Intracoronary ACh testing has been approved by the local ethics committee of the Academic Medical Centre, and the protocol follows the guidelines of Amsterdam UMC for human research.
1. Preparation of the ACh stock solution
<ol>
	<li>Mix the 20 mg of ACh with the 2 mL of solvent provided with the package (Table of Materials).</li>
	<li>Add 1 mL of the ACh solution to 499 mL of 0.9% NaCl to create the stock solution, corresponding to a 20 &#181;g/mL dose.</li>
	<li>Fill a sterile dish with 50 mL of the stock solution, which will be used to prepare the syringes under sterile conditions.</li>
</ol>
2. Preparation of the syringes containing ACh for intracoronary injection
<ol>
	<li>Prepare five 10 mL syringes under sterile conditions for the intracoronary injection according to the Supplementary Material. 
	&#8203;NOTE: It is advised to prepare each syringe sequentially after administration and not all at once to avoid confusion when administering the syringes</li>
</ol>
3. Diagnostic coronary angiography
<ol>
	<li>Anesthetize the puncture site of the right radial artery (usually 2 mL of lidocaine) or the area of the puncture site of the right femoral artery (usually 15 mL of lidocaine) when the femoral artery is the preferred route of catheterization. 
	NOTE: By puncturing the skin with the needle, the successfulness of the local anesthesia can be assessed.</li>
	<li>Puncture the artery with a cannula using the Seldinger wire technique, then insert a wire through the cannula. After removing the cannula, insert a sheath (6F) over the wire. Perform coronary angiography under sterile conditions.</li>
	<li>Advance the wire into the ascending aorta through the sheath and subsequently position the diagnostic catheter (6Fr) above the aortic valve. Withdraw the wire and attach the contrast syringe to the catheter. 
	NOTE: The contrast agent used is iodixanol Injectable Contrast Medium.</li>
	<li>Position the tip of the diagnostic catheter for the left coronary artery in the left main coronary artery. Confirm the position of the catheter by injecting 2 mL of the contrast agent.</li>
	<li>Exclude the presence of obstructive coronary artery disease by perming coronary angiography. Inject ~5-10 mL of the contrast agent to visualize the coronary arteries in different views. 
	&#8203;NOTE: Most often, the following projections provide the best overview: LAO 40&#176; and RAO 35&#176; 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; for the left coronary artery.</li>
</ol>
4. Preparations for Doppler flow assessment
NOTE: Here, ComboWire was used as the Doppler guidewire with the ComboMap system (Table of Materials)
<ol>
	<li>Connect the Doppler guidewire to the system and zero the pressure on the console and the aortic pressure on the hemodynamic system.</li>
	<li>Advance the Doppler guidewire through the guiding catheter into the ostium of the coronary artery with the pressure sensor of the Doppler guidewire placed just distally of the tip of the guiding catheter. Press normalization (Norm) on the system to equalize the two pressures.</li>
	<li>After normalization, reintroduce the wire via the microcatheter into the proximal or mid LAD and acquire a stable flow signal. 
	NOTE: The Doppler guidewire can alternatively be introduced more distally into the LAD or in the CX when acquiring an adequate signal in the proximal LAD is not feasible, such as in the case of tortuous anatomy or when another position is otherwise required, for instance, proximal from a myocardial bridge.</li>
	<li>Document the position of the wire using fluoroscopy.</li>
	<li>Start ACh testing as described in section 5 after exclusion of any relevant epicardial stenosis (&#8805;50%) by visual assessment.</li>
</ol>
5. Intracoronary administration of ACh
<ol>
	<li>Administer the first syringe containing 2 &#181;g of acetylcholine as described in the Supplementary Material into the LCA through the guiding. Inject this in 60 s with continuous monitoring of the ECG, average peak velocity (APV), and the patient&#39;s symptoms (e.g., angina and/or dyspnea).
	<ol>
		<li>Flush the ACh that remains in the guiding into the left coronary artery at a similar injection speed to the injection itself with contrast using the contrast syringe.</li>
		<li>Perform coronary angiography of the left coronary artery by injecting 10 mL of the contrast agent into the catheter in the same projection as done for the baseline coronary angiography. Bookmark and print a 12-lead ECG after every dose of ACh or when symptoms and/or ECG changes occur.</li>
