Methods
Animal experiments were performed in 16 male 3-6 months-old pigs with a mean weight of 44.0 ± 4.7 kg. All animal care and experimental procedures were in accordance with German national legislation on animal protection and approval was given by the Ministry of Agriculture, the Environment and Rural Areas of Land Schleswig-Holstein, Germany (V 312-72241.123–34). The animals were anesthetized using the following sedation, relaxation, and narcosis regimen: ketamine 10% with a dose of 0.25 mL/kg, xylazine 2% in a dose of 0.15 mL/kg, atropine sulfate 1% in a dose of 0.06 mL/kg. After endotracheal intubation anesthesia was continued with constant isoflurane (1.5-2 vol%) inhalation and oxygen (50 vol%) with a fresh gas flow rate of 1 L/min.
A software-controlled device was constructed for standardized automatic tissue dissection (Figure 1). After fixation at the operating table and insertion of the selected dissection tool, the device allowed identical excisions with fixed tissue contact times. Therefore, the dissector blade cut 5 cm in horizontal direction starting at an edge of the defined tissue sample and then moved forward for 1 cm redoing the same movements backwards until a 5 × 3 cm tissue sample was excised. In this study, we used the Ultracision Harmonic Scalpel HSA07 (Ethicon Endo-Surgery, Inc, Nordestedt, Germany.) on power level 5 for UC and the Erbotom ICC 350 (ERBE Elektromedizin GmbH Tübingen, Germany) on 60 W (cutting) for ME. These power level settings were chosen as they represent a widely used standard in abdominal surgery. While manual excision was performed in all 16, the automatic device was used only in 9 animals after validation experiments (data not shown).
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Figure 1.
Automatic device for soft tissue dissection. Self-constructed apparatus fixed at the operating table and loaded with ultrasonic scalpel. The instrument can be moved engine-driven into two directions (aluminium tracks).
Using a template, 8 excisions were sketched on each pig's abdominal wall. Then a double step randomization process defined the mode of excision (automatic versus manual) and the tissue dissection tool (UC versus ME) for each sample. Figure 2 illustrates the subsequent excision process. Vertical incisions were performed with a steel scalpel. Afterwards the tissue sample was excised in horizontal direction either by UC or ME, which exactly had to be done at the cutaneous-subcutaneous junction. Tissue samples were then fixed, dehydrated and paraffin embedded (Leica TP1050 Tissue Processor, Leica EG 1140 Embedding Center, Leica Microsystems, Germany). 3 micrometer cross sections of each sample were produced (Sliding microtome, Leica Microsystems, Germany) for Hematoxilin and eosin (HE) and Elastica van Gieson (EvG) staining. Light microscopy was performed by an experienced histopathologist (BK). 7 measure points were used to determine the median depth of necrosis in each sample. Preparation as well as histopathological and morphometric examination of all specimens was performed at BMP Labor für Medizinische Materialprüfung GmbH, Aachen, Germany using standard operating procedures and an accredited quality management system.
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Figure 2.
Schematic illustration of tissue sample and excision planes. a) epidermal layer, b) corium, c) subcutaneous fatty tissue, d) vertical excision lines performed by steel scalpel, e) horizontal excision line performed by either ultrasonic scalpel (UC) or monopolar electrocautery (ME).
The influence of the variables dissection mode (automatic versus manual) and dissection tool (ME versus UC) onto the depth of necrosis was evaluated using a two-way analysis of variance for repeated measures, including the interaction effect of the two factors as well. Effects were found to be significant if p-values were less than 0.05. All analyses were performed using the free software R (version 2.8, http://www.r-project.org.