The authors thank Nina Bispham for her technical assistance. This work was supported by the American Heart Association (12POST11920023), and the NIH (K23DK088833, K23DK087859).

This protocol follows the guidelines of the Colorado Multiple Institutional Review Board (COMIRB).
1. Preparation for Testing Session
<ol>
	<li>Participants should follow these restrictions for most accurate measurements: 12 hr fast from food and caffeine, 12 hr restraint from exercise, 12 hr restraint from smoking, if applicable, &#62;4 half-life restraint from medications if possible (may not be feasible in a population such as CKD patients), and pre-menopausal women should be tested in days 1-7 of the menstrual cycle to minimize hormonal influences.</li>
	<li>Prepare 500 ml of the dissociation buffer by adding 2 ml of 0.5 M ethylenediaminetetraacetic acid (EDTA), 0.05 g of heparin (180 USP units/ mg), and 2.5 g of bovine serum albumin to 476.8 ml&#160;of phosphate buffered saline (PBS) at pH of 7.4. This can be stored at 4 &#176;C for several months.</li>
	<li>Turn on ultrasound, computer, and the Non-Invasive Hemodynamic Workstation (NIHem; arterial stiffness equipment). Connect cables outputting the ultrasound to the computer R-wave trigger box.</li>
</ol>
2. Collection and Processing of Vascular Endothelial Cells
<ol>
	<li>A trained nurse or physician performs the collection (steps 2.2-2.5, 2.7) and a researcher collects and processes the wires (steps 2.6, 2.8-2.19)</li>
	<li>Prep the antecubital site with a&#160;topical antiseptic, apply a tourniquet, locate vein, and cannulate with an 18 G&#160;catheter. Place a heplock adapter on the end of the IV.</li>
	<li>Put on sterile gloves and put sterile fenestrated drapes over the site.</li>
	<li>Place 2 J-wires on the drapes. Pull the arc of the &#34;J&#34; to uncoil the &#34;J&#34; shape from both wires.</li>
	<li>Uncap the heplock and feed J-wire into vein approximately 8 cm. Push back and forth several times before removing the wire. Avoid gross blood on the wire.</li>
	<li>Use wire cutters to snip the wires so they fit in a 50 ml conical tube containing ~30 ml of dissociation buffer</li>
	<li>Repeat step 2.5 for second wire.</li>
	<li>Repeat step 2.6 for second wire. Return tube to wet laboratory.</li>
	<li>Clasp the wires with a pair of forceps and hold the wires inside of the tube, but above the solution. For 10 min, use a motorized pipetter to repeatedly collect the dissociation buffer from the 50 ml conical tube and release it so it runs down the length of the wires to rinse and vibrate the wires, then shake off excess fluid from wires into tube.</li>
	<li>Centrifuge for 7 min at 400 x g and 4 &#176;C.</li>
	<li>Prepare the formaldehyde solution in foil covered tube by combining 100 ml formaldehyde solution + 900 ml PBS.</li>
	<li>Slowly remove tube from centrifuge, turn on the vacuum pump, place a pipette tip on end of suction hose and leave ~400 ml in the tube, vacuuming off the rest without disturbing the pellet.</li>
	<li>Cover with foil and pipette 1 ml formaldehyde solution into the tube to fix the sample. Do not resuspend. Incubate for 10 min at room temperature.</li>
	<li>Prepare 8 slides by labeling with subject and study visit information and drawing an oval on each slide with a pap pen.</li>
	<li>Add 15 ml PBS, resuspend, and centrifuge for 5 min at 400 x g and 4 &#176;C.</li>
	<li>Repeat step 2.15, add 12 ml PBS, resuspend, and centrifuge for 6 min at 400 x g and 4 &#176;C.</li>
	<li>Slowly remove tube from centrifuge, turn on the vacuum pump, place a pipette tip on end of suction hose and leave ~2 ml in the tube, vacuuming off the rest without disturbing the pellet.</li>
	<li>Resuspend and pipette evenly across the 8 slides in the oval areas.</li>
	<li>Place in incubator at 37 &#176;C for 5 hr and then store at -80 &#176;C until ready for analysis (samples will be fine for many years).</li>
