Initial Reperfusion Period Research Articles

Влияние пероксиредоксина 6 на нефроны в ранний реперфузионный период после ренальной ишемии у крыс

Renal reperfusion injury develops rapidly after ischemia relief. It is with the onset of reperfusion that a cascade of pathological processes is launched, which means that measures to protect the kidney have to be taken as early as in this period. The aim of the paper was to study the effect of exogenous peroxiredoxin 6 (Prx6) on the morphofunctional state of nephrons in the initial reperfusion period following ischemia. Materials and methods. The right kidney of rats was subjected to 45-minute ischemia with prior left-sided nephrectomy and examined after 2, 5 and 24 hours of reperfusion. Exogenous Prx6 was administered intravenously 15 minutes before ischemia. Results. The research showed that nephrectomy has no effect on the morphology of a single kidney, but leads to an increase in urea and creatinine in the blood within 24 hours. We noted signs of morphological and functional damage to nephrons after 2 hours of reperfusion, which tend to increase in the course of 24 hours. In addition, we observed a rise in blood creatinine and urea concentrations, an increase in the areas of renal corpuscles, glomeruli, and Bowman’s capsule, as well as dystrophic changes in nephrocytes and an increase in the immunosignal area of the kidney injury molecule-1 (KIM-1). When exogenous Prx6 was used, we observed normalization of the size of nephron components, a decrease in KIM-1 immunosignal and an improvement in the kidney’s excretory function both in the early reperfusion period and after 24 hours. Thus, exogenous Prx6 reduces damage to nephrons during the early reperfusion period, which improves their compensatory and adaptive properties in ischemia-reperfusion injury.

Lung thermography during the initial reperfusion period to assess pulmonary function in cellular ex vivo lung perfusion.

Thermography is a noninvasive technology to detect low temperatures in poorly circulated areas. In ex vivo lung perfusion (EVLP), lungs are rewarmed to body temperature during the initial 1h. Currently, the effect of graft thermal changes during the rewarming phase on pulmonary function is unknown. In this study, we evaluated the correlation of lung surface temperature with physiological parameters, wet/dry ratio, and transplant suitability in Lund-type EVLP. Fifteen pigs were divided into three groups: control group (no warm ischemia) or donation after circulatory death groups with 60 or 90min of warm ischemia (n= 5, each). Thermal images of the lower lobes were continuously collected from the bottom of an organ chamber using infrared thermography throughout EVLP. At 8min, lung surface temperatures of nonsuitable cases were significantly lower than in suitable cases (25.1±0.6 vs. 27.8±1.2°C, p<0.001), while there was no difference in lung surface temperatures between the two groups at 0-4min and 12-120min. There was a significant negative correlation between lung surface temperatures at 8min and wet/dry ratio at 2h in the lower lobes (R =-0.769, p<0.001, cutoff=26°C, area under the curve=1.0). A lung surface temperature of <26°C was significantly correlated with poor pulmonary function and transplant nonsuitability. A lung surface temperature of ≥26°C at 8min is a good early predictor of transplant suitability in cellular EVLP and might be applicable in clinical EVLP.

Inhalation of carbon monoxide reduces skeletal muscle injury after hind limb ischemia-reperfusion injury in mice

BackgroundThe purpose of this study was to determine if inhaled carbon monoxide (CO) can ameliorate skeletal muscle injury, modulate endogenous heme oxygenase-1 expression, and improve indexes of tissue integrity and inflammation after hind limb ischemia reperfusion. MethodsC57BL6 mice inhaling CO (250 ppm) or room air were subjected to 1.5 hours of ischemia followed by limb reperfusion for either 3 or 6 hours (total treatment time, 4.5 or 7.5 h). After the initial period of reperfusion, all mice breathed only room air until 24 hours after the onset of ischemia. Mice were killed at either the end of CO treatment or at 24 hours' reperfusion. Skeletal muscle was subjected to histologic and biochemical analysis. ResultsCO treatment for 7.5 hours protected skeletal muscle from histologic and structural evidence of skeletal muscle injury. Serum and tissue cytokines were reduced significantly (P < .05) in mice treated with CO for 7.5 hours. Tubulin, heme oxygenase, and adenosine triphosphate levels were higher in CO-treated mice. ConclusionsInhaled CO protected muscle from structural injury and energy depletion after ischemia reperfusion.

