1.	Preparation of the placenta
<ol>
<li>The placenta should be acquired from the delivery room as soon as possible after delivery to minimize clot formation, desiccation and overall deterioration of the tissue.</li>
<li>Care should be taken to ensure that the placenta is not exposed to formalin or other fixatives.</li>
<li>Standard BSL2 precautions for human tissue should be maintained and personnel should wear a mask, gown, gloves and shoe covers.</li>
<li>Place the placenta in a large plastic or metal bowl that has precut holes to allow for the drainage of fluid. This bowl should be placed inside a larger stainless steel bowl to prevent spillage.</li>
<li>Wash the placenta with warm saline.</li>
<li>For better placental visualization, use Metzenbaum scissors to excise the inter-twin amniotic membranes being sure not to injure the placenta itself. Leave a &frac12; cm cuff of inter-twin membrane in place. (Figure 1A).</li>
</ol>
2.	Catheterization of the placental vessels
<ol>
<li>Identify the cords of the respective twins. The obstetricians usually identify each twin by the number of clamps placed on each cord. Be sure to confirm which cord supplied the presenting twin (twin A).</li>
<li>One at a time, keeping track of which cord is which, transect each cord just below the clamps (Figure 1B). Gently milk clot out from the vessels (Figure 1C). At the cut edge of the cord there will be one vein and at least one, usually two, arteries. Arteries are identified by their relatively smaller diameter (compared to the vein) and surrounding muscular cuff (Figure 1D).</li>
<li>Use a standard radial arterial line guide wire, 0.46 mm diameter and 80 cm long (Arrow, Reading PA, USA), with a soft tip to gain access to an arterial orifice on the cut edge of twin A's cord (Figure 1E).</li>
<li>The guide wire should be placed a minimum of 3 cm into the vessel. Gentle counter tension on the nonvascular portion of the cut edge of the cord using a Kelly clamp greatly facilitates advancement of the wire. Minor turning of the cord to straighten the tortuous vessels may help with wire advancement. Care should be taken to avoid false passages. </li>
<li>Using the Seldinger technique, thread a 20 cm long 0.6-1.19 mm inner diameter polyethylene catheter (Intramedic, Batavia IL, USA) with a bluntly beveled distal end over the wire into the artery (Figure 1F). Care must be taken not to dislodge the wire while advancing the catheter. Remove the wire after catheter is seated at least 3 cm into the vessel.</li>
<li>Alternatively, some groups have had success without use of a guidewire and direct cannulation of the umbilical vessels with umbilical artery catheters. Although, this technique works well in our hands the use of a guidewire has decreased the time it takes us to complete the procedure.</li>
<li>Attach an 18 G blunt tip needle (BD Biosciences, Franlik Lakes, NJ, USA) to the proximal end of the tubing. Ensure proper placement of the catheter by injecting a small amount of saline into the vessel and observing clearing of distal arteries. Gentle finger sweeps on the vessels while injecting saline may help facilitate movement of any residual clots. </li>
<li>Carefully repeat steps 2.3 to 2.6 for the vein of twin A with attention to avoid dislodging the arterial catheter.</li>
<li>Tie a piece of umbilical tape 1 cm distal to the cut edge around the cord and catheters. Ensure a tight seal to avoid back flow.</li>
<li>Repeat step 2.3 to 2.9 for the cord of twin B (the non-presenting twin) such that there are catheters in an umbilical artery and vein of each twin secured with umbilical tape and both circulations have been infused with saline (Figure 2).</li>
</ol>
3.	Injection of colored dye into the placental vessels
<ol>
<li>Pathology dye, barium suspensions, gelatin or other viscous colored dyes can be used to visualize the placental vasculature. Choose four contrasting colors with which to inject the arteries and veins of twins A and B. The colors for the artery and vein for each twin should be readily distinguishable. Superficial anastomoses should be detectable by dye mixing and the formation of a different (blended) easily detectable color.</li>
<li>Use a 3ml syringe to hand inject the artery of twin A with dye at low pressure. Gently sweep with one or two fingers along the vessels in the direction of dye flow to ensure filling of the entire vascular tree. If a superficial vascular anastomosis is identified, manual occlusion of the feeding vessel can be employed to allow the rest of the higher resistance vascular bed to fill (Figure 3A).</li>
<li>Use a separate 3ml syringe to hand inject the artery of twin B (Figure 3B). </li>
<li>Repeat steps 3.2 and 3.3 for the venous injections.</li>
<li>If dye leaks from an injured vessel, repair should be performed with a fine suture and a needle driver. Take care not to suture ligate the whole vessel.</li>
<li>Using a high-resolution digital camera take an image after each injection. After all injections are complete, remove the specimen from the bowls, place against a color neutral background and re-image (Figure 4). Document the number and type of connections seen between the twins' circulations. </li>
</ol>
4.	Representative Results:
Artery to artery anastomoses (AAs) have been shown to be "protective" as they are associated with a significant decreased risk of TTTS and with improved outcomes for both twins5. Figure 4 demonstrates an injected MC twin placenta. An AA had been prospectively identified by in utero Doppler ultrasound. This finding was confirmed by the mixing of twin A arterial (yellow) and twin B arterial (blue) dye in a communicating arterial structure (green, arrow). An AV anastomosis with artery from twin A (yellow) and vein from twin B (orange) is also demonstrated (arrow head). Figure 5 and 6 depict photographs of two other placental specimens we injected.
<img src="/files/ftp_upload/2837/2837fig1.jpg" alt="Figure 1"/> Figure 1. (A) The amniotic membrane is carefully removed from the fetal surface of the shared MC placenta with Metzenbaum scissors. (B) A clean edge of the umbilical cord of twin A is exposed by proximal transection with Metzenbaum scissors. (C) Clot is milked from the cord by manual compression. (D) The umbilical arteries (arrowheads) and vein (arrow) of twin A are identified. (E) After placement of a Kelly clamp on the avascular portion of the cut surface of the cord for traction, a guide wire is placed in the umbilical artery of twin A and gently advanced through the tortuous vessel. (F) A small catheter is then advanced over the guidewire into the vessel lumen using the Seldinger technique.
<img src="/files/ftp_upload/2837/2837fig2.jpg" alt="Figure 2"/> Figure 2. Catheters have been placed in one umbilical artery and in the umbilical vein of each twin. These catheters are secured in place with umbilical tape ties around the entire cord. The correct positioning of each catheter has been confirmed and intraluminal clots have been cleared by the injection of warmed normal saline.
<img src="/files/ftp_upload/2837/2837fig3.jpg" alt="Figure 3"/> Figure 3. (A) The arterial circulation of twin A is injected with yellow dye. Contrast clearly passes across the vascular equator into the arterial circulation of twin B. (B) The arterial circulation of twin B is injected with blue dye. Admixing of the yellow dye and blue dye results in green dye within the demonstrated AA anastomosis (arrow).
<img src="/files/ftp_upload/2837/2837fig4.jpg" alt="Figure 4"/> Figure 4. The vein of twin A (red) and the vein of twin B (orange) have been injected. The AA anastomosis (arrow) and an AV anastomosis, with flow directed from twin A toward twin B, (arrowhead) are delineated.
<img src="/files/ftp_upload/2837/2837fig5.jpg" alt="Figure 5"/> Figure 5. Example of MC twin placenta that underwent laser therapy after postnatal injection. The donor twin's artery and vein are in yellow and green respectively. The recipient artery and vein are in blue and orange respectively. No residual connections between the circulations were detected.
<img src="/files/ftp_upload/2837/2837fig6.jpg" alt="Figure 6"/> Figure 6. Example of MC twin placenta after injection. The left sided twin's artery and vein are in yellow and red respectively. The right-sided twin's artery and vein are in blue and orange respectively. An AA (arrowhead) and VV (arrow) are visible on the placental surface.

