Name: testing acetylcholine followed by adenosine for invasive diagnosis of coronary vasomotor disorders
Uploaded: 2021-02-03T14:30:00
Description: Watch this Scientific Journal Video about Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders at JoVE.com

This project was supported by the Berthold-Leibinger-Foundation, Ditzingen, Germany.

Intracoronary ACh testing has been approved by the local ethics committee and the protocol follows the guidelines of our institution for human research. A previous JoVE article covered a protocol showing preparation of the ACh solutions as well as preparation of the syringes for intracoronary injection of ACh15.
1. Preparation of the ACh solutions and preparation of the syringes for intracoronary injection of ACh
<ol>
	<li>Please refer to a previously published JoVE a...

<ol>
	<li>Burneikait&#279;, 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=Cardiac+shock-wave+therapy+in+the+treatment+of+coronary+artery+disease:+systematic+review+and+meta-analysis.">Cardiac shock-wave therapy in the treatment of coronary artery disease: systematic review and meta-analysis.</a> Cardiovascular Ultrasound. 15 (1), 11 (2017).</li><li>Tajti, 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=Update+in+the+Percutaneous+Management+of+Coronary+Chronic+Total+Occlusions.">Update in the Percutaneous Management of Coronary Chronic Total Occlusions.</a> JACC. Cardiovascular Interventions. 11 (7), 615-625 (2018).</li><li>Sharma, S. 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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>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>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>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=Ischemia+and+No+Obstructive+Coronary+Artery+Disease:+Prevalence+and+Correlates+of+Coronary+Vasomotion+Disorders.">Ischemia and No Obstructive Coronary Artery Disease: Prevalence and Correlates of Coronary Vasomotion Disorders.</a> Circulation. Cardiovascular Interventions. 12 (12), 008126 (2019).</li><li>Suda, 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=Coronary+Functional+Abnormalities+in+Patients+With+Angina+and+Nonobstructive+Coronary+Artery+Disease.">Coronary Functional Abnormalities in Patients With Angina and Nonobstructive Coronary Artery Disease.</a> Journal of the American College of Cardiology. 74 (19), 2350-2360 (2019).</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=Stratified+Medical+Therapy+Using+Invasive+Coronary+Function+Testing+in+Angina:+The+CorMicA+Trial.">Stratified Medical Therapy Using Invasive Coronary Function Testing in Angina: The CorMicA Trial.</a> Journal of the American College of Cardiology. 72 (23), 2841-2855 (2018).</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. (114), (2016).</li><li>Sara, J. D., 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=Prevalence+of+Coronary+Microvascular+Dysfunction+Among+Patients+With+Chest+Pain+and+Nonobstructive+Coronary+Artery+Disease.">Prevalence of Coronary Microvascular Dysfunction Among Patients With Chest Pain and Nonobstructive Coronary Artery Disease.</a> JACC. Cardiovascular Interventions. 8 (11), 1445-1453 (2015).</li><li>Kunadian, V., 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=An+EAPCI+Expert+Consensus+Document+on+Ischaemia+with+Non-Obstructive+Coronary+Arteries+in+Collaboration+with+European+Society+of+Cardiology+Working+Group+on+Coronary+Pathophysiology+&amp;+Microcirculation+Endorsed+by+Coronary+Vasomotor+Disorders+International+Study+Group.">An EAPCI Expert Consensus Document on Ischaemia with Non-Obstructive Coronary Arteries in Collaboration with European Society of Cardiology Working Group on Coronary Pathophysiology &amp; Microcirculation Endorsed by Coronary Vasomotor Disorders International Study Group.</a> European Heart Journal. 41 (37), 3504-3520 (2020).</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=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> Journal of the American College of Cardiology. 59 (7), 655-662 (2012).</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> The Lancet. 351 (9110), 1165-1169 (1998).</li><li>Sun, 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=Coronary+microvascular+spasm+causes+myocardial+ischemia+in+patients+with+vasospastic+angina.">Coronary microvascular spasm causes myocardial ischemia in patients with vasospastic angina.</a> Journal of the American College of Cardiology. 39 (5), 847-851 (2002).</li><li>Waxman, S., Moreno, R., Rowe, K. A., Verrier, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+primary+coronary+dilation+induced+by+transatrial+delivery+of+nitroglycerin+into+the+pericardial+space:+a+novel+approach+for+local+cardiac+drug+delivery.">Persistent primary coronary dilation induced by transatrial delivery of nitroglycerin into the pericardial space: a novel approach for local cardiac drug delivery.</a> Journal of the American College of Cardiology. 33 (7), 2073-2077 (1999).</li><li>Fearon, W. F., Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+Assessment+of+the+Coronary+Microvasculature:+The+Index+of+Microcirculatory+Resistance.">Invasive Assessment of the Coronary Microvasculature: The Index of Microcirculatory Resistance.</a> Circulation. Cardiovascular Interventions. 10 (12), (2017).</li><li>Om, S. Y., 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=Diagnostic+and+Prognostic+Value+of+Ergonovine+Echocardiography+for+Noninvasive+Diagnosis+of+Coronary+Vasospasm.">Diagnostic and Prognostic Value of Ergonovine Echocardiography for Noninvasive Diagnosis of Coronary Vasospasm.</a> JACC. Cardiovascular Imaging. 13 (9), 1875-1887 (2020).</li></ol>

