Ischemia Reperfusion-induced Injury Research Articles

Neuroprotective Effects of Diazoxide and Its Antagonism by Glibenclamide in Pyramidal Neurons of Rat Hippocampus Subjected to Ischemia-Reperfusion-Induced Injury

Mitochondrial ATP-sensitive potassium channel opener, diazoxide, is shown to have protective effect on the heart and brain following ischemia-reperfusion-induced injury (IR/II). However, the detailed effect of diazoxide and its antagonist on neuronal death, mitochondrial changes, and apoptosis in cerebral IR/II has not fully studied. IR/II was induced in rats by the 4-vessel occlusion model. Neuronal cell death and mitochondrial changes in CA1–CA4 pyramidal cells of the hippocampus were studied by light and electron microscopy, respectively. Apoptosis was assessed by measuring the amount of protein expressed by Bax and Bcl-2 genes. In light microscopy studies, the number of total and normal cells were increased only following 18 mg/kg of diazoxide. Lower doses (2 and 6 mg/kg) failed to change the cell numbers. All three doses of glibenclamide (1, 5, and 25 mg/kg) decreased the number of total and normal cell populations. In electron microscopy studies, different doses of diazoxide and glibenclamide prevented and aggravated the IR-induced morphological changes, respectively. Western blot analysis showed that diazoxide and glibenclamide inhibited and enhanced Bax protein expression respectively. Regarding Bcl-2 expression, only diazoxide showed a significant enhancement of gene expression. In conclusion, the results show that diazoxide can exhibit neuroprotective effects against IR/II in hippocampal regions, possibly through the opening of mitochondrial ATP-sensitive K+ channels.

Activated Protein C: An Emerging Therapeutic Agent in the Prevention of Ischemia-Reperfusion Injury

Free flap necrosis continues to be a significant problem in microvascular surgery. Despite improved microsurgical techniques and equipment, flap loss remains the major operative complication. Although ischemia-induced reperfusion injury remains a significant etiologic factor in flap loss, there is continued interest in endothelial mechanisms that regulate microvascular injury and thrombosis. In recent years, activated protein C (APC) has emerged as a promising therapy in counteracting microcirculatory injury. APC is an anticoagulant that is also involved in signaling pathways that modulate inflammation, apoptosis, and vascular permeability. This article presents the mechanism of action of APC and the benefits of this therapeutic agent, including a possible role in the prevention of free flap ischemia-reperfusion injury.

Combined use of WEB2170 and HBO therapy can reduce ischemia and reperfusion injury to the skeletal muscle in a rabbit model

Ischemia/reperfusion injury is regarded as the main cause of failure in revascularization of limbs and transfer of free flaps in the so called nonreflow phenomenon. This type of damage is caused by the production of free radicals, above all, of neutrophils that release great quantities of extracellular superoxide through the action of a membrane enzyme. In our study we used 40 white rabbits. Rabbit rectus femoris muscle is perfused by a single artery and vein and is therefore a valuable model for study of ischemia-induced reperfusion injury of skeletal muscle. The objective of this study was to individualize a valid method of protection for the muscle from damage by ischemia-induced reperfusion injury. We have tested the effectiveness of WEB2170, a PAF antagonist, of hyperbaric oxygen therapy one (HBO), and of combined employment of WEB2170 and HBO. The results show that both PAF and HBO play important protective roles against damage from ischemia/reperfusion injury, and that the combined employment of both therapies has a synergistic effect. We propose therefore a new therapeutic protocol for the prevention of damage resulting from ischemia/reperfusion injury with the simultaneous employment of this PAF and HBO.

