CBA/J, DBA/2J and BALB/c mice were obtained from Charles River, Sulzfeld, Germany, and maintained in our animal facilities in Berlin and Magdeburg, Germany with a 12 h light/dark cycle with water and food ad libitum. Experimental procedures were approved by the German authorities (LaGeSo Berlin 0062/03 and Landesverwaltungsamt Sachsen-Anhalt AZ: 2-868 University of Magdeburg). We performed our experiments using the well-established combination CBA/J x DBA/2J, which has been recently described as a suitable IUGR model . Mating of CBA/J females with BALB/c males served as a control combination as it represents a normal pregnancy. Two months old CBA/J females were mated with 2-4 months-old BALB/c or DBA/2J males, checked twice a day for vaginal plugs and separated from the males if pregnant. The day of the vaginal plug was considered as day 0 of pregnancy. Animals were treated with mixed air (20.9% O2, pharmaceutical compressed air) or CO as indicated below.
Mice were placed in a 98-liter Plexiglas animal chamber (A-Chamber, BioSpherix, NY, USA) and exposed to CO (50 or 125 parts per million, ppmmixed with air) during either days 3 to 5 or 3 to 8 of pregnancy as explained elsewhere . Control mice were maintained in a similar chamber without CO, only receiving the mixed air. The gas flow into the Plexiglas chambers was maintained continuously at a rate of 12 liter/min. CO at a concentration of 5% (50.000 ppm) in balanced air (20.9% oxygen) was mixed with compressed air to obtain a final concentration of 50 or 125 ppmbefore being delivered into the exposure chamber. The compressed air came from a 7 bar in house air-supply system and the CO from a high-pressure bottle (Linde Gas Therapeutics GmbH, Unterschleißheim, Germany). CO concentration was controlled by varying the flow rate of CO using a flowmeter (Q-Flow, Vögtlin Instruments, Switzerland) before delivering to the chamber. Because the flow rate (12 liter/min) is primarily determined by the air flow, only the CO flow was changed to deliver the final concentration to the exposure chamber. A CO monitor (X-am 2000 Multi-gas Monitor, Dräger, Germany) was used to measure CO levels in the chamber. Gas samples were introduced to the monitor through a port in the side of the chamber and were analyzed by electrochemical detection (DrägerSensor XXS CO - 68 10 882, Dräger, Germany). Concentration levels were measured periodically and there were no fluctuations in the CO concentration after the chamber had equilibrated (approximately 10 min). The chambers were housed in a fume hood during the experiments and the room was equipped with a CO alarm. In the chambers, gas flowed at controlled rates to obtain the desired CO exposure conditions.
Experimental settings: Animal groups
For analysing the effect of CO on pregnancy outcome in mice known to develop IUGR, CBA/J females mated with DBA/2J males (n = 8/ group) were treated with different CO doses (50 and 125 ppm) as follows:
a) 125 or 50 ppmof CO were applied during implantation window (days 3-5 postcoitum, dpc)
b) 50 ppmof CO were applied during implantation and early placenta development (3-8 dpc)
CBA/J animals previously mated with BALB/c males (normal pregnancy) were treated with 50 ppmCO to study the effect of this gas in normal pregnancies. This was mainly due to discard toxic effects of CO in the normal pregnancy combination.
Implantation rate as well as the percentage of fetal death was analyzed at day 14 of pregnancy after sacrificing the animals by calculating the percentage of non-viable implantations to the total number of implantations (viable + non-viable) multiplied by 100. Placentas and fetuses were weighted. Tissue samples were obtained for flow cytometry, real-time-PCR, histopathology and immunohistochemistry studies.
In a second experiment, females of the IUGR combination (n = 8) were treated with 50 ppmof CO during days 3-8 being the animals sacrificed directly after CO exposure (day 8) to analyze the immediate effect of CO application at trophoblast level.
Measurement of Carboxyhemoglobin (COHb)
Blood was taken from the heart of the anesthetized animal and COHb as well as total hemoglobin (tHb) were analyzed by a blood gas analyzer (ABL 520, Radiometer, Copenhagen).
Cells isolated from spleen, lymph nodes or decidua (18) were incubated for 1 hour with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin at 37°C with 5% CO2for stimulation of cytokine secretion. To allow intracellular accumulation of secreted proteins 2 μM of monensin was added and incubated for further 3 hours. Cells were washed and incubated with antibodies against surface markers for 10 min at 4°C in darkness. For fixation, paraformaldehyde solution (PFA) at a concentration of 1% (p/v) was used, and cells were incubated overnight (ON) at 4°C. For detecting intracellular proteins, cells were washed and antibodies diluted in saponin 0.1% (p/v) were incubated for 20 min at 4°C in darkness. After the incubation time, cells were washed with saponin solution. The labelled cells were finally resuspended in FACS buffer and analyzed in a FACS Calibur (Becton Dickinson) cytometer. The lymphocyte population was gated based on size and granularity and used for further analysis. When only analysing extracellular markers, incubation with PMA, ionomycin and monensin was avoided. Antibodies used included: FITC-labelled anti-CD4; PE-labelled anti CD25, TNF-α, IFN-γ and IL-17; PE-Cy7-labelled anti CD3 (all purchased from BD Biosciences, Heidelberg, Germany).
