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Ann Thorac Surg 2000;69:237-242
© 2000 The Society of Thoracic Surgeons
a Department of Cardiovascular Research, The Rayne Institute, St Thomas’ Hospital, London, England, United Kingdom
b Department of Cardiac Surgical Research, The Rayne Institute, St Thomas’ Hospital, London, England, United Kingdom
Address reprint requests to Dr Featherstone, Cardiovascular Research, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, England
e-mail: rfeather{at}rayne.umds.ac.uk
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Abstract |
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Methods. Function was measured in five groups of perfused, ventilated rat lungs. Group 1 lungs underwent control perfusion (60 minutes) without storage. Groups 2 through 5 underwent the following prestorage protocols: group 2, 20 minutes of perfusion; group 3, 10 minutes of perfusion, 5 minutes of cessation of ventilation and perfusion (ischemia), and 5 minutes of reperfusion; group 4, 5 minutes of perfusion, 10 minutes of ischemia, and 5 minutes of reperfusion; and group 5, 2 periods of 5 minutes of ischemia and 5 minutes of reperfusion. Lungs were then flushed with, and immersed (6 hours) in modified bicarbonate buffer (4°C). Lung function was reassessed during 40 minutes of reperfusion (37°C). Subsequently we examined preconditioning by stopping ventilation or perfusion separately.
Results. After reperfusion, lungs in group 2 had a compliance of 0.015 ± 0.002 mL/cm H2O (mean ± SE, n = 10), significantly lower than lungs in group 1 (0.063 ± 0.002 mL/cm H2O). Ischemic preconditioning was protective, with lungs in groups 3, 4, and 5 having compliances greater (p < 0.05) than those in group 2. Preconditioning by cessation of ventilation alone was also effective.
Conclusions. Preconditioning attenuates deterioration in lung compliance on reperfusion to a degree dependent on the protocol used.
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Introduction |
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There have been reports of preconditioning protection in the lung. One, which was in the cat in vivo, demonstrated that a 10-minute ischemic preconditioning followed by 10 minutes of reperfusion led to a reduction in the neutrophilia consequent to 2 hours of warm ischemia and 2 hours of reperfusion [6]. In another study, in isolated rat lungs, it was reported that preconditioning improved gas exchange after hypothermic storage for 24 hours in University of Wisconsin solution [7].
On the basis of these findings, we were interested in determining the possible extent of protection that preconditioning would offer in the lung. In the heart, the precise protocol used to initiate preconditioning protection has been shown to influence the effectiveness of the protection provided [5]. Therefore, in this study we investigated the most effective approach to induce preconditioning protection in the lung. Given the complicating factor that, in the lung, cessation of perfusion or ventilation may have differential effects on pulmonary metabolism [8], we also compared the effect of halting either perfusion or ventilation separately, or together, as a means of preconditioning this tissue.
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Material and methods |
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Lung preparation
Lungs were obtained from male Wistar rats weighing 230 to 330 g. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research (USA), the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, published by the National Institutes of Health (NIH publication 85-23, revised 1985) and with the "Guidance on the Operation of the Animals (Scientific Procedures) Act 1986" published by Her Majesty’s Stationery Office, London, England.
Rats were anesthetized by injection of pentobarbitone (2 mL/kg of a 60 mg/mL solution). After tracheal intubation, the animals were ventilated at 80 breaths/min by a Harvard small animal ventilator with a positive end-expiratory pressure of 1 to 2 cm H2O. After laparotomy, heparin (500 IU) was injected into the inferior vena cava, and 7 mL of blood was withdrawn for subsequent addition to the perfusion buffer. The thorax was opened, and the pulmonary artery and left atrium were cannulated. Perfusion with modified bicarbonate buffer (BB; composition in mmol/L: NaCl, 118.5; KCl, 3.8; KH2PO4, 1.2; NaHCO3, 25.0; CaCl2, 2.0; MgSO4, 1.2; and glucose, 10.0) mixed with rat whole blood (4:1, vol/vol, sanguineous BB), at a flow rate of 15 mL/min maintained by a peristaltic pump, was started at this stage. The lungs were ventilated throughout the setup procedure and never underwent a period without perfusion of longer than 60 seconds. The lungs were then removed and suspended in a chamber maintained at 37°C. The sanguineous BB was held in a heated reservoir and gassed with 100% CO2whenever it exceeded a pH of 7.3; the output of a pH meter was used to control a valve on tubing supplying CO2 to the perfusate reservoir. Samples of perfusate entering and leaving the lung were collected for pH measurement. Inasmuch as the lung exhales CO2, the perfusate becomes more alkaline as it passes through the lung; this pH increase is a measure of gas exchange by the lung [9, 10]. We used this measure because the recirculating perfusate was not deoxygenated before it entered the lung, whereas gassing with CO2 was performed at this stage, thus creating a perfusate to alveolar air gradient for this gas. Furthermore, whereas transpulmonary pH gradients were available for all experimental groups, this was not the case for dissolved oxygen in the perfusate. Buffer leaving the lungs through the left atrial cannula was returned to the reservoir and recycled. Oxygenation of the perfusate was by the isolated lungs, which were ventilated with room air.
