The authors have no acknowledgements.

The method reported was utilised in a study that received ethical approval from the Bournemouth University Research Ethics Committee on the 9th February 2016 (Reference 10571).
1. LSCI Set up
<ol>
	<li>Connect the moorFLPI LSCI rear panel to its three sockets (power supply, Universal Serial Bus (USB) and IEEE1394) for the system to function.</li>
	<li>Assemble the Desktop Support Arm using 4 screws with the moorFLPI LSCI turned upside down, and fixed to the support arm.</li>
	<li>Rotate the mounting bracket for downward imaging when attached. 
	NOTE: The LSCI has three controls: (1) zoom adjuster &#8211; with the imager set in a position, less magnification can be adjusted to maximum and minimum settings for small and larger fields of view respectively. To ensure repeatability, an indexing ring label is provided; (2) focus adjuster &#8211; this is dependent on the measurement distance and must be adjusted after image position has been set. To ensure repeatability, an indexing ring label is provided; and (3) a polarizer &#8211; a linear polarizing filter is available to minimize specular reflection from exposed organs &#8211; the rotating mount can be turned through 360&#176;.</li>
	<li>Install the software to control the camera. The software is divided into two modules providing a measurement and review function.</li>
</ol>
2. Participant Preparation
<ol>
	<li>Ensure the assessment is performed in a temperature controlled room (22 &#177; 1 &#176;C) and that the participants are seated for 10 minutes prior to testing to adapt to room temperature.</li>
	<li>Avoid a strong source of artificial light and the sunlight shining on the participant or the LSCI as ambient light could affect the moorFLPI near infrared laser source operating at 785 nm. 
	NOTE: A simple test to confirm whether ambient light levels are acceptable is by opening the imaging setup window and obstructing the laser. If the image is almost completely black then no further steps are required; if there is still too much ambient light present, further action is required.</li>
	<li>Ensure the participants are relaxed throughout the assessment, with their feet flat on the ground, if seated, and avoid conversations.</li>
	<li>Place 8 cm2 of adhesive opaque patch (AOP) (e.g., Leukotape) on the skin area to mask the blood flow. This is done to account for the drawback of LSCI in terms of high sensitivity to artifact movement, and signal backscatter will be used for measurement of microcirculatory blood flow.</li>
</ol>
3. Microcirculation Image Measurement
<ol>
	<li>Select &#39;Spatial Processing&#39; for 25 frames per second capture at 152 x 113 pixels.</li>
	<li>Choose &#39;Live image measurement&#39; and adjust the position of the imager 20 cm away from the participant followed by adjusting the zoom, focus and polarizer for minimal specular reflection. The image should appear quite &#39;flat&#39; and featureless.</li>
	<li>Set an exposure time of 20 ms for high sensitivity to small changes and low flux.</li>
	<li>Use a display rate of 25 Hz and a time constant of 0.3 s to account for rapid blood flow changes, and to achieve optimum contrast through reducing the image noise.</li>
	<li>Create two equal size (2 cm2) regions of interest (ROI), named ROI 1 and ROI 2. Align ROI 2 so it is within the 8 cm2 AOP. Take care so that ROIs are not interchanging but kept close within 2-4 cm to reduce the need for re-centering if any mechanical movement results in ROI 2 no longer being in the AO area. 
	NOTE: Blood flow measurement will be less accurate in low and high intensity areas, so it is important to have an optimal gain setting. The gain value ranges between 0 - 200. An optimal gain setting value is achieved in range of 70 - 80.</li>
	<li>Perform flux measurements relative to a reference baseline. In the case of this methodology, introduce &#39;the rest&#39; stage as a reference baseline. Therefore, express a &#39;fast&#39; and &#39;slow&#39; stage blood flow as a percentage change from a baseline, &#39;rest&#39; stage.</li>
	<li>Record blood flow measurement in video format and save for offline analysis using an image review module.</li>
</ol>
4. Offline Analysis
NOTE: The moorFLPI Image Review Software allows the opening of a video to perform analysis.
<ol>
	<li>Calculate the mean flux within ROIs following a series of recordings of mean blood flow. ROI 1 is the real measurement of skin blood flow and ROI 2 is the backscattered laser speckle skin signal from the AOP.</li>
	<li>Calculate the mean blood flow by subtracting ROI 2 from ROI 1 (skin blood flow). 
	ROI 1 - ROI 2 = Mean Blood Flow</li>
</ol>

