Name: is my mouse pregnant? high-frequency ultrasound assessment
Uploaded: 2021-03-18T14:00:00.000+00:00
Duration: 2 min 39 s
Description: Watch this Scientific Journal Video about Is My Mouse Pregnant? High-Frequency Ultrasound Assessment at JoVE.com

All the steps in this protocol follow the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and have been approved by the Institutional Animal Care and Use Committee of New York University Grossman School of Medicine.
1. Mating of mice for timed pregnancies
<ol>
	<li>Pair the appropriate female mouse (usually a heterozygote) in a cage with the appropriate male mouse (usually a heterozygote) for overnight mating.</li>
	<li>Separate the mice the next morning. Alternatively, continuously mate the female and male mice, thus increasing the chances of pregnancy. 
	NOTE: However, an accurately timed pregnancy cannot be assured with the alternative, and staging of mouse embryos by ultrasound is not clear, especially when the disease process results in intrauterine growth retardation. If the embryos carrying the gene variant are assumed to be small, look for the larger wildtype littermates to gauge the gestational age.
	<ol>
		<li>Optional: Look for the vaginal plug. If there is no vaginal plug, the female mouse has not been mated. If there is a vaginal plug, it is still likely that the female mouse does not become pregnant.</li>
	</ol>
	</li>
	<li>Time the day after the overnight mating as E0.5.</li>
	<li>Perform imaging at E6.5&#8211;E.8.5 to determine pregnancy, and re-mate the mice if the mouse is not pregnant (see step 1.1). 
	NOTE: At this stage, the female dam is not obviously pregnant to the eye; hence, the ultrasound imaging allows early determination and re-mating.</li>
</ol>
2. Anesthesia and preparation of mouse
<ol>
	<li>Place the pregnant mouse in the anesthetic induction chamber.</li>
	<li>Mix isoflurane with either room air or medical oxygen, at a concentration of 2%&#8211;3% at 1 L/min flow rate to induce sedation of the pregnant mouse in the induction chamber. 
	NOTE: Sedation typically occurs within 1&#8211;2 min. The mouse will be lying still, and her breathing will have slowed.</li>
	<li>Quickly transfer the mouse to the imaging platform. The imaging platform typically has heating elements as well, which can help keep the mouse warm.</li>
	<li>Place the nose of the mouse into the anesthetic nosecone.</li>
	<li>Quickly re-route the isoflurane/oxygen mixture to the imaging platform nosecone. Maintain isoflurane at 2%&#8211;3% at 1 L/min flow.</li>
	<li>Determine the level of sedation by paw pinch, corneal reflex, level of respiration, and any movement. 
	NOTE: The corneal reflex can be initially determined by applying moisturizing ointment to the eyes to keep them from drying out while the mouse is anesthetized (the mouse does not close her eyes).</li>
	<li>With the mouse lying supine (on its back), tape the paws to the imaging platform&#8217;s electrocardiogram (EKG) pads. 
	NOTE: However, an EKG is not necessary for this kind of imaging.</li>
	<li>Remove the fur from the pregnant dam&#8217;s abdomen as follows:
	<ol>
		<li>Wet the abdominal fur thoroughly with 70% ethanol, including up to the lateral edges. Do not apply so much that there is run-off onto the platform. 
		NOTE: Ethanol works better as a shaving lubricant than water.</li>
		<li>Use the razor blade to carefully shave the abdomen. Be careful not to cut the nipples.</li>
		<li>Wipe the shaved fur off the abdomen with gauze or some wipes.</li>
		<li>Alternatively, use a depilatory cream after using the fur clippers to remove most of the fur.</li>
	</ol>
	</li>
</ol>
3. Transabdominal imaging of the (presumed) pregnant mouse
<ol>
	<li>After the abdomen is shaved, reduce the isoflurane to 1%&#8211;1.5%, still maintaining a flow rate of 1 L/min. Monitor the level of sedation by the level of respiration and any movements as well as paw pinch and/or corneal reflex. 
	NOTE: The heart rate of the anesthetized mouse will typically be 400&#8211;500/min, depending on the core (rectal) temperature. For the purpose of a rapid pregnancy check, do not warm the mouse to a physiological core temperature; the heart rate will be closer to 400&#8211;450/min, but may drop further with an irregular rhythm should the mouse become cold with prolonged imaging.
