Mitochondrial defects by intracellular calcium overload versus endothelial cold ischemia/reperfusion injury

Abstract Questions as to the critical stress factor and primary targets of cold ischemia/reperfusion (CIR) injury were addressed by comparing mitochondrial defects caused by (1) CIR injury and (2) intracellular Ca²⁺ overload. CIR was simulated in transformed human umbilical vein endothelial cell cultures (tEC) by 8 h cold anoxia in University of Wisconsin solution and reoxygenation at 37°C. Intracellular Ca²⁺ concentrations were changed by permeabilization of suspended cells with digitonin in culture medium (RPMI, 0.4 mM Ca²⁺). Binding of free Ca²⁺ by ethylene glycol‐bis(β‐aminoethylether)‐N,N,N',N'‐tetraacetic acid in RPMI or mitochondrial incubation medium served as controls. Extracellular Ca²⁺ protected the cell membrane against permeabilization. Mitochondrial functions were determined before and after permeabilization of the cell membrane. After CIR, mitochondrial respiratory capacity declined, but oxygen consumption remained coupled to adenosine triphosphate (ATP) production. In contrast, Ca²⁺ overload caused uncoupling of mitochondrial respiration. High intracellular Ca²⁺ overload, therefore, does not reproduce cold ischemia/reperfusion injury in endothelial cells.     

Augmentation of cAMP improves muscle-flap survival and tissue inflammation in response to ischemia/reperfusion injury

The authors hypothesized that augmenting skeletal muscle adenosine 3',5'-cyclic monophosphate (cAMP) levels could reduce tissue inflammation and improve muscle survival in response to ischemia/ reperfusion (I/R) injury. Gracilis muscle flaps in male Wistar rats were subject to 4 hr of ischemia followed by 3 hr of reperfusion, to assess neutrophil accumulation and microvessel tone, or by 24 hr to evaluate percentage of muscle survival. Animals were grouped as follows: positive (saline) or negative (sham) control, or with infused cAMP elevating agents (8 Bromo-cAMP (8 Br-cAMP) or forskolin). Radioimmunoassay demonstrated significant increases in tissue cAMP levels throughout 3 hr of reperfusion with forskolin, while the 8 Br-cAMP-treated group showed only a temporary increase. Compared with vehicle-infused controls, forskolin administered 5 min prior to reperfusion and repeated as an infusion during the first 45 min of reperfusion, resulted in reduced neutrophil adherence and transmigration, and muscle edema with sustained vasodilatation. The percentage of muscle survival using nitro-blue tetrazolium staining demonstrated enhanced muscle-flap preservation with forskolin. There was no beneficial change in the presence of 8 Br-cAMP These observations suggested that sustained elevation of the cAMP pathway may reduce ischemia-reperfusion injury by decreasing neutrophil-mediated injury and improving vessel tone. Elucidation of the cAMP pathway may provide novel opportunities to modulate ischemia/ reperfusion injury.     

T cells as mediators in renal ischemia/reperfusion injury

Inflammation has been established to contribute substantially to the pathogenesis of ischemia/reperfusion (I/R) with a central role for particular cells, adhesion molecules, and cytokines. Until recently, most of the research trying to unravel the pathogenesis of I/R injury has been focused on the role of neutrophils. However, recent studies have brought evidence that T cells and macrophages are also important leukocyte mediators of renal and extrarenal (liver) I/R injury. In vivo depletion of CD4+ cells but not CD8+ cells in wild-type mice was protective in I/R of the kidney. A marked preservation of liver function was also found after I/R in T-cell deficient athymic mice. Blocking the b130/CD28 costimulatory pathway by CTLA-4 Ig (recombinant fusion protein) ameliorated renal dysfunction and decreased mononuclear cell infiltration in I/R of the kidney. b130-1 expression was found limited to the membrane of the endothelial cells of the ascending vasa recta, resulting in trapping of CD28-expressing CD4 T cells. This trapping of leukocytes results in the upstream congestion in the ascending arterial vasa recta, generating the since more than 150 years described medullary vascular congestion of the kidney soon after ischemic injury. It seems worthwhile to study a combination therapy using anti-inflammatory/anti-adhesion molecules in the early phase of I/R.     

