Name: assessment of maternal vascular remodeling during pregnancy in the mouse uterus
Uploaded: 2015-12-05T15:00:00.000+00:00
Duration: 6 min 55 s
Description: Watch this Scientific Journal Video about Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus at JoVE.com

The authors would like to thank all members of the Colucci lab for helpful discussions. This work was funded by The Wellcome Trust [094073/Z/10/Z], The Centre for Trophoblast Research and The British Heart Foundation.

All procedures discussed in this manuscript are in accordance with UK Home Office regulations and are approved by the Cambridge Ethical Review Panel.
1. Specimen Collection
<ol>
	<li>Set up timed matings using 8-12 week old female C57BL/6 mice with adult males (up to 2 females per male) in the afternoon. Check for the presence of vaginal plugs as a sign of copulation early in the morning on the following days. The presence of a plug in the morning marks gd0.5.</li>
	<li>On gd9.5, euthanize the animal by cervical dislocation and open the abdominal cavity. Note that euthanasia by CO2 may cause vasodilatation.</li>
	<li>Use dental floss to gently ligate the uterine arteries (Figure 1, dashed arrows). Tie the floss around the arteries at the tip of each uterine horn, proximal to the ovaries, as well as around the cervix. 
	Note: Do not rupture the vessels as this may result in collapsed arteries.</li>
	<li>Dissect the uterus quickly using dissection scissors, excising the whole uterus distally to the three ligation points at the tip of the uterine horns and the cervix.</li>
	<li>Place the uterus on a prepared polystyrene piece so that both horns are aligned along the same long axis in opposite directions from the cervix. Gently stretch it and pin into place using 2-3 25 G needles.</li>
	<li>Immerse the polystyrene into a prepared 50 ml conical tube. Top up to 50 ml with 10% formalin. Fix for 5-6 hr at room temperature.</li>
	<li>Discard the formalin solution and replace with 50 ml 1x phosphate buffered saline (PBS) for 5 min.</li>
	<li>Excise the two uterine horns proximally to the ligation point&#160;at each end and proximally to the cervix in the center, trim away any excess fat around the implantation sites and wash twice in PBS for 5 min to remove traces of formalin.</li>
	<li>Store the samples in 70% ethanol at 4 &#176;C (for up to two weeks) until processing. 
	CAUTION: Ethanol is highly flammable.</li>
	<li>Use an automated processing system using the parameters indicated in Table 1. Embed the horns individually in paraffin so that they can be cut along the plane perpendicular to the long axis of the uterus. This ensures that the maximum area of each implantation site will be exposed for analysis in the middle of the block (Figure 2A). 
	OPTIONAL: This is a convenient time point for randomization of the blocks if desired.</li>
	<li>Using a microtome, cut serial sections of 7 &#181;m thickness from the blocks. Stain one section at every 49 &#181;m interval with hematoxylin and eosin (H&#38;E, see section 2.1). Store the remaining sections at room temperature for SMA staining and other downstream applications.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:99px;">Solution</td>
			<td style="width:43px;">Time</td>
			<td style="width:97px;">Temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Ethanol</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Ethanol</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Ethanol</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Ethanol</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Ethanol</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Ethanol</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>1 hr</td>
			<td>40 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraffin</td>
			<td>1 hr</td>
			<td>63 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraffin</td>
			<td>1 hr</td>
			<td>63 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraffin</td>
			<td>1 hr</td>
			<td>63 &#176;C</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Paraffin</td>
			<td>1 hr</td>
			<td>63 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: Settings for automated tissue processing. Overview of the setting used for the processing of uterine tissue
2. Stereological Assessment
<ol>
	<li>H&#38;E Staining
	<ol>
		<li>Deparaffinize sections by immersing slides mounted in a slide rack into a 100% xylene bath for 10 min at room temperature. 
		CAUTION: Xylene is flammable and a suspected carcinogen that is toxic through contact and inhalation. This work must be conducted in a fume hood, using personal protective equipment (PPE).</li>
		<li>Immerse slides in bath containing clean xylene for a further 10 min.</li>
		<li>Sequentially immerse slides in baths containing 100% ethanol, 95% ethanol, 70% ethanol and purified water for 5 min each.</li>
		<li>Immerse slides in 50% Gill No 3 hematoxylin solution, diluted with purified water, for 5 min. 
		CAUTION: Hematoxylin is harmful by ingestion and contact to eyes. Wear appropriate PPE when handling.</li>
		<li>Wash slides in a staining trough under running tap water for 10 min.</li>
