1 Materials and methods
1.1 Materials
1.1.1 Experimental animals: white rats.
1.1.2 Experimental materials: 1% pentobarbital sodium, BN52021 (Sigma product), 100 mM mannitol + 22.4 mM sodium chloride solution, 10% formaldehyde solution, minor surgical instruments, PBS solution, 3% hydrogen peroxide, anti-ICAM-1 monoclonal antibody (primary antibody), biotinylated IgG (b-SAM IgG), Streptavidin-HRP (DAKO product), 0.05% DAB solution, hematoxylin.
1.2 Methods
1.2.1 Experimental Groups
1.2.1.1 Group of Effects of BN52021 on Flap Inflammatory Response
SD rats 72 The rats, weighing 250-300 g, regardless of sex, were randomly divided into 2 groups: treatment group and control group. Each group was further divided into 3 groups, namely: 3 h group, 6 h group, and 9 h group. Design an arbitrary skin flap with a length-to-width ratio of the pedicle at the head end of 4:1 (6.0 cm×1.5 cm) (Figure 1). The treatment group was given [BN52021 0.5 mg+100 mM mannitol+22.4 mM 0.5 h before surgery Sodium chloride] [4] 2 ml subcutaneous injection; the control group was only given [100 mM mannitol + 22.4 mM sodium chloride] 2 ml subcutaneous injection. Then, specimens were collected 3, 6, and 9 hours after surgery, and the flap was divided into proximal, middle, and distal parts for immunohistochemical pathological examination.
Figure 1 Design diagram of any skin flap on the back of mice
1.2.1.2 Group of BN52021 affecting the survival ability of any skin flap in mice
30 SD rats, body weight 250-300 g, male or female, randomly divided into 2 groups: treatment group and control group, 15 in each group. Among them, the treatment group was given local subcutaneous injection of skin flap [BN52021 0.5 mg+100 mM mannitol+22.4 mM sodium chloride] [4] 2 ml/animal 30 minutes before surgery, and the control group was only given local subcutaneous injection of skin flap [100 mM Mannitol + 22.4 mM sodium chloride] 2 ml/bird.
1.2.2 Immunohistochemical staining method (LSAB method): The specific steps of the LSAB method are: paraffin embedding → sectioning → 3% hydrogen peroxide to inhibit endogenous peroxidase → microwave antigen retrieval, 95 ℃ , 10 min → wash with PBS, add anti-ICAM-1 monoclonal antibody (primary antibody) → biotinylated IgG (b-SAM IgG, secondary antibody) → Streptavidin-HRP complex (Streptavidin-HRP) → 0.05 %DAB+0.03%H2O2, color development→hematoxylin understaining→conventional resin sealing.
1.2.3 Image processing: Auto cad 14 image processing system.
1.2.4 Statistical processing method: t-test.
2 Results
2.1 The expression of ICAM-1 on the surface of vascular endothelial cells in each section of the skin flap in the treatment group was higher than that of ICAM on the surface of vascular endothelial cells in the corresponding section of the skin flap in the control group. The expression of -1 is significantly reduced.
2.2 After using the platelet activating factor specific receptor antagonist BN52021 to treat the local skin flap, the survival ability (area) of the skin flap was significantly greater than that of the control group, P<0.001 (see Table 1, Figure 2) .
Table 1 Effect of BN52021 on mouse skin flap survival area (±s)
Group n survival rate
Treatment group 15 0.598±0.041
Control group 15 0.318±0.017
P<0.001
Figure 2 Effect of BN52021 on the survival ability of any skin flap in mice
3 Discussion
3.1 Finding factors that promote the survival ability of skin flaps has always been an important issue in plastic and reconstructive surgery. Flap transplantation is a very important method in plastic and reconstructive surgery, and arbitrary skin flaps are the most commonly used clinical flaps. Generally speaking, the length-to-width ratio of any surviving flap is 1:1 to 2, and it can be used on the face. Reaching 1:3~4, however, this ratio is far from meeting the needs of clinical treatment. The purpose of this experiment is to observe whether the PAF receptor antagonist BN52021 can reduce the inflammatory response of ischemic flap tissue, thereby improving the local hemodynamics of the flap, thereby promoting the survival of the flap, and infer that platelet activating factors are present in the skin. role in valve transplantation.
