The results about the microvascular response to particulate air pollution are reproduced with permission from Environmental Health Perspectives24. The validated meteorological and air quality data were kindly provided by The Belgian Royal Meteorological Institute and the Flemish Environmental Agency. The retinal image analysis software was obtained from Dr. N. Ferrier (Madison School of Engineering and the Fundus Photograph Reading Center, Department of Ophthalmology and Visual Sciences, University of Wisconsin&#8211;Madison). Tijs Louwies and Eline Provost are supported with a VITO fellowship. Eline Provost holds an aspirant research fellowship of the Flemish Scientific Fund. Tim S. Nawrot is holder of a European Research Council starting grant.

The Ethics Board of Hasselt University and University Hospital Antwerp approved the studies. Participants gave their written informed consent to participate.
1. Instrument Setup
<ol>
	<li>Remove the black protection shells from the digital retinal camera and the main block of the unit.</li>
	<li>Open the battery compartment and place the battery in the camera. Do not disconnect the wire connecting the battery and the main unit.</li>
	<li>Screw the camera onto the main unit and connect the two wires. Connect the main unit to the power grid and to the computer with the supplied USB cables.</li>
	<li>Start the main unit by switching the on/off button to &#8220;on&#8221;. Start the camera by switching the on/off button to &#8220;on&#8221;.</li>
	<li>Start the computer. This will prevent connection errors between the main unit and the computer.</li>
</ol>
2. Capturing Photograph
<ol>
	<li>Start the Retinal Imaging Control Software (and fill in the required password). The software is a part of the digital retinal camera (see materials table for link).</li>
	<li>Start the study by clicking the &#8220;Study&#8221; icon in the upper left part of the screen. For a new patient, fill in all the details such as patient ID, patient name, date of birth, etc. If the patient is already in the system, fill in the &#8220;patient ID&#8221; and use the &#8220;Search history list&#8221;. Double-click on the name of the patient to start the study.</li>
	<li>Ask the patient to take a seat before the camera, place his/her chin on the chin rest and the forehead against the forehead rest and &#8220;lock&#8221; the head to take a photograph.</li>
	<li>Ask the patient to look straight into the lens of the camera. Move the camera in the horizontal (X-Y) plane to the right or left eye.</li>
	<li>Use the chin rest to position the cornea of the patient inside the two circles that appear on the camera display. Fine-tune by using the wheel on the joystick.</li>
	<li>Move the camera forward, backward, and sideways in the X-Y plane in order to position the pupil of the patient within the circles. Make sure the pupil forms a continuous circle. By doing so, the iris of the patient will be split up in two pieces.</li>
	<li>Use the &#8220;back trigger&#8221; on the joystick to switch from the cornea to the retina. At this stage, the patient should observe a green light. Ask the patient to look at the green light.</li>
	<li>Focus the camera by aligning the two lines that pop up when the wheel at the base of the joystick is turned. Turn the wheel until the two lines form a continuous line.</li>
	<li>Use the green light to place the eye in the optimal position for a photograph. If required, move the light using the arrow buttons at the right side of the camera. Position the green light in a way that the optic disc is centered on the camera display.</li>
	<li>Search for 2 white spots which appeared after switching to the retina (in step 2.7). To find the spots, move the unit in the X-Y plane. The spots are visible as a blurry stain. Move the unit forward or backwards until blurry stains turn into bright, white spots. The brighter and rounder the spots, the better the quality of the picture is. Position the spots until both are visible. Use the little wheel on the joystick to bring the spots to the middle of the camera display.</li>
	<li>Confirm that the two lines (from step 2.8) form a continuous line. The optic disc is centered on the camera display and is flanked by two bright, white spots. Take the retinal photograph by firing the button on top of the joystick.</li>
	<li>Save the photograph by pressing the &#8220;Study Complete&#8221; button on the lower right corner of the computer screen. Completing the study will automatically save the pictures in a map and close the study.</li>
</ol>
3. Analysis of Retinal Photograph
<ol>
	<li>Determine the scale ratio by measuring the distance between the center&#160;of macula (fovea) and the center&#160;of the optic disc (blind spot). Anatomically this distance is determined to be 4,500 &#181;m or 2.5x&#160;the diameter of the optic disc, with the latter being approximately 1,800 &#181;m. Ensure the distance is measured in pixels. Calculate the scale ratio by dividing 4,500 by the distance (in pixels) between the macula and blind spot.</li>
