This project was supported by Grant Numbers HL96593 from NIH and D56HP20783 from HRSA/ HHS. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or HRSA / HHS.

Note: The acquisition process for obtaining capillary images has previously been published and is accomplished using a still digital camera with a corresponding image acquisition and analysis computer program. 1121 This lab utilizes still images for analysis, not videos, simplifying image acquisition for analysis. The following describes the new technique for quantitating the capillaries from the images.
1. Image Enhancement Process
<ol>
	<li>Obtain digital images with a monochrome digital camera. Calibrate the images to an object of known size by taking a picture of an object of known length such as a ruler with the camera. This process allows the computer program to measure and count capillaries accurately after processing. Ideally for the highest precision, a reticule (scientifically manufactured piece of glass with a ruler etched into it) should be used. Measure the number of pixels in a 1 mm square box using a computer program. 
	Note: The key to the reproducibility and standardization of this protocol depends largely upon proper placement of the 1 mm2 box that is counted.</li>
	<li>Use contrast enhancement tools to darken the capillaries and lighten the background, which will maximize visualization of the capillaries. Initial differentiation of capillaries from background is important for proper cropping of the image in later steps. Do this by adjusting the image using a &#8220;best fit histogram.&#8221; To do this click on: capture, intensity, image histogram, best fit.</li>
	<li>Crop a region of interest (ROI) for capillary counting as a new image (select, regions of interest). Use a 1 mm2 box, which was determined by calibrating the images at 530 pixels equaling 1 mm. check that the cropped image places the apex of the capillary loops at the very top of the image.</li>
	<li>Flatten the image so all future image adjustments will be evenly applied to the image. To do this click on: process tab, 2D filters, flatten, BG intensity of &#8220;bright,&#8221; object width to &#8220;75,&#8221; apply.</li>
	<li>Raise the contrast of the image so capillaries are maximally visualized. To do this click on: adjust tab, display, raise contrast to 75.</li>
	<li>Despeckle the image to smooth the edges of the capillaries by clicking on process tab, 2D filters, despeckle, kernel size 7 x 7, apply.</li>
	<li>Finalize the image contrast so capillaries are black and the background is white. Perform this step by adjusting the histogram to the &#8220;best fit&#8221; model. 
	Note: Please refer to Figure 2 in Representative Results for an example of what the processed image should look like following these steps.</li>
</ol>
2. Performing Capillary Counts / Quantitating Capillary Density
<ol>
	<li>On each image, manually select one part of a well-defined capillary using the &#8220;target object&#8221; feature to be recognized as objects to be counted by the program. Then, select a small part of background, using the &#8220;background&#8221; feature, as a reference to areas that need to be disregarded by the counting algorithm. 
	Note: The combination of these highlights causes all capillaries to be highlighted while disregarding the background noise. The quantitation (counting) protocol utilizes computer functions to differentiate parts of the image based on color and morphology.</li>
	<li>Use the count function to instantly count all capillaries in the image with the imaging equipment. Set the minimum diameter of counted objects to 5 pixels in order to avoid counting background noise as capillaries.</li>
	<li>For each individual, count the average of 3 -&#160;4 images in order to obtain a more reliable assessment. 
	Note: Please refer to Figure 3 in Representative Results for an example of what the image should look like during the counting procedure.</li>
</ol>
3. Creating and Using Macros to Automate Image Processing
Note: To save time, macros can be created in order to automatically perform a specific sequence of processes on one or many images. These sequences can be customized in order to make the image modifications quicker. In essence, these macros remember how the images are processed, and perform all the steps quickly and with no user input. Performing counts on 12 capillary images takes this lab between 20 to 30 min with the macros (2 to 3 min per picture), as opposed to about 8 min per picture without the macros. Therefore using the macros is 3 to 5 times more efficient than manually processing each individual image.
<ol>
	<li>In order to create a macro, select &#8220;record macro&#8221; and on one image perform the steps and processes desired, as described in steps 1 and 2 above. Name the macro based on which image processing steps were performed for future reference. When using the macro on future pictures, simply click &#8220;run macro&#8221; and the program will automatically apply the recorded enhancements to the image(s) desired. 
	Note: This lab uses a macro to perform all but one of the steps in Section 1 of the methods in a few seconds. The only step that requires user input is choosing where to crop the image, Step 1.2.</li>
</ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-automatic+assessment+of+skin+capillary+density:+proof+of+principle+and+validation.">Semi-automatic assessment of skin capillary density: proof of principle and validation.</a> Microvasc Res. 90, 192-198 (2013).</li><li>Allen, J., Howell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+imaging:+techniques+and+opportunities+for+clinical+physiological+measurements.">Microvascular imaging: techniques and opportunities for clinical physiological measurements.</a> Physiol Meas. 35, R91-R141 (2014).</li><li>Boettcher, M., Gloe, T., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiautomatic+quantification+of+angiogenesis.">Semiautomatic quantification of angiogenesis.</a> J Surg Res. 162, 132-139 (2010).</li><li>Wild, R., Ramakrishnan, S., Sedgewick, J., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+angiogenesis+and+tumor+vessel+architecture+by+computer-assisted+digital+image+analysis:+effects+of+VEGF-toxin+conjugate+on+tumor+microvessel+density.">Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.</a> Microvasc Res. 59, 368-376 (2000).</li><li>Tresadern, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulating+nailfold+capillaroscopy+sequences+to+evaluate+algorithms+for+blood+flow+estimation.">Simulating nailfold capillaroscopy sequences to evaluate algorithms for blood flow estimation.</a> Conf Proc IEEE Eng Med Biol Soc. , 2636-2639 (2013).</li><li>Anderson, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computerized+nailfold+video+capillaroscopy--a+new+tool+for+assessment+of+Raynaud's+phenomenon.">Computerized nailfold video capillaroscopy--a new tool for assessment of Raynaud's phenomenon.</a> J Rheumatol. 32, 841-848 (2005).</li><li>Neubauer-Geryk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+reactivity+of+skin+microcirculation+in+response+to+L-arginine+in+later-onset+type+1+diabetes.">Decreased reactivity of skin microcirculation in response to L-arginine in later-onset type 1 diabetes.</a> Diabetes Care. 36, 950-956 (2013).</li><li>Pazos-Moura, C. C., Moura, E. G., Bouskela, E., Torres-Filho, I. P., Breitenbach, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nailfold+capillaroscopy+in+diabetes+mellitus:+morphological+abnormalities+and+relationship+with+microangiopathy.">Nailfold capillaroscopy in diabetes mellitus: morphological abnormalities and relationship with microangiopathy.</a> Braz J Med Biol Res. 20, 777-780 (1987).</li><li>Antonios, T. F., Singer, D. R., Markandu, N. D., Mortimer, P. S., MacGregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+skin+capillary+rarefaction+in+essential+hypertension.">Structural skin capillary rarefaction in essential hypertension.</a> Hypertension. 33, 998-1001 (1999).</li><li>Kaiser, S. E., Sanjuliani, A. F., Estato, V., Gomes, M. B., Tibirica, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antihypertensive+treatment+improves+microvascular+rarefaction+and+reactivity+in+low-risk+hypertensive+individuals.">Antihypertensive treatment improves microvascular rarefaction and reactivity in low-risk hypertensive individuals.</a> Microcirculation. 20, 703-716 (2013).</li><li>Cheng, C., Diamond, J. J., Falkner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+capillary+rarefaction+in+mild+blood+pressure+elevation.">Functional capillary rarefaction in mild blood pressure elevation.</a> Clinical and Translational Science. 1, 75-79 (2008).</li><li>Debbabi, H., Bonnin, P., Levy, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+blood+pressure+control+with+perindopril/indapamide+on+the+microcirculation+in+hypertensive+patients.">Effects of blood pressure control with perindopril/indapamide on the microcirculation in hypertensive patients.</a> Am J Hypertens. 23, 1136-1143 (2010).</li><li>He, F. J., Marciniak, M., Markandu, N. D., Antonios, T. F., MacGregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+modest+salt+reduction+on+skin+capillary+rarefaction+in+white,+black,+and+Asian+individuals+with+mild+hypertension.">Effect of modest salt reduction on skin capillary rarefaction in white, black, and Asian individuals with mild hypertension.</a> Hypertension. 56, 253-259 (2010).</li><li>Murray, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+measurement+location+on+reliability+of+quantitative+nailfold+videocapillaroscopy+in+patients+with+SSc.">The influence of measurement location on reliability of quantitative nailfold videocapillaroscopy in patients with SSc.</a> Rheumatology (Oxford). 51, 1323-1330 (2012).</li><li>Ingegnoli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+different+capillaroscopic+measures+for+identifying+nailfold+microvascular+alterations.">Feasibility of different capillaroscopic measures for identifying nailfold microvascular alterations.</a> Semin Arthritis Rheum. 38, 289-295 (2009).</li><li>Debbabi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+skin+capillary+density+in+treated+essential+hypertensive+patients.">Increased skin capillary density in treated essential hypertensive patients.</a> Am J Hypertens. 19, 477-483 (2006).</li><li>Serne, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+skin+capillary+recruitment+in+essential+hypertension+is+caused+by+both+functional+and+structural+capillary+rarefaction.">Impaired skin capillary recruitment in essential hypertension is caused by both functional and structural capillary rarefaction.</a> Hypertension. 38, 238-242 (2001).</li><li>Shore, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillaroscopy+and+the+measurement+of+capillary+pressure.">Capillaroscopy and the measurement of capillary pressure.</a> Br J Clin Pharmacol. 50, 501-513 (2000).</li><li>Rieder, M. J., O'Drobinak, D. M., Greene, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computerized+method+for+determination+of+microvascular+density.">A computerized method for determination of microvascular density.</a> Microvasc Res. 49, 180-189 (1995).</li><li>Vermeulen, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+angiogenesis+in+solid+human+tumours:+an+international+consensus+on+the+methodology+and+criteria+of+evaluation.">Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation.</a> Eur J Cancer. 32A, 2474-2484 (1996).</li><li>Cheng, C., Daskalakis, C., Falkner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Assessment+of+Microvascular+and+Endothelial+Function.">Non-invasive Assessment of Microvascular and Endothelial Function.</a> Journal of Visualized Experiments. , (2012).</li><li>Antonios, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximization+of+skin+capillaries+during+intravital+video-microscopy+in+essential+hypertension:+comparison+between+venous+congestion,+reactive+hyperaemia+and+core+heat+load+tests.">Maximization of skin capillaries during intravital video-microscopy in essential hypertension: comparison between venous congestion, reactive hyperaemia and core heat load tests.</a> Clin Sci (Lond). 97, 523-528 (1999).</li></ol>

