The authors would like to thank Koby Sheffy, PhD for his insightful review of this work and William Sotka for ongoing support and technical assistance.
This work was supported in part by grants from the National Institutes of Health (K12 HL087746, RC2HL103400, 1U01HL100397), and the California Tobacco Related Disease Research Program of the University of California (18XT-0098).

I.Prepare the Patient for an Endo-PAT Study
<ol>
 <li>Prior to the study, ensure the patient has fasted for at least 4 hours, and has refrained for at least 8 hours from caffeine, tobacco, vitamins or medications that might affect vascular tone. The patient may wish to use the restroom prior to the study.</li>
 <li>The Endo-PAT study should be conducted in a quiet, dimly lit, temperature-controlled exam room to reduce fluctuations in vascular tone.</li>
 <li>Cell phones or paging devices should be silenced, and restrictive clothing that could interfere with blood flow to the arms should be removed. The patient should also remove watches, rings, or other jewelry on the hands or fingers.</li>
 <li>Inspect the patient's fingers for any deformities or injuries that could affect the study. Do not place the probes on a finger that is cut or injured. Fingernails should not extend more than 5mm or 1/5 of an inch beyond the tip of the finger tissue. Trim or file fingernails if necessary to avoid damaging the internal membranes of the PAT probes and displacing the finger from the sensing region of the probe.</li>
 <li>The index finger is recommended for the study; however, if this finger is unsuitable, a different digit (except the thumb) may be used, as long as the same finger is used on both hands.</li>
 <li>The patient should be supine and comfortable for 15 minutes so as to attain a cardiovascular steady-state. Place the two arm supporters along each of the patient's sides.</li>
 <li>Measure the blood pressure using the control arm (the arm that is not occluded during the Endo-PAT study.</li>
 <li>Place a blood pressure cuff on the arm to be occluded during the Endo-PAT study. Apply the cuff snugly, but without excess pressure. Do not inflate the cuff at this time.</li>
</ol>
II.Prepare the Endo-PAT System for Study
<ol>
 <li>Launch the Endo-PAT 2000 software and click the "Patient Information" icon on the tool bar to create a new patient file.</li>
 <li>Complete the Patient Information dialog box, including patient ID, name (optional), age, gender, height, weight, systolic and diastolic blood pressures. Optional fields allow for free text comments. Select your name from the pre-defined list in the Patographer name field. If your name has not been inserted add it the list and select.</li>
 <li>Select two new PAT probes and connect to the pneumo-electrical tubing. To connect the probes, insert the connector tab into the probe slit and gently press the connector onto the probe until it clicks into place.</li>
 <li>Place the connected probes into the sockets of the arm-supports and press the "Deflate" button on the top of the Endo-PAT 2000 device.</li>
</ol>
III.Conduct an Endo-PAT Study
<ol>
 <li>Place the patient&#8217;s index fingers completely into the probes, confirm with the patient that he or she can feel the very end of the probes, and press the "Inflate" button on the top of the Endo-PAT 2000 device.</li>
 <li>Place a foam anchor ring at the base of the adjacent middle finger. Ensure that the foam ring and the PAT sensor do not touch. Otherwise the ring may mechanically interfere with the sensor.</li>
 <li>Create an approximately 7-10cm loop with the pneumo-electrical tubing. The loop should extend from the PAT sensor and return to the foam ring on the adjacent finger while the rest of the tubing that connects to the EndoPAT device is pointing out tubing to the tip of the finger. </li>
 <li>Position the patient's arms so the forearms are supported on the arm supports and the fingers dangle freely off the edge of the support. Make sure the probes are not in contact with any object, including the arm support, foam ring, tubing, the mattress or another finger.</li>
