Name: model surgical training: skills acquisition in fetoscopic laser photocoagulation of monochorionic diamniotic twin placenta using realistic simulators
Uploaded: 2018-03-21T14:00:00
Description: Watch this Scientific Journal Video about Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators at JoVE.

The authors would like to thank the people who have helped with building the models, providing materials, and facilitating training workshops in Singapore and Bangkok: Dr. Ying Woo Ng, Prof. Yoke Fai Fong, Sommai Viboonchart, Ginny Chen, Cecile Laureano, Pei Huang Kuan, Mei Lan Xie, Prof. Jerry KY Chan. Materials were supported by the Obstetrics and Gynaecology departments of Faculty of Medicine Siriraj Hospital, Bangkok and the National University Hospital, Singapore, and by National Medical Research Council (Singapore) grant NMRC/CSA/043/2012.

The collection of human placenta from term deliveries was approved by the Domain Specific Review Board of the NUH of Singapore (DSRB C/00/524) and by the Siriraj Institutional Review Board (SIRB 704/2559) of Siriraj Hospital in Bangkok. In all cases, patients gave separate informed written consent for the use of the collected specimen. The pig bladders were collected from a local butcher in Singapore and were a kind donation from Dr. Ying Woo Ng (NUH). The non-human primate (NHP) placentas were waste material collected f...

The skills practiced on a fetoscopy simulator and on the tissue models encompass the majority of technical abilities required for SFLP. The advantages of training on these models include learning to simultaneously handle the ultrasound probe and fetoscope, familiarity with handling the straight and curved fetoscopes, practicing the systematic examination of the vascular equator along the whole length of the inter-twin membrane to identify anastomosing vessels on high-fidelity MCDA placenta, and learning the correct techn...

