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Ann Thorac Surg 1995;60:1723-1728
© 1995 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Successful 24-Hour Rabbit Heart Preservation by Hypothermic Continuous Coronary Microperfusion With Oxygenated University of Wisconsin Solution

Division II, Department of Surgery, Kobe University School of Medicine, Kobe, Japan

Accepted for publication July 28, 1995.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. This study assessed whether a combination of hypothermic continuous coronary microperfusion and oxygenated University of Wisconsin Solution (UWS) improves postischemic functional recovery and minimizes myocardial tissue edema.

Methods. Isolated rabbit hearts were divided into four groups (n = 6 each): group I (immediate reperfusion), group II (simple cold storage in UWS), group III (hypothermic continuous coronary microperfusion with UWS), and group IV (hypothermic continuous coronary microperfusion with oxygenated UWS). Hearts in groups II, III, and IV were preserved for 24 hours. Preischemic and postischemic cardiac function was measured using a Langendorff apparatus.

Results. Hearts in group I showed complete functional recovery, whereas cardiac output in group II was inadequate. In groups III and IV, the percentage recovery rate (post/pre) of cardiac output was 57.0% ± 3.1% and 82.2% ± 9.1%, respectively (p < 0.05). In groups III and IV, perfusion pressures at the end of 24-hour preservation increased from the initial 5 mm Hg to 12.3 ± 2.7 and 8.3 ± 1.4 mm Hg (p < 0.05), respectively. In groups I, III, and IV, the percentage tissue water content was 82.8 ± 1.0, 86.7 ± 1.7, and 83.8 ± 1.6, respectively (p < 0.05 for group III versus groups I and IV). There was a significant correlation between the percentage tissue water content and coronary perfusion pressure at the end of the 24-hour preservation (r = 0.60, p = 0.040) and a significant inverse correlation between percentage tissue water content and percentage recovery rate of cardiac output (r = -0.69, p = 0.014). In ultrastructural examination, myocardial tissue edema was limited and mitochondria were well preserved in group IV.

Conclusion. We conclude that the combination of a hypothermic continuous coronary microperfusion technique and oxygenation of UWS was the procedure of choice for reducing tissue edema and improving both the coronary microcirculation and functional recovery during 24-hour heart preservation.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The University of Wisconsin solution (UWS) has proved to be effective for prolonged organ preservation [1, 2]. However, 24-hour heart preservation by simple cold immersion in UWS has been problematic [3]. Application of a coronary artery perfusion technique increases the efficacy of UWS and allows prolonged ischemic time for hypothermic heart preservation [4–7]. Prolonged preservation of the heart would diminish emergency operations and prevent graft failure by allowing human lymphocyte antigen typing to be performed, both of which improve the quality of life postoperatively. However, this technique has not been applied to clinical situations because of the complexity of the device. In addition, the technique is controversial as it may produce tissue edema because of a disturbance in the balance of Starling's forces [8] and intracellular edema because of prolonged hypothermic ischemia [9]. Tissue edema induces extravascular compression of the coronary microcirculation and reduces the benefit of the perfusion technique. These negative factors have offset the beneficial effects of the perfusion technique in the past. The osmolarity of UWS is the same as that of a conventional cardioplegic solution. However, the solution has novel components of impermeants (lactobionate and raffinose) and an oncotic agent (pentafraction) that are effective in suppressing tissue edema during preservation. Therefore, the present study was designed to investigate our hypothermic continuous coronary microperfusion (CCMP) technique using UWS and an oxygen supply to minimize microvascular dysfunction. This technique results in improved functional recovery after 24-hour heart preservation.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Animals and Reagents
Hearts were obtained from Japanese white rabbits weighing between 3 and 3.5 kg. All animals were kept in clean cages and were provided with regular food and sterile water. They received humane care in compliance with the ``Principles of Laboratory Animal Care'' formulated by the Institute of Laboratory Animal Resources and the ``Guide for the Care and Use of Laboratory Animals'' published by the National Institutes of Health. Animals were sedated with intramuscular ketamine hydrochloride (3 mg/kg). General anesthesia was induced by intravenous sodium pentobarbital (50 mg/kg). The animals were intubated through a tracheotomy. After sternotomy, heparin (1,000 U/kg) was administered intravenously. Hearts were then excised rapidly and immersed in 4°C Krebs-Henseleit bicarbonate buffer solution.

