Exogenous adenosine accelerates recovery of cardiac function and improves coronary flow after long-term hypothermic storage and transplantation.

Impaired coronary flow during postischemic reperfusion may limit functional recovery. In the present studies we used the heterotopically transplanted rat heart and the isolated working rat heart to assess whether adenosine, given during reperfusion, could improve either the rate or the extent of postischemic recovery. Hearts were arrested (2 minutes at 4 degrees C) with the St. Thomas' Hospital cardioplegic solution and stored by immersion in the same solution for 8 hours at 4 degrees C. Hearts were then transplanted into the abdomen of homozygous recipients. Immediately before reperfusion, adenosine (0.5 ml of a 1 mumol/L solution, equivalent to 0.13 micrograms) was injected into the left ventricle (control rats received an equivalent amount of saline). Hearts were reperfused in vivo for 30 minutes or 24 hours, after which they were excised and perfused (Langendorff) for 20 minutes for the assessment of function. They were then freeze clamped and taken for metabolic analysis. After 50 minutes of reperfusion, left ventricular developed pressure was 75 +/- 5 mm Hg (4 mm Hg end-diastolic pressure) in the adenosine group versus 61 +/- 4 mm Hg in the control group (p less than 0.05); however, after 24 hours function was identical in the two groups (52 +/- 4 versus 52 +/- 3 mm Hg). After 50 minutes of reperfusion coronary flow was greater in the adenosine group (11.0 +/- 0.4 versus 9.7 +/- 0.4 ml/min in control rats; p less than 0.05), a difference that was sustained for 24 hours (12.8 +/- 0.3 versus 11.4 +/- 0.4 ml/min in control rats; p less than 0.05). Adenosine triphosphate and creatine phosphate contents recovered to similar extents in control and adenosine groups after both 50 minutes and 24 hours of reperfusion. In further studies with an identical storage protocol (8 hours at 4 degrees C), hearts were not transplanted but were reperfused with crystalloid medium in the Langendorff mode for 15 minutes (creatine kinase leakage measured) and in the working mode for 180 minutes. In an attempt to mimic the heterotopic transplant protocol, adenosine (1 mumol/L) was included in the perfusion fluid for the first 2 minutes of reperfusion. Similar results to those of the transplant studies were obtained, with coronary flow being consistently improved in the adenosine group; however, this benefit was lost after only 2 hours of reperfusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heat shock improves recovery and provides protection against global ischemia after hypothermic storage

Background. Improved methods of donor heart preparation before preservation could allow for prolonged storage and permit remote procurement of these organs. Previous studies have shown that overexpression of heat-shock protein 72 provides protection against ischemic cardiac damage. We sought to determine whether rats subjected to heat stress with only 6-hour recovery could acquire protection to a subsequent heart storage for 12 hours at 4°C.

Methods. Three groups of animals (n = 10 each) were studied: control, sham-treated, and heat-shocked rats (whole-body hyperthermia 42°C for 15 minutes). After 12-hour cold ischemia hearts were reperfused on a Langendorff column. To confirm any differences in functional recovery, hearts were then subjected to an additional 15-minute period of warm global ischemia after which function and lactate dehydrogenase enzyme leakage were measured.

Results. Heat-shocked animals showed marked improvements compared with controls in left ventricular developed pressure (63 ± 4 mm Hg versus 44 ± 4 mm Hg, p < 0.05) heart rate × developed pressure (13,883 ± 1,174 beats per minute × mm Hg versus 8,492 ± 1,564 beats per minute × mm Hg, p < 0.05), rate of ventricular pressure increase (1,912 ± 112 mm Hg/second versus 1,215 ± 162 mm Hg/second, p < 0.005), rate of ventricular pressure decrease (1,258 ± 89 mm Hg/second versus 774 ± 106 mm Hg/second, p < 0.005). Diastolic compliance and lactate dehydrogenase release were improved in heat-shocked animals compared with controls and sham-treated animals. Differences between heat-shocked animals and control or sham-treated animals were further increased after the additional 15-minute period of warm ischemia. Western blot experiments confirmed increased heat-shock protein 72 levels in heat-shocked animals (> threefold) compared with sham-treated animals and controls.

