No funding sources for the work.

The study was approved by the Ethics Committee Review Board of Peking University Third Hospital and conformed to the Declaration of Helsinki.
1. Settings
NOTE: The icVEP hardware necessitates a reexamination of stimulus conditions for favoring the M-ON pathway using a standard video card with an 8-bit digital-to-analog converter per electron gun.
<ol>
	<li>Click the Test CFG button and select icvep-bc-8.cfg.</li>
	<li>Click the System button, select Configuration &#8594; Test Configuration and then click the Edit Stimulus button. Please ensure that the frame rate is 60 Hz, luminance of the display&#39;s static background is 51 cd/m2, and total cycles are 20.</li>
	<li>To differentiate OAG patients and control subjects, ensure the following conditions: sinusoidal temporal signals of 10 Hz (6 frames per cycle) and 15% positive-contrast (bright, Contrast 7.50%, Luminance Offset 7.50%, Contrast Offset 0.00%).</li>
</ol>
2. Examination
<ol>
	<li>Select the Eye to Test and ensure that the spatial pattern is a 24 x 24 array of isolated-checks to subtend a 11&#176; visual field, with a 2 x 2 array fixation cross without sinusoidal temporal signals, in order to cue facilitated and careful fixation on the center of the screen (Figure 2)9.</li>
	<li>Place the gold cup electrodes filled with electrolytic water-soluble paste at the following midline sites on the scalp based on the international 10-20 system (Figure 3)24. Ensure that the testing distance is 114 cm.</li>
	<li>Click the Start Test button. One run last for 2 s: the first second of this period presents half of the test contrast level (7.50%) as an adaptation condition, and the following second presents the full test contrast (15.0%).</li>
	<li>Note the prompt Error:Outlier from the program and repeat the run when noise is detected and when the electroencephalography (EEG) epoch is rejected.</li>
	<li>Note the EEG data that is displayed on the operator&#39;s monitor when the run is determined to be valid and when the operator is prompted to click Stop Test button in order to reject the data based on reliability.</li>
</ol>
3. Automatic data processing using software
NOTE: The data is calculated by a discrete Fourier transform after EEG signals are recorded.
<ol>
	<li>Note that once the data is accepted, the program will instruct the operator by a Ding sound and automatically initiate the next run until a set of 8 valid runs are accumulated.</li>
	<li>Note that each run produces a fundamental frequency component (FFC), and if one of the FFCs is an outlier relative to the remaining 7, the program will discard that FFC and will prompt the operator to repeat the run until 8 qualified runs are collected.</li>
	<li>Wait for the program to calculate the mean FFC and radius of a 95% confidence circle using the T2circ statistic25 that is automatically produced from the 8 FFCs within a few seconds.</li>
	<li>Ensure that the individual and mean FFC values, confidence circle, and signal-to-noise ratio (SNR) are automatically displayed on the monitor within less than 1 min after the end of the test (Figure 4A).</li>
</ol>
4. Flowchart for assessing reliability of results
<ol>
	<li>Ensure that the refractive error is corrected to adapt for a distance of 114 cm.</li>
	<li>Ensure that intraocular pressure (IOP) is &#8804; 30 mmHg on the day of examination.</li>
	<li>Ensure that pupil diameters are &#8805; 2 mm and without mydriasis.</li>
	<li>Ensure that each subject rest and is quiet &#8805; 30 min before examination.</li>
	<li>To avoid the influence of a study curve, first check the right eye, then left eye; then, check the right and left eyes again, and record this second result.</li>
	<li>Initiate a retest after at least a 30 min rest when the R-value (nosing ring radius) between both eyes shows a difference of &#62; 0.2, which means the result is unreliable as mood swings.</li>
</ol>