Preischemic Functional Assessment of Isolated Hearts by the Langendorff Apparatus
We used the isolated working heart model described by Neely and associates [10] and Hearse and co-workers [11]. An experimental protocol is shown in Figure 1. The ascending aorta of the isolated heart was cannulated to the Langendorff column containing recirculating, ultrafiltered (5 µm), oxygenated (95% O₂/5% CO₂) Krebs-Henseleit bicarbonate buffer solution (NaCl, 118 mmol/L; KCl, 4.7 mmol/L; CaCl₂, 2.5 mmol/L; MgSO₄, 1.2 mmol/L; KH₂PO₄, 1.2 mmol/L; NaHCO₃, 25.0 mmol/L; glucose, 11.1 mmol/L; pH 7.4 to 7.5) at 37°C, allowing 80 cm H₂O Langendorff nonworking perfusion for 15 minutes. The left atrium then was cannulated through the pulmonary vein and converted to the working mode. The working perfusion was performed at a left atrial pressure of 15 cm H₂O, and the heart was required to eject spontaneously through the aortic cannula against a pressure of 80 cm H₂O for 15 minutes. During the last 2 minutes of the working mode, aortic flow (AF; mL/min) was measured by collecting overflow volume from the aortic chamber; coronary flow (CF; mL/min) was measured by collecting coronary effluent. Cardiac output (CO; mL/min) was calculated as the sum of the aortic and coronary flows. Peak aortic pressure (mm Hg) was measured at a point in the aortic cannula. The heart was suspended within a water-jacketed chamber maintained at 37°C throughout the procedure. After the measurement of preischemic cardiac function, the heart was cooled to 4°C within a water-jacketed chamber and arrested with a drip injection of 4°C St. Thomas Hospital cardioplegic solution (NaCl, 91.6 mmol/L; KCl, 14.8 mmol/L; CaCl₂, 1.2 mmol/L; MgSO₄, 1.2 mmol/L; MgCl₂, 15.0 mmol/L; KH₂PO₄, 1.2 mmol/L; NaHCO₃, 25.0 mmol/L; procaine-HCl, 1.0 mmol/L) for 2 minutes at a pressure of 80 cm H₂O through a sidearm of the aortic cannula. This was followed by 4°C UWS (ViaSpan; E.I. du Pont de Nemours & Company, Wilmington, DE: KH₂PO₄, 1.2 mmol/L; MgSO₄, 1.2 mmol/L; adenosine, 5.0 mmol/L; glutathione, 3.0 mmol/L; raffinose, 30 mmol/L; allopurinol, 1.0 mmol/L; K lactobionate, 100.0 mmol/L; pentastarch, 5%; insulin, 40 IU/L; osmolarity, 320 mOsm/L; pH 7.4) for 2 minutes in prolonged preservation groups.

View larger version (21K): [in this window] [in a new window]   Fig 1. . Experimental protocol for 24-hour heart preservation. (AF = aortic flow; CCMP = continuous coronary microperfusion; CF = coronary flow; CO = cardiac output; pAP = peak aortic pressure; STs = St. Thomas Hospital solution; UWs = University of Wisconsin solution.)

View larger version (33K): [in this window] [in a new window]   Fig 2. . Hypothermic continuous coronary microperfusion technique. (RV = right ventricle; UW = University of Wisconsin.)

Reperfusion and Postischemic Functional Assessment
After 24-hour preservation, the hearts were reattached to the Langendorff apparatus, initially reperfused with a drip infusion of 4°C oxygenated Krebs-Henseleit bicarbonate buffer solution, and gradually rewarmed to 20°C over 15 minutes. The hearts were then converted to the Langendorff nonworking mode at 37°C for 30 minutes, followed by working mode for 15 minutes. During the last 2 minutes of working mode, AF, CF, CO, and pAP were measured (see Fig 1). After assessment of cardiac function, atrial tissue was removed and total biventricular weight was measured.