Conclusions. Heat shock 6 hours before heart removal resulted in marked expression of heat-shock protein 72 and protected isolated rat hearts by increased functional recovery and decreased cellular necrosis after 12-hour cold ischemia in a protocol mimicking that of heart preservation for transplantation. Protection was further confirmed after an additional 15-minute period of warm ischemia.     

Improved recovery of cardiac function after hypothermic ischemic storage with ouabain

We investigated the hypothesis that ouabain would reduce energy expenditure in the hypothermic, ischemic heart by inhibiting membrane-bound sodium/potassium-activated adenosine triphosphatase and lead to improved function on reperfusion. Additionally, we compared ouabain with another potential adjunct, the calcium channel blocker verapamil. The isolated rabbit heart was used as a model, and three experimental groups were studied after 1, 6, 12, and 24 hours of 4 degrees C ischemia. Hearts in group I were stored in a standard high potassium solution; hearts in groups II and III were stored in the same solution supplemented with verapamil (2 mg/L) and ouabain (3 mg/L), respectively. After ischemia, all hearts were reperfused for 45 minutes on a modified Langendorff apparatus, and left ventricular function was measured before freeze-clamping the heart for metabolite determination. At 1 and 6 hours, hearts in all groups functioned well, but the group III hearts had higher levels of adenosine triphosphate, phosphocreatine, total adenine nucleotides, and glycogen. After 12 hours of ischemia, function was significantly better in group III hearts (p less than 0.01) compared with that of hearts in groups I and II. Group III hearts also exhibited higher levels of high energy phosphates and glycogen. After 24 hours of storage, all hearts functioned poorly, and there was a marked decline in measured metabolites. Although we could show no improvement with the addition of verapamil, ventricular function was improved after storage in a high potassium hypothermic solution containing ouabain. Because ouabain inhibits the hydrolysis of adenosine triphosphate by sodium/potassium-activated adenosine triphosphatase, this result suggests that the glycoside maintains energy-rich phosphates necessary for optimal resumption of cardiac function.     

Poloxamer 188 improves neurologic outcome after hypothermic circulatory arrest.

Poloxamer 188, an amphipathic copolymer with cytoprotective properties, was investigated as a means of improving neurologic outcome after a prolonged period (150 minutes) of deep hypothermic circulatory arrest. Dogs were perfusion cooled and surface cooled to 10 degrees C, the heart was arrested for 150 minutes, and then the dogs were rewarmed and weaned from bypass. Seven dogs were treated with poloxamer 188 before and after deep hypothermic circulatory arrest. Six control dogs were treated with saline. Surviving dogs were evaluated for 1 week after deep hypothermic circulatory arrest for neurologic deficits or behavioral changes. Neurologic outcome was graded by the following system: grade 1, death within the observation period; grade 2, comatose; grade 3, holds head up; grade 4, sits up; grade 5, stands; grade 6, normal in both behavior and gait. There were no deaths in the seven poloxamer 188-treated animals versus three deaths in the six control dogs. Poloxamer 188-treated dogs also manifested significantly less neurologic dysfunction after deep hypothermic circulatory arrest than did the control group (p less than 0.003). This study shows that poloxamer 188 has a significant impact in improving neurologic outcome after exceptionally long periods of deep hypothermic circulatory arrest.     