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C., Seliger, S., Zemon, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+evoked+potential+assessment+of+the+effects+of+glaucoma+on+visual+subsystems.">Visual evoked potential assessment of the effects of glaucoma on visual subsystems.</a> Vision Research. 38, 1901-1911 (1988).</li><li>Zemon, V., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminance+contrast+mechanisms+in+humans:+visual+evoked+potentials+and+a+nonlinear+model.">Luminance contrast mechanisms in humans: visual evoked potentials and a nonlinear model.</a> Vision Research. 46, 4163-4180 (2006).</li><li>Xiang, F., Lingling, W., Xia, D., Tong, D., Aihua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Isolated-Check+Visual+Evoked+Potential+in+Early+Stage+of+Open-Angle+Glaucoma+Patients.">Applications of Isolated-Check Visual Evoked Potential in Early Stage of Open-Angle Glaucoma Patients.</a> Chinese Medical Journal. 131 (20), 2439-2446 (2018).</li><li>Victor, J. D., Mast, J. <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+statistic+for+steady-state+evoked+potentials.">A new statistic for steady-state evoked potentials.</a> Electroencephalography and Clinical Neurophysiology. 78 (5), 378-388 (1991).</li><li>Badr, A. A., Zemon, V. M., Greenstein, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-+versus+P-function:+Relationship+to+visual+field+loss+in+patients+with+open+angle+glaucoma.">M- versus P-function: Relationship to visual field loss in patients with open angle glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 44 (13), 50 (2003).</li><li>Kamantigue, M. E., Joson, P. J., Chen, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+visual+field+defects+on+standard+automated+perimetry+by+screening+C-20-1+frequency+doubling+technology+perimetry.">Prediction of visual field defects on standard automated perimetry by screening C-20-1 frequency doubling technology perimetry.</a> Journal of Glaucoma. 15, 35-39 (2006).</li><li>Richard, P. M., Donald, L. B., Paul, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Categorizing+the+stage+of+glaucoma+from+pre-diagnosis+to+end-stage+disease.">Categorizing the stage of glaucoma from pre-diagnosis to end-stage disease.</a> American Journal of Ophthalmology. 141 (1), 27 (2006).</li><li>Brandt, J. D., Beiser, J. A., Kass, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-parametric+analysis+of+experiments.">A non-parametric analysis of experiments.</a> Psychonomic Science. 1, 125-126 (1964).</li><li>Salim, S., 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=Influence+of+pupil+size+and+other+test+variables+on+visual+function+assessment+using+visual+evoked+potentials+in+normal+subjects.">Influence of pupil size and other test variables on visual function assessment using visual evoked potentials in normal subjects.</a> Documenta Ophthalmologica. 121 (1), 1-7 (2010).</li><li>Nebbioso, M., Steigerwalt, R. D., Pecori-Giraldi, J., Vingolo, E. 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C., 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+Nature+of+Macular+Damage+in+Glaucoma+as+Revealed+by+Averaging+Optical+Coherence+Tomography+Data.">The Nature of Macular Damage in Glaucoma as Revealed by Averaging Optical Coherence Tomography Data.</a> Translational Vision Science &amp; Technology. 3 (1), (2012).</li><li>Hood, D. 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All the authors have nothing to disclose.