Myocardial Tissue Water Content
After this experimental protocol, tissue water content (tWC) was measured. Transmural specimens (approximately 2 g) from the left ventricle were harvested and the wet weight was obtained. The dry weight of the specimen was then measured after 48 hours of desiccation at 80°C. Percentage tWC was calculated using the following formula: [(wet weight - dry weight/wet weight)] x 100.

Histopathology of the Myocardium
Myocardial specimens from each group were obtained from the left ventricular free wall in the endocardial zone for evaluation of ultrastructural findings. The specimens were first fixed in Karnovsky solution for 2 hours at 4°C. Postfixation was performed for 1 hour at 4°C in 1% osmium tetroxide buffered to pH 7.4, and the sections were rinsed in the same buffer containing 0.22 mol/L sucrose. The blocks were dehydrated in graded series of ethanol and routinely embedded in Epon. Ultrathin sections were cut, mounted on copper grids, and stained with uranyl acetate. The specimens were examined with a Hitachi H-600 electron microscope.

Statistical Analysis
All results are expressed as the mean ± standard deviation. Data from different groups were compared using the repeated measures analysis of variance. Differences between groups were tested by analysis of variance. When the analysis of variance indicated significance, the differences were tested using the Scheffé F test. A value of p less than 0.05 was considered statistically significant.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Recovery of Cardiac Function
Pre- and postischemic AF, CF, and CO were divided by total biventricular dry weight (mL • min^-1 • [g • tissue]^-1). The preischemic and postischemic cardiac function and percentage recovery (post/pre) of pAP, AF, CF, and CO are shown in Table 1. There were no significant differences in preischemic functions between each group. The hearts in group I showed complete functional recovery, whereas in group II, AF was not obtained against 80 cm H₂O. Percentage recovery of CO in groups III and IV was 57.0 ± 3.1 and 82.2 ± 9.1, respectively (p < 0.01); percentage recovery of CF was 56.4 ± 8.6 and 79.3 ± 8.6, respectively (p < 0.01). Thus, the functional recovery in group IV was significantly better than that in groups II and III (Table 1).

View larger version (16K): [in this window] [in a new window]   Fig 3. . Perfusion pressure at the end of 24-hour preservation (open circles). Closed circles = mean ± standard deviation of six samples in each group; dotted line = 5 mm Hg of initial perfusion pressure. (*p < 0.05 by analysis of variance with repeated measures and Scheffé F test.)

Relation Between Percentage Recovery of Tissue Water Content, Perfusion Pressure at the End of the Preservation, and Percentage Recovery of Cardiac Output
We hypothesized that minimizing tissue edema should decrease perfusion pressure at the end of the 24-hour preservation and result in an increase in percentage of CO after reperfusion. In fact, we noted a significant correlation between percentage tWC and perfusion pressure at the end of preservation (r = 0.60, p = 0.04) and a significant inverse correlation between percentage tWC and percentage CO (r = -0.69, p = 0.014) (Figs 4, 5). These data suggest that postischemic functional recovery was improved when perfusion pressures remained low.

View larger version (22K): [in this window] [in a new window]   Fig 4. . Relation between percentage tissue water content (%tWC) and perfusion pressure at the end of 24-hour heart preservation. Open circles = values in group III; closed circles = values in group IV.

View larger version (17K): [in this window] [in a new window]   Fig 5. . Relation between percentage tissue water content (%tWC) and percentage recovery of cardiac output (%CO). Open circles = values in group III; closed circles = values in group IV.