		<li>Immerse slides in 1% acid/alcohol solution (1% 10 M hydrochloric acid in 70% ethanol) for 10 sec and wash in a staining trough under running tap water.</li>
		<li>Immerse slides in eosin solution for 30 sec and wash in a staining trough under running tap water for 10 min.</li>
		<li>Within a fume hood, sequentially immerse slides in baths containing 70% ethanol, 95% ethanol and 100% ethanol for 5 min each.</li>
		<li>Immerse slides in bath containing xylene for 10 min.</li>
		<li>Mount coverslips using a xylene-based mounting medium. Dry horizontally and thoroughly in a fume hood prior to visualizing the slides under a microscope.</li>
	</ol>
	</li>
	<li>Stereological Assessment of Vessel Size and Remodeling Status
	<ol>
		<li>For each implantation site, determine which sections are close to the midsagittal point. The chorioallantoic attachment between the fetus and developing placenta serves as a good indicator of the midsagittal point.</li>
		<li>Select 3 sections at 49 &#181;m intervals close to the midsagittal point for the analysis. Scan selected slides. In order to avoid including veins, which have been shown to be more peripherally located in the implantation sites16, restrict the analysis on the central 2/4 of each implantation site (Figure 2A).</li>
		<li>To assess lumen size, draw around the inside of the vessels and record the area of this shape. For the total vessel size, draw around the outer wall of the vessel, including endothelial cells, pericytes and intramural leukocytes. 
		Note: The ratio of total vessel size to lumen is a proxy for the remodeling status of a vessel. As the vessels coil through the plane that the section was cut along, there may be multiple cross-sections of the same artery in one slide. Avoid measuring the same artery multiple times.</li>
		<li>Record the measurements from the 5 vessels in each implantation site with the largest and roundest lumens. Measure each implantation site in triplicate (three sections spaced 49 &#181;m apart from each other). The mean of these 15 measurements represents the mean diameter of the largest vessels.</li>
		<li>To avoid overrepresentation of larger vessels in some samples, measure the same number of vessels in each implantation site.</li>
	</ol>
	</li>
</ol>
3. Qualitative Assessment Using Immunohistochemistry
<ol>
	<li>Deparaffinization and Rehydration
	<ol>
		<li>Deparaffinize sections by immersing slides mounted in a heatproof slide rack into a 100% xylene bath for 10 min at room temperature.</li>
		<li>Immerse slides in bath containing clean xylene for a further 10 min.</li>
		<li>Sequentially immerse slides in baths containing 100% ethanol, 95% ethanol and 70% and purified water for 5 min each.</li>
	</ol>
	</li>
	<li>Antigen Retrieval
	<ol>
		<li>Prepare a 10 mM sodium citrate buffer in purified water and adjust to pH 6 using 10 M hydrochloric acid.</li>
		<li>Fill a heatproof container that can hold 600 ml with 400 ml sodium citrate buffer and place in a domestic pressure cooker containing sufficient water to submerge half of the container. Loosely attach the lid of the pressure cooker and heat until boiling.</li>
		<li>Place slide rack into the sodium citrate buffer and tightly fix the lid of the pressure cooker. Once pressurized, allow to boil for 3 min.</li>
		<li>Depressurize the pressure cooker and remove the inner container. Allow slides to cool in the sodium citrate buffer for 10 min at room temperature.</li>
		<li>Wash slides in a bath containing purified water for 5 min.</li>
		<li>Encircle the sections using a hydrophobic barrier pen.</li>
		<li>Wash slides by submerging in PBS for 5 min.</li>
	</ol>
	</li>
	<li>Blocking Endogenous Peroxidase and Non-specific Binding
	<ol>
		<li>Incubate sections with 3% hydrogen peroxide diluted in purified water in a humidified chamber for 30 min at room temperature. 
		CAUTION: Hydrogen peroxide is highly irritant to eyes, skin and upon ingestion. Wear appropriate PPE.</li>
		<li>Tap off excess hydrogen peroxide solution and wash slides twice by submerging in Tris buffered saline (TBS) for 2 min each.</li>
		<li>Incubate sections with mouse immunoglobulin blocking reagent from the mouse on mouse kit prepared according to manufacturer&#39;s instructions.</li>
		<li>Wash slides twice in TBS for 2 min each.</li>
	</ol>
	</li>
	<li>Immunodetection of Smooth Muscle Actin
	<ol>
		<li>Prepare antibody diluent solution (provided in kit) and incubate with sections for 5 min at room temperature, or according to manufacturer&#39;s instructions.</li>
		<li>Dilute 1:100 mouse anti-human smooth muscle actin (DAKO M0851) in antibody diluent solution. Dilute mouse IgG2a isotype control in antibody diluent solution, ensuring that the final immunoglobulin concentration is equivalent in both solutions.</li>
		<li>Tap off excess antibody diluent solution and cover sections with either diluted antibody or isotype control solutions. Incubate in a humidified chamber for 30 min at room temperature.</li>
		<li>Wash slides twice in TBS for 2 min each.</li>
