This project is supported in part by grant KKF-15-1.

NOTE: Intracoronary acetylcholine testing has been approved by the local ethics committee and the protocol follows the guidelines of our institution for human research.
1. Preparation of the Acetylcholine Solutions (See Materials Table)
<ol>
	<li>Mix the 20 mg acetylcholine with the 2 ml solvent provided with the package.</li>
	<li>Add the 2 ml ACH solution to 98 ml of NaCl 0.9 %. This corresponds to a dose of 0.2 mg/ml and is called stock solution 1. Add 9 ml of stock solution 1 to 91 ml of NaCl 0.9%, This corresponds to a dose of 18 &#181;g/ml and is called stock solution 2.</li>
	<li>Prepare 3 perfusion syringes (each of 50 ml) labeled as &#34;high&#34;, &#34;medium&#34; and &#34;low&#34;. Fill the perfusion syringe 1 labeled &#34;high&#34; with 40 ml of stock solution 2.</li>
	<li>Fill the perfusion syringe 2 (labeled &#34;medium&#34;) with 8 ml of the perfusion syringe 1 (high) and add 32 ml of NaCl 0.9%. This corresponds to a dose of 3.6 &#181;g/ml.</li>
	<li>Fill the perfusion syringe 3 (labeled &#34;low&#34;) with 4 ml of the perfusion syringe 2 (medium) and add 36 ml of NaCl 0.9%. This corresponds to a dose of 0.36 &#181;g/ml.</li>
</ol>
2. Preparation of the Syringes for Intracoronary Injection of Acetylcholine
<ol>
	<li>Prepare 5 syringes for the intracoronary injection (4 syringes of 5 ml [#1, #2, #3, #4] and 1 syringe of 10 ml).</li>
	<li>Fill the 5 ml syringe #1 with 6 ml from the perfusion syringe labeled &#34;low&#34; (see step 1.5). This corresponds to a dose of approximately 2 &#181;g.</li>
	<li>Fill the 5 ml syringe #2 with 6 ml from the perfusion syringe labeled &#34;medium&#34;. This corresponds to a dose of approximately 20 &#181;g.</li>
	<li>Fill the 5 ml syringe #3 with 5.5 ml from the perfusion syringe labeled &#34;high&#34;. This corresponds to a dose of approximately 100 &#181;g.</li>
	<li>Fill the 10 ml syringe with 11 ml from the perfusion syringe labeled &#34;high&#34;. This corresponds to a dose of approximately 200 &#181;g.</li>
	<li>Fill the 5 ml syringe #4 with 4.5 ml from the perfusion syringe labeled &#34;high&#34;. This corresponds to a dose of approximately 80 &#181;g. Put this syringe aside and only use it for assessment of the right coronary artery.</li>
</ol>
3. Diagnostic Coronary Angiography
<ol>
	<li>Inject local anaesthesia either in proximity to the right radial artery (usually 2 ml of mepivacain) or in proximity to the right femoral artery (usually 15 ml of mepivacain).</li>
	<li>Confirm the success of local anesthesia is by pricking the anesthetized-skin with the needle and asking the patient if pain is still present.</li>
	<li>Puncture the artery according to the Seldinger technique13 with a cannula, then insert the wire through the cannula and remove it. Insert the sheath (usually 5F) over the wire. Perform coronary angiography under sterile conditions.</li>
	<li>Advance the wire through the sheath to the ascending aorta and place the diagnostic catheter above the aortic valve. Then remove the wire and connect the catheter with the contrast syringe. 
	NOTE: The contrast agent contains Iomeprol, Trometamol, hydrochloric acid and water.</li>
	<li>Place the diagnostic catheter for the left coronary artery in the left main stem by slightly pulling and turning the catheter. Confirm correct positioning of the catheter by injecting 2 ml of contrast agent.</li>
	<li>Perform coronary angiography according to the Judkins technique14 with manual injections of approximately 10 ml of contrast to visualize the coronary arteries in different views. 
