中性粒细胞活化在缺血-再灌注损伤皮瓣微循环改变中的作用及地塞米松的调理

目的：研究皮瓣微循环超微结构的改变及活化中性粒细胞在其中的作用，阐明地塞米松保护皮瓣的作用机理。方法：Wistar大鼠分成4组，形成岛状皮瓣后，Ⅰ组：原位缝合；Ⅱ组：阻断静脉回流6h；Ⅲ组：阻断静脉回流10h；Ⅳ组：阻断静脉回流10h，去除血管夹时腹腔注射地塞米松5mg／kg。取材进行电镜和髓过氧化物酶(MPO)的检测。结果：Ⅰ组皮瓣微循环内皮细胞结构完整，无中性粒细胞粘附。术后MPO值一直为0。Ⅱ组内皮细胞肿胀，部分脱落，有一定数量活化中性粒细胞粘附于管壁。术后l、3天MPO值明显上升。Ⅲ组：内皮细胞坏死脱落。术后l天MPO值几乎是Ⅱ组的2倍。Ⅳ组内皮细胞结构基本完整，管壁少见活化中性粒细胞粘附。结论：缺血一再灌注损伤皮瓣微循环有显著的组织学改变，MPO值术后显著增加，且随阻断静脉回流时间延长而增加。中性粒细胞的活化在这一组织学改变过程中起重要作用。地塞米松保护皮瓣的机理与减轻了中性粒细胞的活化水平有关。     

脂质体携载金属硫蛋白对皮瓣继发静脉缺血再灌注损伤的作用

目的 观察脂质体包裹金属硫蛋白(MT)对岛状皮瓣继发静脉缺血再灌注损伤的保护作用。方法 在大鼠下腹部岛状皮瓣继发静脉缺血再灌注损伤模型上测定皮瓣即刻、继发缺血再灌注30min、7d后的丙二醛(MDA)含量、髓过氧化物酶(MPO)活性，继发缺血再灌注30min时血浆内皮素(ET)、乳酸脱氢酶(LDH)水平，7d后皮瓣MT的含量。结果 脂质体携载MT组明显降低皮瓣MDA、MPO、血浆ET、LDH含量，增加7d后皮瓣组织MT含量，提高皮瓣成活率。结论 脂质体包裹MT对皮瓣继发静脉缺血再灌注损伤具有良好的保护作用。     

不同类型皮瓣缺血再灌注损伤中性粒细胞活化水平的对比研究

目的研究中性粒细胞在阻断动脉供血及静脉回流皮瓣缺血再灌注损伤中不同活化水平,阐明阻断静脉回流皮瓣耐受缺血较阻断动脉供血皮瓣耐受缺血弱的原因.方法在不同时间点取材,测量皮瓣髓过氧化物酶(MPO)含量;计数皮瓣蒂部血管活化中性粒细胞数量.结果正常皮瓣组MPO一直未测到,阻断动脉供血组再灌注4h后MPO开始增加,24h达到高峰;阻断静脉回流组再灌注4h时MPO开始增加,24h达到高峰,最高值是动脉供血组的1.5倍;蒂部血管内活化中性粒细胞数量,阻断静脉回流组明显大于阻断动脉供血组.结论阻断静脉回流组皮瓣中性粒细胞活化时间较阻断动脉供血组早,活化数量多.中性粒细胞活化程度的不同是导致阻断静脉回流组皮瓣耐受缺血较阻断动脉供血组弱的重要原因.     

