This material is the result of work supported with resources and the use of the facilities at the Clement J. Zablocki Veterans Affairs Medical Center in Milwaukee, Wisconsin.

The Institutional Review Board of the Clement J. Zablocki Veterans Affairs Medical Center approved the protocols. Written informed consent was waived because invasive cardiac monitoring and TEE are routinely used in all patients undergoing cardiac surgery in our institution. Patients with relative or absolute contraindications for TEE, those undergoing repeat median sternotomy or emergency surgery, and those with atrial or ventricular tachyarrhythmias were excluded from participation.
1. Anesthesia
<ol>
	<li>Provide each patient with intravenous midazolam (1 to 3 mg) and fentanyl (50 to 150 &#181;g) for conscious sedation before surgery.</li>
	<li>Use local anesthesia (subcutaneous 1% lidocaine) for insertion of intravenous and radial artery catheters. Test the quality of the local anesthesia with a pinprick.</li>
	<li>Ensure that the patient receives supplemental oxygen using a nasal cannula (2 to 4 L/min).</li>
	<li>Place a central venous or pulmonary artery catheter using local anesthesia (subcutaneous 1% lidocaine) under sterile conditions through the right or left internal jugular vein with ultrasound guidance based on appropriate clinical indications.</li>
	<li>Induce anesthesia using intravenous fentanyl (5 mcg/kg), propofol (1 to 2 mg/kg), and rocuronium (0.1 mg/kg). Maintain anesthesia using inhaled isoflurane (end-tidal concentration of 1%) in an air-oxygen mixture, fentanyl (1 to 2 &#181;g/kg/h), and rocuronium (0.05 mg/kg) titrated to effect using neuromuscular monitoring.</li>
	<li>Suction the stomach using an oral-gastric tube.</li>
	<li>Place ultrasound jelly in the patient&#8217;s hypopharynx. Lift the jaw anteriorly and advance a TEE probe into the esophagus with gentle pressure to overcome resistance of the hypopharygeus muscle.</li>
</ol>
2. Transesophageal Echocardiography
<ol>
	<li>Perform a comprehensive TEE examination following American Society of Echocardiography/Society of Cardiovascular Anesthesiologists guidelines42 in each patient.</li>
	<li>Place a pulse-wave Doppler sample volume between the tips of the mitral leaflets to record trans-mitral blood flow velocity in the mid-esophageal four-chamber TEE imaging plane (Figure 1).</li>
	<li>Identify the early LV filling and atrial systole blood flow waveforms of trans-mitral blood flow velocity, and measure their corresponding peak velocities and velocity-time integrals (VTIE and VTIA, respectively) using the echocardiography equipment&#8217;s integrated software package (Figure 1).</li>
	<li>Calculate the atrial filling fraction (&#946;) as the ratio of atrial to total LV filling: 
	<img alt="Equation 1" src="/files/ftp_upload/58374/58374eq1.jpg" /></li>
	<li>Measure the maximum diameter of the LV outflow tract immediately below the aortic valve in the mid-esophageal aortic valve long axis TEE view during mid-systole (Figure 2A).</li>
	<li>Calculate the area of the LV outflow tract assuming circular geometry as the product of &#960;/4 and the square of the diameter (see step 2.5 above).</li>
	<li>Obtain a deep transgastric long axis TEE view, and place a pulse-wave Doppler sample volume in the distal LV outflow tract to record a blood flow velocity envelope (Figure 2B) at the same level where the diameter was measured (see step 2.5 above); integrate the area of this waveform using the echocardiography equipment&#8217;s software package to obtain VTI.</li>
	<li>Multiply the resulting velocity-time integral (VTI) of the LV outflow track blood flow velocity waveform (Figure 2B) by the area of the outflow track (see step 2.6) to obtain stroke volume (SV).</li>
	<li>Record video clips of the mid-esophageal bicommissural and LV long axis TEE imaging planes, respectively42. Be sure to include several cardiac cycles in each recording.</li>
	<li>Visually inspect slow-motion images of the video clips (see step 2.9 above) after the ECG T-wave to choose the maximum opening of the mitral valve leaflets.</li>
	<li>Measure the distance between the mitral leaflets (Figures 3A and 3B) using the echocardiography equipment&#8217;s &#8220;caliper&#8221; function.</li>
	<li>Calculate the mitral valve diameter (D) as the average of the major (transcommissural anterior-lateral-posterior-medial) and minor (anterior-posterior) lengths.</li>
	<li>Calculate VFT using the formula: 
	<img alt="Equation 2" src="/files/ftp_upload/58374/58374eq2.jpg" /></li>
	<li>Perform all quantitative echocardiographic measurements in triplicate at end-expiration.</li>
</ol>
3. Experimental Design
<ol>
	<li>Determine VFT, indices of LV diastolic function, and hemodynamics during steady-state conditions 30 minutes before and 15, 30, and 60 minutes after cardiopulmonary bypass (CPB) in 10 patients with normal preoperative LV ejection fraction under coronary artery surgery to test the hypothesis that CPB transiently decreases VFT39.</li>
	<li>Test the hypothesis that LV pressure-overload hypertrophy produced by aortic valve stenosis reduces VFT by examining (in one group of 8 patients undergoing aortic valve replacement) for severe aortic stenosis and comparing observations to another group of 8 patients with normal LV wall thickness undergoing coronary artery surgery40. Measure VFT, LV diastolic function, hemodynamics, and end-diastolic posterior wall thickness during steady-state conditions 30 minutes before CPB.</li>
	<li>Test the hypothesis that abnormal diastolic blood flow entering the LV affects trans-mitral LV filling efficiency in 8 patients with aortic valve stenosis and moderate aortic insufficiency versus 8 patients with aortic stenosis who do not have regurgitant valves38. Measure VFT and other parameters as described above (step 3.2).</li>
	<li>Test the hypothesis that advanced age is associated with a reduction in LV filling efficiency quantified using VFT in 7 octogenarians (82 &#177; 2 years) compared to 7 younger patients (55 &#177; 6 years)41 undergoing coronary artery surgery. Ensure that both groups have normal preoperative LV ejection fraction. Measure VFT and other parameters as described above (step 3.2).</li>
</ol>
4. Statistics
<ol>
	<li>Present the data as mean &#177; standard deviation.</li>
	<li>Evaluate data using analysis of variance (ANOVA) followed by Bonferroni&#8217;s modification of Student&#8217;s t-test.</li>
	<li>Use linear regression analysis to determine the relationships between VFT and end-diastolic posterior wall thickness and between VFT and age.</li>
	<li>Reject the null hypothesis when p &#60; 0.05.</li>
</ol>