		<li>If diagnostic criteria are not met, continue with the administration of the second syringe containing 20 &#181;g of acetylcholine as described in the Supplementary Material using the same technique as described in steps 5.1.1-5.1.3. Pause for 3 min between every dose.</li>
		<li>If diagnostic criteria are not met after the second dose, continue with the administration of the third syringe containing 100 &#181;g of acetylcholine as described in the Supplementary Material using the same technique as described in steps 5.1.1-5.1.3. Pause for 3 min between every dose. 
		NOTE: At dose 3 most patients will report some symptoms, ischemic ECG changes can occur, and some epicardial diameter reduction may occur. In some cases, the speed of the manual injection will have to be slowed down. For instance, at doses 3 and 4, bradycardia may occur, and the speed of the injection will have to be slowed down. An extended injection over 1-3 min may also be feasible.</li>
		<li>If diagnostic criteria are not met after the third dose, continue with the administration of the fourth syringe containing 100 &#181;g of acetylcholine as described in the Supplementary Material using the same technique as described in steps 5.1.1-5.1.3. Pause for 3 min between every dose. 
		NOTE: At dose 4 most patients will report some symptoms, ischemic ECG changes can occur, and some epicardial diameter reduction may occur. In some cases, the speed of the manual injection will have to be slowed down. For instance, at doses 3 and 4, bradycardia may occur, and the speed of the injection will have to be slowed down. An extended injection over 1-3 min may also be feasible.</li>
	</ol>
	</li>
	<li>When diagnostic criteria according to COVADIS are not met after step 5.1.6 in the LCA, continue with RCA as the target vessel. Inject 80 &#181;g of ACH (10 mL, syringe #RCA) in 60 s into the right coronary artery while continuously monitoring the ECG and the patient&#39;s symptoms. 
	NOTE: Clinical practice on testing of the RCA can vary among centers as routine testing of the RCA is advocated according to the JCS guidelines as multi-vessel epicardial coronary vasospasm is associated with worse prognosis2,15. In contrast, vasospasm provocation protocols from European and United states centers do not routinely test the RCA16. Acetylcholine can induce bradycardia and can be resolved by decreasing the speed of the injection to prevent prolonged bradycardia and/or asystole.
	<ol>
		<li>Flush the remaining ACh in the guiding into the left coronary artery at a similar injection speed to the injection itself with contrast using the contrast syringe. Perform coronary angiography of the RCA (perform angiography in the same projection as the baseline angiography).</li>
		<li>Inject 200 &#181;g of intracoronary NTG into each target vessel after the test, or when severe symptoms (e.g., angina and/or dyspnoea)), ischemic ECG changes or epicardial spasm occurs. Continuously monitor the APV and patient&#39;s symptoms to monitor the effect of NTG.</li>
		<li>Perform coronary angiography of the target vessel after 1 min or when the APV has returned to baseline, and no symptoms are present to document reversion of spasm</li>
	</ol>
	</li>
</ol>
6. Rechallenge
<ol>
	<li>In the case of a positive diagnosis, wait for 3 min after NTG administration until the patient is free of symptoms, ECG changes have resolved, and APV values have returned to baseline before starting the rechallenge.</li>
	<li>Inject 10 mL of the syringe containing the acetylcholine dose that previously induced vasospasm as described in the Supplementary Material into guiding. Inject this in 60 s with continuous monitoring of the ECG, APV and the patient&#39;s symptoms (e.g., angina and/or dyspnea).</li>
	<li>Flush the remaining ACh in the guiding into the left coronary artery at a similar injection speed to the injection itself with contrast using the contrast syringe. Image the target vessel in the same projection as done in the baseline angiography after injection of the 10 mL.</li>
	<li>Perform coronary angiography of the left coronary artery by injecting 10 mL of contrast into the catheter in the same projection as done for the baseline coronary angiography. Bookmark and print a 12-lead ECG after every dose of ACh or when symptoms and/or ECG changes occur.</li>
	<li>Inject 200 &#181;g of intracoronary NTG into each target vessel after the test or when severe symptoms (i.e., angina and/or dyspnea), ischemic ECG shifts, or epicardial spasm occurs.</li>
	<li>Perform coronary angiography of the target vessel after 1 min or when the APV has returned to baseline, and no symptoms are present to document reversion of spasm</li>
</ol>

