CXC chemokine expression and synthesis in skeletal muscle during ischemia/reperfusion

BACKGROUND: The chemokines keratinocyte-Derived Cytokine (KC) and macrophage inflammatory protein (MIP)-2, murine equivalents of human interleukin 8, have been implicated in remote injury after acute hind limb ischemia/reperfusion (I/R). These studies were designed to determine whether the cytokines responsible for remote tissue injury are also synthesized and accumulate in the ischemic or reperfused hind limb. METHODS: B6, 129SF2/J mice were subjected to either 3 hours of unilateral hind limb ischemia alone (IA) or 3 hours of ischemia followed by 4 or 24 hours of reperfusion (I/R). After IA or I/R, experimental and control (nonischemic) contralateral hind limbs were harvested for analysis of protein content, messenger RNA (mRNA), tissue edema, and viability. RESULTS: IA did not increase KC or MIP-2 mRNA or protein levels. In contrast, I/R resulted in a 15- and 10-fold increase in KC mRNA after 4 and 24 hours of reperfusion, respectively. KC protein levels were increased 10-fold after 4 hours of reperfusion and 30-fold after 24 hours (vs IA or sham; P < .001). MIP-2 mRNA transiently increased 42-fold after 4 hours of reperfusion but decreased to basal levels after 24 hours of reperfusion. Despite the relative increase in MIP-2 mRNA by 4 hours of reperfusion, significantly increased (8- to 10 fold) MIP-2 protein levels were not detected until 24 hours of reperfusion only in the reperfused limbs. Tissue edema was increased significantly (P < .01) compared with sham after just 4 hours of reperfusion and remained increased at 24 hours. Tissue viability decreased 52% after 4 hours of reperfusion and did not change significantly by 24 hours. CONCLUSIONS: Skeletal muscle is a site of significant ongoing chemokine synthesis during reperfusion. The persistent increase in muscle chemokine levels at 24 hours of reperfusion was not associated with increased edema or injury. The role of these chemokines during reperfusion may be further investigated by local or oral administration of chemokines or chemokine receptor antagonists. CLINICAL RELEVANCE: I/R injury remains an important clinical problem across a variety of surgical specialties. In the critical care arena, serum levels of proinflammatory cytokines have been useful in predicting the mortality associated with acute respiratory distress syndrome and sepsis. In this article, the data presented indicate that murine skeletal muscle produces potent proinflammatory neutrophil and macrophage chemokines during reperfusion, but not during ischemia. These findings suggest that measurement of tissue and/or serum levels of chemokines during reperfusion may be an important adjunct to predicting tissue injury along with ongoing inflammation during the clinical course of reperfusion injury. Within the vascular system, severe inflammatory responses are usually associated with thrombotic events. New techniques to noninvasively image thrombin activation (by using magnetic resonance imaging) in reperfused limbs may coincide with the pattern of murine skeletal muscle chemokine expression in humans. The data suggest that reperfusion is when chemokine mRNA and protein synthesis increase. Within the time periods studied in these experiments, the chemokine component of the inflammatory response remained in the reperfused, rather than the systemic nonreperfused, tissue. This observation may underestimate the degree of the systemic response to ischemia because the single mouse hind limb represents only 7% of the mouse total body area, whereas the human limb represents nearly 18% of the adult body area. Despite this shortcoming, these data provide potential temporal and quantitative information regarding the location and magnitude of chemokine synthesis in skeletal muscle during reperfusion.     

Protection of carbon monoxide-releasing molecule against lung injury induced by limb ischemia-reperfusion