The acquisition of a new, minimally-invasive surgical technique often employs the traditional surgical apprenticeship model in which an individual learns from observing an expert surgeon operate on a live patient and eventually performs the technique under close supervision1. This time-honored model often limits the passage of knowledge from mentor to individual trainee and relies heavily on the availability of resources such as training funds and patient case-load2. Fetoscopic surgery is an example of a high-risk minimally-invasive surgery, performed on a preterm individual during pregnancy in which there are risks to both the mother and fetus. As with any surgical procedure, higher complication rates arise at the initial steep slope of the learning curve. Thus, surgeries are usually performed by the most senior or skilled surgeon in order to meet the critical volume of cases to optimize patient outcomes3.
Good fetoscopy skills are important for the future of fetal therapy, which strives to be minimally invasive, even with respect to the correction of structural defects4,5,6. Fetoscopic surgery is technically challenging and there are inherent risks to patient safety associated with practicing and developing new skills in the real-life theater environment. Even established surgeons require time and consistent practice on multiple patients to acquire expertise, skills in troubleshooting when difficulties arise, and the instinct to predict and avoid pitfalls in a new and complex procedure. There is less tolerance for suboptimal outcomes usually associated with novice proceduralists7. While it is important not to compromise patient safety during the initial implementation of fetoscopic surgery, there is also a need to enhance the efficiency with which skills and expertise are acquired by all proceduralists, particularly in smaller clinical units just beginning to practice fetoscopy. An alternative system complementary to traditional apprenticeship is needed to meet the challenges of limited training funds and a small patient base on which to master these highly specialized procedures. Procedural learning curves can be shortened, and complications reduced by training on high-fidelity machines or cadaveric animal models, with dedicated traditional mentoring or distant proctorship and procedure-focused stepwise learning8,9,10,11. Familiarization with the fetoscope manipulation, intrauterine orientation of the vascular equator, and laser coagulation before performing the actual surgery has the potential to reduce operative complications12,13. This training may shorten the learning curve for new operators as they master basic skills on a realistic tissue model.
Monozygotic twinning occurs with uniform frequency worldwide affecting 3-5 per 1,000 pregnancies, and the 75% of monozygotic twins with monochorionic diamniotic (MCDA) placentation are at significant risk for TTTS, which currently complicates about 10-15% of MCDA pregnancies, or 1-3 per 10,000 births14. The incidence is expected to increase with the frequency of in vitro fertilization (IVF) in which there is a 2 to 12-fold increase in monozygosity15,16,17,18,19. TTTS arises from unidirectional inter-fetal blood flow via deep intraplacental AVA. Untreated, this carries a 60-100% mortality and significant morbidity for surviving fetuses20,21,22.
Selective fetoscopic laser coagulation (SFLP) is the only curative intervention aimed at the rescue of both twins via fetoscopic identification and ablation of the offending AVA, and is considered the standard of care in TTTS stages II-IV (~ 93% of all cases) in pregnancies at &#60; 26 weeks of gestation, with clinical studies in progress to determine if it should also be applied to selected stage I disease23,24,25. SFLP carries an overall perinatal survival of ~ 70% with a higher likelihood of more advanced gestation and higher birth weights at delivery26,27 and is considered superior to other interventions as it directly rectifies the underlying pathology of TTTS28,29,30. The intervention itself is not without complications, and laser-treated TTTS is associated with recurrence (0-16%), perinatal mortality (~ 35%), and a 5-20% chance of long-term neurologic handicap23. Acquisition of the correct skills, building expertise over a steep learning curve, adherence to international standards of fetoscopic practice, and maintaining surgical dexterity are essential to providing the best outcomes in this complex disease13,31,32,33. This is often dependent on financial and human resources and a critical volume of cases that may take significant time to acquire34. Established fetal therapy centers are currently concentrated in Western Europe and North America, but the predicted population boom (and thus new pregnancies) will mostly affect Asia and Africa35,36. Therefore, an increase in the incidence of fetal anomalies amenable to intrauterine treatment can be expected in these lower-resource populations. The dissemination of specialized services such as fetoscopic surgery is a challenge that needs to be addressed as a regional priority37. New fetal therapy centers in these regions must reliably provide SFLP services to meet the needs of their communities, but significant investment and time is needed for new centers to achieve equivalent outcomes as established ones38,39,40,41.
Departing from the resource-heavy apprenticeship model will facilitate a much-needed dissemination of skills and expertise to communities in which there is a great demand for it. The traditional surgical apprenticeship is still relevant but less practical for many smaller clinical units, as it is time- and resource-consuming and limits the passage of knowledge and skills to one trainee at a time. Simulator training under proctorship is more applicable on a wider scale and facilitates the passage of knowledge and skills passed from one expert to multiple persons through workshops and regular skills training on reliable tissue models13,42,43. It has been suggested that, because of its rarity, TTTS treatment should be accumulated in high-volume fetal centers to improve its outcomes. Yet, there is also a need to establish new fetal care centers to improve patient access to treatment. Emerging fetal care centers, like the National University Hospital in Singapore (NUH), will need to adhere to certain guidelines in order to maintain their surgical outcomes, i.e., Siriraj-NUH proctorship system as seen in Figure 137.
In this article we will describe a model-based system with which new proceduralists can undergo skills training in tandem under the guidance of an expert proctor, and by which skills can be practiced to maintain surgical dexterity during long intervals between patients. We will share practical points from our experiences at the Siriraj Hospital in Bangkok and the NUH in Singapore in initiating fetal therapy6,44,45.