Management of patients with angina and unobstructed coronary arteries is often demanding and sometimes frustrating. An important step during the work-up of these patients is that the underlying pathophysiological mechanism(s) for the patient&#39;s symptoms are adequately investigated. This is challenging as often not only one mechanism is responsible and various aetiologies including cardiac and non-cardiac as well as coronary and non-coronary need to be taken into account.
Frequently patients...

In recent years interventional cardiology has made substantial progress in various areas. This not only comprises interventional treatment of the heart valves using transcatheter aortic valve replacement and edge-to-edge repair of the mitral and tricuspid valve, but also coronary interventions1,2,3,4,5,6. Among the latter are advances in techniques for treatment of chronic total occlusions as well as calcified lesions using rotablation and shock wave therapy. In addition to these rather structural coronary interventional procedures invasive diagnostic procedures (IDP) have now been established in search of functional coronary disorders (i.e., coronary spasm and microvascular dysfunction)7. The latter comprise a heterogeneous group of conditions frequently but not exclusively occurring in patients with angina pectoris and unobstructed coronary arteries. The main mechanisms underlying these vasomotor disorders are impaired coronary vasodilatation, enhanced vasoconstriction/spasm as well as enhanced coronary microvascular resistance. The latter is often due to obstructive microvascular disease8. Anatomically, coronary vasomotor disorders may occur in the epicardial arteries, the coronary microcirculation or both. The Coronary Vasomotor Disorders International Study group (COVADIS) has published definitions for the diagnosis of these disorders9,10 and recent guidelines of the European Society of Cardiology (ESC) on the management of patients with chronic coronary syndrome have made recommendations for adequate patient assessment depending on the clinical condition11. Moreover, recent publications have delineated the various endotypes that can be derived from an IDP12,13. Such an approach has a benefit for the individual patient as randomized studies have shown better quality of life in patients undergoing an IDP followed by stratified medical therapy according to the test result compared to usual care by the general practitioner14. Currently, there is a debate about the most appropriate protocol for testing of such vasomotor disorders. The aim of this article is to describe a protocol where acetylcholine (ACh) provocation testing in search of coronary spasm is followed by Doppler wire-based assessment of coronary flow reserve (CFR) and hyperemic microvascular resistance (HMR) using adenosine (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cannula 0,95 x 50 mm (arterial punction)</td>
			<td>BBraun</td>
			<td>4206096</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula 23 G 0,6 x 25 mm (local anesthesia)</td>
			<td>BBraun</td>
			<td>4670025S-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Coronary angiography suite (AXIOM Artis MP eco)</td>
			<td>Siemens</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Contrast agent Imeron 350 with a 10 mL syringe for contrast injection</td>
			<td>Bracco Imaging</td>
			<td>30699.04.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Diagnostic catheter (various manufacturers)</td>
			<td>e.g. Medtronic</td>
			<td>DXT5JR40</td>
			<td></td>
		</tr>
		<tr>
			<td>Glidesheath Slender 6 Fr</td>
			<td>Terumo</td>
			<td>RM*RS6J10PQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin 5,000 IU (25,000 IU / 5 mL)</td>
			<td>BBraun</td>
			<td>1708.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mepivacaine 10 mg/mL</td>
			<td>PUREN Pharma</td>
			<td>11356266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride solution 0.9 % (1 x 100 mL)</td>
			<td>BBraun</td>
			<td>32000950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 2 mL (1x) (local anesthesia)</td>
			<td>BBraun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL (1x) (Heparin)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride (vial of 20 mg acetylcholine chloride powder and 1 ampoule of 2 mL diluent)</td>
			<td>Bausch &#38; Lomb</td>
			<td>NDC 240208-539-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula 20 G 70 mm (2x)</td>
			<td>BBraun</td>
			<td>4665791</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyceryle Trinitrate 1 mg/mL (5 mL)</td>
			<td>Pohl-Boskamp</td>
			<td>07242798</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride solution 0.9 % (3 x 100 mL)</td>
			<td>BBraun</td>
			<td>32000950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 2 mL (1x)</td>
			<td>BBraun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 5 mL (5x)</td>
			<td>BBraun</td>
			<td>4606051V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL (1x)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 50 mL (3x)</td>
			<td>BBraun</td>
			<td>4187903</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 6 mg/2 mL</td>
			<td>Sanofi-Aventis</td>
			<td>30124.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>ComboMap Pressure/Flow System</td>
			<td>Volcano</td>
			<td>Model No. 6800 (Powers Up)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure/Flow Guide Wire</td>
			<td>Volcano</td>
			<td>9515</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride solution 0.9 % (1 x 100 mL)</td>
			<td>BBraun</td>
			<td>32000950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL (3x)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