The authors have no competing financial interests or other conflicts of interest pursuant to this work.

The current results illustrate that VFT can be reliably measured during cardiac surgery using the TEE techniques described here. Previous descriptions of VFT used transthoracic echocardiography in conscious subjects, but this approach cannot be utilized when the chest is open. We used intraoperative TEE to determine VFT in the anesthetized patients undergoing cardiac surgery during which changes in LV filling dynamics are often encountered as a result of ischemia-reperfusion injury or surgical interventions. Our findings...

Fluid mechanics is a critical yet often underappreciated determinant of left ventricular (LV) filling. A three-dimensional rotational body of fluid, known as a vortex ring, is generated whenever a fluid traverses an orifice1,2,3. This vortex ring improves the efficiency of fluid transport compared with a continuous linear jet4. Movement of blood through the mitral valve during early LV filling causes a vortex ring to form5,6,7,8 and facilitates its propagation into the chamber by preserving fluid momentum and kinetic energy9. These actions enhance LV filling efficiency4,10,11,12,13. The ring not only inhibits blood flow stasis in the LV apex14,15,16,17 but also directs flow preferentially beneath the anterior mitral leaflet7,18, effects that decrease the risk of apical thrombus formation and facilitate filling of the LV outflow track19, respectively. Contrast echocardiography17, Doppler vector flow mapping6,20,21, magnetic resonance imaging7, and particle imaging velocimetry9,22,23,24 have been used to demonstrate the appearance and behavior of trans-mitral vortex rings under normal and pathological conditions. The left atrial-LV pressure gradient, the degree of diastolic mitral annular excursion, the minimum LV pressure achieved during diastole, and the rate and extent of LV relaxation are the four major determinants of the duration, size, flow intensity, and position of the trans-mitral ring2,12,25,26,27,28,29.
Vortex ring development is most often quantified with a dimensionless parameter (vortex formation time; VFT) based on fluid ejection from a rigid tube3, where VFT is defined as the product of the time-averaged fluid velocity and the duration of ejection divided by the orifice diameter. The optimal size of a vortex ring is achieved when VFT is 4 in vitro because trailing jets and energetic limitations prevent it from attaining a larger size3,4. Mitral valve VFT has been approximated clinically using transthoracic echocardiography8,30,31. Based on analysis of trans-mitral blood flow velocity and mitral valve diameter (D), it can be easily shown8 that VFT = 4 &#215; (1-&#946;) &#215; EF &#215;&#945;3, where &#946; = atrial filling fraction, EF = LV ejection fraction, and &#945; = EDV1/3/D, where EDV = end-diastolic volume. Ejection fraction is the ratio of stroke volume (SV) and EDV, allowing this equation to be simplified to VFT = 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). Because VFT is dimensionless (volume/volume), this index allows direct comparison between patients of varying size without adjustment for weight or body surface area8. Optimal VFT ranges between 3.3 and 5.5 in healthy subjects8, and results are consistent with those obtained in fluid dynamics models3,32. VFT was shown to be &#8804; 2.0 in patients with depressed LV systolic function, findings that are also supported by theoretical predictions8. Reductions in VFT independently predicted morbidity and mortality in patients with heart failure30. Elevated LV afterload33, Alzheimer&#39;s disease34, abnormal diastolic function19, and replacement of the native mitral valve with a prosthesis35 have also been shown to decrease VFT. Measurement of VFT may also be useful to identify blood flow stasis or thrombosis in patients with acute myocardial infarction36,37.
Our group is interested in factors that affect LV filling efficiency during cardiac surgery38,39,40,41. We use standard two-dimensional and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables required to calculate VFT. In this report, we describe this methodology in detail and review our findings to date.