A Biphasic Response to Nitric Oxide Donation in an Ex Vivo Model of Donation After Cardiac Death Renal Transplantation

Donation after cardiac death (DCD) donors are vital to maximize the organ donor pool. Reperfusion injury (RI) is an important sequela in DCD organs due to warm and cold ischemia. RI manifests clinically as a high incidence of delayed graft function (DGF) and primary non-function (PNF) compared with donation after brain death organs. The importance of nitric oxide (NO) in the generation of reperfusion injury is pivotal. Using an exvivo porcine model of kidney transplantation the effects of reperfusion with and without NO supplementation on initial renal blood flow and function were compared. Real-time hemodynamic measurements were recorded and biochemical samples taken at set time-points. Molecular markers of reperfusion injury were also measured. Sodium nitroprusside was chosen as the NO donor. Results showed that NO donation initially improved renal blood flow significantly over controls; at the end of reperfusion this benefit was lost. In addition, there was an improvement in creatinine clearance, fractional excretion of sodium and renal oxygen consumption. There were observed to be higher levels of urinary nitrite/nitrate excretion, but no difference in isoprostane levels. This study represents a good model for the initial reperfusion period in large animal renal transplantation. The improvement in renal blood flow observed in the NO supplemented groups represents NO mediated vasodilatation. Later in reperfusion, accumulation of nitrogenous free radicals impairs renal blood flow. Clinically, NO supplementation during initial reperfusion of DCD kidneys improves renal blood flow but should be considered with caution due to potential deleterious effects of nitrogenous compound accumulation.

Effect of postconditioning with electrical stimulation of vagus never on myocardial ischemia reperfusion injury in rats

Objective To evaluate the effect of postconditioning with electrical stimulation of vagus never on myocardial ischemia reperfusion injury in rats. Methods Forty male Sprague-Dawley rats aged 8 weeks and weighing 250 g-350 g were randomly allocated into four groups (n=10 in each group):sham group (S group), ischemia reperfusion group (IR group), ischemia preconditioning group( IPC group)and postconditioning with electrical stimulation of vagus never group( POES group). The myocardial ischemia reperfusion model was preparated by ligation of left anterior descending coronary artery (LAD) for 30 min followed by 120 min reperfusion. During process of ischemia and reperfusion, HR and MAP were recorded and the rate pressure product (RPP) at each measuring point was calculated as the index of myocardial oxygen consumption. Arrhythmia was recorded during ischemia and initial period of reperfusion. At the end of reperfusion, blood samples were collected to assay serum concentrations of CK-MB, and cTnI. At the end of experiment, the infarct sizes was assessed from excised hearts by Evans blue and triphenyltetrazolium chloride (TTC) staining. Results The infarct size (IS%) was (72±9)% in IR group, (36±13)% in IPC group and (47±12)% in POES group, respectively, and serum concentration of cTnI was(0.99±0.14) μg/L in IR group, (0.37±0.08) μg/L in IPC group and (0.40±0.08) μg/L in POES group. Serum concentration of CK-MB was(110±13) μg/L in IR group, (38±8) μg/L in IPC group and(42±7)μg/L in POES group. Compared to the IR group, the IS% and serum concentrations of cTnI and CK-MB in IPC and POES groups were significantly reduced, rate of ventricular arrhythmia during initial period of reperfusion in the IPC and POES groups were also significantly reduced. Compared to IPC group, the IS% was significantly increased in the POES group. Conclusion Postconditioning with electrical stimulation of vagus never can attenuate myocardial ischemia reperfusion injury in rats. However, cardioprotection provided by postconditioning with electrical stimulation of vagus never is weaker than that by ischemia preconditioning. Key words: Ischemia reperfusion injury; Ischemia preconditioning; Postconditioning; Electric stimulation of vagus never