	NOTE: Usually LAO 40&#176; and RAO 35&#176; are used for the right coronary artery and LAO 45&#176;/CRAN 25&#176;, RAO 30&#176;/CRAN 30&#176; and RAO 20&#176;/CAUD 30&#176; are used for the left coronary artery.</li>
	<li>Start acetylcholine testing after exclusion of any relevant epicardial stenosis (&#8805;50%) by visual assessment.</li>
</ol>
4. Intracoronary Injection of Acetylcholine
<ol>
	<li>Inject 6 ml of the 2 &#181;g syringe #1 into the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient&#39;s symptoms (e.g. chest pain, shortness of breath, dizziness). Perform coronary angiography of the left coronary artery (usually a RAO 20&#176;/CAUD 30&#176; projection is best projection) after injection of the 6 ml.</li>
	<li>Perform coronary angiography by injecting 10 ml of contrast manually via the contrast syringe into the catheter. The 12-lead-ECG should be recorded and printed after every dose of acetylcholine. A pause of 1 min should lie between every dose.</li>
	<li>Inject 6 ml of the 20 &#181;g syringe #2 into the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient&#39;s symptoms. Perform coronary angiography of the left coronary artery after injection of the 6 ml as mentioned above.</li>
	<li>Inject 5.5 ml of the 100 &#181;g syringe #3 into the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient&#39;s symptoms. Perform coronary angiography of the left coronary artery after injection of the 5.5 ml as mentioned above. 
	NOTE: Most patients report some symptoms, show ECG changes or epicardial vasoconstriction at this dose. Sometimes the speed of the manual injection needs to be slowed down. As mentioned below different protocols for ACH-testing are in use with a different speed of injection. A slower injection over a period of 3 min compared to the injection within 20 sec may also be feasible.</li>
	<li>If no spasm (i.e. &#62;90% vasoconstriction compared to the relaxed state of the vessel after 200 &#181;g nitroglyercine injection) occurs at the 100 &#181;g dose continue with the 200 &#181;g (10 ml syringe) ACH dose. Inject the 11 ml within 20 sec with continuous monitoring of the ECG and the patient&#39;s symptoms.</li>
	<li>Perform coronary angiography of the left coronary artery after injection of the 11 ml as mentioned above (section 3). 
	NOTE: Although the 200 &#181;g dose has been applied in many patients it should be mentioned that appropriate doses of intracoronary acetylcholine remain to be fully defined. Some authors suggest that 100 &#181;g should be the maximum dose for the left coronary artery. 
	NOTE: Often bradycardia occurs and the speed of the injection needs to be slowed down.</li>
	<li>Inject 80 &#181;g ACH (4.5 ml, syringe #4) into the right coronary artery if no abnormal findings are seen during testing of the left coronary artery. Inject this within 20 sec with continuous monitoring of the ECG and the patient&#39;s symptoms. Often bradycardia occurs and the speed of the injection needs to be slowed down to prevent prolonged bradycardia and/or asystole.</li>
	<li>Perform coronary angiography of the right coronary artery after injection of the 4.5 ml (usually a LAO 40&#176;/0&#176; projection is best) as mentioned above (section 3).</li>
	<li>Inject intracoronary nitroglycerine with a dose of 200 &#181;g into each tested artery after the test or when severe symptoms (i.e. severe chest pain or severe shortness of breath), ischemic ECG shifts or epicardial spasm occurs.</li>
	<li>Image the vessel as mentioned in step 4.2 after one minute to document reversion of spasm.</li>
</ol>

