Tian X L, Jiang B, Yan H
Department of Plastic and Burns Surgery, the First Affiliated Hospital of Southwest Medical University, Luzhou 646000, China.
Zhonghua Shao Shang Za Zhi. 2019 Jan 20;35(1):48-53. doi: 10.3760/cma.j.issn.1009-2587.2019.01.009.
To observe the effects of platelet-rich plasma (PRP) on the survival of ultra-long dorsal random flaps in rats. Sixteen male Sprague Dawley rats aged 6 to 8 weeks (the same below) were sacrificed to collect whole blood of 9 to 10 mL from each rat, and PRP was prepared by modified APPLE method. The platelet count of retained whole blood and PRP detected by automated blood cell analyzer showed that PRP was made successfully. The other thirty-two rats were collected and divided into PRP group and control group according to the random number table, with 16 rats in each group. One rectangular ultra-long random flap with area of 8 cm×2 cm was made on the back of each rat and replanted in situ. The equidistant 3 points were designed on both sides of the flap of each rat. Rats in PRP group were injected with 0.1 mL PRP from dermis and subcutaneous tissue of each injection point, while rats in control group were injected with the same volume of normal saline. Eight rats in each group were sacrificed at post operation hour (POH) 24 and on post operation day (POD) 7. On POD 7, survival of flaps of rats in 2 groups was observed, and the survival rates of flaps were calculated. On POD 7, the proximal, middle, and distal flaps of rats in 2 groups were collected, and histological changes of the flaps of rats in 2 groups were observed with hematoxylin-eosin staining. At POH 24 and on POD 7, flaps in 3 to 4 cm to pedicles were taken to detect mRNA expressions of vascular endothelial growth factor (VEGF), platelet-derived growth factor AA (PDGF-AA) and PDGF-BB by real-time fluorescent quantitative reverse transcription polymerase chain reaction, and to determine content of nitric oxide by nitrate reductase method. Data were processed with test. (1) On POD 7, flaps of rats in PRP group were dry without purulent exudate, and covered with scab, and the pink new skin emerged after scab fell off. On POD 7, flaps of rats in control group were with a large amount of inflammatory exudates, 1/2 to 2/3 of flaps at the distal were with necrosis and covered by scab which was not easy to be stripped. The survival rate of flap of rats in PRP group was (67±6)%, significantly higher than (52±10)% of rats in control group (=1.94, <0.05). (2) There were no obvious inflammatory cell infiltration and a number of microvessels, and fibrous tissue arranged neatly in the proximal flaps of rats in PRP group. There were a few of inflammatory cell infiltration and a number of microvessels, and fibrous tissue arranged slightly disorderly in the middle flaps of rats in PRP group. There were many more inflammatory cell infiltration and microvessels, a small amount of vascular embolism, and fibrous tissue arranged disorderly in the distal flaps of rats in PRP group. There were a large number of inflammatory cells infiltration and a few of microvessels, and fibrous tissue arranged disorderly in the proximal, middle, and distal flaps of rats in control group. (3) At POH 24 and on POD 7, mRNA expressions of VEGF, PDGF-AA, and PDGF-BB of rats in PRP group were significantly higher than those of rats in control group (=6.46, 5.61, 2.88, 10.18, 6.10, 7.67, <0.001). (4) At POH 24, content of nitric oxide in flap of rats in PRP group was (5.0±0.9) μmol/g, significantly higher than (3.4±0.9) μmol/g of rats in control group (=19.14, <0.001). On POD 7, content of nitric oxide in flap of rats in PRP group was (3.3±0.8) μmol/g, which was close to (3.0±0.6) μmol/g of rats in control group (=2.93, >0.05). PRP can improve the survival rate of ultra-long dorsal random flap in rats, which may be related to regulation of angiogenesis related factors, increase of nitric oxide content, and inhibition of excessive apoptosis of cells of PRP, so as to alleviate ischemical reperfusion injury and improve microcirculatory disturbance.
观察富血小板血浆(PRP)对大鼠超长背部随意皮瓣存活的影响。取16只6至8周龄的雄性Sprague Dawley大鼠(下同),处死后每只大鼠采集9至10 mL全血,采用改良APPLE法制备PRP。通过自动血细胞分析仪检测留存全血及PRP的血小板计数,结果显示PRP制备成功。另取32只大鼠,按随机数字表法分为PRP组和对照组,每组16只。于每只大鼠背部制作一个面积为8 cm×2 cm的长方形超长随意皮瓣并原位回植。在每只大鼠皮瓣两侧设计等距的3个点。PRP组大鼠于每个注射点的真皮及皮下组织注射0.1 mL PRP,对照组大鼠注射相同体积的生理盐水。每组分别于术后24小时(POH)和术后第7天(POD)处死8只大鼠。于POD 7观察两组大鼠皮瓣存活情况并计算皮瓣存活率。于POD 7采集两组大鼠皮瓣近心端、中间段及远心端组织,苏木精-伊红染色观察两组大鼠皮瓣组织学变化。于POH 24及POD 7取距蒂部3至4 cm处皮瓣,采用实时荧光定量逆转录聚合酶链反应检测血管内皮生长因子(VEGF)、血小板源性生长因子AA(PDGF-AA)及PDGF-BB的mRNA表达,采用硝酸还原酶法测定一氧化氮含量。数据采用检验处理。(1)POD 7时,PRP组大鼠皮瓣干燥无脓性渗出物,覆盖痂皮,痂皮脱落后可见粉红色新生皮肤。POD 7时,对照组大鼠皮瓣有大量炎性渗出物,远心端1/2至2/3皮瓣坏死,覆盖痂皮且不易剥离。PRP组大鼠皮瓣存活率为(67±6)%,显著高于对照组的(52±10)%(=1.94,<0.05)。(2)PRP组大鼠皮瓣近心端无明显炎性细胞浸润,有较多微血管,纤维组织排列整齐。PRP组大鼠皮瓣中间段有少量炎性细胞浸润,有较多微血管,纤维组织排列稍紊乱。PRP组大鼠皮瓣远心端炎性细胞浸润及微血管较多,有少量血管栓塞,纤维组织排列紊乱。对照组大鼠皮瓣近心端、中间段及远心端均有大量炎性细胞浸润,微血管较少,纤维组织排列紊乱。(3)POH 24及POD 7时,PRP组大鼠VEGF、PDGF-AA及PDGF-BB的mRNA表达均显著高于对照组(=6.46、5.61、2.88、10.18、6.10、7.67,<0.001)。(4)POH 24时,PRP组大鼠皮瓣一氧化氮含量为(5.0±0.9)μmol/g,显著高于对照组的(3.4±0.9)μmol/g(=19.14,<0.001)。POD 7时,PRP组大鼠皮瓣一氧化氮含量为(3.3±0.8)μmol/g,与对照组的(3.0±0.6)μmol/g接近(=2.93,>0.05)。PRP可提高大鼠超长背部随意皮瓣的存活率,其机制可能与调控血管生成相关因子、增加一氧化氮含量及抑制PRP细胞过度凋亡,从而减轻缺血再灌注损伤、改善微循环障碍有关。