Name: the 4-vessel sampling approach to integrative studies of human placental physiology in vivo
Uploaded: 2017-08-02T12:30:00
Description: Watch this Scientific Journal Video about The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo at JoVE.com

First and foremost, we sincerely thank the mothers who participated in this project. Next, we acknowledge all the personnel that assisted and facilitated the sampling procedure, the Anesthesiologist, the nurse anesthesiologist and the surgical nurses. The project would not have been possible without funding from the South-Eastern Norway Regional Health Authority and Norwegian Advisory Unit on Women's Health, Oslo University and local funding provided by Oslo University Hospital.

The study was approved by the data protection officials at Oslo University Hospital and the Regional Committee for Medical and Health Research Ethics, Southern Norway 2419/2011. All participants signed a written informed consent at inclusion.
1. Preparations
NOTE: A timeline for the procedures is outlined in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55847/55847fig1.jpg" /> 
<stro...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+arachidonic+acid+in+plasma+and+tissues+of+patients+near+term+undergoing+elective+or+emergency+Caesarean+section.">The distribution of arachidonic acid in plasma and tissues of patients near term undergoing elective or emergency Caesarean section.</a> Br J Obstet Gynaecol. 85 (2), 119-123 (1978).</li><li>Haberey, P. P., Schaefer, A., Nisand, I., Dellenbach, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+and+importance+of+fetal+lactate+in+the+human+placenta+-a+new+hypothesis.">The fate and importance of fetal lactate in the human placenta -a new hypothesis.</a> J Perinat Med. 10 (2), 127-129 (1982).</li><li>Prendergast, C. 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M., Henriksen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+glucose+transfer:+a+human+in+vivo+study.">Placental glucose transfer: a human in vivo study.</a> PLoS One. 10 (2), 0117084 (2015).</li><li>Holme, A. M., Roland, M. C., Henriksen, T., Michelsen, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+uteroplacental+release+of+placental+growth+factor+and+soluble+Fms-like+tyrosine+kinase-1+in+normal+and+preeclamptic+pregnancies.">In vivo uteroplacental release of placental growth factor and soluble Fms-like tyrosine kinase-1 in normal and preeclamptic pregnancies.</a> Am J Obstet Gynecol. 215 (6), 781-782 (2016).</li><li>Paasche Roland, M. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uteroplacental+unit+as+a+source+of+elevated+circulating+prorenin+levels+in+normal+pregnancy.">Uteroplacental unit as a source of elevated circulating prorenin levels in normal pregnancy.</a> Am J Obstet Gynecol. 155 (6), 1223-1226 (1986).</li><li>Myatt, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategy+for+standardization+of+preeclampsia+research+study+design.">Strategy for standardization of preeclampsia research study design.</a> Hypertension. 63 (6), 1293-1301 (2014).</li><li>Kiserud, T., Rasmussen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+repeat+measurements+affect+the+mean+diameter+of+the+umbilical+vein+and+the+ductus+venosus.">How repeat measurements affect the mean diameter of the umbilical vein and the ductus venosus.</a> Ultrasound Obstet Gynecol. 11 (6), 419-425 (1998).</li><li>Burton, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+sample+collection+for+placental+research.">Optimising sample collection for placental research.</a> Placenta. 35 (1), 9-22 (2014).</li><li>Illsley, N. P., Wang, Z. Q., Gray, A., Sellers, M. C., Jacobs, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+preparation+of+paired,+syncytial,+microvillous+and+basal+membranes+from+human+placenta.">Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta.</a> Biochim Biophys Acta. 1029 (2), 218-226 (1990).</li><li>Staff, A. C., Ranheim, T., Khoury, J., Henriksen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+contents+of+phospholipids,+cholesterol,+and+lipid+peroxides+in+decidua+basalis+in+women+with+preeclampsia.">Increased contents of phospholipids, cholesterol, and lipid peroxides in decidua basalis in women with preeclampsia.</a> Am J Obstet Gynecol. 180 (3), 587-592 (1999).</li><li>Catalano, P. M., Thomas, A. J., Avallone, D. A., Amini, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthropometric+estimation+of+neonatal+body+composition.">Anthropometric estimation of neonatal body composition.</a> Am J Obstet Gynecol. 173 (4), 1176-1181 (1995).</li><li>Ellis, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body-composition+assessment+in+infancy:+air-displacement+plethysmography+compared+with+a+reference+4-compartment+model.">Body-composition assessment in infancy: air-displacement plethysmography compared with a reference 4-compartment model.</a> Am J Clin Nutr. 85 (1), 90-95 (2007).</li><li>Haugen, G., Kiserud, T., Godfrey, K., Crozier, S., Hanson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+and+umbilical+venous+blood+supply+to+the+liver+in+the+human+fetus+near+term.">Portal and umbilical venous blood supply to the liver in the human fetus near term.</a> Ultrasound Obstet Gynecol. 24 (6), 599-605 (2004).</li><li>Acharya, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+validation+of+uterine+artery+volume+blood+flow+measurement+by+Doppler+ultrasonography+in+pregnant+sheep.">Experimental validation of uterine artery volume blood flow measurement by Doppler ultrasonography in pregnant sheep.</a> Ultrasound Obstet Gynecol. 29 (4), 401-406 (2007).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate-glutamine+cycle+and+exchange+in+the+placenta-fetus+unit+during+late+pregnancy.">Glutamate-glutamine cycle and exchange in the placenta-fetus unit during late pregnancy.</a> Amino Acids. 47 (1), 45-53 (2015).</li><li>Tuckey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+synthesis+by+the+human+placenta.">Progesterone synthesis by the human placenta.</a> Placenta. 26 (4), 273-281 (2005).</li><li>Simmons, M. A., Meschia, G., Makowski, E. L., Battaglia, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+metabolic+response+to+maternal+starvation.">Fetal metabolic response to maternal starvation.</a> Pediatr Res. 8 (10), 830-836 (1974).</li><li>Simmons, M. A., Jones, M. D., Battaglia, F. C., Meschia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+effect+on+fetal+glucose+utilization.">Insulin effect on fetal glucose utilization.</a> Pediatr Res. 12 (2), 90-92 (1978).</li><li>Bujold, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+supporting+that+the+excess+of+the+sVEGFR-1+concentration+in+maternal+plasma+in+preeclampsia+has+a+uterine+origin.">Evidence supporting that the excess of the sVEGFR-1 concentration in maternal plasma in preeclampsia has a uterine origin.</a> J Matern Fetal Neonatal Med. 18 (1), 9-16 (2005).</li><li>Jansson, T., Aye, I. L., Goberdhan, D. 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The placenta 4-vessel sampling method is relevant for three main purposes. First, it can be used to study how specific substances are taken up by the placenta on the maternal side and possibly transferred to the umbilical circulation and the fetus, as demonstrated by our glucose and amino acid studies. Second, the method is highly relevant to study substances produced by the placenta and released to the maternal or the fetal circulations, as demonstrated by the progesterone results. Third, it may be useful to study how t...