不同类型皮瓣缺血再灌注损伤中性粒细胞活化水平的对比研究

目的 研究中性粒细胞在阻断动脉供血及静脉回流皮瓣缺血再灌注损伤中不同活化水平。阐明阻断静脉回流皮瓣耐受缺血较阻断动脉供血皮瓣耐受缺血弱的原因。方法 在不同时间点取材。测量皮瓣髓过氧化物酶(MPO）含量；计数皮瓣蒂部血管活化中性粒细胞数量。结果正常皮瓣组MPO-直未测到，阻断动脉供血组再灌注4h后MPO开始增加，24h达到高峰；阻断静脉回流组再灌注4h时MPO开始增加，24h达到高峰。最高值是动脉供血组的1．5倍；蒂部血管内活化中性粒细胞数量，阻断静脉回流组明显大于阻断动脉供血组。结论阻断静脉回流组皮瓣中性粒细胞活化时间较阻断动脉供血组早，活化数量多。中性粒细胞活化程度的不同是导致阻断静脉回流组皮瓣耐受缺血较阻断动脉供血组弱的重要原因。     

缺血再灌注对大鼠岛状浅筋膜瓣超微结构的损伤及Verpamil的作用观察

探讨Ｖｅｒｐａｍｉｌ对大鼠皮瓣超微结构缺血再灌注损伤的保护作用。建立ＳＤ大鼠腹壁浅筋膜岛状皮瓣模型，缺血５小时再灌注２０分钟，在电子显微镜下观察其超微结构的变化，并用Ｖｅｒｐａｍｉｌ作保护性对比。损伤以细胞膜和线粒体为主，Ｖｅｒｐａｍｉｌ组的损伤明显减轻。实验结果提示，Ｖｅｒｐａｍｉｌ对皮瓣的超微结构具有保护作用。     

阻断CD18介导的白细胞粘附对岛状皮瓣成活的影响

研究白细胞白细胞粘附在缺血再灌注损伤中的作用。方法，应用大鼠腹部岛状皮瓣模型，检测了皮瓣过氧化酶和丙二醛含量，观察了皮瓣的成活情况。结论ＣＤ１８介导 白细胞粘附参与了皮瓣再灌注损伤过程，抗ＣＤ１８单抗阻断白细胞附能减轻白细胞介导的组织损伤，并对岛状皮瓣具有保护作用。     

地塞米松防治皮瓣缺血-再灌注损伤及其机制

目的研究地塞米松对中性粒细胞凋亡与坏死的调控,阐明地塞米松防治皮瓣缺血-再灌注损伤的机制. 方法 30只Wistar大鼠腹部制备3 cm×6 cm 岛状皮瓣,分成3组(n＝10).A组正常皮瓣组;B组阻断静脉8 h,腹腔注射生理盐水作为对照组;C组阻断静脉8 h,腹腔注射地塞米松5 mg/kg,作为治疗组.术后7 d观察皮瓣成活面积;以Annexin V及Propidium双标记,流式细胞仪检测全血中性粒细胞凋亡、坏死水平;电镜观察凋亡、坏死中性粒细胞的形态.并于术后1 d,从各组大鼠皮瓣中央取材,电镜观察吞噬细胞吞噬凋亡中性粒细胞形态.阻断血管前,再灌注损伤后0、 3、6、12 及 24 h,以双夹心ELISA法测量血浆肿瘤坏死因子α(tumor necrosis factor α,TNF-α)及白细胞介素10(interleukin 10,IL-10)浓度. 结果皮瓣成活面积A、C组大于B组(P<0.05),A、C组差异无统计学意义(P＞0.05).术后1、3 d,B组全血中性粒细胞凋亡含量明显低于A、C组, 6 d高于A、C组.术后1、3、6 d,中性粒细胞坏死水平B组高于A、C组.术后1 d,皮瓣中吞噬细胞吞噬凋亡中性粒细胞的数量C组明显高于B组(P<0.05).B组血浆TNF-α于再灌注1 h达到高峰,IL-10于再灌注3 h达到最低.C组TNF-α峰值明显低于B组,6 h即明显下降;IL-10再灌注1h达最低,3 h时明显上升,其浓度明显高于B组. 结论地塞米松防治皮瓣缺血再灌注损伤的机制在于调理了中性粒细胞的凋亡水平,减少了中性粒细胞的坏死数量,平衡了中性粒细胞分泌细胞因子.     