Measurements of lung physiology
Study 1
A differential pressure transducer attached to a sidearm of the tracheal cannula measured tracheal pressure. Another pressure transducer connected to the inside of the sealed perfusion chamber measured changes in chamber pressure caused by filling and emptying of the lung during ventilation. Injection of a known volume of air into the lung by syringe before commencing each experiment calibrated this transducer in terms of tidal volume. The ratio of tidal volume to tracheal pressure for a constant value of tracheal pressure was taken as a measure of lung compliance. A third pressure transducer was connected, by a sidearm, to the tube flowing into the pulmonary artery cannula; this pressure, divided by the perfusate flow rate, measured the vascular resistance. The output of each of the three pressure transducers was recorded using a four-channel chart recorder.
Study 2
Pressure transducers were connected to the tracheal, arterial, and venous cannulas. Tracheal pressure and tidal volume, calculated by integration of the flow through a pneumotachograph connecting the trachea to the ventilator, were measured. As in study 1, the ratio of tidal volume to tracheal pressure for a constant value of tracheal pressure measured lung compliance. The difference in pressures between the pulmonary artery and venous cannulas divided by the perfusate flow rate measured vascular resistance. All pressure measurements were recorded using a MacLab 8s analog-to-digital converter connected to a Power Macintosh computer running the MacLab chart software (CAD Instruments, Hastings, UK).
Experimental protocol
Details of the experimental protocols for both studies are shown in Figure 1. Whenever cessation of ventilation was part of a preconditioning protocol, ventilation was stopped at end-exhalation, and the trachea was closed with the lung deflated. During an initial 20-minute period during which lungs underwent control perfusion with sanguineous BB or one of the precondiioning protocols, baseline lung function measurements were taken. The lungs were then flushed with 30 mL of BB storage solution, infused at a pressure of 30 cm H2O. The first 10 mL of the flush was at room temperature (20° to 25°C) to reduce cold-induced vasoconstriction by sudden infusion of 4°C storage solution [11]; the remaining 20 mL was infused at 4°C. Typically the whole 30-mL flush was completed in 150 seconds. The flushed lungs were stored inflated and immersed in the storage solution, with the vasculature open; they were maintained at 4° to 6°C throughout the storage period. After the storage period, lungs were removed from the storage solution, reattached to the perfusion circuit, and reperfused (at 37°C) with sanguineous BB for 40 minutes, using blood obtained from a second rat.
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Study 2
To determine the importance of ventilation or perfusion on the initiation of the preconditioning stimulus, an additional five groups were studied (Fig 1): group 1, control aerobic perfusion (60 minutes) without storage; group 2, 20 minutes of control perfusion before flush and storage for 6 hours in BB (ischemic control); group 3, 2 cycles of 5 minutes of ischemia (cessation of ventilation and perfusion) and 5 minutes of reperfusion before flush and storage for 6 hours in BB; group 4, 2 cycles of 5 minutes of cessation of ventilation with continued perfusion and 5 minutes of restored ventilation before flush and storage for 6 hours in BB; and group 5, 2 cycles of 5 minutes of cessation of perfusion with continued ventilation and 5 minutes of restored perfusion before flush and storage for 6 hours in BB. Each experimental group consisted of lungs from 5 to 7 animals.
Determination of wet to dry weight ratios
At the end of the 40-minutereperfusion period, the lungs were removed from the perfusion chamber, the surface fluid was blotted off, and they were then weighed. The lungs were then stored in an oven for 24 hours at 80°C and reweighed.