 <li>Ask the patient to refrain from moving the fingers, as this will create mechanical artifacts. It is important for the patient to be relaxed throughout the study. Explain to the patient that during the test you will inflate the arm cuff, and during that time they may feel some discomfort, numbness, or tingling.</li>
 <li>Click the "Standby" icon on the Endo-PAT's computer interface. Adjust the time base to 1 minute and adjust the signal gain on the screen to maximize signal clarity. Inspect the tracings of the PAT signals from the two probes to confirm that they are free of artifactual signals. If artifactual signals are present, veriify that the probes are not touching anything and that the patient is not moving the fingers.</li>
 <li>To begin the study, click the "Go" icon on the computer interface. Start the stopwatch, by clicking the "Start/Stop Timer" icon. This will initiate a five minute count down for the baseline recording period. After five minutes, stop the stopwatch by clicking the "Start/Stop Timer" icon.</li>
 <li>Tell the patient that you are going to inflate the cuff for the occlusion phase and that he or she should stay relaxed and not move the fingers.</li>
 <li>Rapidly inflate the blood pressure cuff to a supra-systolic pressure of 60mmHg above the patient's systolic pressure or 200mmHg, whichever is higher and start the stopwatch again. Complete cessation of blood flow to the hand is verified by the absence of a PAT signal from the occluded arm. To confirm occlusion increase the gain on the screen of the channel of the occluded side to 20,000 while keeping the gain of the contra-lateral side constant. Decrease the time base of both channels to 30 seconds. Verify that you do not observe any signals at a periodicity that matches the signal from the control arm as this indicates an incomplete occlusion. If this is the case then further inflate the cuff until no signals are seen. The cuff may be inflated to a maximum of 300mmHg.</li>
 <li>This will initiate a five minute count down for the arterial occlusion recording period. Toward the end of the occlusion period tell the patient you are going to release the cuff and that they should continue to refrain from moving their fingers. After exactly five minutes, deflate the cuff abruptly as quickly as possible and stop the stopwatch by clicking the "Start/Stop Timer" icon.</li>
 <li>Click the "Start/Stop Timer" icon again to initiate a five-minute post occlusion recording period. Stop the timer after five minutes and click the "Test Stop" icon to complete the study. The probes will automatically deflate.</li>
 <li>Remove the probes, tape, and foam rings from the patient's fingers and disconnect the PAT probes from the pneumo-electrical tubing. Discard the used probes.</li>
</ol>
IV.Review and Analysis
<ol>
 <li>Load the study file to the screen using the load icon. To run the automatic analysis, click the "magician stick" icon. The occlusion period will be highlighted in blue and the test result will be displayed, including the Reactive Hyperemia Index (RHI) and Heart Rate (HR), in the right hand column of the screen.</li>
 <li>To review additional data, including study parameters, calculated variables, patient information, and measures of signal quality, click the "Open Results of Last Calculation" icon. This will open a spread sheet with study parameters and results for all analyses performed to date, with the last line in the table containing data from the most recent analysis.</li>
</ol>
V.Representative Results
A representative Endo-PAT screen of a study performed on an individual with normal endothelial vasodilator function is shown in Figure 1. A representative screen of an Endo-PAT study performed on an individual with endothelial vasodilator dysfunction is shown in Figure 2.
<img src="/files/ftp_upload/2167/2167fig1.jpg" alt="Figure 1" /> 
 Figure 1: Normal Endothelial Vasodilator Function. Representative recording of an individual with normal endothelial vasodilator function, characterized by an increase in the signal amplitude after cuff release relative to baseline.
<img src="/files/ftp_upload/2167/2167fig2.jpg" alt="Figure 2" /> 
 Figure 2: Endothelial Vasodilator Dysfunction. Representative recording of an individual with endothelial vasodilator dysfunction.