地塞米松对中性粒细胞死亡过程的调控

目的　研究地塞米松对中性粒细胞死亡的调控 ,阐明地塞米松防治皮瓣缺血再灌注损伤的机理。方法　 5 0只大鼠等分成 5组。Ⅰ组 :正常皮瓣组 ;Ⅱ组 :阻断动脉 8h ,生理盐水对照组 ;Ⅲ组 :阻断静脉 8h ,生理盐水对照组 ;Ⅳ组阻断动脉 8h ,地塞米松 5mg kg组 ;Ⅴ组 :阻断静脉 8h ,地塞米松 5mg kg组。术后观测皮瓣成活面积 ,检测全血中中性粒细胞凋亡、坏死水平。光镜观察中性粒细胞凋亡及坏死形态 ,电镜观察吞噬细胞吞噬凋亡中性粒细胞形态。结果　Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组的皮瓣成活面积分别是 ( 99 8± 2 5 ) %、( 18 8± 1 8) %、( 9 0± 0 8) %、( 98 2± 2 4) %、( 96 2± 2 2 ) %。中性粒细胞凋亡含量术后早期Ⅱ、Ⅲ组明显低于另外 3组 ,但第 6天则高于另外 3组。坏死水平术后Ⅱ、Ⅲ组高于另外 3组。皮瓣中吞噬细胞吞噬凋亡粒细胞的数量Ⅳ、Ⅴ组明显高于Ⅱ、Ⅲ组。结论　地塞米松防治皮瓣缺血再灌注损伤的原因在于调理了中性粒细胞凋亡水平 ,减少了其坏死数量 ,增加吞噬细胞吞噬凋亡中性粒细胞的水平。     

外源性锌对缺血再灌注损伤皮瓣超微结构的影响

目的:观察外源性锌对缺血再灌注(ischemia-reperfusionIR)损伤皮瓣的保护作用。方法:48只大鼠随机分为对照组(n=16)、缺血再灌注组即IR组(n=16)和补锌缺血再灌注组即补锌-IR组(n=16)。在大鼠以腹壁浅血管为蒂的岛状皮瓣缺血再灌注模型上,用硫代巴比妥法和比色法分别测定皮瓣组织中丙二醛(MDA)含量和髓过氧化物酶(MPO)活性。应用透射电镜观察皮瓣缺血再灌注损伤后超微结构的改变,并观察皮瓣成活率。结果:补锌-IR组在再灌注1h和24h,皮瓣组织中MDA含量分别较IR组降低11.3%、33.2%,MPO活性分别较IR组降低17.9%、21.4%。补锌-IR组皮瓣的超微结构改变较IR组明显减轻,皮瓣成活率较IR组升高27.2%。结论:外源性锌能显著减轻缺血再灌注损伤皮瓣中组织细胞超微结构的病理改变,对皮瓣缺血再灌注损伤产生一定的保护作用。     

阻断CD_(18)介导的白细胞粘附对岛状皮瓣成活的影响

目的研究白细胞及白细胞粘附在缺血再灌注损伤中的作用。方法应用大鼠腹部岛状皮瓣模型,检测了皮瓣髓过氰化酶(MPO)和丙二醛(MDA)含量,观察了皮瓣的成活情况。结果与正常时相比,缺血8小时及再灌注1小时皮瓣 MPO 和 MDA 水平明显增高,而以抗 CD_(18)单抗阴断白细胞粘附可明显减轻这种变化,并有效地提高皮瓣的存活面积。结论 CD_(18)介导的白细胞粘附参与了皮瓣再灌注损伤过程,抗 CD_(18)单抗阻断白细胞粘附能减轻白细胞介导的组织损伤,并对岛状皮瓣具有保护作用。     

