Riesgos en los centros educativos

¹MM. Aljunaidy, ¹LK. Harris, ¹JD. Aplin, ²A. Robson & ²JN. Bulmer

¹Maternal and Fetal Health Research Centre, University of Manchester, Manchester Academic Health Science Centre, St Mary‘s Hospital

²Department of Cellular Pathology, University of Newcastle, Royal Victoria Infirmary

Email: [email protected] Abstract

During pregnancy, spiral arteries and extracellular matrix (ECM) in the decidua and upper segments of the myometrium are remodelled. This vascular remodelling (VR) involves losing their vascular smooth muscle cells (VSMC) and their endothelium; and becoming wider, so the increased amount of blood flow can pass gently to the fetus. Uterine natural killer (uNK) cells were proved to play an important part in initiating VR by disrupting and breaking the VSMC and ECM. This study is done to investigate if uNK cells can have any effect on other arteries such as chorionic arteries so they can be proposed as a good experimental model to study VR as they are readily more accessible and easier to be isolated. Chorionic arteries taken from term placentas and myometrial arteries from non-pregnant pre-menopausal women were incubated for 72 hours with factors secreted by first trimester uNK cells. Immunoperoxidase and immunofluorescence staining of the arterial sections by using SMC marker (α-SMA), elastin, collagen-IV and laminin were done. Then, microscopy and image analysis were performed. The preliminary results showed that uNK medium can slightly affect the vascular wall of chorionic arteries in contrast with the severe disorganization it can cause in the myometrial spiral arteries wall. This suggested that uNK medium can not affect chorionic arteries.

Keywords:

Chorionic, myometrial, remodelling and spiral. 1. Introduction

In pregnancy, vascular remodelling (VR) of spiral arteries and extracellular matrix (ECM) in the decidua and upper segments of the myometrium happens. It involves disappearing in their vascular smooth muscle cells (VSMC) and their endothelium; and becoming wider [(Harris, 1966), (Pijnenborg, 2006) and (Pijnenborg, 1998)]. In some pregnancy complications such as intrauterine growth restriction (IUGR) and pre-eclampsia the vascular remodelling is incomplete (Moffett, 2006) (Fig.1). We believe the understanding the mechanism of that will help us to find prevention or cure from these life-threatening illnesses. Uterine natural killer (uNK) cells were suggested to play an important part in initiating VR by disrupting and breaking the VSMC and ECM [(Smith, 2009) and (Harris, 2010)]. This study is done to investigate if uNK cells can have any effect on other arteries such as the chorionic arteries so they can be proposed as a good experimental model to study vascular remodelling as they are more available and easier to be isolated than the spiral arteries.

2. Methodology:

2. Methods

Chorionic arteries taken from term placentas and myometrial arteries from non-pregnant pre- menopausal women were incubated for 72 hours with factors secreted by first trimester uNK cells. Immunoperoxidase and immunofluorescence staining of the arterial sections by using SMC marker (α-SMA), elastin, collagen-IV and laminin were performed. Then, microscopy and image analysis were made.

2.1. Tissue collection and ethical approval

Normal term placenta (n=2), first trimester decidua (n=3) obtained at pregnancy termination, myometrium (n=3) obtained from non-pregnant, pre-menopausal women undergoing hysterectomy (for medical or social reasons) were collected with North West Research Ethics Committee approval (term placenta: 08/H1010/55, first trimester decidua: 08/H1010/28, non- pregnant myometrium: 08/H1011/71) and written informed consent. Tissue was transported in sterile phosphate buffered saline (PBS) and used within 30 min of collection.

2.2. uNK cell isolation and culture: method development

uNK cells were isolated at Newcastle University at the beginning of the project (Vassiliadou, 1998), and later we tried to establish a similar protocol in the University of Manchester. Briefly, decidual tissue was digested with collagenase (1 ml), DNAase I (0.1 mg/ml) and BSA (1 mg/ml). Immunomagnetic beads were used to isolate the uNK cells which were cultured for 48 h in RPMI 1640 with 10% fetal bovine serum (Vassiliadou, 1998) (Sigma, UK). To isolate uNK cell conditioned medium, cells were cultured for 24 h; medium was collected, centrifuged at 1500 rpm for 5 min to remove cell debris and stored at -20°C until future use. preparing uNK cells at Newcastle University generated cells which are 80-90% viable (by trypan blue exclusion) after 48 hours culture (Lash, 2010).We modified the published protocol, adding ammonium chloride to lyse the significant numbers of contaminating erythrocytes, exposing only 107 cells to the magnetic beads rather than using the whole cells, and leaving the cells to adhere for only 1 hour rather than overnight. Both of the uNK cells (either prepared in Manchester or in Newcastle) were used in the experiments and gave the same results as showing later on the section of the results.