</ol>
3. Assessment of FMDBA and aPWV
<ol>
	<li>Have research subject change into disposable shorts and have him/her lie supine in a quiet, dim, climate controlled room.</li>
	<li>Place the appropriate number of ECG for the specific ultrasound and arterial stiffness device (this procedure uses the Non-Invasive Hemodynamic Workstation [NIHem] to measure arterial stiffness, which requires 4 electrodes), and blood pressure cuff on subject.</li>
	<li>After 20 min, begin blood pressure readings. Perform at least 3, and repeat until measurements are within 5 mmHg, resting 2 min between each reading.</li>
	<li>Begin tonometry by palpating for the brachial artery pulse and placing the tonometer to record brachial waveforms using the software program.</li>
	<li>Repeat for the radial, femoral and carotid arteries.</li>
	<li>Measure the distance to each of these sites from the supersternal notch using a tape measure (brachial, radial and carotid) and custom ruler/caliper (femoral).</li>
	<li>Calculate carotid-brachial, carotid-radial, and carotid-femoral (aPWV) using the software program.</li>
	<li>Place forearm blood pressure cuff just distal to the olecranon process and record at least 10 cardiac cycles of baseline brachial artery ultrasound images and blood flow velocity measurements, with a vascular software set to trigger mode. A mechanical arm can be used to steady the ultrasound probe if desired.</li>
	<li>Inflate forearm blood pressure cuff to 250 mmHg and begin timer. Instruct participant to remain very still.</li>
	<li>Begin recording velocities with a vascular software set to trigger mode when the timer reads 4:45. Trigger release the cuff at 5:00 and change the ultrasound to record B-mode (diameter) images when the clock reads 5:10.</li>
	<li>Continue recording until the clock reads 7:00.</li>
	<li>Record at least 10 cardiac cycles of baseline brachial artery ultrasound images with a vascular software set to trigger mode.</li>
	<li>Take subject&#39;s blood pressure. If systolic blood pressure &#62;100 mmHg, place 0.4 mg of sublingual nitroglycerin under the subject&#39;s tongue and begin timer, unless the patient has another contraindication.</li>
	<li>Begin recording B-mode (diameter images) when the clock reads 3:00 with the vascular software set to trigger mode.</li>
	<li>Stop recording when the clock reads 8:00.</li>
	<li>Monitor blood pressure until it returns to baseline</li>
</ol>
4. Preparing Human Umbilical Vein Endothelial Cell (HUVEC) Control Slides
<ol>
	<li>Grow HUVECs to passage 5-6 and ~80% confluency.</li>
	<li>Trypsinize with 3 ml of trypsin or whatever is necessary for the dish/flask.</li>
	<li>Neutralize trypsin using an equal volume of trypsin neutralizing solution.</li>
	<li>Centrifuge at 200 x g for ~5 min and remove trypsin and neutralizing solution by vacuum.</li>
	<li>Resuspend in ~10 ml PBS to wash.</li>
	<li>Centrifuge at 200 x g ~5 min. Remove PBS.</li>
	<li>Remove PBS and fix in 1800 &#956;l PBS + 200 &#956;l formaldehyde.</li>
	<li>Resuspend in PBS (~10 ml).</li>
	<li>Centrifuge at 200 x g ~5 min. Remove PBS, resuspend in an appropriate volume to add ~200 &#956;l per slide.</li>
	<li>Store slides at -80 &#176;C until ready for analysis (samples will be fine for many years).</li>
</ol>
5. Staining of Vascular Endothelial Cells
<ol>
	<li>Take slides out of -80 &#176;C freezer and wait 5 min at room temperature (this procedure is for a batch of 10 slides, including one HUVEC control slide).</li>
	<li>Wipe away excess water with a delicate task wipe (don&#39;t touch center of slide).</li>
	<li>Re-hydrate the slides by adding altered PBS to each slide and leave them for 10 min.</li>
	<li>While the slides are standing, prepare 5% donkey serum and other solutions.
	<ol>