Ohiective: To observe the role and mechanism of Coreleasing molecule (CORM)-2 in lung injury induced by ischemia-reperfusion (IR) of hind limbs in rats.Methods: A rat model of lung injury induced by IR of hind limbs was established.A total of 40 Sprague Dawley (SD) rats were randomly divided into 5 groups (n=8):sham,shanl+CORM-2,IR,IR+CORM-2and IR+dimethyl sulfoxide (DMSO).Rats in the IR group received hind limb ischemia for 2 hours and reperfusion for 2 hours,rats in the sham group underwent sham surgery without infrarenal aorta occlusion.rats in the IR+CORM-2 group and in the sham+CORM-2 group were given CORM-2 (10 pmol/kg in travenous bolus) 5 minutes before reperfusion or at the corresponding time points.while rats in the IR+DMSO group was treated with the same dose of vehicle (DMSO) at the same time.The lung tissue structure,polymorphonuclear neutrophil (PMN) count,wet-to-dry weight ratio (W/D),malondialdehyde (MDA) content,myeloperoxidase (MPO) activity,intercellular adhesion molecule-1 (ICAM-1) expression,I κ Bo degradation and nuclear factor (NF)-κB activity in the lungs were assessed.Results: As compared with the sham group.lung PMNs number,W/D,MDA content,MPO activity,ICAM-1 expression and NF-κB activity significantly increased in the IR group,but the level of I κ Bo decresed (P＜0.01).Compared with the IR group,lung PMNs number,W/D,MDA content.MPO activity and ICAM-1 expression significantly decreased in the IR+COMR-2 group (P＜0.01),while the level of I κ Ba increased.Conclusions: These data demonstrate that CORM-2 attenuates limb IR-induced lung injury through inhibiting ICAM-1 protein expression.NF-κ B pathway and the leukocytes sequestration in the lungs following limb IR in rats,suggesting that CORM-2 may be used as a therapeutic agent against lung injury induced by limb IR.     

The evolution of ischemic spinal cord injury in function, cytoarchitecture, and inflammation and the effects of adenosine A2A receptor activation

OBJECTIVE: Spinal cord ischemia/reperfusion injury involves multiple factors that may be modulated by adenosine A 2A receptor activation. This study defines injury progression in terms of function, cytoarchitecture, and inflammation and assesses whether adenosine A 2A receptor activation by ATL-146e limits injury progression. METHODS: Mature swine were divided into 3 groups: sham thoracotomy, IR (30 minutes of ischemia followed by reperfusion), and ATL (ischemia/reperfusion with ATL-146e administration for the first 3 hours of reperfusion). Subgroups were killed at 0, 3, 6, 12, 24, and 48 hours after reperfusion. Function was followed up with Tarlov scores. Spinal cord tissue was evaluated for neuronal viability, microtubule-associated protein-2 immunohistochemistry, and neutrophil sequestration (myeloperoxidase assay). Spinal cord tissue, cerebrospinal fluid, and serum were evaluated for tumor necrosis factor-alpha by enzyme-linked immunosorbent assay. RESULTS: Function was significantly impaired at 24, 36, and 48 hours in the IR group compared with the sham and ATL groups ( P < .05). Neuronal viability and microtubule-associated protein-2 staining were significantly preserved in the sham and ATL groups compared with the IR group at 24 and 48 hours ( P < .05). Spinal cord myeloperoxidase levels were significantly higher in the IR group than in the sham and ATL groups at 24 and 48 hours. Although negligible in serum and cerebrospinal fluid, tumor necrosis factor-alpha levels in the spinal cord peaked significantly higher in the IR group compared with the sham and ATL groups at 6 and 24 hours ( P < .05). CONCLUSIONS: Spinal cord ischemia/reperfusion induced changes in neutrophil sequestration, microtubule-associated protein-2 expression, and neuronal viability within 24 hours of reperfusion. Spinal cord tumor necrosis factor-alpha increased significantly by 6 to 12 hours after reperfusion. Adenosine A 2A receptor activation attenuates spinal cord inflammation, which may be critical for the preservation of neuronal function and cytoarchitecture after ischemia/reperfusion.     

Ischemic preconditioning attenuates the lipid peroxidation and remote lung injury in the rat model of unilateral lower limb ischemia reperfusion

BACKGROUND: Ischemia and reperfusion of the skeletal muscle tissue may cause remote lung injury. We aimed to evaluate the protective effect of ischemic preconditioning (IP) on the lung during unilateral lower limb ischemia reperfusion (IR). METHODS: Four groups of rats were used in this study: (i) the sham group (sham, n = 6) served as time controls, they remained anesthetized for the whole duration of the study; (ii) the ischemia and reperfusion group (IR, n = 10) underwent 4 h of left lower limb ischemia followed by 2 h of reperfusion; (iii) the ischemic preconditioning group (IP, n = 10), the left lower limbs of rats were exposed to three cycles of IP (10 min of ischemia followed by 10 min of reperfusion); and (iv) the ischemic preconditioning plus ischemia reperfusion group (IP/IR, n = 10) underwent IP followed by IR as in the IP and IR groups. Plasma and tissue samples were taken at the end of the study period for determination of lung tissue myeloperoxidase activity (MPO) and polymorphonuclear leukocyte count (PMNL), histological lung injury score and plasma thiobarbituric acid reactive substances (TBARS) level. RESULTS: PMNL count and MPO activity in the lung tissue, and plasma TBARS level were higher in the IR group compared with other groups while there were no differences between the sham and the IP and between the sham and the IP/IR groups. Histological lung injury score was higher in the IR group than in the IP/IR and sham groups. The plasma TBARS level in the IP group was significantly lower than in the IP/IR group. CONCLUSION: IP pretreatment reduces lipid peroxidation and lung injury caused by lower limb IR.     