Increased survival of island skin flaps by systemic heparin in rats

The effects of systemic heparin on the survival of island skin flaps have been evaluated. Island flaps measuring 3 x 6 cm were created in the abdomen/groin of rats based on an inferior epigastric neurovascular pedicle. The venous drainage from the flap was occluded for various lengths of time ranging from 5 to 10 hours in the control rats and from 8 to 15 hours in the heparin-treated group of rats. The rats in the treated group received 1 ml of heparin solution (300-400 U/kg) through the contralateral saphenous vein immediately prior to the onset venous occlusion and an equivalent dose every 4 hours during venous occlusion. The group of control rats received 1 ml of saline solution. Flap survival was evaluated daily for 7 days. The flaps of control rats demonstrated a gradual decline of the survival rate after 6 hours of venous occlusion and no survival after 10 hours of venous occlusion. The flaps of heparin-treated rats exhibited complete survival until 12 hours of venous occlusion and no survival after 13 hours of venous occlusion. Thus, systemic heparin prolonged tissue tolerance to venous occlusion from 10 to 13 hours. The flaps of control rats demonstrated a significant accumulation of inosine plus hypoxanthine to 260% of normal after 8 hours of venous occlusion. The flaps of heparin-treated rats exhibited little alteration of inosine plus hypoxanthine, and contained a greater concentration of glucose and lactate than the flaps of control rats after 8 hours of venous occlusion. The results demonstrate that systemic heparin can increase tissue tolerance to venous occlusion and improve significantly the flap survival following subsequent reperfusion.     

Improved reflow and viability in reperfused ischemic rat island groin flaps using dexamethasone

Reperfusion injury and the no-reflow phenomenon are manifestations of a complex series of events that culminate in an acute inflammatory reaction within reperfused tissue. Antiinflammatory therapy attenuates ischemia-reperfusion injury. The purpose of this study is to evaluate the effects of dexamethasone on blood flow and viability of ischemic rat island groin flaps. Thirty-three mature male Sprague-Dawley rats were divided into 3 groups. The flaps were subjected to 8 hr of ischemia in the control and treatment groups. Fluorescein studies were performed, and flap viability was assessed. Twenty-four hour fluorescence (after reperfusion) correlated very closely with the percentage of eventual flap viability. The blood flow and viability of the treatment group flaps were significantly better than those in the ischemic control group. Dexamethasone significantly improved ischemic flap survival in the rat 8 hr ischemic island groin flap model in our study. © 1995 Wiley-Liss, Inc.     

岛状皮瓣缺血再灌注损伤的血液流变学改变及地塞米松的调理研究

目的 探讨岛状皮瓣缺血再灌注损伤中血液流变学的变化及地塞米松的调理作用机制。方法 采用Wister大鼠60只，等分为缺血再灌注损伤模型。对照组（Ⅰ组）腹腔注射生理盐水2ml/kg，实验组（Ⅱ组）腹腔注射地塞米松5mg/kg。术后7天观察皮瓣成活面积；电镜观察皮瓣超微结构，计数中性粒细胞的坏死数量；以血液流变检测仪检测红细胞流变学，以微循环检测仪及扫描电镜检测中性粒细胞的流变学。结果 皮瓣成活面积Ⅱ组显著大于Ⅰ组；术后1、2天时，中性粒细胞的坏死数量Ⅱ组显著少于Ⅰ组（P＜0.05）；全血低切黏度、红细胞聚集指数、Casson屈服应力及中性粒细胞的黏附性Ⅰ组明显高于Ⅱ组（P＜0.05）；中性粒细胞的变形性Ⅰ组则比Ⅱ组低。结论 皮瓣缺血再灌注损伤可引起红细胞聚集性增强，中性粒细胞黏附性增加、变形性能力下降和坏死数量增多；地塞米松可有效调理上述指标，减少中性粒细胞的坏死数量，改善皮瓣血运。     

Secondary venous ischemic injury associated with neutrophil infiltration and lipid peroxidation: amelioration of injury by cyclosporin A in a rat inguinal island flap

