Effects of butanedione monoxime and temperature on prolonged cardiac storage.

BACKGROUND: The optimal temperature for cardiac allograft storage remains controversial. We conjectured that supplementation of the potent cardioprotective agent 2,3-butanedione monoxime with calcium may improve allograft storage and make the precise storage temperature less critical. METHODS: Hearts were harvested from Sprague-Dawley rats (250 to 350 g), mounted on a Langendorff apparatus, and instrumented with an intraventricular balloon. Hearts were flushed and stored with either unmodified University of Wisconsin solution (UWS) or UWS supplemented with 10 mmol/L of 2,3-butanedione monoxime and calcium 0.1 mmol/L (BDM). Hearts were then subjected to 12 hours of storage at one of five temperatures (0 degree, 4 degrees, 8 degrees, 12 degrees, or 16 degrees C) in a complete 2 x 5 factorial design (n = 6/group). Data are reported either as a percentage of the prestorage results or as an absolute value (mean +/- standard deviation). RESULTS: Recovery of developed pressure (p < 0.0001), coronary flow (p < 0.0001), and diastolic volume (p < 0.001) were significantly enhanced, whereas creatine kinase (p < 0.0001) and lactate dehydrogenase release (p < 0.0001) were reduced in the BDM versus the UWS groups. In both the BDM and UWS storage groups, recovery was better at temperatures of 8 degrees C or less than at 12 degrees C or more. The single preferred temperature was 4 degrees C, significantly better than 0 degree C with unmodified UWS, while similar to 0 degree and 8 degrees C with BDM. Adenine nucleotide values were decreased equally in the BDM and UWS hearts, but preservation was enhanced at 0 degree C compared with all warmer temperatures. CONCLUSIONS: We conclude that 4 degrees C is the preferred temperature for prolonged cardiac storage with UWS and that the inclusion of 2,3-butanedione monoxime with calcium 0.1 mmol/L markedly enhances recovery for storage temperatures of 8 degrees C or less.

The beneficial effects of heat-shock for prolonged hypothermic storage.

Heat-shock has been reported to induce tolerance for subsequent ischemia. We wished to determine whether thermal stress is protective for hypothermic storage. Three groups of Sprague-Dawley rats were studied (n = 8 per group). Control animals received routine care (CONT) while anaesthetized rats were either warmed to 42 degrees C for 20 min (HEAT) or maintained at room temperature (SHAM). Twenty-four hours later, hearts were mounted on a Langendorff apparatus with an intraventricular balloon. Hearts were then flushed and stored in UW solution for 8 hr at 0 degrees C and reperfused for 45 min. Data are reported as a percentage of the prestorage results or as the absolute value (mean +/- SD). Recovery of developed pressure was significantly greater (P < 0.05) in the heat-shocked animals (79.5 +/- 10.2%) than in the SHAM (63.6 +/- 17.2%) or CONT groups (59.0 +/- 10.8%). Coronary flow was similarly enhanced (P < 0.05) in the HEAT group (86.8 +/- 5.5%) vs the CONT (77.0 +/- 12.3%) or SHAM hearts (74.5 +/- 10.2%). Diastolic compliance as assessed by evaluation of the end-diastolic pressure-volume curves was reduced in all groups (P < 0.0001) but not different among groups. Cardiac creatine kinase release during reperfusion was greater in the CONT and SHAM groups (CONT: 726.9 +/- 297.8 IU/g; SHAM: 548.9 +/- 420.9 IU/g) than in the heat-shocked rodents (282.3 +/- 175.5 IU/g, P < 0.05 HEAT vs CONT). Cardiac biopsies were performed sequentially in separate animals (n = 6 per group). Tissue levels of ATP and total adenine nucleotides were greater in the heat-shocked or SHAM hearts following reperfusion compared with controls (P < 0.05). Thermal stress provides additional protection for prolonged hypothermic storage.

Cardiac storage with University of Wisconsin solution, calcium, and magnesium.

