No funding sources.

1. Preparation of the placenta at delivery
<ol>
	<li>Label the umbilical cords of the twins with one (for the first-born) or two (for the second-born) clamps.</li>
	<li>Inspect the maternal and fetal surface of the placenta for completeness or disruption.</li>
	<li>Record the following data: type of cord insertion (central, eccentric, marginal or velamentous), number of blood vessels in the umbilical cord (usually one vein and two arteries, sometimes only one artery) and color difference between both placental shares. A section of the dividing membranes can be sent to Pathology to confirm the type of chorionicity.</li>
	<li>The placenta can then be placed in a plastic bowl and refrigerated until the final examination (best within one week) and color dye injection.</li>
	<li>The placenta must not be frozen or fixed (do not use formalin).</li>
</ol>
2. Catheterization of the umbilical vessels
<ol>
	<li>Wash the placenta with warm water or saline.</li>
	<li>Trim the peripheral membranes, remove the inter-twin dividing membrane and peel off the amnions (for better visualization of the vascular anastomoses and better quality of the placental pictures).</li>
	<li>Transect each umbilical cord at approximately 5 cm distance from the cord insertion.</li>
	<li>Gently squeeze out blood clots from the umbilical vessels and placental vessels.</li>
	<li>The umbilical vein is usually easy to identify due to its larger diameter, compared to the smaller diameter of the two umbilical arteries.</li>
	<li>Cannulate the umbilical vein with an appropriately sized catheter. Avoid false passages.</li>
	<li>Cannulate one umbilical artery with a smaller catheter. Use tweezers to widen the lumen of the umbilical artery. Avoid false passages. Only one of the 2 umbilical arteries needs to be catheterized since an anastomosis (of Hyrtl) connects the 2 arteries near the cord insertion.</li>
	<li>Repeat both steps for the other umbilical cord.</li>
	<li>Placement of the catheter can be facilitated by gentle back and forth massaging of the umbilical vessels. Any type of catheter can be used for this procedure. We choose to use (and recycle) the catheters used at our neonatology ward for umbilical catheterization in neonates.</li>
	<li>Tie a piece of tape around both cords to avoid back flow of the colored dye during dye injection.</li>
</ol>
3. Injection with colored dye
<ol>
	<li>Connect a 20 ml syringe filled with colored dye to each catheter.</li>
	<li>Any viscous colored dye can be used to visualize the placental angio-architecture. Use contrasting colors to allow good visualization of the anastomoses (dark colors for the arteries, bright colors for the veins).</li>
	<li>Gently inject (with low pressure) the colored dye in the vein while an assistant gently pushes the dye to allow the colored dye to fill all placental vessels, also the smallest ones.</li>
	<li>Pay particular attention to the small vessels near the vascular equator (the vascular equator is the place where the anastomoses from either twin connect with each other).</li>
	<li>Repeat the previous steps to inject colored dye into the artery. Of note: arteries may be more difficult to inject and require more patience.</li>
	<li>Repeat both steps for the other umbilical cord.</li>
</ol>
4. Evaluation and documentation of the placenta after colored dye injection
<ol>
	<li>Carefully examine the vascular equator and record the number and types of anastomoses.</li>
	<li>Place a measuring tape on the placenta to measure the diameters and placental shares on the digital picture.</li>
	<li>Use a high-resolution digital camera and take pictures of the injected placenta. Make sure that the pictures are taken perpendicular to the placenta.</li>
</ol>
5. Representative Results:
The placental angio-architecture in monochorionic twins varies according to the type of monochorionic twin pregnancy. Injection studies have demonstrated that AA, AV and VV anastomoses are present in respectively 80%, 95% and 20% of uncomplicated monochorionic twin pregnancies1 (Figure 1). AA anastomoses are considered to protect against the development of TTTS and TAPS6. Injections studies have shown that AA anastomoses occur in only 20% of TTTS placentas and 10% of TAPS placentas1,2,7 (Figure 2). In TTTS placentas treated with fetoscopic laser surgery, the scars caused by laser coagulation of the vascular anastomoses can be seen on the placental surface (Figure 3 and 4). TAPS placentas are characterized by the presence of only a few minuscule AV anastomoses2 (Figure 5). The placental share of the TAPS recipient is often plethoric, whereas the placental share of the donor is pale (Figure 6). In monochorionic twin pregnancies with birth weight discordance, the growth restricted fetus often has a velamentous cord insertion and a much smaller placental share (Figure 7). AA anastomoses are often present in monochorionic placentas from twins with birth weight discordance8. Monochorionic-monoamniotic placentas have a characteristic angio-architecture with a short distance between both cord insertions (Figure 8). The incidence of AA anastomoses in monoamniotic-monochorionic placentas is virtually 100% and prevents the development of TTTS in monoamniotic twins9. The figures in this article show the characteristic findings of monochorionic placentas injected at our center with color dye. We routinely use darker colors (blue or green) for arteries and lighter colors (yellow, pink or orange) for veins.