Secondary venous ischemia caused by anastomotic failure is one of the major causes of failure after free tissue transfers and replantations. The effects of cyclosporin A (CsA) on secondary ischemic injury associated with neutrophil infiltration and lipid peroxidation were evaluated in a rat inferior epigastric island skin flap model. Primary ischemia was produced by arteriovenous occlusion for 2 hours. Twenty-four hours later, secondary venous ischemia was produced by 5 hours of venous occlusion. Nonischemic (n = 5), primary ischemic (n = 5), and secondary ischemic control groups (n = 10), and four treatment groups (n = 10) were created. Treatment groups received either 15 or 30 mg per kilogram per day oral CsA for 3 days before flap elevation, or 15 or 30 mg per kilogram intravenous CsA at 4 hours of secondary venous ischemia. Flap survival area, malondialdehyde (MDA) content, and myeloperoxidase (MPO) activity were assayed for each group. The mean flap survival area of the high-dose posttreatment group was significantly higher than the secondary ischemic control group (29% +/- 39% vs. 3% +/- 8%; p < 0.05, Student's t-test). The MDA and MPO levels of each treatment group were significantly lower than the secondary ischemic control group at hours 1 and 24 (p < 0.0001, Student's t-test). The lowest MDA and MPO levels were achieved in the high-dose posttreatment group. Results suggest that CsA may improve flap survival after secondary venous ischemia by attenuating neutrophil infiltration and by reducing lipid peroxidation.     

The role of oxygen-free radicals in ischemic tissue injury in island skin flaps

The contribution of free radical-mediated reperfusion injury to the ischemic damage caused by total venous occlusion of island skin flaps was investigated in a standardized rat model. Control flaps subjected to 8 hours of total venous occlusion showed complete, full thickness necrosis when followed for 7 days following release of the vascular occlusion. Treatment with superoxide dismutase, a scavenger of superoxide radicals, prior to and immediately following the onset of reperfusion, significantly enhanced island flap survival from 0/11 (0%) to 8/15 (53%), p less than 0.005, and from 0/9 (0%) to 6/12 (50%), p less than 0.02, respectively. These findings are consistent with the hypothesis that oxygen free radicals generated at the time of reperfusion following a period of ischemia contribute significantly to the ultimate damage caused by ischemic injury. Such findings are consistent with similar reported observations on other tissues and suggest a means by which ischemic tissue injury might be therapeutically modified, even after the period of ischemia.     

Effect of superoxide dismutase for improvement of survival of ischemic reperfused island skin flap]

Using a rat model, we evaluated the effect of SOD on the survival of ischemic reperfused island skin flaps. In experiment 1, the oxygen free radical concentration in the flaps was measured by the technique of ESR. The results showed that the oxygen free radical concentration in ischemic reperfused flaps was significantly higher than in the corresponding control flaps (P less than 0.001). In experiment 2, the flaps were perfused with SOD (2000 U in 1 ml saline) before reperfusion after 8 hours of ischemia. Seven days after operation, the area of flap survived in the test group was significantly larger than in the control group (P less than 0.0005). The obtained data demonstrated that the generation of oxygen free radical increases with time during ischemia reperfusion in island skin flap and the role of oxygen free radical in tissue injury following ischemia and reperfusion. The use of SOD can enhance the survival of ischemic island skin flap.     

The effect of a free-radical scavenger, N-2-mercaptopropionylglycine, on the survival of skin flaps

Oxygen free radicals may have an important role in tissue injury, which occurs on reperfusion of previously ischemic skin flaps. Therefore, therapy directed against the toxic effects of reactive oxygen species may protect skin flaps from ischemia/reperfusion injury. Various scavengers of oxygen free radicals have previously been reported to be effective in ameliorating ischemia/reperfusion injury. In the present study, N-2-mercaptopropionylglycine (MPG), a free-radical scavenger, was evaluated for its effectiveness in limiting the extent of necrosis resulting from ischemia/reperfusion injury in rat skin. Island skin flaps were elevated in the abdomen and groin based on an inferior epigastric neurovascular pedicle. The venous drainage from the flap was occluded for 7 hours, and reperfusion was established. The majority of flaps in the control group exhibited complete necrosis on Day 7 postoperatively. Treatment with systemic MPG (20 mg/kg of body weight) significantly improved flap survival from 22 to 71% (p less than 0.01) when administered at the time of reperfusion. However, MPG administered 1 hour after reperfusion did not influence the survival of the flaps. The results suggest that MPG may exert its beneficial effects on flap survival by scavenging oxygen free radicals formed at the time of reperfusion following prolonged ischemia.     