Neuroprotective effects of magnesium-sulfate on ischemic injury mediated by modulating the release of glutamate and reduced of hyperreperfusion

This study examined the neuroprotective effects of magnesium-sulfate (MgSO4) on the cerebral blood flow (CBF) and extracellular glutamate concentration in an eleven vessel occlusion (11VO) rat model. Twenty-one male Sprague–Dawley rats (250–350g) were used for the 11VO ischemic model, which was induced by a 10-min transient occlusion. The animals were divided into 3 groups, including ischemic-induced animals (ischemia group), ischemic-induced and MgSO4 treated animals (MgSO4 group), and sham animals for comparison. The real-time extracellular glutamate concentration was measured using a microdialysis biosensor, and the CBF was monitored by laser Doppler flowmetry. Neuronal cell death in the hippocampal region was observed 72h after ischemia by several stains (Nissl, DAPI, NeuN, and cleaved caspase3). A significant decrease in %CBF was observed in both the ischemia and MgSO4 groups, such as ~10% during the ischemic period. However, the MgSO4 group showed a significant decrease in the initial reperfusion %CBF compared to the ischemia group. A significantly lower level of glutamate release was observed in the MgSO4 group than in the ischemia group during the ischemic and reperfusion episode. Our staining results revealed a significant decrease in neuronal cell death in the hippocampus in the MgSO4 group compared to the ischemia group. These results suggest that MgSO4 is responsible for the protection of neuronal cells by suppressing the release of extracellular glutamate under ischemic conditions and the CBF response during the initial reperfusion period.

Leukocyte and Platelet Depletion Improves Blood Flow and Function in a Renal Transplant Model

Donation after cardiac death (DCD) donors are an important source of organs for transplantation. Due to warm and cold ischemic injury, DCD kidneys undergo a significant reperfusion insult when transplanted. This is manifested clinically as a high incidence of delayed graft function (DGF) and primary non-function (PNF). The importance of leukocytes in the generation of reperfusion injury is pivotal. Using an ex vivo porcine model of kidney transplantation, the effects of reperfusion with leukocyte and platelet depleted blood (LDB) and whole blood (WB) on renal blood flow and function were compared. Hemodynamic measurements were recorded, and biochemical, hematological, and histologic samples taken at set time-points. Reperfusion with LDB improved renal blood flow significantly compared with WB reperfusion. In addition, there was a significant improvement in creatinine clearance and renal oxygen consumption, but not fractional excretion of sodium, acid-base homeostasis, urinary nitric oxide (NO), or 8-isoprostane levels. This study represents a good model for the initial reperfusion period in renal transplantation. Improvement in only some functional markers and neither urinary NO nor 8-isoprostane levels indicates that improved blood flow alone is not sufficient to reverse the severe ischemic insult endured by DCD kidneys.

The key of ischemia postconditioning：delayed transient acidosis during initial period of reperfnsion

Ischemia posteonditioning is recently introduced as a new strategy to attenuate isehemiaJreperfusion injury, which is conducted several short cycles of reperfusion-reoeelusion before subsequent reperfusion. It is recently presumed that the key of isehemia postconditioning is to attenuate myocardial ischemia-reperfusion injury by producing a delayed transient acidosis during initial period of reperfusion. Key words: Ischemic postconditioning ; Acidosis ; Myocardial protection

Basic Control of Reperfusion Effectively Protects Against Reperfusion Injury in a Realistic Rodent Model of Acute Limb Ischemia