<table border="0" cellpadding="0" cellspacing="0" style="width:1253px;" width="1253">
	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:85px;width:215px;">Fetoscopic Simulator</td>
			<td style="width:215px;">Maternal-Fetal Medicine unit, Department of Obstetrics and Gynaecology, Siriraj Hospital, Bangkok, Thailand</td>
			<td style="width:367px;">NA.</td>
			<td style="width:457px;">Siriraj Fetoscopic Simulator. Customised model of monochorionic anterior/posterior placenta and anastomses produced at the Siriraj Hospital in Bangkok.</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;width:215px;">Laparoscopy tower with light source, camera and video recorder</td>
			<td style="width:215px;">Olympus Singapore</td>
			<td style="width:367px;">Olympus Visera Elite system (Olympus Singapore) with camera OTV-S190 and light source CLV-S190 set at medium intensity (level 0) and video recorder&#160;</td>
			<td style="width:457px;">Laparoscopy tower for fetoscopy and recording of practice</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Voluson E8 ultrasound machine with 4CD probe</td>
			<td style="width:215px;">GE Healthcare Singapore</td>
			<td style="width:367px;">GE Voluson E8; transabdominal 4CD curved transducer (2-5MHz)&#160;</td>
			<td style="width:457px;">Ultrasound system for guidance of fetoscope introduction and manipulation</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Minature straight forward telescope 0o (2mm) for posterior placenta</td>
			<td style="width:215px;">KARL STORZ GmbH &#38; Co KG, Tuttlingen, Germany</td>
			<td style="width:367px;">11630AA</td>
			<td style="width:457px;">Fetoscope. 0&#176; lens, diameter 2mm, length 26cm, autoclavable, fibre optic light transmission incorporated. To use with operating sheath 11630KF.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:86px;width:215px;">Operating sheath, straight with pyramidal obturator.&#160;</td>
			<td style="width:215px;">KARL STORZ GmbH &#38; Co KG, Tuttlingen, Germany</td>
			<td style="width:367px;">11630 KF</td>
			<td style="width:457px;">Size 9 Fr with working channel 1 mm, for use with 11630AA; working channel for laser fibres up to 400&#181;m core.</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:215px;">Multichannel miniature straight forward telescope 0&#176; set straight for posterior placenta</td>
			<td style="width:215px;">KARL STORZ GmbH &#38; Co KG, Tuttlingen, Germany</td>
			<td style="width:367px;">11506AAK</td>
			<td style="width:457px;">Fetoscope. 0&#176; lens, diameter 3.3 mm, length 30cm , 30,000 pixels, integrated channels, autoclavable, fibre optic light transmission incorporated.&#160;</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:215px;">Multichannel miniature straight forward telescope 0&#176; set curved&#160; for anterior placenta</td>
			<td style="width:215px;">KARL STORZ GmbH &#38; Co KG, Tuttlingen, Germany</td>
			<td style="width:367px;">11508AAK</td>
			<td style="width:457px;">Fetoscope. 0&#176; lens, diameter 3.3 mm, length 30cm , 30,000 pixels, integrated channels, autoclavable, fibre optic light transmission incorporated.&#160;</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:215px;">Dornier diode laser with 400um or 600um laser fibre</td>
			<td style="width:215px;">Medilas D Multibeam, Dornier MedTech Asia, Singapore</td>
			<td style="width:367px;">S/N D60-353</td>
			<td style="width:457px;">Laser photocoagulation system. Diode (30-60 W)&#160;</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:215px;">Laser fibre&#160;</td>
			<td style="width:215px;">400-600&#181;m laser fiber</td>
			<td style="width:367px;">Disposable LG type D01-6080-BF-0;LOT 1024/0613</td>
			<td style="width:457px;">Use the provided ceramic cutter to refashion the tip of the fibre once coagulated after burning to maintain the sharp focus of the laser.&#160;</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:215px;">Large plastic container with ultrasound transparent skin;</td>
			<td style="width:215px;">NA</td>
			<td style="width:367px;">NA.</td>
			<td style="width:457px;">Container is a simple houshold item with a watertight lid that cn be locked in place. The silicon rubber &#34;skin&#34; produced inhouse allows US visualisation of the placenta within the container. Can be used as a simulator for vascular laser coagulation.&#160;</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:215px;">Pig bladder and small mid-gestation placenta&#160;</td>
			<td style="width:215px;">NA</td>
			<td style="width:367px;">NA.</td>
			<td style="width:457px;">Obtained from the local butcher. Elastic tissue that can be stretched when filled with large volume of fluid; can incorporate a small human/NHP placenta and used as a simulator for laser coagulation&#160;</td>
		</tr>
	</tbody>
</table>

model surgical training: skills acquisition in fetoscopic laser photocoagulation of monochorionic diamniotic twin placenta using realistic simulators

