of this measure in a general population sample of twins confirmed that both the video-referenced items and the RSB Total Score (video-referenced items plus non-video-referenced items) displayed unimodal, continuous distributions, strong internal consistency, marked preservation of individual differences, and extremely high heritability. In addition, video-referenced items were particularly sensitive to quantifying incremental changes in social communication, a major element of RSB, over the course of early childhood development. Scores on the vrRSB clearly differentiated children with and without ASD and these data comprise an initial validation of this promising method for quantifying early RSB&#8212;cross-sectionally, over time, and as a function of early intervention.

Here we present a protocol for administration of the video-referenced rating of reciprocal social behavior (vrRSB) using a paper or online version. This survey quantifies RSB, a prerequisite for social competence, in toddlers through &#34;video-referenced&#34; items whereby a subject is compared to a reference child shown in a three-minute video.

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency&#8212;which is foundational for both social learning and ...

Using Electroencephalography Measurements and High-quality Video Recording for Analyzing Visual Perception of Media Content

This article explores a method to detect differences in visual perception in humans. The method used is based on the psychological (or "cognitive") function of eyeblinks. Participants' eyeblinks are detected and acquired while watching videos specifically created for the investigation. The detection and acquisition of eyeblinks are carried out with the help of a 20-channel electroencephalographic (EEG) wireless device. The international 10-20 system for electrode placement is followed. A high-definition (HD) video camera is used to record participants' facial expressions, for contrast purposes. Instead of using pre-existing media content, purpose-made video content has been created following specific criteria of interest for this investigation, with stimuli enabling researchers to manage the precise parameters of interest. Otherwise, results could be contaminated with uncontrolled variables. The synchronization of the presentation of video stimuli with EEG recordings needs to be done in milliseconds. Analysis of collected data is performed with robust software for working with big matrices. Statistically significant differences in eyeblink rate related to media professionalization and editing style are found with the reported experimental procedures.

We present detection, acquisition, and analysis of eyeblink rates while watching media content.

This article explores a method to detect differences in visual perception in humans. The method used is based on the psychological (or "cognitive") ...

Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The purpose of the method is to document the effect of various potential anxiolytic treatments on laboratory rodent models. The EPM test is based on the rodents&#39; proclivity toward protected, enclosed dark spaces and unconditioned fear of open spaces and heights, and their innate intense motivation to explore novel environments. The EPM test is a widely used behavioral test for investigating the anxiolytic or anxiogenic responses of rodents given drugs that are known to affect behavior. Observation demonstrating a decreased proportion of time spent on closed arms, an increased proportion of time spent on open arms, a reduced number of entries to closed arms, and an elevated number of entries to open arms measured by the EPM test may reflect reduced anxiety levels. Using this method, the effect of exogenous ketone supplements on anxiety-related behavior is tested in Sprague Dawley (SPD) rats. Exogenous ketone supplements are chronically fed to the rats for 83 days or subchronically and acutely orally gavaged, daily for 7 days, before conducting the EPM test. Behavioral data collection is performed using the SMART video tracking system by a blinded observer at the end of the treatments. The main findings indicate that the EPM test is an effective method to detect the ketone supplement-induced anxiolytic effect and can be considered a sensitive measure to assess changes in anxiety behavior associated with drug- or metabolic-based therapies.

Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios.

The overall goal of this study is to describe the methodology of the elevated plus maze (EPM) test in combination with a video tracking software. The ...

Calculating Heart Rate Variability from ECG Data from Youth with Cerebral Palsy During Active Video Game Sessions

The aim of this study was to generate a method for calculating heart rate variability (HRV) from electrocardiogram (ECG) waveforms. The waveforms were recorded by a HR monitor that participants (youth with cerebral palsy (CP)) wore during active video game (AVG) sessions. The AVG sessions were designed to promote physical activity and fitness (aerobic performance) in participants. The goal was to evaluate the feasibility of AVGs as a physical therapy (PT) intervention strategy. The maximum HR (mHR) was determined for each participant and the Target Heart Rate Zone (THRZ) was calculated for each of three exercise phases in the 20 min AVG session: (warm-up at 40-60% mHR, conditioning at 60-80% mHR, and cool down at 40-60% mHR). Each participant played three 20 min games during the AVG session. All games were played while sitting on a bench because many youth with CP cannot stand for extended periods of time. Each game condition differed with participants using hand icons only, hand and feet icons together or feet icons only to collect objects. The objective of the game (called KOLLECT) is to collect objects to gain points and avoid hazards to not lose points. Hazards were used in the warm-up and cool down phases only to promote slower, controlled movement to maintain HR in the target heart rate zone (THRZ). There were no hazards in the conditioning phase to promote higher levels and more intense physical activity. Analytic methods were used to generate HRV (selected time-domain and frequency-domain measures) from ECG data to examine aerobic workload. Recent applications of HRV indicate that short-term measurements (5 min bouts) are appropriate and that HRV biofeedback may help improve symptoms and the quality of life in a variety of health conditions. Although HR is a well-accepted clinical measure to examine aerobic performance and intensity in PT interventions, HRV may provide information of the autonomic system functions, recovery and adaptation during AVG sessions.

This protocol describes a method for calculating Heart Rate Variability (HRV) from electrocardiogram (ECG) waveforms. Waveforms from continuous heart rate (HR) recordings during active video game (AVG) sessions were used to measure the aerobic performance of youth with cerebral palsy (CP).

The aim of this study was to generate a method for calculating heart rate variability (HRV) from electrocardiogram (ECG) waveforms. The waveforms were ...

Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic Brain Injury

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, considerable variability has been reported for the specific parameters used in different studies that have employed this model, making it difficult to harmonize and interpret the results between laboratories. For example, variability has been reported regarding the size and location of the craniectomy, how the Luer lock hub is placed relative to the craniectomy, the atmospheric pressure applied to the dura and the duration of the pressure pulse. Each of these parameters can impact injury severity, which directly correlates with the incidence of PTE. This has been manifested as a wide range of mortality rates, righting reflex times and incidence of convulsive seizures reported. Here we provide a detailed protocol for the method we have used to help facilitate harmonization between studies. We used FPI in combination with a wireless EEG telemetry system to continuously monitor for electrographic changes and detect seizure activity.&#160; FPI is induced by creating a 5 mm craniectomy over the left hemisphere, between the Bregma and Lambda and adjacent to the lateral ridge. A Luer lock hub is secured onto the skull over the craniectomy. This hub is connected to the FPI device, and a 20-millisecond pressure pulse is delivered directly to the intact dura through pressure tubing connected to the hub via a twist lock connector. Following recovery, rats are re-anesthetized to remove the hub. Five 0.5 mm, stainless steel EEG electrode screws are placed in contact with the dura through the skull and serve as four recording electrodes and one reference electrode. The electrode wires are collected into a pedestal connector which is secured into place with bone cement. Continuous video/EEG recordings are collected for up to 4 weeks post TBI.

Here we present a protocol to induce severe TBI with the lateral fluid percussion injury (FPI) model in adult, male Wistar rats. We also demonstrate the use of a wireless telemetry system to collect continuous video-EEG recordings and monitor for epileptiform discharges consistent with post-traumatic epileptogenesis.&#160;