ST2, an IL-1R family member, attenuates inflammation and lethality after intestinal ischemia and reperfusion

Ischemia reperfusion injury is characterized by local and systemic inflammation leading to considerable mortality. Previously, we have reported that soluble T1/ST2 (sST2), a member of the IL-1 receptor gene family, inhibits LPS-induced macrophage proinflammatory cytokine production. Here, we report the therapeutic effect of sST2-Fc in a murine model of intestinal ischemia reperfusion-induced injury. Administration of sST2-Fc fusion protein i.v., 10 min before reperfusion, reduced the production of TNF-alpha dose-dependently in the intestine and in the lungs. The sST2-Fc treatment with the highest dose (100 mug) resulted in inhibited vascular permeability, neutrophilia, and hemorrhage in the intestine and the lungs compared with controls treated with normal IgG. This was associated with down-regulated tissue levels of proinflammatory cytokines, markedly reduced serum TNF-alpha levels, and increased survival of mice from the sST2-Fc-treated group after ischemia and reperfusion injury. The beneficial effect of sST2-Fc treatment was associated with elevated IL-10 production in intestine and lung. sST2-Fc was not able to prevent the inflammatory response associated with intestinal ischemia and reperfusion in IL-10-deficient mice, suggesting that sST2 exerts its anti-inflammatory effect in a IL-10-dependent manner. These results also demonstrate that sST2-Fc may provide a novel, complementary approach in treating ischemic reperfusion injury.

05 Effect of pentoxifylline on ischemia reperfusion-induced heart injury in rats

05 Effect of pentoxifylline on ischemia reperfusion-induced heart injury in rats

The effects of the nitric oxide donor SIN-1 on ischemia-reperfused cutaneous and myocutaneous flaps.

Ischemia-reperfusion injury causes tissue damage that leads to a decrease in bioavailability of nitric oxide. The authors hypothesized that an exogenous supply of nitric oxide will have beneficial effects on survival of skin and skeletal muscle subjected to ischemia-reperfusion injury. By using the nitric oxide donor SIN-1 (3-morpholino-sydnonimine) the effects of direct intraarterial infusion of an exogenous source of nitric oxide in reperfused flaps was studied. Bilateral island buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs, for a total of 32 flaps. Flaps were subjected to 6 hours of ischemia followed by 18 hours of reperfusion. Flaps on one side of each animal were randomized to be treated with the nitric oxide donor (treatment group). The contralateral side was treated with an equivalent volume of saline vehicle (infusion control) SIN-1, or saline was administered as a continuous direct intraarterial infusion at the onset of reperfusion and continued during the observation period. Outcomes measured were tissue neutrophil accumulation by using myeloperoxidase assay and tissue survival (intravenous fluorescein and nitroblue tetrazolium for skin and muscle, respectively). In both skin and myocutaneous flaps, SIN-1 treatment caused a significant improvement in survival and a decrease in neutrophil accumulation. Nitric oxide may play an important role in the pathophysiologic process of ischemia-induced reperfusion injury in skin and skeletal muscle. Nitric oxide donors may be a promising family of therapeutic agents for the prevention of ischemia-induced reperfusion injury in cutaneous and myocutaneous flaps.

Role of CR2- and CD19-regulated natural antibodies in intestinal ischemia reperfusion-induced injury

Role of CR2- and CD19-regulated natural antibodies in intestinal ischemia reperfusion-induced injury

Inhibition of ischemia reperfusion-induced intestinal injury and systemic inflammation by a novel complement regulatory protein

Inhibition of ischemia reperfusion-induced intestinal injury and systemic inflammation by a novel complement regulatory protein

Prevention of renal ischemia–reperfusion-induced injury in rats by picroliv

Picroliv is a potent antioxidant extracted from the roots and rhizome of Picrorhiza kurrooa. It has been shown to impart significant hepatoprotective activities, partly by modulation of free radical-induced lipid peroxidation. Lipid peroxidation and reactive oxygen species are associated with tissue injury in post-ischemic acute renal failure. The efficacy of picroliv was assessed in an in vivo model of renal ischemia–reperfusion injury (IRI) in rats at a dose of 12 mg/kg orally for 7 days. The animals were killed at various times after reperfusion. Increased lipid peroxidation and apoptotic cell number reflected the oxidative damage following renal IRI. Picroliv-pretreated rats exhibited lower lipid peroxidation, improved antioxidant status, and reduced apoptosis, indicating better viability of renal cells. Immunohistochemical studies revealed that picroliv pretreatment attenuated the expression of intercellular adhesion molecule-1 in the glomerular region. These results suggested that picroliv pretreatment protects rat kidneys from IRI, perhaps by modulation of free radical damage and adhesion molecules.