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome (TTTS), but is technically challenging and can lead to significant complications. Acquiring and maintaining the necessary surgical skills require consistent practice, a critical caseload, and time. Training on realistic surgical simulators can potentially shorten this steep learning curve and enables several proceduralists to acquire procedure-specific skills simultaneously. Here we describe realistic simulators designed to allow the user familiarity with the equipment and specific steps required in the surgical treatment of TTTS, including fetoscopic handling, approaches to anterior and posterior placenta, recognition of anastomoses, and efficient coagulation of vessels. We describe the skills that are especially important in conducting placental laser coagulation that the surgeon can practice on the model and apply in a clinical case. These models can be adapted easily depending on the availability of materials and require standard fetoscopy equipment. Such training systems are complementary to traditional surgical apprenticeships and can be useful aids for fetal medicine units that provide this clinical service.

The basic requirements for a fetoscopy simulator are a transparent &#34;skin&#34; that enables ultrasound visualization of the placenta within the model and a representative model of the MCDA placenta. The simulator illustrated here was developed at Siriraj Hospital (Bangkok), and is a closed system that incorporates a silicon replica of a mid-gestation monochorionic placenta (Figure 1). The consistent use of this model should increase the confidence of the n...

Watch this Scientific Journal Video about Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators at JoVE.

Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators

Practicing the specific skills required for fetoscopic laser coagulation of monochorionic placental anastomoses on realistic models can aid less experienced surgeons in overcoming the steep learning curve associated with this procedure that is now regarded as the standard of care for twin-twin transfusion syndrome.

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome ...

