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Tolerance of the isolated perfused lung to hyperthermia
J
THORAC CARDIOVASC SURG 1991;101:732-9
Tolerance of the isolated perfused lung to hyperthermia With the use of in vivo isolated lung pedusion for targeting antitumor therapy in the treatment of lung cancer, tolerance of normal lung tissue to the tumoricidal conditions becomes the limiting factor. This study was pedormed to determine the short-term tolerance of the lung to hyperthermia. Isolated dog lung lobes were pedused with autologous blood or an artificial salt solution at constant flow. Measurementsof lung weight, extravascular water, vascular volume, serotonin uptake, urea permeability surface area product, perfusion pressure, and lung compliance were made with the temperature at about 37° C. The temperature was then set at between 37° and 45° C, and at the end of the subsequent 2 hours the measurements were repeated. When the temperature was less than about 44.4° C, hyperthermia had no detectable influence on the measured variables. Thus on the time frame consistent with in vivo perfusion therapy the normal lung appears to tolerate a fairly severe hyperthermia.
David A. Rickaby, PhD, Jeffrey F. Fehring, BS, Michael R. Johnston, MD, and Christopher A. Dawson, PhD, Milwaukee, Wis., and Denver, Colo.
Antitumor therapy is generally an attempt to create cytotoxic conditions under whichthe relatively moresusceptible tumor cells are killed while damage to normal cellsis kept within acceptablelimits. Regional perfusion isa meansof targetingtherapyto a particularorganwhile avoiding or minimizing the exposure of uninvolved organs.":' In vivo lung perfusion is a particularlyattractiveregional perfusion approachfor the treatment oflung cancers because, with the use of extracorporeal perfusion technology, lung circulationcan be effectively separated from systemic circulation. Optimal tumoricidal conditionscan be established withinthe lungwhile minimizing systemic toxicity associated with systemic exposure. I, 4 With isolatedlung perfusion the toxicity to normal lung tissue rather than systemic toxicity is the factor limiting the severity of the cytotoxic conditions having practical
utility. Thus optimization of the therapeutic regimen requires knowledge of normal lung tissuetoxicity. Perfusate temperature is an easily controlled variable, and hyperthermia has been used to interferewith tumor growth'"? and to enhance the therapeutic index of chemotherapy and radiation therapy.10-13 In vitro studies have demonstrated synergistic effects of hyperthermia and chemotherapeutic agents.lv'? The maximum temperature and duration in whole body hyperthermia is limited by the tolerance of organs other than the lungs with the result that the thermal tolerance of the lungs cannot be determinedduring whole body hyperthermia. Therefore we performed the present study to begin to evaluate the tolerance of the normal lung to elevated temperaturesinanticipation ofthe useofhyperthermia as an adjunct therapy in in vivo isolated lung perfusion. Methods
From the Department of Physiology, Medical College of Wisconsin, Milwaukee,Wis.,Research Service151,ZablockiVeteransAdministration Medical Center, Milwaukee,Wis., and the Department of Surgery, University of Colorado Health Sciences Center, Denver, Colo. Supported by Public Health Service Grant UOI-CA46088. Received for publication Oct. 30, 1989. Accepted for publication March 12, 1990. Address for reprints: Christopher A. Dawson, PhD, Research Service 151, Zablocki VA Medical Center, 5000 W. National Ave., Milwaukee, WI 53295.
12/1/21376
732
The experiments were performed with an isolated dog left lower lobe preparation previously described in detail.l'' Twentythree mongrel dogs (20.1 ± 3.1 kg body weight [SO]*) were anesthetized with pentobarbital sodium (30 mg/kg intravenously), heparinized (1250 IV/kg), and exsanguinated through a carotid artery. During the exsanguination procedure, 250 ml of saline solution containing 10% dextran (Rheomacrodex, molecular weight 40,(00) was infused. The exsanguination time was 18.3 ± 3.2 minutes (SO). The time from the end of exsanguination to the beginning of reperfusion of the isolated lobes was 15.2 ± 2.6 minutes (SD). Approximately 900 ml of perfu*SD = Standard deviation.
Volume 101 Number 4 April 1991
Tolerance of lung to hyperthermia
45
16
44
14
43 ~
u
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37
2
36 -20
0 -20
0
20
40
60
Time (min)
80
100
120
Fig. 1. Time course for the temperature changes. The 23 lung lobes are grouped according to temperature range. For the control there were six lobes with temperatures from 36.9° C to 38.0° C. Medium refers to seven lobes with temperatures from 39.2° C to 42.9° C, and high refers to 10 lobes with temperatures 43.6° C to 44.9° C. The values are means ± SE. Zero is the time that the heat exchanger temperature was changed. Control measurements, referred to as the before values in Table II, were made just before time O. The after values were obtained just before 120 minutes.
sate, either the autologous blood or salt solution containing KCI 4.7 mmol/L, CaCl2 2.51 rnmol/L, MgS04 1.19 mmol/L, KH2P04 2.5 mmol/L, NaCl 118 mmol/L, NaHC03 25 mmol/L, glucose 5 mmol/L, and 4.5% dextran (average molecular weight 79,100) was used to prime the recirculating perfusion system. The left lower lung lobe was removed, and the lobar artery and vein were attached to the perfusion apparatus, which consisted of a pump, a temperature-controlled perfusate reservoir, and a heat exchanger. The perfusate was pumped from the reservoir, through the heat exchanger, and into the cannulated lobar artery. The perfusate drained from the cannulated lobar vein into the reservoir. The heat exchanger consisted of a heated water bath (1.3 L capacity) in which the perfusate passed through four parallel coils of Tygon tubing (Norton Performance Plastics, Wayne, N.J.) (2.38 mm inner diameter with 0.79 mm wall thickness) with a total volume of approximately 35 ml and a total surface area of about 560 cm 2. The temperature of the water bath was about 1.20° C ± 0.49° C (SO) higher than the lobar artery perfusate temperature at equilibrium. On passage from the lobar artery through the lobe to the lobar vein, the perfusate temperature decreased by about 0.54° ± 0.04° C (SO). The temperatures reported in the Results section are the temperatures of the venous effluent. During an initial period of 7.9 ± 3.0 minutes (SO) after the initiation of the perfusion, the flow was increased slowly to a final mean value of 5.0 ± 0.3 ml/sec (SO), and the venous pressure was set at 1.6 ± 0.7 torr (SO), both of which were then held constant throughout the remainder of the study for each lobe. When the lobes were perfused with the salt solution, the residual lobar blood volume (about 7 ml; Table I) was allowed to mix with the circulating perfusate. Previous experiments indicated that this resulted in a more stable preparation than if all the blood was eliminated by extensive washing of the lobar
t: 1*-
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- - Blood Perfused 44.5 to 44.9 °C. Blood Perfused 36.8 to 44.4
20
40
60
80
100
120
Time (min) Fig. 2. Time course for changes in the arterial-venous pressure drop (Pa-P v) during perfusion. Values are means ± SE. Asterisks indicate that value is significantly increased from initial values, p < 0.Q1.
vascular bed with the perfusate before the recirculating perfusion. In addition, the lobar bronchus was attached to the ventilation system consisting of a piston respirator with adjustable tidal volume and a water overflow valve to maintain end-expiratory airway pressure. The lobes were ventilated at a frequency of about 8 breaths/min with a tidal volume of 75 to 100 mI, depending on the lobe size, with an end-expiratory pressure of about 3 torr. The gas mixture was composed of approximately 16% oxygen and 5.8% carbon dioxide in nitrogen, which maintained perfusate oxygen tension at 103 ± 3.5 torr (SO) and carbon dioxide tension at 39.5 ± 4.0 torr. Blood samples were obtained during perfusion for pH measurements at 37° C. A temperature correction of0.0147 pH unit per degree Celsius for blood or 0.0061 ° C for the salt solution was used to correct for the difference between the electrode temperature and the temperature of the perfusate in the perfusion system. Once the perfusion was begun, each lobe was perfused at a control temperature (36.6° to 38.0° C) for 31.7 ± 6.8 minutes (SO), during which time initial control measurements were made. The lobes were then perfused for an additional 2 hours. During this period the heat exchanger temperature was set at a constant temperature from about 37° to 46° C. When the heat exchanger temperature was altered by changing to a new preheated water source, it took about 3 minutes for the water bath temperature to change to the new temperature. The time course for the perfusate temperature change can be seen in Fig. 1. Lobar arterial and venous pressures, measured relative to the height of the venous cannula at approximately the top surface of the lobes, were recorded continuously. Airway pressure was also recorded continuously and used to detect changes in lung compliance. Measurements ofvascular and extravascular volume, serotonin uptake, and urea permeability surface area product were made during the control period and at the end of the perfusion period with multiple indicator dilution techniques. The permeability surface area product was measured only in the lobes perfused with salt solution. To make the measurements the ventilator was stopped at end expiration and a bolus containing iodine 125 human serum albumin (HSA) (0.15 ILCi), tritiated water
The Journal of Thoracic and Cardiovascular Surgery
7 3 4 Rickaby et al.