According to the diagnostic criteria suggested by COVADIS9, vasospastic angina can be diagnosed if the following criteria apply during ACh provocation testing: transient ECG changes indicating ischemia, reproduction of the patient&#180;s usual anginal symptoms and &#62; 90% vasoconstriction of an epicardial vessel&#160;as confirmed during coronary angiography (Figure 2).
Spas...

Watch this Scientific Journal Video about Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders at JoVE.com

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

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 ...

The invasive diagnostic procedure, the so-called IDP, allows to identify functional coronary diseases, which are frequent mechanisms of angina in patients with unobstructed coronary arteries. The IDP provides a comprehensive evaluation of the dilatory and constrictive component of coronary vasomotion using intracoronary flow and pressure measurements. This is done at rest and during hyperemia, and also coronary spasm testing is performed.There are different subtypes of functional coronary diseases, which are associated with different prognosis and treatment recommendations. The IDP helps to make the diagnosis and to find the optimal treatment. Begin by placing a guiding catheter suitable for the left coronary artery into the left main artery, and deliver another 5, 000 international units of heparin.Advance the Doppler flow pressure wire through the guiding catheter into the left main artery, and after flushing to avoid any contrast in the catheter, calibrate the distal pressure sensor of the Doppler flow pressure wire. Place the tip of the wire in the proximal mid portion of the vessel, usually the left anterior descending artery, and perform thoracoscopy to record the wire position. Press record to record the signals on the system and assess and optimize the Doppler in ECG signal quality as necessary.Once the recording has been started, inject six milliliters of the lowest acetylcholine concentration into the left coronary artery over a period of 20 seconds. Afterwards, flush the catheter with three to four milliliters of saline while performing continuous monitoring of the patient's symptoms, the 12-lead ECG, and the coronary blood flow velocity. Next, perform coronary angiography by injecting 10 milliliters of contrast agent through the catheter.After one minute, inject six milliliters of the medium acetylcholine concentration before flushing with three to four milliliters of saline and recording the average peak velocity, the 12-lead ECG, and the patient's symptoms as demonstrated. Perform coronary and geography of the left coronary artery as previously demonstrated, and if no spasm occurs, continue with injecting 5.5 milliliters of the highest acetylcholine concentration, followed by three to four milliliters of saline. If no epicardial spasm occurs at the 100 microgram dose, continue with the 200 microgram dose while continuously monitoring the ECG and the patient symptoms.When symptoms, ischemic ECG shifts, or epicardial spasm occurs, inject 200 micrograms of nitroglycerin and perform coronary angiography after one minute to document the reversion of the spasm. After the average peak velocity has returned to baseline and the patient's symptoms have returned to normal, press Base to capture the baseline values of the average peak velocity. Quickly inject a bolus of 3.5 milliliters of adenosine solution followed by a brief 10 milliliter saline flush.Press the Peak Search button three heartbeats after the injection to initiate a peak search. The system will calculate and display the values for fractional flow reserve, coronary flow reserve, and hyperemic microvascular resistance. Repeat for at least two consistent measurements, then retract the wire and obtain a final angiogram to exclude any vessel injury.According to the diagnostic criteria epicardial spasm can be diagnosed if transient ECG changes indicating ischemia, reproduction of the patient's usual anginal symptoms, and at least 90%vasoconstriction of an epicardial vessel as confirmed during coronary angiography can be observed. Spasm of the coronary microvasculature can be diagnosed if the patient's symptoms and ischemic ECG alterations occurred during provocation testing in the absence of epicardial vasospasm. Impaired microvascular vasodilation can be diagnosed by interpreting the CFR and HMR measurements following adenosine injections.Depending on the cutoff values applied, a reduced CFR is defined as less than 2.0 or 2.5 or less. For HMR, data on the optimal cutoff values is scarce, but an increased microvascular resistance is currently defined as an HMR greater than 1.9 or greater than 2.4. In order to obtain an informative IDP, it is very important to ensure the quality of the Dopplar signal is appropriate and also to ensure that the 12-lead ECG during acetylcholine testing is interpretable.This is required for an optimal IDP. The IDP can be expanded by additional tests such as the acetylcholine re-challenge after anti-vasospastic drug administration in order to evaluate its efficacy in the individual patient.

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FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

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 ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

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 ...

Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration

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

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

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

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

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

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

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

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.