<ol>
	<li>Mahe, G., Rousseau, P., Durand, S., Leftheriotis, G., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+speckle+contract+imaging+accurately+measures+blood+flow+over+moving+skin+surfaces.">Laser speckle contract imaging accurately measures blood flow over moving skin surfaces.</a> Microvas Res. 81 (2), 183-188 (2010).</li><li>Rousseau, P., 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=Increasing+the+&amp;#34;region+of+interest&amp;#34;+and+&amp;#34;time+of+interest&amp;#34;,+both+reduce+the+variability+of+blood+flow+measurements+using+laser+speckle+contrast+imaging.">Increasing the &amp;#34;region of interest&amp;#34; and &amp;#34;time of interest&amp;#34;, both reduce the variability of blood flow measurements using laser speckle contrast imaging.</a> Microvas Res. 82, 88-91 (2011).</li><li>Hecht, N., Woitzik, J., Dreier, J., Vajkoczy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraoperative+monitoring+of+cerebral+blood+flow+by+laser+speckle+contrast+analysis.">Intraoperative monitoring of cerebral blood flow by laser speckle contrast analysis.</a> Neurosurg Focus. 27 (4), 1-6 (2009).</li><li>Mahe, G., Durand, S., Humeau-Heurtier, A., Leftheriotis, G., Abrham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+experimental+conditions+on+noncontact+laser+recordings+in+microvascular+studies.">Impact of experimental conditions on noncontact laser recordings in microvascular studies.</a> Microcirculation. 19 (8), 669-675 (2012).</li><li>Cheng, H., Duong, T. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+laser-speckle-imaging+analysis+method+and+its+application+to+retinal+blood+flow+imaging.">Simplified laser-speckle-imaging analysis method and its application to retinal blood flow imaging.</a> Opt Lett. 32 (15), 2188-2190 (2007).</li><li>Doherty, J., McNamara, P., Clancy, N. T., Enfield, J. G., Leahy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+instruments+for+investigation+of+microcirculatory+blood+flow+and+red+blood+cell+concentration.">Comparison of instruments for investigation of microcirculatory blood flow and red blood cell concentration.</a> J Biomed Opt. 14 (3), 034025 (2009).</li><li>Briers, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+speckle+contrast+imaging+for+measuring+blood+flow.">Laser speckle contrast imaging for measuring blood flow.</a> Opt Appl. 37 (1), 139-152 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> moorFLPI User Manual. (8), (2012).</li><li>Mahe, G., Durand, S., Humeau-Heurtier, A., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Experimental+Conditions+on+Noncontact+Laser+Recordings+in+Microvascular+Studies.">Impact of Experimental Conditions on Noncontact Laser Recordings in Microvascular Studies.</a> Microcirculation. 19, 669-675 (2012).</li><li>Bahadori, S., Immins, T., Wainwright, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+calf+neuromuscular+electrical+stimulation+and+intermittent+pneumatic+compression+on+thigh+microcirculation.">The effect of calf neuromuscular electrical stimulation and intermittent pneumatic compression on thigh microcirculation.</a> Micro Res. 111, 37-41 (2017).</li><li>Omarjee, L., 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=Optimisation+of+movement+detection+and+artifact+removal+during+laser+speckle+contrast+imaging.">Optimisation of movement detection and artifact removal during laser speckle contrast imaging.</a> Miscrovas Res. 97 (1), 75-80 (2015).</li></ol>

The authors have nothing to disclose.