Interaction between neutrophils and endothelial cells following ischemia/reperfusion]

The adherence of activated neutrophils to endothelial cells during ischemia/reperfusion injury is mediated by inside-out signal transduction. Subsequently, outside-in signal transduction occurs following ligation of adhesion molecules with their ligands triggering respiratory bursts of neutrophils. In addition, neutrophil elastase enhances CC- and CXC-chemokine production by monocytes and macrophages. MCP-1, a CC-chemokine, enhances tissue factor production by macrophages and increases ICAM-1 expression on endothelial cells. Chemotaxis and respiratory bursts of neutrophils are augmented by CXC-chemokines. Furthermore, neutrophil elastase inactivates anticoagulants including antithrombin III, heparin cofactor II, and thrombomodulin, suggesting that neutrophil elastase aggravates microcirculatory disturbance after ischemia/reperfusion. Thus neutrophil elastase modulates the interation of neutrophils and endothelial cells during ischemia/reperfusion injury. Taken together with these observations, a therapeutic regimen with antibodies against adhesion molecules in combination with neutrophil elastase inhibitor and anticoagulants may attenuate ischemia/reperfusion injury.     

心磷脂与缺血/再灌注损伤

心磷脂（CL）是维持线粒体能量代谢所必需的一种磷脂，参与了众多重要生理过程。此文对CL的结构、合成和转化；在维持线粒体正常功能中的作用；缺血／再灌注（I／R）损伤中的改变以及和线粒体功能的关系等作了逐一阐述，说明CL与I／R损伤导致的细胞死亡关系密切，CL量或性质的改变在细胞凋亡中处于中心地位。     

Evidence that both cyclosporin and azathioprine prevent warm ischemia reperfusion injury to the rat liver

The present work was undertaken to study whether the immunosuppressive agents cyclosporin (CyA) and azathioprine (AZA) ameliorate hepatic injury after warm ischemia. A temporary, normothermic liver ischemia was induced in female Sprague-Dawley rats. The rats were treated with CyA (10 mg/kg per day p.o.), AZA (8 mg/kg per day p.o.), or vehicles for 4 days before surgery. Seven-day survival rates after 60 min of ischemia improved significantly with CyA (76.2%, P<0.005) and AZA (78.6%, P<0.001) treatment, compared with 43.0% for the control group. The highest levels of serum aminotransferases in the treatment groups tended to be lower than those in the control group. The peak values for the percentage of liver necrosis, an indicator of the extent of hepatic necrosis, in the animals treated with CyA (26.1%±7.2%, mean±SEM) and AZA (32.1%±5.7%) were significantly lower than in the control group (47.4%±3.7%). Lipid peroxidative damage after reperfusion, assessed as the hepatic malondialdehyde (MDA) concentration, was significantly suppressed by pretreatment with CyA and AZA. Histological findings coincided with other parameters. This study demonstrates that both AZA and CyA have beneficial effects on normothermic liver ischemia in rats. It is suggested that the diminished lipid peroxidative damage with these agents might be one of the mechanisms responsible for this.     

上调肝脏HO-1抑制肥大细胞脱颗粒减轻大鼠肝脏缺血再灌注损伤

目的 探索HO-1对肝脏缺血再灌注损伤中肥大细胞脱颗粒的影响。方法 将20只SD大鼠随机分成4组：假手术组（Sham组）,缺血再灌注损伤组（I/RI组）,HO-1诱导剂钴原卟啉组(CoPP组,术前24h给予CoPP,5 mg/kg)及HO-1抑制剂锌原卟啉组(ZnPP组,术前24h给予ZnPP,20 mg/kg)。建立大鼠缺血再灌注损伤模型,各组于再灌注后2h收集标本。RT-PCR检测肝脏组织HO-1 mRNA表达,Western blot检测肝脏组织HO-1蛋白表达;测定血清中ALT、AST水平;肝脏组织甲苯胺蓝染色检测肥大细胞脱颗粒数量,HE染色评价肝脏组织损伤情况。结果 与Sham组相比,I/RI组、CoPP组、ZnPP组大鼠组织HO-1 RNA和蛋白表达增加,血清ALT、AST水平升高,肥大细胞脱颗粒数量增多,肝脏细胞损伤加重。CoPP组与I/RI组相比,HO-1 mRNA和蛋白表达增加,血清ALT、AST水平减低,肥大细胞脱颗粒数量减少,肝细胞损伤减轻。ZnPP组与I/RI组相比,HO-1 mRNA和蛋白表达减少,血清ALT、AST水平升高,肥大细胞脱颗粒数量增多、肝细胞损伤严重。组间比较差异具有统计学意义(P＜0.05)。结论 HO-1过表达能减轻肝脏I/RI,其机制可能与抑制肝脏组织中肥大细胞脱颗粒有关。     