<ol>
	<li>Prinzmetal, M., Kennamer, R., Merliss, R., Wada, T., Bor, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angina+pectoris.+I.+A+variant+form+of+angina+pectoris;+preliminary+report.">Angina pectoris. I. A variant form of angina pectoris; preliminary report.</a> Am J Med. 27, 375-388 (1959).</li><li>Cheng, T. O., Bashour, T., Kelser, G. A., Weiss, L., Bacos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+angina+of+Prinzmetal+with+normal+coronary+arteriograms.+A+variant+of+the+variant.">Variant angina of Prinzmetal with normal coronary arteriograms. A variant of the variant.</a> Circulation. 47 (3), 476-485 (1973).</li><li>Yasue, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+coronary+artery+spasm+by+acetylcholine+in+patients+with+variant+angina:+possible+role+of+the+parasympathetic+nervous+system+in+the+pathogenesis+of+coronary+artery+spasm.">Induction of coronary artery spasm by acetylcholine in patients with variant angina: possible role of the parasympathetic nervous system in the pathogenesis of coronary artery spasm.</a> Circulation. 74 (5), 955-963 (1986).</li><li>Bell, M. R., Berger, P. B., Holmes, D. R., Mullany, C. J., Bailey, K. R., Gersh, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Referral+for+coronary+artery+revascularization+procedures+after+diagnostic+coronary+angiography:+evidence+for+gender+bias?.">Referral for coronary artery revascularization procedures after diagnostic coronary angiography: evidence for gender bias?.</a> J Am Coll Cardiol. 25 (7), 1650-1655 (1995).</li><li>Patel, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+diagnostic+yield+of+elective+coronary+angiography.">Low diagnostic yield of elective coronary angiography.</a> N Engl J Med. 362 (10), 885-895 (2010).</li><li>Pocock, S. J., Henderson, R. A., Seed, P., Treasure, T., Hampton, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+of+life,+employment+status,+and+anginal+symptoms+after+coronary+angioplasty+or+bypass+surgery.+3-year+follow-up+in+the+Randomized+Intervention+Treatment+of+Angina.">Quality of life, employment status, and anginal symptoms after coronary angioplasty or bypass surgery. 3-year follow-up in the Randomized Intervention Treatment of Angina.</a> Circulation. 94 (2), 135-142 (1996).</li><li>Ong, P., Athanasiadis, A., Borgulya, G., Mahrholdt, H., Kaski, J. C., Sechtem, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+a+pathological+response+to+acetylcholine+testing+in+patients+with+stable+angina+pectoris+and+unobstructed+coronary+arteries.+The+ACOVA+Study+(Abnormal+COronary+VAsomotion+in+patients+with+stable+angina+and+unobstructed+coronary+arteries).">High prevalence of a pathological response to acetylcholine testing in patients with stable angina pectoris and unobstructed coronary arteries. The ACOVA Study (Abnormal COronary VAsomotion in patients with stable angina and unobstructed coronary arteries).</a> J Am Coll Cardiol. 59 (7), 655-662 (2012).</li><li>Furchgott, R. F., Zawadzki, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+obligatory+role+of+endothelial+cells+in+the+relaxation+of+arterial+smooth+muscle+by+acetylcholine.">The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.</a> Nature. 288 (5789), 373-376 (1980).</li><li>Shimizu, H., Lee, J. D., Ogawa, K., Hara, A., Nakamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+artery+vasoreactivity+to+intracoronary+acetylcholine+infusion+test+in+patients+with+chest+pain+syndrome.">Coronary artery vasoreactivity to intracoronary acetylcholine infusion test in patients with chest pain syndrome.</a> Intern Med. 31 (1), 22-27 (1992).</li><li>Montalescot, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2013+ESC+guidelines+on+the+management+of+stable+coronary+artery+disease:+the+Task+Force+on+the+management+of+stable+coronary+artery+disease+of+the+European+Society+of+Cardiology.">2013 ESC guidelines on the management of stable coronary artery disease: the Task Force on the management of stable coronary artery disease of the European Society of Cardiology.</a> Eur Heart J. 34 (38), 2949-3003 (2013).</li><li>JCS Joint Working Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+diagnosis+and+treatment+of+patients+with+vasospastic+angina+(Coronary+Spastic+Angina)+(JCS+2013).">Guidelines for diagnosis and treatment of patients with vasospastic angina (Coronary Spastic Angina) (JCS 2013).</a> Circ J. 78 (11), 2779-2801 (2014).</li><li>Ong, P., Athanasiadis, A., Sechtem, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+coronary+vasomotor+responses+to+intracoronary+acetylcholine+provocation.">Patterns of coronary vasomotor responses to intracoronary acetylcholine provocation.</a> Heart. 99 (17), 1288-1295 (2013).</li><li>Seldinger, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheter+replacement+of+the+needle+in+percutaneous+arteriography;+a+new+technique.">Catheter replacement of the needle in percutaneous arteriography; a new technique.</a> Acta radiol. 39 (5), 368-376 (1953).</li><li>Judkins, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+coronary+arteriography.+I.+A+percutaneous+transfemoral+technic.">Selective coronary arteriography. I. A percutaneous transfemoral technic.</a> Radiology. 89 (5), 815-824 (1967).</li><li>Mohri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angina+pectoris+caused+by+coronary+microvascular+spasm.">Angina pectoris caused by coronary microvascular spasm.</a> Lancet. 351 (9110), 1165-1169 (1998).</li><li>Chandrasekar, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+cardiac+catheterization+in+the+current+era:+a+single-center+experience.">Complications of cardiac catheterization in the current era: a single-center experience.</a> Catheter Cardiovasc Interv. 52, 289-295 (2001).</li><li>Nakao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperventilation+as+a+specific+test+for+diagnosis+of+coronary+artery+spasm.">Hyperventilation as a specific test for diagnosis of coronary artery spasm.</a> Am J Cardiol. 80 (5), 545-549 (1997).</li></ol>