大鼠骨骼肌缺血再灌注时血红素氧合酶-1的表达

目的：观察大鼠缺血再灌注时骨骼肌中血红素氧合酶－1（HO－1）表达的变化，方法：夹闭大鼠股动脉造成下肢缺血模型，分别采集假手术（S）组，单纯缺血（I）4小时组及缺血4小时再灌注（R）2、4、8、16和24小时组的比目鱼肌，检测其组织学和丙二醛（MDA）含量变化，通过Northern印迹、Western印迹及免疫组织化学分析，观察HO－1表达的变化。结果：S组和I组未见HO－1mRNA表达；R2组可见其表达信号，且随着再灌注时间延长表达信号逐渐增强，到R8组时达到高峰，之后渐减弱，R24组时已消失。蛋白表达的变化与mRNA变化基本一致，免疫组织化学显示，R组HO－1阳性信号主要出现在骨骼肌细胞浆内，S组和I组均未见阳性信号。与S组相比，R各组MDA含量显著增高（P＜0．05），但R8组较R4组显著减低（P＜0．05）。结论：肢缺血再灌注可诱发骨骼肌HO－1的表达，其表达可能具有保护作用。     

Increased ischemia-reperfusion blood flow impairs the skeletal muscle contractile function.

BACKGROUND: The ultimate aim of replantations and transplantations of skeletal muscle is to improve impaired function. The purpose of this study was to examine the contribution of varying durations of ischemia to postischemic blood flow in the skeletal muscle and the contribution of modulation of postischemic blood flow to skeletal muscle function and viability, using an ischemic revascularized hind limb model in rats. MATERIALS AND METHODS: Warm ischemia produced by vascular pedicle clamping was sustained for 90 min, 3 h, or 6 h. Postischemic blood flow was measured by a Doppler flowmeter or microsphere technique. In another series of experiments of 3-h ischemia, either saline or N(G)-methyl-l-arginine acetate (l-NMMA) was infused for the first 2 h of reperfusion. Postischemic blood flow was also measured. Muscle contractile function and viability were determined after 24 h of reperfusion. RESULTS: Postischemic blood flow was significantly increased during the first 10 min of reperfusion in the 90-minute ischemic group and during the first 2 h in the 3-h ischemic group compared with contralateral control blood flow. No significant increase in postischemic blood flow was noted in the 6-h ischemic group. Postischemic blood flow was significantly decreased by the l-NMMA infusion. Contractile function and viability of the tibialis anterior muscle and contractile function of the gastrocnemius muscle in the l-NMMA group were significantly increased. CONCLUSION: Reperfusion blood flow increased time dependently until 3 h of warm ischemia. Hyperemia deteriorated skeletal muscle contractile function, although it was well preserved by l-NMMA infusion to restrict the postischemic hyperemia.     

Protective effect of sivelestat,a neutrophil elastase inhibitor,on ipsilateral and contralateral testes after unilateral testicular ischaemia–reperfusion injury in rats

What’s known on the subject? and What does the study add? Following ischemic damage, reperfusion may cause further injury paradoxically in the ischemic tissue, known as reperfusion injury. Decreased blood flow causes hypoxia, leading to increased levels of lactic acid, hypoxanthine, and lipid peroxides in ischemic tissues and subsequent increase in blood flow after lipid peroxidation produces reactive oxygen species. In addition, several experimental studies and clinical trials demonstrated that unilateral testicular torsion has a detrimental effect also to the contralateral testis. Although the basic pathological mechanism underlying testicular ischemia/reperfusion injury has not been completely understood, it has been shown that reactive oxygen species formed during ischemia/reperfusion play the key role in this process. In the international literature there is no information available regarding the effects of neutrophil elastase inhibitors such as sivelestat sodium aminoacetate tetrahydrate on the ischemia/reperfusion injury of the testis. In this study we investigated the effects of sivelestat in the testes bilaterally, after unilateral testicular ischemia/reperfusion injury using an experimental unilateral testicular ischemia/reperfusion rat model. We found that sivelestat reduces the oxidative stress and partially prevents the testicular damage both in the ischemic and in the contralateral testis.