3.2 Platelet-activating factor (hereinafter referred to as PAF) is an extremely strong inflammatory mediator [1], which mainly acts by binding to specific receptors.
PAF activates leukocytes in circulation, causing leukocytes to chemotaxis, aggregation and degranulation while releasing many other inflammatory mediators such as: 5-HT, leukotrienes, prostaglandins, free radicals and vasoactive intestinal peptide, etc., causing cell damage, Increased vascular permeability, inflammatory cell infiltration, plasma concentration and tissue edema [2,3].
3.3 During the transplantation of ultra-long arbitrary skin flaps, tissue ischemia and hypoxia (especially in the middle and terminal sections) are inevitable, leading to an increase in the concentration of PAF in the flap tissue [5], which activates White blood cells and lymphocytes lead to increased local vascular permeability, edema, blood flow resistance, and inflammatory cell infiltration in the flap, seriously affecting the viability of ischemic tissue. It is difficult to establish and control experimental conditions by using methods such as indium-labeled autologous leukocyte reinfusion or detection of local myeloperoxidase activity in skin flaps to monitor the degree of local inflammatory reaction in tissues [1, 5]. The application of immunohistochemistry technology to detect changes in ICAM-1 expression indirectly reflects the degree of local inflammatory reaction in skin flaps. It is convenient, intuitive, and reliable, and provides a new method for monitoring the inflammatory reaction in local flap tissues.
3.4 The chemotaxis of circulating leukocytes to areas of inflammation or tissue damage is a highly coordinated and finely regulated process, and most of them occur in postcapillary venules. The process of circulating white blood cells to the inflammatory site is inseparable from the firm adhesion between white blood cells and vascular endothelial cells. The firm adhesion between white blood cells and vascular endothelial cells is a key step in the inflammatory response [4]. Leukocyte adhesion and infiltration caused by ischemia-reperfusion injury mainly rely on the interaction between CD11b/CD18 on leukocytes and ICAM-1 on endothelial cells. ICAM-1 is basically expressed on the surface of leukocytes, lymphocytes, fibroblasts, endothelial cells and epithelial cells. PAF is known to be an extremely strong activating factor that can increase the expression of ICAM-1 and promote the interaction between leukocytes and post-capillary microorganisms. The strong adhesion between venous endothelial cells causes inflammatory infiltration of leukocytes. It is generally believed that the level of free ICAM-1 in the blood can reflect the level of adherent ICAM-1; while the level of adherent ICAM-1 The level can reflect the degree of tissue inflammatory response [6].
3.5 In this experiment, the horseradish peroxidase staining reaction of flap tissue treated locally with BN52021 was significantly lower than that of the control group, indicating that PAF receptor antagonists can block a series of inflammation mediated by PAF. reaction, reduce the expression of ICAM-1, reduce the degree of leukocyte infiltration in the local flap, and can successfully reduce the inflammatory response of the flap tissue [2, 4]. This study introduces the influence of inflammatory reaction on flap survival ability into the flap survival mechanism, deepens the understanding of the role of PAF in flap survival in the field of plastic and reconstructive surgery, further enriches the relevant flap survival mechanism, and has certain implications for clinical practice. guiding significance.
3.6 During any flap transplantation process, the local PAF level of the flap increases, leading to a series of inflammatory reactions, seriously affecting the local hemodynamics of the flap and affecting the survival of the flap. In this experiment, a single dose of BN52021 was used to treat the local skin flap, blocking the pathophysiological reaction mediated by PAF and significantly improving the survival ability of the skin flap.