Endopeptidase inhibitors decrease myocardial ischemia/reperfusion injury in an in vivo rabbit model

Periods of ischemia followed by reperfusion of the ischemic tissue are associated with myocardial damage and ventricular arrhythmia. Angiotensin converting enzyme inhibitors limit the occurrence of these arrhythmias. The protective effects of angiotensin converting enzyme inhibitors may be due to inhibition of bradykinin (BK) degradation, rather than inhibition of angiotensin II formation. Other enzymes which catabolize BK include the endopeptidases EP24.11 and EP24.15. The purpose of this study was to determine if inhibitors of EP24.11 and EP24.15 decrease ischemia/reperfusion injury and if this protection is mediated by BK receptors. Rabbits were anesthetized and prepared for recording of cardiovascular parameters. The chest was opened and a left ventricular artery occluded for 30 min, followed by a 2-hr reperfusion period. Infarct size was determined using triphenyl tetrazolium chloride staining immediately after reperfusion. The enzyme inhibitors, ramiprilat, N-[1-(R,S)-carboxy-3-phenylpropyl]-Phe-pAB, and N[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-pAb, singly and in combinations were administered 3 min before reperfusion. Compared to saline (32.1 +/- 2.1), ramiprilat (18.3 +/- 2.8) and the EP inhibitors (14.4 +/- 1.4 for the combination) significantly decreased infarct size, with the greatest decrease occurring when all three inhibitors were combined (10.6 +/- 1.5). The protective effect of the EP inhibitors was blocked by the BK2 receptor antagonist, HOE 140 (30.1 +/- 2.6). Enzyme assays demonstrated EP24.11 and EP24.15 in the rabbit heart. We conclude that the EP inhibitors decreased ischemia/reperfusion injury by protecting BK from metabolism and that a combination of inhibitors provides superior protection to that given by a single agent.     

A fish oil diet minimizes hepatic reperfusion injury in the low-flow, reflow liver perfusion model

In this study, the effects of fish oil treatment on hepatic reperfusion injury in a low-flow, reflow perfusion model were investigated. Rats were fed powdered diets containing either 5% corn or 5% encapsulated fish oil for 13 to 15 days. Average daily food intake in both control and fish oil groups was about 20 g per rat, and weight gain averaged 9 g/rat/d. Livers were perfused at flow rates around 1 muL/g/min for 75 minutes, which caused cells in pericentral regions to become anoxic because of insufficient delivery of oxygen. When normal flow rates (about 4 mL/g/min) were restored for 40 minutes, reperfusion injury occurred. Upon reflow, lactate dehydrogenase (LDH) release increased from basal levels around 1 to 50 IU/g/h in livers from control rats, whereas fish oil treatment minimized values to 16 IU/g/h Rates of bile production reached 23 muL/g/h during reperfusion in livers from controls and 38 muL/g/h in the fish oil-treated group. Oxygen uptake was about 110 mumol/g/h during the reperfusion period in livers from both groups. Malondialdehyde (MDA), an end product of lipid peroxidation, was released into the effluent perfusate at rates around 80 nmol/g/h during reperfusion in controls, and values were not affected by fish oil treatment, Portal pressure, an indicator of hepatic microcirculation, increased from basal levels of 3 to 10.5 cm H2O during reperfusion in controls, but was only elevated to 8.3 cm H2O in the fish oil-treated group. In addition, Trypan Blue distribution time, another indicator of hepatic microcirculation, was reduced significantly by 44% by fish oil treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of injury and the role of neutrophils in reperfusion injury of rat lung

Using a rat lung model, we sought to characterize the time course for ischemia-reperfusion injury and the role of neutrophils in the development of injury. Adult male Long-Evans rats underwent left thoracotomy with dissection and clamping of the left pulmonary artery, bronchus, and vein for 90 min, resulting in complete left lung ischemia. The lungs were then ventilated and reperfused for up to 4 hr. Time-matched sham animals underwent the identical thoracotomy and hilar dissection, but the lungs were not rendered ischemic. Using vascular permeability of 125I-labeled bovine serum albumin as a measure of reperfusion injury, a bimodal pattern of injury was observed. Compared to sham controls, animals undergoing ischemia-reperfusion demonstrated a significant early phase of lung injury at 30 min of reperfusion (P < 0.0001), followed by partial recovery. A second peak of lung injury was noted after 4 hr of reperfusion (P < 0.001). Myeloperoxidase activity in reperfused lung tissue, a measure of neutrophil sequestration, increased during the reperfusion time course. To determine the role of neutrophils in the development of lung reperfusion injury, additional animals undergoing the identical ischemia-reperfusion protocol received either rabbit anti-rat neutrophil serum or preimmune serum the day prior to operation. Profound neutropenia (< 75/mm3 blood) was confirmed by differential leukocyte counts. Neutropenia had no protective effect against microvascular permeability at 30 min of reperfusion, but there was a significant reduction in lung injury at 4 hr (P < 0.005). We conclude that, during lung ischemia-reperfusion, there is a bimodal pattern of injury, consisting of both neutrophil-independent and neutrophil-mediated events.     