	<ol>
		<li>Importantly, make sure that the respirations are moderate in depth and regularity, not erratic or agonal (gasping: deep, slow, and erratic, with deep intercostal and subcostal retractions). 
		NOTE: The respiratory rate of the non-ventilated, anesthetized mouse will typically be 60&#8211;100/min. See https://ahcs.ninds.nih.gov/ACUC_pages/pg_003_anesth_animals.html and https://az.research.umich.edu/animalcare/guidelines/guidelines-anesthesia-and-analgesia-mice.</li>
	</ol>
	</li>
	<li>Apply ultrasound (acoustic coupling) gel to the abdomen generously.</li>
	<li>Place the imaging transducer on the abdomen to orient it in a horizontal plane: orient the probe to obtain a left-right orientation on the imaging system; the &#8220;dot&#8221; or ridge indicated on the side of the imaging probe should be facing right. 
	NOTE: Sliding the imaging probe to the mouse&#8217;s right should shift the corresponding image on the ultrasound system to the mouse&#8217;s right (imagine looking &#8220;up&#8221; from the mouse&#8217;s tail&#8212;the mouse&#8217;s right will be left on the monitor).
	<ol>
		<li>Typically, use the imaging system&#8217;s transducer mount and rail-manipulator system. Here, &#8220;free hand&#8221; imaging has been used in which the imaging transducer is hand-held (two hands are steadier than one) and which allows for more rapid movements around the abdomen. These movements, as will be outlined below, include both rotational and translational movements. However, this requires more practice.</li>
	</ol>
	</li>
	<li>Identify the bladder on the screen (Video 1).</li>
	<li>Scanning caudally from the bladder, identify the vagina. Then, scanning slowly and smoothly in a cranial direction, identify the bifurcation of the vagina into the left and right uterine horns (Figure 2; Video 1).</li>
	<li>Begin the survey of the uterus (left and right uterine horns)13 (Figure 3; Video 2). 
	NOTE: Up to mid-gestation (E10.5 or E11.5), the mouse embryos will be positioned along the right and left peripheries. As they grow, the more distal portions of the uterus and their corresponding embryos will turn outwards and posteriorly. As the embryos grow further (E15.5 and later, generally), the mouse fetuses will be positioned almost randomly in various directions, and it becomes difficult to &#8220;track&#8221; a uterus from proximal to distal.
	<ol>
		<li>Scan quickly simply to check if a mouse is pregnant or not. This rapid method requires only recognition of a gravid uterus and mouse embryos.</li>
		<li>Alternatively, take extra time to enumerate the embryos (live, dead, resorbed) in each uterine horn. 
		NOTE: In general, a live embryo will exhibit distinct organs such as a heart, limbs, head with ventricles, and eyes. A dead embryo takes on a homogeneous, &#8220;mushy&#8221; appearance unless just dead. Resorbed embryos have a pinpoint echogenic spot in the middle of a gravid-looking uterus (Figure 4, Figure 5, Figure 6, Figure 7, Figure 8).</li>
		<li>To determine if an embryo is truly alive, look for the heartbeat and/or blood flow. 
		NOTE: Color Doppler flow mapping can aid in determining the presence of blood flow, both in the embryo and in the umbilical cord. In general, this may be applied to embryos older than E8.5.</li>
		<li>Recognize potential artifacts that may mimic a gravid uterus (Figure 9, Figure 10). In addition, as bowel gas and other ultrasound &#8220;shadow&#8221; artifacts may obscure segments of the uterine horn, perform the imaging from multiple vantage points to ensure adequate visualization of the uterus.</li>
	</ol>
	</li>
	<li>After the survey is complete, wipe the gel from the abdomen with gauze or wipes. Try to remove as much as possible as the gel tends to cool the mouse down.</li>
	<li>Untape the paws, and remove the mouse from the anesthetic nosecone.</li>
	<li>Gently move the mouse back into her cage. She should wake up and start moving around within a minute or so. 
	NOTE: Genetically modified mice may take a little longer to recover from anesthesia and need to be watched more closely.</li>
</ol>

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The authors have nothing to disclose.