		<li>Dilute biotinylated anti-mouse secondary antibody and incubate sections for 10 min at room temperature, or according to manufacturers instructions.</li>
		<li>Wash slides once each with TBS and PBS for 2 min, respectively.</li>
		<li>Prepare 3,3&#39;-Diaminobenzidine (DAB) solution according to manufacturer&#39;s instructions. Cover sections with DAB solution and incubate for up to 4 min, ensuring to monitor sections to avoid excess color development. 
		CAUTION: DAB is flammable and a suspected carcinogen that is toxic through contact and inhalation. Wear appropriate PPE when handling.</li>
		<li>Immerse slides in purified water once desired intensity of staining is reached.</li>
		<li>Counterstain with 50% Gill No. 3 hematoxylin for 20 sec&#160;and wash in a staining trough under running tap water until water runs clear.</li>
	</ol>
	</li>
	<li>Dehydration and Mounting
	<ol>
		<li>Within a fume hood, sequentially immerse slides in baths containing 70% ethanol, 95% ethanol and 100% ethanol for 5 min each.</li>
		<li>Immerse slides in bath containing xylene for 10 min.</li>
		<li>Mount coverslips using a xylene-based mounting medium. Dry horizontally and thoroughly in a fume hood prior to visualizing the slides under a microscope</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The protocol presented here is designed to provide a reproducible method by which to assess the vascular changes that are necessary for successful pregnancy. Critical for the success of this evaluation is the quality of the tissue sections. Both accurately determining the gestational age of the implantation sites as well as reliably ligating the uterine arteries are crucial steps. Changes in parameters such as time of fixation, tissue-processing protocol, etc. may also affect the result. For an unbiased stereological assessment, it is important to measure the same number of vessels in each implantation site. It is recommended to measure 5 vessels per implantation site and each implantation site in triplicate. The mean of these 15 measurements represents the mean size of the 5 largest vessels.
Further to the data from alymphoid Rag2-/-IL2rg-/- mice shown here, we found this protocol useful for a number of models of inadequate maternal immune function, including one of inhibited NK cells14. Defects in arterial remodeling have also been shown in models of impaired trophoblast invasion18 and the techniques described here may be helpful to assess the vasculature in these cases. We optimized and validated this protocol for the investigation of decidual vascular remodeling at mid-gestation in mouse, but it is also conceivable to adapt this protocol to work in other model systems (e.g. rats) or even in other organs. While we find that the NK-dependent remodeling can be robustly assessed at gd9.5, it may be amended for other time points such as gd12.5, when the maternal vasculature is completely remodeled. At this time point, endovascular trophoblast invasion is deeper16 and this time point may be more suitable for investigations of the role of trophoblast for vascular changes. If additional quantitative readouts are desired, investigators can also increase the number of sections stained for SMA and assess the percentage of partially remodeled vessels within each implantation site.
A limitation of this protocol is the descriptive nature of histological examinations. Functional assays, however, are only slowly becoming available (such as Doppler ultrasound19). These techniques require technical expertise and much higher expenses for acquiring and maintaining equipment, but have the advantage of providing a longitudinal in vivo readout for the effect of the changes that can be observed histologically. A possible drawback of all stereological examinations is the subjectivity of the investigators. To this end, using two independent examiners that analyze all samples independently can help to avoid this issue. While the protocol outlined here gives insight into how much blood can reach the fetomaternal interface, it may be advantageous to combine it with the complementary approach of assessing the total amount of blood at any given time in the vasculature through plastic casts16.
The strength of this protocol is that it combines two independent approaches. Vascular change is first quantitatively assessed by stereology and the result is then validated qualitatively using immunohistochemistry. The reliability of the results can be further strengthened by randomizing the samples. The samples prepared for this protocol can easily be used to also investigate other parameters of pregnancy such as decidualisation, development of the mesometrial lymphoid aggregate of pregnancy (MLAp) or immunodetection of cells of interest, to rigorously assess a pregnancy phenotype at midgestation.
Arterial remodeling is a critical step for uncomplicated pregnancies in women and failure of these changes underpins the great obstetrical syndromes (GOS), namely pre-eclampsia, fetal growth restriction and stillbirth. We present here techniques to carefully assess a pregnancy phenotype in mouse models that mimic some characteristics of the pathophysiology of GOS.