According to the National Institutes of Health, USA, the placenta is the least understood organ in the human body 1,2,3. It is difficult to access and study the human placenta in vivo without imposing unethical risks on the ongoing pregnancy. Studies of placental function in the human are therefore largely based on in vitro and ex vivo models. The majority of previous in vivo studies of placental transport and metabolism have been performed in animals 4,5,6. However, as placental structure and functions vary considerably between species, extrapolation of results from animals to humans must be done with caution. Only a few smaller human in vivo studies have investigated placental and fetal uptake and transport under normal physiological conditions, and none have explored the integrated transfer of several compounds 7,8,9,10,11,12,13. These fundamental studies illustrate that in vivo studies of the human placenta are feasible, and that they may serve several purposes. First, current concepts of placental functions mainly derived from in vitro, ex vivo and animal studies may be tested in a human setting and thus provide novel and more specific insight into the human placenta. Second, properties of the dysfunctional placenta associated with aberrant fetal growth, preeclampsia, maternal diabetes, metabolic syndrome and other maternal metabolic disturbances may be better characterized. Third, human in vivo studies provide an opportunity to develop diagnostic and predictive tools of placental function.
On this background we aimed to establish a comprehensive collection of physiological data to investigate human placental function in vivo. During a planned cesarean section, we exploit the intraabdominal access to the uterine vein to collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta (the 4-vessel sampling method). These samples are used to calculate the paired arteriovenous concentration differences of nutrients and other substances 14. In addition, we measure volume blood flow on both sides of the placenta by ultrasound. Consequently, placental and fetal uptake of any compound may be quantified. Further, it is possible to determine substances released by the placenta to the maternal and fetal circulations 15,16,17. When combined with clinical parameters of mother and child, and analyses of placental and other relevant tissues, this method has the exciting potential to integrate many aspects of placental functions in vivo in the same mother-fetus pairs.