Table I. Measured variables related to hemoglobin content data Variables
Wet weight (gm) Blood included Bloodfree Wet weight/dry weight Blood included Bloodfree Residual blood volume (ml) Extravascular volume gravimetric (ml) Extravascular volume indicator dilution (ml) Vascular volume (ml)
Before
After
39.7 ± 3.6 32.7 ± 3.8
44.7 ± 3.2 34.8 ± 3.1
4.58 4.52 6.8 23.9
± ± ± ±
0.03 0.05 1.2 1.8
4.72 4.60 9.5 27.2
± ± ± ±
0.05 0.11 1.3 2.5t
22.3 ± 1.6
25.8 ± 1.5*
35.2 ± 3.6
34.8 ± 3.1
Values are means ± standard error; n = 6. Extravascular volume from 3HOH and [1251]HSA indicator dilution method orthe bloodfree wet weight minus the bloodfree dry weight for the gravimetric method. Vascular volume from [ 1251]HSA dilution. For the gravimetricvalues, before refers tothe valuesobtained from the left upper lobe orfrom the fractional upper lobe values times the initial left lower lobe weight. The temperatures studied for these six lungs were 37.3', 39.2°,39.8°,42.9°,44.7°,and 44.7° C, and no significant correlations with temperature were found. *p < 0.01. tp < 0.05.
(5 JLCi), and carbon 14 seroto~in (0.3 JLCi [5 nmol]) or [14C] urea (0.5 JLCi) was rapidly introduced into the arterial cannula with an injector situated in the inflow tubing. The outflowwas simultaneouslydivertedfrom the reservoirto the samplingtubes of a Gilson Escargot fraction collector (Gilson Medical Electronics, Inc., Middleton, Wis.). The sampling rate was set at 1.67 samples/sec. Sampling tubes were prefilled with 9 ml of coldethanol to precipitatethe [125] HSA for gamma scintillation counting and to clear the samples for hydrogen 3 and 14C measurement by liquid scintillation counting. At the end of the experiment the lobe was removed and the arterial and venous cannulas were connected directly. A bolus containing [125I]HSA was then injected and samples were collectedin the same manner as when the lung lobe was in place to obtain the mean and variance of the tubing transit times. The mean transit time (t) of [I25I]HSA or 3HOH was calculated as follows:
t
=f o
T
tC(t) dt /
IT
C(t) dt
0
and the variance «(12) of the outflowconcentrationcurveswas calculated from the following: (12
=f T(t-t)2C(t) dt / IT C(t) dt o
0
where C is the concentration of [I25I]HSA or 3HOH, and T is the time (t) at which C had decreased to 1%of its peak value. The lobar t or cl- was calculated by subtracting the respective values oft or (12 obtained when the lobe was removed from the perfusioncircuit from the t or (12 obtained with the lobein place.
The vascular volume was the product of the lobar t for [I25I]HSA and the perfusate flow rate. The volumeof distributionof 3HOH wasthe lobar t for 3HOH timesthe flow rate. The extravascular volume accessible to 3HOH was the difference between vascular volume and the volume of distribution for 3HOH. The relativedispersion of the lobar transport function19 was the lobar (1/t for the [125I]HSA. The fraction of injected serotonin taken up by the lung during passage of the bolus (U) was calculated as follows: U=
Q
f T [CR(t) - Cs(t)] dt o
where Q is flow rate in millilitersper second,CRand Cs are the concentrationsof [125I]HSA and serotonin, respectively, divided by the amounts injected. . The permeability surface area product for urea was calculated as follows: PS = -
where
E;
~ 1-
[f'
Q In
(1- Ei)
Cs(t) dt /
f'
CR(t)
dt]
and Cs is the normalized urea concentration and p is the time that CR reached its maximal value.-" Each lobewasweighedbeforebeingattached to the perfusion system and after being removedfrom the perfusionsystem.At the end of the experiment the lobes were lyophilized.to determine dry weight. The left upper lobes were removed immediately after the chest was openedand werealso weighedand lyophilized. The wet-to-dry weight ratios of the upper lobeswere used as an estimate of the preperfusionwet-to-dryweightratios of the left lower lobes. After perfusionthe blood-freeweight and dry weightsof six of the blood-perfused lobeswere estimated with the hemoglobin method. For this purpose, a sample of blood drawn from the reservoirat the end of the experimentwas used to determinethe bloodconcentration of hemoglobin. The lyophilized lobeswere ground to a powder that was extracted with Drabkin's reagent (200 ml/gm). The mixture was centrifuged for 20 minutesand filtered (0.22 JLm filter),and the opticaldensityof the filtratewas determined at 545 nm. The processwas repeated on the precipitate until absorbance of the filtrate fell below0.025. The residual blood volume in the lobe was determined by dividing total hemoglobin in the lobe by the bloodhemoglobin concentration. Preperfusionlobar residual bloodvolumesper gram of left lower lobe were estimated with the same method on the left upper lobes and blood samples drawn at the end of exsanguination. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciencesand publishedby National Institutesof Health (NIH Publication No. 80-23, revised 1978).
Results The time courses for the temperature changes are shown in Fig. I. For this graph the data for the bloodperfused lobes and the lobes perfused with salt solution
Volume 101 Number 4 April 1991
Tolerance of lung to hyperthermia 7 3 5
Table II. Measuredvariables in blood and lung lobes perfused with Krebs-Ringer bicarbonate solution Blood p.-Pv(torr) 36.8°-44.4 ° C 44.5°-44.9° C Qev (ml) 36.8°-44.4° C 44Y-44.9° C QL (ml) 36.8°-44.4° C 44.5°-44.9° C
Salt solution
Before
After
n
Before
7.7 ± 0.5 9.0 ± 0.3
7.3 ± 0.5 12.8 ± 0.6*
12 5
5.6 ± 0.7
5.8 ± 0.8
6
20.5 ± 1.3 23.6 ± 0.3
23.7 ± 1.2* 27.6 ± 0.8t
10 4
19.7 ± 1.6
21.0 ± 1.9
6
33.2 ± 2.9 30.7 ± 1.9
33.8 ± 2.5 30.5 ± 1.6
11 5
34.0 ± 3.5
31.9 ± 4.1
6
0.46 ± 0.03 0.42 ± 0.03
0.48 ± 0.03 0.53 ± 0.02*
12 4
0.38 ± 0.02
0.40 ± 0.02
6
86.2 ± 1.8 91.0 ± 2.4
88.3 ± 1.1 88.1 ± 2.7
10
91.8 ± 1.1
90.6 ± 1.1
6
1.50 ± 0.15
2.01 ± 0.28t
6
After
n
RD 36.8°-44.4° C 44.5°-44.9° C U 5-HT (%) 36.8°-44.4° C 44.5°-44.9° C PS Urea (ml/sec) Wet Weight (gm) 36.8°-44.4 ° C 44.5°-44.9° C Wet/dry Weight 36.8°-44.4° C 44.5°-44.9° C C (mI/cmH20) 36.S°-44.4° C 44.5°-44.9° C pH 36.8°-44.4° C 44.5-44.9° C
4
37.5 ± 2.5 39.3 ± 1.2
42.6 ± 2.4* 49.7 ± 4.7
12 5
40.2 ± 3.8
41.5 ± 3.7
6
4.30 ± 0.13 4.52 ± 0.38
4.88 ± 0.09* 5.46 ± 0.59t
12 4
4.96 ± 0.06
5.16 ± 0.14
6
36.8 ± 4.1 35.6 ± 8.7
23.3 ± 2.8* 21.6 ± 4.6t
12 5
37.9 ± 2.4
22.2 ± 2.1*
6
7.39 ± om 7.39 ± 0.02
7.38 ± om 7.37 ± 0.04
11 5
7.45 ± 0.01
7.40 ± 0.01*
6
Values are means ± standard error; n = number of experiments in which each variable was measured. Before refers to measurements made during the control period at 37.2 ± 0.4" C (SD) before the subsequent 2-hour exposure to either hyperthermic or normothermic perfusion. After refers to measurements made at the end of perfusion. Pa, Lobar artery pressure; Pv, lobar venous pressure; Qev, extravascular volume; QL, vascular volume; RD, lobar relative dispersion; U 5-HT, serotonin uptake; PS urea, urea permeability surface area product; C, lobar compliance. 'Indicates after value significantly different from before value, p < 0.01. tp < 0.05.