	<li>Open the retinal vessel analysis software &#8220;IVAN&#8221;. 
	NOTE: The software is created at the University of Wisconsin at Madison. Detailed information on the usage of IVAN is taken from the manual.</li>
	<li>Fill in the scale ratio and proceed through the configuration.</li>
	<li>Verify that three yellow rings appear on the retinal photograph. The scale ratio determines the radius of the inner circle and encloses the optic disc. Verify that the middle point of the inner ring is on the middle point of the optic disc. If this is not the case, adjust the location of the circle by using the cursor keys. The radii of the middle and outer circles are 2x&#160;and 3x&#160;larger than the radius of the inner circle, respectively. In this way, zones A and B are created at a fixed distance from the optic disc.</li>
	<li>Check that the retinal image has the optic disc in the center of the photograph. This ensures a sharp focus of the image in zone B and this will facilitate the grading process (Figure 4A).</li>
	<li>Observe that the software automatically detects the blood vessels and assigns these vessels as venules (Figure 4B).</li>
	<li>Distinguish the vessels between arterioles and venules based on physiological differences. Arterioles will be indicated in red and venules in blue (Figure 4C). Use the following guidelines to identify each vessel:
	<ol>
		<li>Determine the vessel color. Arterioles have a lighter orange-red color with a strong central light reflection. Veins have darker purple-red color with little or no central light reflex.</li>
		<li>Determine the course of the vessel. Arterioles tend to be straighter and smoother in outline; they are more regular in both path and outline. Venules are generally more tortuous, and more irregular in outline and diameter. Venules are broader in diameter at the disc margin than the corresponding arterioles.</li>
		<li>Identify the vessel by looking at the annotation of the preceding vessel. In principle, arterioles alternate with venules. Therefore, if a distinct venule is measured, the next vessel is more likely to be an arteriole.</li>
		<li>Define the crossing pattern. As a general rule, arterioles do not cross arterioles and venules do not cross venules. If a vessel of unknown identity crosses a venous branch within or distal to Zone B, then the unknown vessel is an arteriole. If it crosses an arteriolar branch within or distal to Zone B, then it is a venule.</li>
		<li>Identify smaller branches by tracing them proximally to their branching from a parent vessel, the identity of which may be evident from the first two guidelines. Use angles between vessels to differentiate crossings and branchings. 
		NOTE: Crossings are frequently almost perpendicular (90&#176;) or, if the two vessels are coursing in parallel, the angle of the crossing may be very shallow (less than 30&#176;). Branchings are usually somewhat less than perpendicular (with the angle between the two branches from 30&#176; to 45&#176;).</li>
		<li>Select the full length of the vessel in the grading zone. Make sure that the standard deviation of the selected vessel does not exceed the value of 10. Smaller standard deviations indicate a good measurement.</li>
	</ol>
	</li>
	<li>Use the software tools to select vessels that were not selected by the software itself. The same rules apply for these vessels as for the vessels automatically selected by the software.</li>
	<li>Determine Central Retinal Arteriolar and Venular Equivalent (CRAE and CRVE) automatically in IVAN.</li>
	<li>Calculate the CRAE and CRVE from their respective branching daughter vessels using revised formulas of Parr and Hubbard28. 
	NOTE: The relationship between trunks and branches, with empirically derived branching coefficients, is given in following two formulas to approximate vessel equivalents. IVAN uses the six largest arterioles and venules for calculating CRAE and CRVE. The formulas are applied in an iterative procedure to pair up the six largest arterioles (or venules) until the central arteriolar (or venular) vessel equivalent is obtained. 
	Arterioles: <img alt="Equation 1" fo:content-width="1in" src="/files/ftp_upload/51904/51904eq1.jpg" /> (1) 
	Venules: <img alt="Equation 2" fo:content-width="1in" src="/files/ftp_upload/51904/51904eq2.jpg" /> (2) 
	where w1, w2, and W are the widths of the narrower branch, the wider branch, and the parent trunk, respectively. 
	NOTE: Assume that in a retinal photograph the six largest arterioles are 120, 110, 100, 90, 80, and 70 &#181;m wide. Put 120 and 70 into equation (1), as well as 110 and 80, and 100 and 90. After the first iteration there are three values: 122.2, 120.0, and 118.4. Perform the next iteration by pairing 122.2 and 118.4, yielding 149.8. Carry over the middle number (120.0) to the final iteration. Pair 149.8 and 120.0 to yield 168.7 for CRAE.</li>
</ol>