The authors have no conflicts of interest.

Nailfold capillaroscopy shows promise as a clinically useful tool in the future for various oncology, cardiovascular, and rheumatologic disease applications. The image acquisition process is fairly consistent among researchers, yet there are currently multiple methods for image processing and analysis. Methods currently include computerized and manual capillary counts. Manual counts are problematic as they are time-consuming, and subject to user subjectivity and fatigue. Current computer based methods require a high leve...

Microvascular imaging is a rapidly growing field with many potential clinical applications.2 For example, oncologists are using microvessel imaging to determine the extent of tumor angiogenesis, yielding valuable information concerning the state of the tumor and insight into possible treatment options. 34 However, nailfold capillaroscopy is perhaps the most cost efficient and widely applicable form of microvascular imaging. Researchers are using video nailfold capillaroscopy to study blood flow rates and investigate capillary morphology.5 6 Both video and still-picture nailfold capillaroscopy are adjuncts to care for diagnosing and treating Raynaud&#8217;s phenomenon and various connective tissue diseases such as systemic sclerosis.2
Nailfold capillaroscopy has various potential cardiovascular applications as well. Current research using nailfold capillaroscopy suggests that diabetes mellitus Type 1 and Type 2 patients exhibit a high prevalence of abnormal capillary morphology, yet have unchanged capillary densities compared to non-diabetic individuals.7-8 Capillaroscopy has also been studied experimentally in hypertension. Structural capillary rarefaction leading to a reduced capillary density has been demonstrated in hypertensive individuals compared to non-hypertensive individuals.9-10 In contrast to these older hypertensive patients (mean age 40 and above) who exhibit structural rarefaction, more recent research has demonstrated that younger hypertensive patients (mean age under 40 years old) have functional rarefaction without structural rarefaction.11 This suggests that functional rarefaction occurs before and may progress over time to structural rarefaction.
Interestingly, hypertensive patients treated with specific antihypertensive drugs such as Perindopril/Indapamide displayed normal capillary density and endothelial function after treatment, while those treated with ACE (angiotensin-converting-enzyme) inhibitors or diuretics maintained a low capillary density despite comparable blood pressure control.12 This suggests that some antihypertensive medications may normalize capillary density by reversing the capillary rarefaction caused by hypertension. In addition, other researchers have shown that a reduction in salt intake leads to reversal of both functional and structural capillary rarefaction in hypertensive individuals.13
Despite the various potential clinical applications of this technology, there is little standardization in technique for quantitating capillary density images.2 To date, researchers have found that capillary density results are reproducible from both an intra-observer and inter-observer perspective only if the exact same area is being counted each time. 1,14 15 Of note, previous researchers have largely performed capillary counts manually using the naked eye, 9 16 17 18 which is a slow and subjective process.
Standardized, computer based algorithms for quantitation of capillary images theoretically provide more efficient and reproducible data analysis with less subjectivity, facilitating clinical applications of capillaroscopy. Some researchers have indeed used computer-based programs to quantitate the data from nailfold capillaroscopic pictures. 1,6 19 20 However, only one publication to date describes reliability of a complex software program available for quantitating capillaroscopy data,1 and this program is complicated as previously noted above by the requirement to count the exact same visual field. Here, we present a simpler, reliable protocol for quantitating capillaries using a standardized algorithm which allows for the use of multiple visual fields. The use of multiple visual fields not only simplifies the procedure, but also permits the assessment of normal biological variation in capillary count.
The aim of this study is to describe a reproducible and efficient computer based algorithm which standardizes the capillary quantitation process. While these methods are not fully automated they require very little user input, and provide rapid and reliable quantitation of the pictures.