BACKGROUND: Previous investigations from this institution and others support the role of University of Wisconsin solution for prolonged hypothermic cardiac storage. Modification of the divalent cation concentrations has been beneficial for cardioplegic investigations and may enhance cardiac recovery after extended preservation. METHODS: To investigate this hypothesis, isolated rodent hearts were obtained from Sprague-Dawley rats and mounted on a Langendorff apparatus. Rat hearts were flushed (15 ml/kg) and stored (30 ml) for 8 hours at 0 degree C in unmodified University of Wisconsin solution (n = 16/group) or University of Wisconsin solution with calcium concentrations of 0.025 to 10 mmol/L or magnesium concentrations of 10 to 20 mmol/L (six to eight hearts/group). Finally, combinations of calcium and magnesium were examined. Rat hearts were studied before storage and after 45 minutes of reperfusion with an intraventricular balloon. RESULTS: Developed pressure (mean +/- standard deviation) was increased with calcium 0.1 mmol/L (University of Wisconsin solution: 69.2% +/- 7.0%; Ca++ 0.1 mmol/L: 78.9% +/- 6.1%, p < 0.05), whereas only the addition of the highest calcium concentration (10 mmol/L) was significantly harmful (developed pressure: 58.3% +/- 8.4%, p < 0.05; creatine kinase release: 408 +/- 200 versus 170 +/- 104 IU/gm, p < 0.05; lactate dehydrogenase release: 103 +/- 43 versus 37 +/- 26 IU/gm, p < 0.05). Coronary flow recovered to control values with magnesium 15 mmol/L, which was significantly greater than that achieved with unmodified University of Wisconsin solution (97.1% +/- 14.6% versus 72.1% +/- 8.4%, p < 0.05). Of the calcium-magnesium combinations tested, developed pressure was increased compared with unmodified University of Wisconsin solution with calcium 0.1 and magnesium 20 mmol/L (76.8% +/- 6.4%, p < 0.05). Diastolic function was reduced in all groups (p < 0.0001) and not significantly different between groups. CONCLUSIONS: The experiments indicated that recovery after storage with University of Wisconsin solution is enhanced with the addition of calcium and magnesium. The addition of high concentrations of calcium (> or = 2.5 mmol/L) appears harmful.

Adenosine pretreatment for prolonged cardiac storage. An evaluation with St. Thomas' Hospital and University of Wisconsin solutions.

Adenosine pretreatment has been shown to be beneficial in several models of ischemia-reperfusion. We wished to evaluate whether adenosine pretreatment is cardioprotective for prolonged cardiac storage and whether the presence of adenosine in the storage media affects the results. Isolated rodent hearts were obtained from Sprague-Dawley rats, mounted on a Langendorff apparatus, instrumented with an intraventricular balloon, and ventricularly paced at 300 beats/min. Four groups of hearts were studied in a 2 x 2 factorial experiment (n = 8 to 12 per group). Hearts were subjected to normal perfusion or to solution supplemented with adenosine 50 mumol/L for 10 minutes followed by adenosine-free perfusion for 10 minutes. Hearts then were stored for 8 hours at 0 degrees C in either University of Wisconsin solution (adenosine 5 mmol/L) or St. Thomas' Hospital II solution (adenosine free). Adenosine pretreatment increased tissue levels of adenosine triphosphate before storage (p = 0.04). Nonfunction was less common after storage (1/19 versus 6/20 hearts, p < 0.05), and diastolic function was better preserved in the adenosine groups in the reperfusion phase (p = 0.01). The beneficial effects of adenosine pretreatment were independent of which storage solution was used. Developed pressure was increased (p < 0.05) and release of creatine kinase and lactate dehydrogenase was reduced (p < 0.0001) in hearts treated with University of Wisconsin solution compared with those treated with St. Thomas' Hospital solution. These studies suggest that adenosine pretreatment improves recovery after prolonged hypothermic storage and that the presence of adenosine in the preservation solution does not alter the results. The experiments provide further evidence that extended myocardial protection is better enhanced with University of Wisconsin solution than with St. Thomas' Hospital II solution.

Cardiac storage with University of Wisconsin solution and a nucleoside-transport blocker.