<table border="0" cellpadding="0" cellspacing="0" width="1323">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:557px;">Maternal body composition</td>
			<td style="width:173px;"></td>
			<td style="width:107px;"></td>
			<td style="width:487px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Impedance scale</td>
			<td>Tanita</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound measurements&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sequoia 512 ultrasound machine</td>
			<td>Acuson</td>
			<td></td>
			<td>equipped with a curved transducer with colour and pulsewave Doppler (frequency bandwidth 2-6 MHz)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blood samples</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arerial cannula</td>
			<td>BD Medical</td>
			<td>682245</td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20cc Eccentric Luer Tip Syringe without Needle</td>
			<td>Termo</td>
			<td>SS-20ES</td>
			<td>or similar. 3 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cc Eccentric Luer Tip Syringe without Needle</td>
			<td>Termo</td>
			<td>SS-10ES</td>
			<td>or similar. 9 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5cc 6% Luer Syringe without Needle</td>
			<td>Termo</td>
			<td>SS-05S1</td>
			<td>or similar. 2 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arterial blood gas syringe&#160;</td>
			<td>Radiometer Medical</td>
			<td></td>
			<td>or similar. 4 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile winged needle connected to flexible tubing, 21 gauge</td>
			<td>Greiner Bio-One</td>
			<td>450081</td>
			<td>(intended for single use).3 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer tube 6 mL EDTA&#160;</td>
			<td>Greiner Bio-One</td>
			<td>456043</td>
			<td>or similar. Label with sample site. 10 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer tube 5 ml LH Lithium Heparin Separator</td>
			<td>Greiner Bio-One</td>
			<td>456305</td>
			<td>or similar. Label with sample site. 5 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer tube 6 mL Serum Clot Activator&#160;</td>
			<td>Greiner Bio-One</td>
			<td>456089</td>
			<td>or similar. Label with sample site. 5 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:557px;">Vacutainer tube 3 ml&#160; 9NC Coagulation sodium citrate 3,2%</td>
			<td>Greiner Bio-One</td>
			<td>454334</td>
			<td>or similar. Label with sample site. 5 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryogenic vials, 2.0 mL</td>
			<td>Corning</td>
			<td>430488</td>
			<td>or similar. Label with sample site, serum/type of plasma and ID. 90 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Marked trays to transport the syringes</td>
			<td></td>
			<td></td>
			<td>to transport the blood samples in the operation theatre</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rocker</td>
			<td></td>
			<td></td>
			<td>for gentle mixing of the samples</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td></td>
			<td></td>
			<td>in styrofoam box</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>in appropriate container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Placenta samples</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal tray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td></td>
			<td></td>
			<td>in styrofoam box</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal ruler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>or similar. Dilute 10 M to&#160; 1M before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA stabilization solution</td>
			<td>Sigma</td>
			<td>R0901-500ML&#160;</td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optimal Cutting Temperature (O.C.T.) compound</td>
			<td>vwr</td>
			<td>361603E</td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryogenic vials, 2.0 mL</td>
			<td>Corning</td>
			<td>430488</td>
			<td>or similar. Label with sample site. content and ID. 10 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge tubes, conical bottom 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>227,285</td>
			<td>or similar. Label with &#34;RNA later&#34;, sample site and ID. 2 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>in appropriate container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal body composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Measuring tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the 4-vessel sampling approach to integrative studies of human placental physiology in vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel sampling method is applicable in clinical practice and we have successfully obtained blood samples from 209 mother/infant-pairs. In 128 of these we also achieved to measure volume blood flow. Complete 4-vessel sampling and good quality flow measurements of both maternal and fetal vessels were obtained in 70 mother-fetus pairs (Figure 3). In addition, we have so far collected blood and placenta samples from 30 preeclamptic patients. We have previo...