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<li>Nurkiewicz, T. R., Porter, D. W., Barger, M., Castranova, V., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particulate+matter+exposure+impairs+systemic+microvascular+endothelium-dependent+dilation%5BTitle%5D)+AND+%22Environmental+Health+Perspectives%22%5BJournal%5D)">Particulate matter exposure impairs systemic microvascular endothelium-dependent dilation.</a> Environmental Health Perspectives. 112, 1299-1306 (2004).</li>
<li>Nurkiewicz, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+microvascular+dysfunction+and+inflammation+after+pulmonary+particulate+matter+exposure%5BTitle%5D)+AND+%22Environmental+Health+Perspectives%22%5BJournal%5D)">Systemic microvascular dysfunction and inflammation after pulmonary particulate matter exposure.</a> Environmental Health Perspectives. 114, 412-419 (2006).</li>
<li>Barath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+vascular+function+after+exposure+to+diesel+exhaust+generated+at+urban+transient+running+conditions%5BTitle%5D)+AND+%22Particle+and+Fibre+Toxicology%22%5BJournal%5D)">Impaired vascular function after exposure to diesel exhaust generated at urban transient running conditions.</a> Particle and Fibre Toxicology. 7, (2010).</li>
<li>Tornqvist, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+endothelial+dysfunction+in+humans+after+diesel+exhaust+inhalation%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+and+Critical+Care+Medicine%22%5BJournal%5D)">Persistent endothelial dysfunction in humans after diesel exhaust inhalation.</a> American Journal of Respiratory and Critical Care Medicine. 176, 395-400 (2007).</li>
</ol>

The authors declare they have no actual or potential competing financial interests.