The aim of this study was to introduce a novel technique for the measurement and analysis of alternating speeds of blood microcirculation in a single experiment using the LSCI. The measurements can be affected by ambient light, vibration and participant movement including breathing and twitching. The steps outlined in the protocol have all been designed to minimize those effects and obtain reliable and repeatable measurements of blood microcirculation.
It is emphasized that every step within the protocol is critical for an accurate measurement of blood microcirculation, the technique introduced was discovered following sequential testing of all possible setting options and combinations available including: time constant, zoom setting, exposure time, display rate, gain and flux image palette. Results were analyzed and repeated using live video display and offline analysis to find the optimum imaging setup. This was essential as the image processing software uses the fact that high perfusion produces rapid variation in laser speckle pattern, and as a result a low contrast area of well-defined speckle is produced in the video image. Perfusion image is then created in a color-coded map of microcirculatory perfusion.
Experimental area and participation preparation is found to be essential, and this can be controlled by avoiding work near sources of daylight (window) or strong sources of artificial light, as these may interfere with the moorFLPI near infra-red laser source. The protocol also introduced an AOP as it was acknowledged that the environmental vibration and movement of participant both generate signals that are indistinguishable from blood flow. The AOP proved to be a simple yet effective choice, offering a thin but opaque, light and accessible option which had a microscopically rough surface area to avoid significant specular reflection. Preliminary research by Omarjee et al.11 highlights a potential limitation by which Leukotape creates a reflection signal amplitude different to that of the skin and varies significantly between subjects; however Mahe et al.1 found no drastic difference between participants. Although Leukotape is more accessible than other bespoke, bilayer adhesive patches, the accuracy of Equation (1) measurement could be enhanced by using an alternative AOP.
The offline analysis section highlighted the importance of sizes of ROIs, and their location within the area of interest. Initially, a bigger ROI 1, approximately 8 cm2, was tried which overlaid the ROI 2. This methodology soon became un-reliable due to artifact movement resulting in the ROI 2 moving and the live experiment had to be stopped in order to re-center the ROI 2. Another short coming was that due to ROI 1 overlaying the AOP, the mean flux no longer took the area under AOP into an account, as there was no longer a backscattered signal. This meant a large section of blood microcirculation was being overlooked and therefore resulting flux data were not correct. Therefore, a methodology in which two ROIs of 2 cm2, with an AOP of 8 cm2 and no interaction between ROI 1 and ROI 2 (but kept within 2-4 cm of each other), provides a reliable and repeatable analysis technique for measurement of blood microcirculation.

Laser Speckle Contrast Imaging (LSCI) is a proven non-contact, real time monitoring method to analyse blood flow microcirculation1,2,3,4,5,6,7. The LSCI used in this paper is moorFLPI Full Field. The measuring of blood flow perfusion in large areas at high spatial and temporal resolution using a phenomenon called &#34;laser speckle&#34; is one of the main advantages of this device6. The real-time assessment of microcirculation is done through captured patterns via a camera using scattered speckle patterns. Given that the moorFLPI LSCI is intended for clinical and physiological research, the image processing software works on the fact that high perfusion produces rapid variation in the laser speckle pattern, which is then integrated by the Charged Coupled Device (CCD) to produce an area of low contrast8. The contrast is quantified and the resulting flux is colour coded to produce a perfusion image8.
Unfortunately, the LSCI is very sensitive to environmental vibration, artifact and movement of the subject area9. To date this has provided challenging when alternating flow states have been studied. This paper explains details of the explicit technique outlined in a recent study10 where a Neuromuscular Electrical Stimulation Device was used to measure blood microcirculation when there was movement of the limb being examined.

