The authors would like to thank Zeiss Mexico for the unrestricted support to use the Cirrus 6000 with AngioPlex equipment.

This protocol was approved by Sala Uno&#39;s human research ethics committee. See Video 1 for sections 1 and 2 and the Table of Materials for details about the equipment used in this study.
1. Retinal analysis in the OCTA device
<ol>
	<li>Select the menu for retinal analysis in the OCTA device.</li>
	<li>Select a 3 x 3 mm retinal map; select superficial if the OCTA device measures different capillary plexuses.</li>
	<li>Select vessel length density (or its equivalent, e.g., skeletonized vascular density).</li>
	<li>Measure vessel length density in mm-1 in a 3 x 3 mm retinal map. 
	NOTE: The map is divided into two regions: center (within a 1 mm circle, concentric to the foveal center) and inner (outside the 1 mm center circle, Figure 3). The equipment also measures a full density (within the 3 mm circle) and subdivides the inner region into four fields: superior, inferior, temporal, and nasal (Figure 4). Each region is specified so that the vessel length densities are measured automatically. The instruments display the values for center, inner, and full densities and for superior, temporal, inferior, and nasal fields of the inner density.</li>
	<li>Return to the menu for retinal analysis.</li>
	<li>Select a 3 x 3 mm retinal map; select superficial if the OCTA device measures different capillary plexuses.</li>
	<li>Select perfusion density (or its equivalent, e.g., vessel density).</li>
	<li>Measure perfusion density in % in a 3 x 3 mm retinal map. 
	NOTE: The map is divided into two regions: center (within a 1 mm circle, concentric to the foveal center) and inner (outside the 1 mm center circle). The equipment also measures a full density (within the 3 mm circle) and subdivides the inner region into four fields: superior, inferior, temporal, and nasal. Each region is specified so that the perfusion densities are measured automatically. The instruments display the values for center, inner, and full densities and for superior, temporal, inferior, and nasal fields of the inner density.</li>
	<li>Verify that the density maps have a signal strength &#62; 7; then, verify that the maps have no measurement errors resulting from artifacts or eye movements.</li>
	<li>Register the values of center vessel length density, center perfusion density, inner vessel length density, inner perfusion density, superior vessel length density, superior perfusion density, inferior vessel length density, inferior perfusion density, temporal vessel length density, temporal perfusion density, nasal vessel length density, and nasal perfusion density in a spreadsheet.</li>
</ol>
2. Calculation of the coefficients of determination using a spreadsheet
<ol>
	<li>Select the variables to be evaluated (e.g., center vessel length density and center perfusion density). Select the values of both variables for a defined group (e.g., group 1).</li>
	<li>In the toolbar, click on insert.</li>
	<li>Click on the recommended charts button in the graphs section. Wait for a scatter chart to appear as a suggestion in a window. Click the OK button to accept the suggestion.</li>
	<li>Inspect the scatter chart of the data. Right-click on the series to display an options menu.</li>
	<li>Select the add trendline option. Wait for a linear trendline to be added to the chart and for a menu on the right side of the screen.</li>
	<li>Displace the menu downwards to find the Display R-squared value on chart option. Select this option to display the R-squared value on the chart. Select the R-squared-value.</li>
	<li>Select Home on the toolbar and then click on the copy button.</li>
	<li>Prepare a chart of coefficients of determination on a new page.</li>
	<li>Select a destination cell (e.g., center coefficient of determination for group 1). Click on the right mouse button. Select paste with keep source formatting.</li>
	<li>Prepare a new chart to show the percentage of perfusion density changes explained by changes in vessel density.</li>
	<li>Select the cell with the coefficient of determination in the previous chart. Click on the right mouse button. Select copy.</li>
	<li>Select a destination cell in the new chart (e.g., center in group 1). Click on the right mouse button. Select paste.</li>
	<li>Select the cell with the pasted value; then, in the toolbar, select home | percent style in the number menu.</li>
	<li>Select increase decimal in the number menu and click it once. 
	NOTE: The resulting number is the percentage of changes in perfusion density explained by the changes in vessel density.</li>
	<li>Prepare another table to show the percentage of perfusion density explained by the changes in vessels larger than capillaries.</li>
	<li>Select a destination cell (e.g., center in group 1). Subtract the last result from 1.</li>
	<li>Select this cell. Select home in the toolbar.</li>
	<li>Select percent style in the number menu.</li>
	<li>Click once on increase decimals in the number menu.</li>
	<li>Format the charts to display the contribution of capillaries (vessel density) and vessels larger than capillaries to the changes in perfusion density.</li>
	<li>Repeat the procedure to obtain the values of inner vessel/perfusion densities and superior, inferior, temporal, and nasal vessel/perfusion densities in group 3.</li>
</ol>
3. Comparison of the coefficients of determination
<ol>
	<li>Compare the coefficients of determination in three groups: 1, healthy people; 2, patients with arterial hypertension without hypertensive retinopathy; and 3, patients with type 2 diabetes mellitus without diabetic retinopathy. In group 3, also compare the coefficients of determination between fields: superior, inferior, temporal, and nasal.</li>
</ol>
4. Compare the percentage differences in the contribution of capillaries and vessels larger than capillaries to perfusion density, between groups and between fields in group 3