Retina image analysis is proposed as a convenient tool for studying microvascular responses in epidemiological studies. When the operator is experienced, it takes less than 5&#160;min&#160;to take a fundus picture. Furthermore, this unobtrusive procedure for visualizing the microcirculation can be used for participants from early age up to old age.
Literature is increasing with respect to the associations between morphological changes in the retinal vasculature (for example change in vessel caliber, geometric pattern, etc.) and modifiable lifestyle and environmental risk factors15,16. Experimental and epidemiological works show that short-term and long-term air pollution exposure is strongly associated with cardiovascular morbidity and mortality. However, a convenient technique such as retinal fundus photography has been used very little to study microcirculatory effects that may be induced by air pollutants.
The different steps that are required for obtaining a high-quality fundus picture are explained in this video protocol. Subsequently, the methodology is given for obtaining arteriolar and venular caliber measurements, and more specifically the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE)13,28. The results of the repeated measures analysis showed that the within-rater results for CRAE and CRVE are highly reproducible for pictures that were taken within a timeframe of four days. These findings are in line with the recent observations reported by McCanna and colleagues. The latter authors reported that CRAE and CRVE values are stable over a period of one month. They reported correlations for pairs of study visits of 0.9 and the correlations decreased slightly with an increasing length of time interval31.
Subsequently, it is shown in a panel study with healthy adults that the retinal microcirculation can respond rapidly to particulate matter air pollution. More specifically, a decrease in CRAE that relates to an increased short-term exposure to PM10 and BC is reported24. Narrowing of retinal arterioles is a proxy for estimating the risk of cardiovascular disease and cardiovascular mortality32-35. It is envisioned that retinal microvasculature can be used to probe for cardiovascular effects of air pollution. In this respect, Adar and colleagues reported for the first time about the short-term effects of air pollution on the human retinal microvasculature in a cross-sectional analysis of the MESA cohort21. The microvascular changes reported by Louwies et al. (2013) complement those found by Adar et al. (2010). The latter authors reported a 0.4 &#181;m-decrease in CRAE (95% CI: -0.8, -0.04) per 9-&#956;g/m&#179; increase in average PM2.5 on the previous day. Based on repeated measurements, Louwies et al. (2013) reported an estimate of -1.2 &#181;m (95% CI: &#8211;1.61, &#8211;0.61) and it is suggested that the larger effect size may be due to greater variation in PM and BC exposure concentrations in this panel study24.
Pulmonary inflammation and low-grade, systemic inflammation have been associated with&#160;exposure to air pollution36. Systemic inflammation has also been linked with endothelial dysfunction37,38. This process may affect the reactivity of retinal blood vessels39. It is assumed that inflammatory responses lead to altered endothelial activity, which may be reflected in the narrowing of the arteriolar calibers. The findings from the panel study suggest that this might occur in a timeframe of less than 24 hr because exposure to PM10 was inversely associated with CRAE during all the hourly exposure windows. The observations are in line with the known impact of air pollution on health. Short-term animal studies with exposure to peak levels of air pollutants have revealed that the microvasculature can be affected40,41. In addition, human intervention studies in controlled environment have shown that endothelial function is impaired upon exposure to diesel exhaust42,43.
In conclusion, many developmental and anatomical similarities exist between retinal blood vessels and the microvasculature of the heart, lungs, and brain10. Therefore, the retinal blood vasculature is considered a surrogate tissue for the systemic microcirculation. A change in retinal blood vessels may be a valuable predictor for cardiovascular disease development. The convenient and unobtrusive analysis of retinal images is now considered useful for population-based studies with a focus on cardiovascular epidemiology. This protocol paper should encourage more research groups to use fundus photography to study microvascular effects of environmental and lifestyle factors.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m and includes smallest resistance arteries, arterioles, capillaries, and venules. These vessels make up a large part of the circulatory system and play an important role in maintaining cardiovascular health. The vessel diameter of 150 &#181;m is a physiological and a physical limit. The rheological properties of vessels with a diameter less than 150 &#181;m differ from large arteries. Furthermore, most of the autoregulatory resistance changes occur downstream from 150 &#181;m in vascular beds exhibiting blood flow autoregulation1. The microcirculation has two important functions. The primary function is to provide cells with oxygen and metabolic substrates in order to match tissue demand and to drain waste products and carbon dioxide. Alterations in the number of exchange vessels and the microvascular flow patterns reduces the effective exchange surface area and may lead to suboptimal tissue perfusion and a failure to meet metabolic demand2. Further, the hydrostatic pressure drops within the vascular bed and the microcirculation plays a role in regulating the overall peripheral resistance3.
