[Gaseous oxygen for protection and conditioning of organs during ischemia]. / Gasförmiger Sauerstoff zur Protektion und Konditionierung von Organen in Ischämie.

During organ ischemia, oxygen (O2) is the first "substrate", which is depleted. However, during ischemic storage in hypothermia (0-4 degrees C), a sufficient oxygenation is attainable by means of gaseous O2. The results of organ preservation were (mostly) better than those obtained with other methods at the respective times. O2 can be supplied via organ surfaces: Applying high O2-pressures (3040-15,200 hPa), ileum and lungs or hearts had some functions after 48 and 72 h storage, respectively; life-supporting functions regained kidneys and pancreas after 48 and 22 h storage, respectively. At normobaric conditions, intestine supplied with O2 via its lumen had during ischemic storage an aerobic metabolism and a better post-ischemic function. Using the "two-layer-method" (TLM), pancreas was stored for 96 h and after 90 min anaerobic warm ischemia (aWI) for 48 h with life-supporting functions after transplantation (Tx). Ischemic organs can be persufflated normobarically with gaseous O2 via their vessels. Hearts, skeletal muscles and kidneys in normothermia or frogs' spinal cords-remained viable for many hours. In hypothermia, kidneys damaged by 30 or 60 min aWI could be preserved for 48 and 24 h, respectively, with life-supporting functions after Tx. Hearts subjected to several hours of aerobic ischemia performed post-ischemically better. Livers aerobically stored for 48 h, or for 24 or 4 h after 30 or 60 min aWI, respectively, exhibited greatly improved post-ischemic functions. After 60 min aWI and 2 h persufflation for reconditioning, livers could be stored for another 22 h period of anaerobic ischemia. With normobaric O2-persufflation or TLM during ischemia, energy supply in form of ATP and its demand-meeting utilisation during hypothermia are apparently guaranteed, so that even longer periods of ischemia for Tx-related measures can be overcome. Not only the maintenance of cell and organ integrity or of cellular functions, but also the repair of damaged structures and functions have become possible with less expenditures and risks than with perfusion. The composition of the solutions for preservation or reconditioning of the ischemic organs is pivotal.

No beneficial effects of taurine application on oxygen free radical production after hemorrhagic shock in rats.

Oxygen free radical generation contributes to the reinfusion damage after hemorrhagic shock. Taurine has been proposed to have radical scavenging properties under certain experimental conditions. Therefore the present study was undertaken to investigate if taurine would be able to attenuate adverse effects of shock/resuscitation in male rats (fasted over night). Under pentobarbital anesthesia, hemorrhagic shock (HS) was induced for 1 h by bleeding of the animal [mean arterial blood pressure (MAP) = 40 mm Hg] followed by shed blood reinfusion and another 1 h period of resuscitation. Rats were divided into two groups: Treated rats (n = 6) were injected with taurine (40 mg/kg body mass) prior to withdrawal of shed blood; untreated rats (n = 9) received respective volumes of a normal saline solution. In untreated animals, free radical induced lipid peroxidation was documented by an increase of malondialdehyde (MDA) in the systemic circulation (nmol/ml; HPLC measurement) from 1.06 +/- 0.08 during normotension (NT) to 1.35+/- 0.18** 1 h after resuscitation (RS). Accordingly, plasma levels of alanine aminotransferase (ALT) (11 +/- 2; 35 +/- 12; 94 +/- 44 U/l, NT; HS; RS) and ammonia (120 +/- 39; 532 +/- 161; 224 +/- 101 micrograms/dl) changed significantly during the experimental protocol. Hepatic ATPase-content as an indicator of energetic status of the liver fell from 4.8 +/- 0.83 to 0.56 +/- 0.27 after HS and recovered to only 2.7 +/- 1.6 mumol/g after RS. Leukocyte infiltration of the liver was followed by tissue levels of myeloperoxidase (MPO) which did not change during HS, but rose during RS (37.9 +/- 18.5; 38.6 +/- 16.4; 77.5 +/- 24; arbitrary units), documenting an inflammatory reaction after HS. Taurine treated rats showed levels of MDA not different from untreated rats after RS; also no differences were observed concerning enzyme concentrations and ammonia levels. The liver tissue levels of ATP and MPO revealed no differences between the two groups during the various periods of the experiment. Liver tissue perfusion, as measured by Laser Doppler flowmetry, also did not show significant differences between both groups. MAP was significantly higher in the taurine-treated rats during the first 40 min of resuscitation. It is concluded that even a relatively high dose of taurine failed to attenuate the impact of oxygen free radicals and did not improve the recovery of the rats during the early resuscitation period.