Isoflurane-sevoflurane adminstration before ischemia attenuates ischemia-reperfusion-induced injury in isolated rat lungs.

The effects of volatile anesthetics on ischemia-reperfusion (IR)-induced lung injury are not clear. The authors investigated the effects of preadministration of isoflurane and sevoflurane on IR-induced lung injury in an isolated buffer-perfused rat lung model. Isolated rat lungs were designated into four groups: control group (n = 6): perfusion for 120 min without ischemia; IR group (n = 6): interruption of perfusion and ventilation for 60 min followed by reperfusion for 60 min; sevoflurane (SEVO)-IR (n = 6) and isoflurane (ISO)-IR (n = 6) groups: 1 minimum alveolar concentration (MAC) isoflurane or sevoflurane was administered for 30 min, followed by 60 min ischemia, then 60 min reperfusion. The authors measured the coefficient of filtration (Kfc) of the lung, lactate dehydrogenase (LDH) activity, tumor necrosis factor alpha, and nitric oxide metabolites (nitrite + nitrate) in the perfusate and the wet-to-dry lung weight ratio. IR caused significant increases in the coefficient of filtration (approximately sevenfold at 60 min of reperfusion compared with baseline; P < 0.01), the wet-to-dry lung weight ratio, the rate of increase of lactate dehydrogenase activity, and tumor necrosis factor a in the perfusate, and caused a significant decrease in nitric oxide metabolites in the perfusate. Administration of 1 MAC isoflurane or sevoflurane before ischemia significantly attenuated IR-induced increases in the coefficient of filtration and the wet-to-dry lung weight ratio, inhibited increases in the rate of increase of lactate dehydrogenase activity and tumor necrosis factor alpha in the perfusate, and abrogated the decrease in nitric oxide metabolites in the perfusate. No difference was found between the SEVO-IR and ISO-IR groups. Isoflurane and sevoflurane administered before ischemia can attenuate IR-induced injury in isolated rat lungs.

BGP-15, a nicotinic amidoxime derivate protecting heart from ischemia reperfusion injury through modulation of poly(ADP-ribose) polymerase

The protective effect of O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15) against ischemia–reperfusion-induced injury was studied in the Langendorff heart perfusion system. To understand the molecular mechanism of the cardioprotection, the effect of BGP-15 on ischemic–reperfusion-induced reactive oxygen species (ROS) formation, lipid peroxidation single-strand DNA break formation, NAD + catabolism, and endogenous ADP-ribosylation reactions were investigated. These studies showed that BGP-15 significantly decreased leakage of lactate dehydrogenase, creatine kinase, and aspartate aminotransferase in reperfused hearts, and reduced the rate of NAD + catabolism. In addition, BGP-15 dramatically decreased the ischemia–reperfusion-induced self-ADP-ribosylation of nuclear poly(ADP-ribose) polymerase(PARP) and the mono-ADP-ribosylation of an endoplasmic reticulum chaperone GRP78. These data raise the possibility that BGP-15 may have a direct inhibitory effect on PARP. This hypothesis was tested on isolated enzyme, and kinetic analysis showed a mixed-type (noncompetitive) inhibition with a K i = 57 ± 6 μM. Furthermore, BGP-15 decreased levels of ROS, lipid peroxidation, and single-strand DNA breaks in reperfused hearts. These data suggest that PARP may be an important molecular target of BGP-15 and that BGP-15 decreases ROS levels and cell injury during ischemia–reperfusion in the heart by inhibiting PARP activity.

Losartan attenuates myocardial ischemia-induced ventricular arrythmias and reperfusion injury in spontaneously hypertensive rats.