Reperfusion injury is insufficiently addressed in current clinical management of acute limb ischemia. Controlled reperfusion carries an enormous clinical potential and was tested in a new reality-driven rodent model. Acute hind-limb ischemia was induced in Wistar rats and maintained for 4 hours. Unlike previous tourniquets models, femoral vessels were surgically prepared to facilitate controlled reperfusion and to prevent venous stasis. Rats were randomized into an experimental group (n=7), in which limbs were selectively perfused with a cooled isotone heparin solution at a limited flow rate before blood flow was restored, and a conventional group (n=7; uncontrolled blood reperfusion). Rats were killed 4 hours after blood reperfusion. Nonischemic limbs served as controls. Ischemia/reperfusion injury was significant in both groups; total wet-to-dry ratio was 159+/-44% of normal (P=0.016), whereas muscle viability and contraction force were reduced to 65+/-13% (P=0.016) and 45+/-34% (P=0.045), respectively. Controlled reperfusion, however, attenuated reperfusion injury significantly. Tissue edema was less pronounced (132+/-16% versus 185+/-42%; P=0.011) and muscle viability (74+/-11% versus 57+/-9%; P=0.004) and contraction force (68+/-40% versus 26+/-7%; P=0.045) were better preserved than after uncontrolled reperfusion. Moreover, subsequent blood circulation as assessed by laser Doppler recovered completely after controlled reperfusion but stayed durably impaired after uncontrolled reperfusion (P=0.027). Reperfusion injury was significantly alleviated by basic modifications of the initial reperfusion period in a new in vivo model of acute limb ischemia. With this model, systematic optimizations of according protocols may eventually translate into improved clinical management of acute limb ischemia.

Diastolic Ca2+ overload caused by Na+/Ca2+ exchanger during the first minutes of reperfusion results in continued myocardial stunning

The pathogenesis of myocardial stunning caused by brief ischemia and reperfusion remains unclear. The aim of the present study was to investigate the underlying mechanism of myocardial stunning. An isolated cell model of myocardial stunning was firstly established in isolated rat ventricular myocytes exposed to 8 min of simulated ischemia and 30 min of reperfusion, the cardiomyocyte contractile function was used to evaluate myocardial stunning. A diastolic Ca(2+) overload without significant changes in systolic Ca(2+) and the amplitude of Ca(2+) transient during the first 10 min of reperfusion played an important role in the occurrence of myocardial stunning. Decreasing Ca(2+) entry into myocardial cells with low Ca(2+) reperfusion was a very efficient way to prevent myocardial stunning. Diastolic Ca(2+) overload was closely related to the reverse mode of Na(+)/Ca(2+) exchanger (NCX) rather than L-type Ca(2+) channel. The activity of the reverse mode of NCX was found significantly higher at the initial time of reperfusion, and KB-R7943, a selective inhibitor of the reverse mode of NCX, administered at first 10 min of reperfusion rather than at the time of ischemia significantly attenuated myocardial stunning. In addition, NCX inhibition also attenuated the Ca(2+) oscillation and cardiac dysfunction when field stimulus was stopped at first 10 min of reperfusion. These data suggest that one of the important mechanisms of triggering myocardial stunning is diastolic Ca(2+) overload caused by activation of the reverse mode of NCX of cardiomyocytes during the initial period of reperfusion following brief ischemia.

Oxidant Stress during Simulated Ischemia Primes Cardiomyocytes for Cell Death during Reperfusion

Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.

Necrosis and apoptosis: Sequence of liver damage following reperfusion after 60 min ischemia in rats

This study evaluated the time-dependent modes of cell death that occur during the course of reperfusion after 60 min ischemia. The serum ALT level increased immediately after reperfusion, peaked at 6 h and then declined gradually thereafter. This was supported by the H&E staining of the liver tissues taken at 2 h reperfusion, which revealed massive peri-portal necrosis. The succinate driven mitochondrial-swelling rate, release of cytochrome c into the cytoplasm, increase in caspase-3 activity and TUNEL stained tissue were measured to determine the changes in the biochemical markers of apoptosis. The biochemical markers of apoptosis increased by 2 h of reperfusion, peaked at 6 h and remained elevated throughout the 24 h reperfusion period. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), inhibited MPT opening, the release of cytochrome c and caspase-3 activation. This indicates that necrotic death occurs particularly in the peri-portal region in the initial period of reperfusion, and delayed apoptotic death occurs primarily in the peri-central region in the liver tissues undergoing I/R.