Microscopic analysis of NADH fluorescence during aerobic and anaerobic liver preservation conditions: A noninvasive technique for assessment of hepatic metabolism.

Gaseous insufflation of oxygen via the venous vascular system is thought to be an useful tool for preventing anoxic tissue injury during extended time periods of ischemic preservation and for allowing for an improved recovery of organ function after transplantation. The present study aimed at the application of a noninvasive technique for monitoring effectiveness and homogeneity of gaseous areation by using an epiillumination microscopic technique for assessment of tissue nicotinamide adenine dinucleotide (NADH) fluorescence. Rat livers were flushed with and stored in University of Wisconsin solution at 4 degrees C for 48 h (n = 20). In half of the experiments (n = 10) gaseous oxygen was applied subsequent to organ harvest. Using ultraviolet-excitation high-resolution microscopy and computer-assisted image analysis liver surfaces were scanned for NADH intensity and spatial heterogeneity at 1, 24, and 48 h preservation time. Livers simply stored without aeration served as controls (n = 10). NADH intensity data were compared with corresponding data of tissue adenosine triphosphate (ATP) concentrations determined enzymatically. NADH fluorescence already differed at 1 h preservation between the two groups with significantly lower values in the aerobically stored livers. NADH fluorescence further decreased between 1 and 24 h preservation and remained low until 48 h, whereas in the anaerobically stored livers NADH fluorescence was found to be constantly high over the entire observation period. Aerobic storage resulted in rather homogeneous tissue oxygenation with an intrahepatic variation of NADH fluorescence <20%. In parallel, oxygen persufflation appropriately restored tissue ATP content within 1 to 24 h of preservation, while the simply stored livers exhibited pronounced depletion of ATP. We demonstrate for the first time that by means of retrograde gaseous oxygenation, ischemic livers can be readily and effectively oxygenated. Our study further indicates that the noninvasive microscopic analysis of tissue NADH fluorescence may be an useful tool for estimating efficiency of strategies in organ preservation.

Successful transplantation of porcine livers after warm ischemic insult in situ and cold preservation including postconditioning with gaseous oxygen.

BACKGROUND: In order to reduce the shortage of viable donor livers for organ transplantation, a method has been developed that allows even predamaged livers from nonheartbeating donors to be used as transplantable organs. METHODS: Porcine livers were harvested 45 min after cardiac arrest of the nonheparinized donor, preflushed with heparinized saline solution, and subsequently rinsed with University of Wisconsin solution, to which superoxide dismutase was added as an oxygen free radical scavenger. Thereafter, the livers were persufflated with gaseous oxygen via the venous vascular system while immersed in University of Wisconsin solution at 4 degrees C for 4 to 5 hr. RESULTS: After orthotopic transplantation, all of the livers conditioned with gaseous oxygen were able to support life-sustaining function during the whole observation period of 7 days post transplantation, while no survival of the recipients could be obtained without the described treatment. CONCLUSIONS: The present study establishes a new perspective for the use of ischemically altered livers from nonheartbeating donors for organ transplantation under clinical circumstances.

Cellular signal level of cyclic AMP and functional integrity of the small bowel after ischemic preservation: an experimental pilot study in the rat.

The intestinal mucosa is one the tissues most sensitive to ischemia. Anoxia of the gut is known to result in an early impairment of cellular permeability and transcapillary barrier function upon reperfusion. In vitro, an increased permeability of endothelial cell monolayers could be shown to be related to a decrease in cellular content of cyclic AMP (cAMP). Thus, the present study was aimed at investigating the role of the cellular cAMP second messenger signal in the context of intestinal ischemia/reperfusion injury after cold preservation. Segments of the upper jejunum were isolated from Wistar rats with vascular pedicle and flushed with 10 ml of UW preservation solution. The intestinal lumen was rinsed with 10-15 ml of UW solution and the organ was stored immersed in UW solution at 4 degrees C for 4 or 18 h. After 18 h of cold ischemic storage structural and functional integrity of the preparation was tested by perfusion via the vascular system with modified Krebs-Henseleit buffer and the intestinal lumen with saline solution (containing 200 mg % of galactose) for 30 min. In half of the experiments, dibutyryl-cAMP a membrane permeable cAMP analogue, was admixed to the flush solution (2 mM). It was found that tissue levels of cAMP linearily decreased to 34% during 18 h of ischemic preservation in UW. Addition of dibutyryl cAMP significantly improved postischemic recovery of the intestinal preparations by decreasing cellular loss of lactic dehydrogenase (18.2 +/- 4.6 vs. 7.6 +/- 2.6 U/I) and improving intestinal absorbtion of galactose from the luminal circuit (0.18 +/- 0.14 vs. 0.36 +/- 0.14 mg %) after 30 min of oxygenated reperfusion, but was not effective to reduce transcapillary water loss into the gut lumen. It is concluded that the anoxia-related decrease of the cellular cAMP level may represent a codeterminator influencing postischemic recovery of the small bowel and that the control of the cAMP signal of ischemic intestines might improve the quality of cold preservation of the gut prior to transplantation.