Watch this Scientific Journal Video about The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo at JoVE.com

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

The overall goal of this procedure is to study human placental function in vivo using several methods, which together ensure the collection of comprehensive and integrative data of human fetal placental physiology. This method can help answer key questions about human placental physiology, such as the role of placenta in maternal-fetal interplay. The main advantage of this technique is that it makes it possible to test current concepts of placental transfer, uptake, and secretion in humans in vivo.Extrapolation of experimental studies to humans is hampered by the large differences between animals and humans in placental anatomy, duration of pregnancy, and pregnancy complications. Though this method can provide insight into placental metabolism and transfer of nutrients, it can also be applied to study placental release of compounds both in healthy and in complicated pregnancies, such as preeclampsia. This method requires a highly coordinated cooperation of several medical specialists, nurses, and researchers.The key to success is strict planning while all the time keeping the mother and her baby in focus. This chart illustrates the timeline and the personnel required to obtain the samples. Each individual assisting in the sampling is represented by a different color.This protocol consists of the combined use of three methods to study human placental physiology. First volume blood flow in the uterine artery and umbilical vein are performed by ultrasound. Second blood samples are collected from the incoming and outgoing vessels on the maternal and fetal sides of the human placenta during cesarean section.Third placenta tissue samples are collected for analysis. To begin, assist the woman into a semi-supine position, tilted slightly laterally opposite to the region of interest, in order to avoid compression of the aorta and vena cava. During a period of fetal quiescence, visualize the umbilical vein in a sagittal or oblique transection of the fetal abdomen and measure the internal vessel diameter in a straight portion of the intra-abdominal umbilical vein before any visible branches.Use regular B-mode and visual the vessel in a perpendicular insonation angle for diameter measurements and keep several optimal frames for later measurements to minimize the effect of pulsatile diameter changes. Repeat the measurements five to 10 times. At the same site, adjust the probe and use Doppler ultrasound to get an insonation angle as low as possible in order to measure the time-averaged maximum velocity or TAMX.Obtain the velocity over a period of three to five seconds. Next, use Doppler ultrasound to visualize the uterine artery as it crosses the external iliac artery immediately after it branches from the internal iliac artery. Adjust the probe at this site to get a low insonation angle and measure TAMX.Obtain the velocity as the mean velocity of three heart cycles. Follow the vessel distally to get a correct angle for diameter measurements as close to the sites of TAMX measurements as achievable. It is challenging to measure volume blood flow in the uterine artery because it is unlikely to get a perpendicular angle at the same site as TAMX is measured.Vessels that branch off between the two sites of measurements may result in exclusion of the diameter measurements. To carry out four-vessel blood sampling before the onset of surgery, prepare the solutions, tubes, and equipment. Then give a briefing and hand the equipment to all personnel that will assist with the sampling.Place an arterial line in the radial artery in addition to the standard peripheral intravenous access. The antecubital vein is preferable because it is easier to draw samples from this site. Next, after opening the abdominal cavity, use a retractor to lift the abdominal wall and expose the main branches of the uterine veins on the anterolateral sides of the uterus.Identify a uterine vein branch on the same side as the placenta whenever possible or the most prominent vein plexus of the placenta is located in the uterine midline. When a suitable uterine vein has been identified, instruct the assisting personnel to draw blood samples from the radial artery and antecubital vein. Simultaneously, at a 30 degree angle, insert a butterfly needle on a blood gas syringe in the uterine vein and collect blood through gentle aspiration.While carefully securing the IV position of the butterfly needle, let an assistant replace the filled blood gas syringe with a 20 milliliter syringe. Immediately follow with a 10 milliliter syringe. From the radial artery, the antecubital vein, discard the first five milliliters, and then aspirate three milliliters into a heparin syringe for blood gas analyses.Then aspirate 30 milliliters from each site. When the child is born, begin with a syringe for