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The video development was supported by Itamar Medical Co. Dr Cooke is the inventor of patents owned by Stanford University for diagnostic and therapeutic applications of the NOS pathway from which he receives royalties.

The major cause of morbidity and mortality worldwide is atherosclerotic vascular disease, leading to stroke, myocardial infarction, heart failure, renal insufficiency, aneurysm rupture or embolism, intermittent claudication and gangrene 1,2. Endothelial dysfunction is one of the earliest events in the pathophysiological process leading to these atherosclerotic disorders 3. Furthermore endothelial dysfunction contributes to the progression of disease, by facilitating inflammation and thrombosis. 4-6. The traditional cardiovascular risk factors are associated with endothelial vasodilator dysfunction 7-11. Endothelial vasodilator dysfunction can be detected in seemingly healthy individuals that are at risk for developing cardiovascular disease 12. Furthermore, the finding of endothelial vasodilator dysfunction is predictive of major adverse cardiovascular events as well as mortality 13-16. The vasodilators released by the endothelium include vasodilator prostanoids such as prostacyclin, peptides such as adrenomedullin and atrial natriuretic peptide, and small molecules such as endothelium dependent hyperpolarizing factor, carbon monoxide and nitric oxide 17. In addition, the effect of hyperpolarizing currents generated in the endothelium can be transmitted to underlying vascular smooth muscle of smaller vessels, relaxing them 18. An indirect assessment of the generation of these endothelial vasodilator influences can be gained by studying vascular reactivity. The most prevalent method to assess endothelial regulation of vascular reactivity non-invasively has been duplex ultrasonography to detect flow-mediated vasodilation of the brachial artery 19. The tractive force of fluid flow stimulates the endothelium to release vasodilators, most prominently nitric oxide 20. This phenomenon can be observed by ultrasound in the brachial artery during increases in forearm blood flow induced by reactive hyperemia 19. This technology has been widely utilized to document the association of endothelial vasodilator dysfunction with cardiovascular risk factors; the relationship of endothelial vasodilator dysfunction to various biomarkers, such as C-reactive peptide, or asymmetric dimethylarginine (the endogenous antagonist of nitric oxide synthase); and the correction of endothelial vasodilator function with nutritional and lifestyle modifications as well as with the use of angiotensin converting enzyme inhibitors, angiotensin receptor blockers, statins, insulin sensitizing agents or aspirin 21-23. The assessment of flow-mediated vasodilation by brachial artery ultrasound requires expensive equipment, is highly operator-dependent, the response has a very small dynamic range and the signal-to-noise ratio is low. New approaches to address these problems with assessment of endothelial vasodilator function are needed 24. The Endo-PAT 2000 is a new approach to assess endothelial vasodilator function in a rapid non-invasive manner. The technique provides values for the calculation of a Reactive Hyperemia Index (RHI), which gives an indication of the endothelial vasodilator function. The RHI is the post-to-pre occlusion PAT signal ratio in the occluded arm, relative to the same ratio in the control arm, and corrected for baseline vascular tone. Studies using the EndoPAT have shown that the RHI score reflects NO-bioavailability 25. The RHI correlates with the measurement of endothelial vasodilator function in the coronary arteries 26 and with brachial FMD 27. Patients with a greater degree of cardiovascular disease exhibit a lower score 28 and values are also lower in other conditions associated with impaired endothelial function and risk of cardiovascular disease 29-33. Notably, RHI values appear to be predictive of cardiovascular outcomes 35. A low RHI (indicating endothelial dysfunction) can be reversed with treatment 36. In conclusion, we have demonstrated how to perform a reliable and reproducible test for endothelial vasodilator function with the Endo-PAT 2000. The test is noninvasive, easy to perform, and is a useful research tool. Its utility in clinical monitoring of endothelial function and in tailoring disease management is under investigation.

assessing endothelial vasodilator function with the endo-pat 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

Watch this Scientific Journal Video about Assessing Endothelial Vasodilator Function with the Endo-PAT 2000 at JoVE.com

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

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Medicine

43.3K ビュー.  Stanford University. 内皮機能を評価するための非侵襲的な手順は、遠藤- PAT 2000を使用して示されています。

ビデオ: Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT...

ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery in clinical research

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed...

assessing mitochondrial function in cerebral vascular endothelial cells

The day before the mitochondrial functional assessment assay, hydrate an extracellular flux sensor cartridge with 150 microliters of extracellular flux calibrant solution, and incubate the plate overnight at 37 degrees Celsius without carbon...

Assessing Mitochondrial Function in Cerebral Vascular Endothelial Cells

This video demonstrates the assessment of the mitochondrial functioning of cerebral vascular endothelial (CVE) cells using an extracellular flux assay. CVE cells are plated in an extracellular flux cell culture plate. Extracellular flux sensors are hydrated before loading and calibration into the bioanalyzer. The plate containing cells is then loaded for...

ultrasound assessment of endothelial function: a technical guideline of the flow-mediated dilation test

Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

quantitative mri of endothelial permeability and (dys)function in atherosclerosis

Quantitative MRI of Endothelial Permeability and Dysfunction in Atherosclerosis

We have developed an accurate, non-invasive, and easy-to-use method to quantify endothelial permeability and dysfunction in the arteries using Magnetic Resonance Imaging (MRI), named qMETRIC. This technique enables assessing vascular damage and cardiovascular risk associated with atherosclerosis in preclinical models and...

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis

non-invasive assessment of microvascular and endothelial function

Non-invasive Assessment of Microvascular and Endothelial Function

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial...

microfluidics in assessing platelet function

Author Spotlight: Microfluidic-Based Assessment of Platelet Function Under Physiologic Flow for Advancing Hemostatic Resuscitation and Transfusion Medicine

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics in Assessing Platelet Function

assessing murine resistance artery function using pressure myography

Assessing Murine Resistance Artery Function Using Pressure Myography

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure...

evaluation of bioenergetic function in cerebral vascular endothelial cells

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial...

assessing internalized porphyromonas gingivalis in host endothelial cells

In this video, we demonstrate an assay to assess the survival of Porphyromonas gingivalis inside human endothelial cells. The measure of survival and growth of internalized Porphyromonas gingivalis is evaluated by the number of colonies on the blood agar...

Assessing Internalized Porphyromonas gingivalis in Host Endothelial Cells

Next, scrape the lysed cells, and collect the lysate in a centrifuge tube. Dilute the lysate 1 to 1 with BHI medium, and then prepare serial dilutions starting at 1 to 100. Plate 200 microliters of the suitable concentrations on blood agar plates. Allow the bacteria to grow for seven days in an anaerobic chamber, and then count the...

assessing the osteogenic differentiation of 3d stromal-endothelial cells by monitoring the alkaline phosphatase activity

Assessing the Osteogenic Differentiation of 3D Stromal-Endothelial Cells by Monitoring the Alkaline Phosphatase Activity  

electric cell-substrate impedance sensing for the quantification of endothelial proliferation, barrier function, and motility

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are...

quantifying the mechanical properties of the endothelial glycocalyx with atomic force microscopy

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and...

assessing leukocyte-endothelial interactions under flow conditions in an ex vivo autoperfused microflow chamber assay

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

a versatile, behavioral method to investigate thyroid hormone effects on cerebellar function

Author Spotlight: Accurately Assessing Thyroid Hormone-Driven Motor Alterations in Mouse

Here we present a protocol for a versatile behavior test developed recently, the ladder beam test. This test has the advantage of detecting subtle cerebellar ataxia caused by a defect of thyroid hormone action in the central nervous system over the conventional behavior tests assessing motor performance.

Assessing Motor Performance in TH-Related Murine Models Using the Ladder Beam Test

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assessment of vascular function in patients with chronic kidney disease

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...

the knob supination task: a semi-automated method for assessing forelimb function in rats

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional...

assessing motor function in the thyroid hormone-related mouse model using the ladder beam test

Assessing Motor Function in the Thyroid Hormone-Related Mouse Model Using the Ladder Beam Test

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

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.

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 ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.
Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.
Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...