View larger version (170K): [in this window] [in a new window]   Fig 6. . Ultrastructural findings in the myocardium from groups I (A), II (B), III (C), and IV (D) after preservation. (x20,000 before 14% reduction.) (A) Myocardium with normal structure and well-preserved mitochondria granules. (B) Severely injured myofilaments and disrupted mitochondria. (C) Intracellular edema with injured myofilaments and disrupted mitochondria. (D) Reduced intracellular edema and almost normal structure of myofilaments.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

Hypothermic perfusion techniques for long-term heart preservation have been reported by many investigators [4–7, 20]. This method improves cardiac preservation and successfully extends the preservation time beyond that possible with simple hypothermic immersion. A continuous perfusion technique can supply the novel components of UWS with uniform cooling and washout of toxic metabolites, thereby preventing intracellular acidosis. However, there are major disadvantages to this approach, including the complexity of the device and myocardial edema. Thus, we have constructed a simple perfusion circuit with one roller pump and a filter. University of Wisconsin solution was oxygenated by bubbling and equilibrated with 95% O₂ and 5% CO₂.

Myocardial tissue edema occurs at two levels: interstitial and intracellular. Interstitial edema is due to an imbalance of the Starling forces: a disparity between hydrostatic and oncotic pressures [8]. In contrast, intracellular edema occurs from a failure of the adenosine triphosphate-dependent sodium-potassium pump in the cytoplasmic membrane [9] and results in impairment of mitochondrial and other cellular organelles. Our data show that myocardial tissue edema impairs the coronary microcirculation, resulting in poor functional recovery. Minimizing both interstitial and intracellular edema maintains the coronary microcirculation and contributes to better functional recovery. Interstitial edema could be prevented by applying a low hydrostatic pressure and a high oncotic pressure. However, continuous perfusion pressure has to be set carefully to minimize tissue edema while delivering adequate microvascular perfusion. The use of low-pressure perfusion is generally believed to be a critical factor in maintaining the balance of Starling forces and successfully perfusing isolated hearts [21]. Manciet and associates [22] have studied the effects of low-pressure perfusion on microvascular perfusion during a 24-hour hypothermic preservation. They concluded that a perfusion pressure of 13 mm Hg is as effective as a perfusion pressure of 80 mm Hg and that the absence of effective osmolarity in the vasculature might result in excessive interstitial edema and microvascular compression. Wicomb and Collins [5] performed hypothermic microperfusion with UWS for preserving rabbit hearts for 24 hours and accomplished excellent functional recovery. They set a very low constant flow with a rate of 3 to 6 mL per gram of heart per 24 hours to minimize tissue edema. The normal CF is 8% of CO, corresponding to 6.7 to 8.0 mL • kg^-1 • min^-1. A hypothermic arrested heart requires about 5% of the oxygen of a normothermic working heart [23, 24]. Therefore, a rabbit weighing 3 kg requires a CF of 1 to 1.2 mL/min. Because our perfusion device can supply 0.8 to 1.0 mL/min at 5 mm Hg, we set the initial perfusion pressure to this setting with UWS.

Postischemic CO was obtained using the hypothermic CCMP technique with the original UWs (group III), whereas CO was not obtained in simple immersed hearts (group II). However, the recovery rate of group III was poor, and tWC was significantly elevated with intracellular edema because of hypoxic cellular damage. This result suggested that suppression of intracellular edema was more important for long-term heart preservation than for short-term preservation. The hypothermic CCMP technique provided high-energy phosphate precursors (adenosine and phosphate), but prolonged ischemia decreases adenosine triphosphate production. We attempted to promote aerobic metabolism and minimize the failure of the adenosine triphosphate-dependent sodium-potassium pump by oxygenating the UWS (group IV). This modification significantly reduced intracellular edema, as assessed by ultrastructural findings, and decreased tWC. This group had a significantly improved recovery of cardiac function after preservation. Therefore, oxygenation of UWS in combination with our hypothermic CCMP technique resulted in successful 24-hour heart preservation, with reduced myocardial tissue edema and improved functional recovery.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We thank Dr Hiroshi Itoh for helpful suggestions regarding the histologic techniques used in this study.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr K. Okada, Division II, Department of Surgery, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650, Hyogo, Japan.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Chojiro Yamashita Masayoshi Okada Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Okada, K. Articles by Okada, M. Search for Related Content PubMed PubMed Citation Articles by Okada, K. Articles by Okada, M.