To assess the efficacy of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)methyl]imidazole, potassium salt), an angiotensin II receptor antagonist, on acute myocardial ischemia, 36 four-month-old spontaneously hypertensive rats were used. The animals underwent 45 min of left coronary artery occlusion and 1 h of reperfusion and were randomly assigned to control and losartan-treated groups (2, 5, and 10 mg/kg, intravenously). Losartan was administered 15 min before ischemia. Electrocardiograms (lead II) were monitored continuously throughout the experiment. To assess the anti-infarct effect of losartan, the area at risk was determined by methylene blue dye and the infarct size was determined by nitroblue tetrazolium chloride staining. The areas of risk and infarct were measured by computerized planimetry. Results demonstrated that the low and intermediate doses (2 and 5 mg/kg) of losartan significantly decreased the incidence of ventricular fibrillation and mortality during the ischemic period induced by left coronary artery occlusion. However, a significant reduction in infarct size, calculated as a percentage of the area at risk, was noted in all three losartan-treated groups (control: 41.5% +/- 5.2%, losartan, 2 mg/kg: 11.2% +/- 5.8%, 5 mg/kg: 8.5% +/- 2.7% and 10 mg/kg: 13.7% +/- 1.6%). The results suggest that losartan may be useful in the treatment of ventricular arrhythmias induced by acute myocardial infarction and attenuation of reperfusion injury in hypertension.

Spinal Cord Gray Matter Layers Rich in NADPH Diaphorase-Positive Neurons Are Refractory to Ischemia–Reperfusion-Induced Injury: A Histochemical and Silver Impregnation Study in Rabbit

Silver impregnation analysis of neuronal damage and concurrent histochemical characterization of NADPH diaphorase-positive neuronal pools in the rabbit lumbosacral segments was performed during and after transient spinal cord ischemia. Strongly enhanced staining of NADPH diaphorase-positive neurons and their processes appeared in the superficial dorsal horn (laminae I–III), the pericentral region (lamina X) of lower lumbar segments, the lateral collateral pathway, and mainly in neurons of the sacral parasympathetic nucleus in the S2 segment at the end of 40 min of abdominal aorta ligation or 1 day after reperfusion. Despite the development of extensive neuronal degeneration in the central gray matter (laminae IV–VII) between 1 and 4 days after ischemia, a number of nonnecrotizing neurons localized in the areas corresponding with the distribution of NADPH diaphorase-positive neurons was detected, suggesting a selective resistance of these classes of neurons against transient ischemic insult. While the precise mechanism of the observed resistance is not known, it is postulated that region-specific synthesis of nitric oxide and its vasodilatatory effect during the period of incomplete spinal ischemia may account for the observed selective resistance of these spinal cord neurons to transient ischemia.

Mitochondrial Glutathione Redox and Energy Producing Function during Liver Ischemia and Reperfusion

The glutathione in mitochondria is thought to play a crucial role in protecting mitochondria against ischemia–reperfusion-induced injury. However, little is known about the mitochondrial redox system. This study was conducted to clarify changes of mitochondrial glutathione redox during liver ischemia and reperfusion and its role on energy producing function. Rats were divided into three groups each of which were treated respectively with saline (Control), buthionine sulfoximine (BSO), which induces a rapid decrease in tissue glutathione concentrations, and γ-glutamylcysteine ethyl ester (GCE), which conversely induces a rapid increase in tissue glutathione concentrations before induction of ischemia. Liver ischemia was induced for 120 min, and blood reflow was subsequently restored for 60 min. Total and mitochondrial glutathione concentrations, mitochondrial respiratory function, and tissue adenine nucleotide were determined after both the ischemic and the reperfusion periods. In all groups, concentrations of the reduced form of glutathione (GSH) gradually decreased during ischemia and reperfusion. On the other hand, significant increases in mitochondrial GSH were apparent after reperfusion despite significant decreases during ischemia in the control and GCE groups. Total and mitochondrial GSH in the BSO and GCE groups were significantly lower and higher, respectively, compared with the control throughout the experiment. Recovery of the mitochondrial energy producing function and cellular adenine nucleotide after reperfusion were dependent on GSH concentrations. We conclude that mitochondrial GSH concentrations dramatically change in a different manner from cytosolic concentrations after reperfusion, and that recovery of the mitochondrial energy-producing function might be closely associated with mitochondrial GSH concentrations.

Assessment of ischemia-induced reperfusion injury in the pig latissimus dorsi myocutaneous flap model.

