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Vedanta Mehta MSc*, Khalil N. Abi Nader MD*, Donald M. Peebles MD FRCOG,
Elizabeth Benjamin MD, Victoria Wigley MSc, Belen Torondel PhD, Elisa Filippi MD,
SW Steven Shaw MD, Michael Boyd MSc, John Martin PhD FRCP, Ian Zachary PhD,
Anna L. David PhD MRCOG
*These authors contributed equally to this work.

Seventeen time-mated Romney breed mid-gestation pregnant ewes (80-102 days of gestation, term=145 days) carrying singleton (n=10) or twin pregnancies (n=7) were studied until the end of gestation. With the ewe turned up, the gestational ages were confirmed using an Acuson 128 XP10 ultrasound scanner with a C3 3.5 MHz curvilinear abdominal transducer (Siemens, Bracknell, UK) to examine fetal size as described.
1
Animals were acclimatized for one week before procedures were performed. All work was conducted in accordance with the UK Animals (Scientific Procedures) Act (1986).
After starving overnight, pregnant ewes (n=11, 5 twins, 6 singleton gestations) underwent general anaesthesia induced with thiopental sodium 20mg/kg IV (Thiovet, Novartis
Animal Health UK Ltd, Hertfordshire, UK) and maintained with 2-2.5% isoflurane in oxygen (Isoflurane-Vet, Merial Animal Health Ltd, Essex, UK) after intubation.
Maternal serum and plasma samples were collected. A detailed ultrasound examination of the fetus was performed including fetal measurements. Umbilical artery Doppler measurements, pulsatility index (PI) and resistance index (RI) were acquired. For longterm maternal hemodynamic monitoring (n=5), the ewe’s neck was incised 1cm to the left of the trachea and the left carotid artery was identified and ligated with an 0 silk tie
(Mersilk, Ethicon Ltd, Edinburgh, UK). A small incision was made in the vessel wall with scissors and a 1.2 mm outer diameter PA-D70 hemodynamic catheter (Data

Sciences International, Tilburg, Netherlands) was then inserted into the carotid artery lumen proximal to the ligation. The catheter tip was passed proximally towards the aorta until a clear blood pressure waveform was obtained on the acquisition computer. The catheter within the carotid artery was secured with 0 silk ties (Mersilk, Ethicon Ltd,
Edinburgh, UK).
The catheter’s telemetric transmitter was buried in a pouch created under the skin of the neck and the neck skin was closed using 2-0 PDS (Ethicon, St.
Stevens-Woluwe, Belgium). A laparotomy was then performed, the uterine arteries
(UtAs) were identified bilaterally and mobilised immediately proximal to the first bifurcation. A 6mm 6PS transit time flow probe (Transonic Systems Inc., NY, USA), which can measure blood flow with an absolute accuracy of +10% and a relative accuracy of +2% was placed (n=11) around the main UtA on each side. The cabling from each probe was then exteriorized onto the ewe’s right flank, and the skin buttons were secured to the skin, as described.
2
Four to ten days later, the sheep underwent a second general anaesthetic and laparotomy.
A short 2cm section of the main UtA proximal to the previously placed flow probe was freed from the visceral peritoneum on both sides. Adenoviruses (Ad) containing the human VEGF-A
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or

-galactosidase (lacZ) genes were injected into the lumen of the main UtA using a 23 Gauge needle; Ad.VEGF-A
165 into one UtA, and Ad.lacZ into the contralateral UtA as a control. Before vector injection, each free UtA section was occluded digitally at its most proximal part and the vector was then injected distal to the occlusion but proximal to the flow probe over 1 min; occlusion was maintained for a further 4 min to maximize tissue transduction. The operators were unaware as to which

side had received Ad.VEGF-A
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vector. The ewe received standard analgesia and antibiotic prophylaxis and the abdominal incision was closed.
2
Adventitial neovascularization was assessed using immunohistochemistry against von
Willebrand factor (vWF). Paraffinised UtA sections were dewaxed and endogenous peroxidise activity was blocked with 0.6% Hydrogen peroxide for 15 minutes. Antigen retrieval was performed using 0.1% trypsin (215240, BD Biosciences, UK) digestion at
37°C for 10 minutes. The sections were then blocked with 5% Normal goat serum
(X0907, Dako, Glostrup, Denmark) at room temperature for 30 minutes. Polyclonal rabbit anti-human vWF (1:400, A0082, Dako, Glostrup, Denmark) was used as the primary antibody and incubated overnight at 4°C, followed by a biotinylated polyclonal goat anti-rabbit IgG secondary antibody (1:100, E0432, Dako, Glostrup, Denmark) for 1 hour at room temperature. Negative controls were obtained by omitting the primary antibody. Following one wash with PBS supplemented with 0.1% bovine serum albumin and two washes with PBS, the sections were incubated with an ABC solution (PK4000,
Vector Laboratories, Peterborough, UK) for one hour. The slides were washed again with
PBS and the sites of antibody binding were visualised by 3,3’ diaminobenzidine (D5905,
Sigma Aldrich) staining until colour development. The sections were counter-stained with Mayer’s Haematoxylin solution. The slides were then dehydrated by passing through an ascending alcohol gradient, treated with xylene and mounted in DPX. The positively stained adventitial blood vessels with a distinct lumen were counted under a light microscope (Nikon Eclipse E600, Amstelveen, Netherlands). Each UtA section was