OBJECTIVE

To investigate the effect of a neutrophil elastase inhibitor, sivelestat sodium hydrate, on testicular ischaemia–reperfusion (IR)‐injury.

MATERIAL AND METHODS

Eight‐week‐old male Sprague–Dawley rats were divided into four groups: sham‐operated control rats; IR rats (group IR); and IR rats that received intra‐abdominal administration of 15 mg/kg or 60 mg/kg sivelestat (group IR15 and group IR60, respectively). Right testicular vessels were clamped for 90 min in groups IR, IR15 and IR60. Sivelestat had been administered 45 min after the induction of the ischaemia in groups IR15 and IR60. In subpopulations of IR, IR15 and IR60 rats, reperfusion was performed after ischaemia for 2 h (groups IR‐A, IR15‐A and IR60‐A, respectively) or 48 h (groups IR‐B, IR15‐B and IR60‐B, respectively). At the end of the reperfusion period, blood samples were aspirated from both spermatic veins of each rat and testosterone was evaluated. Then both testes from all rats were collected and tissue levels of malondialdehyde (MDA), myeloperoxidase (MPO), and heat‐shock protein‐70(HSP‐70) were evaluated. Testicular tissue samples were also processed for histological evaluation and TUNEL staining.

RESULTS

MDA, MPO and HSP‐70 levels in the ischemic testis were significantly higher in the IR group compared with the control group. MDA and HSP‐70 in the contralateral testis were significantly higher in the IR group compared with the control group. Bilateral testosterone levels were lower in all rat groups in comparison with the control group. Bilateral testicular samples in group IR showed extensive histopathologic degenerative alterations and increased percentage of apoptotic cells. Sivelestat treatment lowered the MDA concentration and the percentage of apoptotic cells bilaterally and ameliorated the testicular histological pattern bilaterally.

CONCLUSIONS

Unilateral testicular ischaemia causes significant contralateral testicular damage. Sivelestat may be a novel adjunct tool for reducing oxidative stress and partially preventing bilateral testicular damage.     

Hind limb ischemia-reperfusion in the leptin receptor deficient (db/db) mouse

BACKGROUND: Diabetic patients have high incidence of peripheral vascular disease and limb loss after acute extremity injury. Experiments were designed to test the hypothesis that acute tissue injury in leptin receptor deficient (Db) diabetic (type2) mice would be more severe than in non-diabetic mice. METHODS: Db and wild type (Wt) mice were subjected to 3 h of ischemia followed by either 4 or 24 h of reperfusion (3/4 IR, 3/24 IR). Muscle analyzed for tissue viability (mitochondrial activity), cytokines (KC-murine equivalent of human IL-8, TNFalpha, IL-6), growth factor, and histological evaluation (neutrophils/uninjured muscle fibers). Tissue perfusion was detected during basal and reperfusion conditions using laser Doppler imaging. RESULTS: Mitochondrial activity and histological evaluation for tissue injury did not differ in the Db versus Wt mice at the time intervals studied. When compared with their respective sham animals, both Db and Wt mice had similarly increased levels of KC, IL-6, and VEGF after 3/24 IR. TNFalpha levels increased in Db but not Wt mice after IR. Although absolute increases in TNFalpha and KC were higher in Db mice, VEGF levels were actually lower in the Db mice. CONCLUSION: The patterns of tissue perfusion, cytokines, and growth factors were different in Db versus Wt mice. At the acute time intervals studied, these differences did not correlate with an expected greater degree of acute muscle injury in Db mice.     

Protective effect of ischaemic preconditioning against ischaemia-induced reperfusion injury of skeletal muscle: how many preconditioning cycles are appropriate?

