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 ...

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18.6K Aufrufe.  Oregon Health and Science University. Insgesamt retinalen Blutflusses wird durch Doppler optische Kohärenztomographie und halbautomatische Grading-Software gemessen.

Serie: Doppler Optical Coherence Tomography of Retinal Circulation

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Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

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The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
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Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

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 ...