中性粒细胞与心肌缺血/再灌注损伤

背景 现已明确,炎症过程是心肌缺血/再灌注损伤(ischemia/reperfusion injury,I/RI)最重要的致病因素之一,而中性粒细胞是炎症反应的核心介导者.针对中性粒细胞的这种致病作用,部分研究者提出了抗中性粒细胞治疗,但是治疗效果却不尽相同,甚至大相径庭.更有研究者指出,中性粒细胞在心肌I/RI中尚发挥着一定的有益作用.针对这种现状,我们在此将中性粒细胞与心肌I/RI作一综述. 目的 评价中性粒细胞在心肌I/RI致病机制中的作用,探索抗中性粒细胞治疗的方向.内容 包括中性粒细胞对心肌I/RI的致病作用,抗中性粒细胞治疗的现状及其当前存在矛盾之处. 趋向 通过全面理解中性粒细胞在心肌I/RI中的作用,为今后进行适度的抗中性粒细胞治疗提供参考,并为今后发展多靶向联合措施治疗心肌I/RI提供思路.     

丙泊酚与肝脏缺血/再灌注损伤

缺血/再灌注对肝脏造成损伤.众多资料显示丙泊酚对肝脏缺血/再灌注损伤有保护作用,这一保护作用与其抗氧化,阻断钙超载,减轻炎性细胞导致的损伤有关.肝脏缺血/再灌注也影响了丙泊酚的代谢.     

Melatonin ameliorates renal ischemia/reperfusion injury

BACKGROUND: We studied whether melatonin is able to reduce organ damage during renal ischemia/reperfusion via its effects on the oxidative response in early and late reperfusion. MATERIALS AND METHODS: Renal ischemia/reperfusion injury (I/R) was induced in two groups of rats by 75 min occlusion of the left renal artery and vein and right nephrectomy, followed by reperfusion. The formation of reactive oxygen species was evaluated in the early reperfusion phase (60 min) by lipid peroxidation products and glutathione assay. In the late reperfusion phase (24 h) tissue neutrophil infiltration, inducible nitric oxide synthase (iNOS) gene expression, and histopathology were evaluated. Groups received either systemic melatonin (MEL) or normal saline (NS). There were two nonischemic sham control groups, one with and another without melatonin (S+MEL and S). RESULTS: Creatinine was higher in the NS group at all times. A reduction in glutathione and increases in lipid peroxidation products and myeloperoxidase activity induced by I/R indicated renal injury involving reactive oxygen formation. Melatonin reversed this oxidant response and reduced the rise in creatinine and iNOS expression. Seven-day group survivals were 5/10 for NS, 8/10 for MEL, and 10/10 for both Sham groups. CONCLUSIONS: Exogenous melatonin is able to preserve renal functional status following I/R-induced injury by increasing glutathione and reducing lipid peroxidation in the early reperfusion phase, without any apparent effect on neutrophil infiltration in the late reperfusion phase.     