We investigated the efficacy of ischaemic preconditioning (IPC), consisting of repeated brief episodes of vascular occlusion followed by reperfusion, as protection against ischaemia-reperfusion injury of skeletal muscle, using a rat amputation-like model. Wistar rats underwent temporary amputation at the level of the femur, excluding the femoral vessels. The femoral artery and vein were clamped for 4h, using a micro-clamp, in the groups exposed to ischaemia. The rats were randomly divided into eight groups: a control (C) group (n = 7) with non-amputated and non-ischaemic hind limbs; a sham control (SC) group (n = 7) with amputated but non-ischaemic hind limbs; an ischaemia-reperfusion (IR) group (n = 7) with amputated and ischaemic hind limbs; and five IPC groups (n = 7 in each) with hind limbs that were subjected to 4h of ischaemia after one to five cycles of brief ischaemia and reperfusion for 10 min each, respectively. All rats were sacrificed 24h after reperfusion. The viability of the anterior tibial muscles was evaluated using nitroblue tetrazolium staining. The total viable area ratio (T-VAR) of the muscle tissue was calculated in each animal as follows: T-VAR\total viable area/total slice areae 100%. The T-VAR values of the eight groups were as follows: C group, 100% +/- 0%; SC group, 100% +/- 0%; IR group, 73.5% +/- 1.7%; IPC1 group, 79.4% +/- 6.5%; IPC2 group, 70.5% +/- 6.2%; IPC3 group, 90.6% +/- 2.8%; IPC4 group, 90.0% +/- 1.6%; and IPC5 group, 87.8% +/- 1.8%. The T-VARs in the IPC3, IPC4 and IPC5 groups were significantly higher (alpha < 0.01) than those in the IR group. In contrast, there were no significant differences between the T-VARs of the IPC1 and IPC2 groups and those of the IR group. In conclusion, three to five cycles of IPC could protect skeletal muscle against ischaemia. 2002 The British Association of Plastic Surgeons.     

Acute lung injury following reperfusion after ischemia in the hind limbs of rats.

In this study, we proposed that oxygen free radicals participate in the acute pulmonary injury that follows limb ischemia/reperfusion. Using an established model of hind limb ischemia, reproducible lung injury occurred after reperfusion. Lung microvascular permeability was measured with 125I-BSA and increased two-fold after 30 minutes of reperfusion. Pulmonary injury was blocked with DMSO, DMTU, allopurinol, indomethacin, and SOD plus catalase. The degree of pulmonary neutrophil sequestration as assessed by tissue myeloperoxidase activity was significantly diminished in animals pretreated with antioxidants. Pretreatment with indomethacin did not attenuate the neutrophil sequestration within the pulmonary parenchyma. These data suggest that increased lung microvascular permeability and neutrophil accumulation occur following hind limb ischemia/reperfusion. Therapeutic interventions with oxygen radical inhibitors blocked this process, while the prostaglandin inhibitor, indomethacin, only reduced lung permeability.     

Duration of extremity tourniquet application profoundly impacts soft-tissue antibiotic exposure in a rat model of ischemia-reperfusion injury

IntroductionExtremity tourniquet (TNK) application is an effective means of achieving compressible hemorrhage control in the emergency prehospital and clinical trauma setting. Modern United States military medical doctrine recommends TNK use to prevent lethal hemorrhage from extremity injury, followed by systemic prophylactic antibiotics to prevent wound infection. Because tissue pharmacokinetics of prophylactic antimicrobials during and after TNK-induced limb ischemia are largely unknown, this study was conducted to empirically determine the relationship between TNK application time and soft tissue antibiotic exposure in order to guide medical personnel in the management of extremity trauma.Materials and MethodsHind limbs of anesthetized male Sprague Dawley rats were exsanguinated, and ischemia maintained by a pneumatic cuff placed at the level of the mid femur on one limb; the non-ischemic contralateral limb served as comparison tissue. Systemic prophylactic antibiotics (cefazolin, moxifloxacin, or ertapenem) were administered intravenously before or after TNK release following 2 or 4 h of ischemia with subsequent re-dosing every 12 h for 3 days. Free antibiotic in the interstitial fluid (ISF) of the tibialis anterior muscle of both hind limbs was recovered via microdialysis during ischemia and over three periods during reperfusion: immediately following TNK release, at 24 h post TNK release, and at 72 h post TNK release. Plasma and ISF free drug concentrations were determined by high-performance liquid chromatography.ResultsTourniquet application prevented delivery of prophylactic antibiotics into distal soft tissue for the duration of ischemia, and caused a profound reduction in skeletal muscle drug exposure for up to 72 h following TNK release. A progressive decline in tissue antibiotic exposure during reperfusion was observed as TNK times increased from 2 h to 4 h. The timing and severity of reduced drug distribution in post-ischemic skeletal muscle varied substantially among the three antibiotic classes evaluated.ConclusionsProlonged tourniquet application can significantly reduce distribution of prophylactic antibiotics into soft tissue during and after ischemia, potentially impairing prophylaxis of extremity wound infection. Our findings support the examination of alternative approaches to wound infection prophylaxis under conditions of delayed casualty evacuation when occlusive hemorrhage control measures are utilized.     