<table>
	<tbody>
		<tr>
			<td>Echocardiography Machine</td>
			<td>Philips Ultrasound, Bothall, WA</td>
			<td>iE33</td>
			<td></td>
		</tr>
		<tr>
			<td>Transesophageal Echocardiography Probe</td>
			<td>Philips Ultrasound, Bothall, WA</td>
			<td>X7-2t</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical Software</td>
			<td>AnalystSoft, Walnut, CA</td>
			<td>StatPlus:mac Pro</td>
			<td></td>
		</tr>
	</tbody>
</table>

noninvasive determination of vortex formation time using transesophageal echocardiography during cardiac surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular (LV) filling compared with a continuous linear jet. Vortex ring development is most often quantified with vortex formation time (VFT), a dimensionless parameter based on fluid ejection from a rigid tube. Our group is interested in factors that affect LV filling efficiency during cardiac surgery. In this report, we describe how to use standard two-dimensional (2D) and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables needed to calculate VFT. We calculate atrial filling fraction (&#946;) from velocity-time integrals of trans-mitral early LV filling and atrial systole blood flow velocity waveforms measured in the mid-esophageal four-chamber TEE view. Stroke volume (SV) is calculated as the product of the diameter of the LV outflow track measured in the mid-esophageal long axis TEE view and the velocity-time integral of blood flow through the outflow track determined in the deep transgastric view using pulse-wave Doppler. Finally, mitral valve diameter (D) is determined as the average of major and minor axis lengths measured in orthogonal mid-esophageal bicommissural and long axis imaging planes, respectively. VFT is then calculated as 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). We have used this technique to analyze VFT in several groups of patients with differing cardiac abnormalities. We discuss our application of this technique and its potential limitations and also review our results to date. Noninvasive measurement of VFT using TEE is straightforward in anesthetized patients undergoing cardiac surgery. The technique may allow cardiac anesthesiologists and surgeons to assess the impact of pathological conditions and surgical interventions on LV filling efficiency in real time.

The current technique allowed us to reliably measure VFT during cardiac surgery under a variety of clinical conditions by obtaining each determinant from blood flow and dimensional recordings in standard TEE imaging planes. A pulse-wave Doppler sample volume was placed at the tips of the mitral leaflets in the mid-esophageal four-chamber view to obtain the trans-mitral blood flow velocity profile necessary to calculate atrial filling fraction (&#946;; Figure 1</strong...

Watch this Scientific Journal Video about Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery at JoVE.com

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...

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7.8K Views.  Clement J. Zablocki Veterans Affairs Medical Center. We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

Video: Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

An Anatomical Study of Nerves at Risk During Minimally Invasive Hallux Valgus Surgery

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional orientation and localization of structures that are not directly visible to the surgeon. This is especially critical for structures at risk like nerves or blood vessels. Optimization of the handling of cadaveric material and the combination of multiple techniques compensate for the limited availability of adequate specimens. The described protocol combines anatomical plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to help localize relevant structures, such as nerves, arteries, veins and to correctly position the portals during MIS procedures. Depiction of these structures in anatomy textbooks can differ from what is encountered in the surgical field; and for this reason, new anatomical studies with a surgical orientation are needed. However, this is a complex, time-consuming technique requiring specific training. The anatomical references described with the so-called 'clock method' provide the surgeon with an easy and reproducible system to locate the path of the nerves at risk in Hallux Valgus MIS procedures. This model can be extrapolated to many other minimally invasive surgical procedures.

Minimally invasive surgical (MIS) procedures rely on anatomical references to localize structures not directly visible to the surgeon. This manuscript describes a combined method of plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to locate the structures at risk during MIS procedures.

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional ...

Robotized Testing of Camera Positions to Determine Ideal Configuration for Stereo 3D Visualization of Open-Heart Surgery

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room and the surgical field is restricted. It is a sterile environment, and the physical space is crowded with surgical staff and technical equipment. In this setting, unobscured capture and realistic reproduction of the surgical procedures are difficult. This paper presents a method for rapid and reliable data collection of stereoscopic 3D videos at different camera baseline distances and distances of convergence. To collect test data with minimum interference during surgery, with high precision and repeatability, the cameras were attached to each hand of a dual-arm robot. The robot was ceiling-mounted in the operating room. It was programmed to perform a timed sequence of synchronized camera movements stepping through a range of test positions with baseline distance between 50-240 mm at incremental steps of 10 mm, and at two convergence distances of 1100 mm and 1400 mm. Surgery was paused to allow 40 consecutive 5-s video samples. A total of 10 surgical scenarios were recorded.

The human depth perception of 3D stereo videos depends on the camera separation, point of convergence, distance to, and familiarity of the object. This paper presents a robotized method for rapid and reliable test data collection during live open-heart surgery to determine the ideal camera configuration.

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...