Findings from previous investigations conducted at this institution and others have suggested that University of Wisconsin solution (UWS) is preferable for the prolonged hypothermic storage of hearts before transplantation. The benefit seen with UWS may in part be related to the inclusion of adenosine (5 mmol/L) in the UWS. To investigate whether further manipulations of adenosine metabolism might enhance myocardial protection, studies were initially conducted using cultured myocytes, followed by confirmatory experiments using isolated rat hearts. Cultured human ventricular myocytes (7 to 8 dishes/group) were stored for 12 hours at 0 degrees C in unmodified UWS or UWS supplemented with increasing concentrations (1 to 100 mumol/L) of the nucleoside-transport blocker p-nitrobenzylthioinosine. The adenosine triphosphate concentrations were found to be enhanced with nucleoside-transport inhibition, with the best results achieved with the 1- and 3-mumol/L groups (control, 3.37 +/- 0.41 nmol/micrograms DNA; UWS, 2.89 +/- 1.31 nmol/micrograms DNA; 1 mumol/L, 5.91 +/- 3.23 nmol/micrograms DNA; 3 mumol/L, 7.86 +/- 3.45 nmol/micrograms DNA; p < 0.05 versus control or UWS group). Isolated rodent hearts from Sprague-Dawley rats were prepared on a Langendorff apparatus with an intraventricular balloon and subsequently stored for 8 hours at 0 degrees C in unmodified UWS (13 hearts/group) or UWS supplemented with 1 or 3 mumol/L of p-nitrobenzylthioinosine (9 to 10 hearts/group).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac storage with UW solution and glucose.

Previous investigations from our institution using an isolated human cardiomyocyte model concluded that glucose supplementation of University of Wisconsin solution (UWS) was beneficial with respect to adenine nucleotide and protein recovery. We wished to confirm these results using an isolated heart model. Rodent hearts were frozen in liquid nitrogen (control) or flushed and stored in UWS for 8 hours at 0 degrees C or UWS supplemented with 10, 20, or 30 mmol/L glucose. Experimental hearts were assessed at end-storage or after 45 minutes of reperfusion on a Langendorff apparatus. Adenine nucleotides were assessed by high performance liquid chromatography. In parallel experiments, ventricular function was assessed before and after storage in Langendorff-perfused hearts instrumented with a left ventricular balloon. Glucose supplementation was associated with greater poststorage (20 and 30 mmol/L glucose) and postreperfusion (10, 20, and 30 mmol/L glucose) adenosine triphosphate levels than unmodified UWS. Developed pressure (expressed as a percentage of control values) was increased with 10 mmol/L glucose (75.2% +/- 7.9%, mean +/- standard deviation) compared with unmodified UWS (64.6% +/- 6.6%; p < 0.05). Coronary flow was greater with 10 (72.6% +/- 10.7%) or 20 mmol/L (71.2% +/- 12.5%) versus 0 mmol/L glucose (58.6% +/- 12.1%, p < 0.05). The data support previous in vitro findings and suggest that the addition of 10 mmol/L glucose to UWS is associated with enhanced recovery after prolonged hypothermic storage.

Comparison of two experimental models for assessment of cardiac preservation.

Previous studies from this institution using human cell cultures have suggested that University of Wisconsin solution is preferred for prolonged hypothermic storage for cardiac transplantation. The primary objective of this study was to evaluate the effectiveness of extended cardiac preservation with University of Wisconsin solution by assessing the time-related changes of purine metabolites using two different models of cold storage. Isolated rat hearts (n = 6/group) or human ventricular myocyte cultures (n = 7 dishes/group) were assessed after 0, 6, 12, and 24 hours in University of Wisconsin solution at 0 degrees C using high-performance liquid chromatography. Adenosine triphosphate content decreased from 18.1 +/- 5.4 to 9.6 +/- 2.7 mumol/g dried weight by 12 hours and to 1.0 +/- 0.6 mumol/g by 24 hours (p < 0.0001 by analysis of variance) in the rat model. Adenosine triphosphate content decreased from 0.64 +/- 0.42 to 0.14 +/- 0.11 nmol/micrograms DNA at 6 hours and to 0.04 +/- 0.03 nmol/micrograms DNA by 24 hours (p < 0.00001) in the cardiomyocytes. Inosine monophosphate content increased from 0.1 +/- 0.2 to 10.8 +/- 1.0 by 24 hours (p < 0.0001) in the rat studies. Inosine monophosphate values tended to increase up to 12 hours (p = 0.06) in the cell cultures and then declined. Adenosine concentration increased from 0.3 +/- 0.3 to 2.3 +/- 0.9 mumol/g at 6 hours and declined thereafter (p < 0.0005) in the rodent hearts. Adenosine concentration increased from 0.03 +/- 0.02 to 1.53 +/- 0.72 nmol/micrograms DNA at 6 hours (p < 0.0001) in the cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro assessment of the effects of glucose added to the University of Wisconsin solution on myocyte preservation.