are combined, since the temporal pattern was similar in the twogroups. The lobes havebeendivided into control, medium, and high temperature groups for this graph to give a perspective on the time course over the range of temperatures studied. The results of the measurements made before and at the endof the experimental periodare shown in Table II. There were few differences that could be ascribed to hyperthermia. When the temperaturewasbelow approximately 440 C we could detect no correlation between temperature and any of the measured variables. Above 44.4° C in the blood-perfused lungs there was a significant increase in perfusion pressurenear the end of perfusion, as shown in Fig. 2. Therefore we have divided the results intotwogroups, oneaboveand onebelow 44.40 C. The only other temperature-dependent result was a significant increasein relative dispersion when the temperaturewasabove44.4° C. Overalltherewasa smallbut
significant (p < 0.05)correlation (r = 0.574)betweenthe relative dispersion and the changes in perfusion pressure that occurred during perfusion. This suggests that increases invascularresistance wereassociated withmore heterogeneous perfusion, and decreases were associated with more homogeneous perfusion. In the high temperature group the averageweightgain of the lung lobeswas considerably greaterthan inthe lowertemperaturegroup. However, this mean is heavily weighted by the one lobe atthehighesttemperaturestudied, 44.90 C, whichgained 27 gm and was clearly edematous. Severalchangesoccurredin the lobesduring perfusion, which were independent of temperature. In the bloodperfused lobes these included an increasein wet weight, extravascular volume and wet-to-dry weightratio, and a decrease in lung compliance. In lungs perfusedwith salt solution similartendencies werenoted,although onlythe decrease in compliance was significant. The decrease in
The Journal of Thoracic and Cardiovascular Surgery
7 3 6 Rickaby et al.
2.0 1.5 ........ Ul <,
11.0
70
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60 50
E
40
~
30
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20
0.5
10 0.0
36
38
40 42 TEMP (OC)
44
46
0 36
38
40 42 TEMP (OC)
44
46
Fig. 3. Summary of the urea permeability surface area product (PS) changes in the lungs perfused with salt solution. Measurements were made before (circles) and at the end (arrowheads) of the 2-hour experimental perfusion period. Vertical lines connect before and after values for each lobe studied.
Fig. 4. Summary of wet weight (Wt) of the lungs measured before being attached to perfusion system (circles) and after being removed from perfusion system (arrowheads). Vertical lines connect the before and after values for each lobe studied. Closed symbols are for the blood-perfused lung lobes, and open symbols are for the lung lobes perfused with salt solution.
compliance was almost linear with time, with no significant difference between blood-perfused lobes and those perfused with salt solution. For the lobes perfused with salt solution we added the measurement of the urea permeability surface area product, which was significantly greater at the end of perfusion than at the beginning (Table II, Fig. 3). Figs. 3 to 6 summarize the pulmonary vascular variables that were significantly altered by perfusion. These include lung weight, wet-to-dry weight ratio, extravascular water, and urea permeability surface area product. BlOOd volume and serotonin uptake were not influenced by perfusion and are summarized only in Table II. Table I summarizes the gravimetric data, wherein the influence of the residual blood volume was taken into account. These data suggest that the increase in lung weight included an increase in extravascular water as opposed to simply an increase in residual blood volume. In addition, it can be noted that the indicator dilution extravascular volume was a substantial fraction of the gravimetric estimate of extravascular water, and the increase in the gravimetric extravascular volume was paralleled by an increase in indicator dilution extravascular volume.
ery will be possible if substantial acute lung injury is produced during perfusion. Even if injury is not detected during the acute phase, it is conceivable that the conditions established during perfusion could result in impaired recovery of lung function during resuscitation (the subacute recovery phase). Iflung tolerance is acceptable, as judged from the responses measured during the first two phases, the issue of chronic toxicity can be dealt with and criteria established for evaluating the long-term outcome. Experimental evaluation including all three phases can be an expensiveproposition, and any protocol to evaluate the last two phases requires that acute toxicity does not terminate the study during perfusion. We performed the present studies on isolated perfused dog lung lobes in vitro in an attempt to evaluate the acute tolerance of the lungs to elevated temperatures and thus to establish conditions that are potentially useful for in vivo study. A number of variables exist that could he used to evaluate the status of the lung. It appears that the ability of the isolated perfused lung to maintain constant weight is a summary criterion that reveals the impact of changes in other variables on the viability of the lungs. In other words, pulmonary .edema is the final common end point of acute lung injury of diverse causes. Therefore we have used the changes in lung weight occurring during perfusion as an index of lung injury. The use of lung weight as a criterion has at least two potential disadvantages. First, lung weight is not easily measured during in vivo perfusion; second, in the absence of a hemodynamic cause, endothelial injury with increased permeability is the antecedent of edema. Thus an increase in weight occurs as a secondary event both mechanistically and temporal-
Discussion For the evaluation of in vivo perfusion conditions it is useful to consider the acute, subacute, and chronic phases resulting from the surgical procedure. It is necessary that during perfusion (the acute phase) the normal lung tissue is able to tolerate the therapeutic conditions that are established. It is unlikely that resuscitation and/or recov-
Volume 101 Number 4
Tolerance of lung to hyperthermia 7 3 7
April 1991
8
~
6
z-
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4
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1 ~
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30
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10
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5 O+-----+---+----t----+-------1 36 38 40 42 46 44 TEMP (OC)
0 36
38
40 42 TEMP (OC)
44
46
Fig. 5. Summary of the wet-to-dry weight ratios measured on the left upper lobe immediately after the chest was opened (circles) and on the left lower lobe after being removed from the perfusionsystem (arrowheads). Vertical lines connect the before and after values for each lobe studied. Closed symbols are for blood-perfused lung lobes, and open symbols are for the lung lobes perfused with salt solution.