Experiments were conducted to assess ischemia-induced reperfusion injury in the pig latissimus dorsi myocutaneous flap model. Forty Yorkshire pigs (19.5 +/- 0.6 kg) were assigned to groups A, B, C, and D (n = 10 pigs). Bilateral 8 x 13 cm latissimus dorsi myocutaneous flaps were constructed in each pig, and one flap was assigned to ischemic treatment and the contralateral flap served as a nonischemic control. The treatment flaps in groups A, B, C, and D were subjected to 2, 4, 6, and 8 hours of warm global ischemia, respectively. Pigs in groups A, B, C, and D were divided into two subgroups (n = 5 pigs), and extents of skin and muscle necrosis in control and treatment flaps were assessed with the fluorescein and nitroblue tetrazolium dye stain tests, respectively, after 2 and 7 days of reperfusion. Significantly (p < 0.01) greater extents of skin and muscle necrosis were observed in latissimus dorsi myocutaneous flaps subjected to 4, 6, or 8 hours of ischemia compared with their contralateral controls. Extents of skin and muscle necrosis also increased significantly (p < 0.01) with increases in ischemia time in treatment flaps. Of particular importance was the observation that there was no significant difference in the extent of skin or muscle necrosis between 2 and 7 days of reperfusion in all control and treatment groups. This observation indicates that 2 days of reperfusion time is adequate to assess the maximum extent of skin and muscle ischemia-induced reperfusion injury in pig latissimus dorsi myocutaneous flaps. Furthermore, it was observed that 1-cm segments of latissimus dorsi muscle were not too thick to allow the use of the nitroblue tetrazolium dye stain test for assessment of muscle viability, as judged by the highly correlated (r = 0.98, n = 40) linear relationship between assessment of muscle viability from one transverse cut surface of muscle segments and by weighing total viable and nonviable muscles dissected from the flaps according to the nitroblue tetrazolium dye stain on both transverse cut surfaces. It is important to note that the maximum length of the latissimus dorsi myocutaneous flap model for ischemia-induced reperfusion injury research should not exceed the maximum length of skin viability in the nonischemic control in order to avoid the complication of skin necrosis due to excessive length of skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Deferoxamine Attenuates Ischemia-Induced Reperfusion Injury in the Skin and Muscle of Myocutaneous Flaps in the Pig

The dose effect of deferoxamine treatment in attenuation of ischemia-induced reperfusion injury in the skin and muscle of latissimus dorsi myocutaneous flaps was studied in pigs weighing 19.7 +/- 0.5 kg. The latissimus dorsi myocutaneous flaps were subjected to 4, 6, or 8 hours of warm global ischemia. The length and area of viable and nonviable skin and muscle were assessed 48 hours after the ischemic insult by using the fluorescein and nitroblue tetrazolium dye tests, respectively. It was observed that perioperative deferoxamine treatment (250 mg/kg IV) was effective (p < 0.05) in attenuation of ischemia-induced reperfusion injury in the skin but not in the muscle of latissimus dorsi myocutaneous flaps subjected to 4, 6, or 8 hours (n = 10) of ischemia compared with the saline-treated control (n = 10). In a separate study, it was observed that preoperative deferoxamine treatment (250 mg/kg per day x 2 days, IM) plus perioperative deferoxamine treatment (250 mg/kg IV) was effective (p < 0.05) in attenuation of muscle ischemia-induced reperfusion injury in latissimus dorsi myocutaneous flaps subjected to 4 hours of ischemia and 48 hours of reperfusion (n = 10) compared with the saline treated control (n = 10). Morphologic studies with light and electron microscopy also provided evidence to indicate that preoperative plus perioperative deferoxamine treatment, but not perioperative deferoxamine treatment alone, remarkably reduced ischemia-induced reperfusion injury in the skeletal muscle of latissimus dorsi myocutaneous flaps compared with the saline-treated control. It is concluded that deferoxamine is effective in the attenuation of ischemia-induced reperfusion injury in the skin and muscle of pig latissimus dorsi myocutaneous flaps, but a longer period and/or higher dose of deferoxamine treatment is required for the muscle than for the skin. The pharmacologic actions and metabolism of deferoxamine relating to mitigation of ischemia-induced reperfusion injury in the pig skin and muscle are discussed.