BACKGROUND: University of Wisconsin solution (UWS) has been successfully used for liver transplantation and may be beneficial for hypothermic cardiac storage. The addition of glucose may enhance myocardial preservation. METHODS AND RESULTS: Cultured human ventricular myocytes (eight dishes per group) were stored at 0 degree C for 12 hours in either unmodified UWS or UWS with glucose (1, 3, 10, 30, or 100 mmol/l). Cells were assayed for protein by spectrofluorometry and adenine nucleotides by high performance liquid chromatography after storage. Protein recovery, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and total adenine nucleotides (ATP+ADP+AMP) were all depleted after storage (p < 0.0001 by ANOVA). Protein recovery (p < 0.005), ATP (p < 0.05), and ADP (p < 0.05) were increased with glucose administration compared with unmodified UWS. Improvement was maximal using 30 mmol/l (protein, 0 mmol/l = 0.48 +/- 0.14 and 30 mmol/l = 0.65 +/- 0.11 mg per dish; ATP, 0 mmol/l = 3.08 +/- 0.63 and 30 mmol/l = 4.32 +/- 0.90 nmol/mg protein; ADP, 0 mmol/l = 3.76 +/- 0.80 and 30 mmol/l = 4.63 +/- 0.38 nmol/mg protein, mean +/- SD). Total adenine nucleotides tended to increase at any glucose concentration (p = 0.07 by ANOVA) and were significantly better with 30 mmol/l glucose (0 mmol/l = 7.61 +/- 1.58 and 30 mmol/l = 9.62 +/- 1.08 nmol/mg protein). CONCLUSIONS: Increasing the glucose concentration from 0 to 30 mmol/l improved adenine nucleotide and cellular protein preservation in this in vitro assessment.

The limits of cardiac preservation with University of Wisconsin solution.

Previous studies from this institution have suggested that University of Wisconsin solution is preferred for prolonged cardiac storage and preserves high-energy phosphates better than other storage fluids. University of Wisconsin solution contains adenosine (5 mmol/L), which may maintain the concentration of myocardial adenine nucleotides. Cultures of human adult myocytes were grown from left ventricular biopsy specimens obtained from patients undergoing coronary bypass procedures. Cells (seven to nine dishes per group) were rinsed of culture medium and stored at 0 degrees C in University of Wisconsin solution. Cells were analyzed for adenine nucleotide content after 1, 6, 12, and 24 hours of storage by high-performance liquid chromatography (units = nmol/microgram DNA) and compared with control samples (0 hour). Adenosine concentration increased from 0.03 +/- 0.02 (mean +/- standard deviation) to 1.77 +/- 1.03 by 1 hour (p less than 0.0001, analysis of variance) and remained increased thereafter. Adenosine was largely degraded to inosine (0 hours, 0.03 +/- 0.03; 6 hours, 0.88 +/- 0.56; p less than 0.001) and hypoxanthine (0 hours, 0.01 +/- 0.01; 6 hours, 0.15 +/- 0.09; p = 0.004). Measured levels of xanthine and uric acid were extremely low at all time intervals. Adenosine triphosphate levels were maintained at 1 hour (0 hours, 0.64 +/- 0.38; 1 hour, 0.67 +/- 0.45) but declined thereafter (6 hours, 0.21 +/- 0.21; 12 hours, 0.11 +/- 0.09; 24 hours, 0.04 +/- 0.03; p less than 0.0001). Levels of adenosine diphosphate (p = 0.007) and adenosine monophosphate (p less than 0.05) decreased to approximately 25% of original values by 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged preservation with University of Wisconsin Solution.

Previous studies from this institution using human cell cultures have suggested that University of Wisconsin Solution may be preferred for prolonged cardiac storage. University of Wisconsin Solution (UWS) contains adenosine (5 mmole/liter) which could maintain adenine nucleotides better than other storage fluids. Human cardiomyocytes were isolated from left ventricular biopsies. Cells (seven to nine dishes/group) were rinsed of culture media and placed in one of four solutions: Stanford cardioplegia, phosphate-buffered saline, modified EuroCollins', or UWS. Metabolites were assessed using high-performance liquid chromatography (units = nmole/micrograms DNA) after 24 hr of storage at 0 degrees C and compared to baseline controls (BASE). Adenosine triphosphate (P less than 0.0001, ANOVA), adenosine diphosphate (P less than 0.0001), and adenosine monophosphate (P less than 0.01) decreased with each solution compared to BASE but were maintained best with UWS (P less than 0.05). Adenosine increased in the UWS cells only (BASE, 0.029 +/- 0.118; UWS, 1.836 +/- 1.110; P less than 0.0001, ANOVA). Adenosine in the UWS cells was largely degraded to inosine (UWS, 1.013 +/- 0.779; BASE, 0.034 +/- 0.032; P less than 0.0001) and hypoxanthine (UWS, 0.124 +/- 0.091; BASE, 0.005 +/- 0.005; P less than 0.001). University of Wisconsin Solution does preserve adenine nucleotides better than other storage fluids and may improve the clinical results of cardiac transplantation.

