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。     

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。     

硫酸锌对皮瓣缺血再灌注损伤保护作用的研究

目的 观察外源性锌对皮瓣缺血再灌注（ischemia—reperfusion，IR）损伤的保护作用，并探讨其机制。方法 48只大鼠随机分为非-IR组、IR组和补锌-IR组。在大鼠以腹壁浅血管为蒂的岛状皮瓣缺血再灌注模型上，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。观察免疫组化切片中金属硫蛋白（metallothionein，MT）的表达，并对切片进行图像分析。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，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％（P〈0．05），MPO活性分别较IR组降低14．2％、22．7％（P〈0．05），MT含量分别较IR组高41．5％、44％（P〈0．01）。在补锌-IR组掀起皮瓣即刻的标本中有一定量的MT表达。MT表达在皮瓣组织多种细胞的细胞浆中。补锌-IR组皮瓣的超微结构改变较IR组减轻，皮瓣成活率较IR组升高27．2％（P〈0．05）。结论 外源性锌能诱导皮瓣内MT产生，MT通过细胞保护作用对皮瓣缺血再灌注损伤产生一定的保护作用。     

地塞米松对阻断静脉的岛状皮瓣缺血再灌注损伤的保护作用

目的：寻求对岛状皮瓣阻断静脉回流致致血再灌注损伤的有效保护药物。方法：采用大鼠岛状皮瓣缺血再灌注损伤模型,观察治疗组与对照组皮瓣皮活率。组织形态学改变。结果：对照组静脉阻断8h皮瓣大部分坏死,而地塞米松治疗组则可明显延长皮瓣耐受静脉阻断,时间,再灌注8h内给药均可起良好的保护性作用,地塞米松5mg/kg保护效果最佳,再增加剂量并不能进一步提高皮瓣保护效果。结论：地塞米松对阻断皮瓣静脉回流所致的岛状皮瓣缺血再灌注损伤有良好的保护性作用。     

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。     

热应激预处理对皮瓣缺血再灌注损伤的影响及机制

目的探索减轻皮瓣缺血再灌注损伤的有效措施。方法采用大鼠腹部岛状皮瓣,制作活体原位热缺血模型,观察热缺血再灌注后皮瓣的成活率、皮瓣组织形态学改变,检测皮瓣超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、HSP_(70)表达。结果缺血8h 后再灌注,实验组皮瓣存活率明显高于对照组;与对照组相比,实验组皮瓣组织中 SOD 活性较高而 MDA 水平较低;电镜显示,实验组皮瓣毛细血管内膜较完整,细胞肿胀轻,线粒体结构较稳定。结论热应激预处理,能减轻缺血再灌注对皮瓣的损伤,对缺血再灌注皮瓣具有保护作用,其机制可能与热应激预处理抗自由基损害作用及维护细胞膜结构稳定有关。     

热应激预处理对皮瓣缺血再灌注损伤的影响及机制

目的　探索减轻皮瓣缺血再灌注损伤的有效措施。方法　采用大鼠腹部岛状皮瓣,制作活体原位热缺血模型,观察热缺血再灌注后皮瓣的成活率、皮瓣组织形态学改变,检测皮瓣超氧化物歧化酶（ Ｓ Ｏ Ｄ） 活性、丙二醛（ Ｍ Ｄ Ａ） 含量、 Ｈ Ｓ Ｐ７ ０ 表达。结果　缺血８ｈ 后再灌注,实验组皮瓣存活率明显高于对照组;与对照组相比,实验组皮瓣组织中 Ｓ Ｏ Ｄ 活性较高而 Ｍ Ｄ Ａ 水平较低;电镜显示,实验组皮瓣毛细血管内膜较完整,细胞肿胀轻,线粒体结构较稳定。结论　热应激预处理,能减轻缺血再灌注对皮瓣的损伤,对缺血再灌注皮瓣具有保护作用,其机制可能与热应激预处理抗自由基损害作用及维护细胞膜结构稳定有关。     