The retina is a layered tissue lining the interior of the eye. Its main function is to convert the incoming light into a neural signal that is further propagated to the visual cortex for processing visual information. The function of the retina is to see the outside world and all the ocular structures involved in this process are optically transparent. This makes the retinal tissue accessible for non-invasive imaging of the microvasculature4. Retinal imaging is being used to identify diseases of the eye. For example, an advanced form of macular degeneration can lead to vision loss because of abnormal blood vessel growth into the macula. These blood vessels tend to be more permeable and subject to bleeding and leaking of blood and proteins within or below the retina. The latter events are responsible for the irreversible damage to the photoreceptors. Development of glaucoma correlates with a damaging of ganglion cells and their axons. The effect of this process leads to cupping of the optic disc, which can be observed in retinal images5. Diabetic retinopathy is caused by hyperglycemia that leads to damage in&#160;the retinal vessel walls. This can result in ischemia, the growth of new blood vessels and a change in the vascular geometric network. Furthermore, the blood-retinal barrier may be subject to breakdown, causing leakage of dilated hyperpermeable capillaries and aneurysms6.
Retinal microvasculature shows homology with the microvascular beds found in the heart, lungs, and brain7. It is established that systemic diseases that affect the microcirculation of the brain can cause parallel changes in the retina. Arteriolar narrowing and enhanced arteriolar light reflex of the retina is associated with vessel abnormalities, white matter lesions and lacunes that are caused by cerebral small vessel disease8. A significant relationship was discovered between narrower retinal venules, an altered retinal microvascular network and the occurrence of Alzheimer disease. It is suggested that brains of patients have an altered cerebral microvasculature that is also observable in the retina9.
Evidence is also increasing about the correlation between retinal vascular changes and coronary heart disease10,11. The ratio between the diameter of retinal arteries and retinal veins (A/V) has been shown to be a sensitive proxy to reflect hypertension and atherosclerosis12. A narrowing of the arteries and widening of the veins, leading to a decreased A/V ratio, corroborates risk of stroke and myocardial infarction13. Hypertension can cause direct retinal ischemia and retinal infarcts that become visible as cotton wool spots and deep retinal white spots14. Serre and Sasongko recently summarized the literature and they concluded that exposure to lifestyle and environmental risk factors (e.g., diet, physical activity, smoking, and air pollution) can induce morphological changes in the retinal microvascular bed15. Importantly, such retinal changes have been associated with cardiovascular risk factors, even before clinical manifestations of diseases16.
Significant increases in the incidence of cardiovascular morbidity and mortality have been attributed to long- and short-term exposures to particulate matter air pollution17,18. Research indicates that particulate matter (PM), an important fraction of air pollution, contributes to the development of cardiovascular disease and induces cardiovascular events19,20. An impairment of the function of the microvascular bed is thought to play a role in the observed associations. In this respect, an association between exposure to air pollution and arteriolar narrowing in the retina has been reported by Adar and colleagues21. The retinal arteriolar caliber was narrower and venular caliber was wider among the 4607 participants of the Multi-Ethnic Study of Atherosclerosis (MESA) that were living in areas with increased long- and short-term exposure to PM2.5 (particulate matter &#8804; 2.5 &#956;m in diameter)21. Systemic inflammation caused by chronic air pollution exposure may result in wider venular diameters22. This corroborates&#160;the studies that report the impact of smoking on the retinal microvascular bed23. A recent publication reports on the association between short-term air pollution exposure and microvascular changes in healthy adults (22-63 years of age) measured with retinal fundus photography24. An increase in PM10 (particulate matter &#8804; 10 &#956;m in diameter) and BC (black carbon, a combustion by-product that can be used as a proxy for traffic-related diesel exhaust) was associated with a decrease in arteriolar caliber24,25.
In this scientific video protocol, the procedures are described to collect fundus pictures of the eye, to perform image analysis to obtain arteriolar and venular vessel calibers, and to calculate Central Retinal Arteriolar Equivalent (CRAE) and Central Retinal Venular Equivalent (CRVE). Retinal imaging is gaining increased attention because the retina is the only tissue that allows an unobtrusive analysis of the microvasculature and images can be collected from early age up to old age26,27. CRAE and CRVE appear to be sensitive parameters that reflect the impact of modifiable lifestyle and environmental cardiovascular disease risk factors on the microvasculature. In the manuscript, the repeatability of the vessel analysis is demonstrated. Furthermore, the applicability of retinal microvasculature analysis in epidemiological studies is shown by summarizing our findings obtained in a repeated-measures design with a focus on the impact of particulate air pollution exposure24.

