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Fig. 1 Schematic illustration of the HIPEC procedure. Saline solution and fluid containing cisplatin were heated up through a water bath (37 or 42 °C) and circulated continuously into the abdominal cavity (perfusion rate: 180 ml per hour), through an inlet catheter into the right and an outlet catheter into the left lower abdomen. The temperature and flow are constantly monitored, and the outflow fluid is trapped by a drainage bag.
Fig. 2 Overview about the peritoneal carcinomatosis index (PCI). (A) Total and PCI of the respective organs was recorded according to the principle of Jacquet and Sugarbaker et al.(1996), which has been adapted for the animal model. PCI lesion size score: 0 = no tumor; 1 = tumor ≤ 1 mm; 2 = tumor > 1 mm ≤ 3 mm; 3 = tumor > 3 mm. (B) Exemplary photographic representation of the main tumor localizations. There are up to five intra-abdominal tumor sites, which are preferentially located on the large curvature of the stomach, peri-hepatic, peri-splenic, mesenteric and peritoneally on the abdominal wall.
Fig. 3 In vivo detection of RH-30 tumors by MR imaging. RH-30 tumors were detected by MR imaging of the abdominal cavity(A) without (native) and (B) with contrast agent.(C) For evaluation of the fluid distribution in the abdominal cavity during peritoneal lavage, MR images were also taken during HIPEC treatment. MR imaging confirmed the observed tumor dissemination depicted in Figure 2. Tumor lesions are indicated by red asterisks.
Fig. 4 Evaluation of changes in cell morphology and proliferation marker expression after HIPEC treatment. (A)Hematoxylin and eosin staining revealed no cell morphological changes in RH-30 tumors. (B) Immunohistochemical analysis of the proliferation marker Ki-67 (brown) showed no obvious changes in protein expression through the individual treatments. (C) The detailed examination of Ki-67 protein expression and determination of the Ki-67 proliferation index using a Ki-67 quantifier module of a pathology software (Cognition Master Professional Suite: Ki67 Quantifier, VMscope GmbH, Berlin, Germany) showed a slight reduction in the proliferation capacity.
Fig. 5 Investigation of markers for apoptosis induction after HIPEC treatment. (A) Apoptosis marker expression of the activated variant of Caspase 3 (black) revealed no HIPEC-specific apoptosis induction. The nuclei were counterstained with hematoxylin (blue). (B) Terminal desoxynucleotidyl transferase (TUNEL)-labelled DNA fragmentation as a sign of apoptosis induction (green fluorescence signal) is visible in the marginal areas of the HIPEC treated tumors in a dose and temperature dependent manner. The nuclei were counterstained in blue with DAPI.