Recovery of glomerular and tubular function in autotransplanted dog kidneys preserved by hypothermic storage or machine perfusion. Relation of initial function to long-term function

Sixteen male dogs had split renal function studies prior to unilateral nephrectomy and autotransplant. Kidneys were preserved for 24 hr by either simple hypothermic storage in Collins C2 solution (SHS) or machine-perfused (MP) on a Waters machine (MOX 100) with plasmanate perfusate. Renal function studies were repeated at 1 hr and at 7, 14, and 28 days, and the statistical relationship between initial and 1-month function was determined for a number of parameters. All MP kidneys functioned immediately, whereas 1/3 of SHS kidneys had delayed function. Recovery was more rapid in MP kidneys and was essentially complete by 14 days, at which time MP kidneys had higher rates of creatinine clearance and sodium reabsorption. However, by one month 3/7 MP kidneys (P = 0.15 compared with SHS) had lower creatinine clearance rates than at 2 weeks, and para-aminohippurate (PAH) clearance and fractional sodium reabsorption were significantly decreased. During the same period SHS kidneys either showed continued improvement or maintained stable function. Thus, by one month there were no differences between the groups in clearances of creatinine and PAH, plasma creatinine and blood urea nitrogen concentrations, or fractional reabsorption of sodium, potassium, and water. For SHS kidneys, the 1-hr creatinine clearance and the absolute rate of sodium reabsorption were strong predictors of the eventual function of the kidneys at one month (r = 0.93 and r = .83, P less than 0.05, respectively). No such correlations were found in MP kidneys (r = less than .01, P greater than 0.9 for both variables). The data show that MP results in significantly better function early after transplant, but this advantage does not persist, and that SHS kidneys early function is a good predictor of long-term recovery, but this is not true for MP kidneys.     

Microperfusion techniques for long-term hypothermic preservation.

BACKGROUND: The aim of this study was to compare several methods of hypothermic heart preservation. METHODS: We preserved isolated pig hearts for 24 hours in cold cardioplegia (4 degrees C), using either continuous microperfusion (Group I) or simple storage (Group II), and with a new preservative solution (NPS, groups IA and IIA) vs St. Thomas' solution (groups IB and IIB). The main characteristics of the NPS include (1) prevention of cell swelling with polyethelene glycol (PEG), (2) low calcium and magnesium, and (3) presence of metabolic substrates, such as glucose, insulin, pyruvate, aspartate, alanyl-glutamine, and membrane stabilization compounds such as ethanol and chlorpromazine. RESULTS: The 4 above groups were compared with hearts harvested and immediately reperfused (control group). During preservation, only Group IB showed significant edema (40% +/- 8.4% water gain). Adenylate charge was 25% to 50% higher in microperfused Groups IA and IB (0.678 +/- 0.049 and 0.795 +/- 0.071, respectively) as compared with simple-storage groups IIA and IIB (0.605 +/- 0.048 and 0.524 +/- 0.160, respectively). Ultrastructural analysis showed that tissue injury occurred mainly in Group IIB (altered mitochondria, chromatin clumping). Functional data showed better recovery of NPS groups as compared with St. Thomas groups: coronary flow was identical in Group IB and control (57.8 +/- 22 and 56.6 +/- 14 ml/min/100 g, respectively), and in IA > IB (p < 0.001) and IIA > IIB (p < 0.01); the rate pressure products were higher in NPS groups compared with St. Thomas groups (IA > IB, p < 0.01); IIA > IIB, p < 0.05). CONCLUSIONS: The microperfusion method associated with the NPS provides excellent protection in long-term hypothermic heart preservation.     

Hypothermic reconditioning after cold storage improves postischemic graft function in isolated porcine kidneys

Delayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by short‐term hypothermic machine perfusion immediately prior to reperfusion. A total of 18 kidneys from female German Landrace pigs was flushed with Histidine‐Tryptophan‐Ketoglutarate solution and cold‐stored for 18 h (control). Some grafts were subsequently subjected to 90 min of hypothermic reconditioning by hypothermic machine perfusion with (HR+O₂) or without (HR?O₂) oxygenation of the perfusate. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n = 6, respectively). Renal function upon reperfusion was significantly enhanced by HR+O₂ with more than threefold increase in renal clearances of creatinine and urea. HR+O₂ also led to significantly higher urinary flow rates and abrogated the activation of caspase 3. By contrast, HR?O₂ was far less effective and only resulted in minor differences compared to control. It is derived from the present data that initial graft function can be significantly improved by 2 h of oxygenated machine perfusion after arrival of the preserved organ in the transplantation clinic.     