<table border="0" cellpadding="0" cellspacing="0" style="width:1117px;" width="1117">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:260px;">moorFLPI LSCI</td>
			<td style="width:188px;">Moor Instruments</td>
			<td style="width:152px;">Not Available - Online Link Provided in descreption</td>
			<td style="width:517px;">moorFLPI is an instrument designed for the measurment of blood flow within microvasculature by using infra red laser speckle contrast analysis.&#160; https://gb.moor.co.uk/</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:260px;">moorFLPI Image Review Module</td>
			<td style="width:188px;">Moor Instruments</td>
			<td style="width:152px;">No Available - Online Link Provided</td>
			<td style="width:517px;">Used with moorFLPI, user can record and measure changes in blood flow by changerating a colour coded map of tissue perfusion.&#160; https://gb.moor.co.uk/</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:260px;">Leukotape</td>
			<td style="width:188px;">BSN Medical</td>
			<td style="width:152px;">72978-10</td>
			<td style="width:517px;">Medical tape with microporous surface. http://www.bsnmedical.co.uk/fileadmin/z-countries/United_Kingdom/PDF/L/Leukotape_K_A46PP_low_res_11112013.pdf</td>
		</tr>
	</tbody>
</table>

a novel approach to overcome movement artifact when using a laser speckle contrast imaging system for alternating speeds of blood microcirculation

The laser speckle contrast imager (LSCI) provides a powerful yet simple technique for measuring microcirculatory blood flow. Ideal for blood dynamic responses, the LSCI is used in the same way as a conventional Laser Doppler Imager (LDI). However, with a maximum skin depth of approximately 1 mm, the LSCI is designed to focus on mainly superficial blood flow. It is used to measure skin surface areas of up to 15 cm x 20 cm. The new technique introduced in this paper accounts for alternating speeds of microcirculations; i.e. both slow and fast flow flux measurement using the LSCI. The novel technique also overcomes LSCI&#39;s biggest shortcoming, which is high sensitivity to artifact movement. An adhesive opaque patch (AOP) is introduced for satisfactory recording of microcirculatory blood flow, by subtracting the LSCI signal from the AOP from the laser speckle skin signal. The optimal setting is also defined because the LSCI is most powerful when flux changes are measured relative to a reference baseline, with blood microcirculatory flux expressed as a percentage change from the baseline. These changes may be used for analyzing the status of the blood flow system.

The LSCI experimental set up is outlined in Figure 1 with functional tools identified. A typical participant preparation for a measurement of blood flow on an area of the anterior thigh is illustrated. Adjustable mounting bracket allows rotation of the LSCI for the measurement of blood flow within microvasculature of any particular skin area. Figure 2 outlines an example of a typical raw speckle image and converted speckle image with bespoke settings outlined in the protocol for measurement of blood microcirculation.
Figure 3 shows an example of skin area and placement of AOP (step 3.1), LSCI raw imaging setup (step 3.2), a live image for slow blood flow (step 3.3) and a live image for a fast blood flow (step 3.4) achieved in a continuous data recording of an alternating flux using moorFLPI LSCI. Palette color-coding setting allows differentiation between flux levels. With the standard palette of 16 colors, low flux is seen as blue, medium flux values are seen as green and high flux values are seen as orange and red.&#160;
Skin blood flow is expressed in laser speckle perfusion units (LSPU). Figure 4 shows the graphical representation of ROI 1, ROI 2 and AOP on an area of skin. The mean blood flow is calculated in offline analysis using data from ROI 1 and ROI 2, and Equation (1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56415/56415fig1.jpg" /> 
Figure 1: moorFLPI LSCI experimental set up. Desktop support arm, cable outputs, position controls (zoom adjuster, focus adjuster and polarizer), AOP and laptop for configuration of image setting. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56415/56415fig2.jpg" /> 
Figure 2: Raw speckle image prior to data recording. 2.1 - 2.2) Flux and raw speckle images of a badly configured setting resulting in a high gain with poor visibility which will result in a less accurate blood flow measurement. 2.3 - 2.4) System configured as per protocol, resulting in a correct gain with maximum visibility for a reliable result. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56415/56415fig3.jpg" /> 
Figure 3: An overview of setup and record measurement using moorFLPI LSCI. 3.1. An area of skin (thigh) with a 2 cm2 AOP to account for artifact movement. 3.2) A raw speckle &#39;flat and featureless&#39; image indicating good backscatter light intensity with optimal setting. 3.3) A live image recording of a slow blood flow. 3.4) A live image recording of a fast blood flow. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56415/56415fig4.jpg" /> 
Figure 4: Graphical representation of the ROI 1, ROI 2 and AOP layout on an area of the skin. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Rest (Baseline Reference) (LSPU)</td>
			<td colspan="4">Slow Blood Flow (LSPU)</td>
			<td colspan="4">Moderate Blood Flow (LSPU)</td>
			<td colspan="4">Fast Blood Flow (LSPU)</td>
		</tr>
		<tr>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Blood Flow Increase % from Baseline</td>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Blood Flow Increase % from Baseline</td>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Blood Flow Increase % from Baseline</td>
		</tr>
		<tr>
			<td>157.9</td>
			<td>35.1</td>
			<td>122.8</td>
			<td>178.5</td>
			<td>41.6</td>
			<td>136.9</td>
			<td>10.9</td>
			<td>216.9</td>
			<td>44.6</td>
			<td>172.3</td>
			<td>33.5</td>
			<td>418.9</td>
			<td>77.5</td>
			<td>341.4</td>
			<td>94.2</td>
		</tr>
	</tbody>
</table>
Table 1: Mean flux in LSPU for ROI 1 and ROI 2 at baseline, slow, moderate and fast Blood Flow. Blood flow increase is expressed as a percentage change from a baseline stage.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56415/56415fig5.jpg" /> 
Figure 5: Example of ROI 1 and ROI 2 positioning on a skin surface area (thigh). A 16-color palette outlines the lavers of perfusions. Data is recorded for ROI 1 and ROI 2 in perfusion units and subtracted as explained in equation 1 for measurement of blood microcirculation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation at JoVE.com