Prevention of reperfusion injury of an ischemic flap: an experimental study]

Oxygen-derived free radicals are important mediators of tissue injury in experimental island skin flaps that have been subjected to prolonged ischemia (vascular occlusion) followed by reperfusion. In this study, the role of oxygen free radical scavenger, SOD, and a herb, salvia miltiorrhiza, in the protection of cellular damages during total ischemia and reperfusion was study in the epigastric island skin flaps in experimental rats with electron microscopy and the assessment of survival of the flaps. Control flaps subjected to 10 hours of total vascular occlusion showed a high incidence of necrosis when followed for 7 days following release of the vascular occlusion. Treatment with superoxide dismutase and salvia miltiorrhiza prior to the onset of reperfusion significantly enhanced island flap survival to 72.5% (P < 0.001) and to 64.2% (P < 0.05), respectively. The conclusions are: 1. Reperfusion for 10 hours following ischemia for 8 hours in the epigastric island flaps of the rats greatly exaggerated the original injury. 2. SOD and salvia miltiorrhiza may protect the flaps from such injury considerably and enhanced flap survival.     

Dual preconditioning: effects of pharmacological plus ischemic preconditioning on skin flap survival

To enhance skin flap viability, pharmacological and ischemic preconditioning methods were investigated intensively. This study was designed to determine whether combined local dexamethasone administration and pedicle clamping would result in an additive enhancement of skin flap survival in the rat model. Twenty-eight male Sprague-Dawley rats were included in dexamethasone injection, clamping, clamping plus dexamethasone injection, and control groups. A rectangular random skin flap (3 x 11 cm) was outlined as bipedicled on the back of the animals. The dexamethasone or saline injection points in the flap were standardized. In the dexamethasone injection group, after raising the flaps, a total of 2.5 mg/kg dexamethasone was injected into the flaps. In the ischemic preconditioning group, 1 hour after saline injection, the cranial pedicle was clamped for 20 minutes and then 40 minutes reperfusion was performed. The clamping-plus-dexamethasone injection group was the same as the clamping group except dexamethasone was injected instead of the saline. In the control group, saline was injected instead of dexamethasone. Regardless of the group, all flaps were cut at the cranial side at the end of the 2 hours and were sutured back. On day 7, the surviving area was significantly greater in all experimental groups compared with the control group (p<0.05). Furthermore, the clamping-plus-dexamethasone group demonstrated the highest flap viability.     

The effect of prostaglandin E1 versus ischemia-reperfusion injury of musculocutaneous flaps

The purpose of this study was twofold. To evaluate whether prostaglandin El can increase the survival of the flap, and to determine its function against ischemia-reperfusion injury in musculocutaneous flaps. Thirty-five Sprague-Dawley rats weighing 250 to 350 g were analyzed. The transverse rectus abdominis musculocutaneous flap was used in all rats. The rats were divided into three groups: group 1 (N = 15), the control group with 4-hour ischemic injury and intraflap injection of normal saline followed by reperfusion; group 2 (N = 15), prostaglandin E1 intraflap injection of 1 microg immediately after ischemic injury and reperfusion 4 hours later; and group 3 (N = 5), the sham-operated group. Analysis consisted of flap skin survival area measurements, immunohistochemical study using anti-intercellular adhesion molecule (anti-ICAM-1) monoclonal antibody, and histological evaluation including endothelium-sticking leukocytes at 24 hours and 5 days after reperfusion. The group treated with prostaglandin E1 showed immunohistochemical findings with decreased expression of ICAM-1 on the surface of the endothelium, and histology showed significant (p < 0.01) reduction of leukocyte adhesion at 24 hours and 5 days after reperfusion. These two factors were considered to play a role against ischemia-reperfusion injury, and led to improved survival of the flap. These results suggest that prostaglandin E1 may increase flap survival and may have a protective mechanism against ischemia-reperfusion injury by decreasing leukocyte-endothelial cell adhesion through decreased expression of ICAM-1.