Recipient treatment with trimetazidine improves graft function and protects energy status after lung transplantation.

BACKGROUND: Ischemia-reperfusion injury remains an important obstacle to successful lung transplantation. Trimetazidine is an anti-ischemic drug that restores the ability of ischemic cells to produce energy and reduces the generation of oxygen-derived free radicals. The aim of this study was to assess the protective effect of trimetazidine after prolonged ischemia in lung transplantation. METHODS: Rat single-lung transplantation was performed in 4 experimental groups (n = 5 each). In all groups, transplantation was performed after 18 hours of cold (4 degrees C) ischemia. All donor lungs were flushed with low-potassium dextran-glucose (LPDG) solution that also contained 500 microg/liter prostaglandin estradiol (E(1)). Groups studied included: Group I: flush solution was administered containing 10(-6) mol/liter trimetazidine (TMZ), neither donor nor recipient treatment given; Group II: donors were treated with 5 mg/kg intravenous TMZ 10 minutes prior to harvest, but the flush solution did not contain TMZ; Group III: recipients treated with 5 mg/kg intravenous TMZ 10 minutes before reperfusion, and flush solution contained 10(-6) mol/liter trimetazidine; Group IV: ischemic control group. After 2 hours of reperfusion, oxygenation was measured and lung tissue was frozen and assessed for adenosine triphosphate (ATP) content, myeloperoxidase (MPO) activity and thiobarbituric acid-reactive substances (TBARS). Peak airway pressure (PawP) was recorded throughout the reperfusion period. RESULTS: Group III showed significantly higher levels of ATP content (11.1 +/- 5.01 pmol vs Group I, 3.36 +/- 1.8 pmol, p = 0.008; vs Group II, 4.7 +/- 1.9 pmol, p = 0.03; vs Group IV, 0.7 +/- 0.2 pmol, p = 0.008), better oxygenation (442.5 +/- 26.5 mm Hg, vs Group I, 161.06 +/- 54.5 mm Hg; vs Group II, 266.02 +/- 76.9 mm Hg; vs Group IV, 89.4 +/- 14.7 mm Hg, p = 0.008) and reduced lipid peroxidation (TBARS) (0.15 +/- 0.03 nmol/g; vs Group I, 1.04 +/- 0.76 nmol/g; vs Group II, 0.69 +/- 0.4 nmol/g; vs Group IV, 2.29 +/- 0.4 nmol/g, p = 0.008). PawP and MPO activity were comparable in the 4 study groups. CONCLUSION: Recipient treatment with TMZ provided significant protection of energy status, better oxygenation and reduced lipid peroxidation. Our data suggest that TMZ may be an important adjunct in the prevention of post-transplant lung ischemia-reperfusion injury.     

Effect of sodium aspartate on the recovery of the rat heart from long-term hypothermic storage.

