We thank Junming Yan, consultant of Johnson&#38;Johnoson, who is responsible for research grants.This work was funded by the Jilin Provincial Department of Science and Technology (20220402076GH).

This research procedure closely adhered to the rules of human research ethics committee of China-Japan Union Hospital of Jilin University. Patients who underwent radiofrequency ablation of atrial fibrillation were searched on the Carto system (3D mapping system). Then, the PACS system was used to determine whether the patient underwent left atrial CT examination before surgery to ascertain that each selected patient had left atrial CT images for comparison. Soundstar is the ICE catheter used in this study, and a Cartosound module is available in the 3D mapping system. Each patient provided written informed consent before ICE 3D modeling.
1. Preparation before scanning
<ol>
	<li>Check patient information, such as accurate heart rate, rhythm, blood pressure, and oxygen saturation. Let the patient be supine with the arms on both sides and the thighs slightly abducted.</li>
	<li>Provide deep conscious sedation with fentanyl (200 &#181;g/mL) to all patients throughout the procedure. Select the right femoral vein as the puncture point, which is disinfected and paved for local anesthesia with 2% lidocaine.</li>
	<li>Set up the ICE catheter: Connect the ICE catheter tail line with the 3D mapping system and an ultrasonic machine. Open the Study interface in the 3D mapping system and select P500 in Connected Machines.</li>
	<li>Place the ICE catheter (Diameter: 10f; ultrasound mode: B-mode) inside the femoral vein. During the pushing process, monitor the ultrasound in real-time to ensure that the catheter is in a safe space.</li>
	<li>Pass the ablation catheter to the right atrium to develop the right atrium and coronary sinus model. Place the coronary sinus electrode by combining it with ICE guidance.</li>
	<li>After the ICE catheter enters the right atrium, ensure that the left atrium and left atrial appendage structure are displayed on the 3D mapping system display screen using the short and long axes of the left atrium while excluding the left atrial thrombus.</li>
	<li>Determine the optimal puncture site under ICE guidance after inserting the atrial septal puncture needle. Then, perform transseptal puncture.
	<ol>
		<li>Use the Home view to confirm that the ICE reached the right atrium. Then the software displays the left PV section. Click Right Bend to demonstrate the inferior vena cava and atrial septum.</li>
		<li>Move the puncture needle sheath to point in the four o&#39;clock direction. Retract the needle sheath to the oval fossa while monitoring ICE, and the &#34;tent sign&#34; is visible.</li>
		<li>Adjust the ICE catheter to reveal the left PV as a &#34;rabbit ear sign.&#34; Under ICE guidance, slowly withdraw the needle sheath to the lower edge of the oval fossa.</li>
		<li>Rotate the puncture needle clockwise to break through the oval fossa. Then, inject heparin saline through the atrial septum puncture needle. 
		&#8203;NOTE: Salt blisters were observed in the LA, indicating successful puncture of the atrial septum.</li>
	</ol>
	</li>
</ol>
2. 3D modeling of the left atrium and pulmonary vein
NOTE: The ICE constructs the left atrium model in two directions.
<ol>
	<li>After pushing the ICE catheter into the femoral vein, pass it through the inferior and superior vena cava and enter the right atrium.</li>
	<li>Push the ultrasound catheter to the middle of the right atrium on the short axis and rotate clockwise. Now, the ultrasound fan points toward the one o&#39;clock (Home view) direction depicting the right atrium and the right ventricle.</li>