The authors have nothing to disclose.

It is feasible to implement the acetylcholine test in daily clinical routine in the catheterisation laboratory. Apart from the preparation of the ACH solutions there are several technical issues that have to be resolved before starting the test including radiolucent ECG leads for continuous 12-lead ECG registration. This is essential to be able to detect transient ischemic ECG changes during the test. Moreover, it is important to know that the ACH solutions can only be used for 2 hr. After that they should be newly prepa...

Angina pectoris is the hallmark of coronary artery disease and the concept of an epicardial stenosis causing myocardial ischemia and exercise-induced angina has been established for many years. However, many patients with angina pectoris do not have the typical triad of retrosternal pain, onset during exercise and relief by nitroglycerine or rest. Frequently patients report angina pectoris at rest or a combination of exertional and resting angina as well as shortness of breath upon exertion as a possible angina equivalent. In 1959 Prinzmetal was the first to introduce the concept of transient spasm of the coronary arteries causing angina at rest associated with ST-segment elevation on the electrocardiogram (ECG) but with preserved exercise capacity1. This hypothesis was confirmed later using coronary angiography and it appeared that coronary spasm can be present in patients with epicardial stenoses or normal coronary arteries2. In the 1980ies intracoronary acetylcholine provocation testing (ACH-test) for the detection of coronary artery spasm was established in Japan and subsequently the interested in clinical research for coronary spasm increased3.
However, after introduction of percutaneous coronary intervention in 1977 and the first stent implantation in 1986 the interest in coronary vasomotor disorders declined considerably at least in Europe and the United States. This may be also due to the invasive nature of the ACH-test (due to the short half-life of ACH it can only be administered into the coronary arteries for the assessment of coronary spasm). Still, many patients with signs and symptoms of myocardial ischemia do not have any relevant epicardial stenosis on coronary angiography4,5,6. In such patients intracoronary acetylcholine provocation testing is useful to detect a clinically relevant coronary vasomotor disorder and institute appropriate medical therapy7.
Acetylcholine is a neurotransmitter in the parasympathetic nervous system. It acts via nicotinergic as well as muscarinergic (mAChR) receptors. The latter are important for vascular homeostasis and acetylcholine binds on mAChR as a non-selective agonist. Activation of these receptors at the endothelial level leads to nitric oxide mediated vasodilatation whereas activation of mAChR on the vascular smooth muscle cells leads to vasoconstriction8. Depending on the integrity of the endothelium and the reactivity of the smooth muscle cells the net effect of intracoronary acetylcholine administration is vasodilatation or vasoconstriction. The physiologic response of coronary arteries in response to acetylcholine in human beings is not fully understood but it has been reported that vasodilatation as well as vasoconstriction of up to 25% of the vessel diameter may be physiologic as shown in patients with normal coronary arteries and no angina pectoris9.
Intracoronary acetylcholine provocation testing has been recommended by the European Society of Cardiology guidelines10 as well as the Japanese Circulation Society guidelines11 for assessment of epicardial and/or microvascular spasm in patients with angina pectoris and unobstructed coronary arteries. Intracoronary acetylcholine provocation testing has been established in daily clinical routine in the catheterization laboratory of our institution in 2003. Since 2006 a standardized protocol has been followed12. ACH-testing is generally performed in all patients with signs and symptoms of myocardial ischemia yet no relevant epicardial stenosis (&#60;50%) on coronary angiography. ACH-testing is performed immediately after diagnostic coronary angiography according to the protocol described below. Ergonovine is another agent that is used for spasm provocation testing with a different mechanism of action. Detailed information on ergonovine testing can be found elsewhere12.