We have investigated the reported ability of aspartate to enhance greatly the cardioprotective properties of the St. Thomas' Hospital cardioplegic solution after prolonged hypothermic storage. Rat hearts (n = 8 per group) were excised and subjected to immediate arrest with St. Thomas' Hospital cardioplegic solution (2 minutes at 4 degrees C) with or without addition of monosodium aspartate (20 mmol/L). The hearts were then immersed in the same solution for 8 hours (4 degrees C) before heterotopic transplantation into the abdomen of homozygous rats and reperfusion in vivo for 24 hours. The hearts were then excised and perfused in the Langendorff mode (20 minutes). Addition of aspartate to St. Thomas' Hospital cardioplegic solution gave a small but significant improvement in left ventricular developed pressure, which recovered to 82 +/- 3 mm Hg compared with 70 +/- 2 mm Hg in control hearts (p less than 0.05). However, coronary flow and high-energy phosphate content were similar in both groups. In subsequent experiments hearts (n = 8 per group) were excised, arrested (2 minutes at 4 degrees C) with St. Thomas' Hospital cardioplegic solution containing a 0, 5, 10, 20, 30, 40, or 50 mmol/L concentration of aspartate, stored for 8 hours at 4 degrees C, and then reperfused for 35 minutes. A bell-shaped dose-response curve was obtained, with maximum recovery in the 20 mmol/L aspartate group (cardiac output, 48 +/- 5 ml/min versus 32 +/- 5 ml/min in the aspartate-free control group; p less than 0.05). However, additional experiments showed that a comparable improvement could be achieved simply by increasing the sodium concentration of St. Thomas' Hospital cardioplegic solution by 20 mmol/L. Similarly, if sodium aspartate (20 mmol/L) was added and the sodium content of the St. Thomas' Hospital cardioplegic solution reduced by 20 mmol/L, no significant protection was observed when recovery was compared with that of unmodified St. Thomas' Hospital cardioplegic solution alone. In still further studies, hearts (n = 8 per group) were perfused in the working mode at either high (greater than 80 ml/min) or low (less than 50 ml/min) left atrial filling rates. Under these conditions, if functional recovery was expressed as a percentage of preischemic function, artifactually high recoveries could be obtained in the low-filling-rate group. In conclusion, assessment of the protective properties of organic additives to cardioplegic solutions requires careful consideration of (1) the consequences of coincident changes in ionic composition and (2) the characteristics of the model used for assessment.     

Prolonged hypothermic cardiac storage for transplantation. The effects on myocardial metabolism and mitochondrial function.

Cardiac storage for transplantation is currently limited to 6 hours. To better understand the metabolic changes that occur during hypothermic (4 degrees C) storage, we monitored the morphologic and metabolic changes in the canine myocardium at 0, 12, and 24 hours of storage in University of Wisconsin solution. Attempts to isolate cardiac mitochondria resulted in a progressive decline in the yield (milligrams of mitochondria per gram of heart tissue), which decreased (p less than 0.05) from 9.2 +/- 0.4 at 0 hours (control) to 4.0 +/- 0.3 after 12 hours and further decreased (p less than 0.05) to 1.9 +/- 0.2 after 24 hours of cold storage. Mitochondrial state 3 respiration fell to 64% of control after 12 hours and 28% of control after 24 hours of cold storage (p less than 0.05). Citrate synthetase activity, but not cytochrome C oxidase activity, was significantly depressed after 12 and 24 hours of cold storage. Adenosine triphosphate content decreased to 67% of control after 12 hours and 50% of control after 24 hours. After 12 hours of storage, sufficient adenosine diphosphate and monophosphate were present to permit some restoration of adenosine triphosphate, provided mitochondrial function was normal after transplantation. However, restoration of mitochondrial function and adenosine triphosphate levels sufficient to support myocardial contractility was unlikely after 24 hours of storage. This study suggests that a return of adequate cardiac function after transplantation may be possible after 12 hours of cold storage in University of Wisconsin solution but not after 24 hours of cold storage.     

Theophylline improves functional recovery of isolated rat lungs after hypothermic preservation