Ischemic preconditioning improves oxygenation of exercising muscle in vivo

BACKGROUND: Ischemic preconditioning (IP) improves tissue tolerance to prolonged ischemia. In this study, we investigated the functional effect of IP on skeletal muscle of rat hind limb by means of near-infrared spectroscopy (NIRS) and by measuring myeloperoxidase (MPO) activity. MATERIALS AND METHODS: Adult male Sprague Dawley rats were divided into four separate protocol groups according to different preparations prior to 2 h of global ischemia: a group of ischemic reperfusion without any preparation (I/R), ischemic reperfusion with ischemic preconditioning (IP+IR), ischemic reperfusion with adenosine infusion (ADO+I/R), and sham operation. Ischemia and ischemic preconditioning were induced by clamping infrarenal abdominal aorta and left common iliac artery. For each rat, an exercise test of gastrocnemius muscles was performed by stimulating sciatic nerve before and after global ischemia while performing NIRS. MPO activity of ischemic muscles was also measured. RESULTS: Half-resaturation time after exercise and MPO activity were significantly improved in IP+IR and ADO+I/R groups. Difference of oxyhemoglobin during exercise was also improved in the IP+IR group. CONCLUSION: This study has demonstrated that IP provides the protective effect on in vivo skeletal muscle oxygenation during exercise.     

Thromboxane mediates the ischemia-induced neutrophil oxidative burst

Indirect evidence exists that the reperfusion of ischemic tissue activates white blood cells. Thus local and systemic reperfusion injuries are prevented by making animals leukopenic or by inhibiting white blood cell lung entrapment by blocking thromboxane A2 generation. This study tests directly whether ischemia and reperfusion activates neutrophils, as measured by their oxidative burst, and whether thromboxane mediates this event. Anesthetized rats underwent 4 hours of bilateral hind limb tourniquet ischemia followed by 60 minutes of reperfusion. Plasma thromboxane B2 levels increased to 2750 pg/ml at 5 minutes of reperfusion, higher than the sham control (n = 36) value of 370 pg/ml (p less than 0.01). In untreated ischemic animals (n = 30) the intracellular H2O2 production of circulating neutrophils, as assayed flow cytometrically by dichlorofluorescein oxidation, increased from a preischemic value of 133 to a peak of 251 femtomoles dichlorofluorescein/neutrophil at 5 minutes of reperfusion (p less than 0.01). Treatment of neutrophils with phorbol myristate acetate (PMA) 10(-7) mol/L led to a 91% increase in neutrophil H2O2 production before ischemia, and 5 minutes after reperfusion there was an enhanced response to PMA of 222% (p less than 0.01). Pretreatment of animals with the thromboxane-synthetase inhibitor OKY 046 (n = 36) prevented ischemia-induced thromboxane generation, neutrophil H2O2 production (p less than 0.05), as well as the enhanced response to PMA stimulation (p less than 0.05). Treatment with the thromboxane-receptor antagonist SQ 29,548 (n = 36) did not affect the increase in plasma thromboxane levels after ischemia but was as effective as OKY 046 in preventing the ischemia-induced increase in neutrophil H2O2 production and the enhanced response to PMA stimulation. These data indicate that lower-torso ischemia leads to neutrophil activation, manifest by H2O2 production, an event mediated by thromboxane.     