Different settings of icVEP can stimulate different M-cell pathways and create different EEG signals. Under conditions of high temporal frequency (15 Hz) luminance contrast of icVEP (16% positive-contrast), a study involving 15 OAG patients and 14 normal observers showed a sensitivity of 73.33% and specificity of 100%22. However, one-half of these patients had advanced OAG. Therefore, for early stage OAG, the sensitivity could not be estimated due to the small sample size.
Tsai&#39;s study showed a sensitivity of 78% (conditions of 15% positive-contrast and 10 Hz temporal modulation) and specificity of 100%, with an accuracy of 94% from the ROC curve. These results improved upon Greenstein&#39;s study because of the lower contrast and spatial frequency found in earlier glaucoma patients. Nevertheless, there were less than 11 early stage OAG patients among 18 glaucoma patients (17 open-angle, 1 angle-closure) and 16 controls in the study9.
In the current study, the OAG patients were only those in early stages and included a much larger sample size, which suggests that icVEP is indeed useful for detecting OAG in the &#34;real&#34; early-stage. About 70% of early stage OAG eyes were detected by icVEP, and the SNR of patients was greatly different than that of normal subjects.
A recent study showed that pupil size can affect icVEP results in normal subjects. icVEP values were influenced by pupillary constriction and dilation as well as optical blur33. This suggests that when obtaining icVEP measurements, the influence of pupil size and optical blur should be kept in mind for accurate interpretations. In the current study, pupil size was measured, and it was ensured that all values fell in the normal range. Furthermore, all EEG signals may have been affected by emotions, which yields mostly false positive errors. The current study ensured an IOP of &#8804; 30mmHg on the day of examination to avoid mood swings caused by high pressure. All patients rested for &#8805; 30 min before each examination, and reexamination was also performed to avoid mood effects.
SNR was defined as the ratio of the mean amplitude of the FFC to the radius of the 95% confidence circle. An SNR of &#62; 1 indicated a significant response at the 0.05 level, which implied normal electrophysiological activity in the optic nerve. An SNR of &#8804; 1 indicated a response similar to or weaker than the background noise at the 0.05 level, implying abnormal electrophysiological activity in the optic nerve. However, an SNR of 0.93 was optimal for discrimination of early stage OAG patients and control subjects in the current study using a ROC curve. Therefore, a SNR criterion of 0.93 may distinguish the severity of GON in early stage OAG patients for this study.
More than 50% M cells were in the macular region; thus, if the fovea was stimulated, there was likely a strong signal resulting in SNR &#62; 1. Therefore, the 2 x 2 array fixation cross on the center of the screen without sinusoidal temporal signals was able to cue-facilitate careful fixation as well as avoid false negative errors with poor fixation34. Moreover, recent SD-OCT studies proved that RGCs in the macular region become damaged even in early stages of glaucoma, because proteolysis and secondary axotomy after damage to the optic nerve head may result in RGC apoptosis35,36,37,38.
Analysis of central 16 test points in the current study based on pattern deviations in HFA corresponded to the 5&#176;-10&#176; of Bjerrum areas, where almost one-half of M-cells are distributed10,11,12,13,14. This study showed the numbers of abnormal test points in which different possibility criteria were negatively correlated with SNR (negative R-value); though, only when p &#60; 0.5% was the correlation significant, suggesting that icVEP was able to detect functional abnormalities and reflect the severity of central visual field loss in early stage OAG.
It has been reported that the responses to stimulation of the P-cell and M-ON pathway are severely disrupted in early stages of glaucoma, even without functional involvement of the central visual field test26. However, a limitation of this study is that the icVEP test requires patients with a BCVA value of larger than 0.3, spherical refraction between -6 and +3 diopters, and transparent ocular media. The study only shows the usefulness of icVEP in early OAG eyes with better visual acuity. Therefore, further studies are needed to create better stimulations and define more accurate criteria for OAG eyes with poorer visual acuity. This will help determine if the icVEP can serve as the optimal functional test for discriminating glaucoma suspects as well as pre-perimetrical and early-stages of OAG. Furthermore, another limitation is that the study does not account for differences between dominant and non-dominant eyes. Differences between those pathways and testing of these two eyes may affect the EEG signals. Above all, the flowchart will be improved after further studies are performed.
In summary, icVEP is able to detect glaucomatous visual function abnormalities in almost 70% of early stage OAG patients, with a specificity about 95%. The measured functions correlate with both the severity of central 11&#176; visual field loss of standard automated perimetry and decreases in RNFL thickness as detected by OCT. Therefore, icVEP can serve as a useful and objective electrophysiological visual field functional test for diagnosing early stage OAG.