The exchange of nutrients, gases and waste products during eutherian gestation is mediated by the placenta. In the female reproductive tract, the uterine arteries are the main conduits of blood to the uterus. After implantation, these branch into specialized vessels called spiral arteries that coil through the decidua basalis towards the fetoplacental unit. Diameter and elasticity of these spiral arteries, which are surrounded by leukocytes, in particular uterine natural killer (uNK) cells, dictate volume and velocity of blood available to the placenta1,2. Correct hemodynamic changes as a result of remodeling of the spiral arteries are critical for pregnancy success.
While the underlying mechanisms differ in detail between human and murine pregnancy, the final result of the remodeling process in both species is dilated, high conductance vasculature that loses its smooth muscle layer. In mice, uNK cell-derived interferon (IFN-)&#947; is necessary to induce these changes at around mid-gestation3-5. Mice that lack either NK cells or components of the IFN-&#947; signaling pathway fail to undergo these changes and this is associated with reduced fetal growth6,7, stressing the importance of an adequate blood supply to the fetoplacental unit. During early human pregnancy, the interstitial and endovascular invasion of extravillous trophoblast and their interaction with uNK cells are important for inducing vascular changes and promoting fetal growth8-10. uNK cells can orchestrate human trophoblast invasion through chemokines11 and cytokines such as granulocyte macrophage - colony stimulating factor (GM-CSF)12. Shallow trophoblast invasion is associated with reduced arterial remodeling and pregnancy complications such as pre-eclampsia9.
This protocol is based on the pioneering work of Anne Croy who described the important role of the immune system and particularly NK-derived IFN-&#947; for successful vascular remodeling through immunohistochemistry and elegant transfer experiments5,13. Here we describe a fast and economical way to assess the remodeling status of spiral arteries. Although our lab routinely assesses this at mid-gestation (gestation day (gd)9.5)14, the remodeling process takes place between gd8.5 and 12.515. The advent of automated slide scanners has greatly facilitated the assessment of vessel and lumen areas from sections and we found this to be a more reproducible approach than measuring vessel diameters. The addition of immunohistochemical detection of SMA allows for a straightforward validation of the results obtained from the stereological assessment. As with all stereological techniques, it is recommended to perform the assessment as a blinded experiment by at least two examiners. To this end, a randomization step can be introduced when serially cutting the samples so that the investigators assessing the slides do not know from which experimental group the samples come.
The overall goal of this protocol is to provide a reliable tool to carefully assess the remodeling of spiral arteries in mice with defined maternal and paternal genotypes, in the context of mouse models for pregnancy-related complications. The results stress the dependence on uNK cells of this process, which is essential for normal fetal growth.