Age-related changes in contact photosensitivity differ among mouse strains.

There are differences among mouse strains in the age-related changes in reactivity to the contact photosensitizer tetrachlorosalicylanilide (TCSA). We found a tendency to lower reactions in older mice, with some strains showing declines from an early age (BALB/cJ, MRL/MpJ +/+, MRL/MpJ lpr/lpr and SJL/J). Others had increasing reactions until about 30-50 weeks of age before declining (DBA/1J, C3H/HeJ, and A/J) and one strain (C57BL/6J) had increased reactivity with age. There are also differences in the role of cyclophosphamide-sensitive T-suppressor cells in these age-related changes. In some mouse strains, BALB/cJ, C57BL/6J, A/J, DBA/1J and C3H/HeJ, age-related changes in reactivity to TCSA are independent of changes in cyclophosphamide-sensitive suppressor cells. In other strains, MRL/MpJ +/+, MRL/MpJ lpr/lpr and SJL/J, the development of cyclophosphamide-sensitive suppressor cells is responsible for the initial, though not later, stages of the age-related decline in reactivity.

Assessment by mouse model of the ultraviolet A protective effect of topical sunscreens.

We have developed an animal model to assess the ultraviolet A (UVA) protective effect of topical sunscreens with the use of BALB/cJ mice in which contact photosensitivity to 3, 3', 4', 5 tetrachlorosalicylanilide had been induced. The mice were sensitized on the clipped dorsal skin and challenged on the ears. Changes in ear thickness after challenge were used to measure the degree of photosensitivity. The efficacy of two doses of each topical sunscreen was assessed by the degree of suppression of the contact photosensitivity response at challenge. Control studies were performed with the base of each sunscreen. Some but not all sunscreens that contained UVA-absorbing chemicals showed active suppression of contact photosensitivity in this test system. Several sunscreens gave greater suppression at 5 microliters/cm2 than at 2 microliters/cm2, which suggests a dose-related effect. One sunscreen, however, gave greater suppression at 2 microliters/cm2. Several of the bases tested also suppressed the contact photosensitivity response. An unexpected finding was an enhancement of the contact photosensitivity reaction by two of the bases tested.

Optimization of tetrachlorosalicylanilide and ultraviolet A doses at sensitization and challenge for contact photosensitivity in the mouse.

We studied contact photosensitivity (CPS) to tetrachlorosalicylanilide (TCSA) in BALB/cJ mice with various doses of TCSA and UVA at sensitization and challenge. From these studies we recommend the following protocol: sensitization on days 0 and 1 with 50 microliters of 1% TCSA followed by 3 J/cm2 of UVA, and challenge on day 7 with 10 microliters of 3% TCSA followed by 6 J/cm2 of UVA. This gave an ear swelling response of 27.4 +/- 0.6 x 10(-3) (mean +/- standard error). We also demonstrated that there is reciprocity between volume and concentration of TCSA at sensitization but not between TCSA and UVA doses at sensitization.

Circadian rhythms are suppressed in hyperproliferative mouse epidermis.

We examined the chronically hyperproliferative epidermis of the asebia (ab/ab) mouse for circadian rhythms in cell proliferation and in the rate of DNA synthesis, which is related to S phase duration. The curve for the circadian rhythm in cell proliferation for asebia epidermis was suppressed and distorted in comparison to that for BALB/cJ epidermis and in comparison to a composite curve produced by averaging the results from ten other published studies.

Studies of the action spectrum for induction of photosensitivity to tetrachlorosalicylanilide in mice.

We have developed a new system, using ear swelling in mice, to study the action spectrum for the induction of photosensitivity to tetrachlorosalicylanilide (TCSA). Using narrow-band interference filters, we produced data for action spectra between 250 nm and 421 nm with doses of 0.5 J/cm2 and 0.2 J/cm2. We found that the higher dose generally produced greater reactions. The action spectrum was similar to the absorption spectrum for TCSA in the UVA region. The reduced responses in the UVB and UVC regions may be due to immunological suppression produced by these parts of the spectrum.