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 border="0" cellpadding="0" cellspacing="0" width="923">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">Vial of 20 mg acetylcholine chloride powder and 1 Ampoule of 2 mL diluent</td>
			<td style="width:151px;">Bausch &#38; Lomb&#160;</td>
			<td style="width:165px;">NDC 24 208-539-20</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 x 100 ml NaCl 0.9 %</td>
			<td>BBraun</td>
			<td>3200950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 x syringe 50 ml each</td>
			<td>BBraun</td>
			<td>4187903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;1 x 2 ml syringe</td>
			<td>BBraun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;1 x 10 ml syringe</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 x cannula 20 G 70mm</td>
			<td>BBraun</td>
			<td>4665791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;5 x 5 ml syringe</td>
			<td>BBraun</td>
			<td>4606051V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Contrast agent Imeron 350 with a 10ml syringe for contrast injection</td>
			<td>Bracco Imaging</td>
			<td>30699.03.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

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15.5K 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. ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake

Different methods can be used in research to assess dietary intake, many of which are still paper-based. Written estimated food diaries are often utilized in clinical trials, despite being a burden for both study participants and researchers. This method requires participant literacy, it is time consuming, labor intensive, and can easily lead to under-reporting. With advancements in technology, there is a growing interest in electronic diaries that automate the dietary assessment process. These are focused on improving accuracy, reducing both time and cost and providing users with a visual and more enjoyable experience. The methodology presented here aimed to validate the &#39;Boden Food Plate&#39;, a novel web-based platform for self-recording of food and drink items, compared to a traditional estimated food diary. The application was also rated on a satisfaction scale by study participants using a paper-based questionnaire. Sixty-seven participants completed the dietary measures on both the three-day electronic and paper food diaries. For the analysis, only dietary data completed at both study time points (baseline and week six) was utilized. Despite small mean differences between dietary data collection methods, Bland Altman analysis showed fairly wide 95% limits of agreement between the electronic platform and the written estimated food diary and there were few cases which did not fall within the 95% confidence intervals. Overall, participants found the electronic food diary to be more fun than the paper method and as easy to use as hard copy diaries. The new platform has potential as a self-recording tool for the collection of dietary data, particularly when utilized in clinical trial settings. However, further validation studies are needed to improve the validity of this novel electronic dietary data collection tool.

The &#39;Boden Food Plate&#39; is an electronic food diary designed to be an interactive and fun method to collect dietary intake using visual depictions. The purpose of this study was to validate the web-based application against a traditional three-day estimated food diary method.

Different methods can be used in research to assess dietary intake, many of which are still paper-based. Written estimated food diaries are often utilized ...

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.

Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

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 ...