<table><tbody><tr><td>C57BL/6 mice (8-12 weeks old)</td><td>Charles River</td><td></td><td></td></tr><tr><td>Dental floss</td><td></td><td></td><td></td></tr><tr><td>Polystyrene</td><td></td><td></td><td>Used for holding tissue in place</td></tr><tr><td>Straight dissection scissors</td><td>EMS</td><td>72940</td><td></td></tr><tr><td>25 G needles</td><td>BD&amp;nbsp;</td><td>300600</td><td>Used for holding tissue in place</td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma</td><td>HT501128-4L</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P3813-10PAK</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>32221-2.5L</td><td></td></tr><tr><td>Paraffin</td><td>Sigma</td><td>327204-1KG</td><td></td></tr><tr><td>Xylene</td><td>Fisher Scientific</td><td>X/0250/17</td><td></td></tr><tr><td>Automated tissue processor</td><td>Sakura</td><td>6032</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2245</td><td></td></tr><tr><td>Slide rack</td><td>Sigma</td><td>Z710989</td><td></td></tr><tr><td>Coplin jar</td><td>Sigma</td><td>S5891</td><td></td></tr><tr><td>Purified water</td><td></td><td></td><td></td></tr><tr><td>Hematoxylin solution, Gill No. 3</td><td>Sigma</td><td>GHS332-1L</td><td></td></tr><tr><td>Eosin Y solution</td><td>Sigma</td><td>HT110116-500ML</td><td></td></tr><tr><td>Fume hood</td><td></td><td></td><td></td></tr><tr><td>Xylene-based mounting media</td><td>VWR</td><td>361254D</td><td></td></tr><tr><td>Slide scanner</td><td>Hamamatsu</td><td>NanoZoomer</td><td></td></tr><tr><td>Slide scanner software</td><td>Hamamatsu</td><td>NDP viewer</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma</td><td>32320</td><td>Make 10 mM solution and adjust pH to 6</td></tr><tr><td>Pressure cooker</td><td></td><td></td><td>Used for antigen retrieval</td></tr><tr><td>Heating plate</td><td></td><td></td><td>Used for antigen retrieval</td></tr><tr><td>Hydrophobic barrier pen</td><td>Vector</td><td>H-4000</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Fisher Scientific</td><td>H/1800/15</td><td></td></tr><tr><td>Tris buffered saline</td><td>Sigma</td><td>T6664-10PAK</td><td></td></tr><tr><td>Mouse on mouse kit</td><td>Vector</td><td>BMK-2202</td><td>Significantly reduces background</td></tr><tr><td>Mouse anti-human smooth muscle actin</td><td>DAKO</td><td>M0851</td><td>Cross-reacts with mouse</td></tr><tr><td>IgG2a isotype control</td><td>DAKO</td><td>X0943</td><td></td></tr><tr><td>Humidified chamber</td><td>Sigma</td><td>H6644</td><td></td></tr><tr><td>3,3&amp;rsquo;-Diaminobenzidine solution</td><td>Sigma</td><td>D4293-5SET</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>H/1050/PB17</td><td></td></tr></tbody></table>

