This study is supported by NIH grant RO1 013516 and a grant form Optovue.

1. Protocol Text
<ol>
	<li>Patients are scanned by RTVue Fourier-domain optical coherence tomography (OCT) system (Optovue Inc., Fremont, CA, USA) using the circumpapillary double-circular scan (CDCS) and the 3D optic disc scan.
	<ol>
		<li>The CDCS pattern consists of two concentric circles around the optic nerve head. The inner ring diameter is 3.40 mm and the outer ring diameter is 3.75 mm. This pattern transects all branch retinal arteries and veins emanating from the optic nerve head. The double circles are performed 6 times in a single scan to cover about 2 cardiac cycles. To calculate Flow velocity, Doppler shift and Doppler will be estimated on vessel detected in the OCT image. (Figure 1a, Figure 1b)</li>
		<li>A "dual angle" protocol is used to acquire Doppler OCT scans. In the "dual-angle" protocol, 3 scans are obtained with the OCT beam passing through the superonasal portion of the pupil and 3 scans through the inferonasal portion.</li>
		<li>The quality of each scan is evaluated for signal strength, motion error, and Doppler angle. The technician is required to re-do any scan that did not pass the quality check. A total of 6 acceptable CDCS scans are performed for each eye. The "dual-angle" protocol ensures that, for each vessel, at least half of the scans provide good Doppler angles.</li>
		<li>The 3D disc scan pattern is a raster scan covering a 6x6 mm region around the optic disc. It is only done once and provides a detailed en face image of this area. (Figure 1b)</li>
		<li>A color photograph of the optic disc is also imported to help distinguish arteries and veins.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/3524/3524fig1a.jpg" /> 
 Figure 1a. Measuring total blood flow with the circumpapillary double-circular scan and the 3D disk scan using DOCTORC.
<ol class="lalpha">
	<li>The Doppler angle is the angle between probe beam and normal vector of the vessel, and the Doppler signal is the Doppler frequency shift proportional to the flow velocity component parallel to the axis of the probe beam. Thus flow velocity can be estimated from Doppler angle and Doppler signal. However, both Doppler angle and Doppler signal become unreliable when they are below the noise level. On the other hand, when Doppler angle is large, The Doppler shift will be out of the measureable range. When the Doppler angle is appropriate, about 5-15 degrees for the FD-OCT system used in this study, the flow velocity can be correctly estimated by Doppler shift and angle.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/3524/3524fig1b.jpg" /> 
 Figure 1b. 
<ol start="2" type="a">
	<li>The Doppler angle in each vessel is measured by the relative positions of the vessel lumina in the two concentric circular scans. The axial component of the flow velocity is obtained by the Doppler shift measured from the relative phase of adjacent OCT axial scans. The total flow velocity is calculated from the axial flow component and the Doppler angle. The flow in each vessel is then calculated by integrating the total velocity profile over vessel cross-sectional area. The vessel pattern is matched to the en face image of the 3D disk scan and fundus photograph to identify each vessel as either a vein or an artery. The total retinal flow is calculated by summing flow in the major retinal veins, i.e., those with luminal diameters above 33 &mu;m.</li>
</ol>
<ol start="2">
	<li>All scans of the same eye are exported as raw data using the RTVue software. The raw OCT images, including both Doppler and intensity images, are first tested for image quality using "Doppler optical coherence tomography of retinal circulation" (DOCTORC) software.
	<ol>
		<li>Eye movements are measured by the pooled standard deviation of the inner limiting membrane and the maximum difference between two frames that is estimated by the bulk motion.</li>
		<li>Signal strength (reflectance and estimation of average Doppler angle) are also calculated.</li>
		<li>According to the average eye movement and signal strength of the repeated scans, the data is classified as either "good" or "poor" in quality. Only good quality data are graded.</li>
	</ol>
	</li>
	<li>For acceptable scans, an automated segmentation algorithm is applied to each OCT image for vessel detection.
	<ol>
		<li>An automated algorithm that matches vessels is used to locate the same vessel in each frame. The frames on the same circle are registered, and two averaged frames of the inner ring and outer ring are created. Both reflectance and Doppler images are averaged.</li>
		<li>For an overview, the detected vessel is projected as a line segment onto the en face fundus image calculated from the 3D disc scan.</li>
	</ol>
	</li>
	<li>Graders review each vessel on a small portion of the averaged inner and outer ring frames overlaid with a circle that represents the automated segmentation result.
	<ol>
		<li>The grader judges if the location, vessel diameter, and vessel type (vein/artery) matching the vessel on the two rings are correct according to the OCT image, en face OCT image, and disc photograph of the same eye.</li>
		<li>The grader is allowed to change any of the above values if he thinks it is necessary. The grader also judges the quality of the Doppler signal in the vessel and gives a subjective confidence score for the vessel label on each scan.</li>
		<li>A confidence score of 0-5 is automatically given to each vessel based on Doppler signal strength in the vessel area. It is then manually corrected by the grader based on the strength of the vessel Doppler signal, regularity of vessel boundary, agreement between inner and outer rings of vessel size, and sign agreement of Doppler shift between inner and outer rings.</li>
	</ol>
	</li>
	<li>After all vessels are verified and corrected, an automated algorithm is applied to calculate the blood flow of each vein with a method adapted from our previous publication.1
	<ol>
		<li>The Doppler signal is integrated over the vessel area and then averaged among all frames. Then the Doppler flow is calculated as the summed Doppler signal divided by the Doppler angle.</li>
		<li>For each vessel, 6 scans are evaluated on subjective confidence scores, Doppler angles, and Coefficient of variation of the Doppler angles. The flow of a vein is considered valid only if more than 1 scan passes the quality check. For vessels that pass the quality check, the flow is averaged among valid scans.</li>
		<li>For vessel with invalid results, the flow is estimated using the vessel area and the average flow speed from valid veins. The average flow speed is calculated by summing the flows in the valid veins and dividing by the summed areas of those veins. The estimate of flow starts from the average flow speed and is then corrected for the dependence of flow velocity on vessel area. Larger vessels have a higher average flow velocity. Thus the correction is made based upon the slope of average velocity versus vessel diameter, 2.13, that we previously reported.2</li>
		<li>The calculated flow of valid veins and the estimated flow of invalid veins are added to determine the total retinal blood flow.</li>
		<li>The total retinal blood flow result is evaluated based on the valid venous area percentage, eye movement, and signal strength.</li>
		<li>Venous area and total arterial area are also obtained by adding the vessel areas. Assuming the total retinal blood flow is same in arteries and veins, the arterial and venous velocities are calculated by dividing total blood flow with arterial area and venous area.</li>
	</ol>
	</li>
</ol>
2. Representative Results
Normal and glaucomatous eyes are selected from the Advanced Imaging for Glaucoma Study (AIGS, <a href="http://www.aigstudy.net" target="_blank">www.aigstudy.net</a>). 48 eyes were scanned by the "dual-angle" protocol and produced scans that passed the image quality check. Using the DOCTORC software, valid flow measurements could be obtained from 83% of the eyes.
To evaluate the reproducibility of the DOCTORC system, another small dataset with 20 eyes was graded by 3 graders. This dataset was also used to train and test graders. 2 graders used the semi-automated DOCTORC software and 1 used an earlier totally manual software employed in previous publications.2,3 The total retinal blood flows (Table 1) determined by the two graders using DOCTORC software are similar to one another and to flow rates determined by the other grader using the manual software. Only 65% of the eyes had valid results because some of the data were not based on dual angle protocol, but single angle protocol.2 The single angle protocol includes 5 Doppler scans obtained with the OCT beam passing through the center of the pupil. Therefore the Doppler angle is more often small and therefore a greater portion of vessels are usually not gradable.
For all graders, the inter-grader reproducibility, as measured by the coefficient of variation, is similar for both glaucomatous and normal eyes (Table 2). Likewise, the reproducibility measurements for the two methods, DOCTORC and the Manual software,1-5 are similar (Table 2). For three graders, a good correlation exists between total blood flow and the pattern standard deviation from visual field tests (Figure 2) for glaucomatous eyes.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td colspan="3">DOCTORC software Manual software3</td>
		</tr>
		<tr>
			<td>Condition</td>
			<td>Grader 1</td>
			<td>Grader 2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Normal</td>
			<td>47.0&plusmn;9.1</td>
			<td>48.7&plusmn;7.2</td>
			<td>48.0&plusmn;6.5</td>
		</tr>
		<tr>
			<td>Glaucoma</td>
			<td>36.5&plusmn;5.5</td>
			<td>36.7&plusmn;5.9</td>
			<td>34.9&plusmn;5.1</td>
		</tr>
	</tbody>
</table>
Table 1. Total Retinal Blood Flow using 2 Different Software.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td colspan="2">Coefficient of Variation</td>
		</tr>
		<tr>
			<td>Glaucoma (7 eyes)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Grader 1 vs. Grader 2(DOCTORC)</td>
			<td>9.58%</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DOCTORC vs. manual method3</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Grader 1</td>
			<td>8.00%</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Grader 2</td>
			<td>9.74%</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Normal (6 eyes)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Grader 1 vs. Grader 2 (DOCTORC)</td>
			<td>5.99%</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DOCTORC vs. manual method</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Grader 1</td>
			<td>8.87%</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Grader 2</td>
			<td>9.98%</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 2. Reproducibility of Total Retinal Blood Flow Measurements.
<img alt="Figure 2" src="/files/ftp_upload/3524/3524fig2.jpg" /> 
 Figure 2. Correlation between Total Retinal Blood Flow and Visual Field in Glaucoma. a. Grader 1 using DOCTORC software. Visual field loss is summarized by pattern standard deviation (p=0.048). b. Grader 2 using DOCTORC software. Visual field loss is summarized by pattern standard deviation (p=0.032).