Placenta samples were fixed in 96% ethanol and embedded in paraffin as described by Saint-Marie (see ). For detecting VEGF, paraffin samples were dewaxed with xylol (2 × 20 min) followed by a series of incubations in ethanol (100%, 95% and 75%, 10 min each), with a final incubation of 5 min in distilled water. The sections were washed with Tris buffered saline solution (TBS, pH = 7.40) for 10 min and treated with 3% hydrogen peroxide in methanol for 30 min at room temperature (RT) to block the endogenous peroxidase activity. After washing, the tissues were exposed to 5% BSA in TBS for 20 min at RT for protein blocking, stained with the primary antibody (goat anti-VEGF, Santa Cruz, San Diego, USA) diluted 1:100 in 5% BSA in TBS and incubated ON at 4°C. The tissues were washed and further stained with the secondary antibody (rabbit anti-goat, Dako, Germany) diluted in 5% BSA in TBS for 1 h at RT. After washing, the samples were incubated for 30 min with an AB-Complex/HRP solution (Dako, Germany). Finally, they were developed with AEC+ Substrate Chromogen (Dako), counterstained with Hematoxylin and mounted. Negative controls were obtained by replacing the first antibody with 5% BSA or goat diluted serum. For complement staining, 8 μm thick sections were cut, fixed and incubated with goat anti-mouse C3 (Cappel) 1:200, diluted in 5% BSA in TBS for 60 min at RT. As secondary antibody, we employed rabbit anti-goat biotinylated 1:200 for 60 min at RT. After washing, the samples were incubated for 30 min with an AB-Complex/HRP solution, developed with AEC+ Substrate Chromogen, counterstained with Hematoxylin and mounted.
Paraffin-embedded placenta sections were dewaxed and hydrated as already described. This was followed by a permeabilization of the samples with 0.1 M citrate buffer. The detection of apoptotic cells was performed following manufacturers instructions using the in situCell Death Detection Kit, POD (Roche Diagnostic Systems, Mannheim, Germany) but changing the incubation temperature to RT instead of 37°C. Amino-9-ethylcarbazole/substrate chromogen (DakoCytomation, Eching, Germany) and a counterstaining with Hemalaun (Roth, Karlsruhe, Germany) was used for visualization of apoptotic cells. Negative controls were performed by using only the label solution. All sections were analyzed under the light microscope by two independent observers without knowledge of the samples. The numbers of TUNEL+cells per mm2of placental tissue were evaluated with a scaled eyepiece, pre-calibrated with a slide micrometer.
Protein isolation and western blot analysis
Frozen placenta pieces were homogenized in lysis buffer (1% NP-40, 0,1 mg/ml n-Dodecil beta maltoside, 10 mM NAO3V, 1 M Tris pH 7.5, 5 m NaCl, 500 mM NaF, 500 mM EDTA pH 7,5, 100 mM PMSF) for 45 min. After isolation, homogenates were centrifuged at 12.000 rpm for 20 min at 4°C and the supernatant containing the proteins was transferred to a fresh tube. Protein concentration was assessed using the Pierce BCA Protein Assay (Thermo Thermo Fisher Scientific, Bonn, Germany) as indicated by the manufacturer. Protein samples were kept at -80°C and while working with them, they were always kept on ice. For Western Blot analysis, 20 μg of protein (for Bag-1, sFlt-1 and sEng analysis) or 50 μg of protein (for VEGF analysis) were transferred into a 8% (sFlt-1), 10% (Bag-1 or sEng) or 15% (VEGF) polyacrylamide gel and a SDS-PAGE in denaturizing conditions was performed at 100 V. After the electrophoresis proteins were transferred into PVDF membranes in transfer buffer containing 20% methanol (v/v), 0,19 M glycine and 0.025 M Tris-base pH 8,3. For protein detection, membranes were incubated with primary antibodies for 2 h with rabbit polyclonal antibody against Bag-1 (1:500), or overnight (ON) with goat polyclonal antibody against VEGF (1:100), sFlt-1 (1:200) or sEng (1:200) all from Santa Cruz, Biotechnology, San Diego, USA. After three washing steps with TBST (TBS with 0.5% Tween) for 5 min each, the membranes were then incubated with an anti rabbit HRP-conjugated (Thermo Fisher Scientific) or anti goat biotin-conjugated (Dako) antibody diluted 1:2000 for 1 h at RT and then with avidin-horseradish peroxidase complex (ABC complex, Biozol). GAPDH or β-Actin was used as loading control. The chemiluminescence signal was generated by using luminol (A8511-5G, Sigma-Aldrich), 4-hydroxycinnamic acid (p-coumaric acid; C9008-25G, Sigma Aldrich), and hydrogen peroxide (Merck, Darmstadt, Germany). The intensity of the bands was quantified by using the GeneSnap®Software, Version 4.01c from Syngene.
RNA isolation, cDNA synthesis and Real-time RT- PCR
RNA extraction was performed using Trizol®Reagent (Invitrogen, Darmstadt, Germany). Briefly, frozen placentas (100 mg) were treated with 1 ml Trizol®and disaggregated using an Ultra-Turrax T25 homogenizer. Isolation of RNA, cDNA synthesis and RT-PCR was performed as described elsewhere . For Bag-1 Bcl, Bclxl, Bax, RT-PCR was performed using TaqMan technology. For amplifying VEGF, PGF, SDF-1α and HIF-1α, RT-PCR was performed using SYBR green technology (Applied Biosystems, Warrington, UK), both with the i-Cyler (Biorad, Munich, Germany). β-Actin was used as house keeping gene. The amount of mRNA was calculated as 2-ΔCtin both cases. Primer sequences are available upon request.