A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation

This study introduces a novel technique for the measurement and analysis of alternating speeds of blood microcirculation in a single experiment using a laser speckle contrast imager.

The laser speckle contrast imager (LSCI) provides a powerful yet simple technique for measuring microcirculatory blood flow. Ideal for blood dynamic ...

a-novel-approach-to-overcome-movement-artifact-when-using-laser

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.3K Views.  Bournemouth University. This study introduces a novel technique for the measurement and analysis of alternating speeds of blood microcirculation in a single experiment using a laser speckle contrast imager.

Video: A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to clearly describe the acromion marker cluster (AMC) method of determining scapular kinematics when using a passive marker motion capture system, with consideration for the sources of error which could affect the validity and reliability of measurements. The AMC method involves placing a cluster of markers over the posterior acromion, and through calibration of anatomical landmarks with respect to the marker cluster it is possible to obtain valid measurements of scapular kinematics. The reliability of the method was examined between two days in a group of 15 healthy individuals (aged 19-38 years, eight males) as they performed arm elevation, to 120&#176;, and lowering in the frontal, scapular and sagittal planes. Results showed that between-day reliability was good for upward scapular rotation (Coefficient of Multiple Correlation; CMC = 0.92) and posterior tilt (CMC = 0.70) but fair for internal rotation (CMC = 0.53) during the arm elevation phase. The waveform error was lower for upward rotation (2.7&#176; to 4.4&#176;) and posterior tilt (1.3&#176; to 2.8&#176;), compared to internal rotation (5.4&#176; to 7.3&#176;). The reliability during the lowering phase was comparable to results observed during the elevation phase. If the protocol outlined in this study is adhered to, the AMC provides a reliable measurement of upward rotation and posterior tilt during the elevation and lowering phases of arm movement.

This report presents details of how to adopt the acromion marker cluster method of obtaining scapular kinematics when using a passive marker motion capture device. As has been described in the literature, this method provides a robust, non-invasive, three-dimensional, dynamic and valid measurement of scapular kinematics, minimizing skin movement artifact.

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

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

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

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

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

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