阻断CD18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，检测了皮瓣髓过氧化酶（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ＣＤ１８单抗阻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ＣＤ１８介导的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。     

大鼠背部对称性岛状皮瓣的一种新型缺血再灌注损伤动物模型

目的 研究一种新型的缺血再灌注损伤皮瓣的动物模型.方法 在解剖研究的基础上,于10周龄雄性Fisher大鼠背部制备对称性皮瓣,形成不同缺血时间的缺血再灌注损伤模型,进行两侧皮瓣存活率、微小血管成像评估及组织学检查.结果缺血6h的大鼠背部对称性皮瓣缺血再灌注损伤模型,其左右两侧皮瓣存活率、平均微小血管数及组织学检查差异均无统计学意义(P>0.1).结论 以旋髂深血管为蒂的大鼠背部对称性岛状皮瓣(缺血6h)是一种较好的缺血再灌注损伤皮瓣模型,适用于药物、细胞及细胞因子等对缺血再灌注损伤的实验研究.     

盐酸罂粟碱对任意皮瓣微循环影响的实验研究

目的:探讨局部使用盐酸罂粟碱对任意皮瓣微循环重建的影响.方法:设计大鼠背部3cm×10cm长方形任意皮瓣,蒂部皮下注射盐酸罂粟碱,观察术后变化.结果:存活率提高,差异有显著性.结论:①盐酸罂粟碱注射液能够诱导任意皮瓣真皮下血管网血管生成;②盐酸罂粟碱注射液能够改善任意皮瓣缺血再灌注损伤保护皮瓣内多种细胞,从而对皮瓣缺血再灌注损伤产生一定的保护作用.     

钙拮抗剂对大鼠腹部皮瓣抗氧化作用的研究

Objective　To observe the protective effect of calcium antagonist on the ischemia - reperfusion injury in rat abdominal skin flap.Methods　33 SD rats were randomized into three groups. The abdominal island flap based on the inferior epigastric vessels was used as the ischemia - reperfusion model. In the control group, normal saline was given intraperitoneally for 3 consecutive days preoperatively, while Verapamil (Ver) and Tetrandcin (Tet) were given in the other two groups respectively. Samples from the flap were harvested during ischemia and reperfusion periods to measure the changes of superoxide dismutase （SOD） activity,glutathione peroxidase （GSH - Px） activity and malonyl dialdehyde （MDA） content. Results　SOD activity and GSH - Px activity were much higher in the Ver and Tet groups than in the control group, while the MDA content was remarkably decreased in these two groups.Conclusions　Calcium antagonist, Verapamil and Tetrandcin, plays an important role in oxygen free radical scavenging and in reducing lipid peroxidation.     

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.     

Postconditioning attenuates ischemia-reperfusion injury in rat skin flap

Reperfusion injury by the abrupt restoration of circulation after the prolonged ischemia has been remained unsolved problem in the reconstructive microsurgery. We tested the hypothesis that a procedure of intermittent interruption of reperfusion, i.e., postconditioning (post-con) attenuates ischemia/reperfusion (I/R) injury of rat epigastric skin flap. A complete 4 hours of ischemia was generated by occlusion of the pedicle of dissected flap. The post-con procedure was started at the end of ischemia. A cycle of 15 seconds of full reperfusion, followed by 15 seconds of complete reocclusion was repeated six times (3 min of total intervention) prior to the unlimited reperfusion. Flap necrosis area of post-con group was compared with sham (no ischemic exposure) and control (4 hours of ischemia followed by full reperfusion without intervention) groups at postreperfusion day 5. Histology and MPO activities of flaps were evaluated. The post-con group showed significantly reduced flap necrosis at the end of 5 days of reperfusion compared with the control. Decreased inflammatory cell infiltration and MPO activity indicated post-con attenuated acute inflammatory reaction caused by I/R. This study reports for the first time that ischemic post-con effectively attenuates skin flap I/R injury. With further study, post-con may eventually be clinically applicable for the I/R injury as an "after-injury strategy."     