Fig. 6. Summary of indicator dilution extravascular water (Q",) measurements. Measurements were made before (circles) and at the end (arrowheads) of the 2-hour experimental perfusion period. Vertical lines connect before and after values for each lobe studied. Closed symbols are for bloodperfused lungs, and open symbols are for lungs perfused with salt solution.
ly. Therefore in the present study we also used indicator dilution methods that can be readily applied in viv04, 20-24 and that- may also address the events leading to edema before the accumulation of excess fluid. The rationale behind this study included the concept that correlations between the direct index ofinjury obtainable in vitro (lung weight) and some variables that also can be measured in vivo would provide a basis for establishing useful measurements to be made in future in vivo isolated perfusion studies. To date, in vivo lung perfusions have been limited to about 1 hour. I, 4 In the present study the total perfusion time was about 2lf2 hours with the intent of encompassing the presently acceptable range of in vivo perfusion times. We did not attempt to optimize all possible lung perfusion conditions. For the present study that would have been impractical. Instead we used methods that have been used with reasonable success in the past.l'' Accepting the fact that deterioration with perfusion time occurs in any in vitro isolated perfused lung preparation.P our major interest was in whether this deterioration was measurably accelerated by temperatures in excess of about 37 0 C. Because one of the advantages of isolating lung perfusion in vivo is the potential for altering perfusate composition to gain therapeutic advantage.l't 26 we also examined whether the utilization of a nonblood perfusate would alter the range of temperatures tolerated by the preparation. The experiments were performed on dog lung lobes because the dog has proved to be a useful animal model in the development of in vivo lung perfusion procedures. I, 4
The results indicate that in the isolated perfused dog lung temperatures as high as 44 0 C for approximately 2 hours produced little short-term damage detectable by the measured variables beyond that produced by the isolated perfusion procedure. Lung perfusion was associated with some apparently detrimental changes, irrespective of temperature. These included a small increase in weight and a progressive decrease in compliance. These changes do not appear to have been of sufficient magnitude to mask any added influence of hyperthermia had it occurred. The most notable impact of hyperthermia on the variables studied was a tendency for an increase in perfusion pressure in the blood-perfused lungs when the temperature was 44.5° C and above. The mechanism responsible for this increase is not known, but hardening of the erythrocytes by the high temperature is consistent with the results.i? Otherwise, there was little to distinguish the higher temperature experiments, except that the one lung that was perfused at the highest temperature studied, 44.9° C, gained substantially more than the average weight gain. The increase in lung weight and extravascular water volume that occurred regardless of temperature can be compared with what might be expected for a lung lobe from a 20 kg dog on the basis of normal lung fluid transport. The normal lung lymph flow rate appears to be about 1 mljhr for the left lower lobe. 28 Assuming that this rate approximates the net fluid transport out of the pulmonary capillaries in vivo, we can compare the normal fluid transport with the rate of weight gain in the isolated lungs. The average weight gain was about 5 gm over the
The Journal of Thoracic and Cardiovascular Surgery
7 3 8 Rickaby et al.
2Y2-hour perfusion period, and the increase in extravascular volume was about 3 gm over the 2-hour experimental period in the blood-perfused lobes. These rates are greater than expected in vivo, even if there were no lymphatic drainage or pleural effusion from the isolated lobes. This appears typical for isolated perfused lungs. 25 The reasons are not completely understood. They are of interest not only for possible relevance to in vivo isolated perfusion, but also in regard to problems of lung storage before transplantation.P'" We added the measurement of the permeability surface area product for the lobes perfused with salt solution, and the results are consistent with an increase in permeability for small hydrophilic molecules. In these lungs, however, there was little weight gain. Unfortunately, this variable was not measured in the blood-perfused lungs. It is conceivable that a larger increase in urea permeability might have been observed in conjunction with the larger weight gain, but this remains to be examined. We suspect that the decrease in lung compliance with perfusion time may be the result of progressive microateleetasis, because the increases in lung fluid volume that occurred during the perfusion period appear to be too small to have had a measurable influence on lung mechanics. This question, however, remains to be resolved. Serotonin uptake has been considered an indicator of pulmonary endothelial cell function.l': 23, 24, 32 particularly with endothelial injury associated with oxidant stress." which has been associated with hyperthermia in studies on the liver. 34 We could detect no significant changes in the serotonin uptake in the present study. This suggests that metabolic functions involved in the serotonin uptake process were not disrupted to the extent that significant interference with serotonin uptake could be observed. The fact that there appeared to be an increase in permeability surface area product for urea with no change in serotonin uptake suggests that the processes controlling urea permeability and serotonin uptake are not tightly linked or that serotonin uptake is relatively insensitive to such changes. The extravascular volume accessible to the 3HOH was quite close to the gravimetrically determined extravascular volume in this preparation, and it tracked the increase with perfusion time. This suggests that this measurement may have utility in in vivo studies in which whole lung gravimetric measurements are impractical. For in vivo perfusion the use of a nonblood perfusate has a number of potential advantages. The ability to manipulate substrate composition and the absence of plasma proteins are two useful features. The latter is advantageous because many chemotherapeutic agents bind to plasma proteins. The elimination of these proteins
from the perfusate would allow for much smaller vascular concentrations to be used, with the result that effective pulmonary-systemic crossover after reconnection of the pulmonary circuit would be virtually eliminated. In the present study we could detect no disadvantages to the particular salt solution used. There was no attempt to find an optimal perfusate. However, the lobes appeared to tolerate both the particular solution used and the hyperthermia as well as the blood-perfused lungs. The tendency was for a smaller weight gain than with the blood-perfused lungs. The salt solution did contain approximately 1% autologous blood, which we suspect is an important addition to this particular perfusate, based on previous observations. The observations made in this study cannot be taken as evidence that hyperthermia up to about 44 0 C had no deleterious influence, It is not practical to evaluate all the variables that might be important. We chose a few that have been used as measures oflung injury in the past. The changes in these measures associated with lung injury are well documented. However, their sensitivity to those aspects of the injury that determine the long-term outcome is not known. The important test in the future will be to determine whether the hyperthermic exposure alters recovery oflung function invivo. The present studies suggest that such studies are feasible and that acuteinjury during the perfusion period is not likely to be the limiting factor in such studies.
1.
2.
3.
4.
5. 6. 7. 8. 9.
REFERENCES Minchin RF, Johnston MR, Aiken MA, Boyd MR. Pharmacokinetics of doxorubicin in isolated lung of dogs and humans perfused in vivo. J Pharmacol Exp Ther 1984; 229:193-8. Roseman JM, Tench D, Bryant LR. The safe use of cisplatin in hyperthermic isolated limb perfusion systems. Cancer 1985;56:742-4. Grace RH, Scott KWM. Successful treatment of multiple liver metastases by liver perfusion. Br Moo J 1987;295: 637-8. Johnston MR, Christensen CW, Minchin RF, et al. Isolated total lung perfusion as a means to deliver organ-specific chemotherapy: long-term studies in animals. Surgery 1985; 98:35-44. Cavaliere R, Ciocatto EC, Giovanella BC, et al. Selective heat sensitivity of cancer cells. Cancer 1967;20:1351-81. Dickson JA. Hyperthermia in the treatment of cancer. Cancer Chemother Rep 1974;58:294-6. Suit HD, Shwayder M. Hyperthermia: potential as an anti-tumor agent. Cancer 1974;34:122-9. OVergaard J. Effect of hyperthermia on malignant cells in vivo. A review and a hypothesis. Cancer 1977;39:2637-46. Giovanella BC, Morgan AC, Stehlin JS, Williams LJ.