Non-pregnant Pre-eclampsia Pregnant

Fig. 1: Vascular remodelling in normal and complicated pregnancies. The remodelling is restricted to the decidua and incomplete in pre-eclampsia. (Moffett, 2006)

2.3. Isolating chorionic plate arteries and exposing them to the uNK cells

Chorionic plate arteries from term placentas (n=2) were dissected and cultured with uNK cells for 72 hours.

2.4. Myometrial spiral artery culture and exposing them to the uNK cells

Spiral arteries were dissected from myometrial biopsies (n=3) under sterile conditions and cultured in a 1:1 ratio of DMEM/Ham‘s F12 containing 10% fetal bovine serum at 37 o C and in 5% CO2/95% air. Following dissection, some arteries were cultured with control medium (20% (v/v); CA), the rest of the arteries were cultured with uNK cell -conditioned medium (20% (v/v); UA). After 72 h, the arteries were placed in a frozen embedding matrix (OCT) (Raymond A Lamb/UK) and stored at -20o C. Arterial sections (10μm) were prepared using LEICA CM3050 cryostat, mounted on coated slides (Thermo/CA) and stored at -80°C.

2.5. Fluorescent staining of frozen sections

Tissue sections were removed from the freezer (- 80°C) and left for 15 min at room temperature to warm up. Then, the sections were fixed with paraformaldehyde (4% (v/v) in PBS) for 15 min and washed with PBS. For permeablisation Triton-X solution (0.1% in PBS) was applied for 10 min. After washing the sections, 30 μl of primary antibody diluted in PBS was added to each tissue section for one hour. After three 5 min PBS washes, 30 μl of diluted secondary antibody was added and the slides incubated in a dark place for one hour in room temperature. Three 5 min washes with PBS were done then sections were mounted with vectashield mounting medium containing propidium iodide (Vector/CA).

2.6. Immunohistochemistry of frozen tissue sections

Frozen sections were warmed up to room temperature, fixed and permeabilised as described in the fluorescent staining protocol. Before blocking non-specific binding sites with 5% BSA, slides were incubated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Primary antibodies diluted to the required concentrations with TBS, were added to the tissue sections and stored overnight at 4o C. On the following day, the sections were washed with TBS for 3 X 5 min; the secondary antibodies were applied and left for 30 min at room temperature. Following 3X5 min washes in TBS; avidin-peroxidase was used at a concentration of 1:200 in TBS and left for 30 min at room temperature. After more TBS washes (3X5 min) the tissue sections were stained and mounted.

2.7. Microscopy and image analysis

2.7.1. Immunohistochemistry images

A Q Imaging Qicam FAST 1394 camera connected to a Leitz Dialux 22 microscope was used to take images of the tissue sections and digital image capture: Image-Pro Plus 6.0 programme was used for the image analysis.

2.7.2. Immunofluorescence images

A Q Imaging FAST 1394 camera linked to Olympus 1X70 microscope was used to capture the images of the tissue sections and digital image capture: Q Capture Pro 6.0 software was used for image analysis.

Fig. 2:Isolation of uNK cells and preparation of uNK medium. A and B: Decidual tissue was minced into fine pieces about 1 mm³ under sterile conditions in RPMI 1640 medium containing 100 U/ml penicillin, 1 mg/ml streptomycin and 2 mM L-glutamine (Sigma, UK). C: tissue was digested twice for 30 min with 1 mg/ml type II collagenase and 0.1 mg/ml DNase. D: The resulting uNK cells were selected using immunomagnetic beads coated with anti-CD56 antibodies. E: uNK cells were transferred to fresh RPMI 1640 with 10% fetal bovine serum cultured for 48 h at 37ºC in 5% co2. F: The cell-free conditioned medium which contains secreted substances from the uNK cells was centrifuged to remove contaminating cells and stored at -20°C until use.

3. Results

Secreted substances from uNK cells (uNK medium) had little effect on the vascular wall of chorionic arteries (Fig. 3) in contrast with the severe disorganization it caused in the myometrial spiral arteries wall (Fig. 4).

Control uNK medium

Fig. 3: chorionic arteries were exposed to the uNK medium for 72 h showed no important changes in the vascular wall after staining the tissue sections for collagen IV (COL), laminin (LAM) and α-smooth muscle actin (α-SMA). Scale bars: A-D, G and H: 100 µm. E, F, I and J: 25 µm.

4. Discussion

Our preliminary findings suggest that uNK medium can not affect chorionic arteries. The reason of that might be the difference in the structure of chorionic arteries and myometrial arteries. In the former the smooth muscle cells are disorganized while they are organized in