<table><tbody><tr><td>Canon CR-2 nonmydriatic retinal camera&amp;nbsp;</td><td>Hospithera (Brussels, Belgium)</td><td></td><td>http://www.usa.canon.com/cusa/healthcare/products/eyecare/digital_non_mydriatic_retinal_cameras/cr_2. Any other retinal camera with comparable resolution and specifications can be used for the analysis of the retinal microvasculature. Compatibility should&amp;nbsp; be checked before starting a study.</td></tr><tr><td>IVAN: Vessel Measurement Software</td><td></td><td></td><td>This software can be used without charge for scientific purpose. It can be obtained by contacting Dr. Nicola Ferrier (Madison School of Engineering and the Fundus Photograph Reading&lt;br/&gt; Center, Department of Ophthalmology and Visual Sciences, University of Wisconsin&amp;ndash;Madison). http://directory.engr.wisc.edu/me/faculty/ferrier_nicola. Phone: (608) 265-8793,&lt;br/&gt; Fax: (608) 265-2316 or e-mail: ferrier@engr.wisc.edu&lt;br/&gt; &amp;nbsp;</td></tr></tbody></table>

fundus photography as a convenient tool to study microvascular responses to cardiovascular disease risk factors in epidemiological studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Repeatability of CRAE and CRVE Determination
A panel of 61 individuals between 22 to 56 years old and free of clinically diagnosed cardiovascular diseases was recruited for studying technical repeatability and within-rater variability of Central Retinal Arteriolar Equivalent (CRAE) and Central Retinal Venular Equivalent (CRVE) determinations. The fundus of the right eye of each individual was photographed twice within a time period of 5&#160;min&#160;using a retinal camera (Figures 1 and 2). This procedure was done on 4&#160;consecutive days, approximately at the same time of the day. The average coefficients of variation &#177; standard deviation of the CRAE and CRVE of pictures taken within the 5-min period were 1.76 &#177; 1.71 and 1.78 &#177; 1.51, respectively. Average &#177; standard deviation of CRAE and CRVE values were 151.31 &#177; 13.53 and 213.20 &#177; 18.44, respectively. No significant differences were observed for CRAE and CRVE values obtained on 4&#160;consecutive days.
CRAE and CRVE values of the right eye were averaged to one CRAE and CRVE value per day. Subsequently, repeatability of the measurements was evaluated by means of the Intraclass Correlation Coefficient (ICC), a dimensionless statistic bounded by 0 and 1 that describes the reproducibility of repeated measures in a population. The measurements were done by a single rater. Hence, a one-way random effects model allowed to estimate the within-rater variability29. The ICC was 0.919 (95% CI: 0.883, 0.946) and 0.898 (95% CI: 0.854, 0.932) for CRAE and CRVE, respectively. These ICC values are well above the threshold of 0.6, which is considered to be clinically significant and the estimates both fall within the broad category as being &#8220;almost perfect&#8221; in reliability30.
Panel Study to Investigate the Effect of Particulate Air Pollution
The study was conducted between January 2012 and May 2012 and included 84 individuals. Participants were 22 to 63 years old and free of clinically diagnosed cardiovascular diseases before and during the study period. One photograph of the fundus of the right eye was taken using a retinal camera on each of three separate examination days. The reader is referred to the paper of Louwies and collaborators for detailed information on how air pollution data was collected24. During the course of the study period, the ambient PM10 and BC levels were high in Belgium due to westbound atmospheric transport of polluted air from Eastern Europe. This is visualized in a time lapse video (Supplementary information). Air pollution concentrations were assigned to each participant for 2, 4, and 6 hr&#160;preceding the retinal exam. Air pollution levels were calculated on the day of the clinical visit from midnight until the time of the retinal exam. Air pollution levels were also assigned for the previous day and two days before the retinal exam. These concentrations are summarized as: lag2h, lag4h, lag6h, lag 24h, and lag 2d. Pollutant-specific, exposure-response analysis using mixed models was performed. Details of these analyses can be found in the original publication24. There was an inverse association between CRAE and air pollution concentrations (measured as PM10 and BC concentrations) in the hourly and daily exposure windows before the clinical examination.&#160;A decrease in CRAE of 0.93 &#956;m (95% CI: &#8211;1.42, &#8211;0.45; p = 0.0003) was observed for each 10-&#956;g/m&#179; increase in average PM10 during 24 hr preceding the examination (Figure 3). Shorter hourly PM10 exposure windows and PM10 concentrations averaged over the previous 2 days also revealed a significant decrease of the CRAE values. A decrease in CRAE of 1.84 &#181;m (95% CI: &#8211;3.18, &#8211;0.51; p = 0.008) was also found for each 1-&#956;g/m&#179; increase in BC 24 hr before the examination. No additional significant associations were observed between CRAE and the other calculated BC exposure windows. A decrease in CRVE of 0.86 &#181;m (95% CI: &#8211;1.42, &#8211;0.30; p = 0.004) was observed for every 10-&#956;g/m&#179; increase in PM10 in the 24 hr exposure window before the retinal picture was taken. Shorter exposure windows revealed additional significant effects (Figure 3). A negative association between CRVE and BC exposure during the 24 hr before the examination was observed. However, the effect did not reach the level of statistical significance (&#8211;1.18 &#956;m; 95% CI: &#8211;3.11, 0.75; p = 0.23).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51904/51904fig1highres.jpg" width="500" /> 
Figure 1.&#160;Retina picture and example of a retinal camera. Annotated retinal fundus picture of the right eye of a healthy volunteer (Left) and a picture of a non-mydriatic digital retinal camera (Right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51904/51904fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51904/51904fig2highres.jpg" width="500" /> 
Figure 2.&#160;Screen shot of IVAN software. Example of a picture that is processed with the IVAN software. The software identifies the vasculature and calculates diameters. The operator supervises the results and identifies arteries (shown in red) and venules (shown in blue). CRAE and CRVE are then calculated automatically. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51904/51904fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51904/51904fig3highres.jpg" width="500" /> 
Figure 3.&#160;Association between air pollution and retinal vessel calibers. Estimated change in mean CRAE and CRVE (95% CI) in association with a 10-&#181;g/m&#179; increase in PM10 (Left) or a 1-&#181;g/m&#179; increase in BC (Right) for different exposure lags. The data were obtained from a panel of 84 persons. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51904/51904fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51904/51904fig4highres.jpg" width="500" /> 
Figure 4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51904/51904fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental video.&#160;Time lapse video of air pollution concentrations during the panel study.