The most important first step in the imaging is to identify the vagina and then to determine the bifurcation of the uterine horn to the left and right. By following each uterine horn, the imager is less likely to mis-identify loops of the bowel as the uterus. Moreover, understanding the variations in the appearance of the bowel (with/without fecal matter) is important to distinguish these from the uterus; occasionally, fecal &#8220;balls&#8221; in bowel loops may mimic a gravid (pregnant) uterus. Although other authors have described the diagnosis of pregnancy and staging of mouse embryonic development17,18,19 including the detection of resorbed embryos20, this study is the first to outline the steps and potential pitfalls in imaging the gravid murine uterus.
The imager must recognize potential artifacts that may mimic an early pregnancy or gravid uterus or that may interfere with the imaging of the uterus and embryos. Following the uterine horns laterally will reduce the likelihood of mistaking other organs and artifacts in the abdomen for the uterus (and small embryos). Potential artifacts that may be mistaken for uterus, embryos, and/or obstructive items include the bowel and bowel gas, feces, spleen, liver, and stomach.
This method requires general anesthesia, and we are careful to limit: 1) time of imaging and 2) frequency of pregnancy checks, to reduce any chance of intrauterine loss due to anesthesia. Although anesthetics and analgesics appear to be safe overall during pregnancy21, significant exposure may have consequences on mouse embryonic growth22. As mouse knockout models often demonstrate prenatal or early perinatal death, exposure of the embryos to general anesthesia during this imaging may (at least theoretically) increase their risk of demise or influence their biology in unknown ways. While an absolute time limit is unknown, we try to limit each imaging session to no more than 15 minutes, and to 2&#8211;3 imaging sessions (maximum) per pregnancy. The &#8220;ALARA principle&#8221; is prudent here: As Low As Reasonably Achievable.
This method allows for more efficient breeding as well as rapid determination of intrauterine demise. This is especially important in experiments using knockout models that die early; other examples include toxicological studies. While a few studies have detailed the weight gain during pregnancy, it is quite clear that the weight gain early (prior to E8.5) is small and may not be different from diurnal weight changes. Furthermore, the data were derived from first-time pregnant mice only and may not reflect the confounding effects of multi-gravid mice10,11. Timed pregnancies may not be evident early on, and especially with genetically-engineered mice, intrauterine losses may be common or even affect the entire litter. Thus, simply because a mouse does not deliver a litter does not mean she was never pregnant. Mice can be re-mated in a week if the female is not pregnant; otherwise, researchers will simply have to wait it out to see if the female has become pregnant. After skills are developed to do more than simply check for pregnancy, this method will also allow mapping and monitoring the embryos as the pregnancy progresses. In this way, the optimal timing for embryo harvest can be determined if tissues must be harvested prior to demise23.

The mouse is the preferred mammalian model for many human diseases and biological processes1,2,3,4. Research in developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints5,6,7,8. Moreover, optimal and effective breeding of model mice requires an assessment of timed pregnancies, particularly when investigators are studying the effects of a gene mutation on development. Typically, investigators mate heterozygous mice overnight, look for a vaginal plug early the next morning, and hope that a pregnancy ensues9. Determining intrauterine loss typically starts with checking a newborn litter for Mendelian ratios of genotypes, then working backwards by sacrificing pregnant mice at various gestational stages, and recovering the embryos. Investigators may determine weight gain as a metric of a positive pregnancy10,11; however, especially with genetically-engineered mice, the litters may be very small and subsequently resorbed when there is intrauterine loss due to which the weight gain may not be obvious (particularly early in pregnancy, ~E6.5&#8211;8.5). A mouse may appear falsely pregnant due, for example, to a benign abdominal tumor. In essence, one works &#8220;blind&#8221;.
High-resolution ultrasound biomicroscopy allows for direct visualization of the gravid uterus and developing mouse embryos12,13,14,15,16. Although we had initially developed methods to assess embryonic mouse cardiovascular physiology16,17, we recognized the utility of this imaging modality to streamline our mouse breeding. Specifically, we no longer had to wait to &#8220;see&#8221; if a mouse were pregnant, based on either the obvious weight gain or delivery of a litter; we could determine the gravid state and re-mate mice quickly if the dam was not pregnant. Moreover, intrauterine losses could also be easily imaged, and a timeline of loss could be determined without sacrificing the mouse (see Figure 1 for a schematic). Time, valuable model mice, and funds can thus be saved.