Posttraumatic hypothermia in the treatment of axonal damage in an animal model of traumatic axonal injury

Twenty-one-day-old BALB/c mice were shaved on the back to synchronize hair growth. On day 30 or 31, when at least 90% of mice exhibited hair regrowth in the shaved area, 1,25(OH)2D3 was applied topically to the shaved area daily for 5 days. On the 6th day, cyclophosphamide (Cytoxan, CTX) was injected i.p. to induce hair loss in the shaved area. Alopecia was induced in a dose-dependent manner by CTX treatment within 1 to 2 weeks. This effect was reduced significantly if mice were pre-treated with 1,25(OH)2D3, though only slight protection was observed in female mice. Interestingly, this 1,25(OH)2D3-mediated protection against hair loss was attenuated in male mice but became more significant in female mice when they were inoculated with the EMT-6 murine mammary tumor prior to treatment. More importantly, topical treatment with 1,25(OH)2D3 alone was able to inhibit EMT-6 tumor growth in both male and female BALB/c mice. Furthermore, 1,25(OH)2D3 pre-treatment also augmented the anti-tumor effect of CTX. Our results demonstrate that topical application of 1,25(OH)2D3 can protect against CTX-induced alopecia both in tumor-free and in tumor-bearing mice in a sex-dependent manner. Moreover, 1,25(OH)2D3 was shown, either alone or in combination with CTX, to inhibit tumor growth.     

Controlled reperfusion and pentoxifylline modulate reperfusion injury after single lung transplantation

OBJECTIVE: Rodent models have suggested that initial low-pressure reperfusion of transplanted lungs reduces injury after ischemia. We investigated this phenomenon and the use of pentoxifylline in a porcine model of left single lung transplantation. METHODS: Donor lungs were preserved with Euro-Collins solution for a mean ischemic time of 18.4 hours. Neutrophil trapping in the graft, pulmonary artery pressure, and gas exchange were assessed over a 12-hour period. Partial occlusion of the contralateral pulmonary artery allowed manipulation of the pulmonary artery pressure in the transplanted lung. Group A (n = 5) was perfused at a mean pulmonary artery pressure of 20 mm Hg, group B was reperfused at a mean pulmonary artery pressure of 45 mm Hg for 10 minutes before reducing the pressure to the same as group A, and group C was reperfused at a mean pressure of 20 mm Hg for 10 minutes, then increased to a mean of 45 mm Hg for the remainder of the experiment. Group D was reperfused as in group A with the addition of intravenous pentoxifylline. RESULTS: Leukocyte sequestration was observed in the first 10 minutes after reperfusion in groups A, B, and C, with maximal sequestration at 2 minutes. Significantly more sequestration was observed in the first 6 minutes in group B than in groups A and C, which were similar. Pentoxifylline significantly reduced leukocyte sequestration. Pulmonary venous oxygen tension in the allograft lung was worst in group B. Groups A and C were similar, but group D was superior to all other groups (p < 0.001). CONCLUSIONS: Low-pressure reperfusion, even when limited to the first 10 minutes, modulates reperfusion injury possibly through a leukocyte-dependent mechanism. The addition of pentoxifylline in the recipient confers significant additional benefit.     

Thoracoscopic lobectomy in the animal model

To evaluate the possibility of a pure thoracoscopic lobectomy by preparation and selective division of hilar structures we performed left cranial lobectomies in 5 G?ttingen mini-pigs. The vessels and the main bronchus were isolated and divided by an Endo-GIA stapler. As an alternative technique we used clips or endoscopic ligation. Inside the thoracic cavity the resected lobes were divided into 2 or 3 parts by the Endo-GIA. They could be extracted without destroying the tissue therefore making macroscopic examination possible. The intraoperative blood loss was minimal and all the pigs survived the operation. Two pigs were sacrificed initially, the remaining three one month later. These three showed no evidence of pleural fistula or atelectasis in remaining lung tissue either macroscopically or histologically. It appears that thoracoscopic selective lobectomy is technically possible at least in pig studies. Further studies will show whether thoracoscopic lobectomy in patient with malignancy is as effective as open radical thoracotomy techniques and if endoscopic mediastinal division is possible.     

