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 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.
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-)γ is necessary to induce these changes at around mid-gestation3-5. Mice that lack either NK cells or components of the IFN-γ 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-γ 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.
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
Solution | Time | Temperature |
70% Ethanol | 1 hr | 40 °C |
100% Ethanol | 1 hr | 40 °C |
100% Ethanol | 1 hr | 40 °C |
100% Ethanol | 1 hr | 40 °C |
100% Ethanol | 1 hr | 40 °C |
100% Ethanol | 1 hr | 40 °C |
100% Ethanol | 1 hr | 40 °C |
Xylene | 1 hr | 40 °C |
Xylene | 1 hr | 40 °C |
Xylene | 1 hr | 40 °C |
Paraffin | 1 hr | 63 °C |
Paraffin | 1 hr | 63 °C |
Paraffin | 1 hr | 63 °C |
Paraffin | 1 hr | 63 °C |
Table 1: Settings for automated tissue processing. Overview of the setting used for the processing of uterine tissue
2. Stereological Assessment
3. Qualitative Assessment Using Immunohistochemistry
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&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).
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.
Figure 2: Stereological assessment of arterial remodeling at mid-gestation. (A) Example assessment of luminal and total vessel areas on H&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 µm (left), 50 µm (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.
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 µm. WT: wildtype.
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 authors have nothing to disclose.
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.
C57BL/6 mice (8-12 weeks old) | Charles River | ||
Dental floss | |||
Polystyrene | Used for holding tissue in place | ||
Straight dissection scissors | EMS | 72940 | |
25G needles | BD | 300600 | Used for holding tissue in place |
50 ml conical tubes | Falcon | 352070 | |
Formalin solution, neutral buffered, 10% | Sigma | HT501128-4L | |
Phosphate buffered saline | Sigma | P3813-10PAK | |
Ethanol | Sigma | 32221-2.5L | |
Paraffin | Sigma | 327204-1KG | |
Xylene | Fisher Scientific | X/0250/17 | |
Automated tissue processor | Sakura | 6032 | |
Microtome | Leica | RM2245 | |
Slide rack | Sigma | Z710989 | |
Coplin jar | Sigma | S5891 | |
Purified water | |||
Hematoxylin solution, Gill No. 3 | Sigma | GHS332-1L | |
Eosin Y solution | Sigma | HT110116-500ML | |
Fume hood | |||
Xylene-based mounting media | VWR | 361254D | |
Slide scanner | Hamamatsu | NanoZoomer | |
Slide scanner software | Hamamatsu | NDP viewer | |
Sodium citrate tribasic dihydrate | Sigma | 32320 | Make 10 mM solution and adjust pH to 6 |
Pressure cooker | Used for antigen retrieval | ||
Heating plate | Used for antigen retrieval | ||
Hydrophobic barrier pen | Vector | H-4000 | |
Hydrogen peroxide | Fisher Scientific | H/1800/15 | |
Tris buffered saline | Sigma | T6664-10PAK | |
Mouse on mouse kit | Vector | BMK-2202 | Significantly reduces background |
Mouse anti-human smooth muscle actin | DAKO | M0851 | Cross-reacts with mouse |
IgG2a isotype control | DAKO | X0943 | |
Humidified chamber | Sigma | H6644 | |
3,3’-Diaminobenzidine solution | Sigma | D4293-5SET | |
Hydrochloric acid | Fisher Scientific | H/1050/PB17 |