纤维连接蛋白连接片段-1肽段减轻大鼠移植肝缺血再灌注损伤

目的 探讨纤维连接蛋白连接片段-1(CS1)肽段对大鼠肝移植缺血再灌注损伤的影响及其机制.方法 用Wistar大鼠作为供、受鼠,制作肝移植模型.CS1组供鼠分别于术前3d、取肝时和移植前注射CS1肽段,供肝获取后于UW液中保存18h,肝移植术后3d每天注射CS1肽段;对照组用随机肽段替代CS1,其他操作同CS1组.术后6、24、72 h检测受鼠血清转氨酶水平和肝组织病理改变.免疫组织化学染色显示肝脏中的炎症细胞和肝窦内皮细胞.多聚酶链反应检测肝组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和血管内皮细胞生长因子(VEGF) mRNA的表达水平.结果 术后24 h,对照组坏死肝组织面积约占总面积的(25.7±5.4)％,而CS1组为(12.6±3.6)％(P＜0.05).对照组血清转氨酶水平也低于对照组(P＜0.05).术后CS1组移植肝中枯否细胞和中性粒细胞数目均少于对照组(P＜0.05).两组冷缺血18h的供肝中,肝窦内皮细胞均失去正常形态,仅少数内皮细胞特异性标志物阳性.术后72 h,CS1组肝窦内皮细胞基本恢复正常形态,SE-1表达恢复,而对照组肝窦内皮细胞恢复欠佳.术后6和24 h时,CS1组肝组织中TNF-α mRNA的水平低于对照组(P＜0.0)5);术后24 h时,对照组VEGF mRNA的表达高于CS1组(P＜0.05);两组IL-1βmRNA水平的差异无统计学意义(P＞0.05).结论 用CS1肽段处理供、受鼠可以减少炎症因子mRNA的表达,保护肝窦内皮细胞,减轻移植肝缺血再灌注损伤.     

内毒素血症加重实验大鼠肝缺血再灌注损伤

目的探讨内毒素血症对肝缺血再灌注(I／R)损伤的影响。方法对24只大白鼠于术前1h静脉注射0．8mg／kg的LPS，检测肝门血流阻断30min的情况下复流后2h、6h、12h和24h4个时间点血清ALT与AST值，用ELISA法检测了复流后2h和6h2个时间点的循环TNF-α水平和肝组织中TNF-α含量，于复流后24h取缺血部分肝组织行组织病理学检查。24只未注射LPS的I／R肝损伤模型大鼠作为对照组。结果实验组大鼠术后各时刻ALT和AST升高幅度均明显大于对照组，并且下降缓慢；循环和肝组织中的TNF-α也明显高于对照组，复流后24h的肝组织存在片状坏死，伴大量多形核细胞浸润。结论内毒素通过升高循环和肝组织局部的TNF-α加重了缺血后再灌注肝损伤。     

Contribution of T lymphocytes to rat renal ischemia/reperfusion injury

BACKGROUND: This study aimed to elucidate the early involvement of T lymphocytes in renal ischemia/reperfusion injury. METHODS: Athymic nude rats (F344/N_Jcl-nu) and control F344/Jcl were subjected to 45 min unilateral renal ischemia. To determine whether the observed differences might be derived from the T lymphocyte presence, T lymphocytes from the spleens of F344/Jcl were injected into F344/N_Jcl-nu via tail vein at the initiation of reperfusion. Immunohistochemical analysis was performed for CD3, the proliferative cell nuclear antigen (PCNA), vimentin, and E-cadherin. T lymphocytes were obtained from the green fluorescent protein transgenic (GFP) rats, and transplanted to F344/N_Jcl-nu 10 min before reperfusion. The animals were euthanized 15 min after reperfusion. RESULTS: F344/N_Jcl-nu showed less retention of both Cr and BUN at 24 and 48 h after reperfusion, compared with F344/Jcl. F344/N_Jcl-nu received T lymphocyte transplantation showed significantly higher retention of both Cr and BUN 24, 48, and 72 h after reperfusion than those without T lymphocyte. A rapid infiltration of T lymphocytes into proximal tubular epithelial cells and tubular lumen was observed using T lymphocytes with green fluorescent protein. In contrast, T lymphocytes were observed with much less frequency 24 h after ischemia. The number of PCNA-positive proximal tubular cells 24 h after the initiation of reperfusion was significantly smaller in the T lymphocyte transplantation group compared with the non-transplantation group. The vimentin positivity and cytoplasmic staining of E-cadherin were also more prominent in the transplantation group. CONCLUSION: These findings demonstrate a rapid renal T lymphocyte infiltration, which propagate renal functional deterioration.