Adenosine A 2A receptor activation reduces inflammation and preserves pulmonary function in an in vivo model of lung transplantation

Reperfusion injury continues to significantly affect patients undergoing lung transplantation. Isolated lung models have demonstrated that adenosine A 2A receptor activation preserves function while decreasing inflammation. We hypothesized that adenosine A 2A receptor activation by ATL-146e during the initial reperfusion period preserves pulmonary function and attenuates inflammation in a porcine model of lung transplantation. Mature pig lungs preserved with Viaspan (Barr Laboratories, Pomona, NY) underwent 6 hours of cold ischemia before transplantation and 4 hours of reperfusion. Animals were treated with (ATL group, n = 7) and without (IR group, n = 7) ATL-146e (0.05 microg kg -1 . min -1 ATL-146e administered intravenously for 3 hours). With occlusion of the opposite pulmonary artery, the animal was maintained for the final 30 minutes on the allograft alone. Recipient lung physiology was monitored before tissue evaluation of pulmonary edema (wet-to-dry weight ratio), myeloperoxidase assay, and tissue tumor necrosis factor alpha by means of enzyme-linked immunosorbent assay. When the ATL group was compared with the IR group, the ATL group had better partial pressure of carbon dioxide (43.8 +/- 4.1 vs 68.9 +/- 6.3 mm Hg, P < .01) and partial pressure of oxygen (272.3 +/- 132.7 vs 100.1 +/- 21.4 mm Hg, P < .01). ATL-146e-treated animals exhibited lower pulmonary artery pressures (33.6 +/- 2.1 vs 47.9 +/- 3.5 mm Hg, P < .01) and mean airway pressures (16.25 +/- 0.08 vs 16.64 +/- 0.15 mm Hg, P = .04). ATL-146e-treated lungs had lower wet-to-dry ratios (5.9 +/- 0.39 vs 7.3 +/- 0.38, P < .02), lower myeloperoxidase levels (2.9 x 10 -5 +/- 1.2 x 10 -5 vs 1.3 x 10 -4 +/- 4.0 x 10 -5 DeltaOD mg -1 . min -1 , P = .03), and a trend toward decreased lung tumor necrosis factor alpha levels (57 +/- 12 vs 96 +/- 15 pg/mL, P = .06). The ATL group demonstrated significantly less inflammation on histology. Adenosine A 2A activation during early reperfusion attenuated lung inflammation and preserved pulmonary function in this model of lung transplantation. ATL-146e and similar compounds could play a significant role in improving outcomes of pulmonary transplantation.

Superior mesenteric artery occlusion models shock-induced gut ischemia-reperfusion

Background Superior mesenteric artery occlusion (SMAO) is a simple and reproducible model of shock-induced gut ischemia/reperfusion, but some argue that it is not clinically relevant. The purpose of the current study was to compare SMAO to a standard model of controlled hemorrhage (CH) and uncontrolled hemorrhage (UH). Methods Rats had femoral lines and a jejunal mucosal laser Doppler placed followed by SMAO (60 min of ischemia, no resuscitation), controlled hemorrhage (40 mm Hg for 60 min, 2:1 resuscitation shed blood and lactated Ringers), or uncontrolled hemorrhage (liver injury, 3:1 resuscitation with lactated Ringers). Base deficit, lactate, and jejunal mucosal flow (as a percentage of baseline) were recorded during ischemia and for 120 min after reperfusion. Jejunal tissue was harvested for morphological evaluation. Comparison among groups was by analysis of variance (ANOVA), and significance was set at P < 0.05. Results Mucosal blood flow was similar among groups at the onset of reperfusion (CH, 16.9 ± 5.0% versus UH, 10.9 ± 3.1% versus SMAO, 13.9 ± 6.2%) and during the initial period of reperfusion. By 120 min, however, flow in CH (75.4 ± 2.5%) was significantly higher that in either UH (36.4 ± 13.1%) or SMAO (31.7 ± 8.4%). Histological injury was less with CH, while base deficit was significantly higher in CH at the onset of reperfusion (−24 ± 2 versus UH, −10 ± 3 and SMAO, −6 ± 3 mM/L) but comparable by the end (CH, −17 ± 4 versus UH, −16 ± 3 and SMAO, −17 ± 2 mM/L). Conclusion SMAO is a clinically relevant model of shock-induced gut ischemia/reperfusion.