<ol>
	<li>Knuuti, J., 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=2019+ESC+Guidelines+for+the+diagnosis+and+management+of+chronic+coronary+syndromes.">2019 ESC Guidelines for the diagnosis and management of chronic coronary syndromes.</a> European Heart Journal. 41 (3), 407-477 (2020).</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> Circulation Journal: Official Journal of the Japanese Circulation Society. 78 (11), 2779-2801 (2014).</li><li>Ong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+standardization+of+diagnostic+criteria+for+microvascular+angina.">International standardization of diagnostic criteria for microvascular angina.</a> International Journal of Cardiology. 250, 16-20 (2018).</li><li>Beltrame, J. F., 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=International+standardization+of+diagnostic+criteria+for+vasospastic+angina.">International standardization of diagnostic criteria for vasospastic angina.</a> European Heart Journal. 38 (33), 2565-2568 (2017).</li><li>Ford, T. J., 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=1-Year+Outcomes+of+angina+management+guided+by+invasive+coronary+function+testing+(CorMicA).">1-Year Outcomes of angina management guided by invasive coronary function testing (CorMicA).</a> JACC: Cardiovascular Interventions. 13 (1), 33-45 (2020).</li><li>Bairey Merz, C. N., Pepine, C. J., Walsh, M. N., Fleg, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia+and+no+obstructive+coronary+artery+disease+(INOCA):+Developing+evidence-based+therapies+and+research+agenda+for+the+next+decade.">Ischemia and no obstructive coronary artery disease (INOCA): Developing evidence-based therapies and research agenda for the next decade.</a> Circulation. 135 (11), 1075-1092 (2017).</li><li>Sun, H., Fukumoto, Y., Ito, A., Shimokawa, H., Sunagawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+microvascular+dysfunction+in+patients+with+microvascular+angina:+analysis+by+TIMI+frame+count.">Coronary microvascular dysfunction in patients with microvascular angina: analysis by TIMI frame count.</a> Journal of Cardiovascular Pharmacology. 46 (5), 622-626 (2005).</li><li>Kiyooka, T., Kobayashi, Y., Ikari, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+of+vasospastic+angina+in+which+the+ergonovine+provocation+test+with+intracoronary+isosorbide+dinitrate+and+nicorandil+was+effective+in+the+diagnosis+of+microvascular+spasm.">A case of vasospastic angina in which the ergonovine provocation test with intracoronary isosorbide dinitrate and nicorandil was effective in the diagnosis of microvascular spasm.</a> Cardiovascular Intervention and Therapeutics. 29 (4), 344-349 (2014).</li><li>Takahashi, J., 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=Prognostic+impact+of+chronic+nitrate+therapy+in+patients+with+vasospastic+angina:+multicentre+registry+study+of+the+Japanese+coronary+spasm+association.">Prognostic impact of chronic nitrate therapy in patients with vasospastic angina: multicentre registry study of the Japanese coronary spasm association.</a> European Heart Journal. 36 (4), 228-237 (2015).</li><li>Ferrari, 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=Expert+consensus+document:+A+'diamond'+approach+to+personalized+treatment+of+angina.">Expert consensus document: A 'diamond' approach to personalized treatment of angina.</a> Nature Reviews Cardiology. 15 (2), 120-132 (2018).</li><li>Redfield, M. 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=Isosorbide+mononitrate+in+heart+failure+with+preserved+ejection+fraction.">Isosorbide mononitrate in heart failure with preserved ejection fraction.</a> The New England Journal of Medicine. 373 (24), 2314-2324 (2015).</li><li>Seitz, A., 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=Acetylcholine+rechallenge:+A+first+step+towards+tailored+treatment+in+patients+with+coronary+artery+spasm.">Acetylcholine rechallenge: A first step towards tailored treatment in patients with coronary artery spasm.</a> Journal of the American College of Cardiology: Cardiovascular Interventions. 15 (1), 65-75 (2022).</li><li>Crea, F., Lanza, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+microvascular+angina:+The+need+for+precision+medicine.">Treatment of microvascular angina: The need for precision medicine.</a> European Heart Journal. 37 (19), 1514-1516 (2016).</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=Intracoronary+acetylcholine+provocation+testing+for+assessment+of+coronary+vasomotor+disorders.">Intracoronary acetylcholine provocation testing for assessment of coronary vasomotor disorders.</a> Journal of Visualized Experiments: JoVE. (114), e54295 (2016).</li><li>Han, S. 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=Impact+of+multi-vessel+vasospastic+angina+on+cardiovascular+outcome.">Impact of multi-vessel vasospastic angina on cardiovascular outcome.</a> Atherosclerosis. 281, 107-113 (2019).</li><li>Feenstra, R. G. T., 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=Principles+and+pitfalls+in+coronary+vasomotor+function+testing.">Principles and pitfalls in coronary vasomotor function testing.</a> EuroIntervention: Journal of EuroPCR in Collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology. , (2021).</li><li>Seitz, A., Beck, S., Pereyra, V. M., Bekeredjian, R., Sechtem, U., Ong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+acetylcholine+followed+by+adenosine+for+invasive+diagnosis+of+coronary+vasomotor+disorders.">Testing acetylcholine followed by adenosine for invasive diagnosis of coronary vasomotor disorders.</a> Journal of Visualized Experiments: JoVE. (168), e62134 (2021).</li></ol>