Open-angle glaucoma (OAG) is a chronic, irreversible disease and one of the leading causes of blindness. Previous studies have shown that visual field tests, which are the current gold standard for glaucomatous visual loss detection, are based on conventional standard automated perimetry (SAP) cannot detect early glaucomatous functional loss until 20%-40% of retinal ganglion cells (RGCs) are damaged1,2. Furthermore, SAP has also been shown to have only moderate test-retest reliability, because it is a subjective psychophysical test and a time-consuming task for patients3.
Objective electrophysiological visual field functional measures have better test-retest reliability when detecting glaucoma. Such measures include the multifocal visual evoked potential (mfVEP) and pattern electroretinogram (pERG). However, the pERG cannot provide topographic information, and the mfVEP is more time-consuming than SAP4,5,6,7,8. Fortunately, the isolated-check visual evoked potential (icVEP) was recently designed as an additional technique to detect glaucomatous damage earlier and faster9.
In the retina, there are several RGC subpopulations such as magnocellular cells (M-cells), parvocellular cells (P-cells), and bistratified cells. They represent parallel pathways for visual information being transmitted to the brain (Figure 1)9,10. To govern the separate perceptions of brightness and darkness, the dichotomy of ON and OFF pathways has been established11,12. Magnocellular ON (M-ON) cells are considerably larger than magnocellular OFF (M-OFF) cells, while M-cells are considerably larger than P-cells in humans13,14. The M-cell pathway mainly conveys low spatial frequency/high temporal frequency information15. Thus, cells involved in the M-ON pathway are sensitive to low levels of luminance contrast and not sensitive to chromatic information with larger diameter axons, which are preferentially damaged in early glaucoma16,17. Therefore, the icVEP produces low spatial frequency/high temporal frequency bright stimuli and records cortical activity primarily initiated by afferents (such as those found in the M-ON pathway) for the early detection of glaucoma18,19,20,21,22,23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CR-2 AF Digital Non-Mydriatic Retinal Camera</td>
			<td>Canon U.S.A., Inc., Melville, NY, USA</td>
			<td></td>
			<td>Stereoscopic fundus photographs</td>
		</tr>
		<tr>
			<td>DGH 500 PachetteTM</td>
			<td>DGH Technology, Exton, PA, USA</td>
			<td></td>
			<td>ultrasound pachymetry</td>
		</tr>
		<tr>
			<td>HFA II 750i</td>
			<td>Carl Zeiss Meditec Inc., Dublin, German</td>
			<td></td>
			<td>Humphrey Field Analyzer II</td>
		</tr>
		<tr>
			<td>Neucodia novel electrophysiological instrument</td>
			<td>Huzhou Medconova Medical Technology Co.Ltd., Zhejiang province, P.R. China</td>
			<td></td>
			<td>icVEP</td>
		</tr>
		<tr>
			<td>Spectralis SD-OCT</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td></td>
			<td>OCT</td>
		</tr>
	</tbody>
</table>

assessing early stage open-angle glaucoma in patients by isolated-check visual evoked potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