Volume 101 Number 4
Tolerance of lung to hyperthermia
April 1991
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Selective lethal effects of supranormal temperatures of mouse sarcoma cells. Cancer Res 1973;33:2568-78. Herman TS, Teicher BA, Holden SA, Collins LS. Interaction of hyperthermia and radiation in murine cells: hypoxia and acidosis in vitro, tumor subpopulations in vivo.Cancer Res 1989;49:3338-43. Kim JH, Hahn EW, Ahmed SA. Combination hyperthermia and radiation therapy for malignant melanoma. Cancer 1982;50:478-82. Leith JT, Miller RC, Gerner EW, Boone MLM. Hyperthermic potentiation. Biologicalaspects and applications to radiation therapy. Cancer 1977;39:766-79. Wallner KE, Li GC. Effect of drug exposure duration and sequencing on hyperthermic potentiation of mitomycin-C and cisplatin. Cancer Res 1987;47:493-5. Hahn GM, Braun J, Har-Kedar I. Thermochemotherapy: synergism between hyperthermia (42-43 degrees) and Adriamycin (or bleomycin) in mammalian cell inactivation. Proc Nat Acad Sci 1975;72:937-40. Marmor JB, Kozak D, Hahn G M. Effects of systemically administered bleomycin or Adriamycin with local hyperthermia on primary tumor and lung metastases. Cancer Treat Rep 1979;63:1279-90. Lanks KW, Shah V, Chin NW. Enhancing hyperthermic cyctotoxicity in L929 cells by energy source restriction and insulin exposure. Cancer Res 1986;46:1382-87. Honess DJ, Bleehen NM. Thermochemotherapy with cisplatinum, CCNU, BCNU, chlorambucil and melphalan on murine marrow and two tumours: therapeutic gain for melphalan only. Br J RadioI1985;58:63-72. Rickaby DA, Dawson CA, Linehan JH. Influence of embolism and imipramine on kinetics of serotonin uptake by dog lung. J Appl Physiol 1984;56:1170-7. Dawson CA, Bronikowski TA, Linehan JH, Hakim TS. Influence of pulmonary vasoconstriction on lung water and perfusion heterogeneity. J Appl Physiol 1983;54:654-60. Brigham KL, Kariman K, Harris TR, Snapper JR, Bernard GR, Young SL. Correlation of oxygenation with vascular permeability-surface area but not with lung water in humans with acute respiratory failure and pulmonary edema. J Clin Invest 1983;72:339-49. Gillis CN, Catravas JD. Altered removal of vasoactive substances in the injured lung: detection of lung microvascular injury. Ann NY Acad Sci 1982;384:458-74. Brigham KL, Snell JD, Jr, Harris TR, et al. Indicator dilu-
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tion lung water and vascular permeability in humans. Circ Res 1979;44:523-30. Morel DR, Dargent F, Bachmann M, Suter PM, Junod AF. Pulmonary extraction of serotonin and propranolol in patients with adult respiratory distress syndrome. Am Rev Respir Dis 1985;132:479-84. Gillis CN, Pitt BR, Weidemann HP, Hammond GL. Depressed prostaglandin E 1 and 5-hydroxytryptamine removal in patients with adult respiratory distress syndrome. Am Rev Respir Dis 1986;134:739-44. Pearse DB, Brower RG, Adkinson NF Jr, Sylvester JT. Spontaneous injury in isolated sheep lungs: role of perfusate leukocytes and platelets. J Appl Physiol 1989;66:1287-96. Demetrakopoulos GE, Linn B, Amos H. Rapid lossof ATP by tumor cellsdeprived of glucose: contrast to normal cells. Biochem Biophys Res Commun 1978;82:787-94. Greene R, Hughes JMB, I1iffLD, Pineo GF. Red cell flexibility and pressure-flow relations in isolated lungs. J Appl PhysioI1973;34:169-75. Staub NC. Pulmonary edema. Physiol Rev 1974;54:678811. Miyamoto Y, Lajos TZ, Bhayana IN, Bergsland J, Celik CF. Beneficial effects of prostaglandin E] on autoperfused heart-lung preservation. J Heart Transplant 1988;7:135-9. Hachida M, Morton DL. Lung function after prolonged lung preservation. J THORAC CARDIOVASC SURG 1989; 97:911-9. Wahlers T, Haverich A, Fieguth HG, Schafers HJ, Takayama T, Borst HG. Flush perfusion using Euro-Collins solution vs cooling by means of extracorporeal circulation in heart-lung preservation. J Heart Transplant 1986;5:89-98. Dawson CA, Christensen CW, Rickaby DA, Linehan JH, Johnston MR. Lung damage and pulmonary uptake of serotonin in intact dogs. J Appl Physiol 1985;58: 1761-6. Dawson CA, Linehan JH. Biogenic Amines. In: Massaro D, ed. Lung biology in health and disease-Lung cell biology. Vol 41. New York: Marcel Dekker; 1989:1091139. Skibba JL, Quebbeman EJ, Kalbfleisch JH. Nitrogen metabolism and lipid peroxidation during hyperthermic perfusion of human livers with cancer. Cancer Res 1986; 46:6000-3.
THORAC CARDIOVASC SURG 1991;101:732-9
Tolerance of the isolated perfused lung to hyperthermia With the use of in vivo isolated lung pedusion for targeting antitumor therapy in the treatment of lung cancer, tolerance of normal lung tissue to the tumoricidal conditions becomes the limiting factor. This study was pedormed to determine the short-term tolerance of the lung to hyperthermia. Isolated dog lung lobes were pedused with autologous blood or an artificial salt solution at constant flow. Measurementsof lung weight, extravascular water, vascular volume, serotonin uptake, urea permeability surface area product, perfusion pressure, and lung compliance were made with the temperature at about 37° C. The temperature was then set at between 37° and 45° C, and at the end of the subsequent 2 hours the measurements were repeated. When the temperature was less than about 44.4° C, hyperthermia had no detectable influence on the measured variables. Thus on the time frame consistent with in vivo perfusion therapy the normal lung appears to tolerate a fairly severe hyperthermia.
David A. Rickaby, PhD, Jeffrey F. Fehring, BS, Michael R. Johnston, MD, and Christopher A. Dawson, PhD, Milwaukee, Wis., and Denver, Colo.
Antitumor therapy is generally an attempt to create cytotoxic conditions under whichthe relatively moresusceptible tumor cells are killed while damage to normal cellsis kept within acceptablelimits. Regional perfusion isa meansof targetingtherapyto a particularorganwhile avoiding or minimizing the exposure of uninvolved organs.":' In vivo lung perfusion is a particularlyattractiveregional perfusion approachfor the treatment oflung cancers because, with the use of extracorporeal perfusion technology, lung circulationcan be effectively separated from systemic circulation. Optimal tumoricidal conditionscan be established withinthe lungwhile minimizing systemic toxicity associated with systemic exposure. I, 4 With isolatedlung perfusion the toxicity to normal lung tissue rather than systemic toxicity is the factor limiting the severity of the cytotoxic conditions having practical
utility. Thus optimization of the therapeutic regimen requires knowledge of normal lung tissuetoxicity. Perfusate temperature is an easily controlled variable, and hyperthermia has been used to interferewith tumor growth'"? and to enhance the therapeutic index of chemotherapy and radiation therapy.10-13 In vitro studies have demonstrated synergistic effects of hyperthermia and chemotherapeutic agents.lv'? The maximum temperature and duration in whole body hyperthermia is limited by the tolerance of organs other than the lungs with the result that the thermal tolerance of the lungs cannot be determinedduring whole body hyperthermia. Therefore we performed the present study to begin to evaluate the tolerance of the normal lung to elevated temperaturesinanticipation ofthe useofhyperthermia as an adjunct therapy in in vivo isolated lung perfusion. Methods
From the Department of Physiology, Medical College of Wisconsin, Milwaukee,Wis.,Research Service151,ZablockiVeteransAdministration Medical Center, Milwaukee,Wis., and the Department of Surgery, University of Colorado Health Sciences Center, Denver, Colo. Supported by Public Health Service Grant UOI-CA46088. Received for publication Oct. 30, 1989. Accepted for publication March 12, 1990. Address for reprints: Christopher A. Dawson, PhD, Research Service 151, Zablocki VA Medical Center, 5000 W. National Ave., Milwaukee, WI 53295.
12/1/21376
732
The experiments were performed with an isolated dog left lower lobe preparation previously described in detail.l'' Twentythree mongrel dogs (20.1 ± 3.1 kg body weight [SO]*) were anesthetized with pentobarbital sodium (30 mg/kg intravenously), heparinized (1250 IV/kg), and exsanguinated through a carotid artery. During the exsanguination procedure, 250 ml of saline solution containing 10% dextran (Rheomacrodex, molecular weight 40,(00) was infused. The exsanguination time was 18.3 ± 3.2 minutes (SO). The time from the end of exsanguination to the beginning of reperfusion of the isolated lobes was 15.2 ± 2.6 minutes (SD). Approximately 900 ml of perfu*SD = Standard deviation.