		<li>Prepare 5% donkey serum by adding 300 &#956;l of donkey serum to 5,700 &#956;l of altered PBS (to a pH of 7.4) for up to 10 slides (increase this amount for more).</li>
		<li>Dilute the primary antibody of interest in 1,000 &#956;l of 5% serum. For example, nitrotyrosine and NADPH oxidase (1:300 and 1:1,500) could be used as markers of oxidative stress.</li>
		<li>Prepare secondary AF568 by diluting 5 &#956;l of AF568 to 1,500 &#956;ll of 5% serum.</li>
		<li>Prepare VE cadherin by diluting 2 &#956;l of VE Cadherin to 1,000 &#956;l of 5% serum.</li>
		<li>Prepare AF488 by diluting 5 &#956;l of VE Cadherin to 1,000 &#956;l of 5% serum.</li>
		<li>Keep AF568, VE cadherin and AF488 under foil through the whole process and then place on rocker in 4 &#176;C refrigerator while preparing slides.</li>
	</ol>
	</li>
	<li>After 10 min of rehydration, dry slides with a delicate task wipe.</li>
	<li>Add 5% donkey serum for 60 min and place a piece of plastic paraffin film over the circled area to ensure complete coverage of the chemical.</li>
	<li>Discard the plastic paraffin film and dry slides with a delicate task wipe. Do not wash. Add primary antibody for 60 min and place a piece of plastic paraffin film over the circled area to ensure complete coverage of the chemical.</li>
	<li>Discard the plastic paraffin film, rinse with altered PBS from squirt bottle and soak in slide columns for 5 min. While the slides are soaking, move them into a dark room. Work in the dark for all remaining steps.</li>
	<li>Dry slides with Kimwipes, add AF568 (secondary antibody) for 45 min and place a piece of plastic paraffin film over the circled area to ensure complete coverage of the chemical. Cover from light.</li>
	<li>Discard the plastic paraffin film in the biohazard waste container. Rinse with altered PBS from squirt bottle, and then soak in columns for 5 min.</li>
	<li>Dry slides with Kimwipes, then add VE Cadherin for 60 min and place a piece of plastic paraffin film over the circled area to ensure complete coverage of the chemical. Cover from light.</li>
	<li>Discard the plastic paraffin film in the biohazard waste container. Rinse with altered PBS from squirt bottle, and then soak in columns for 5 min.</li>
	<li>Dry slides with a delicate task wipe, add AF488 for 30 min and place a piece of plastic paraffin film over the circled area to ensure complete coverage of the chemical. Cover from light.</li>
	<li>Discard the plastic paraffin film in the biohazard waste container. Rinse with altered PBS from squirt bottle, and then soak in columns for 5 min.</li>
	<li>Dry slides with a delicate task wipe and allow slides to dry for 20 min. Cover from light.</li>
	<li>Add one drop only of fluoroshield mounting medium with 4&#39;, 6-diamidino-2-phenylindole hydrochloride (DAPI) to each slide and cover each with a cover slip.</li>
	<li>Place slides in 4 &#176;C refrigerator covered with foil. Imaging needs to be completed within 48 hr.</li>
</ol>
6. Imaging and Analysis of Vascular Endothelial Cells
<ol>
	<li>Prepare microscope for imaging stained endothelial cells according to specifications of the specific microscope. A single blinded technician should analyze any particular protein for a batch of cells.</li>
	<li>Scan slides systematically. Identify endothelial cells by positive staining for VE Cadherin and confirm nuclear integrity by positive staining for DAPI.</li>
	<li>Image 30 cells per slide for later analysis. Repeat for each slide in the stained batch, including the HUVEC.</li>
	<li>Analyze the intensity of the staining for the primary antibody of interest using a qualitative software .</li>
	<li>To minimize the possible confounding effect of differences in intensity staining between different staining sessions, report values as ratio of protein expression in the collected endothelial cells to the same protein expression in the HUVEC.</li>
</ol>

