Crocin enhances the viability of random pattern skin flaps: involvement of enhancing angiogenesis and inhibiting oxidative stress

Abstract

The random pattern skin flap has been an important procedure in plastic and reconstructive surgery to cover various challenging defects. However, distal flap necrosis is problematic. Crocin is a natural carotenoid compound, which have been reported to possess a wide spectrum of properties and induce pleiotropic anti-inflammatory, anti-oxidative and cytoprotective effects. We explored whether crocin enhanced random skin flap survival. Thirty-six male SD rats were distributed to two groups randomly: the crocin and control groups. Flap survival areas were measured 7 days after surgery. Neutrophil numbers and microvascular density were evaluated via haematoxylin and eosin staining, and blood perfusion via laser Doppler imaging. Vascular endothelial growth factor (VEGF) levels were measured by immunohistochemical staining and Western blotting. We also measured the levels of markers of ischaemia-reperfusion injury [malondialdehyde (MDA) and superoxide dismutase (SOD)]. With regard to flap survival area assay, a significant between-group difference of survival area for the experimental flap was evident. As for flap blood flow test in Area II, the crocin group was statistically better than that of the control group. And in the histological result, the mean vessel density and VEGF level were statistically higher when treated with crocin. Crocin also decreased the MDA but increased the SOD level. Crocin thus improved random skin flap viability, enhancing angiogenesis and inhibiting ischaemia-reperfusion injury.

Keywords: Crocin; VEGF; laser Doppler imaging; random pattern skin flap.

Figure 1

Chemical structure of crocin, a prenylated flavonoid glycoside.

Figure 2

A McFarlane flap model was designed (3×9 cm); no axial vessels were incorporated into the flap. A. The modified McFarlane flap model (3×9 cm) was applied to every rat. B. No axial vessels were incorporated into the flap in which the ischemic gradient is proportional to the distance from the base. White arrow refers to the axial vessels. C. Each flap was sutured back to its original position with continuous 4-0 silk sutures.

Figure 3

Crocin improves flap survival area and ameliorates tissue edema. A. Digital photographs of flap survival in the Control and test groups were taken on day 7. B. Flap survival percentages for each group were then quantified and analyzed. Significance: *P<0.01 versus the Control group. Data represent Mean values ± SEM.

Figure 4

Crocin promoted angiogenesis in the ischemic areas of the flap. A. Histological changes in middle area II of the flaps of the test and control groups. Magnifications: ×200 (hematoxylin and eosin stain). The test group showed more angiogenesis. Black arrows refer to new blood vessels. B. The mean vessel densities (MVDs) in each group were quantified and analyzed. *P<0.01 versus the Control group. Data represent Mean values ± SEM.

Figure 5

A. The blood perfusion on day 7 in two groups. B. Mean blood flow of the II zone in the flap in two groups measured by laser Doppler imaging at day 7 after surgery (*P<0.01 vs. the control group).

Figure 6

Crocin Increased Vascular Endothelial Growth Factor Expression. A. VEGF protein levels in the test and control groups (immunohistochemical staining; ×400). B. Optical density values of VEGF expressions are quantified and analyzed for each group. C. Western blotting for VEGF protein expressions in area II tissue in the control and test groups. D. Optical density values of VEGF expressions in each group are quantified and analyzed for each group. *P<0.01 versus the control group. Data represent Mean values ± SEM.

Figure 7

A. SOD activities in the test and control groups. SOD, superoxide dismutase. B. MDA levels in the test and control groups. MDA, malondialdehyde. *P<0.01 versus the control group. Data represent Mean values ± SEM.