Microvascular transfer-related abrogation of capillary flow motion in critically reperfused composite flaps

Capillary flow motion is defined as rhythmic fluctuations of blood flow in the capillaries. Although critical perfusion has been demonstrated to induce capillary flow motion, little is known about the role of capillary flow motion in microvascular free flaps. The aim of this study was to elucidate the tissue-confined incidence and consequence of capillary flow motion in microvascularly transferred composite flaps, using intravital fluorescence microscopy. In Wistar rats, transferred osteomyocutaneous flaps (n = 7), which were exposed to 1 h of ischaemia during the anastomotic procedure followed by 1 h of reperfusion, were subjected to critical perfusion by stepwise reduction of the femoral-artery blood flow to 0.15 ml min(-1), 0.10 ml min(-1) and 0.05 ml min(-1). Pedicled osteomyocutaneous flaps that were not subjected to ischaemia (n=8) served as controls. In pedicled flaps critical perfusion induced capillary flow motion in the muscle, but not in the skin, subcutis and periosteum. In these flaps, the functional capillary density was preserved in all tissues analysed, including the skeletal muscle. Additional sympathetic denervation of the pedicled flaps did not change the incidence or pattern of capillary flow motion. In contrast, after flap transfer capillary flow motion in muscle tissue did not occur during critical perfusion. As a consequence, a shutdown of perfusion of individual capillaries was observed, resulting in a significant reduction (P<0.05) in functional capillary density, not only in the subcutis, skin and periosteum but also in the muscle itself. Thus, our data suggest that the microcirculatory control of pedicled osteomyocutaneous flaps is preserved during critical perfusion by skeletal-muscle capillary flow motion, whereas this protective regulatory mechanism is lost during the initial reperfusion period after flap transfer, probably not because of denervation but because of surgery- and/or ischaemia-reperfusion-associated injury.

Effects of endothelium/leukocytes/platelet interaction on myocardial ischemia--reperfusion injury.

Platelet fibrinogen receptor (GPIIb/IIIa) antagonists clinically improve the effectiveness of thrombolysis or PTCA in treatment of acute myocardial infarction. 7E3Fab, the chimeric Fab fragment of a monoclonal GPIIb/IIIa antibody, reduces the incidence of death, reinfarction or restenosis in patients and may improve blood flow and regional wall motion in reperfused myocardium. Besides inhibition of platelet aggregation, 7E3Fab may block fibrinogen bridging between the polymorphonuclear neutrophil (PMN) adhesion molecule MAC-1 and platelet GP IIb/IIIa, thus attenuating interaction of platelets with PMN. Experimentally, the interaction of platelets with PMN exacerbated postischemic myocardial stunning. In our own studies in isolated guinea pig hearts, human PMN, platelets and fibrinogen where simultaneously infused during the initial reperfusion period after 15 min of global ischemia. FACS analysis of cells in the coronary effluant revealed that 7E3Fab reduced platelet GP IIb/IIIa expression to 10% of baseline. PMN-platelet aggregate formation in the coronary effluate was markedly reduced by 7E3Fab, parallel to a decrease of PMN-platelet aggregates found by in situ double fluorescence microscopy in the postischemic coronary vasculature. The inhibition of PMN-platelet aggregate formation by 7E3Fab treatment coincided with a significant improvement of external heart work, which suffered a 50% reduction after ischemia, reperfusion, and exposure to PMN, platelets and fibrinogen. Obviously, application of 7E3Fab inhibits formation and coronary retention of PMN-platelet aggregates in the postischemic, reperfused myocardium. This effect may contribute to the clinically observed beneficial effects of this adjuvant treatment after myocardial ischemia.