<img alt="Human placental vascular anatomy, diagram highlighting umbilical vessels, colored pathways for study." src="/files/ftp_upload/3208/3208fig1.jpg" data-altupdatedat="1747929244000" data-alt="Placental vascular network, diagram of blood vessels, fetal-maternal nutrient exchange, anatomical study."> 
Figure 1. Monochorionic placenta from a normal, uncomplicated monochorionic twin pregnancy showing several AV anastomoses from green arteries to pink veins (white stars), several VA anastomoses from blue arteries to yellow veins (green stars) and 1 large AA anastomosis (identified by the mixing of the blue and green dye; blue star).
<img alt="Placenta anatomy diagram with highlighted blood vessels and clamp tools, showcasing fetal circulation." src="/files/ftp_upload/3208/3208fig2.jpg" data-altupdatedat="1747929244000" data-alt="Human placenta anatomy; blood vessels color-coded; educational diagram; vascular system."> 
Figure 2. Monochorionic placenta in a TTTS pregnancy treated with serial amnioreduction showing the presence of only AV (white stars) and VA (green stars) anastomoses without an AA anastomosis.
<img alt="Human placental vascular system diagram with color-coded arteries and veins." src="/files/ftp_upload/3208/3208fig3.jpg" data-altupdatedat="1747929244000" data-alt="Placenta anatomy with color-coded arteries and veins, educational diagram for vascular study."> 
Figure 3. TTTS placenta after fetoscopic laser coagulation of the vascular anastomoses using the selective laser technique in which a small residual anastomosis was inadvertently left patent (white star). With the selective laser technique, the vascular anastomoses are first identified and subsequently coagulated one by one.
<img alt="Human placenta with colored latex-injected vessels, anatomical study, vascular structure diagram." src="/files/ftp_upload/3208/3208fig4.jpg" data-altupdatedat="1747929244000" data-alt="Colored vascular anatomy in placenta; diagram, blood vessel network, educational biology tool."> 
Figure 4. TTTS placenta after fetoscopic laser coagulation using the Solomon technique in which, after identification and coagulation of each individual anastomosis, the complete vascular equator is coagulated from one placental margin to the other.
<img alt="Human placenta anatomy; vascular network diagram; biological development study." src="/files/ftp_upload/3208/3208fig5.jpg" data-altupdatedat="1747929244000" data-alt="Human placenta anatomy with vessels, lab study, observed structure and branching pattern analysis."> 
Figure 5. TAPS placenta in which only a few, minuscule VA anastomoses (white stars) are visible along the vascular equator.
<img alt="Placenta morphology study, dissected tissue photo, dual coloration for structural analysis." src="/files/ftp_upload/3208/3208fig6.jpg" data-altupdatedat="1747929244000" data-alt="Two-tone placenta on green background, illustrating placental health and vascular distribution."> 
Figure 6. The maternal side of the TAPS placenta (shown in Figure 5) demonstrates the characteristic color difference between both placental shares.
<img alt="Human placenta showing blood vessel network, dissected tissue educational diagram." src="/files/ftp_upload/3208/3208fig7.jpg" data-altupdatedat="1747929244000" data-alt="Human placenta anatomy with visible blood vessels, surgical dissection, educational biology diagram."> 
Figure 7. Monochorionic placenta of a twin pregnancy with selective intrauterine growth restriction of one twin. The growth restricted fetus has a velamentous cord insertion and a much smaller placental share. The white star indicates an AA anastomosis.
<img alt="Human placental blood vessels network diagram; educational anatomy study, vascular system." src="/files/ftp_upload/3208/3208fig8.jpg" data-altupdatedat="1747929244000" data-alt="Human placenta with colored vascular system; anatomical diagram for educational purposes."> 