<table><tbody><tr><td>Depilatory cream</td><td></td><td></td><td></td></tr><tr><td>Ethanol, 70%</td><td></td><td></td><td></td></tr><tr><td>Fur clippers</td><td></td><td></td><td></td></tr><tr><td>Gauze or KimWipes</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>Medical oxygen (optional)</td><td></td><td></td><td></td></tr><tr><td>Medical tape</td><td></td><td></td><td></td></tr><tr><td>Mouse imaging system (including anesthesia set-up and imaging platform)</td><td>Fujifilm Visual Sonics</td><td>Various</td><td>Any system with 40 MHz center-frequency ultrasound transducer probe</td></tr><tr><td>Razor blade (not a safety razor)</td><td></td><td></td><td></td></tr><tr><td>Scale (to weigh mouse)</td><td></td><td></td><td></td></tr><tr><td>Ultrasound gel</td><td></td><td></td><td></td></tr></tbody></table>

is my mouse pregnant? high-frequency ultrasound assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

This protocol will allow an investigator to determine confidently whether a mouse is pregnant, including during the early stages and to determine whether there are obvious prenatal embryonic or fetal losses without needing to sacrifice the pregnant dam. This protocol is especially useful when breeding genetically engineered mice; typically, heterozygous x heterozygous crosses to yield homozygous offspring leads to failure of proper development, which causes prenatal lethality. Figure 1 depicts a representative situation in which embryos progressively die and then are resorbed through mid-gestation. Figure 2 shows how to find the left and right uterine horns by following the vagina up through its bifurcation. Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, and Video 3 show mouse embryos at various stages of development. Early-stage mouse embryos, dead embryos, or resorbed embryos may resemble other organs in the abdomen or feces in the intestines, or conversely, intestinal loops may mimic the non-gravid uterus. Figure 9 and Figure 10, as well as Video 4 and Video 5, demonstrate such potential imaging artifacts that may mimic the gravid uterus, for which the investigator must be on alert.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61893/61893fig01.jpg" /> 
Figure 1: Schematic diagram of a theoretical pregnant mouse abdomen, imaged at E11.5, then again at E14.5. Up to mid-gestation (E10.5 or E11.5), the mouse embryos will be positioned along the right and left peripheral aspects of the abdomen. As the embryos grow, the more distal portions of the uterus and their corresponding embryos will turn outwards and posteriorly. As the embryos grow further (E15.5 and later, generally), the mouse fetuses will be positioned almost randomly in various directions, and it becomes difficult to &#8220;track&#8221; a uterus from proximal to distal. When there is prenatal lethality in a genetically engineered mouse model, the embryos (open circles) may die; the dead embryos (hatched circles) will eventually become resorbed (solid circles). <a href="https://www.jove.com/files/ftp_upload/61893/61893fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61893/61893fig02.jpg" /> 
Figure 2: Once one finds the vagina (A), immediately to the right of the bladder, sweeping cranially will demonstrate the bifurcation (B) to the right and left uterine horns (D)&#8211;(F). 
Scale bar (A) = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61893/61893fig03.jpg" /> 
Figure 3: Images of non-gravid (non-pregnant) uterus (identified by the rows of arrows). The uterus may vary in thickness: thicker in (A), (B), (E); thin with a central thin echogenic line (C), very thin (D), or may even contain small, cystic structures that should not be mistaken for concepti (B) and (E) especially, although this may be difficult to determine. (A) is a right uterine horn; this is more difficult to follow distally in our experience due to bowel gas. (B)&#8211;(F) are left uterine horns; (F) is quite distal/lateral and so becomes more difficult to image due to increasing bowel gas artifact. Scale bar (A) = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61893/61893fig04.jpg" /> 
Figure 4: Resorbed and dead embryos have distinct appearances. Resorbed embryos, which are very commonly found, are encased within a round (gravid) uterine sac that appears relatively homogeneous except for a central echogenic (very bright) &#8220;spot&#8221;&#8212;arrows in (A) and (B). (C) shows resorbed or dead embryos; there is an entirely homogeneous, &#8220;mushy&#8221; appearance to the uterus, and we see probably 3&#8211;4 dead embryos in this frame. (D) shows a recently dead embryo, which still shows some structures; there also appears to be cellular debris in the amniotic sac. In (E), the dead embryo is much shrunken and still connected to the placenta (&#8220;P&#8221;). (F) shows a homogeneous, &#8220;mushy&#8221; appearance of an embryo that probably died 1&#8211;2 days previously, but is not yet completely resorbed. Scale bar for (A) and (D) = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61893/61893fig05.jpg" /> 