Cold preservation of the small intestine with the new Celsior-solution. First experimental results.

The aim of the present study was to evaluate the potential of Celsior, a recently developed cardioplegic and heart storage solution, to protect the small bowel during ischemic storage. Small bowel segments were isolated from rats, flushed with either UW or Celsior solution, and cold-stored for 18 h at 4 degrees C in the respective solution. After ischemic storage, some preparations were freeze-clamped for analysis of tissue metabolites while other preparations were tested for structural and functional integrity by isolated perfusion in vitro using a previously validated model. After 18 h of ischemic storage no significant differences were seen between Celsior and UW with regard to the development of edema, energy charge, or creatine phosphate, but lactate accumulation was significantly reduced in the Celsior group, although glucose catabolism was not inhibited. Histological evaluation of the cold-stored organs showed no differences with regard to structural integrity between the two groups. Total vascular resistance upon reperfusion was significantly lower in the Celsior group (666 +/- 126 vs 827 +/- 88 MPa s m-3*), as was the intestinal release of LDH (9.7 +/- 4.4 vs 18.2 +/- 4.6 U/l*). Carbohydrate absorption from the intestinal lumen amounted to venous effluent concentrations of 0.58 +/- 0.24 vs 0.18 +/- 0.15 mg% * of galactose in the Celsior and UW groups, respectively. Within the limits of this in vitro pilot study, Celsior provided better postischemic recovery of the small bowel than UW in terms of vascular perfusion characteristics, enzyme release, and carbohydrate absorption and may, thus, be considered a suitable alternative for intestinal organ preservation.

[Differential effect of preservative solutions (UW vs HTK) on mitochondrial redux status and energy metabolism during liver ischemia with oxygen persufflation]. / Differentieller Einfluss von Konservierungslösungen (UW vs HTK) auf mitochondrialen Redoxstatus und Energiestoffwechsel während Leberischämie unter Sauerstoffpersufflation.

Venous-systemic oxygen persufflation (VSOP) was performed in rat livers stored at 4 degrees C in either UW or HTK preservation solution. Since tissue anoxia is associated with a transformation of cellular NAD+ to NADH and the latter fluoresces upon UV-epiillumination, homogeneity and intensity of liver oxygenation could be analysed by intravital microscopic detection of NADH fluorescence. VSOP resulted in a significant decrease of the NADH signal, documenting effective tissue oxygenation in both UW and HTK. This effect was quite homogeneous (spatial variance < 15%). After 48 h of cold storage tissue levels of ATP (mumol/g dry weight) were increased upon VSOP in UW to 17.3 +/- 4.8 but only to 2.9 +/- 0.6 in HTK, while ATP amounted to less than 0.4 without VSOP in either of the groups. It is concluded that VSOP is an appropriate tool to prevent alterations of the hepatic redox status during ischemic preservation in UW and HTK. Metabolic preservation of energy-rich adenine nucleotides seems to be largely improved in combination with UW compared with HTK.

[Revitalizing donor livers after cardiovascular arrest with venous oxygen persufflation]. / Revitalisierung von Spenderlebern nach Kreislaufstillstand mittels venöser Sauerstoffpersufflation.