Protective effects of vitamins A and E pretreatment in venous ischemia/reperfusion injury

It has been suggested that venous ischemia is more injurious to tissue viability than is arterial ischemia of equivalent duration. The precise mechanism of tissue damage due to venous ischemia is still not well-determined. Current research has shown that it is multifactorial, and that lipid peroxides, prostanoid metabolism, and a free radical mechanism are the major contributors. Vitamins A and E are lipid-soluble vitamins that have been suggested to be successful in the treatment of arterial ischemia/reperfusion injury due to their antioxidant properties. In the present study, the authors examined the protective effects of vitamins A and E pretreatment on reperfusion injury induced by venous occlusion of rat epigastric island flaps based on an epigastric artery and vein pedicle. In the first part of the study, to determine critical ischemia time, epigastric island skin flaps (3 x 6 cm) were elevated on their vascular pedicles in 40 male Sprague Dawley rats. Total venous occlusion of the skin flap was produced by ligating all draining veins and clamping the epigastric vein. Arterial inflow was left intact. Rats were randomly assigned to five groups (n=8) for 2, 5, 7, 9, and 10 hr of venous ischemia. Despite the occurrence of widespread reperfusion injury, reflow was established (p<0.005) at 9 hr. In the second part of the study, 20 rats were randomly divided into four groups (n=5). The effects of vitamins A and E following 9 hr ischemia/reperfusion injury were examined. Rats were pretreated with vitamin E, vitamin A and vitamins A+E for 5 days. At the end of the fifth day, each rat had undergone an epigastric island skin flap and venous occlusion, as described above. Mean surviving flap area (percent) and plasma lipid peroxides (TBARs), total thiol content (t-SH), glutathione peroxidase (Gpx), and superoxide dismutase (SOD) were determined for each rat. Results suggest that, in the prevention of venous ischemia/reperfusion injury, vitamin A and vitamin E are not effective when used as single agents; however, when used in combination, they significantly increase surviving flap area by a synergistic effect.     

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.     

Hemoglobin-based oxygen carrier does not improve survival of ischemic rat island groin flaps.

Reducing reperfusion injury to skin flaps is an effective means to improve the survival of the flap. By enhancing oxygen delivery to the microcirculation within the flap, ischemia-reperfusion injury should be decreased, improving the flap's survival. This study evaluated the effects of a hemoglobin-based oxygen carrier (Oxyglobin) on the development of necrosis and survival of ischemic rat island groin flaps. Sprague-Dawley rats were randomly assigned to one of three treatment groups. A groin flap was elevated on each rat and subjected to 9 h of ischemia. Rats in group I were given an intravenous infusion of 0.9% saline prior to elevation of the skin flap. Rats in group II were given an intravenous infusion of Oxyglobin prior to elevation of the skin flap. Rats in group III were given a low-dose intravenous infusion of Oxyglobin following the 9 h of ischemia, just prior to reperfusion. The flaps were monitored for 7 days postoperatively for necrosis. The percentage of flap necrosis was recorded at the end of 7 days. All rats were euthanized at the completion of the study and the flaps were harvested for histopathological analysis. No significant difference was noted in the survival of the flaps or the degree of necrosis in the rats treated with Oxyglobin compared to the control group. Thus, pre-reperfusion treatment with Oxyglobin did not improve the percentage of flap survival, or the degree of severity of necrosis in rat groin flaps subjected to 9 h of ischemia.     