Ischemic limb preconditioning downregulates systemic inflammatory activation

We examined local and systemic antiinflammatory consequences of ischemic preconditioning (IPC) in a rat model of limb ischemia‐reperfusion (I‐R) by characterizing the leukocyte‐endothelial interactions in the periosteum and the expression of adhesion molecules playing a role in leukocyte‐mediated inflammatory processes. IPC induction (2 cycles of 10 min of complete limb ischemia and 10 min of reperfusion) was followed by 60 min of ischemia/180 min of reperfusion or sham‐operation. Data were compared with those on animals subjected to I‐R and sham‐operation. Neutrophil leukocyte‐endothelial cell interactions (intravital videomicroscopy), intravascular neutrophil activation (CD11b expression changes by flow cytometry), and soluble and tissue intercellular adhesion molecule‐1 (ICAM‐1; ELISA and immunohistochemistry, respectively) expressions were assessed. I‐R induced enhanced leukocyte rolling and adherence in the periosteal postcapillary venules after 120 and 180 min of reperfusion. This was associated with a significantly enhanced CD11b expression (by ～80% and 72%, respectively) and moderately increased soluble and periosteal ICAM‐1 expressions. IPC prevented the I‐R–induced increases in leukocyte adherence and CD11b expression without influencing the soluble and tissue ICAM‐1 levels. The results show that limb IPC exerts not only local, but distant antiinflammatory effects through significant modulation of neutrophil recruitment. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 897–902, 2009     

Curcumin and dexmedetomidine prevents oxidative stress and renal injury in hind limb ischemia/reperfusion injury in a rat model

Curcumin and dexmedetomidine have been shown to have protective effects in ischemia–reperfusion injury on various organs. However, their protective effects on kidney tissue against ischemia–reperfusion injury remain unclear. We aimed to determine whether curcumin or dexmedetomidine prevents renal tissue from injury that was induced by hind limb ischemia–reperfusion in rats. Fifty rats were divided into five groups: sham, control, curcumin (CUR) group (200?mg/kg curcumin, n?=?10), dexmedetomidine (DEX) group (25?μg/kg dexmedetomidine, n?=?10), and curcumin–dexmedetomidine (CUR–DEX) group (200?mg/kg curcumin and 25?μg/kg dexmedetomidine). Curcumin and dexmedetomidine were administered intraperitoneally immediately after the end of 4?h ischemia, just 5?min before reperfusion. The extremity re-perfused for 2?h and then blood samples were taken and total antioxidant capacity (TAC), total oxidative status (TOS) levels, and oxidative stress index (OSI) were measured, and renal tissue samples were histopathologically examined. The TAC activity levels in blood samples were significantly lower in the control than the other groups (p?0.01 for all comparisons). The TOS activity levels in blood samples were significantly higher in Control group and than the other groups (p??0.01 for all comparison). The OSI were found to be significantly increased in the control group compared to others groups (p?0.001 for all comparisons). Histopathological examination revealed less severe lesions in the sham, CUR, DEX, and CUR–DEX groups, compared with the control group (p?< 0.01). Rat hind limb ischemia–reperfusion causes histopathological changes in the kidneys. Curcumin and dexmedetomidine administered intraperitoneally was effective in reducing oxidative stress and renal histopathologic injury in an acute hind limb I/R rat model.     

Attenuation of ischemia/reperfusion injury by N-acetylcysteine in a rat hind limb model

BACKGROUND: Ischemia/reperfusion is a complex set of events with severe pathologic consequences. Reperfusion initiates both the local and systemic damage in part through rapid oxygen generation. N-acetylcysteine (NAC) is a scavenger of free radical species, inhibits neutrophil accumulation, acts as a vasodilator and also improves microcirculation. In present study, we examined the protective effect of NAC in a rat hind limb ischemia/ reperfusion model. Dimethyl-sulfoxide (DMSO), a well-known antioxidant was also tested for comparison. MATERIALS AND METHODS: Ischemia was induced for 4 h by vascular clamping and followed by 1 h of reperfusion. Muscle injury was evaluated in 3 groups as a saline group (control), DMSO group, and NAC group. Plasma levels of creatine kinase, lactate dehydrogenase, thiobarbituric acid reactive substances (TBARS), and blood HCO(3), as well as muscle tissue TBARS, were measured at the end of reperfusion. Muscle tissue samples were taken for histological evaluation. RESULTS: DMSO and NAC group showed significant amelioration of plasma CPK (P < 0.05, P < 0.05), plasma TBARS (P < 0.05, P < 0.05), and muscle tissue TBARS (P < 0.05, P < 0.05) compared with the control group. Similarly, neutrophil infiltration in DMSO and NAC groups were significantly less prominent than the control group (P < 0.01, P < 0.01). CONCLUSIONS: These results show that NAC improved effectively ischemia reperfusion injury in a rat hind limb model.     