In vivo functional and MRS metabolic evaluation of the heart graft; application to improvements of cardiac preservation.

Previous MRS studies o11 heart preservation have been mostly conducted oil ex vivo preparations which allow easily concomitant 3~p MRS and functional evaluation. Despite its limitations due to improper evahtation of the contractility, the standard heterotopic transplantation in rat offers close similarity to clinical situations and provides in-vivo blood reperfusion. Therefore this experimental model allows to study the recovery of the heart late after the initial period of reperfusion as opposed to ex vivo perfusion models which are not suitable for delayed study due to the limited stability of the preparation. We aimed to develop a new experimental model in order (i) to study the evolution of function and phosphorous metabolism of the heart graft during prolonged blood reperfusion; and (ii) to evaluate in-vivo the role of L-arginine as an adjuvant to the Centre de R6sonance Magnetique Biologique et M6dicale (CRMBM) heart preservation solution after prolonged cardioplegic arrest.

Controlled reperfusion prevents pulmonary injury after 24 hours of lung preservation

Background. Posttransplantation lung reperfusion injury continues to be a major problem. We have shown that controlling the initial period of reperfusion limits this injury after 2 hours of warm lung ischemia. The effectiveness of this modality, however, is unknown after longer periods of cold ischemia, which more closely mimics the clinical situation. Methods. After baseline measurements, 10 pigs had the left lung flushed with a modified Euro-Collins solution, explanted, stored at 4°C for 24 hours, and transplanted into 10 other pigs. Five (group 1) underwent uncontrolled reperfusion created by removal of the vascular clamps after implantation of the new left lung, mimicking the clinical situation. The other five (group 2) underwent controlled reperfusion, which we performed by taking blood from the femoral artery, mixing it with a crystalloid solution (using a mixer heater) to make the blood hyperosmolar, alkalotic, and substrate-enriched, and pumping it through a leukocyte-depleting filter into the transplanted lung for 10 minutes at a pressure of 20 to 30 mm Hg before removing the pulmonary artery clamp. The right pulmonary artery and bronchus were then ligated, and left lung function was assessed each hour for 4 hours and compared with baseline. Results. Controlled reperfusion (group 2) minimized the reperfusion injury, preserving posttransplant pulmonary compliance (92% ± 1% versus 68% ± 1%; p < 0.001), reducing the rise in pulmonary vascular resistance (27% ± 2% versus 166% ± 3%; p < 0.001), improving oxygenation (P o 2, 425 ± 14 versus 82 ± 11 mm Hg; p < 0.001), and lowering myeloperoxidase activity (0.22 ± 0.02 versus 0.45 ± 0.02 ΔOD/mg protein per minute; p < 0.001) and tissue edema (83.0% ± 0.3% versus 84.9% ± 0.3%; p < 0.001) compared with uncontrolled reperfusion, which resulted in an injury so severe that 3 of 5 pigs died before the 4-hour measurements. Conclusions. After 24 hours of cold ischemia uncontrolled reperfusion results in a severe pulmonary reperfusion injury. This injury is almost completely avoided by controlling the composition (modified solution and white blood cell filter) and conditions (pressure) of the reperfusion. Because this experiment mimics the clinical situation, it suggests surgeons should begin to use this modality to limit reperfusion injury after lung transplantation.