<table>
	<tbody>
		<tr>
			<td class="xl65">Image-Pro Premier</td>
			<td class="xl65">Media Cybernetics, Inc</td>
			<td align="right" class="xl63">9.1</td>
			<td class="xl63">Image processing software</td>
		</tr>
	</tbody>
</table>

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.

The goal of this image processing procedure is to differentiate the capillaries from the background image so they can be accurately quantified. Both incomplete image processing and excessive image processing are detrimental to the program's ability to quantify the capillaries. As seen in Figure 3, incomplete image processing makes the capillaries difficult to distinguish from the background. It is critical that the user be able to readily distinguish the border of a capillary since the counting method de...

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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 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 ...

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8.9K Views.  Thomas Jefferson University. 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. 

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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

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.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

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.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

Ablation of Ischemic Ventricular Tachycardia Using a Multipolar Catheter and 3-dimensional Mapping System for High-density Electro-anatomical Reconstruction

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars represent zones of slow conduction that allow the occurrence and maintenance of reentrant circuits. Catheter ablation enables substrate modification of those low voltage areas and thus can help to alter the scar tissue in such a way that arrhythmias cannot appear anymore. Hospitalizations of concerned patients decrease, quality of life and outcome rise. Consequently, VT ablation represents a growing field in electrophysiology, especially for patients with endocardial scars in ischemic heart disease after myocardial infarction. However, ablation of ventricular tachycardia remains one of the most challenging procedures in the electrophysiology lab. Precise scar definition and localization of abnormal potentials are critical for ablation success. The following manuscript describes the use of a multipolar mapping catheter and 3-dimensional (3D) mapping system to create a high density electro-anatomical map of the left ventricle including a precise scar representation as well as mapping of fractionated and late potentials in order to allow a highly accurate substrate modification.

With the following protocol, we provide an approach to Ventricular Tachycardia (VT) ablation using high density mapping with a multipolar catheter and 3D mapping system enhancing the success of the procedure.

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars ...

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a computerized functional skills training program that was paired in half of the participants with a commercially available cognitive training (CCT) program.
Non-impaired older individuals (NC) aged 60+ (n=45) and similarly aged individuals with mild cognitive impairment (MCI; n=50) were randomized to receive 12 weeks of twice-weekly computerized functional skills training (CFST) or 12 weeks of twice-weekly sessions split between CCT and CFST. Skills trained were use of an ATM; internet banking; ticket kiosk; telephone and internet prescription refill; medication management; and internet shopping. As with previous functional capacity assessments, we focus on completion time for each simulation.
51 participants completed the training program, either by mastering all 6 tasks (34) or completing 12 weeks of training. 44 more participants completed 4 or more training sessions so they were also analyzed for improvement up to their last training session. Completion time for all 6 tests significantly improved from the baseline assessment to the final training session in both groups of participants (all p&#60;0.001 with an average improvement in task completion time of 45%). Further, there was no differential improvement in MCI and NC in the 6 tests from baseline to end of training (all t&#60;1.66, all p&#62;0.12). Finally, combined CCT plus CFST did not differ from CSFT alone on any of the percent-change score measures (all t&#60;1.64, all p&#62;0.11).
Both NC and MCI groups evidenced substantial improvements in performance. CCT supplementation led to similar functional gains with half as many training sessions. The NC participants proceeded through the training fairly rapidly even without CCT supplementation; MCI participants required more training but learned equivalently. These findings suggest that even in cases with memory impairments, functional skills can be efficiently learned with training.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.