<ol>
	<li>Rand, L., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complicated+monochorionic+twin+pregnancies:+updates+in+fetal+diagnosis+and+treatment.">Complicated monochorionic twin pregnancies: updates in fetal diagnosis and treatment.</a> Clin Perinatol. 36, 417-430 (2009).</li><li>Sebire, N., Talbert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twin-to-twin+transfusion+syndrome+results+from+dynamic+asymmetrical+reduction+in+placental+anastomoses:+a+hypothesis.">Twin-to-twin transfusion syndrome results from dynamic asymmetrical reduction in placental anastomoses: a hypothesis.</a> in Placenta. , (2001).</li><li>Machin, G. A., Keith, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An atlas of multiple pregnancy: biology and pathology. , (1999).</li><li>Machin, G. A., Feldstein, V. A., van Gemert, M. J., Keith, L. G., Hecher, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+sonographic+demonstration+of+arterio-venous+anastomosis+in+monochorionic+twin+gestation.">Doppler sonographic demonstration of arterio-venous anastomosis in monochorionic twin gestation.</a> Ultrasound Obstet Gynecol. 16, 214-217 (2000).</li><li>Denbow, M. L., Cox, P., Taylor, M., Hammal, D. M., Fisk, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+angioarchitecture+in+monochorionic+twin+pregnancies:+Relationship+to+fetal+growth,+fetofetal+transfusion+syndrome,+and+pregnancy+outcome.">Placental angioarchitecture in monochorionic twin pregnancies: Relationship to fetal growth, fetofetal transfusion syndrome, and pregnancy outcome.</a> American Journal of Obstetrics and Gynecology. 182, 417-426 (2000).</li><li>Taylor, M. J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+detection+of+arterio-arterial+anastomoses+in+monochorionic+twins:+feasibility+and+clinical+application.">Doppler detection of arterio-arterial anastomoses in monochorionic twins: feasibility and clinical application.</a> Human Reproduction. 15, 1632-1636 (2000).</li><li>Berm&#250;dez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+types+and+twin-twin+transfusion+syndrome.">Placental types and twin-twin transfusion syndrome.</a> American Journal of Obstetrics and Gynecology. 187, 489-494 (2002).</li><li>Lewi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intertwin+anastomoses+in+monochorionic+placentas+after+fetoscopic+laser+coagulation+for+twin-to-twin+transfusion+syndrome:+is+there+more+than+meets+the+eye.">Intertwin anastomoses in monochorionic placentas after fetoscopic laser coagulation for twin-to-twin transfusion syndrome: is there more than meets the eye.</a> Am J Obstet Gynecol. 194, 790-795 (2006).</li><li>Wee, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+deep+arterio-venous+anastomoses+within+monochorionic+placentae+by+vascular+casting.">Characterisation of deep arterio-venous anastomoses within monochorionic placentae by vascular casting.</a> Placenta. 26, 19-24 (2005).</li><li>Paepe, M. E. D. e., Burke, S., Luks, F. I., Pinar, H., Singer, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+placental+vascular+anatomy+in+monochorionic+twin+gestations.">Demonstration of placental vascular anatomy in monochorionic twin gestations.</a> Pediatr Dev Pathol. 5, 37-44 (2002).</li></ol>