Recent studies showed that the accuracy of icVEP for glaucoma diagnosis ranges from 91%-100%9,22,26. Cross-sectional studies in China are presented here to further evaluate the potential diagnostic value of icVEP for early stage OAG.
Subjects 
Subjects were OAG patients and healthy volunteers recruited by the Department of Ophthalmology, Peking University Third Hospital during 2015 and 2016. Inclusion criteria for OAG patients included the following: 25-75 years of age; best-corrected visual acuity (BCVA) &#60; 0.3 (logarithm of the minimum angle of resolution, log MAR); spherical refraction between -6 and +3 diopters; and transparent ocular media. Additionally, patients showed the presence of OAG (subjects with open-angle, visual field defects corresponding to glaucomatous optic neuropathy [GON], and having normal or elevated IOP without secondary causes), in which the IOP was medically well-controlled and had reliable visual field test results (false positive errors &#8804; 20%, false negative errors &#8804; 20%, fixation losses &#8804; 30%) that showed early glaucomatous visual field defects on SAP.
Inclusion criteria for control subjects included the following: no ocular abnormalities, especially no GON in any eye; and a normal IOP that was never elevated over 21 mmHg. Exclusion criteria included the following: diabetes or any other systemic disease; history of ocular or neurologic disease; unequal pupil diameters and pupil diameters of &#60; 2.0 mm; poor fixation; current use of medications that can affect visual field sensitivity (i.e., ethambutol, hydroxychloroquine, chlorpromazine); and previous history of intraocular surgery or refractory surgery.
Examinations for OAG diagnosis 
For all patients, spectacle corrections were used to decrease possible effects of a blur on visual field sensitivity. At least two reliable SAP tests were performed by the Humphrey Field Analyzer II 30-2 SITA standard program at baseline. The second reliable visual field result obtained was used in this study to minimize learning effects27. An early stage of glaucomatous visual field loss was defined as a mean deviation (MD) of &#8805; -6.00 dB, and with at least one of the following: 1) there existed a cluster of &#8805; 3 points in an expected location of the visual field depressed &#60; 5% level, at least one of which was &#60; 1% level on pattern deviation plot; 2) corrected pattern standard deviation or pattern standard deviation significant were at p &#60; 0.05; 3) glaucoma hemifield test result was &#34;outside normal limits&#34;28.
Baseline examination consisted of tests for visual acuity and refraction, pupil-diameter-measurement with a ruler in natural light, slit-lamp biomicroscopy, gonioscopy, Goldmann applanation tonometry (GAT), and dilated stereoscopic fundus examination in all subjects.
The baseline IOP was measured by GAT during glaucoma service (8 A.M. to 11 A.M. local time) on the day after receiving icVEP test reports. Each patient was also subjected to a central corneal thickness (CCT) measurement using ultrasound pachymetry under topical anesthesia29. An average of five consecutive readings was recorded.
Stereoscopic fundus photographs were obtained from each patient after pupil dilation and evaluated in a masked fashion by two experienced doctors. Discrepancies between the two doctors were either resolved by consensus or adjudication of a third experienced doctor. GON was defined as at least one of the following: 1) the rim-to-disc ratio was &#60; 0.1 in the upper or lower rims; 2) there existed retinal nerve fiber layer (RNFL) defects; 3) optic disc showed splint hemorrhages30,31.
Each patient was also subjected to an optical coherence tomography (OCT) test to confirm RNFL defects corresponding to both stereoscopic photographs and HFA results. The changing of RNFL thickness in temporal superior (TS) quadrant and temporal inferior (TI) quadrant were calculated as follows: changing of RNFL thickness = RNFL thickness value - standard value from database of normal people (Figure 4B).
Statistical analysis 
One eye was randomly selected to be analyzed when both eyes met the inclusion criteria. All the data needed to be established within 3 months for each subject. The SPSS 22.0 statistical package with statistical tests was used as follows: independent sample t-test was used for normally distributed variables; Mann-Whitney U test was used for numeric variables that were not normally distributed; and binomial variables were compared with a Chi-squared test or Fisher&#39;s exact test, when necessary. Receiver-operating-characteristic (ROC) curve analysis was used to estimate prediction accuracy for the presence of glaucomatous damage32. Pearson correlation coefficient was used to analyze correlations between SNR and parameters on OCT as well as between SNR and abnormalities in the central 11&#176; field on SAP. If p &#60; 0.05, differences were considered significant.
Results 
A total of 44 OAG patients and 39 control subjects were included with complete data. None of these subjects complained during the icVEP test. All 83 subjects were Chinese (48 males and 35 females) with an average age of 48.54 &#177; 16.70 years old (range of 25-74 years). No statistical differences existed in age, sex, right/left eye, BCVA, spherical equivalent, or pupil diameter between patients and controls (Table 1, p &#62; 0.05), but SNR was significantly lower in patients than in controls (Table 1, p &#60; 0.05).
Regarding the icVEP results, there were 30 eyes of early OAG patients that were SNR-positive (68.18%) and only two eyes in the control group (5.13%). Using an SNR criterion of 1, icVEP showed a sensitivity of 68.18% and specificity of 94.87% for diagnosing early OAG (calculating an accuracy of 67/83 [80.72%]). However, ROC analysis indicated that an a priori SNR criterion of 0.93 was optimal for discrimination between patients and control subjects (Figure 5). Using an SNR criterion of 0.93, the specificity of the test reached 100% with a sensitivity of 65.90% (calculating an accuracy of 82.10%).