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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=Reversed+neurovascular+coupling+on+optical+coherence+tomography+is+the+earliest+detectable+abnormality+before+clinical+diabetic+retinopathy.">Reversed neurovascular coupling on optical coherence tomography is the earliest detectable abnormality before clinical diabetic retinopathy.</a> Journal of Clinical Medicine. 9 (11), 3523 (2020).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

The contribution of vessels larger than capillaries to perfusion density changes in retinal vascular diseases before the development of retinopathy. It decreased in the inner region of patients with arterial hypertension and varied between fields in patients with diabetes. There are direct methods for measuring vascular reactivity in the retina, which depend on the exposure to a stimulus14,15. The measurement proposed in this paper uses two metrics, automatically generated by OCTA devices, to estimate the contribution of vessels larger than capillaries to the percentage of the evaluated area with circulation.
The critical step in the method is obtaining adequate measurements of vessel and perfusion densities in the 3 x 3 mm map. Images with a signal strength &#62; 7 and without artifacts produce reliable numbers for use in a scatterplot. Although there are protocols for correcting segmentation errors in OCTA measurements16, this study worked only with images with high&#160;quality, without artifacts or measurement errors. The coefficient of determination is calculated using a usual spreadsheet or any other statistical package; the contribution of vessels larger than capillaries requires only subtraction and a conversion to a percent expression.
A limitation of the technique is that it currently evaluates only samples because it requires several subjects to assess the dispersion of changes in the outcome variable. Further studies should address cut points that allow the use of the information in an individual patient or eye. The significance of the results of this method is that it can be of value for detecting population clusters with a particular alteration of retinal circulation, which can then be evaluated with direct, more expensive, or invasive methods.
The change in the percent contribution of vessels larger than capillaries may reflect a compensatory event when a decrease in permeable capillaries induces arteriolar dilatation. It has been reported that capillaries dilate by 1% and arterioles by up to 6% in response to flicker light stimulation17. However, patients with arterial hypertension may not show the same dilatation because of the increased arteriolar constriction, which could explain the reduction in the contribution of vessels larger than capillaries to perfusion density, which was found in this group.
Compensatory changes in vessels larger than capillaries have not received the same attention as capillary density in retinal vascular diseases. However, they may show a condition where the reduction of capillary density is critical and where local hypoxia requires another source of blood flow. There is insufficient data to define whether this finding may occur simultaneously to the loss of neurovascular coupling, reported early in diabetic patients without retinopathy18.
The changes found in this study may not apply to every patient with arterial hypertension or diabetes. Although the estimation proposed is indirect, it revealed differences worth comparing with direct methods and that show the composition of parafoveal circulation at a particular time point. The potential application of this measurement is the future identification of threshold values of capillary dropout that induce arteriolar dilatation in various stages of retinal vascular diseases. Those thresholds have not been reported and might be helpful as biomarkers for disease progression and responses to treatments.
In conclusion, a method is proposed to evaluate the contribution of vessels larger than capillaries, which requires only the usual measurements that the OCTA devices produce and that may go unnoticed with the automatic metrics. The changes found in people with vascular diseases before retinopathy appears suggest reactive vasodilation, which&#160;may be useful to evaluate therapeutic interventions without using other equipment.