<table><tbody><tr><td>Vial of 20 mg acetylcholine chloride powder and 1 Ampoule of 2 ml diluent</td><td>Bausch &amp;amp; Lomb&amp;nbsp;</td><td>NDC 24 208-539-20</td><td></td></tr><tr><td>3x 100 ml NaCl 0.9 %</td><td>BBraun</td><td>3200950</td><td></td></tr><tr><td>3x syringe 50 ml each</td><td>BBraun</td><td>4187903</td><td></td></tr><tr><td>1x 2 ml syringe</td><td>BBraun</td><td>4606027V</td><td></td></tr><tr><td>1x 10 ml syringe</td><td>BBraun</td><td>4606108V</td><td></td></tr><tr><td>2x cannula 20 G 70 mm</td><td>BBraun</td><td>4665791</td><td></td></tr><tr><td>5x 5 ml syringe</td><td>BBraun</td><td>4606051V</td><td></td></tr><tr><td>Contrast agent Imeron 350 with a 10 ml syringe for contrast injection</td><td>Bracco Imaging</td><td>30699.03.00</td><td></td></tr><tr><td>Coronary angiography suite (AXIOM Artis MP eco )</td><td>Siemens</td><td>n/a</td><td></td></tr></tbody></table>

intracoronary acetylcholine provocation testing for assessment of coronary vasomotor disorders

Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the catheterization laboratory which was introduced more than 30 years ago. Due to the short half-life of acetylcholine it can only be applied directly into the coronary arteries. Several studies have demonstrated the safety and clinical usefulness of this test. However, acetylcholine testing is only rarely applied in the U.S. or Europe. Nevertheless, it has been shown that 62% of Caucasian patients with stable angina and unobstructed coronary arteries on coronary angiography suffer from coronary vasomotor disorders that can be diagnosed with acetylcholine testing. In recent years it has been appreciated that the ACH-test not only assesses the presence of epicardial spasm but that it can also be useful for the detection of coronary microvascular spam. In such cases no epicardial spasm is seen after injection of acetylcholine but ischemic ECG shifts are present together with a reproduction of the patient's symptoms during the test. This article describes the experience with the ACH-test and its implementation in daily clinical routine.

Interpretation of the acetylcholine test is based on three criteria. First, the patient is asked throughout the test whether or not symptoms occur. Frequently, patients report a reproduction of their usual symptoms such as chest pain, shortness of breath or other symptoms. This represents an important point for the overall interpretation of the test. Second, a 12-lead-ECG registration is continuously performed throughout the test with a special emphasis on ischemic ECG shifts such as ST-s...

Watch this Scientific Journal Video about Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders at JoVE.com

Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders

Intracoronary acetylcholine testing has been established for the assessment of epicardial coronary spasm more than 30 years ago. Recently, the focus has shifted towards the microcirculation and it has been shown that microvascular spasm can be detected using ACH-testing. This article describes the ACH-test and its implementation in daily routine.

Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the ...

intracoronary-acetylcholine-provocation-testing-for-assessment

Research

JoVE Journal

Medicine

15.6K Views.  Robert-Bosch-Krankenhaus. Intracoronary acetylcholine testing has been established for the assessment of epicardial coronary spasm more than 30 years ago. Recently, the focus has shifted towards the microcirculation and it has been shown that microvascular spasm can be detected using ACH-testing. This article describes the ACH-test and its implementation in daily routine.