BACKGROUND: Raising intracellular cyclic adenosine monophosphate levels protects lungs from ischemia-reperfusion injury. We hypothesized that the phosphodiesterase inhibitor theophylline would protect lungs during storage. METHODS: Rat lungs were perfused with modified bicarbonate buffer mixed with rat blood (4:1 vol/vol) (37 degrees C) and ventilated (80 breaths/min). After 20 minutes of perfusion during which vascular resistance and airway compliance were measured, lungs were flushed with and then immersed in bicarbonate buffer (4 degrees C) alone or containing theophylline (30 to 1,000 micromol/L). After 6 hours of storage, lung function was reassessed during 40 minutes of reperfusion. RESULTS: Lungs stored in the presence of theophylline had improved lung function on reperfusion. After 40 minutes of reperfusion, pulmonary compliance was 0.008+/-0.004 mL/cm H2O, 0.022+/-0.010, 0.037+/-0.007, 0.044+/-0.006, and 0.073+/-0.003 mL/cm H2O, and vascular resistance was 3.84+/-0.40 cm H2O x min x mL(-1), 3.64+/-0.78, 2.12+/-0.35, 2.22+/-0.25, and 1.90+/-0.38 cm H2O x min x mL(-1) in lungs stored in the presence of 0, 30, 100, 300, or 1,000 micromol/L theophylline, respectively. Similar improvements were obtained for wet to dry weight ratio and gas exchange. CONCLUSIONS: Theophylline merits investigation as a potentially beneficial addition to solutions for the flushing and storage of human lungs for transplantation.     

Cardioprotection by cariporide after prolonged hypothermic storage of the isolated working rat heart.

BACKGROUND: Inhibition of the sodium-hydrogen (Na(+)-H(+)) exchanger decreases the extent of ischemia-reperfusion injury in the myocardium. Inhibition may also improve preservation of hearts stored for transplantation. Our aim was to characterize the dose response and to determine optimal timing for administering cariporide, an Na(+)-H(+) exchange inhibitor, during prolonged hypothermic storage. METHODS: We used the rat isolated working-heart model to measure cardiac function. To determine the optimal dose of cariporide, hearts received either no treatment (control) or incremental doses of cariporide (1, 3.2, 10, or 30 micromol/liter) before storage and during reperfusion. Hearts were arrested with and stored in an extracellular-based cardioplegic solution at 2 to 3 degrees C for 6 hours. To determine optimal timing, we arrested a group of hearts with and stored them in a cariporide-supplemented (10 micromol/liter) cardioplegic solution but did not pre-treat them with cariporide. Finally, we treated a separate group of hearts with 10 micromol/liter cariporide before, during, and after storage. RESULTS: Recovery of cardiac function in control hearts was poor. The cardioprotective effect of cariporide was dose dependent, with maximal protection observed at a concentration of 10 micromol/liter. Storing hearts in a cariporide-supplemented cardioplegic solution did not result in better recovery of cardiac function compared with cariporide given before storage and during reperfusion. Moreover, recovery of cardiac function was significantly worse in hearts that had not been pre-treated with cariporide. CONCLUSIONS: Sodium-hydrogen-exchange inhibition with cariporide significantly protects the hypothermic ischemic rat heart, increasing cardiac function after reperfusion. The timing of cariporide administration is an important determinant of this cardioprotection.     

Perfluorohexyloctane improves long-term storage of rat pancreata for subsequent islet isolation

Pancreas oxygenation by means of the hyperoxygen carrier perfluorodecalin (PFD) has been established to prevent ischemically induced damage from cold-stored pancreata. However, large-scale studies did not confirm the promising results that had been observed in smaller donor populations. This study assessed whether islet isolation from pancreata stored for prolonged periods can be improved by utilizing the new oxygen carrier perfluorohexyloctane (F6H8) characterized by lower gravity and higher lipophilicity than PFD. Subsequent to 24 h of storage in either oxygenated PFD or F6H8, the rat pancreata were assessed for the intrapancreatic partial oxygen pressure (pO₂) and subsequently processed with current standard procedures. The intrapancreatic pO₂ was nearly identical in rat pancreata stored either in PFD or F6H8. Nevertheless, rat islet isolation outcome was significantly increased in terms of yield, integrity, in vitro function and post-transplant outcome after transplantation in diabetic nude mice when F6H8 was used as oxygen carrier. This proof-of-concept study demonstrated in rats that islet isolation performed after long-term storage of oxygenated pancreatic tissue can be significantly improved if PFD was replaced by F6H8.