Fig. 15

Application of conventional unilateral versus BEFAF model to explore the effects of prophylactic/therapeutic surgical flap treatment on tissue susceptibility to secondary ischemia. Experimental design: the SIEV and SIEA of the pedicle are clamped with a single atraumatic non-serrated S&T 11 mm vascular clamp for 2 h to induce primary ischemia (PI). After 2 h the clamp is released, and the flap is allowed to reperfuse for 6 h (R1) prior to repeated SIEV and SIEA clamping to induce a secondary ischemia (SI) by arteriovenous occlusion (AVO) lasting for 4 h. Once the SI is alleviated, the secondary reperfusion (R2) begins until the endpoint (POD 8). Experimental and control treatments are introduced prior to R1 and R2 events by intradermal injection into the distal flap region. A In a traditionally used ischemic flap model, a single fasciocutaneous flap is created and rotated around a pivotal axis point (the base of the pedicle may remain intact or may be severed to minimize a residual microvascular inflow) per animal. Power analysis suggests that if the flap survival percentage response within each subject after SI is normally distributed with standard deviation of 10, and assuming the attrition rate is 10%, then if the true difference in the experimental and control means is 20 as to be calculated by independent two-tailed Student’s T-test, we would need to study 7 experimental and 7 control subjects (total N = 14 animals) to be able to reject the null hypothesis that the population means of two groups are equal with probability (power) of 0.9. The Type I error probability associated with this test of this null hypothesis is 0.05. B Left panel. To perform the similarly powered experiment with twice as few animals per group, two 3 × 9 cm BEFAF flaps were created within each animal: one flap was assigned to the treatment, and the opposite flap – to the control group. Experimental treatment solutions were prepared from blood collected through cardiac puncture of previously sacrificed inbred Lewis strain rats (N = 12). Mixed arteriovenous blood was collected in 4.5 mL BD Vacutainer® Sodium Citrate (0.105 M, 3.2%) glass tubes that were centrifuged at 400 × g for 10 min at 4°C to separate plasma from red blood cells. Plasma with a buffy coat layer was collected into sterile 5 mL Eppendorf tube and spun at 1,000 × g for 10 min to sediment platelets and leukocytes. Half of supernatant was collected into 2 mL Eppendorf tube (platelet poor plasma, PPP). The remaining supernatant was gently mixed with the pellet and transferred into a separate 2 mL Eppendorf tube (platelet rich plasma, PRP). PPP and PRP solutions were activated by adding 1 M CaCl₂ to the final concentration of 22.2 mM and incubating the tubes in a dry bath at 37°C for 1 h. Growth factors (GFs) were released from the precipitated platelets into the liquid fraction after final centrifugation step at 10,000 × g for 10 min. Supernatants were passed through sterile 0.22 µm pore size syringe filter, combined into a single aPPP or aPRP stock solution and stored at -20°C until further use. Pooling was intended to eliminate or minimize the heterogeneity of aPRP or aPPP content so that all flaps would receive an equal concentration of GF-enriched or control filtrate. 250 μL of aPRP was injected to the distal portion of inner right-side flap (R), while the 250 μL of aPPP (control) was injected in the similar location of the left-side flap (L) prior to the start of R1 and R2 events. At POD 8, the paired flaps were photographed, harvested, and divided into equal proximal (P), medial (M) and distal (D) regions that in turn were subdivided into left, central, and right segments. Right panel. The left segment of P, M or D regions was flash frozen, crushed into powder, lysed, pooled from seven 10-month old retired breeder Lewis strain male rats (mean weight = 542 g), and used for protein separation by lithium dodecyl-sulfate polyacrylamide gel electrophoresis (LDS-PAGE) at 130 V constant in 4–12% gradient NuPAGE gels followed by protein transfer onto 0.22 μm nitrocellulose membranes at 30 V constant for 90 min, and protein expression analysis by immunoblotting (IB) using the primary antibodies against indicated total or phosphorylated (p-) forms of antigens of interest and horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent proteins bands were visualized by a Kodak Image Station 440CF equipped with the Caresteam image analysis software (Kodak, New York, NY) following 5 min strip incubation with 20 ml SuperSignal™ West Dura Extended Duration Substrate (TFS, # 34076)