blood gas analysis and immediately aspirate blood from the umbilical artery without clamping the umbilical cord or delivering the placenta. Follow with three 10 milliliter syringes if possible.When the arterial samples are secured, clamp and cut the cord before sampling from the umbilical vein, obtaining all umbilical samples with the placenta in situ. The four-vessel method requires coordinated blood sampling in addition to quick handling of samples from five different sites simultaneously. Pay attention to the umbilical artery, which is susceptible to contraction, hemolysis, and coagulation.Perform a final inspection of the sampling site on the uterine vein before starting to close the abdomen. Place all the blood gas syringes on ice and use a blood gas analyzer to analyze the samples within five minutes. Immediately transfer the blood samples to vacutainers and rock the plasma tubes for one to two minutes before putting them on ice.Centrifuge the plasma samples as soon as possible and within 30 minutes at 2, 500 times g and six degrees Celsius for 20 minutes. Then carefully aliquot the supernatants to two milliliter cryotubes. Analyze the blood samples for the desired substances and carry out calculations of placental transfer according to the text protocol.Place the placenta flat down on a ice-chilled dissection tray as soon as possible after it has been delivered. Record the weight of the tissue as well as the longest diameter and the diameter 90 degrees to the first measurement. Then record any gross pathology and the number of vessels in the cord.Place the placenta with the maternal surface facing up and identify four to five sampling sites randomly located in each quadrant of the placenta. Use scissors to remove the decidua, cutting away three to five millimeters from the maternal surface. Then collect a one to two centimeters cubed piece of villous tissue from each site.With 50 milliliters of cold, one molar PBS, gently wash the collected tissue. Then divide the tissue into several pieces from each sampling site and aliquot it. Add aliquots of 0.1 to 0.5 centimeters cubed tissue samples to five cryotubes and snap freeze the samples in liquid nitrogen.Add small pieces, 0.1 to 0.2 centimeters cubed in size, to previously prepared tubes containing 25 milliliters of RNA stabilization solution. For histological studies, add 0.5 centimeters cubed pieces to five cryotubes with 0.5 milliliters of OCT, top off with OCT, then mix the contents of the tubes. Freeze the samples at negative 80 degrees Celsius until further analysis.Blood samples from 209 mother-infant pairs have been successfully obtained using the four-vessel sampling method. Volume blood flow was measured in 128 of the samples. Complete four-vessel sampling and good quality flow measurements of both maternal and fetal vessels were obtained in 70 mother-fetus pairs.The four-vessel approach has been used to study placental transfer as exemplified by glucose, shown here. Significant arteriovenous glucose differences were found on both sides of the placenta, demonstrating an in vivo uteroplacental uptake of 0.29 millimoles per liter and a fetal uptake of 0.54 millimoles per liter. A median umbilical venous flow of 196.2 milliliters per minute was measured.Employing the Fick's Principle, a median fetal glucose uptake from the umbilical circulation of 0.10 millimoles per minute was calculated. Using the four-vessel method, the placental uptake of glutamic acid was studied, which is important as a fuel in metabolic pathways and for fetal-placental interconversion of amino acids. Placental uptake of glutamic acid from both the umbilical and the maternal circulation was observed.The in vivo release of progesterone by the placenta at term was also measured and a significant progesterone release into maternal circulation was found, demonstrating how the placental four-vessel sampling method can be used to detect substances released by the placenta. While attempting this procedure, it is important to underscore that volume blood flow measurements should be done by a fetal medicine specialist with experience in this type of sonography in order to obtain accurate measurements. Following this procedure, the blood samples can be analyzed to study placental transfer and uptake or release of the substance of interest, whether it is nutrients, metabolites, signaling molecules, exosomes, waste compounds, drugs, or toxins.Further, analysis of the placenta samples can answer additional questions concerning transfer regulation and placental metabolism. Altogether, this protocol demonstrates how coordinated collections of volume blood flow measurements, blood, and tissue samples can be achieved to gain an integrity in vivo model to study human placental physiology. After watching this video, an obstetrician should have a good understanding of how to safely collect arteriovenous blood samples on both sides of the human placenta during cesarean section.

