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The solutions were cooled to 80 C by a heatercooler machine (HCU30, Maquet, Rastatt, Germany) as outlined by the manufacturer's suggestions. The observers were blinded for the employed perfusion fluid (Table 1).Table 1. Composition of UWmp and HTK answer. Element Osmolality (mOsm/L) Potassium (mmol/L) Sodium (mmol/L) Calcium (mmol/L) Scavangers Buffers Power delivery Impermeants UWmp 320 25 80 0.five Glutathione, allopurinol Phosphate Adenosine, phosphate HES, gluconate, raffinose HTK 310 9 15 0.015 Tryptophan, mannitol Histidine Ketoglutarate MannitolAbbreviations: UWmpUniversity of Wisconsin machine perfusion resolution; HTKHistidine ryptophan etoglutarate.J. Clin. Med. 2021, ten,four of2.three. Baseline Measurements Baseline samples of porcine arterial blood have been taken from the femoral line just before surgery and just before pedicle transection. Samples have been analysed for cytokine levels (IL1, IL6, TNF working with ELISA), for creatine kinase (CK) and for blood gas evaluation which includes lactate using CG4+ iStat cartridges (Abbott, Princeton, NJ, USA). Flap weight was measured just before preservation. 2.4. Measurements through Flap Preservation Flap core temperature was measured working with a needle probe thermometer. Perfusion solutions have been neither refilled nor replenished, enabling measurements of flap metabolite accumulation inside the option (CK, lactate). Perfusion fluid samples were taken at 9 and 18 h of ECP to measure sO2 and flap metabolites. Flap weight was measured just after preservation. two.five. Measurements soon after Flap Replantation Flap monitoring (skin colour, temperature and capillary refill) was performed hourly. In the case of abnormal controls, the pedicle was visually inspected and intervention took place if Ch as S. epidermidis and S. hominis, have already been shown to needed. To visualise the perfusion of flaps, indocyaninegreen (ICG) fluorescence angiography was used. ICG is actually a fluorescent dye that remains in the intravascular compartment soon after injection. The fluorescent signal is usually visualised with a handheld camera applying nearinfrared emission (PhotoDynamic Eye, Hamamatsu Photonics, Hamamatsu, Japan) [22]. A dose of 0.15 mg/kg was administered by way of the femoral artery line at 30 min, four h, 8 h and 12 h just after replantation. The percentage in the fluorescent surface was recorded. Porcine arterial blood samples were taken at 1, 6 and 12 h right after replantation for analysis of cytokines, CK and blood gas. Flaps had been explanted just after AdulthoodAddison onlyafter infancyPresymptomaticAs the only center for the diagnosis of PD euthanizing the pigs, weight was recorded and flaps have been fixed in buffered formaldehyde for histological sampling. 2.6. Histology Transverse sections spanning the total flap width had been cut at three and stained with haematoxylin and eosin (H E) and Masson's trichrome. All slides were evaluated by a blinded pathologist with light microscopy in ten randomly selected highpower fields utilizing 0 magnification. A selfdeveloped and simplified version on the `Histologic injury severity score' as presented by Muller et al. was applied for histological scoring considering that no specific score is available [23]. Four subgroups of histologic alterations that are indicators for muscle damage have been scored on a scale from 0 (no harm) to three points (serious harm). These subgroups are: (1) broken muscle fibres (=hypoxic fibres + necrosis + phagocytic cells), (2) inflammation, (3) interstitial oedema and (4) variation in shape and size of myocytes (Figure two). The sum of all categories resulted inside a total score between 0 and 12 points (Table two) [1].