The experimental study investigates the resuscitation of livers procured from non heart beating donors by venous systemic oxygen persufflation in a pig transplantation model. In group 1 livers were harvested from donor animals after 60 min of potassium induced cardiac arrest. Livers were then flushed and preserved in 4 degrees C cold University of Wisconsin solution for 4 hrs. Then liver transplantation was performed in recipient animals. Animals of group 2 received the same treatment, but during hypothermic storage we applied venous systemic oxygen gas persufflation (VSOP) via the supra-hepatic vena cava. Livers of group 3 were harvested without warm ischemia and without oxygen treatment during cold ischemic storage and were then transplanted. All recipient animals of group 2 and 3 survived the five day follow-up time. All recipient animals of group 1 died a few hours after liver reperfusion due to circulatory shock or liver failure. The liver quality of group 2 recipient animals was similar to group 3 livers, because serum transaminase levels and blood coagulation test showed no significantly different values between those groups. Our data suggest that livers procured from non heart beating donors after 1 hr of cardiac arrest can be resuscitated and successfully transplanted by therapeutic treatment with VSOP.

Effect of glutamine or glucagon-insulin enriched total parenteral nutrition on liver and gut in 70% hepatectomized rats with colon stenosis.

BACKGROUND: Malnutrition of enterocytes is believed to facilitate the breakdown of the intestinal mucosal barrier, furthering a translocation of enteric bacteria with subsequent severe infection, which has been described after extensive hepatectomy. Glutamine and glucagon insulin are said to attenuate the malnutrition of enterocytes. To determine whether this was true, the effects on the remnant liver and the gut of total parenteral nutrition supplemented by admixtures of glutamine and/or glucagon insulin were investigated in rats subjected to massive hepatectomy and transient intestinal stasis. STUDY DESIGN: Rats underwent a permanent cannulation of the superior caval vein without restraining their mobility, a 70% hepatectomy, and a 24 hour string-ligation stenosis of the colon. A standard total parenteral nutrition solution was infused without or with 2% glutamine and without or with glucagon-insulin supplementation, respectively. RESULTS: Glutamine and glucagon-insulin supplemented total parenteral nutrition increased ileal mucosal DNA concentrations during and after intestinal stasis. Glutamine or glucagon-insulin alone had less pronounced effects. In the liver, the combined supplementation resulted in reduced adenosine triphosphate concentrations and increased mitochondrial adenosine triphosphate synthesis as well as in an early increase in DNA concentrations. CONCLUSION: Glutamine and glucagon-insulin enriched total parenteral nutrition attenuates malnutrition of enterocytes after massive abdominal stress and promotes liver regeneration after extensive hepatectomy.

Biophysical aspects of liver aeration by vascular persufflation with gaseous oxygen.

BACKGROUND: Venous systemic oxygen persufflation of the liver (i.e., gaseous insufflation of oxygen via the venous vascular system) has proven to be an effective tool for preventing anoxic tissue injury during extended time periods of ischemic preservation. It also allows for an improved recovery of the persufflated organ after orthotopic transplantation. METHODS: Biophysical aspects of the persufflation technique with regard to persufflation pressure (9 mmHg versus 18 mmHg) and oxygen concentration (pure oxygen versus air) in the persufflation gas were investigated in rat livers, using epi-illumination microscopic detection of autofluorescence of NADH, which accumulates in anoxic tissue. RESULTS: We demonstrated that a low-pressure persufflation (9 mmHg) is as sufficient as a higher pressure persufflation (18 mmHg) in oxygenating the ischemic organ. Moreover, oxygenation of the liver was found to be complete and rather homogeneous upon the pure oxygen persufflation, irrespective of the insufflation pressure used. In contrast, insufflation of air instead of pure oxygen resulted in insufficient aeration of the liver, even at the higher persufflation pressure of 18 mmHg. CONCLUSIONS: Our results indicate that the oxygen concentration of the persufflation gas rather than the persufflation pressure is a determinant of successful tissue oxygenation during cold storage.

Hypothermic storage alone in lung preservation for transplantation: a metabolic, light microscopic, and functional analysis after 18 hours of preservation.

BACKGROUND: Current lung preservation consists of flushing of the donor organs, with successive hypothermic storage in an inflated state. Recently, hypothermic storage alone was reported to be superior in terms of functional recovery. This study was designed to investigate the metabolic, morphologic, and functional consequences of hypothermic storage alone, in experimental lung preservation. METHODS: Orthotopic left-sided lung transplantation was performed in pigs. Donor lungs were flushed with Euro-Collins solution (n=6) or simply explanted (n=6) and stored for 18 hr at 4 degrees C. After this, left-sided single lung transplantation was performed. Sham-operated animals (n=6) served as control. Morphology and metabolism were analyzed in normal lungs, after ischemia and at the end of reperfusion. Gas exchange and pulmonary hemodynamics of the transplanted organs were measured, after exclusion of the native lung from perfusion and ventilation. RESULTS: Metabolic and morphologic evaluation did not show a significant difference between the groups at the end of ischemia. Lungs preserved by hypothermia alone showed a functional recovery close to sham-operated animals and superior to flushed organs. CONCLUSIONS: Hypothermia alone is a sufficient means of preservation for explanted lungs for at least 18 hr.