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Research

JoVE Journal

Medicine

Roux-en-Y Gastric Bypass Operation in Rats

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12.
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

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

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

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

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

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

Full-root Aortic Valve Replacement by Stentless Aortic Xenografts in Patients with Small Aortic Roots

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial valve might not meet the functional needs. The implantation of a too-small stented pericardial valve, leading to an effective orifice area indexed to a body surface area less than 0.85 cm2/m2, is regarded as prosthesis-patient mismatch (PPM). A PPM negatively affects the regression of left ventricular hypertrophy and thus the normalization of left ventricular function and the alleviation of symptoms. Persistent left ventricular hypertrophy is associated with an increased risk of arrhythmias and sudden cardiac death. In the case of predictable PPM, there are three options: 1) accept the PPM resulting from the implantation of a stented pericardial valve when comorbidities of the patient forbid the more technically demanding operative technique of implanting a larger prosthesis, 2) enlarge the aortic root to accommodate a larger stented valve substitute, or 3) implant a stentless biological valve or a homograft. Compared to classical aortic valve replacement with stented pericardial valves, the full-root implantation of stentless aortic xenografts offers the possibility of implanting a 3-4 mm larger valve in a given patient, thus allowing significant reduction in transvalvular gradients. However, a number of cardiac surgeons are reluctant to transform a classical aortic valve replacement with stented pericardial valves into the more technically challenging full-root implantation of stentless aortic xenografts. Given the potential hemodynamic advantages of stentless aortic xenografts, we have adopted full-root implantation to avoid PPM in patients with small aortic roots necessitating an aortic valve replacement. Here, we describe in detail a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses. Limitations of this technique and alternative options are discussed.

Full-root aortic valve replacement by stentless aortic xenograft is a viable option in patients with small aortic roots. We describe, a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses, and discuss its limitations and alternative options.

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial ...

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the body, an additional extraction procedure is commonly required to release the active constituents from raw herbs. Water decoction is a classical extraction procedure that is still broadly used in the clinical settings. Here, we propose a detailed protocol for er-xian decoction (EXD) in order to apply herbal decoctions to experimental studies. The calculation of an animal-appropriate dose is described, as well as the four main steps of EXD: soaking, water decoction, filtration, and concentration. In addition, serum-containing EXD is introduced to rats as a means of in vitro validation. Here, rats were orally administered EXD for three days. Blood samples were then collected, inactivated, centrifuged, and filtered. The serum, diluted with the culture medium, can be utilized to treat cells or tissues in vitro. For example, EXD was applied to both in vivo and in vitro studies and demonstrated that EXD enhances osteogenesis. This protocol can be used as a reference for the preparation and application of herbal medicines.

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Here, we present the preparation of an er-xian decoction (EXD) in four steps&#8212;soaking, decoction, filtration, and concentration&#8212;and demonstrate the administration of a prepared EXD-containing serum to rats. These methods are applicable to the in vivo and in vitro study of herbal decoctions such as traditional Chinese medicines.

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures prevents a number of cardiac surgeons from incorporating them into their practice. The aim of this protocol is to describe a simplified and user-friendly technique of an aortic valve-sparing root replacement (VSRR) procedure by re-implantation of the aortic valve. Proper selection of patients and limitations of the technique are discussed.
In 54 consecutive patients, normal appearing aortic valves were re-implanted in a commercially available polyester prosthesis with pre-shaped sinuses by a simplified and standardized technique. Placement of the first row of the proximal suture line, choice of the prosthesis size, and adjustment of the height of the commissures of the patient to the fixed height of the sinus portion of the prosthesis were slightly modified from the reference techniques with the aim of increasing its feasibility for use by other cardiac surgeons. Early mortality and morbidity as well as 5-year survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were collected in all patients.
Thirty-day mortality, re-sternotomy for bleeding, re-sternotomy for mediastinitis, and the incidence of stroke were very low, 1.8% for each (1 of 54). No patient required permanent pace-maker implantation. At 5 years, survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were 97.5%, 95.2%, and 91.6%, respectively.
Mid-term results of our standardized technique of re-implantation of the aortic valve for valve-sparing aortic root replacement are very good and compare with more complex techniques reported by experienced surgeons. By following the present protocol of the standardized re-implantation technique, a greater number of cardiac surgeons can perform this procedure with comparable good results.

Valve-sparing aortic root replacement has the advantage of preserving the patient&#39;s own aortic valve. The complexity of the reported techniques to date restricts their use to a limited number of cardiac surgeons. This protocol describes step-by-step a standardized technique reproducible by a greater number of cardiac surgeons.

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...