	<li>Tighten the Tension Knob to achieve locking tension tuning. Then turn to the Home view and click Antecurvature (A) to fully display the tricuspid annulus. Attach to the tricuspid annulus for gating training and use the end-breath phase modeling.</li>
	<li>From the Home view, turn clockwise to the left atrial anterior wall, leading to the appearance of the left atrial appendage.</li>
	<li>Continue the clockwise turn to the left atrium, leading to the appearance of the left upper and lower pulmonary veins, displayed as a &#34;rabbit ear sign.&#34; Then, turn it clockwise and counterclockwise to precisely gather the image of the left pulmonary veins by identifying the anterior and posterior venus boundaries.</li>
	<li>Continue to turn clockwise to the left atrium, establishing the posterior wall, and in the process, the esophagus appears as the &#34;double track sign.&#34;</li>
	<li>Turn to the left atrium along the clockwise direction to observe the right lower pulmonary vein, displayed as a &#34;3-word sign&#34;. Then, turn it clockwise and counterclockwise to precisely capture the image of the right pulmonary veins by identifying the anterior and posterior boundaries.</li>
	<li>On the long axis, click Palintrope (P) to make the tip of the catheter at the same height as the coronary sinus mouth. This supplements the left atrium model. Adjust Left Bend/Right Bend (L/R) to observe the front wall of the long axis of the left atrium. This image captures the anterior wall of the left atrium.</li>
	<li>Mark the important anatomical locations, including pulmonary vein ostia, left atrial appendage, and other vital sites accordingly (Video 1).</li>
</ol>
3. Image acquisition and measurement of the pulmonary vein area
<ol>
	<li>Left atrial CT
	<ol>
		<li>Open the PACS system by double-clicking the icon. Click on Advanced Query to enter the patient name and inspection item. Click OK to find the image.</li>
		<li>Click on Tune to transfer the image to the vue pacs system (picture archiving and communication system).</li>
		<li>Transfer the 3D volume reconstruction image to the work box and click Export Image to save the left atrial posterior anterior (PA), left later (LL), and right lateral (RL) position images within the folder.</li>
		<li>After returning to the previous program, transfer the left atrial arterial phase enhancement sequence to the work box, and click on the image displayed as 3D.</li>
		<li>Double-click on the 3D image, then click on 3D in the toolbar. Select the Resection Tool to remove the ribs, spine, aorta, and other structures to expose the left atrium and pulmonary vein system.</li>
		<li>Expose the pulmonary vein vestibule. Click on Figure in the toolbar and select Area to calculate the cross-sectional area of the pulmonary vein vestibule.</li>
	</ol>
	</li>
	<li>ICE
	<ol>
		<li>Open the 3D mapping system. Then, click on Review Study and enter the name of the patient. Finally, use Search for Current Patient to identify the image.</li>
		<li>Click on OK to open the work interface.</li>
		<li>Click on Study &#62; Continue Study and select the Model and Channel sequences.</li>
		<li>Click on Capture Preferences, then select Region and adjust the image to &#34;Posterior-anterior&#34;, &#34;Left lateral,&#34; &#34;Right lateral,&#34; &#34;Left anterior oblique (LAO),&#34; and &#34;Right anterior oblique (RAO).&#34;</li>
		<li>Click on Picture, select the photo area, and click OK to save the picture.</li>
		<li>Click on the Map option and select Save Map. Then, use Eraser in the toolbar to remove the left and right pulmonary veins.</li>
		<li>Click on the image, select Area Measurement and measure the area of the pulmonary vein vestibule.</li>
	</ol>
	</li>
</ol>