丝裂原活化蛋白激酶在一氧化碳抗大鼠肢体缺血再灌注所致肺损伤中的作用

目的 观察丝裂原活化蛋白激酶(MAPKs)在外源性一氧化碳(C0)抗大鼠肢体缺血再灌注(IR)所致肺损伤中的作用。方法 健康SD大鼠，随机分为4组(每组n=8)：对照组(Con-trol)、Control CO、IR和IR CO组。复制大鼠双后肢缺血及再灌注后肺损伤模型。IR CO和Control CO组在再灌注前1h或相应时间点置含CO的空气中，其余两组呼吸空气。观察大鼠肺组织学、肺组织中中性粒细胞(PMN)数目、肺组织湿重和干重之比(W／D)、丙二醛(MDA)含量以及动物生存情况变化。应用Western blotting检测肺组织中三种磷化MAPKs，即细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)和p38表达的变化。结果 与Contorl组相比。IR组动物死亡率、肺组织PMN数目、W／D、MDA含量以及磷酸化ERK、JNK和p38表达均显著增高；与IR组相比，IR CO组IR组动物死亡率、肺组织中PMN数目、W／D和MDA含量均显著降低、肺损伤减轻，p38表达显著增高，JNK表达显著降低，ERK表达无显著变化。结论 MAPKs信号通路参与了外源性CO抗大鼠肢体IR所致肺损伤作用的分子机制。     

丙泊酚对肢体缺血-再灌注大鼠肾损伤和肺损伤时细胞凋亡的影响

目的从细胞凋亡的角度探讨大鼠肢体缺血-再灌注后肾损伤和肺损伤的发病机制及丙泊酚对其影响的可能机制。方法复制大鼠肢体缺血-再灌注损伤(LIR)动物模型。24只SD大鼠随机分为对照组(C组)、缺血-再灌注组(IR组)、丙泊酚处理组(P组)和10%的脂肪乳对照组(T组)。采用脱氧核苷酸末端转移酶介导的DNA原位末端缺口标记技术(TUNEL)检测细胞凋亡的变化,琼脂糖凝胶电泳法进一步证实组织DNA的改变,同时用免疫组织化学的技术检测肾、肺Caspase-3、Bcl-2蛋白的表达。结果(1)C组肺组织和肾组织未见细胞凋亡现象,IR组凋亡细胞显著增多,P组凋亡细胞数较IR组和T组显著减少而多于C组(P<0.05),T组和IR组比差异无统计学意义。(2)Bcl-2表达在IR组和T组均比C组明显下降(P<0.05),P组的表达比IR组和T组增高(P<0.05)。Caspase-3在IR组和T组的表达显著高于P组和C组(P<0.05),P组和C组及IR组和T组差异有统计学意义。Caspase-3表达与凋亡细胞数呈正相关(r=0.9149,P<0.01);Caspase-3与Bcl-2的表达呈直线负相关(P<0.01)。(3)C组肾、肺组织均无凋亡梯形条带的出现,而IR组、T组有凋亡典型的梯形条带,P组梯形条带不甚清楚。结论肾、肺细胞凋亡的发生可能参与了LIR后肾、肺损伤,丙泊酚的活性成分1,6-二异丙酚通过抑制Caspase-3表达同时上调Bcl-2而减少细胞凋亡,从而减轻LIR后肾和肺的损伤。     

大鼠肢体缺血再灌注早期血栓前状态的研究

目的探讨大鼠急性下肢缺血再灌注早期血栓的形成情况。方法将大鼠随机分为假手术组、下肢缺血再灌注组两组。夹闭大鼠股动脉并用张力带绑扎下肢，建立急性下肢缺血再灌注大鼠模型。观察急性下肢缺血再灌注大鼠血小板膜糖蛋白GPⅡb／Ⅲa和血管内皮细胞血栓调节蛋白（thrombomoclulin，TM）的表达。结果缺血再灌注组大鼠血小板膜糖蛋白GPⅡb／Ⅲa表达高于假手术组（P〈0．05）；缺血再灌注组大鼠血管内皮TM表达低于假手术组（P〈0．05）。结论急性下肢缺血再灌注可能导致大鼠血栓前状态的形成。