The authors declare that they have no competing financial interests.

Obtaining accurate results for FMDBA and aPWV requires acquiring high quality ultrasound images and pressure waveforms, respectively. Central to this is appropriate and continued training and use of each technique by the operator44. In addition, it is critical to control for as many external variables that may influence the results as possible by standardizing the testing session (e.g.,&#160;prior 12 hr fast, climate controlled room, etc.)44,45. As mentioned above, the u...

Chronic kidney disease (CKD) is a major public health concern that has reached epidemic proportions, affecting ~11.5% of the population in the United States alone1. The risk of cardiovascular death or a cardiovascular event in patients with CKD is significantly increased compared with the general population2-4. Although patients with CKD exhibit a high prevalence of traditional cardiovascular risk factors, this only explains part of their increased incidence of cardiovascular disease (CVD)5. Vascular dysfunction is an important nontraditional cardiovascular risk factor gaining increased recognition in the field of nephrology6-9.
While many changes likely contribute to the development of arterial dysfunction, among those of greatest concern are the development of vascular endothelial dysfunction, most commonly assessed as impaired endothelium-dependent dilation (EDD), and stiffening of the large elastic arteries10. Various techniques exist to assess EDD and large elastic artery stiffness, but the most commonly used are brachial artery flow-mediated dilation FMDBA and aortic pulse-wave velocity (aPWV), respectively. Another commonly used technique to assess EDD is measuring forearm blood flow response to pharmacological agents such as acetylcholine using venous occlusion plethysmography11,12. However, this methodology requires catheterization of the brachial artery, which is more invasive than FMDBA and may be contraindicated in patients with CKD. An alternate technique to assess arterial stiffness is to measure the local arterial compliance (the inverse of stiffness) of the carotid artery, although this is not as widely used or validated with clinical endpoints as aPWV13 .
Patients with CKD demonstrate both impaired FMDBA14-16 and increased aortic pulse-wave velocity aPWV13,17,18, even prior to needing dialysis. Importantly from a clinical perspective, both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events and mortality both in patients with CKD19-21, as well as in other populations22-26. These techniques can be applied to studying various populations at risk of CVD, including patients with CKD.
The exact mechanisms by which arterial dysfunction develops in CKD are incompletely understood; however, reduced nitric oxide (NO) bioavailability is a critical contributor27-30 and a common mechanism of both impaired EDD and increased arterial stiffness10,31. In CKD, oxidative stress is increased and contributes to the reduction in NO bioavailability32-34. Oxidative stress is defined as excessive bioavailability of reactive oxygen species (ROS) relative to antioxidant defenses. Physiological stimuli, including inflammatory signaling, promote oxidant enzyme systems (e.g., the oxidant enzyme NADPH oxidase) to produce ROS, including superoxide anion (O2&#9679;-)35. Production of superoxide ultimately leads to reduces bioavailability of nitric oxide (NO).
Impaired NO bioavailability may in turn contribute to the development of CKD, as endothelial dysfunction is an independent predictor of incident CKD36. This is consistent with animal data indicating that eNOS inhibition induces hypertension (systemic and glomerular), glomerular ischemia, glomerulosclerosis, and tubulo-interstitial injury37. Indeed, reduced NO bioavailability appears necessary for the development and progression of experimental kidney disease that mimics human disease, suggesting a key role for endothelial dysfunction in human CKD38,39.
