Two months old, male Wistar rats were anaesthetised by intraperitoneal (ip.) injection of pentobarbital sodium (Repose 50%, Le Vet. Pharma, Oudewater, Netherlands). Stomach area was shaved and disinfected with iodine solution (Braunol; B/Braun, B. Braun Medical, Sempach, Switzerland). After a midline laparotomy, the abdominal aorta was temporarily clipped between the left renal artery and the aortic bifurcation and the abdominal aorta was poked through with a G18 needle into the wall of the inferior vena cava, resulting in the formation of an aortocaval fistula, a short‐cut for the bloodstream. Upon removal of the needle, tissue glue (cyanoacrylate glue, Loctite, Dusseldorf, Germany) was added on the surface of the aorta to seal the surface and to prevent bleeding. After surgery the animals were closed, provided proper wound care, and were administered with buprenorphine (0.05 mg/kg; ip.). Every effort was made to minimize the discomfort of the animals used in this study. Sham operated animals were used for controls underwent sham surgery, the same procedure as the treated groups except creation of the aortocaval fistula. For the development of volume overload‐induced hypertrophy, animals were kept for 4 or 8 months, respectively (n = 5–6 each group) (Figure1A ).
Braunol
Manufactured by B. Braun
Sourced in Germany
Braunol is an aqueous disinfectant solution containing 0.5% hydrogen peroxide and 0.05% polyhexanide. It is intended for the disinfection of skin and mucous membranes.
Automatically generated - may contain errors
8 protocols using braunol
1
Aortocaval Fistula-Induced Cardiac Hypertrophy
2
Skin Biopsy and Cell Isolation
3
Decellularized Porcine Small Intestinal Submucosa Preparation
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The preparation of decellularized small intestinal submucosa (SIS) was performed as previously described11 (link),29 (link),30 (link). In brief, porcine small intestinal segments were isolated from German landrace pigs (18–25 kg) and stored in undiluted Braunol (7.5% povidone-iodine solution in water, B. Braun) at 4 °C. Tunica mucosa and tunica serosa of intestinal segments were mechanically removed, followed by a chemical decellularization in 1% Triton X-100 in 10 mM TRIS, pH 7.5 under continuous shaking (90 rpm) at room temperature for 24 h. Afterwards, SIS was washed with distilled water for 24 h under continuous shaking, followed by washing with phosphate buffered saline (PBS) supplemented with 1 g/L Vancomycin, 100 mg/L Gentamicin, and 2.5 mg/L Amphotericin B under continuous shaking for 10 days at room temperature with daily change of washing buffer. Finally, SIS was sterilized by 150 Gy gamma-ray irradiation and stored in PBS at 4 °C until further use for a maximum of 6 months. Before use, SIS was cut open along the longitudinal axis, fixed in a metal frame with the submucosal side facing up and covered with culture medium.
4
Murine Melanoma Cell Lines Generation
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No animals were sacrificed as murine melanoma cell lines generated in 2019 were used (7 (link)). In brief, tissue samples from primary tumors (ear and tail) from two Tg(Grm1)Cyld−/− and two Tg(Grm1)Cyld+/+ male mice (age, 153 and 217 days) were collected and washed with Braunol® (7.5%; B Braun Meisungen AG), followed by 1X PBS, 70% ethanol and 1X PBS. Tumor tissue was added to a mixture of DMEM (Sigma-Aldrich; Merck KGaA) and collagenase. Following incubation for 3 h at 37°C, the cell suspension was centrifuged (4 min at 600 g, room temperature) and the cells were seeded in T25 flasks, as previously described (7 (link),13 (link)).
5
Anesthesia and Pain Relief for Mouse Abdominal Surgery
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For pain relief, mice received a combination of 0.1 mg/kg buprenorphine (Temgesic; Indivior Schweiz, Baar, Switzerland) and 5 mg/kg carprofen (Rimadyl; Pfizer, New York, NY, USA) subcutaneously 30 min before surgery. Mice were anesthetized in an enclosed container with isoflurane (5% vehicled by oxygen 1 L/min); thereafter, the isoflurane mask was fixed over the nose with continuous treatment of 2%–3% isoflurane vehicled by oxygen 1 L/min. Mice were placed on a surgical board on top of a heating pad (37°C). Thereafter, eyes were covered with vitamin A (retinol) for moisturization. The surgical field was shaved with an electric clipper and disinfected with antiseptic povidone-iodine solution (Braunol; B. Braun, Melsungen, Germany). The abdomen was opened through a longitudinal midline incision.
6
Skin Biopsy and Cell Isolation
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Skin sampling occurred in accordance with the German Act on Organ and Tissue Donation, Removal and Transplantation (Transplantationsge-setz). After obtaining written informed donor consent, an elliptical ~3 × 1.5 cm2 excisional skin biopsy was taken from behind the ear. Donors who were serologically positive for HIV1/2 or had active hepatitis B virus, hepatitis C virus or active syphilis (Clinical Laboratory Improvement Amendments-positive/Treponema pallidum antibodies ≥1:80/fluorescent treponemal antibody IgM/rapid plasma reagin) were excluded. The manufacturing process took place in a European Union GMP grade B clean room facility under laminar air flow (A in B). Biopsy tissue was disinfected using povidone-iodine solution (Braunol and Braunoderm; B. Braun, Melsungen, Germany), washed, dissected and subjected to two-step enzymatic digestion using collagenase (Collagenase NB 6 GMP Grade; Nordmark, Uetersen, Germany), followed by non-animal recombinant trypsin (TrypZean; Sigma-Aldrich, Taufkirchen, Germany). Following filtration and washing/centrifugation, pellets were resuspended in a stem cell-favoring medium.
7
Decellularization of Whole Aortic Valves
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Prior to decellularization, the whole OMVs were disinfected by washing in povidone iodide (Braunol®; B. Braun, Melsungen, Germany) for 5 min, followed by 20 min washing in PBS. The whole valves were subsequently decellularized using sodium deoxycholate (SD; Sigma-Aldrich, St Louis, MO, USA), sodium dodecyl sulphate (SDS ultrapure; Carl Roth, Karlsruhe, Germany) and β-mercaptoethanol (β-ME; AppliChem, Darmstadt, Germany). The detergent treatment was performed under constant shaking at room temperature: Thereafter DNA was cleaved with DNase I (24 h at room temperature under continuous shaking, 30000 IU DNase I per valve, 150 IU/ml, Sigma-Aldrich) using the previously described protocol [13] . Washing was performed in ten 12-h steps, two with distilled water and eight with PBS. The overall length of the decellularization process was 198 h.
8
Carotid Artery Ligation for Atherosclerosis
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Western-type (42% of total calories from fat, 1.5% cholesterol) diet was purchased from Ssniff (Soest, Germany). Animals were started on the western-type diet after tamoxifen treatment and 'wash-out' phase at an age of 10 weeks. Neo-intima formation and accelerated atherosclerosis was induced 4 days later by partial left common carotid artery ligation as described previously. 10 In brief, mice were anaesthetized using intraperitoneal injection of xylazine (5 mg/kg) and ketamine (100 mg/kg). The skin was shaved, disinfected (Braunol, B.Braun, Melsungen, Germany) and a ventral midline incision (5 mm) was made in the neck. The left common carotid artery was exposed by blunt dissection. Excepting the superior thyroid artery, the left internal and external carotid and the occipital artery were ligated with 6-0 silk suture. The incision was closed and the mice received subcutaneous injections of buprenorphine (0.05 mg/kg) post-surgery and every 12 h thereafter for the following 3 days.
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