assessment of maternal vascular remodeling during pregnancy in the mouse uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

Rag2-/-IL2rg-/- mice lack all mature lymphocytes, including NK cells17, and their uterine arteries fail to undergo the vascular changes seen in congenic wildtype C57BL/6 (WT) mice around mid-gestation5,14. As a result of this reduced remodeling Rag2-/-IL2rg-/- mice show significantly smaller luminal surface areas, indicative of overall smaller vessels which can be visualized on H&#38;E-stained sections and quantified stereologically (Figure 2). Furthermore, the relative wall thickness (vessel to lumen ratio) is higher in the spiral arteries in these mice, suggesting reduced supply of blood and higher blood velocity.
This quantitative assessment was confirmed using immunohistochemical detection of SMA. It is characteristic for WT mice to lose most of the SMA within the vascular media of the spiral arteries by mid-gestation. If this NK cell-driven process does not take place, SMA remains in the media of the vessels and can be readily detected immunohistochemically as rings around the blood vessels (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/53534/53534fig1.jpg" /> 
Figure 1: Mouse uterus at gd9.5, schematic overview. Drawing showing the relative positions of the uterine horns, cervix (blue) and main uterine arteries (red). Indicated are the long axis of the uterine horns (arrows) and the ligation points (dashed arrows). The sections for immunohistochemistry are cut along the plane of view.
<img alt="Figure 2" src="/files/ftp_upload/53534/53534fig2.jpg" />
Figure 2: Stereological assessment of arterial remodeling at mid-gestation. (A) Example assessment of luminal and total vessel areas on H&#38;E stained sections from WT females in the central 2/4 of the decidua basalis (demarcated by the vertical black dashed lines) at gd9.5. In the vessels at higher magnification, the red dashed line demarcates total vessel area, yellow dashed line demarcates lumen area. Bar = 500 &#181;m (left), 50 &#181;m (right). The long axis of the uterus is a horizontal line in this orientation. (B) Quantification of luminal areas (left panel) and ratio of luminal area to total vessel area (right panel) from WT (C57BL/6) and Rag2-/-Il2rg-/- mice (on C57BL/6 background). Each data point represents the mean of 15 measurements (5 measurements per section, 3 sections per implantation site). Bars represent mean of n = 11-13 implantation sites from 4 pregnancies per group; p-value from a two-tailed, unpaired Mann-Whitney test. WT: wildtype.
<img alt="Figure 3" src="/files/ftp_upload/53534/53534fig3.jpg" /> 
Figure 3: Immunohistochemical detection of smooth muscle actin in the decidua basalis. Representative SMA staining on WT (top) and Rag2-/-IL2rg-/- mice (on C57BL/6 background, bottom) at gd9.5. Bar = 100 &#181;m. WT: wildtype.

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Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

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Developmental Biology

13.2K Views.  University of Cambridge. This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry. 

Video: Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

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 ...

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 ...

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.

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 ...

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Neuroscience

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 ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Development of a Murine Model for Femoral Artery Anastomotic Stenosis

In vascular surgery, vascular anastomosis is a common reconstructive technique used to restore blood flow. However, anastomotic restenosis is a frequent postoperative complication, primarily caused by surgery-induced vascular injury, intimal hyperplasia, and inflammatory responses. The mouse femoral artery anastomosis model is widely used to investigate the mechanisms of anastomotic restenosis and vascular repair. Microscopically guided end-to-end femoral artery anastomosis allows precise simulation of vascular injury and repair processes following surgery, providing a reliable experimental tool for studying the pathological mechanisms related to restenosis. This study focuses on refining the surgical technique for femoral artery anastomosis in mice. Through refinements in surgical techniques and optimization of technical details, we have achieved a marked increase in the success rate and reproducibility of the model. Specific improvements include refined vascular handling techniques during surgery, the selection of suture materials, and the optimization of suturing methods to minimize anastomotic leakage and postoperative occlusion. The study also emphasizes the observation of intimal hyperplasia, vascular remodeling at the anastomotic site, and long-term vessel patency. Through this research, we provide a concise and efficient operational guide for performing mouse femoral artery anastomosis, offering reliable technical support for experimental studies in vascular surgery. This work lays a solid foundation for subsequent investigations into related mechanisms and evaluations of therapeutic intervention.

Here we present a murine model for femoral artery anastomosis, offering researchers a valuable animal model to study and simulate vascular anastomotic stenosis. This development is crucial for advancing our understanding of the pathophysiology underlying this condition and facilitating more accurate and effective research into vascular diseases.

In vascular surgery, vascular anastomosis is a common reconstructive technique used to restore blood flow. However, anastomotic restenosis is a frequent ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

Summary

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Chapters in this video

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

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

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

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Ultrasonography in Experimental Reproductive Investigations on Rats

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Development of a Murine Model for Femoral Artery Anastomotic Stenosis