Proinflammatory cytokines gene expression in skin flaps with arterial and venous ischemia in rats

Infiltration of inflammatory cells is the crucial element in ischemia-reperfusion injury of the microsurgical flap. Cytokines are a large functional group of polypeptide regulatory molecules that influence the activity of various cell types through autocrine and paracrine mechanisms. In this study, expression of selected proinflammatory cytokines was examined in skin flaps with arterial and venous ischemia in the rat model. Fifty-four Sprague-Dawley rats were used in the study. The ischemia of each flap was induced by clamping its vascular pedicle for 6 hr. The flap was then replaced and allowed to reperfuse. All flaps were biopsied immediately post-event, and at 3, 6 and 18 hr after reperfusion. Expression of tumor necrosis factor (TNF-alpha), interleukin (IL-1beta), and monocyte chemoattractant protein-1 (MCP-1) mRNA was determined by RT-PCR in each case. The same number of skin flaps without ischemia was used for baseline gene expression. Results showed that the TNF-alpha expression was significantly up-regulated in the flaps with arterial ischemia at 6 hr after reperfusion. In the flaps with venous ischemia, MCP-1 expression was increased with its peak expression at 3 hr after reperfusion. IL-1beta expression was increased threefold in the flaps subjected to venous ischemia and following reperfusion in 3 hr, but the peak expression in the flap with arterial ischemia was observed at 18 hr after reperfusion. This study delineated the changes in expression of these proinflammatory cytokines in flaps with arterial and venous ischemia reperfusion injury, and showed that cytokine expression was different in the arterial and venous injuries.     

Improved survival in free skin flap transfers in rats

We have demonstrated previously that 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 radicals in ischemia/reperfusion injury has been investigated in free flap transfers. Groin skin flaps were harvested, stored at room temperature for 21 to 24 hours, and transplanted to the contralateral groin. These free flap transfers normally exhibit a high incidence of complete necrosis. Treatment before the onset of reperfusion with a single dose of superoxide dismutase (SOD), a scavenger of superoxide radicals, increased the survival rate of these skin flaps from 38% in the control group to 76% (p less than 0.025). Tissue levels of SOD were measured before ischemia, after ischemia but before reperfusion, and 30 minutes after reperfusion: untreated flap tissues, which were destined to undergo necrosis, exhibited a significant decrease in SOD activity after reperfusion, whereas SOD-treated flap tissues, destined to survive, demonstrated increased enzyme activity. High levels of tissue SOD activity thus appeared to be associated with improved flap survival. The results have significant clinical implications with regard to organ preservation and transplantation.     

Platelet glycoprotein IIb/IIIa receptor antagonist (abciximab) inhibited platelet activation and promoted skin flap survival after ischemia/reperfusion injury

BACKGROUND: Evidence has shown that platelets play an important role in the pathogenesis of flap failure. Employing a rat inferior epigastric artery skin flap as a flap reperfusion injury model, we investigated whether platelet activation was involved in the skin flap failure and whether administration of abciximab (ReoPro, chimeric 7E3 Fab) could decrease platelet activation/aggregation and promote flap survival. METHODS: Normal saline and abciximab (0.06 mg/kg; 0.2 mg/kg; 1 mg/kg) were injected intravenously into skin flaps 30 min before reperfusion and 1 h after reperfusion (each subgroup n = 6). Platelet activation as demonstrated by P-selectin (CD62P) was analyzed by flow cytometry. P-selectin expression on flap vessels was detected by immunohistochemical staining. Platelet aggregation was induced with adenosine diphosphate (ADP). Laser Doppler flowmetry monitored tissue perfusion. The surviving area was evaluated 7 days postoperatively. RESULTS: CD62P progressively increased after reperfusion. The peak CD62P occurred after reperfusion for 12 h. Immunohistochemical staining showed CD62P significantly deposited on the endothelium after reperfusion. Administration of abciximab (1 mg/kg) effectively improved flap survival rate (P = 0.003), significantly decreased ADP-induced platelet aggregation (P < 0.001), and suppressed CD62P expression on blood platelets (P = 0.002) and its deposition on the flap vessels. CONCLUSION: Abciximab promotion of skin flap survival is due to blocked platelet activation/aggregation and decreased activated-platelet deposition on the vascular endothelium. Thus, administration of a platelet glycoprotein IIb/IIIa receptor antagonist such as abciximab may save the skin flap from reperfusion injury after a long period of ischemia.