The project was approved by the local ethics committee (N°16 in the french registry of ethical committees) of animal experimentation of the National Veterinary School of Alfort and validated by the French Ministry of Research under registration “APAFIS” number 2016113016134972. Sheep were euthanized under general anaesthesia after PIPAC procedure and before sampling. This was performed by a trained team. All precautions were taken to limit anxiety and pain of the animals.
Altogether, 10 non-pregnant multiparous ewes were used. The first three animals were used for preliminary tests and development of the model. Thereafter, PIPAC was carried out as follows: (i) one control female with physiological serum, (ii) three females with a capnoperitoneum at 12mmHg (group 1), and (iii) 3 females with a capnoperitoneum at 20mmHg (group 2). To avoid a potential “day” effect, group 1 and 2 were performed alternatively (2-3 procedures/day). Animal characteristics are described in Table 1.
All PIPAC procedures were performed in the surgery theatre of the Biomedical research center (CRBM) of the National Veterinary School of Alfort.
The anaesthesia was carried out by a trained team. Animals were fasted for 12-16 hours before surgery. After a premedication with ketamine (Imalgen 1000®, Merial, 4 mg/kg IV) and diazepam (Diazepam, TVM, 0.5mg/kg IV), anaesthesia was maintained with an automated ventilator, using isofluorane (2-2.5%) diluted in a mixture of air and oxygen (50/50). Analgesia was ensured by IV injection of fentanyl (Fentadon®, Eurovet Animal Health, 2µg/kg IV) per hour. Per-operating supervision focused on respiratory rate, cardiac frequency, oxygen saturation and arterial pressure.
- PIPAC: surgical procedure
The PIPAC was performed according to the safety rules described by Solaß (2013). All precautions were taken to ensure staff safety: every operator wore a surgical blouse, gloves, protection glasses and a high protection breathing mask.
After clipping the anterior abdominal wall, points were drawn on the skin for trocar localization 6 cm (laparoscopic camera) and 18cm (nebulizer) below the umbilicus. Two 12mm-incisions were made at these localizations (open-laparoscopy) and two 12mm-balloon trocars (Medtronic®, Autosuture 12mm, BTT, Covidien) were inserted, ensuring tightness of the abdomen and steadiness of the pressure (Figure 1). A capnoperitoneum was established and a camera was introduced in the abdomen for a short exploration phase. The nebuliser (MIP®, Reger Medizintechnik, Tottweil, Germany) was connected to the high-pressure injector using a high-pressure injection line (Medrad, Mark 7, Arterion®, Bayer). The distal part of the nebulizer was positioned at a 1cm depth, as measured from the trocar end. The sheep was placed in Trendelenbourg position to raise the rumen and provide a better exposition of the pelvis. Three milligrams of doxorubicin (Mylan®, 2mg/mL) diluted in 50mL saline were nebulized at a flowrate of 30mL/minute with a maximum pressure of 200psi, as usually recommended in human patients . After nebulization, the capnoperitoneum was maintained during 30 minutes. The abdomen was subsequently deflated using an airtight device equipped with a smoke filter and connected to the waste air system in order to avoid contamination of the surgical room with doxorubicin. Thirty additional minutes were allowed for optimum drug penetration in tissues before the animal was euthanized with pentobarbital (Dolethal®, Vetoquinol, 3.6g, i.e., 20ml, IV). A median laparotomy was performed and 9 samples (6 peritoneal, 1 ovarian, 1 omental and 1 ceacal) were collected (Figure 2). One more sample (omentum) was collected just facing the nebulizer. In order to ensure the reproducibility of the sampling for each animal, positions of the peritoneal samples were annotated relatively to their distance to the nebulizer. Samples were immediately frozen in isopentane at -40°C after horizontal inclusion in Optimum Cutting Temperature (Tissue-Tek® O.C.T. Compound, Sakura® Finetek). Blocks were kept frozen at -80°C.
All analyses were performed blindly. The natural fluorescent properties of doxorubicin was used for its localization in the tissues . Samples were handled in a dark room to avoid light exposure that may decrease fluorescence.
Sections (7 µm) were cut using a cryostat (Leica® CM1950), then mounted with 25µL anti-fade mounting medium (Vectashield®, Vector laboratories) that contained with 4,6-diamidino-2phenylindole (DAPI) at 1/1000. They were kept at 4°C until observation.
Analyses were performed with a Carl Zeiss (Germany) AxioObserver Z1 fluorescence microscope equipped with an ApoTome slider and coupled to AxioVision 4.8 software (Zeiss). A complete brightfield view of the section was imaged using a 10x Plan-Neofluar (NA 0.3) objective and 10 square areas of about 200µm side length were randomly selected. Then fluorescence analysis of each area was performed using a Plan Neofluar X40 oil immersion (NA 1.3) objective and an Axiocam MRm camera (Zeiss). Nuclei were identified using DAPI (blue). Doxorubicin positive nuclei (DOXO+) were stained both in orange and blue. Cytoplasm and extracellular stroma fluoresced in orange together with green auto-fluorescence (Figures 3 et 4). The time for image acquisition was similar for each fluorochrome throughout the experiments. Fluorescence setup and image acquisition times are detailed in Table 2. Since all images were in the same horizontal plane, fluorescence was not decreased depending on tissue depth.
Statistical analyses were performed with data collected from the 6 doxorubicin PIPAC-treated sheep. All analyses were performed with SPSS v15.0 and Stata v12.0 software (Stata Corp., College Station, TX, USA). Effect of treatment was analyzed using individual sample location, distance to nebulizer (for peritoneum, distinguishing frontal, proximal and distal samples) and histological type as variables.
Tissue distribution patterns of doxorubicin positive cells were assessed by measuring the ratio of DOXO+/DAPI+ nuclei. For each tissue sample, DAPI + and DOXO + positive cells were counted for each of the 10 square areas and summed up. A Mann-Whitney test was used to analyze the effect of increased intra-peritoneal pressure on the distribution pattern of doxorubicin according to the histological type and location of the sample related to the nebulizer.
Penetration depth of doxorubicin was estimated by measuring the distance between the luminal surface of the tissue and the deepest DOXO+ nuclei that were identified. Samples showing no doxorubicin were removed from analysis. The drug penetration depth was analyzed for each histological type and sample location. Tissue drug penetration was classified in 2 categories: <100µm and ≥100 µm for group comparison. In order to take into account the correlation between samples from the same ewe, a GEE model (Generalized Estimating Equation) was used  to compare penetration depth between the two groups. When one single sample was collected from each animal (ovary, caecum and omentum), drug penetration was compared using a one tailed Chi2 test.