Retinal circulation is the combination of arteriolar, capillary, and venular flow, whose contribution can vary to meet the oxygen needs of the different retinal layers. This circulation does not depend on the autonomous nervous system regulation and has been traditionally evaluated with fluorescein angiography, an invasive method that uses intravenous contrast to delineate retinal vessels. Sequential photographs allow the evaluation of arterial, arteriolar, venular, and venous circulation, as well as sites of capillary damage in retinal vascular diseases1.
A current method to measure the macular circulation is optical coherence tomography angiography (OCTA), which uses interferometry to obtain retinal images and can outline capillaries and larger retinal vessels2. Unlike fluorescein angiography, OCTA imaging is not influenced by macular xanthophyll pigment shadowing, allowing superior imaging of macular capillaries3. Other advantages of OCTA over fluorescein angiography are its noninvasiveness and higher resolution4.
OCTA devices measure the superficial capillary plexus at the parafovea in a 3 x 3 mm map, concentric to the foveal center (Figure 1). The equipment automatically measures vessel length density (the length of capillaries with circulation in the measured area) and perfusion density (the percentage of the measured area with circulation), which includes that of vessels larger than capillaries (Figure 2)5. Vessel density has a substantial contribution to perfusion density under physiological conditions. Some devices measure vessel density as a &#34;skeletonized vascular density&#34; and perfusion density as &#34;vessel/vascular density.&#34; Regardless of the device, there is usually a measurement for length (measured in mm/mm2 or mm-1) and another for the area with circulation (measured in %), which are generated automatically.
Vessel density can change in healthy people when exposed to darkness, flicker light6, or caffeinated drinks7 because of the neurovascular coupling that redistributes blood flow between the superficial, middle, and deep capillary plexuses according to the retinal layer with the highest activity. Any decrease in vessel density caused by this redistribution returns to baseline values after the stimulus ceases and does not represent capillary loss, a pathological change reported before retinopathy appears in vascular diseases such as diabetes8 or arterial hypertension9.
The decrease in capillaries could be compensated partially by arteriolar vasodilatation. Measuring only a percentage or perfused area does not provide any insight into whether there is vasodilatation, which can appear when capillaries reach a minimum threshold. Measuring vessel density would not help detect an increased circulation area resulting from vasodilatation. The contribution of arteriolar circulation to perfusion density can be estimated indirectly using a coefficient of determination between vessel density and perfusion density, and defining the percentage of the area with circulation that corresponds to capillaries or other vessels.
The rationale behind this technique is that regression analysis can identify the extent to which the changes of an independent numeric value result in changes of a dependent numeric value. In macular vessel imaging using OCTA, capillary circulation is an independent variable that influences the area with circulation because there are few larger vessels in the evaluated region. However, the parafovea has larger vessels that can dilate and change the percentage of the area with circulation, which cannot be identified directly by the current automated OCTA metrics. The advantage of using a coefficient of determination is that it measures a relationship between two existing metrics to produce two more: the percentage of the area with circulation that corresponds to capillaries, and the percentage that corresponds to other vessels. Both percentages can be measured directly using a pixel count with imaging software. However, the coefficient of determination can be calculated for a sample with the numbers that the OCTA devices generate automatically10,11.
Pathak et al. used a coefficient of determination to estimate lean muscle and fat mass from demographic and anthropometric measures using an artificial neural network. Their study found that their model had an R2 value of 0.92, which explained the variability of a large portion of their dependent variables12. O&#39;Fee and colleagues used a coefficient of determination to rule out nonfatal myocardial infarction as a surrogate for all-cause and cardiovascular mortality because they found an R2 of 0.01 to 0.21. Those results showed that the independent variable explained less than 80% of the changes of the dependent variables, set as a criterion of surrogacy (R2= 0.8)13.
The coefficient of determination is used to assess the effect of changes of a variable, a group of variables, or a model over the changes of an outcome variable. The difference between 1 and the R2 value represents the contribution of other variables to the changes of the outcome variable. It is uncommon to attribute the difference to a single variable because there are usually more than two contributing to the outcome. However, the proportion of the macular area that has circulation can only originate from the area covered by capillaries and from that covered by larger vessels, as larger vessels dilate more than capillaries. Moreover, reactive vasodilation is considered to most probably originate from retinal arterioles, because a reduced capillary circulation could decrease oxygen supply.
Only two sources contribute to a percentage of area with circulation in the macula: capillaries and vessels larger than them. The coefficient of determination between vessel density and perfusion density determines the contribution of capillaries to the area with circulation, and the remaining changes (the difference between 1 and the R2 value) represent the contribution of the only other variable that represents an area with circulation (that within larger retinal vessels). This paper describes the method of measuring this contribution in healthy people (group 1) and how it changes in patients with retinal vascular diseases: arterial hypertension without hypertensive retinopathy (group 2) and diabetes mellitus without diabetic retinopathy (group 3).