L-arginine treatment in ischemia/reperfusion injury

We tested whether treatment with exogenous L-arginine, the precursor of nitric oxide (NO), could protect the skeletal muscle from ischemia/reperfusion (I/R) injury. A rabbit hindlimb I/R model (2.5 h ischemia/2 h reperfusion) was used. Morphological changes were elucidated by morphometry. Plasma concentrations of malondialdehyde (pMDA), as well as L-arginine and L-citrulline content in the plasma and skeletal muscle were measured. I/R injury in the skeletal muscle was manifested by development of prominent interstitial edema (fraction of interfiber area was 26.23% vs 15.09% in sham operated control, p < .005) and severe microvascular constriction (capillary area was 11.41 microns2 vs 16.92 in control, p <.005). These changes were accompanied by increased pMDA levels, indicating a process of lipid peroxidation in the cell membranes. L-arginine treatment (4 mg/kg/min intravenously, for 1 h, infusion initiated 30 min before reperfusion) caused an intracellular accumulation of this amino acid in the SM. Intracellular concentrations of L-citrulline increased (201.0 mumol/dm3 after reperfusion vs 176.0 before ischemia onset, p < .005), suggesting stimulated endogenous NO synthesis. L-arginine treatment protected capillary constriction (capillary area was 17.64 microns2 vs 11.41 in the untreated animals, p < .0005) and reduced interstitial edema after reperfusion (fraction of interfiber area was 17.80% vs 26.23 in untreated animals, p < 0.005). The protective effect of L-arginine treatment on I/R injury of SM may be related to its ability to prevent microvascular constriction and reduce permeability disorders by the stimulation of endogenous NO production.     

Carbonyl histochemistry in rat reperfusion nerve injury

Free radical mediated, site-specific lipid and protein oxidation has been implicated in the pathophysiology of an ischaemic/reperfusion injury. The aim of the present study was to determine whether carbonyl formation could be detected histochemically in reperfused rat sciatic nerves. We also examined the effects of preischaemic alpha-tocopherol supplementation on carbonyl formation in reperfused nerves. Seven hours of near-complete ischaemia was induced in rat right hindlimb by occlusion of major arteries using microvascular clips. Histochemical detection of carbonyl compounds, applying naphthoic acid hydrazide (NAH) and Fast Blue B (FBB), was undertaken at thigh, knee and calf levels of sciatic, tibial and peroneal nerves. NAH-FBB reactivity was confined to vessels in reperfused nerves. Positively stained epi-, peri- and endoneurial vessels were invariably observed after 2 h of reperfusion at all levels examined. After 24 and 48 h and 7 days of reperfusion, NAH-FBB-positive vessels were more frequently found at knee and calf levels than at the thigh level. Following preischaemic alpha-tocopherol supplementation, no vessels were stained positively with NAH-FBB, except for some epineurial vessels at knee and calf levels after 2 h of reperfusion. Morphometry in endoneurial vessels at the knee level revealed that endothelial cell area in alpha-tocopherol-treated reperfused nerves was significantly less when compared with those in reperfused nerves without alpha-tocopherol. In conclusion, we have demonstrated histochemical evidence of carbonyl formation in vessels, but not with nerve fibres, in ischaemic/reperfused rat sciatic nerves. These abnormalities were prevented with preischaemic supplementation of alpha-tocopherol.     

Hemostatic efficacy of a fibrin sealant dressing in an animal model of kidney injury

Arterial spin tagging techniques have been used to image tissue perfusion in MR without contrast injection or ionizing radiation. Currently, spin tagging studies are performed primarily using single-slice imaging sequences, which are time consuming. This note reports a multislice echo-planar arterial spin tagging technique (Simultaneous Multislice Acquisition with aRterial-flow Tagging, or "SMART"). Multiband RF encoding (Hadamard) is used to provide simultaneous multislice acquisition capability for spin tagging techniques (such as echo planar imaging signal targeting with alternating radio frequency and flow-sensitive alternative inversion recovery). The method is illustrated with a two-slice pulse sequence that was implemented using the FAIR technique to generate two perfusion weighted images simultaneously. Compared with single-slice sequences, this two-slice sequence provided similar image quality, signal-to-noise ratio, and twice the spatial coverage compared with the single-slice technique within the same scan time.     