The authors have nothing to disclose.

The usefulness of the ACh after NTG rechallenge has shown to be two-fold: (1) to unmask the co-existence of microvascular spasm in patients with epicardial spasm and (2) to assess the preventive efficacy of NTG on a per-patient level in order to guide medical therapy12. Regardless of the result of the spasm provocation test, intracoronary NTG is always routinely administered into the target vessel after the test or when severe symptoms, ischemic ECG changes, or epicardial spasm&#160;occur. Adding ...

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina and non-obstructive coronary artery disease (ANOCA) by means of spasm provocation testing with acetylcholine(ACh)1,2,3,4.The recent CorMicA trial demonstrated that identification and concurrent tailored treatment of CAS persistently improve the patient&#39;s quality of life and reduce the burden of angina5. Usually, once CAS is diagnosed, it is regarded as one distinct disease and treated with anti-vasospastic medication, such as calcium antagonists and nitrates1. Although, CAS can be divided into different subtypes with different pathophysiological mechanisms that may require tailored drug treatment6. CAS can occur on an epicardial level, either focally or diffusely, throughout the epicardial coronary arteries or on a microvascular level. The former is defined as vasospastic angina (VSA) and the latter as microvascular angina (MVA) due to microvascular spasm according to the COronary VAsomotor DISorders study group (COVADIS)3,4. Moreover, combinations of endotypes of CAS may co-exist that can further complicate tailored treatment. This is especially important as the occurrence of microvascular spasm may be masked during spasm provocation when a simultaneous epicardial spasm occurs.
As a consequence, treatment of these patients in clinical practice can be cumbersome and initiates a period of trial and error based treatment with various anti-vasospastic or anti-anginal medications. Nitrates, in particular, are often initiated as first-line treatment in short-acting form as rescue medication for acute anginal attacks or in the long-acting form as maintenance therapy. 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. Especially in the case of microvascular or diffuse distal epicardial spasm, the effect of NTG is controversial7,8.&#160;Furthermore, the therapeutic efficacy of chronic NTG treatment has to be weighed against potential side effects, such as severe headaches and a worsened exercise capacity9,10,11.
Recently Seitz et al. demonstrated the clinical usefulness of the ACh rechallenge technique after NTG administration as an add-on procedure to the spasm provocation test12. This is performed after a positive spasm provocation test by readministering the vasospastic dose of ACh in a similar fashion as the vasospastic dose itself 3 min after NTG administration. To this end, the COVADIS criteria are revisited in order to evaluate the preventive effect of NTG, e.g., improvement in symptoms, ischemic ECG changes, and reassessment of the site and mode of spasm by angiography3,4. Moreover, prevention of epicardial spasm&#160;during rechallenge can unmask the co-existence of microvascular spasm.
The purpose of the rechallenge after NTG, therefore, is two-fold: (1) to assess the preventive effect of NTG on the re-occurrence of spasm on a per patients level in order to improve clinical outcomes and tailor treatment immediately after diagnosis that is made during spasm provocation and (2) to assess the co-existence of microvascular spasm in patients with epicardial coronary artery spasm10,13.
A previous publication by Ong et al. extensively covered the spasm provocation test14. In our institute, we use a variation of this protocol where the ACh dosages are administered in 60 s instead of 20 s. The purpose of this paper is to describe the NTG rechallenge as an add-on procedure to the ACh spasm provocation test. This technique can be performed with each type of protocol, as demonstrated by Seitz et al. since the results of the NTG rechallenge did not differ among the participating centers that used different protocols.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cannula (various manufacturers)</td>
			<td>BBraun</td>
			<td>4206096</td>
			<td></td>
		</tr>
		<tr>
			<td>ComboMap system</td>
			<td>Volcano-Philips</td>
			<td>Model No. 6800 (Powers Up)</td>
			<td></td>
		</tr>
		<tr>
			<td>ComboWire XT Guide Wire</td>
			<td>Volcano-Philips</td>
			<td>9515</td>
			<td>Doppler guidewire</td>
		</tr>
		<tr>
			<td>Diagnostic catheter</td>
			<td>Boston scientific</td>
			<td>34356-661</td>
			<td>H749343566610/ MODEL-6F MACH 1 JL3.5</td>
		</tr>
		<tr>
			<td>Diagnostic catheter</td>
			<td>Boston scientific</td>
			<td>34356-686</td>
			<td>H749343566860/MODEL - 6F MACH 1 JR4</td>
		</tr>
		<tr>
			<td>FINECROSS MG Coronary Micro-Guide Catheter</td>
			<td>Terumo</td>
			<td>NC-F863A</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracoronary NTG</td>
			<td>hameln pharma gmbh</td>
			<td>RVG 119982</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine HCL</td>
			<td>Fresenius Kabi</td>
			<td>RVG 51673</td>
			<td></td>
		</tr>
		<tr>
			<td>Miochol-E Acetylcholine chloride</td>
			<td>Bausch &#38; Lomb</td>
			<td>NDC 240208-539-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheath Radialis</td>
			<td>Teleflex</td>
			<td>AA15611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe- 10 mL</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Visipaque</td>
			<td>GE Healthcare</td>
			<td>RVG 17665</td>
			<td>Iodixanol injectable contrast medium</td>
		</tr>
	</tbody>
</table>