Figure 8. Monoamniotic placenta: Note the various AV (green star), VA (blue star) and the 2 AA anastomoses (white stars), and the short distance between both cord insertions.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+anastomoses+in+twin-to-twin+transfusion+syndrome+treated+with+selective+fetoscopic+laser+surgery:+localization,+size,+and+consequences.">Residual anastomoses in twin-to-twin transfusion syndrome treated with selective fetoscopic laser surgery: localization, size, and consequences.</a> Am. J. Obstet. Gynecol. , (2009).</li><li>Lewi, L., Jani, J., Cannie, M. <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>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> Am. J. Obstet. Gynecol. 182, 417-426 (2000).</li><li>van Meir, H., Slaghekke, F., Lopriore, E., van Wijngaarden, W. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoamniotic-versus+diamniotic-monochorionic+twin+placentas:+anastomoses+and+twin-twin+transfusion+syndrome.">Monoamniotic-versus diamniotic-monochorionic twin placentas: anastomoses and twin-twin transfusion syndrome.</a> Am. J. Obstet. Gynecol. 189, 1325-1329 (2003).</li><li>Senat, M. V., Deprest, J., Boulvain, M., Paupe, A., Winer, N., Ville, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+laser+surgery+versus+serial+amnioreduction+for+severe+twin-to-twin+transfusion+syndrome.">Endoscopic laser surgery versus serial amnioreduction for severe twin-to-twin transfusion syndrome.</a> N. Engl. J. Med. 351, 136-144 (2004).</li><li>Lopriore, E., Sueters, M., Middeldorp, J. M., Vandenbussche, F. P., Walther, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemoglobin+differences+at+birth+in+monochorionic+twins+without+chronic+twin-to-twin+transfusion+syndrome.">Haemoglobin differences at birth in monochorionic twins without chronic twin-to-twin transfusion syndrome.</a> Prenat. Diagn. 25, 844-850 (2005).</li><li>Papathanasiou, D., Witlox, R., Oepkes, D., Walther, F. J., Bloemenkamp, K. W., Lopriore, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monochorionic+twins+with+ruptured+vasa+previa:+double+trouble!.">Monochorionic twins with ruptured vasa previa: double trouble!.</a> Fetal Diagn. Ther. 28, 48-50 (2010).</li><li>Weingertner, A. S., Kohler, A., Kohler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+placental+characteristics+in+four+new+cases+of+twin+anemia-polycythemia+sequence.">Clinical and placental characteristics in four new cases of twin anemia-polycythemia sequence.</a> Ultrasound Obstet. Gynecol. 35, 490-494 (2010).</li><li>Lewi, L., Cannie, M., Blickstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+sharing,+birthweight+discordance,+and+vascular+anastomoses+in+monochorionic+diamniotic+twin+placentas.">Placental sharing, birthweight discordance, and vascular anastomoses in monochorionic diamniotic twin placentas.</a> Am. J. Obstet. Gynecol. 197, 587-588 (2007).</li><li>Lewi, L., Gucciardo, L., Huber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcome+and+placental+characteristics+of+monochorionic+diamniotic+twin+pairs+with+early-+and+late-onset+discordant+growth.">Clinical outcome and placental characteristics of monochorionic diamniotic twin pairs with early- and late-onset discordant growth.</a> Am. J. Obstet. Gynecol. , (2008).</li><li>De Paepe, M. E., Friedman, R. M., Poch, M., Hansen, K., Carr, S. R., Luks, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+findings+after+laser+ablation+of+communicating+vessels+in+twin-to-twin+transfusion+syndrome.">Placental findings after laser ablation of communicating vessels in twin-to-twin transfusion syndrome.</a> Pediatr. Dev. Pathol. 7, 159-165 (2004).</li><li>De Paepe, M. E., DeKoninck, P., Friedman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+distribution+patterns+in+monochorionic+twin+placentas.">Vascular distribution patterns in monochorionic twin placentas.</a> Placenta. 26, 471-475 (2005).</li><li>Nikkels, P. G., Hack, K. E., vanGemert, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+twin+placentas+with+special+attention+to+monochorionic+twin+placentas.">Pathology of twin placentas with special attention to monochorionic twin placentas.</a> J. Clin. Pathol. 61, 1247-1253 (2008).</li><li>Quintero, R. A., Martinez, J. M., Lopez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+placental+territories+after+selective+laser+photocoagulation+of+communicating+vessels+in+twin-twin+transfusion+syndrome.">Individual placental territories after selective laser photocoagulation of communicating vessels in twin-twin transfusion syndrome.</a> Am. J. Obstet. Gynecol. 192, 1112-1118 (2005).</li><li>Quintero, R. A., Comas, C., Bornick, P. W., Allen, M. H., Kruger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+versus+non-selective+laser+photocoagulation+of+placental+vessels+in+twin-to-twin+transfusion+syndrome.">Selective versus non-selective laser photocoagulation of placental vessels in twin-to-twin transfusion syndrome.</a> Ultrasound Obstet. Gynecol. 16, 230-236 (2000).</li></ol>