Reduction of proteolysis by venous-systemic oxygen persufflation during rat liver preservation and improved functional outcome after transplantation.

An increase of cytosolic proteolytic activity during ischemic preservation and consecutive tissue degradation have recently been recognized as a major pathogenetic factor for liver injury during ischemia/reperfusion. In the present study, we propose a method for preventing proteolytic tissue disintegration, which results in improved recovery of the liver after transplantation. Livers were harvested from rats and stored for 24 hr at 4 degrees C in University of Wisconsin solution (group A). Others were additionally persufflated with gaseous oxygen via the inferior caval vein during this time (group B). At the end of ischemic preservation, proteolysis was confirmed in group A, with significantly elevated tissue levels of free alanine and free amino groups, whereas proteolysis was prevented in group B. After transplantation, the integrity of the graft was significantly improved in group B, in which there was a 50% reduction of plasma activities of alanine amino-transferase and a twofold increase in hepatic bile production after the onset of reperfusion, as compared with group A. Moreover, venous-systemic oxygen persufflation during cold preservation significantly attenuated the rise in plasma levels of malondialdehyde (MDA) after liver transplantation. In conclusion, venous-systemic oxygen persufflation during ischemic storage prevents tissue proteolysis and reduces parenchymal injury after transplantation in vivo; this technique may, thus, represent a useful adjunct in long-term liver preservation with University of Wisconsin solution.

Assessment of intestinal integrity after ischemic preservation by luminal and vascular perfusion in vitro.

BACKGROUND: In the present study a technique for isolated perfusion of rat intestines in vitro should be tested as an evaluative tool in the assessment of intestinal alterations related to ischemia and reoxygenation. METHODS: Segments of upper jejunum (15 cm) were isolated from Wistar rats with vascular pedicle (superior mesenteric artery, SMA and portal vein). The SMA was cannulated with polyethylene tubing and flushed with 10 ml of University of Wisconsin (UW) preservation solution. The intestinal lumen was rinsed with 10-15 ml of UW solution and the organ was stored immersed in UW solution at 4 degrees C for 4 or 18 h. After cold ischemic storage structural and functional integrity of the preparation was tested by biluminal perfusion with artificial buffer via SMA (5 ml/min modified Krebs-Henseleit buffer, 200 mg% glucose, 5% dextran 78, 0.06 mg% dexamethasone, 7 mg% atropine to counteract paralytic hypersecretion) and the intestinal lumen (0.5 ml/min NaCl 0.9% with 200 mg% of galactose). The in vitro model was validated by perfusion of control preparations harvested without ischemic alteration. It was seen that ischemic preservation of 4 h had only a minor impact on the recovery of cellular ATP content and enzyme release (LDH) upon reperfusion, whereas both parameters were significantly changed after 18 h of preservation. Functional parameters like transmucosal carbohydrate absorption and luminal water balance, however, were significantly impaired already after 4 h of ischemic storage of the gut, thus yielding sensitive criteria for the appreciation of the postischemic integrity of the gut. CONCLUSIONS: It is concluded that the isolated gut preparation, being an inexpensive and technically feasible model, may be a useful tool in experimental research of intestinal ischemia/reperfusion.

Resuscitation of cadaveric livers from non-heart-beating donors after warm ischemic insult: a novel technique tested in the rat.

Clinical liver transplantation has become the therapy of choice in end-stage liver disease, but the limited availability of suitable donor organs still impedes its widespread application. In order to increase the availability of donor organs for liver transplantation, it would be advantageous if ischemically damaged livers could be resuscitated from cadavers in which the heart has stopped beating. A method for doing this has been developed in a rat model. Compared to livers excised from rats in which the heart is still beating, severe deteriorations of tissue integrity and functional performance were evident in predamaged livers after cold preservation without supplementary treatment. A treatment of those livers which included an antioxidant rinse with superoxide dismutase, and venous vascular insufflation of gaseous oxygen during preservation, completely prevented tissue alterations upon reperfusion, and promoted a functional recovery of the livers, making them comparable to organs harvested from heart-beating donors.