All existingBaker et al. Breast Cancer Research 2010, 12:R70 http://breast-cancer-research.com
All existingBaker et al. Breast Cancer Research 2010, 12:R70 http://breast-cancer-research.com/content/12/5/RPage 4 ofcontaminants from skin. Intradermal injections of 100 l of HAMC and HAMC dually infused with VEGF-C and ANG-2 were administered through 25-gauge needles. Equal volumes of saline and bradykinin (1 mg/ml) served, respectively, as the negative and positive controls. Injections were administered in duplicate once hourly for the first 3 hr (255, 195, 135, 75 min) and then at 45, 30 and 15 min. Following injections at 15 min, 100 L of 125 I bovine serum albumin ( 125 I-BSA) (0.364 mg/ml; Perkin Elmer, Ontario, Canada) and 25 ml of Evans blue dye (EB) were introduced through a catheter in the cephalic vein, following which the catheter was flushed with 5 ml of heparinized saline. A 15min time period was then PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29069523 allowed to elapse to allow radioactivity and dye to accumulate at points of inflammation, at which point the animal was killed. Skin encompassing the injection points was removed and photographed with a Nikon digital camera (model CoolPix 4500). Each injection area was then punched out individually using a 2.54-cm cork borer, placed in 10 formalin and measured for radioactivity in a gamma counter (1282 Compugamma CS Universal gamma counter; LLK Wallas, Turku, Finland) to quantify 125IBSA accumulation. Hematoxylin and eosin staining was performed on 7-m paraffin-embedded cross-sections of the injection areas to determine neutrophil infiltration. On the basis of preliminary results, it became necessary to assess inflammation induction over a BelinostatMedChemExpress PXD101 longer period of time, and as a result intradermal injections of HAMC (with and without growth factors) were introduced into nonanesthetized sheep at time points corresponding to 24 and 128 hr prior to being killed.Induction of lymphedema and growth factor therapythe area for 10 min to duplicate the length of time needed to perform the growth factor treatment surgery. The animals were returned to their holding pens after recovery from the anesthetic.Edema assessmentThe hind legs were shaved and leg circumference measurements were taken at a point 10 cm distal to the hock (tarsus). This landmark was highlighted with a skin marker for subsequent measurements postoperatively at the same location. The limb circumferences were divided by the original (presurgical value) and expressed as percentage change over time. To compare the edema outcomes in the various groups, the percentage change in limb circumference was plotted against time, and graphical integration of the area under the curves was calculated using the trapezoidal rule.Quantitative assessment of lymphatic functionLymphedema induction was conducted as previously described by Tobbia et al. [2,7]. Fasting and anesthesia induction were performed as described in the previous section. Surgical areas were shaved, washed with soap and water and disinfected with 70 alcohol. A vertical incision (6-8 cm) was made over the lateral aspect of the popliteal region in the hindlimb and the popliteal fossa was exposed. Following identification of the popliteal node in its surrounding fat pad, the pre- and postnodal lymphatics were ligated and the node was excised. At this time, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27872238 growth factor therapy was introduced. Preprepared HAMC (1 ml preloaded into a 3-ml syringe with a blunted 18-gauge needle) was introduced into the area of nodal excision. To increase the retention of HAMC at this site, the fat pad pocket was then carefully sealed with absor.