Effects of Specific Sodium/Hydrogen Exchange Inhibitor During Cardioplegic Arrest

Background. The accumulation of intracellular sodium during myocardial ischemia couples an inappropriate calcium influx and depressed cardiac recovery during subsequent reperfusion. The effects of the selective sodium/hydrogen exchange inhibitor HOE 694 are evaluated during myocardial ischemia and reperfusion. Methods. Ten isolated rat hearts were subjected to a 2-minute infusion of St. Thomas’ cardioplegia ± 1 μmol/L HOE 694 followed by 50 minutes’ normothermic (37°C) global ischemia. Intracellular sodium accumulation was continuously measured using triple quantum filtered 23Na nuclear magnetic resonance spectroscopy without chemical shift reagents. Hemodynamic variables were assessed before and after ischemia. Results. The addition of 1 μmol/L HOE 694 to St. Thomas’ cardioplegic solution (n = 5) attenuated the accumulation of intracellular sodium after 50 minutes’ ischemia (160.5% ± 9.1% versus 203.4% ± 10.9% [mean ± standard error], HOE 694 versus control, respectively; p = 0.014) and after the initial reperfusion period (first 30 minutes) (288.7% ± 10.2% versus 335.9% ± 10.3%; p = 0.008). HOE 694–treated hearts showed significantly improved postischemic recovery of left ventricular developed pressure (53.5% ± 8.4% versus 26.4% ± 6.6%; p = 0.036) and rate–pressure product (40.2% ± 6.9% versus 13.2% ± 5%; p = 0.014). Postischemic recovery of coronary flow was not significantly different between the two groups (68.6% ± 5.9% versus 55.5% ± 4.6%, HOE 694 versus control, respectively; p = 0.11). Conclusions. The addition of 1 μmol/L HOE 694 to cardioplegic solution attenuates the increase of intracellular sodium during myocardial ischemia and early reperfusion. This is coupled with an improved recovery of contractile function, possibly as a result of decreased sodium and calcium overload of ischemic myocardium.

The response of heat shock proteins 25 and 72 to ischaemia in different kidney zones.

Induction of heat shock proteins (HSPs) following cell injury contributes to the protection of vital cell functions. It was, therefore, of interest to study the effects of transient renal ischaemia on the abundance and distribution of two HSPs, HSP25 and HSP72, in renal tissue using Western-blot techniques. Analyses were performed on the supernatant (HSP25, HSP72) and pellet (HSP25) of homogenates obtained from cortex (CX) and outer (OM) and inner (IM) medulla of the rat kidney immediately after 60 min of ischaemia followed by varying periods of reperfusion. Ischaemia of the left kidney caused HSP25 contents to decrease in CX, OM and IM by 73, 89 and 54% respectively, compared with the corresponding zones of the contralateral control kidney. This initial decrease in supernatant HSP25 was accompanied by an increased abundance of HSP25 in the pellet. Following reperfusion, HSP25 contents in the supernatant gradually increased in CX and OM, reaching, after 24 h, values that were 5.4- and 2.5-fold higher, respectively, than those in the control kidneys. After 7 or 14 days of reperfusion, HSP25 contents had not completely normalised in CX, but had reached control levels in OM. In IM, the HSP25 content remained below control throughout the entire reperfusion period. HSP72 (supernatant) was below the detection limit in the CX of the control kidney. Similar to the level of HSP25, that of HSP72 was also markedly lower in OM and IM immediately after ischaemia. The intrarenal distribution of HSP72 and the sequence of zonal changes in HSP72 contents were similar to those observed for HSP25. These results are compatible with the view that, during ischaemia and the initial reperfusion period, HSP25 migrates from the cytoplasmic compartment (supernatant) into the nucleus and/or associates with cytoskeletal structures. The observation that both HSP25 and HSP72 are transiently induced in CX and OM, but not in IM, may be explained by the fact that, while all kidney cells are exposed to ischaemic stress, only inner medullary cells experience a major postischaemic attenuation of osmotic stress.