ICE-Based 3D Remodeling for the Left Atrium and Pulmonary Vein

<ol>
	<li>Xu, Y., Sharma, D., Li, G., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+remodeling:+new+pathophysiological+mechanism+of+atrial+fibrillation.">Atrial remodeling: new pathophysiological mechanism of atrial fibrillation.</a> Medical Hypotheses. 80 (1), 53-56 (2013).</li><li>George, J. C., Varghese, V., Mogtader, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+echocardiography:+evolving+use+in+interventional+cardiology.">Intracardiac echocardiography: evolving use in interventional cardiology.</a> Journal of Ultrasound in Medicine. 33 (3), 387-395 (2014).</li><li>Jingquan, Z., 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=Intracardiac+echocardiography+Chinese+expert+consensus.">Intracardiac echocardiography Chinese expert consensus.</a> Frontiers in Cardiovascular Medicine. 9, 1012731 (2022).</li><li>Anter, E., 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=Comparison+of+intracardiac+echocardiography+and+transesophageal+echocardiography+for+imaging+of+the+right+and+left+atrial+appendages.">Comparison of intracardiac echocardiography and transesophageal echocardiography for imaging of the right and left atrial appendages.</a> Heart Rhythm. 11 (11), 1890-1897 (2014).</li><li>Rossillo, 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=Novel+ICE-guided+registration+strategy+for+integration+of+electroanatomical+mapping+with+three-dimensional+CT/MR+images+to+guide+catheter+ablation+of+atrial+fibrillation.">Novel ICE-guided registration strategy for integration of electroanatomical mapping with three-dimensional CT/MR images to guide catheter ablation of atrial fibrillation.</a> Journal of Cardiovascular Electrophysiology. 20 (4), 374-378 (2009).</li><li>Okumura, 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=Three-dimensional+ultrasound+for+image-guided+mapping+and+intervention:+methods,+quantitative+validation,+and+clinical+feasibility+of+a+novel+multimodality+image+mapping+system.">Three-dimensional ultrasound for image-guided mapping and intervention: methods, quantitative validation, and clinical feasibility of a novel multimodality image mapping system.</a> Circulation: Arrhythmia and Electrophysiology. 1 (2), 110-119 (2008).</li><li>Liu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+utility+of+intracardiac+echocardiography+in+cardiac+procedures.">The evolving utility of intracardiac echocardiography in cardiac procedures.</a> Journal of Atrial Fibrillation. 6 (6), 1055 (2014).</li><li>Bartel, T., Edris, A., Velik-Salchner, C., M&#252;ller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+echocardiography+for+guidance+of+transcatheter+aortic+valve+implantation+under+monitored+sedation:+a+solution+to+a+dilemma.">Intracardiac echocardiography for guidance of transcatheter aortic valve implantation under monitored sedation: a solution to a dilemma.</a> European Heart Journal. Cardiovascular Imaging. 17 (1), 1-8 (2016).</li><li>Haissaguerre, 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=Spontaneous+initiation+of+atrial+fibrillation+by+ectopic+beats+originating+in+the+pulmonary+veins.">Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins.</a> New England Journal of Medicine. 339 (10), 659-666 (1998).</li><li>Kaseno, 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=The+impact+of+the+CartoSound&#174;+image+directly+acquired+from+the+left+atrium+for+integration+in+atrial+fibrillation+ablation.">The impact of the CartoSound&#174; image directly acquired from the left atrium for integration in atrial fibrillation ablation.</a> Journal of Interventional Cardiac Electrophysiology. 53, 301-308 (2018).</li><li>Enriquez, 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=Use+of+intracardiac+echocardiography+in+interventional+cardiology:+working+with+the+anatomy+rather+than+fighting+it.">Use of intracardiac echocardiography in interventional cardiology: working with the anatomy rather than fighting it.</a> Circulation. 137 (21), 2278-2294 (2018).</li></ol>

The authors have no conflicts of interest to declare.

Intracardiac echocardiography (ICE) is a noncontact three-dimensional reconstruction tool. It determines the appropriate ablation plane and reduces the incidence of pulmonary vein stenosis. Moreover, ICE improves the effectiveness of catheter ablation by assessing the distal position of the ablation catheter and its relative association with the anatomical structures. These structures include the left atrium and pulmonary vein and the diameter and morphology of the pulmonary vein.
ICE-guided a...