<table><tbody><tr><td>Cirrus 6000 with Angioplex</td><td>Carl Zeiss Meditec Inc., Dublin CA</td><td>N/A</td><td>3 x 3 vessel and perfusion density maps</td></tr><tr><td>Excel</td><td>Microsoft</td><td>N/A</td><td>spreadsheet</td></tr><tr><td>Personal computer</td><td>Generic</td><td>N/A</td><td>for running the calculations on the spreadsheet</td></tr></tbody></table>

evaluation of capillary and other vessel contribution to macular perfusion density measured with optical coherence tomography angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

There were 45 subjects in group 1, 18 in group 2, and 36 in group 3. Table 1 shows the distribution of age and densities by group; only vessel and perfusion densities in group 1 were lower than in group 2. The coefficients of determination of center vessel and perfusion densities are shown in Figure 5. There was no significant difference between the groups.
The coefficient of determination between the inner vessel and perfusion densities was 0.818 in group 1, 0.974 in group 2, and 0.836 in group 3. The contribution of vessels larger than capillaries accounted for 18.2% in healthy subjects, 2.6% in patients with arterial hypertension, and 16.4% in patients with diabetes (Figure 6).
In group 3, the coefficients of determination between vessel and perfusion density were 0.722 in the superior field, 0.793 in the inferior field, 0.666 in the temporal field, and 0.862 in the nasal field. Although the inner region had a contribution of vessels larger than capillaries that accounted for 16.4% of perfusion density, this contribution was 27.8% in the superior field, 20.7% in the inferior field, 33.4% in the temporal field, and 13.8% in the nasal field (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63033/63033fig01.jpg" /> 
Figure 1: Distribution of an optical coherence tomography 3 x 3 mm density map of the right eye. The map is centered in the fovea and measures 3 mm in diameter; the center metrics correspond to a region 1 mm in diameter. The inner metrics correspond to the ring between the central 1 mm and the 3 mm diameter circles. The full metrics correspond to the whole area within the boundaries of the map. The inner ring is divided into fields: superior, temporal, inferior, and nasal; the map for the left eye switches the positions of the temporal and nasal fields. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/63033fig01large.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/63033/63033fig02.jpg" /> 
Figure 2: A 3 x 3 mm optical coherence tomography angiography density map of the superficial macular capillary plexus. The device uses the representation of the retinal vessels to measure vessel length density in mm-1 and perfusion density in %. Vessel length density corresponds to the sum of the length of vessels with circulation within the boundaries of the map; perfusion density corresponds to the percent area of the macula with circulation. The broader vessels correspond to arterioles and venules, which are larger than capillaries and have a higher contribution to perfusion density. The vertical magenta and horizontal lines are references of the scan used to center the map. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/63033fig02large.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/63033/63033fig03.jpg" /> 
Figure 3: Vessel length density maps. The OCT device outlines the area with circulation (upper left image), the retinal structure (lower left image), the retinal surface (upper right image) and generates the metrics automatically (lower right image). Maps of (A) a healthy individual and (B) a diabetic patient without retinopathy. The vessels at the level of the superficial capillary plexus are shown in white in the upper left images; there is a larger number of vessels in A than in B, a difference that is confirmed as a reduction in all the densities, especially center density. Interna = inner density; completa&#160;= full density. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/63033fig03large.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/63033/63033fig4v2.jpg" /> 