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Dr. Huang receives grant support, patent royalty, stock options, travel support and lecture fees from Optovue, Inc.; Dr. Tan and Dr. Wang receives patent royalty and grant support from Optovue, Inc.; Dr. Koduru and Dr. Sadda received grant support from Optovue.

Blood flow abnormalities occur in glaucoma and vascular diseases of the retina such as diabetic retinopathy.6-10 Volumetric measurement of retinal blood flow gives valuable information about the disease process.4-6,11,12 DOCTORC provides a practical way to estimate total retinal blood flow based on the measurements in individual vessels determined by Doppler OCT using the double circle scan pattern.1-5
The mean total retinal blood flow measured by Doppler OCT ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>RTVue Fourier Domain optical coherence tomography </td>
 <td>Optovue </td>
 <td>N/A</td>
 <td>Version 6.1.0.21 or higher 
 Installed with blood flow double ring scan pattern </td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Doppler Optical Coherence Tomography of Retinal Circulation at JoVE.com

Doppler Optical Coherence Tomography of Retinal Circulation

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 ...

doppler-optical-coherence-tomography-of-retinal-circulation

Research

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18.6K Views.  Oregon Health and Science University. Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Video: Doppler Optical Coherence Tomography of Retinal Circulation

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Bioengineering

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

Engineering

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Neuroscience

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 ...

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 ...

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 ...

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.

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 ...

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 ...