Volume 101 Number 4 April 1991
Tolerance of lung to hyperthermia
45
16
44
14
43 ~
u
42
E40 Q)
12
>
8
* *--
~10
~41
t-
'2 0...
I
39
0
0...
:r-l-I-I-I- -1-
37
2
36 -20
0 -20
0
20
40
60
Time (min)
80
100
120
Fig. 1. Time course for the temperature changes. The 23 lung lobes are grouped according to temperature range. For the control there were six lobes with temperatures from 36.9° C to 38.0° C. Medium refers to seven lobes with temperatures from 39.2° C to 42.9° C, and high refers to 10 lobes with temperatures 43.6° C to 44.9° C. The values are means ± SE. Zero is the time that the heat exchanger temperature was changed. Control measurements, referred to as the before values in Table II, were made just before time O. The after values were obtained just before 120 minutes.
sate, either the autologous blood or salt solution containing KCI 4.7 mmol/L, CaCl2 2.51 rnmol/L, MgS04 1.19 mmol/L, KH2P04 2.5 mmol/L, NaCl 118 mmol/L, NaHC03 25 mmol/L, glucose 5 mmol/L, and 4.5% dextran (average molecular weight 79,100) was used to prime the recirculating perfusion system. The left lower lung lobe was removed, and the lobar artery and vein were attached to the perfusion apparatus, which consisted of a pump, a temperature-controlled perfusate reservoir, and a heat exchanger. The perfusate was pumped from the reservoir, through the heat exchanger, and into the cannulated lobar artery. The perfusate drained from the cannulated lobar vein into the reservoir. The heat exchanger consisted of a heated water bath (1.3 L capacity) in which the perfusate passed through four parallel coils of Tygon tubing (Norton Performance Plastics, Wayne, N.J.) (2.38 mm inner diameter with 0.79 mm wall thickness) with a total volume of approximately 35 ml and a total surface area of about 560 cm 2. The temperature of the water bath was about 1.20° C ± 0.49° C (SO) higher than the lobar artery perfusate temperature at equilibrium. On passage from the lobar artery through the lobe to the lobar vein, the perfusate temperature decreased by about 0.54° ± 0.04° C (SO). The temperatures reported in the Results section are the temperatures of the venous effluent. During an initial period of 7.9 ± 3.0 minutes (SO) after the initiation of the perfusion, the flow was increased slowly to a final mean value of 5.0 ± 0.3 ml/sec (SO), and the venous pressure was set at 1.6 ± 0.7 torr (SO), both of which were then held constant throughout the remainder of the study for each lobe. When the lobes were perfused with the salt solution, the residual lobar blood volume (about 7 ml; Table I) was allowed to mix with the circulating perfusate. Previous experiments indicated that this resulted in a more stable preparation than if all the blood was eliminated by extensive washing of the lobar
t: 1*-
r
6 4
38
-1"
..- I-I-
733
- - Blood Perfused 44.5 to 44.9 °C. Blood Perfused 36.8 to 44.4
20
40
60
80
100
120
Time (min) Fig. 2. Time course for changes in the arterial-venous pressure drop (Pa-P v) during perfusion. Values are means ± SE. Asterisks indicate that value is significantly increased from initial values, p < 0.Q1.
vascular bed with the perfusate before the recirculating perfusion. In addition, the lobar bronchus was attached to the ventilation system consisting of a piston respirator with adjustable tidal volume and a water overflow valve to maintain end-expiratory airway pressure. The lobes were ventilated at a frequency of about 8 breaths/min with a tidal volume of 75 to 100 mI, depending on the lobe size, with an end-expiratory pressure of about 3 torr. The gas mixture was composed of approximately 16% oxygen and 5.8% carbon dioxide in nitrogen, which maintained perfusate oxygen tension at 103 ± 3.5 torr (SO) and carbon dioxide tension at 39.5 ± 4.0 torr. Blood samples were obtained during perfusion for pH measurements at 37° C. A temperature correction of0.0147 pH unit per degree Celsius for blood or 0.0061 ° C for the salt solution was used to correct for the difference between the electrode temperature and the temperature of the perfusate in the perfusion system. Once the perfusion was begun, each lobe was perfused at a control temperature (36.6° to 38.0° C) for 31.7 ± 6.8 minutes (SO), during which time initial control measurements were made. The lobes were then perfused for an additional 2 hours. During this period the heat exchanger temperature was set at a constant temperature from about 37° to 46° C. When the heat exchanger temperature was altered by changing to a new preheated water source, it took about 3 minutes for the water bath temperature to change to the new temperature. The time course for the perfusate temperature change can be seen in Fig. 1. Lobar arterial and venous pressures, measured relative to the height of the venous cannula at approximately the top surface of the lobes, were recorded continuously. Airway pressure was also recorded continuously and used to detect changes in lung compliance. Measurements ofvascular and extravascular volume, serotonin uptake, and urea permeability surface area product were made during the control period and at the end of the perfusion period with multiple indicator dilution techniques. The permeability surface area product was measured only in the lobes perfused with salt solution. To make the measurements the ventilator was stopped at end expiration and a bolus containing iodine 125 human serum albumin (HSA) (0.15 ILCi), tritiated water
The Journal of Thoracic and Cardiovascular Surgery
7 3 4 Rickaby et al.
Table I. Measured variables related to hemoglobin content data Variables
Wet weight (gm) Blood included Bloodfree Wet weight/dry weight Blood included Bloodfree Residual blood volume (ml) Extravascular volume gravimetric (ml) Extravascular volume indicator dilution (ml) Vascular volume (ml)
Before
After
39.7 ± 3.6 32.7 ± 3.8
44.7 ± 3.2 34.8 ± 3.1
4.58 4.52 6.8 23.9
± ± ± ±
0.03 0.05 1.2 1.8
4.72 4.60 9.5 27.2
± ± ± ±
0.05 0.11 1.3 2.5t
22.3 ± 1.6
25.8 ± 1.5*
35.2 ± 3.6
34.8 ± 3.1
Values are means ± standard error; n = 6. Extravascular volume from 3HOH and [1251]HSA indicator dilution method orthe bloodfree wet weight minus the bloodfree dry weight for the gravimetric method. Vascular volume from [ 1251]HSA dilution. For the gravimetricvalues, before refers tothe valuesobtained from the left upper lobe orfrom the fractional upper lobe values times the initial left lower lobe weight. The temperatures studied for these six lungs were 37.3', 39.2°,39.8°,42.9°,44.7°,and 44.7° C, and no significant correlations with temperature were found. *p < 0.01. tp < 0.05.
(5 JLCi), and carbon 14 seroto~in (0.3 JLCi [5 nmol]) or [14C] urea (0.5 JLCi) was rapidly introduced into the arterial cannula with an injector situated in the inflow tubing. The outflowwas simultaneouslydivertedfrom the reservoirto the samplingtubes of a Gilson Escargot fraction collector (Gilson Medical Electronics, Inc., Middleton, Wis.). The sampling rate was set at 1.67 samples/sec. Sampling tubes were prefilled with 9 ml of coldethanol to precipitatethe [125] HSA for gamma scintillation counting and to clear the samples for hydrogen 3 and 14C measurement by liquid scintillation counting. At the end of the experiment the lobe was removed and the arterial and venous cannulas were connected directly. A bolus containing [125I]HSA was then injected and samples were collectedin the same manner as when the lung lobe was in place to obtain the mean and variance of the tubing transit times. The mean transit time (t) of [I25I]HSA or 3HOH was calculated as follows:
t
=f o
T
tC(t) dt /
IT
C(t) dt
0
and the variance «(12) of the outflowconcentrationcurveswas calculated from the following: (12
=f T(t-t)2C(t) dt / IT C(t) dt o
0
where C is the concentration of [I25I]HSA or 3HOH, and T is the time (t) at which C had decreased to 1%of its peak value. The lobar t or cl- was calculated by subtracting the respective values oft or (12 obtained when the lobe was removed from the perfusioncircuit from the t or (12 obtained with the lobein place.