The overall goal of this surgical training model is to allow novice surgeons to familiarize themselves with the techniques used in selective fetoscopic laser photocoagulation in twin-twin transfusion syndrome, and to refine these techniques prior to surgery in patients. These training models are designed to help fetal surgeons refine their skills in fetoscopic ablation of the communicating vessels which cause twin-twin transfusion syndrome. The main advantage of this process is that the entire procedure can be learned before approaching the patient, which helps to overcome the procedural learning curve when a low case load is a significant limitation.So individuals new to this method will be challenged by the dual use of ultrasound and fetoscope understanding the placenta orientation and navigating the vascular equator. The visual demonstration of these steps is critical as it emphasizes the importance of pre-surgical planning and allows for a better understanding of the surgical process. Begin with setting up the fetoscopy simulator and equipment.First, take note of the 2.0 millimeter fibro straight fetoscope design. There is the lens, which is zero or 30 degrees and has a standard or remote eye cap. Next, it has a three millimeter double lumen operating chief and a sharp obturator tip that gets directly inserted into the amniotic cavity under ultrsound guidance.For operation, remove the obturator and place the fetocope into an operating sheath which makes it semi-flexible and able to curve around to the interior placenta. Then connect the eye cap to the laparoscope camera. Next, position the laparoscope tower and ultrasound machine near each other so that the ultrasound probe can be manipulated while operating the fetoscope.Next, through one of the one way valve ports, fill the fetoscopy simulator to the brim with water. Then, position the simulator on the plastic base as needed. The upper surface of the simulator represents the interior maternal abdomen.Now, use the curvilinear ultrasound probe with aqueous gel to visualize the placenta within the fetoscopy simulator. Identify the placenta location and a window adjacent to it in which to place the fetoscope. Next, select the 0 degrees straight fetoscope for a posterior placenta or the 0 degree curved fetoscope for an anterior placenta.Then, under continuous ultrasound guidance, insert the fetoscope in the operating sheath through the port. Monitor the insertion and depth of the fetoscope by ultrasound and bring the placenta in to view. Adjust the focus of the camera to bring the vision into sharper leaf.Then, systematically explore the vasculature from one end to the other to identify the typical AVA's for ablation. Such anastomosis will be seen runnning along the chorionic surface. The artery is darker and always runs over the vein.Now, reorientate the simulator for a view of the placenta in the opposite orientation and repeat the procedure. For this placenta in a box model, use a standard store bought plastic container that can be locked water tight. Cut out a wide window of the plastic cover and replace it with ultrasound transparent rubber skin stitched to the margins of the cover.This skin forms the interior surface of the simulated maternal abdomen through which the fetoscope is placed. Next, mount a rubber latex sheet along the bottom of the box to prevent sonographic reverberation. At the maternity ward, wash the surface of the donated human placenta with tap water in the sink.Clean all of the blood off of the surface. Then, cut the umbilical cord using strong tissue scissors to a manageable length of about five centimeters. Next, tie the free end of the umbilical cord using suture band or cotton cord tape to prevent blood from running out of the cut end.At the laboratory, to simulate an interior placenta fix the placenta to the refashioned lid of the container with clear plastic thread or a plastic net. To simulate a posterior placenta, hold it in place with a plastic net or small weights. Fix the placenta to the rubber sheet at the bottom of the container after installing either configuration, fill the container with tap water and lock the lid in place.Next, prepare the appropriate fetoscope and it's operating sheath with a working channel. Then, connect the eye cap to the camera. Now, perform an ultrasound assessment of the placenta.For an anterior placenta, find a window into which the fetoscope can be placed such that the lens lies above the center of the placenta. For a posterior placenta, find a window for the fetoscope where the zero degree lens will be perpendicular to the center of the placenta. Now perform direct fetoscopy starting with a two millimeter stab incision into the skin.Then, under continuous ultrasound guidance, insert the operating trocar with its pyramidal obturator into the fluid filled container which replicates the amniotic sac of the recipient twin. To avoid piercing the placenta, advance the fetoscope slowly. Then, slowly remove the pyramidal obturator from the operating trocar.Next, carefully place the lens of the fetoscope into the operating trocar in the channel previously occupied by the obturator. Then, bring the placenta and surface vasculature into sharp focus. Now, insert the laser fiber into the operating side channel.Advance the fiber slowly as the tip nears the end of the sheath, ultimately positioning it five to ten millimeters beyond the operating sheath. If the laser tip advances too far, it may lacerate the vessel. If it doesn't advance far enough, the coagulation effect may be comprimised.The laser fiber tip should be two to three millimeters from the surface of the vessel and should not touch the vessel when the laser is fired. Now, identify the umbilical cord and placental vessels. Review the placental vaculature from end to end using a combination of ultrasound and direct fetoscopic vision.The fetoscope should be directed at a 90 degree angle to the target vessel. When it is perpendicular to the vessel or anastomosis use the foot pedal to fire the laser to coagulate the vessel or anastomosis. Continue coagulating the vessel until in blanches and expect to do this across a one to two centimeter segment for a complete cessation of flow.Afterwards, the vessel should appear flattened and pale. Systematically practice coagulation of vessels from one placental end to the other. Laser coagulate the thick vessels from the periphery towards the center to prevent rupture.A thick turgid vessel can be narrowed down safely in this manner. Also, practice laser coagulating the smaller feeding vessels first before the larger ones to decongest the region and mitigate the chances of a vascular rupture. The basic requirements for a fetoscopy simulator are a transparent skin that enables ultrasound visualization of the placenta within the model and a representative model of the MCDA placenta.Use of this model will familiarize novice surgeons with placental orientation, fetoscope placement and handling of straight and curved fetoscopes. Surgeons should gain ample practice in examining vessels along the full length of the placenta and performing laser coagulations. Complete coagulation is evident through the absence of deep anastomosis.Furthermore, surgeons will become familiar with the common pitfalls such as rapid vascular bleeding and ruptured vessels. After watching this video, you should have a good understanding of placenta orientation on ultrasound and fetoscopic view, how to manipulate the fetoscope correctly to navigate the vascular equator and the safe laser ablation technique. Once mastered, this technique can be done in 40 to 60 minutes, if performed properly.While attempting this procedure, it's important to remember that there is only one instrument and one entry point. There is rarely a chance to correct mistakes and incorrect fetoscopy placement can make it impossible to safely complete laser ablation. To conclude, carefully plan the surgical approach by mapping the placenta on ultrasound and choosing the right entry site.These are the most critical steps to practice on the relevant models before approaching the patient.

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Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...