Figure 4: Vessel length density map in a diabetic patient without retinopathy, analyzed by field. The upper left image outlines the area with circulation; the lower left image shows the retinal structure; the upper right image shows the retinal surface; the lower right image shows the automatically generated metrics. The figure corresponds to the left eye and shows the automatic measurements for the superior, temporal, inferior, and nasal fields of the inner density in the upper left image. Abbreviations: S = superior; T = temporal; I = inferior; N = nasal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/63033fig4v2large.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/63033/63033fig05.jpg" /> 
Figure 5: Comparison of the coefficients of determination between center vessel (mm-1) and perfusion (%) densities in the three groups. There are few capillaries in the center region and almost no vessels larger than capillaries, which explains the slight differences between the groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/63033fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63033/63033fig06.jpg" /> 
Figure 6: Comparison of the coefficients of determination between inner vessel (mm-1) and perfusion (%) densities in the three groups. The contribution of vessels larger than capillaries to perfusion density was lower in patients with arterial hypertension and did not change in patients with diabetes, compared with healthy subjects. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/63033fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63033/63033fig07.jpg" /> 
Figure 7: Comparison of the coefficient of determination between vessel (mm-1) and perfusion (%) densities by field, in group 3. The contribution of vessels larger than capillaries was greater in the temporal field, which was 20 percentage points higher than that of the nasal field. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/63033fig07large.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>Variable</td>
			<td colspan="2">Group 1 (n= 45)</td>
			<td>Group 2 (n=18)</td>
			<td>Group 3 (n= 36)</td>
			<td>p*</td>
		</tr>
		<tr>
			<td colspan="2">Age</td>
			<td>57.16&#177;1.01</td>
			<td>55.89&#177;1.82</td>
			<td>55.33&#177;1.16</td>
			<td>0.495</td>
		</tr>
		<tr>
			<td colspan="2">Center vessel density (mm-1)</td>
			<td>8.86&#177;0.44</td>
			<td>8.12&#177;0.79</td>
			<td>8.66&#177;0.59</td>
			<td>0.713</td>
		</tr>
		<tr>
			<td colspan="2">Inner vessel density (mm-1)</td>
			<td>21.14&#177;0.29</td>
			<td>19.84&#177;0.91</td>
			<td>20.52&#177;0.27</td>
			<td>0.116</td>
		</tr>
		<tr>
			<td colspan="2">Superior vessel density (mm-1)</td>
			<td>20.98&#177;0.35</td>
			<td>20.33&#177;0.82</td>
			<td>20.27&#177;0.34</td>
			<td>0.392</td>
		</tr>
		<tr>
			<td colspan="2">Inferior vessel density (mm-1)</td>
			<td>21.18&#177;0.32</td>
			<td>19.31&#177;1.17</td>
			<td>20.64&#177;0.31</td>
			<td>0.057</td>
		</tr>
		<tr>
			<td colspan="2">Temporal vessel density (mm-1)</td>
			<td>21.06&#177;0.31</td>
			<td>19.95&#177;0.91</td>
			<td>20.50&#177;0.30</td>
			<td>0.229</td>
		</tr>
		<tr>
			<td colspan="2">Nasal vessel density (mm-1)</td>
			<td>21.36&#177;0.30</td>
			<td>19.72&#177;0.99</td>
			<td>20.69&#177;0.36</td>
			<td>0.076</td>
		</tr>
		<tr>
			<td colspan="2">Center perfusion density (%)</td>
			<td>15.74&#177;0.77</td>
			<td>14.54&#177;1.40</td>
			<td>15.13&#177;1.02</td>
			<td>0.734</td>
		</tr>
		<tr>
			<td colspan="2">Inner perfusion density (%)</td>
			<td>39.12&#177;0.48</td>
			<td>38.85&#177;1.58</td>
			<td>37.95&#177;0.49</td>
			<td>0.108</td>
		</tr>
		<tr>
			<td colspan="2">Superior perfusion density (%)</td>
			<td>38.54&#177;0.62</td>
			<td>37.72&#177;1.40</td>
			<td>37.59&#177;0.58</td>
			<td>0.578</td>
		</tr>
		<tr>
			<td colspan="2">Inferior perfusion density (%)</td>
			<td>39.38&#177;0.56</td>
			<td>35.57&#177;2.11</td>
			<td>37.95&#177;0.57</td>
			<td>0.026</td>
		</tr>
		<tr>
			<td colspan="2">Temporal perfusion density (%)</td>
			<td>39.05&#177;0.61</td>
			<td>37.99&#177;1.36</td>
			<td>38.19&#177;0.61</td>
			<td>0.561</td>
		</tr>
		<tr>
			<td colspan="2">Nasal perfusion density (%)</td>
			<td>39.53&#177;0.55</td>
			<td>35.99&#177;1.96</td>
			<td>38.10&#177;0.77</td>
			<td>0.049</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of variable distribution by group (mean &#177; standard error). *One-way analysis of variance.
Video 1: Calculation and comparison of coefficients of determination between variables, using a spreadsheet. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63033/evaluation of capillary and other vessel .mp4" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography at JoVE.com