For the patients, abnormalities in the central 11&#176; visual field test (HFA, pattern deviation, central 16 test points; Figure 4C) were calculated by the numbers of abnormal points with different possibility criteria. With a criterion level of p &#60; 0.5, the amount of abnormal test points in the central 11&#176; visual field was significantly negatively correlated with SNR (p &#60; 0.05, r = -0.332, Table 2). Thickness changing of RNFL in the temporal superior quadrant was significantly positively correlated with SNR (p &#60; 0.05, r = 0.370, Table 2), while SAP-MD, SAP-MD of the other eye, thickness changing of RNFL in the temporal inferior quadrant, and baseline IOP and CCT were all not correlated with SNR (p &#62; 0.05, Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60673/60673fig1.jpg" /> 
Figure 1: Representation of the isolated-check visual evoked potential evaluating the M-cell pathway. Layers 1 and 2 are involved in the magnocellular pathway. Layers 3, 4, 5, and 6 are involved in the parvocellular pathway. The spaces between these six layers are involved in the bistratified cell pathway. RGC = retinal ganglion cell. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60673/60673fig1large.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/60673/60673fig2.jpg" /> 
Figure 2: Bright conditions (positive-contrast) on the screen of isolated-check visual evoked potential. This figure has been modified from a previous publication24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60673/60673fig2large.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/60673/60673fig3.jpg" /> 
Figure 3: Diagram of isolated-check visual evoked potential examination. GND = grounding electrode; Cz = central midline electrode; Pz = parietal midline electrode; Oz = occipital midline electrode. This figure has been modified from a previous publication24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60673/60673fig3large.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/60673/60673fig4.jpg" /> 
Figure 4: Typical results from an early stage open angle glaucoma patient. (A) Abnormal isolated-check visual evoked potential results. (B) Outcomes of peripapillary retinal nerve fiber layer thickness (RNFLT) classification on the report of optical coherence tomography. Changing of RNFLT = RNFLT value (black number). The standard value from a database of normal subjects. (green number in brackets). G = global; N = nasal; T = temporal; NS = nasal superior; TS = temporal superior; NI = nasal inferior; TI = temporal inferior. (C) Central 16 test points of pattern deviation on Humphrey Field Analyzer 30-2 SITA program corresponding to the central 11&#176; visual field. This figure has been modified from a previous publication24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60673/60673fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60673/60673fig5.jpg" /> 
Figure 5: ROC curve. Shown is a ROC curve (blue) for data collected from the signal-to-noise ratios of isolated-check visual evoked potential in open-angle glaucoma patients and control subjects. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60673/60673fig5large.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>OAG patients (n=44)</td>
			<td>Control subjects (n=39)</td>
			<td>p</td>
		</tr>
		<tr>
			<td>Age (year)</td>
			<td>51.59&#177;14.98</td>
			<td>44.72&#177;16.88</td>
			<td>0.053*</td>
		</tr>
		<tr>
			<td>Sex (male/female)</td>
			<td>28/16</td>
			<td>20/19</td>
			<td>0.175$</td>
		</tr>
		<tr>
			<td>Right eyes / Left eyes</td>
			<td>20/24</td>
			<td>19/20</td>
			<td>0.770$</td>
		</tr>
		<tr>
			<td>BCVA (log MAR)</td>
			<td>0.04&#177;0.06</td>
			<td>0.01&#177;0.04</td>
			<td>0.093#</td>
		</tr>
		<tr>
			<td>Spherical Equivalent (D)</td>
			<td>-1.80&#177;2.16</td>
			<td>-1.30&#177;2.00</td>
			<td>0.276#</td>
		</tr>
		<tr>
			<td>Pupil Diameters (mm)</td>
			<td>3.43&#177;0.50</td>
			<td>3.46&#177;0.51</td>
			<td>0.789#</td>
		</tr>
		<tr>
			<td>icVEP-SNR</td>
			<td>0.85&#177;0.53</td>
			<td>1.44&#177;0.57</td>
			<td>0.000#</td>
		</tr>
		<tr>
			<td colspan="4">*Independent-sample t test, $Chi-square test, #Mann-Whitney U test</td>
		</tr>
		<tr>
			<td colspan="4">OAG: open-angle glaucoma, BCVA: best-corrected visual acuity; log MAR: logarithm of the minimum angle of resolution; icVEP: isolated-check visual evoked potential; SNR: signal-to-noise ratio</td>
		</tr>
	</tbody>
</table>
Table 1: Clinical characteristics of OAG patients and control subjects at baseline.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Numbers of abnormal test points</td>
			<td>Mean &#177; Std (n=44)</td>
			<td>r</td>
			<td>p*</td>
		</tr>
		<tr>
			<td>When P&#60;5%</td>
			<td>4.20&#177;2.60</td>
			<td>-0.264</td>
			<td>0.099</td>
		</tr>
		<tr>
			<td>When P&#60;2%</td>
			<td>2.83&#177;2.34</td>
			<td>-0.298</td>
			<td>0.061</td>
		</tr>
		<tr>
			<td>When P&#60;1%</td>
			<td>2.08&#177;2.12</td>
			<td>-0.266</td>
			<td>0.097</td>
		</tr>
		<tr>
			<td>When P&#60;0.5%</td>
			<td>1.48&#177;1.80</td>
			<td>-0.332</td>
			<td>0.037</td>
		</tr>
		<tr>
			<td colspan="4">*Pearson correlation test</td>
		</tr>
		<tr>
			<td colspan="4">icVEP: isolated-check visual evoked potential; SNR: signal-to-noise ratio; SAP: standard automated perimetry</td>
		</tr>
	</tbody>
</table>
Table 2: Correlation between icVEP-SNR and abnormalities in central 11&#176; visual field of SAP in open-angle glaucoma patients.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Mean &#177; Std (n=44)</td>
			<td>r</td>
			<td>p*</td>
		</tr>
		<tr>
			<td>SAP-MD (dB)</td>
			<td>-3.83&#177;1.26</td>
			<td>0.115</td>
			<td>0.457</td>
		</tr>
		<tr>
			<td>SAP-MD of the other eye (dB)</td>
			<td>-4.86&#177;3.94</td>
			<td>-0.15</td>
			<td>0.33</td>
		</tr>
		<tr>
			<td>OCT-Thickness changing of RNFL (&#956;m)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temporal Superior quadrant</td>
			<td>-39.31&#177;29.89</td>
			<td>0.37</td>
			<td>0.016</td>
		</tr>
		<tr>
			<td>Temporal Inferior quadrant</td>
			<td>-43.64&#177;29.83</td>
			<td>-0.22</td>
			<td>0.161</td>
		</tr>
		<tr>
			<td>Baseline IOP (mmHg)</td>
			<td>15.48&#177;2.80</td>
			<td>-0.121</td>
			<td>0.435</td>
		</tr>
		<tr>
			<td>CCT (&#956;m)</td>
			<td>523.24&#177;29.64</td>
			<td>0.171</td>
			<td>0.333</td>
		</tr>
		<tr>
			<td colspan="4">*Pearson correlation test</td>
		</tr>
		<tr>
			<td colspan="4">icVEP: isolated-check visual evoked potential; SNR: signal-to-noise ratio; SAP: standard automated perimetry (HFA 30-2 SITA); MD: mean deviation; OCT: optical coherence tomography; RNFL: retinal nerve fiber layer; IOP: intraocular pressure; CCT: central corneal thickness</td>
		</tr>
	</tbody>
</table>
Table 3: Correlation between icVEP-SNR and relating factors in patients with open-angle glaucoma.