Markers of vascular oxidative stress can be assessed in vascular endothelial cells collected from human research subjects, using a technique originally developed by Colombo et al.40 and modified Seals et al41-43. Using 2 sterile J-wires, cells are collected from the antecubital vein, recovered, fixed, and later positively identified as endothelial cells and analyzed for expression of proteins of interest using immunofluorescence.
We provide here a discussion of this methodology that can be used to a) measure FMDBA; b) measure aPWV; c) measure vascular endothelial cell protein expression of markers of oxidative stress. The focus is on patients with CKD, not requiring chronic dialysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:736px;" width="736">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:109px;">Company</td>
			<td style="width:244px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">J-wire</td>
			<td style="width:109px;">St. Jude</td>
			<td style="width:244px;">404584</td>
			<td>2 per collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Disposable shorts (MediShorts)</td>
			<td>Quick Medical&#160;</td>
			<td>4507</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Non-invasive hemodynamic workstation (NIHem)</td>
			<td style="width:109px;">Cardiovascualr Engineering</td>
			<td style="width:244px;">N/A</td>
			<td>Includes custom ruler.&#160; An alternate system is the Sphygmocor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ultrasound</td>
			<td style="width:109px;">G.E.</td>
			<td>Model: Vivid7 Dimension</td>
			<td>We use a G.E., but there are many companies and models</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vascular software (Vascular Imager)&#160;</td>
			<td style="width:109px;">Medical Imaging Applications</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">R-wave trigger box</td>
			<td style="width:109px;">Medical Imaging Applications</td>
			<td>N/A</td>
			<td>custom made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rapid Cuff Inflation System</td>
			<td style="width:109px;">Hokanson</td>
			<td style="width:244px;">Model: Hokanson E20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Forearm blood pressure cuff</td>
			<td style="width:109px;">Hokanson</td>
			<td style="width:244px;">N/A</td>
			<td>custom cuff with 6.5 x 34 cm bladder&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HUVECs</td>
			<td style="width:109px;">Invitrogren&#160;</td>
			<td>C-015-5C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Donkey serum</td>
			<td style="width:109px;">Jackson&#160;</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Pap pen</td>
			<td style="width:109px;">Research Products International</td>
			<td>195505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">VE Cadherin</td>
			<td style="width:109px;">Abcam</td>
			<td style="width:244px;">ab33168</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">AF568</td>
			<td style="width:109px;">Life Technologies</td>
			<td>A11011</td>
			<td>depends on specifications of microscpe&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">AF488</td>
			<td style="width:109px;">Life Technologies</td>
			<td>A11034</td>
			<td>depends on specifications of microscpe&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Nitrotyrosine antibody&#160;</td>
			<td style="width:109px;">Abcam</td>
			<td>ab7048</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NADPH oxidase antibody</td>
			<td>Upstate</td>
			<td>07-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DAPI&#160;</td>
			<td style="width:109px;">Vector</td>
			<td style="width:244px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Delicate task wipe (Kimwipe)&#160;</td>
			<td style="width:109px;">Fisher Scientific</td>
			<td>06-666-A&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic paraffin film (parafilm)&#160;</td>
			<td style="width:109px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Confocal microscope&#160;</td>
			<td style="width:109px;">Olympus&#160;</td>
			<td>Model: FV1000 FCS/RICS</td>
			<td>many options exist&#160;</td>
		</tr>
	</tbody>
</table>