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.

Interpretation of the ACh-test and rechallenge are based on criteria defined by the COVADIS study group4. A positive diagnosis for CAS is defined as (i) reproduction of the previously reported symptoms such as chest pain, shortness of breath, or other symptoms and (ii) the induction of ischemic ECG changes (ST-segment elevation or depression, or U-waves) in reaction to ACh. (Figure 2). It is therefore important to register a 12-lead-ECG continuously throughout the tes...

Watch this Scientific Journal Video about Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration at JoVE.com

Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration

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) ...

acetylcholine-re-challenge-after-intracoronary-nitroglycerine

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1.2K Views.  Amsterdam UMC. 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 in patients with epicardial spasm and to assess the protective efficacy of nitroglycerine on a per-patient level to guide medical therapy.

Video: Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

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.

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 ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

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 ...

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

Measurement of Tumor T2* Relaxation Times after Iron Oxide Nanoparticle Administration

T2* relaxometry is one of the established methods to measure the effect of superparamagnetic iron oxide nanoparticles on tumor tissues with magnetic resonance imaging (MRI). Iron oxide nanoparticles shorten the T1, T2, and T2* relaxation times of tumors. While the T1 effect is variable based on the size and composition of the nanoparticles, the T2 and T2* effects are usually predominant, and T2* measurements are the most time-efficient in a clinical context. Here, we present our approach to measuring tumor T2* relaxation times, using multi-echo gradient echo sequences, external software, and a standardized protocol for creating a T2* map with scanner-independent software. This facilitates the comparison of imaging data from different clinical scanners, different vendors, and co-clinical research work (i.e., tumor T2* data obtained in mouse models and patients). Once the software is installed, the T2 Fit Map plugin needs to be installed from the plugin manager. This protocol provides step-by-step procedural details, from importing the multi-echo gradient echo sequences into the software, to creating color-coded T2* maps and measuring tumor T2* relaxation times. The protocol can be applied to solid tumors in any body part and has been validated based on preclinical imaging data and clinical data in patients. This could facilitate tumor T2* measurements for multi-center clinical trials and improve the standardization and reproducibility of tumor T2* measurements in co-clinical and multi-center data analyses.

We present a standardized protocol for the quantification of T2* relaxation times of tumors using external software. Multi-echo gradient echo images are acquired and fed into the software to create tumor T2* maps and measure tumor T2* relaxation times.

T2* relaxometry is one of the established methods to measure the effect of superparamagnetic iron oxide nanoparticles on tumor tissues with magnetic ...