荧光示踪法研究逆行岛状皮瓣静脉回流

目的探讨采用荧光示踪法研究逆行岛状皮瓣静脉回流的可行性，并初步观察静脉回流规律。方法20只新西兰大白兔，每只取耳静脉血0．ImL，分离RBC并用FITC标记。流式细胞仪检测已标记的RBC阳性率及荧光强度，倒置荧光显微镜观察其形态。取20只新西兰大白兔，在动物双侧后肢内侧分别建立4cm×3cm隐动、静脉逆行岛状皮瓣模型（n=10）和顺行岛状皮瓣模型（n=10），血管蒂长3cm。将一侧后肢随机设定为实验组，皮瓣制备后注射已标记的RBC悬液5pL；对侧为对照组，不注射示踪剂。实验组按顺行和逆行皮瓣分成两组，即顺行皮瓣组和逆行皮瓣组，每组10个；再根据注入示踪剂途径不同，分为动脉和静脉2个亚组，每亚组5个皮瓣。注射示踪剂5S后取下皮瓣立即冷冻，取连续的3张冰冻切片（5～7pm），其中2张行HE染色和GENMED染色，另]张不染色直接压片，荧光显微镜观察荧光分布。结果流式细胞仪分析FITC标记的RBC阳性率在99％以上，荧光强度均≥10。；倒置荧光显微镜下标记的RBC呈均匀分布的绿色荧光，荧光强度均匀、稳定。冰冻切片显示实验组皮瓣蒂部均出现荧光，对照组未见荧光。顺行岛状皮瓣组荧光主要分布在静脉腔、静脉壁、动脉内膜和外膜；逆行岛状皮瓣组荧光分布在动脉内膜、外膜和静脉壁。结论荧光示踪剂可用于静脉回流研究，顺行岛状皮瓣静脉主要通过静脉腔、静脉壁、动脉内膜和外膜回流；逆行岛状皮瓣静脉主要通过动脉内膜、外膜和静脉壁的“迷宫式途径”回流。

STUDY OF VENOUS DRAINAGE IN RETROGRADE ISLAND FLAPS BY FLUORESCENCE TRACING TECHNIQUE IN A RABBIT MODEL

OBJECTIVE: To investigate the venous drainage in retrograde island flaps by fluorescence tracing technique and to observe the pathway of venous drainage. METHODS: The 0.1 mL venous blood was collected from the marginal ear vein of every rabbit (n=20), respectively, and erythrocytes were separated by centrifugation and then were labeled with FITC. Positive rate and fluorescence intensity of FITC-labeled RBC were detected by flow cytometry. RBC morphous was observed under the inverted fluorescence microscope. Saphenous retrograde island fasciocutaneous flap and antegrade island fasciocutaneous flap (4.0 cm x 3.0 cm in size with vascular pedicle length of 3.0 cm) were successfully established in hind limbs of 20 New Zealand white rabbits. One hind limb of each rabbit was randomly assigned as the experimental group and the contralateral side was assigned as the control. The same flap was established in the control group without any fluorescence tracer. According to retrograde or antegrade flaps, the experimental group was divided into 2 groups with 10 rabbits in each group. And then, according to different pathways of tracer-giving, each group was divided into 2 subgroups of artery and vein, with 5 rabbits in each subgroup. The labeled erythrocytes (5 microL) were injected into artery or vein and then flaps were cut down 5 seconds later. The flaps were immediately frozen and chipped (5-7 microm). Consecutive three frozen sections were made and two of them were stained with HE and GENMED, respectively, but the third one was squashed without staining. All frozen sections were observed under the microscope. RESULTS: Positive rate of FITC-labeled RBC was beyond 99% and fluorescence intensity was more than or equal to 10(3). FITC-labeled RBC showed steady green fluorescence under the inverted fluorescence microscope. Fluorescence appeared in all experimental groups, but none was found in the control groups. In antegrade island flap group, fluorescence appeared mainly in lumen of vein, wall of vein and inner membrane and outer membrane of artery. In retrograde island flap group, fluorescence distributed principally in inner membrane and outer membrane of artery and wall of vein. CONCLUSION: The fluorescence tracing is applicable to the research of venous drainage. Venous drainage in the antegrade island flaps is mainly through lumen of vein, wall of vein and inner membrane and outer membrane of artery. While, venous drainage in retrograde island flaps is principally through inner membrane and outer membrane of artery and wall of vein.