stained in duplicate and counted by two researchers independently, who were blinded to the treatment administered at the time of counting.
The snap-frozen UtA tissues obtained at post-mortem (from the current cohort of sheep as well as a previous short-term cohort, which was sacrificed 4-7 days postadministration of the vector) were pulverised in liquid nitrogen. The powdered tissue was then homogenised using a polytron, in RIPA buffer (R0278, Sigma Aldrich) containing
Complete EDTA-free Protease Inhibitor Cocktail Tablets (11697498001, Roche
Diagnostics, Germany) and Phosphatase Inhibitors 1 and 2 (P2850 and P5726, Sigma
Aldrich), according to the manufacturers’ recommended dosages. The homogenate was spun down in a microfuge at 13,000 rpm for 10 minutes at 4°C. The supernatant was transferred to a clean tube and used for protein estimation using a protein assay kit based on Bradford method (500-0002, Bio-Rad, Herts, UK).
25 μg of total protein was loaded on a 4-12% Bis-Tris Gel (NP0035BOX, Invitrogen,
UK) and transferred onto an Immobilon PVDF membrane (LC2005, Invitrogen, UK).
The membrane was blocked with 5% skimmed milk powder and subsequently probed with a mouse monoclonal anti-eNOS antibody (1:3000, 610296 , BD Transduction
Laboratories), followed by a secondary sheep anti-mouse IgG-HRP conjugate (1:3000,
RPN 4201, GE Healthcare, UK). The membrane was developed on an X-ray Hyperfilm
(28906837, Amersham Life Sciences) using ECL Plus Western blotting detection kit
(RPN 2132, GE Healthcare, UK).

Immunohistochemistry was used to assess changes in the VEGF receptors in UtA sections, namely VEGFR1, VEGFR2 and neuropilin-1. Slides were dewaxed and antigen retrieval was performed by 0.5 mg/ml Pronase (10165921001, Roche Diagnostics,
Germany) treatment at room temperature for 15 minutes (for neuropilin-1) or boiling in citrate buffer for 20 minutes (for VEGFR-1 and VEGFR-2). The sections were then treated with 3% hydrogen peroxide for 10 minutes, washed with PBS, permeabilized with
PBS-Tween 20 (0.1%) for 10 minutes and blocked in 5% rabbit serum (Neuropilin-1 and
VEGFR-2) or goat serum (VEGFR-1). A goat polyclonal neuropilin-1 (1:50, sc-7239
Santa Cruz Biotechnology USA), rabbit polyclonal VEGFR-1 (1:50, sc-316 Santa Cruz
Biotechnology, USA) or mouse monoclonal VEGFR-2 (1:50, sc-6251 Santa Cruz
Biotechnology, USA) primary antibody was applied overnight at 4°C. Next morning, the slides were washed and the appropriate secondary antibody was applied at 1:400 dilution; polyclonal rabbit anti-goat biotinylated IgG (E0466, Dako Glostrup, Denmark) for neuropilin-1, polyclonal rabbit anti-mouse biotinylated IgG (E0464, Dako Glostrup,
Denmark) for VEGFR-2 and polyclonal goat anti-rabbit biotinylated IgG (E0432, Dako
Glostrup, Denmark) for VEGFR-1. The slides were then incubated with ABC solution, stained and fixed as described above for anti-vWF staining.
References:
1. Barbera A, Jones OW, 3rd, Zerbe GO, Hobbins JC, Battaglia FC, Meschia G.
Ultrasonographic assessment of fetal growth: comparison between human and ovine fetus. Am J Obstet Gynecol 1995; 173 (6) : 1765-9.

2. Abi-Nader KN, Mehta V, Wigley V, Filippi E, Tezcan B, Boyd M et al.
Doppler ultrasonography for the noninvasive measurement of uterine artery volume blood flow through gestation in the pregnant sheep. Reprod Sci 2010; 17 (1) : 13-9.