Ischemic and reperfusion injury of cyanotic myocardium in chronic hypoxic rat model: changes in cyanotic myocardial antioxidant system

OBJECTIVE: The objective was to evaluate the effect of left ventricular function on cyanotic myocardium after ischemia-reperfusion and to determine the effect of cyanosis on the myocardial antioxidant system. METHODS: Cyanotic hearts (cyanotic group) were obtained from rats housed in a hypoxic chamber (10% oxygen) for 2 weeks and control hearts (control group) from rats maintained in ambient air. Isolated, crystalloid perfused working hearts were subjected to 15 minutes of global normothermic ischemia and 20 minutes of reperfusion, and functional recovery was evaluated in the two groups. Myocardial superoxide dismutase, glutathione peroxidase, glutathione reductase activity, and reduced glutathione content were measured separately in the cytoplasm and mitochondria at the end of the preischemic, ischemic, and reperfusion periods. RESULTS: Mean cardiac output/left ventricular weight was not significantly different between the two groups. Percent recovery of cardiac output was significantly lower in the cyanotic group than in the control group (56.1% +/- 5.7% vs 73.0% +/- 3.1%, p = 0.001). Mitochondrial superoxide dismutase, mitochondrial and cytosolic glutathione reductase activity, and cytosolic reduced glutathione were significantly lower in the cyanotic group than in the control group at end-ischemia (superoxide dismutase, 3.7 +/- 1.3 vs 5.9 +/- 1.5 units/mg protein, p = 0.012; mitochondrial glutathione reductase, 43.7 +/- 14.0 vs 71.0 +/- 30.3 munits/mg protein, p = 0.039; cytosolic glutathione reductase, 13.7 +/- 2.0 vs 23.2 +/- 4.2 munits/mg protein, p < 0.001; and reduced glutathione, 0.69 +/- 0.10 vs 0.91 +/- 0.24 microgram/mg protein, p = 0.037). CONCLUSIONS: Cyanosis impairs postischemic functional recovery and depresses myocardial antioxidant reserve during ischemia. Reduced antioxidant reserve at end-ischemia may result in impaired postischemic functional recovery of cyanotic myocardium.     

Ischemia/reperfusion injury in flow-through venous flaps

This study was designed to test the activity and feasibility of 5'-deoxy-5-fluorouridine (5'-DFUR) and cisplatin combination therapy in the treatment of advanced gastric cancer. Nineteen patients with inoperable and/or metastatic gastric cancer, which was histologically proven, were orally administered 5'-DFUR 1,200 mg/m2 on days 1 to 4 and days 15-18 combined with 70 mg/m2 of cisplatin being repeated every 4 weeks. Five partial responses (PRs) were achieved. Seven patients had stable disease and 6 progressed on therapy. The overall response rate was 27.7% (95% confidence interval: 9.69% to 53.5%). The median survival duration of all 18 patients was 25 weeks (9-64). The majority of patients had WHO grade I/II toxicity, but there was no treatment-related death. These data support that the combinations of oral 5'-DFUR and cisplatin are well tolerable and have a moderate activity with low toxicity in the treatment of advanced gastric cancer.     

Role of endothelins and nitric oxide in hepatic reperfusion injury in the rat

In epithelial cells, alpha-, beta-, and gamma-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. alpha-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell-matrix and cell-cell contacts, alpha-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell-cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and alpha-catenin. We show that alpha-catenin colocalizes at cell-cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to alpha-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The Kd of the complex was determined to 2-4 x 10(-7) M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of alpha-catenin is involved in this interaction. Complex formation of vinculin and alpha-catenin was challenged in transfected cells. In PtK2 cells, intact alpha-catenin and alpha-catenin1-670, harboring the beta-catenin- binding site, were directed to cell-cell contacts. In contrast, alpha-catenin697-906 fragments were recruited to cell-cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo alpha-catenin, like vinculin, is tightly regulated in its ligand binding activity.     

Tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs

The effects of both recombinant rat tumor necrosis factor-alpha (TNF-alpha) and an anti-TNF-alpha antibody were studied in isolated buffer-perfused rat lungs subjected to either 45 min of nonventilated [ischemia-reperfusion (I/R)] or air-ventilated (V/R) ischemia followed by 90 min of reperfusion and ventilation. In the I/R group, the vascular permeability, as measured by the filtration coefficient (Kfc), increased three- and fivefold above baseline after 30 and 90 min of reperfusion, respectively (P < 0.001). Over the same time intervals, the Kfc for the V/R group increased five- and tenfold above baseline values, respectively (P < 0.001). TNF-alpha measured in the perfusates of both ischemic models significantly increased after 30 min of reperfusion. Recombinant rat TNF-alpha (50,000 U), placed into perfusate after baseline measurements, produced no measurable change in microvascular permeability in control lungs perfused over the same time period (135 min), but I/R injury was significantly enhanced in the presence of TNF-alpha. An anti-TNF-alpha antibody (10 mg/rat) injected intraperitoneally into rats 2 h before the lung was isolated prevented the microvascular damage in lungs exposed to both I/R and V/R (P < 0.001). These results indicate that TNF-alpha is an essential component at the cascade of events that cause lung endothelial injury in short-term I/R and V/R models of lung ischemia.