Figure 5: Early-stage embryos, from approximately E5.5 (A) and E6.5 (B) to E8.5 ((C) and (D)). There are variations in appearances, and the estimated stages here were based on timing of mating as well as the appearance of the embryos themselves. Scale bar (A) = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61893/61893fig06.jpg" /> 
Figure 6: E9.5 embryos are considerably larger than E8.5 embryos and are beginning to take form. Representative images, showing adjacent embryos, are shown in (A) and (B). Scale bars = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61893/61893fig07.jpg" /> 
Figure 7: E10.5 embryos exhibit even clearer organs such as the head, spine, and heart. Representative images, showing adjacent embryos, are shown in all panels; in (D), a dead/resorbing embryo lies adjacent to a live embryo. Scale bar = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61893/61893fig08.jpg" /> 
Figure 8: Older embryos, approximately E12.5 (A), E14.5 (B), and E15.5 (C). Oblique planes of imaging obscure the precise anatomy somewhat, but the heart (arrow) is in the central portion of each embryo; in (C), the myocardium is now more echogenic than the blood. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61893/61893fig09.jpg" /> 
Figure 9: The bowel, which is the organ most likely to be confused with the uterus. In (E), a resorbed embryo (arrowheads) overlies a segment of bowel (arrows). In (F), a non-gravid uterus (arrowheads) overlies a short segment of bowel (arrows) <a href="https://www.jove.com/files/ftp_upload/61893/61893fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/61893/61893fig10.jpg" /> 
Figure 10: Additional imaging artifacts in the gravid abdomen include the kidneys, spleen, and liver. (A) Right kidney; (B) Right kidney with renal artery (arrows); (C) Left kidney; (D) Left kidney with renal artery (arrows); (E) Spleen; (F) Liver overlying a segment of bowel; (G) Kidney overlying segment of bowel; (H) Spleen, liver, and left kidney seen in one plane of imaging. B = bowel; K= kidney; L= liver; S= spleen. Scale bar (A) = 2 mm. <a href="https://www.jove.com/files/ftp_upload/61893/61893fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Is My Mouse Pregnant? High-Frequency Ultrasound Assessment at JoVE.com

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

is-my-mouse-pregnant-high-frequency-ultrasound-assessment

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.0K Views.  New York University Grossman School of Medicine. High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

Video: Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Medicine

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

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

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

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

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

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

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Cancer Research

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

High-frequency Ultrasonography Based Early Pregnancy Characterization: A Technique to Study Embryo Implantation and Pregnancy Progression in Pregnant Murine Model

Source: Peavey, M. C. et al. <a href="https://www.jove.com/video/56207" target="_blank">A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse</a>. J. Vis. Exp. (2017)
In this video, we demonstrate high-frequency ultrasonography to image embryo implantation sites in a pregnant murine model. This imaging technique successfully detects viable pregnancy and characterizes embryo-implantation sites during early murine pregnancy.

Source: Peavey, M. C. et al. A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse. J. Vis. ...

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

Summary

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Chapters in this video

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Murine Fetal Echocardiography

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

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

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Ultrasonography in Experimental Reproductive Investigations on Rats

High-frequency Ultrasonography Based Early Pregnancy Characterization: A Technique to Study Embryo Implantation and Pregnancy Progression in Pregnant Murine Model