Pharmacologic intervention in ischemia‐induced reperfusion injury in the skeletal muscle

This article provides a concise review on the potential causes of ischemia-induced reperfusion (I/R) injury and pharmacologic intervention in the skeletal muscle. Special emphasis is placed on the recent observation of the acute ischemic preconditioning phenomenon for prevention of I/R injury in skeletal muscle. Finally, the mechanism of ischemic preconditioning and its clinical applications for augmentation of skeletal muscle tolerance to prolonged ischemic insult are discussed.

Protective effects of coenzyme Q10 on ischemia-induced reperfusion injury in ischemic limb models.

We examined simple cold preservation of rat limbs in Euro-Collins' solution to elucidate the protective effect of coenzyme Q10 (CoQ10) on the ischemia-induced reperfusion injury in an ischemic extremity replant model. A total of 126 Lewis rat limb replants were performed. Limbs were amputated from donor rats and preserved at 4 degrees C in Euro-Collins' solution and were orthotopically grafted to isogeneic rats by microsurgical technique. In the experimental groups (n = 42), coenzyme Q10 (10 mg/kg) was injected intraperitoneally into the recipients about 1 hour before reperfusion. In the control groups (n = 84), the same dose of solvent was given by the same route. We evaluated vascular patency of anastomoses by direct observation or microangiogram and performed histologic examinations 7 days after replantation. In the control groups, the ischemic limit was 96 hours. Ischemic limbs treated with coenzyme Q10 showed a statistically significant (p < 0.05) improvement in vascular patency after 72 and 96 hours of ischemia. Histologically, bone viability with osteoblastic activity was maintained in coenzyme Q10-treated animals of the 72-hour ischemic group. We conclude that the protective effect of coenzyme Q10 on reperfusion injury is suggested in this replant model.

Future horizons of lung preservation by application of a platelet-activating factor antagonist compared with current clinical standards

With the introduction of platelet-activating factor antagonists, a direct inhibition of ischemia-induced reperfusion injury can be achieved by prevention of platelet activation, reduction of microvascular leakage, and platelet-activating factor-induced bronchoconstriction. At present, two preservation methods are established for clinical lung preservation: (1) donor core cooling by extracorporeal circulation and (2) pulmonary artery flush with Euro-Collins solution and prostacyclin. We compared the quality of organ preservation obtained with these methods to the application of a platelet-activating factor antagonist (WEB 2170; 0.3 mg/kg) for the donor, perfusion solution, and throughout the first 6 hours of reperfusion in combination with prostacyclin (20 ng/kg/min) and Euro-Collins solution (60 ml/kg). Eighteen heterotopic heart and orthotopic left lung transplants were performed in three groups of six dogs each after 6 hours of cold ischemia (group I, donor core cooling; group II, Euro-Collins flush and prostacyclin; group III, Euro-Collins flush, prostacyclin, and WEB 2170). Myocardial preservation was achieved with St. Thomas' Hospital solution (20 ml/kg) in all groups. After transplantation, cardiorespiratory function was assessed at an inspired oxygen fraction of 0.4. After transplantation, superior results were observed in group III, as expressed by significantly improved oxygenation, while cardiac output and pulmonary artery pressures were similar in all groups. We concluded that the use of the platelet-activating factor antagonist WEB 2170 resulted in better lung preservation than current clinical standards.

Combined effect of acute denervation and ischemia on the microcirculation of skeletal muscle

Using direct in vivo videomicroscopy and a fluorescein dye technique, reperfusion injury after 3 h of ischemia was studied in the acutely denervated cremaster muscle of the rat. Compared with normally innervated controls, ischemia-induced reperfusion injury was more severe in the denervated group and included a delay of blood flow recovery, vortex formation, edema, hemorrhage, and vessel spasm. Vessel size was reduced at the arteriole and small artery level, and there was a decrease of reactive hyperemia. The injury mechanism may be related to a loss of active vasomotion and vascular response to vasoactive substances after denervation. The results suggest that shortening the ischemia time of denervated tissues may reduce ischemia-induced reperfusion injury. Similarly, given the same ischemia time, improved tissue reperfusion may be expected if the nerve supply is maintained.