Watch this Scientific Journal Video about Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential at JoVE.com

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

assessing-early-stage-open-angle-glaucoma-patients-isolated-check

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.2K Views.  Peking University Third Hospital. The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

Video: Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Extracting Visual Evoked Potentials from EEG Data Recorded During fMRI-guided Transcranial Magnetic Stimulation

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological effects. Specifically, by creating a transient interference with the normal activity of a target region and measuring changes in an electrophysiological signal, we can establish a causal link between the stimulated brain area or network and the electrophysiological signal that we record. If target brain areas are functionally defined with prior fMRI scan, TMS could be used to link the fMRI activations with evoked potentials recorded. However, conducting such experiments presents significant technical challenges given the high amplitude artifacts introduced into the EEG signal by the magnetic pulse, and the difficulty to successfully target areas that were functionally defined by fMRI. Here we describe a methodology for combining these three common tools: TMS, EEG, and fMRI. We explain how to guide the stimulator&#39;s coil to the desired target area using anatomical or functional MRI data, how to record EEG during concurrent TMS, how to design an ERP study suitable for EEG-TMS combination and how to extract reliable ERP from the recorded data. We will provide representative results from a previously published study, in which fMRI-guided TMS was used concurrently with EEG to show that the face-selective N1 and the body-selective N1 component of the ERP are associated with distinct neural networks in extrastriate cortex. This method allows us to combine the high spatial resolution of fMRI with the high temporal resolution of TMS and EEG and therefore obtain a comprehensive understanding of the neural basis of various cognitive processes.

This paper describes a method for collecting and analyzing electroencephalography (EEG) data during concurrent transcranial magnetic stimulation (TMS) guided by activations revealed with functional magnetic resonance imaging (fMRI). A method for TMS artifact removal and extraction of event related potentials is described as well as considerations in paradigm design and experimental setup.

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...