Watch this Scientific Journal Video about Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies at JoVE.com

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

fundus-photography-as-convenient-tool-to-study-microvascular

Nanyang Technological University

Research

JoVE Journal

Medicine

19.1K Views.  Flemish Institute for Technological Research (VITO).  Retinale beeldanalyse is een onopvallende procedure voor het visualiseren van de microcirculatie. De gevolgen van hart-en vaatziekten risicofactoren kunnen leiden tot veranderingen van het netvlies kalibers vaartuig. De procedures voor fundus foto en stappen te nemen voor het berekenen van het vaartuig kalibers worden beschreven. 

Video: Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

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 ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

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 ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

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 ...

Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

Intradermal Microdialysis: An Approach to Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally invasive technique used to investigate mechanisms of vascular smooth muscle and endothelial function in the cutaneous circulation. This technique allows for the pharmacological dissection of the pathophysiology of microvascular endothelial dysfunction as indexed by decreased nitric oxide-mediated vasodilation, an indicator of cardiovascular disease development risk. In this technique, a microdialysis probe is placed in the dermal layer of the skin, and a local heating unit with a laser Doppler flowmetry probe is placed over the probe to measure the red blood cell flux. The local skin temperature is clamped or stimulated with direct heat application, and pharmacological agents are perfused through the probe to stimulate or inhibit intracellular signaling pathways in order to induce vasodilation or vasoconstriction or to interrogate mechanisms of interest (co-factors, antioxidants, etc.). The cutaneous vascular conductance is quantified, and mechanisms of endothelial dysfunction in disease states can be delineated.

Author Spotlight: A Pharmacodissection Approach to Uncover Mechanisms in Cardiovascular Disease Risk Populations 

Intradermal microdialysis is a minimally invasive technique used to investigate microvascular function in health and disease. Both dose-response and local heating protocols can be utilized for this technique to explore mechanisms of vasodilation and vasoconstriction in the cutaneous circulation.

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally ...

Using Arterial Stiffness and Endothelial Function for Cardiovascular Assessment

Pulse-Wave Velocity, Flow-Mediated Dilation, and Carotid Intima-Media Thickness to Assess Cardiovascular Risk in Population with Metabolic Syndrome

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical settings to assess arterial stiffness, endothelial function, and subclinical atherogenesis. These measurements may reflect vascular disease and atherosclerotic progression, which are major causes of adverse cardiovascular events. These methods are particularly valuable in determining cardiovascular dysfunction among populations with different risk factors, such as diabetes mellitus, hypertension, and other metabolic dysfunction-related conditions. They provide a non-invasive and reliable source of information that complements clinical practice. Early detection, risk assessment, and therapeutic decisions regarding cardiovascular disease can be achieved, ultimately contributing to improved patient outcomes. Traditional tools for evaluating cardiovascular disease do not reveal whether metabolic syndrome affects early subclinical cardiovascular disease in patients with obesity. Recent research has highlighted the importance of including arterial stiffness and endothelial function in a comprehensive cardiovascular assessment. Therefore, the aim of the present study is to describe methods that provide information on early subclinical vascular aging, endothelial dysfunction, and atherogenic disease, enabling vascular-targeted risk stratification among populations with obesity and different metabolic profiles.

Author Spotlight: Assessing the Cardiovascular Profile of Patients with Metabolic Syndrome

Endothelial/vascular aging and atherogenesis are key mechanisms that promote the development of cardiovascular diseases. The present protocol describes methods to evaluate arterial stiffness, endothelial dysfunction, and atherogenesis in patients with related risk factors, which are highly valuable in the cardiovascular research field.

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

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