The vascular volume was the product of the lobar t for [I25I]HSA and the perfusate flow rate. The volumeof distributionof 3HOH wasthe lobar t for 3HOH timesthe flow rate. The extravascular volume accessible to 3HOH was the difference between vascular volume and the volume of distribution for 3HOH. The relativedispersion of the lobar transport function19 was the lobar (1/t for the [125I]HSA. The fraction of injected serotonin taken up by the lung during passage of the bolus (U) was calculated as follows: U=
Q
f T [CR(t) - Cs(t)] dt o
where Q is flow rate in millilitersper second,CRand Cs are the concentrationsof [125I]HSA and serotonin, respectively, divided by the amounts injected. . The permeability surface area product for urea was calculated as follows: PS = -
where
E;
~ 1-
[f'
Q In
(1- Ei)
Cs(t) dt /
f'
CR(t)
dt]
and Cs is the normalized urea concentration and p is the time that CR reached its maximal value.-" Each lobewasweighedbeforebeingattached to the perfusion system and after being removedfrom the perfusionsystem.At the end of the experiment the lobes were lyophilized.to determine dry weight. The left upper lobes were removed immediately after the chest was openedand werealso weighedand lyophilized. The wet-to-dry weight ratios of the upper lobeswere used as an estimate of the preperfusionwet-to-dryweightratios of the left lower lobes. After perfusionthe blood-freeweight and dry weightsof six of the blood-perfused lobeswere estimated with the hemoglobin method. For this purpose, a sample of blood drawn from the reservoirat the end of the experimentwas used to determinethe bloodconcentration of hemoglobin. The lyophilized lobeswere ground to a powder that was extracted with Drabkin's reagent (200 ml/gm). The mixture was centrifuged for 20 minutesand filtered (0.22 JLm filter),and the opticaldensityof the filtratewas determined at 545 nm. The processwas repeated on the precipitate until absorbance of the filtrate fell below0.025. The residual blood volume in the lobe was determined by dividing total hemoglobin in the lobe by the bloodhemoglobin concentration. Preperfusionlobar residual bloodvolumesper gram of left lower lobe were estimated with the same method on the left upper lobes and blood samples drawn at the end of exsanguination. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciencesand publishedby National Institutesof Health (NIH Publication No. 80-23, revised 1978).
Results The time courses for the temperature changes are shown in Fig. I. For this graph the data for the bloodperfused lobes and the lobes perfused with salt solution
Volume 101 Number 4 April 1991
Tolerance of lung to hyperthermia 7 3 5
Table II. Measuredvariables in blood and lung lobes perfused with Krebs-Ringer bicarbonate solution Blood p.-Pv(torr) 36.8°-44.4 ° C 44.5°-44.9° C Qev (ml) 36.8°-44.4° C 44Y-44.9° C QL (ml) 36.8°-44.4° C 44.5°-44.9° C
Salt solution
Before
After
n
Before
7.7 ± 0.5 9.0 ± 0.3
7.3 ± 0.5 12.8 ± 0.6*
12 5
5.6 ± 0.7
5.8 ± 0.8
6
20.5 ± 1.3 23.6 ± 0.3
23.7 ± 1.2* 27.6 ± 0.8t
10 4
19.7 ± 1.6
21.0 ± 1.9
6
33.2 ± 2.9 30.7 ± 1.9
33.8 ± 2.5 30.5 ± 1.6
11 5
34.0 ± 3.5
31.9 ± 4.1
6
0.46 ± 0.03 0.42 ± 0.03
0.48 ± 0.03 0.53 ± 0.02*
12 4
0.38 ± 0.02
0.40 ± 0.02
6
86.2 ± 1.8 91.0 ± 2.4
88.3 ± 1.1 88.1 ± 2.7
10
91.8 ± 1.1
90.6 ± 1.1
6
1.50 ± 0.15
2.01 ± 0.28t
6
After
n
RD 36.8°-44.4° C 44.5°-44.9° C U 5-HT (%) 36.8°-44.4° C 44.5°-44.9° C PS Urea (ml/sec) Wet Weight (gm) 36.8°-44.4 ° C 44.5°-44.9° C Wet/dry Weight 36.8°-44.4° C 44.5°-44.9° C C (mI/cmH20) 36.S°-44.4° C 44.5°-44.9° C pH 36.8°-44.4° C 44.5-44.9° C
4
37.5 ± 2.5 39.3 ± 1.2
42.6 ± 2.4* 49.7 ± 4.7
12 5
40.2 ± 3.8
41.5 ± 3.7
6
4.30 ± 0.13 4.52 ± 0.38
4.88 ± 0.09* 5.46 ± 0.59t
12 4
4.96 ± 0.06
5.16 ± 0.14
6
36.8 ± 4.1 35.6 ± 8.7
23.3 ± 2.8* 21.6 ± 4.6t
12 5
37.9 ± 2.4
22.2 ± 2.1*
6
7.39 ± om 7.39 ± 0.02
7.38 ± om 7.37 ± 0.04
11 5
7.45 ± 0.01
7.40 ± 0.01*
6
Values are means ± standard error; n = number of experiments in which each variable was measured. Before refers to measurements made during the control period at 37.2 ± 0.4" C (SD) before the subsequent 2-hour exposure to either hyperthermic or normothermic perfusion. After refers to measurements made at the end of perfusion. Pa, Lobar artery pressure; Pv, lobar venous pressure; Qev, extravascular volume; QL, vascular volume; RD, lobar relative dispersion; U 5-HT, serotonin uptake; PS urea, urea permeability surface area product; C, lobar compliance. 'Indicates after value significantly different from before value, p < 0.01. tp < 0.05.
are combined, since the temporal pattern was similar in the twogroups. The lobes havebeendivided into control, medium, and high temperature groups for this graph to give a perspective on the time course over the range of temperatures studied. The results of the measurements made before and at the endof the experimental periodare shown in Table II. There were few differences that could be ascribed to hyperthermia. When the temperaturewasbelow approximately 440 C we could detect no correlation between temperature and any of the measured variables. Above 44.4° C in the blood-perfused lungs there was a significant increase in perfusion pressurenear the end of perfusion, as shown in Fig. 2. Therefore we have divided the results intotwogroups, oneaboveand onebelow 44.40 C. The only other temperature-dependent result was a significant increasein relative dispersion when the temperaturewasabove44.4° C. Overalltherewasa smallbut
significant (p < 0.05)correlation (r = 0.574)betweenthe relative dispersion and the changes in perfusion pressure that occurred during perfusion. This suggests that increases invascularresistance wereassociated withmore heterogeneous perfusion, and decreases were associated with more homogeneous perfusion. In the high temperature group the averageweightgain of the lung lobeswas considerably greaterthan inthe lowertemperaturegroup. However, this mean is heavily weighted by the one lobe atthehighesttemperaturestudied, 44.90 C, whichgained 27 gm and was clearly edematous. Severalchangesoccurredin the lobesduring perfusion, which were independent of temperature. In the bloodperfused lobes these included an increasein wet weight, extravascular volume and wet-to-dry weightratio, and a decrease in lung compliance. In lungs perfusedwith salt solution similartendencies werenoted,although onlythe decrease in compliance was significant. The decrease in
The Journal of Thoracic and Cardiovascular Surgery
7 3 6 Rickaby et al.
2.0 1.5 ........ Ul <,
11.0
70
I
I
I
60 50
E
40
~
30
~
I•
20
0.5
10 0.0
36
38
40 42 TEMP (OC)
44
46
0 36
38
40 42 TEMP (OC)
44
46
Fig. 3. Summary of the urea permeability surface area product (PS) changes in the lungs perfused with salt solution. Measurements were made before (circles) and at the end (arrowheads) of the 2-hour experimental perfusion period. Vertical lines connect before and after values for each lobe studied.