No conflicts of interest declared.

TTTS and TAPS are two severe disorders that can occur in monochorionic twins. Both disorders are due to AV anastomoses causing imbalanced intertwin blood flow. In the last 2 decades, fetoscopic laser surgery became available and was shown to be the best method for treatment of TTTS10. The aim of fetoscopic laser treatment is to interrupt the inter-twin circulation through coagulation of the vascular anastomoses on the placental surface. However, the laser treatment for TTTS is far from perfect. Treatment with ...

<table><tbody><tr><td>Umbilical catheters(various dimensions, Length 40 cm)</td><td>Vygon</td><td>1270.08 (8 F) 1270.08 (5 F)1270.04 (4 F)1270.03 (3.5 F)1270.02 (2.5 F)</td><td>Any type of catheter can be used</td></tr><tr><td>20 ml syringes</td><td>BD Biosciences</td><td>300613</td><td></td></tr><tr><td>Color dye(different colors)</td><td>Royal Talens Schoolverf</td><td>36716010 (blue)36715010 (green)36712350 (yellow)36713570 (pink)</td><td>Any viscous color dye can be used</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye at JoVE.com

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

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 ...

accurate-simple-evaluation-vascular-anastomoses-monochorionic

Research

JoVE Journal

Medicine

26.8K Views.  Leiden University Medical Center. Twin-to-twin transfusie syndroom en twin bloedarmoede polycythemia volgorde zijn twee potentieel verwoestende problemen in de perinatale geneeskunde. Beide aandoeningen komen alleen in monochoriale tweeling en het gevolg zijn van onevenwichtige de bloedstroom door de placenta vasculaire anastomosen. Wij bieden een eenvoudig protocol om nauwkeurig te evalueren van de aanwezigheid van vasculaire anastomosen met behulp van kleurstof injectie van de placenta schepen na de geb...

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

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 ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Immunology and Infection

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Developmental Biology

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes1 (i.e. carmine red, India ink) or latex with2 or without3 carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen3. Maeda et al.2 have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound4. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner5. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion5. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.

Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

Neuroscience

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

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 ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

Evaluation of Color Difference in Placenta with Twin Anemia Polycythemia Sequence

Twin anemia polycythemia sequence (TAPS) occurs in 5% of monochorionic twins and is characterized by large inter-twin hemoglobin differences. The postnatal diagnostic criteria for TAPS are based on hematologic parameters and placental characteristics. Placental examination after birth shows that color of the maternal side between placental territories of the anemic and polycythemic twins is remarkably different. The color difference in TAPS placentas is higher compared to monochorionic placentas with acute peripartum feto-fetal transfusion; thus, this is used as an additional diagnostic criterion for TAPS. Software such as ImageJ enables the computer-based measurement of color intensity in TAPS placentas. However, a detailed method for the calculation of color differences between anemic and polycythemic components of TAPS placentas has not yet been described. The protocol presented here provides a step-by-step method for analyzing color differences in the maternal side of TAPS placentas using ImageJ software.

In monochorionic twins with twin anemia polycythemia sequence (TAPS), the donor twin and its corresponding placenta share are pale, while the recipient twin and its placental share have a plethoric aspect. Presented here is a protocol to quantify the color difference in maternal side of TAPS placenta after birth.

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

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