Video: Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)

Improved methods for assessing bulbar impairment are necessary for expediting diagnosis of bulbar dysfunction in ALS, for predicting disease progression across speech subsystems, and for addressing the critical need for sensitive outcome measures for ongoing experimental treatment trials. To address this need, we are obtaining longitudinal profiles of bulbar impairment in 100 individuals based on a comprehensive instrumentation-based assessment that yield objective measures. Using instrumental approaches to quantify speech-related behaviors is very important in a field that has primarily relied on subjective, auditory-perceptual forms of speech assessment1. Our assessment protocol measures performance across all of the speech subsystems, which include respiratory, phonatory (laryngeal), resonatory (velopharyngeal), and articulatory. The articulatory subsystem is divided into the facial components (jaw and lip), and the tongue. Prior research has suggested that each speech subsystem responds differently to neurological diseases such as ALS. The current protocol is designed to test the performance of each speech subsystem as independently from other subsystems as possible. The speech subsystems are evaluated in the context of more global changes to speech performance. These speech system level variables include speaking rate and intelligibility of speech.
The protocol requires specialized instrumentation, and commercial and custom software. The respiratory, phonatory, and resonatory subsystems are evaluated using pressure-flow (aerodynamic) and acoustic methods. The articulatory subsystem is assessed using 3D motion tracking techniques. The objective measures that are used to quantify bulbar impairment have been well established in the speech literature and show sensitivity to changes in bulbar function with disease progression. The result of the assessment is a comprehensive, across-subsystem performance profile for each participant. The profile, when compared to the same measures obtained from healthy controls, is used for diagnostic purposes. Currently, we are testing the sensitivity and specificity of these measures for diagnosis of ALS and for predicting the rate of disease progression. In the long term, the more refined endophenotype of bulbar ALS derived from this work is expected to strengthen future efforts to identify the genetic loci of ALS and improve diagnostic and treatment specificity of the disease as a whole. The objective assessment that is demonstrated in this video may be used to assess a broad range of speech motor impairments, including those related to stroke, traumatic brain injury, multiple sclerosis, and Parkinson disease.

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis ALS

Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations.

Improved methods for assessing bulbar impairment are necessary for expediting diagnosis of bulbar dysfunction in ALS, for predicting disease progression ...

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Quantification of Absolute Myocardial Flow and Resistance by Continuous Thermodilution in Patients with Ischemia and Nonobstructive Coronary Artery Disease (INOCA)

In approximately half of the patients undergoing coronary angiography for angina pectoris or for signs or symptoms suggestive of ischemic heart disease, no obstructive coronary artery disease is angiographically visible. The majority of these patients with angina or ischemia and no obstructive coronary artery disease (INOCA) have an underlying coronary vasomotor dysfunction, and current consensus documents recommend diagnostic invasive coronary vasomotor function testing (CFT). 
During CFT, a variety of vasomotor dysfunction endotypes can be assessed, including vasospastic coronary dysfunction (epicardial or microvascular vasospasm), and/or microvascular vasodilatory dysfunction, including impaired vasodilatory capacity and increased microvascular resistance. The quantification of the continuous thermodilution derived absolute coronary blood flow and resistance might be a better measure compared to the currently used standard physiologic measures. This article provides an overview of this continuous thermodilution method.

Quantification of Absolute Myocardial Flow and Resistance by Continuous Thermodilution in Patients with Ischemia and Nonobstructive Coronary Artery Disease INOCA

Presented here is a protocol to measure absolute myocardial flow and resistance using continuous thermodilution in patients with ischemia and nonobstructive coronary artery disease.

In approximately half of the patients undergoing coronary angiography for angina pectoris or for signs or symptoms suggestive of ischemic heart disease, ...

Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary vasomotor disorders (impaired vasodilatation and/or enhanced vasoconstriction/spasm) represent important functional causes for such a clinical presentation. Although impaired vasodilatation may be assessed with non-invasive techniques such as positron emission tomography or cardiac magnetic resonance imaging, there is currently no reliable non-invasive technique for the diagnosis of coronary spasm available. Thus, invasive diagnostic procedures (IDP) have been developed for the diagnosis of coronary vasomotor disorders including spasm testing as well as assessment of coronary vasodilatation. The identification of the underlying type of disorder (so called endotype) allows the initiation of targeted pharmacological treatments. Despite the fact that such an approach is recommended by the current European Society of Cardiology guidelines for the management of chronic coronary syndromes based on the CorMicA study, comparability of results as well as multicenter trials are currently hampered by major differences in institutional protocols for coronary functional testing. This article describes a comprehensive IDP protocol including intracoronary acetylcholine provocation testing for diagnosis of epicardial/microvascular spasm, followed by Doppler wire-based assessment of coronary flow reserve (CFR) and hyperemic microvascular resistance (HMR) in search of coronary vasodilatory impairment.