No conflicts of interest declared.

Vascular mapping of MC twin placentas by dye injection is a valuable procedure for assessing complicated, clinically-treated cases and for further enhancing our understanding of MC twinning. Coordination with the obstetric team to ensure expeditious acquisition of an intact placental specimen is crucial. Desiccation or prolonged storage of the tissue prior to injection may lead to vessel injury and extravasation of dye. Fixation of the placenta must be avoided, because cross-linked blood clots will block perfusion of int...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent	</th>
			<th>Company	</th>
			<th>Catalogue number	</th>
			<th>Comments(optional)</th>
		</tr>
		<tr>
			<td>Perforated plastic bowl</td>
			<td>QuickMedical
			Medical Equipment and Supplies</td>
			<td>Polarware PA75</td>
			<td>Perforations can be made with a drill</td>
		</tr>
		<tr>
			<td>Stainless steel bowl</td>
			<td>Narang Medical Limited</td>
			<td>HH517</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Fisher</td>
			<td>50983204</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.6-1.9 mm ID polyethylene tubing		</td>
			<td>Intramedic</td>
			<td>7436</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Arterial line guide wire (0.46 mm)			 </td>
			<td>Arrow International</td>
			<td>PW-18080</td>
			<td> Soft tip</td>
		</tr>
		<tr>
			<td>18 G Blunt Needles		</td>
			<td>BD Biosciences</td>
			<td>305180</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Umbilical Tape	</td>
			<td>QC supply</td>
			<td>	140895</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Metzenbaum scissors	</td>
			<td>Cole-Parmer	</td>
			<td>EW-10921-04</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kelly clamp		</td>
			<td>Cole Parmer</td>
			<td>EW-10918-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3 ml syringe		</td>
			<td>Covidien</td>
			<td>1180300555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathology Dye	</td>
			<td>Bradley Products</td>
			<td>	7365-x</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Digital Camera		</td>
			<td>Panasonic</td>
			<td>Lumix-TZ50</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas at JoVE.com