Atrial fibrillation (AF) is commonly associated with atrial remodeling, including mechanical remodeling, electrophysiologic remodeling, and structural remodeling1. Structural remodeling will dramatically affect the anatomy of the atrium. Hence, assessing the left atrial anatomy in AF patients is essential for AF ablation procedures and any procedure targeting the left atrium. For the FAM 3D modeling, the 3D modeling of the heart is reconstructed based on the spatial position change of its position corresponding to the fixed magnetic field by continuously displacing the magnetic catheter in the heart. In contrast, ICE 3D modeling integrates the two-dimensional image in the cardiac cavity with the 3D electroanatomic mapping system by positioning the sensor at the head end of the ICE phase array catheter. Thus, the ultrasonic sector represents 3D modeling to demonstrate the anatomical relationship and real-time catheter position.
Based on our clinical experience, intracardiac echocardiography (ICE) can identify the atrial wall boundary and further establish the 3D remodel. However, most ICE usage during AF ablation or 3D remodeling just provides a brief profile of the atria or pulmonary veins. Originally, ICE was applied to guide the interventional closure of atrial septal defect and patent foramen ovale2. ICE can clarify the location and shape of the atrial septal and has been used for various interventional procedures requiring atrial septal puncture3. These include radiofrequency catheter ablation of atrial fibrillation, mitral valve interventional therapy, etc. ICE can precisely identify pulmonary vein boundaries and atrial walls to establish a more detailed 3D model3. It is unclear whether this method could provide operators with a more precise atrial anatomy assessment, especially for the pulmonary vein antrum and transseptal sites. In this study, we compared the left atrium CT image and 3D remodel established using traditional methods and precise ICE procedures to determine additional information.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CARTO V6</td>
			<td>Johnson&#38;Johnson</td>
			<td>6.0.80.45</td>
			<td></td>
		</tr>
		<tr>
			<td>CARTO V7</td>
			<td>Johnson&#38;Johnson</td>
			<td>7.1.80.33</td>
			<td></td>
		</tr>
		<tr>
			<td>PACS system</td>
			<td>Philips(China) Investment Co.,Ltd</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Soundstar</td>
			<td>Johnson&#38;Johnson</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional modeling of the left atrium and pulmonary veins with a precise intracardiac echocardiography approach

Intracardiac echocardiography (ICE) is a novel tool for estimating cardiac anatomy during pulmonary vein isolation procedures, particularly the left atrium (LA) anatomy and pulmonary vein structures. ICE is widely used to establish a three-dimensional (3D) left atrial structural model during ablation procedures. However, it is unclear whether using ICE in a precise 3D modeling method can provide a more accurate left atrial 3D model and the transseptal approach. This study proposes a protocol to model the left atrium and pulmonary veins with ICE and fast anatomical mapping (FAM) catheter remodeling. It evaluates the accuracy of the models produced using the two methods through observer scoring. We included 50 patients who underwent ICE-based 3D remodeling and 45 who underwent FAM 3D remodeling based on pulmonary vein isolation procedures. The pulmonary vein antrum remodeling is estimated by comparing the antrum area acquired by remodeling and left atrial computed tomography angiography (CTA). The observer scores for the modeling in the ICE and FAM groups were 3.40 &#177; 0.81 and 3.02 &#177; 0.72 (P &#60; 0.05), respectively. The pulmonary vein antrum area obtained using the ICE- and FAM-based methods showed a correlation with the area acquired by left atrium CT. However, the 95% confidence interval bias was narrower in ICE-acquired models than in FAM-acquired models (-238 cm2 to 323 cm2 Vs. -363 cm2 to 386 cm2, respectively) using Bland-Altman analysis. Therefore, precise ICE possesses high accuracy in estimating the left atrial structure, becoming a promising approach for future cardiac structure estimation.

From January 2021 to June 2022, we selected 114 patients who underwent radiofrequency ablation of atrial fibrillation at our hospital. Patients were excluded based on the following criteria: no 3D volume reconstruction image of left atrial CT (n = 11), no transseptal puncture ICE image (n = 4), and incomplete reconstruction of the left atrium and pulmonary vein images (n = 4). Finally, 50 patients with ICE 3D modeling and 45 with FAM 3D modeling as control group was included in this study.
Two...

Watch this Scientific Journal Video about Advancements in Intracardiac Echocardiography for Atrial Anatomy Assessment at JoVE.com

Author Spotlight: Advancements in Intracardiac Echocardiography for Atrial Anatomy Assessment 

Accurate intracardiac echocardiography (ICE) shows significant accuracy in estimating the left atrial structure, a prospective and promising cardiac structure estimation method. Here we propose a protocol for three-dimensional modeling of the left atrium and pulmonary veins with ICE and fast anatomical mapping (FAM) catheter remodeling.

Three-Dimensional Modeling of the Left Atrium and Pulmonary Veins with a Precise Intracardiac Echocardiography Approach

Intracardiac echocardiography (ICE) is a novel tool for estimating cardiac anatomy during pulmonary vein isolation procedures, particularly the left ...