Coronary vasomotion disorders represent frequent functional causes of angina in patients with unobstructed coronaries. The underlying mechanism of angina (endotype) in these patients can be determined by a comprehensive invasive diagnostic procedure based on acetylcholine provocation testing followed by Doppler-derived assessment of the coronary flow reserve and microvascular resistance.

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary ...

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 ...

Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) using acetylcholine (ACh) spasm provocation testing. CAS can further be divided into different subtypes (e.g., focal, diffuse epicardial, or microvascular spasm), each with different pathophysiological mechanisms that may require tailored drug treatment. The evidence behind the role of nitrates in the setting of each CAS subtype is lacking, and the effectivity can vary on a per-patient basis. In order to assess on a per-patient level whether nitroglycerine (NTG) can prevent inducible spasm, the vasospastic ACh dose can be readministered after NTG administration as part of the spasm provocation test. The preventive effect of NTG is assessed by evaluating improvements in the severity of induced symptoms, ischemic ECG changes, and by reassessing the site and mode of spasm on angiography. This technique can therefore be used to assess the nitrate responsiveness on a per-patient level and unmask co-existing microvascular spasm in patients with epicardial spasm that is prevented with NTG. The NTG rechallenge, therefore, allows to further guide targeted therapy for CAS and provide new insights into the pathophysiological mechanism behind vasospastic disorders.

This protocol presents the acetylcholine rechallenge after nitroglycerine as an add-on procedure to spasm provocation testing. The purpose of this technique is to unmask co-existing microvascular spasm&#160;in patients with epicardial spasm&#160;and to assess the protective efficacy of nitroglycerine on a per-patient level to guide medical therapy.

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina attack. Since the pressure-rate product is almost identical at rest and the onset of attack in those patients, the decrease in coronary blood flow rather than increased myocardial oxygen consumption is likely to explain myocardial ischemia, indicating a substantial involvement with coronary microvascular spasm (MVS). Myocardial lactate production, which could be defined as a negative myocardial lactate extraction ratio (ratio of the coronary arterial-venous difference in lactate concentration to arterial concentration), is considered indicative of objective evidence to support the emerging myocardial ischemia. Thus, monitoring of the myocardial lactate production and the emergence of chest pain and ischemic electrocardiographic changes during acetylcholine (ACh) provocation testing is of significant value for detecting the entity of MVS. Practically, 1 min after incremental doses of ACh (20, 50, and 100 &#956;g) are administered into the left coronary artery (LCA), paired samples of 1 mL of blood are collected from the LCA ostium and coronary sinus for measurement of lactate concentration by a calibrated automatic lactate analyzer. Then, the development of MVS could be confirmed by negative myocardial lactate extraction ratio despite the absence of angiographically demonstrable epicardial coronary spasm or before its occurrence throughout ACh provocation testing. In conclusion, assessment of myocardial lactate production is essential and valuable for the diagnosis of MVS.

Myocardial lactate production (coronary arterial-venous difference in serum lactate level) during coronary spasm provocation testing is considered as a highly sensitive marker that reflects acetylcholine-induced myocardial ischemia due to microvascular spasm. This article presents the procedures to assess myocardial lactate production for the diagnosis of coronary microvascular spasm.

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, myocardial infarction, and angina pectoris, which seriously threaten the life and health of people all over the world. However, how to record the dynamic biomechanical characteristics of isolated blood vessels has long puzzled people. Meanwhile, precise positioning and isolation of coronary arteries to measure in vitro dynamic vascular tension changes have become a trend in CAD drug development. The present protocol describes the macroscopic identification and microscopic separation of rat coronary arteries. The contraction and dilation function of the coronary artery ring along the vessel diameter was monitored using the established multi myograph system. The standardized and programmed protocols of coronary ring tension measurement, from sampling to data acquisition, tremendously improve the repeatability of the experimental data, which ensures the authenticity of vascular tension records after physiological, pathological, and drug intervention.

The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, ...

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.