Statistics
The data are displayed as mean ± standard error of the mean. To compare the effects of various treatments on lung function over the time course of reperfusion, trapezoid integration was used to calculate the area under the time-response curve for each variable for each animal. For unstored control lungs, the final 40 minutes of the 60-minute perfusion period was taken as being analogous to the reperfusion period in stored lungs. The individual area under the curve values were then used for statistical comparisons of the various groups. Comparisons among groups were performed by one-way analysis of variance, and if this revealed significant differences, a two-sided Dunnett’s test was used to compare multiple values with controls. In all tests, a p value of less than 0.05 was considered significant.
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Results |
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Storage in BB alone for 6 hours (group 2) resulted in a significant fall in pulmonary compliance to 0.015 ± 0.005 mL/cm H2O after 40 minutes of reperfusion (Fig 2) compared with the control, unstored, lungs (0.066 ± 0.005 mL/cm H2O). In contrast, ischemic preconditioning before storage in BB attenuated the fall in pulmonary compliance, with the best recovery (0.038 ± 0.004 mL/cm H2O) being seen in those lungs subjected to two preconditioning episodes of 5 minutes of ischemia and 5 minutes of reperfusion before the extended storage period (ie, group 5). This is reflected both in the area under the curve values for this variable in the various treatment groups assessed during the 40-minute reperfusion time (Table 1) and the final values measured at the end of the reperfusion period (Fig 2).
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The difference in perfusate pH across the lung fell from 0.20 ± 0.03 in unstored controls to 0.04 ± 0.02 in those stored for 6 hours in BB after 40 minutes of reperfusion (Fig 3A). Slight increases in poststorage gas exchange in preconditioned lungs did not translate to statistically significant improvements compared with lungs stored without preconditioning, nor were the various preconditioning groups different from one another.
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Study 2
In study 1, pulmonary compliance was the only measurement that demonstrated a significant improvement after storage by the preconditioning protocols, thus, only data for this variable are presented for this study. After 20 minutes of control perfusion in group 2 (ischemic controls), compliance was 0.072 ± 0.006 mL/cm H2O. As was the case in study 1, the other groups studied did not differ significantly from this value at this point, and the preconditioning protocols did not result in any detectable change in lung function. The effects of 6 hours of unprotected storage in BB followed by 40 minutes of reperfusion were similar to those seen in study 1 (Fig 4, Table 2), with compliance in group 2 (ischemic controls) falling to 0.026 ± 0.01 mL/cm H2O compared with 0.089 ± 0.007 mL/cm H2O in group 1 (unstored controls). As in study 1, lungs receiving a preconditioning stimulus of two periods of 5 minutes with cessation of both ventilation and perfusion showed a significantly improved recovery of compliance (0.068 ± 0.011 mL/cm H2O). In lungs preconditioned by cessation of ventilation alone (group 4), the compliance at the end of 40 minutes of reperfusion was 0.066 ± 0.011 mL/cm H2O, which was also a significant improvement compared with ischemic controls. Cessation of flow alone as a preconditioning protocol did not improve compliance (0.040 ± 0.010 mL/cm H2O) after 40 minutes of reperfusion (Fig 4). Similar results for area under the curve versus reperfusion time curve are shown in Table 2.
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Comment |
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These data differ in some respects from the findings of Du and colleagues [7], who demonstrated the protective effect of preconditioning in a rat lung transplantation model. In particular, we found no beneficial effect with respect to gas exchange; this may have been caused by differences in methodology, because we used an isolated lung system, stored the lungs in BB, and ventilated the reperfused lungs with room air, whereas in the study by Duand associates [7], lungs were stored in University of Wisconsin solution, implanted in living recipient animals for reperfusion, and ventilated with 100% oxygen. It is conceivable that any of these factors may have an impact on lung function during reperfusion and therefore gas exchange. Indeed, even under the conditions used by Du and coworkers [7], the gas exchange in preconditioned lungs was limited, with oxygenation of the blood being only approximately 30% of that seen in control animals, although this was twice that seen in lungs stored without preconditioning. It is also the case that we used perfusate pH as a surrogate measure for gas exchange; however, this measure has been correlated with oxygenation in other studies [9, 10]. Even with this difference, taken together, our data and the results of Du and colleagues[7] suggest that preconditioning can enhance lung function seen on reperfusion even after several hours of hypothermic storage. Hence, preconditioning of the lung before storage may, subject to confirmatory data from large animal lung transplantation models, represent a new approach in transplantation of this organ.