Our research tries to evaluate whether intracardiac echocardiography-based 3D construction could enhance the accuracy of pulmonary atrium and assessments in arterial anatomy. Intracardiac echocardiography is capable of identifying the arterial wall boundary and creating a 3D model of pulmonary winds. Recently this has been increased, the focus on using S2 often detailed images for accurately determining arterial anatomy.One of the current experimental challenges is accurately imitating the left arterial structure. However, promising results have been obtained use as best arterial imaging and safety deconstruction, which has high accuracy and hold potential for future cardiac structure imaging.

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695 Views.  China-Japan Union Hospital of Jilin University. Our research tries to evaluate whether intracardiac echocardiography-based 3D construction could enhance the accuracy of pulmonary atrium and assessments in arterial anatomy. Intracardiac echocardiography is capable of identifying the arterial wall boundary and creating a 3D model of pulmonary winds. Recently this has been increased, the focus on using S2 often detailed images for accurately determining arterial anatomy.One of the current experimental challenges is accurately imitating...

Video: Author Spotlight: Advancements in Intracardiac Echocardiography for Atrial Anatomy Assessment 

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

Use of a Percutaneous Ventricular Assist Device/Left Atrium to Femoral Artery Bypass System for Cardiogenic Shock

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the left ventricle by draining blood from the left atrium (LA) and returning it to the systemic arterial circulation via the femoral artery. It can provide flows ranging from 2.5-5 L/min depending on the size of the cannula. Here, we discuss the mechanism of action of LAFAB, available clinical data, indications for its use in cardiogenic shock, steps of implantation, post-procedural care, and complications associated with the use of this device and their management.
We also provide a brief video of the procedural component of device therapy, including the pre-placement preparation, percutaneous placement of the device via transseptal puncture under echocardiographic guidance and the post-operative management of device parameters.

The following article describes the stepwise procedure for placement of a device (e.g., Tandemheart) in cardiogenic shock (CS) that is a percutaneous left ventricular assist device (pLVAD) and a left atrial to femoral artery bypass (LAFAB) system that bypasses and supports the left ventricle (LV) in CS.

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the ...

Whole-Kidney Three-Dimensional Staining with CUBIC

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood ...

Enhancing Pulmonary Nodule Diagnosis Using Whole-Lung 3D Reconstruction

Three-Dimensional Reconstruction for the Whole Lung with Early Multiple Pulmonary Nodules

For patients with early multiple pulmonary nodules, it is essential, from a diagnostic perspective, to determine the spatial distribution, size, location, and relationship with surrounding lung tissue of these nodules throughout the entire lung. This is crucial for identifying the primary lesion and developing more scientifically grounded treatment plans for doctors. However, pattern recognition methods based on machine vision are susceptible to false positives and false negatives and, therefore, cannot fully meet clinical demands in this regard. Visualization methods based on maximum intensity projection (MIP) can better illustrate local and individual pulmonary nodules but lack a macroscopic and holistic description of the distribution and spatial features of multiple pulmonary nodules.
Therefore, this study proposes a whole-lung 3D reconstruction method. It extracts the 3D contour of the lung using medical image processing technology against the background of the entire lung and performs 3D reconstruction of the lung, pulmonary artery, and multiple pulmonary nodules in 3D space. This method can comprehensively depict the spatial distribution and radiological features of multiple nodules throughout the entire lung, providing a simple and convenient means of evaluating the diagnosis and prognosis of multiple pulmonary nodules.

Author Spotlight: Advancing 3D Modeling for Enhanced Diagnosis and Treatment of Pulmonary Nodules in Early-Stage Lung Cancer 

This study introduces a three-dimensional (3D) reconstruction method for the entire lung in patients with early multiple pulmonary nodules. It offers a comprehensive visualization of nodule distribution and their interplay with lung tissue, simplifying the assessment of diagnosis and prognosis for these patients.

For patients with early multiple pulmonary nodules, it is essential, from a diagnostic perspective, to determine the spatial distribution, size, location, ...