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

evaluation-capillary-other-vessel-contribution-to-macular-perfusion

Nanyang Technological University

Research

JoVE Journal

Medicine

1.7K Views.  Sala Uno Opthalmology Clinic. Het protocol maakt het mogelijk om het aandeel van een gebied met de circulatie van verwijde bloedvaten te identificeren. Deze verwijding kan een reactie zijn van klinisch pathologische toestand. Deze techniek kan indirect vasodilatatie schetsen met behulp van een gebruikelijke optische coherentietomografie om een groep patiënten te evalueren.Het vinden van vaatverwijding kan indirect een lage oxygenatie onthullen in de stadia van retinale vaatziekten voordat klinische symptomen versc...

Video: Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

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

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The capillaroscopy technique can provide insight into a patient&#8217;s microvascular health, leading to a variety of potentially valuable dermatologic, ophthalmologic, rheumatologic and cardiovascular clinical applications. In addition, tumor growth may be dependent on angiogenesis, which can be quantitated by measuring microvessel density within the tumor. However, there is currently little to no standardization of techniques, and only one publication to date reports the reliability of a currently available, complex computer based algorithms for quantitating capillaroscopy data.1 This paper describes a new, simpler, reliable, standardized capillary counting algorithm for quantitating nailfold capillaroscopy data. A simple, reproducible computerized capillaroscopy algorithm such as this would facilitate more widespread use of the technique among researchers and clinicians. Many researchers currently analyze capillaroscopy images by hand, promoting user fatigue and subjectivity of the results. This paper describes a novel, easy-to-use automated image processing algorithm in addition to a reproducible, semi-automated counting algorithm. This algorithm enables analysis of images in minutes while reducing subjectivity; only a minimal amount of training time (in our experience, less than 1 hr) is needed to learn the technique.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Neuroscience

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

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