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

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 ...

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Research

JoVE Journal

Medicine

14.8K 조회수.  University of California, San Francisco. 출산 후 monochorionic (MC) 트윈 placentas의 혈관 매핑은 쌍둥이 '순환 사이의 혈관 연결에 대한 자세한 시연을위한 수단을 제공합니다. 이러한 연결의 불균형은 트윈 - 투 - 쌍둥이 수혈 증후군을 포함한 MC의 쌍동이의 합병증의 발전에 중추적인 역할을 생각합니다.

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

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&amp;#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

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 ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

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.

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 ...

Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due to sodium retention and hyperlipidemia. To study the pathophysiology of this syndrome, rodent models have been developed based on the injection of toxic substances such as doxorubicin causing podocyte damage. In mice, only few strains are susceptible to this model. In wildtype 129S1/SvImJ mice, the administration of doxorubicin by rapid intravenous injection to the retrobulbar sinus induces experimental nephrotic syndrome that features all the symptoms of human disease including sodium retention and edema. After the onset of proteinuria, mice exhibit increased urinary serine protease activity that leads to the activation of the epithelial sodium channel (ENaC) and sodium retention. Pharmacological inhibition of urinary serine proteases by the treatment with sustained release aprotinin abrogates ENaC activation and prevents sodium retention. This model is ideal to study the pathophysiology of proteasuria, i.e., the excretion of active serine proteases that cause ENaC activation by the proteolysis of its &#947;-subunit. This can be regarded as the primary mechanism of ENaC activation and sodium retention in proteinuric kidney disease.

Here, we describe the induction of experimental nephrotic syndrome in 129S1/SvImJ mice by rapid retrobulbar injection of doxorubicin. We also treat nephrotic mice with sustained release pellets containing aprotinin to inhibit urinary serine protease activity and prevent sodium retention.

Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...

Brachial Artery Catheterization in Swine

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood pressure continuously and collect arterial blood samples to assess arterial blood gas measurements. Arterial blood pressures and arterial blood gases are important physiological parameters to monitor during experimental procedures. In swine, four common methods of arterial catheterization have been described, including catheterization of the carotid, femoral, auricular, and medial saphenous arteries. Each of these techniques have advantages, such as ease of access for the auricular artery, and disadvantages that include deep tissue dissection for carotid artery catheterization. The described alternative method of arterial catheterization in swine, the catheterization of the distal aspect of the brachial artery, is a rapid procedure that requires relatively minimal tissue dissection and provides information that is in line with data collected from other arterial catheterization sites. The procedure uses a medial approach along an oblique plane of the lower brachium, positioned between the olecranon and the flexor aspect of the elbow joint, and this approach allows researchers the major advantage of unimpeded freedom for procedures that involve the caudoventral, caudodorsal back, or hind limbs of the pig. Due to the location of the upper forelimb of the catheterized vessel and potential challenges of effective homeostasis following catheter removal from the artery, this technique may be limited to non-recovery procedures.

The video describes in detail the catheterization of the distal brachial artery in swine. This procedure accurately measures arterial blood pressure and is a simple and fast method to collect samples for arterial blood gas measurements.

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...