The precise mechanism of preconditioning at the cellular level is still controversial, particularly with respect to the role of protein kinase C [13, 14]. What is clear is that one or more mediators released during a period of transient ischemia can cause effects consequent to receptor activation and subsequent activation of G-protein, phospholipase C, and a possible interaction with protein kinase C [14] that confers resistance to subsequent sustained ischemia. Adenosine [15], bradykinin [16], and noradrenaline [17] all mimic the effects of preconditioning ischemia in the heart. It seems likely that although a specific mediator can be used to mimic preconditioning pharmacologically, no single one is crucial for preconditioning in the physiologic situation [18]. However, it would be more clinically acceptable to precondition pharmacologically, and future studies in the lung should seek to identify the molecular mechanisms of preconditioning in the lungs and appropriate pharmacologic preconditioning agents.
In the heart, or indeed any organ other than the lung, preconditioning may be achieved simply by occluding the blood supply to all or part of the tissue. The anoxia consequent to this is believed to play a major role in triggering the metabolic responses that result in preconditioning [19]. In the lung, the production of the preconditioning response is not likely to be so straightforward. For example, it has been reported that making lungs ischemic while continuing ventilation does not lead to a reduction in adenosine triphosphate levels [20]. Other authors report that the lung responds differently to oxygenated ischemia compared with ventilation with 100% nitrogen during continued vascular perfusion [8]. Although oxygenated ischemia was not innocuous to the lungs, producing levels of reactive oxygen species and inducing damage (lipid peroxidation) similar to that seen after anoxia-reoxygenation, the nature of the reactive oxygen species generated during the reperfusion or reoxygenation period appeared to differ. The data from the second study presented here clearly indicate a difference with respect to efficacy of preconditioning of the lung according to whether ventilation or perfusion is halted, cessation of perfusion in isolation being ineffective. However, a brief cessation of ventilation with the lung in a collapsed state, as used here, may itself differ from anoxic ventilation, and mechanical effects of collapse and reinflation may be more important than any degree of hypoxia that may develop [21]. Much work, therefore, remains to be done to obtain a complete understanding of the mechanisms of preconditioning in the lung.
In clinical lung transplantation, severe graft dysfunction occurs in 10% to 20% of transplant recipients in the immediate postoperative period [22]. Consequently, there is a need to design strategies for the alleviation of this phenomenon. The data we present here show a protective effect of preconditioning on lungs subsequently subjected to prolonged hypothermic storage, suggesting that preconditioning may have a role to play in enhancing donor lung function in transplantation.
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Acknowledgments |
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References |
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This article has been cited by other articles:
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  S. Pasupathy and S. Homer-Vanniasinkam Surgical Implications of Ischemic Preconditioning Arch Surg, April 1, 2005; 140(4): 405 - 409. [Abstract] [Full Text] [PDF]
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 C. S. H. Ng, S. Wan, and A. P. C. Yim Pulmonary ischaemia-reperfusion injury: role of apoptosis Eur. Respir. J., February 1, 2005; 25(2): 356 - 363. [Abstract] [Full Text] [PDF]
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 R. L. Featherstone and D. J. Chambers Long-term hypothermic lung preservation: does adenosine A1 receptor antagonism have a role in ischemic preconditioning protection? Interactive CardioVascular and Thoracic Surgery, March 1, 2004; 3(1): 182 - 187. [Abstract] [Full Text] [PDF]
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 M. de Perrot and S. Keshavjee Lung preservation Ann. Thorac. Surg., August 1, 2002; 74(2): 629 - 631. [Full Text] [PDF]
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C. D. Raeburn, J. C. Cleveland Jr, M. A. Zimmerman, and A. H. Harken Organ Preconditioning Arch Surg, November 1, 2001; 136(11): 1263 - 1266. [Abstract] [Full Text] [PDF]
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), or 6 hours of storage in bicarbonate buffer after 5 minutes of preconditioning (
), 10 minutes of preconditioning (
), or two periods of 5 minutes of preconditioning (
). Data are expressed as mean ± standard error of the mean, n = 10 lungs per group. Some error bars have been omitted for clarity. *Significantly different from tissues stored for 6 hours in bicarbonate buffer after 40 minutes of reperfusion; 
significantly different from unstored tissues after 60 minutes of perfusion (Dunnett’s test, p < 0.05).