assessment of vascular function in patients with chronic kidney disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

FMDBA is quantified as the peak change in diameter of the brachial artery following reactive hyperemia. Thus, the diameter at rest is compared to the diameter following the end of a 5 min blood pressure cuff occlusion period (Figure 1). Panel A shows a representative ultrasound image of the brachial artery, and Panel B displays a graph of the R-wave gated change in diameter from cuff release to 2 min following, as obtained using commercially available software. As the change is often quite min...

Watch this Scientific Journal Video about Assessment of Vascular Function in Patients With Chronic Kidney Disease at JoVE.com

Assessment of Vascular Function in Patients With Chronic Kidney Disease

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

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16.1K Views.  University of Colorado, Denver. The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Video: Assessment of Vascular Function in Patients With Chronic Kidney Disease

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer&#039;s Disease

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

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 ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Interventional Diagnostic Procedure: A Practical Guide for the Assessment of Coronary Vascular Function

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery disease (ANOCA). Abnormal coronary function underlies coronary vasomotion syndromes including coronary endothelial dysfunction, microvascular angina, vasospastic angina, post-PCI angina and myocardial infarction with no obstructive coronary arteries (MINOCA). Each of these endotypes are distinct subgroups, characterized by specific disease mechanisms. Diagnostic criteria and linked therapy for these conditions are now established by expert consensus and clinical guidelines.
Coronary function tests are performed as an adjunctive interventional diagnostic procedure (IDP) in appropriately selected patients during coronary angiography. This aids differentiation of patients according to endotype. The IDP includes two distinct components: a diagnostic guidewire test and a pharmacological coronary reactivity test. The tests last approximately 5 minutes for the former and 10-15 minutes for the latter. Patient safety and staff education are key.
The diagnostic guidewire test measures parameters of coronary flow limitation (fractional flow reserve [FFR], coronary flow reserve [CFR], microvascular resistance [index of microvascular resistance (IMR)], basal resistance index, and vasodilator function [CFR, resistive reserve ratio (RRR)]).
The pharmacological coronary reactivity test measures the vasodilator potential and propensity to vasospasm of both the main coronary arteries and the micro-vessels. It involves intra-coronary infusion of acetylcholine and glyceryl trinitrate (GTN). Acetylcholine is not licensed for parenteral use and is therefore prescribed on a named-patient basis. Vasodilatation is the normal, expected response to infusion of physiological concentrations of acetylcholine. Vascular spasm represents an abnormal response, which supports the diagnosis of vasospastic angina.
The purpose of this practical guide is to provide information on the preparation and administration of the IDP in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success. The IDP supports stratified medicine for a personalized approach to health and wellbeing.

The purpose of this practical guide is to provide information on the preparation and administration of an interventional diagnostic procedure in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success.

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery ...

Muscle Function Obtained with Motion Mode Ultrasound and Surface Electromyography during Core Endurance Exercise

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured between fascial borders at a given time point during an exercise. This selected time point produces a one-dimensional image resulting in real-time, live observation of anatomy. Ultrasound used during functional movement can be referred to as dynamic ultrasound; this is feasible and reliable with the use of a linear transducer, elastic belt, and foam block to secure consistent transducer placement. The lateral abdominal wall is commonly investigated using ultrasound due to the overlapping nature of the muscles. Surface electromyography (sEMG) can complement M-mode ultrasound imaging because it measures the electrical representation of muscle activation. There is minimal evidence using M-mode ultrasound and sEMG simultaneously during core exercise. Exercises that challenge the core musculature involve both isometric holds (e.g., side plank), as well as oscillatory extremity movements (e.g., dead bug). In this study, both instruments will be used simultaneously to measure core muscle function during exercise. Ultrasound measurements will be obtained using a linear transducer and ultrasound unit, and sEMG measurements will be acquired from a wireless sEMG system. To make comparisons between participants and exercises, normalization methods using static, exercise starting positions for both instruments will be used. An activation ratio will be used for ultrasound and calculated by dividing the contracted thickness (thickness during a time point of exercise) by the rested (starting position) thickness. Muscle thickness will be measured in centimeters from the superior inferior fascial border to inferior superior fascial border. These methods aim to offer an innovative and practical measurement of muscle function with M-mode ultrasound and sEMG during core endurance exercises.

This protocol uses motion mode ultrasound and surface electromyography simultaneously to measure muscle function of the core. Muscle thickness and activation of the local stabilizers (e.g., transverse abdominis, internal oblique) and global movers (e.g., external oblique) is achievable during specific time points of the side plank and dead bug exercises.

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured ...

Acupoint Application Combined with Acupoint Massage for Treating Constipation in a Patient with Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a common and frequent disease in elderly patients, with a tendency for progressive exacerbation. Constipation that reduces the quality of life and triggers the risk of diseases is a common concomitant symptom in patients with COPD. Currently, western medical treatment does not achieve the desired results for patients. A high recurrence rate accompanies it, whereas traditional Chinese medicine (TCM) has a long history and rich experience in treating chronic diseases. Both acupoint application and acupoint massage are characteristic therapies of TCM, with minor side effects, high safety, simple operation, and outstanding advantages. They are effective in treating constipation for patients with COPD and are considered an ideal alternative therapy for patients with chronic constipation. The purpose of this article is to introduce the method of acupoint application combined with acupoint massage for the treatment of constipation in patients with COPD, including the selection of points, items, treatment time, and operation procedure.

Here we demonstrate acupoint application combined with acupoint massage for treating constipation in a patient with chronic obstructive pulmonary disease (COPD).