Fig. 4. Summary of wet weight (Wt) of the lungs measured before being attached to perfusion system (circles) and after being removed from perfusion system (arrowheads). Vertical lines connect the before and after values for each lobe studied. Closed symbols are for the blood-perfused lung lobes, and open symbols are for the lung lobes perfused with salt solution.
compliance was almost linear with time, with no significant difference between blood-perfused lobes and those perfused with salt solution. For the lobes perfused with salt solution we added the measurement of the urea permeability surface area product, which was significantly greater at the end of perfusion than at the beginning (Table II, Fig. 3). Figs. 3 to 6 summarize the pulmonary vascular variables that were significantly altered by perfusion. These include lung weight, wet-to-dry weight ratio, extravascular water, and urea permeability surface area product. BlOOd volume and serotonin uptake were not influenced by perfusion and are summarized only in Table II. Table I summarizes the gravimetric data, wherein the influence of the residual blood volume was taken into account. These data suggest that the increase in lung weight included an increase in extravascular water as opposed to simply an increase in residual blood volume. In addition, it can be noted that the indicator dilution extravascular volume was a substantial fraction of the gravimetric estimate of extravascular water, and the increase in the gravimetric extravascular volume was paralleled by an increase in indicator dilution extravascular volume.
ery will be possible if substantial acute lung injury is produced during perfusion. Even if injury is not detected during the acute phase, it is conceivable that the conditions established during perfusion could result in impaired recovery of lung function during resuscitation (the subacute recovery phase). Iflung tolerance is acceptable, as judged from the responses measured during the first two phases, the issue of chronic toxicity can be dealt with and criteria established for evaluating the long-term outcome. Experimental evaluation including all three phases can be an expensiveproposition, and any protocol to evaluate the last two phases requires that acute toxicity does not terminate the study during perfusion. We performed the present studies on isolated perfused dog lung lobes in vitro in an attempt to evaluate the acute tolerance of the lungs to elevated temperatures and thus to establish conditions that are potentially useful for in vivo study. A number of variables exist that could he used to evaluate the status of the lung. It appears that the ability of the isolated perfused lung to maintain constant weight is a summary criterion that reveals the impact of changes in other variables on the viability of the lungs. In other words, pulmonary .edema is the final common end point of acute lung injury of diverse causes. Therefore we have used the changes in lung weight occurring during perfusion as an index of lung injury. The use of lung weight as a criterion has at least two potential disadvantages. First, lung weight is not easily measured during in vivo perfusion; second, in the absence of a hemodynamic cause, endothelial injury with increased permeability is the antecedent of edema. Thus an increase in weight occurs as a secondary event both mechanistically and temporal-
Discussion For the evaluation of in vivo perfusion conditions it is useful to consider the acute, subacute, and chronic phases resulting from the surgical procedure. It is necessary that during perfusion (the acute phase) the normal lung tissue is able to tolerate the therapeutic conditions that are established. It is unlikely that resuscitation and/or recov-
Volume 101 Number 4
Tolerance of lung to hyperthermia 7 3 7
April 1991
8
~
6
z-
O
:> ....'"
4
~$n 11
I·
1 ~
I
I}, ~Ii't
OJ
S:
30
1
25 ,-..
I
>
20
I
15
•
I
l~
OJ
a
10
2
5 O+-----+---+----t----+-------1 36 38 40 42 46 44 TEMP (OC)
0 36
38
40 42 TEMP (OC)
44
46
Fig. 5. Summary of the wet-to-dry weight ratios measured on the left upper lobe immediately after the chest was opened (circles) and on the left lower lobe after being removed from the perfusionsystem (arrowheads). Vertical lines connect the before and after values for each lobe studied. Closed symbols are for blood-perfused lung lobes, and open symbols are for the lung lobes perfused with salt solution.
Fig. 6. Summary of indicator dilution extravascular water (Q",) measurements. Measurements were made before (circles) and at the end (arrowheads) of the 2-hour experimental perfusion period. Vertical lines connect before and after values for each lobe studied. Closed symbols are for bloodperfused lungs, and open symbols are for lungs perfused with salt solution.
ly. Therefore in the present study we also used indicator dilution methods that can be readily applied in viv04, 20-24 and that- may also address the events leading to edema before the accumulation of excess fluid. The rationale behind this study included the concept that correlations between the direct index ofinjury obtainable in vitro (lung weight) and some variables that also can be measured in vivo would provide a basis for establishing useful measurements to be made in future in vivo isolated perfusion studies. To date, in vivo lung perfusions have been limited to about 1 hour. I, 4 In the present study the total perfusion time was about 2lf2 hours with the intent of encompassing the presently acceptable range of in vivo perfusion times. We did not attempt to optimize all possible lung perfusion conditions. For the present study that would have been impractical. Instead we used methods that have been used with reasonable success in the past.l'' Accepting the fact that deterioration with perfusion time occurs in any in vitro isolated perfused lung preparation.P our major interest was in whether this deterioration was measurably accelerated by temperatures in excess of about 37 0 C. Because one of the advantages of isolating lung perfusion in vivo is the potential for altering perfusate composition to gain therapeutic advantage.l't 26 we also examined whether the utilization of a nonblood perfusate would alter the range of temperatures tolerated by the preparation. The experiments were performed on dog lung lobes because the dog has proved to be a useful animal model in the development of in vivo lung perfusion procedures. I, 4
The results indicate that in the isolated perfused dog lung temperatures as high as 44 0 C for approximately 2 hours produced little short-term damage detectable by the measured variables beyond that produced by the isolated perfusion procedure. Lung perfusion was associated with some apparently detrimental changes, irrespective of temperature. These included a small increase in weight and a progressive decrease in compliance. These changes do not appear to have been of sufficient magnitude to mask any added influence of hyperthermia had it occurred. The most notable impact of hyperthermia on the variables studied was a tendency for an increase in perfusion pressure in the blood-perfused lungs when the temperature was 44.5° C and above. The mechanism responsible for this increase is not known, but hardening of the erythrocytes by the high temperature is consistent with the results.i? Otherwise, there was little to distinguish the higher temperature experiments, except that the one lung that was perfused at the highest temperature studied, 44.9° C, gained substantially more than the average weight gain. The increase in lung weight and extravascular water volume that occurred regardless of temperature can be compared with what might be expected for a lung lobe from a 20 kg dog on the basis of normal lung fluid transport. The normal lung lymph flow rate appears to be about 1 mljhr for the left lower lobe. 28 Assuming that this rate approximates the net fluid transport out of the pulmonary capillaries in vivo, we can compare the normal fluid transport with the rate of weight gain in the isolated lungs. The average weight gain was about 5 gm over the
The Journal of Thoracic and Cardiovascular Surgery
7 3 8 Rickaby et al.
2Y2-hour perfusion period, and the increase in extravascular volume was about 3 gm over the 2-hour experimental period in the blood-perfused lobes. These rates are greater than expected in vivo, even if there were no lymphatic drainage or pleural effusion from the isolated lobes. This appears typical for isolated perfused lungs. 25 The reasons are not completely understood. They are of interest not only for possible relevance to in vivo isolated perfusion, but also in regard to problems of lung storage before transplantation.P'" We added the measurement of the permeability surface area product for the lobes perfused with salt solution, and the results are consistent with an increase in permeability for small hydrophilic molecules. In these lungs, however, there was little weight gain. Unfortunately, this variable was not measured in the blood-perfused lungs. It is conceivable that a larger increase in urea permeability might have been observed in conjunction with the larger weight gain, but this remains to be examined. We suspect that the decrease in lung compliance with perfusion time may be the result of progressive microateleetasis, because the increases in lung fluid volume that occurred during the perfusion period appear to be too small to have had a measurable influence on lung mechanics. This question, however, remains to be resolved. Serotonin uptake has been considered an indicator of pulmonary endothelial cell function.l': 23, 24, 32 particularly with endothelial injury associated with oxidant stress." which has been associated with hyperthermia in studies on the liver. 34 We could detect no significant changes in the serotonin uptake in the present study. This suggests that metabolic functions involved in the serotonin uptake process were not disrupted to the extent that significant interference with serotonin uptake could be observed. The fact that there appeared to be an increase in permeability surface area product for urea with no change in serotonin uptake suggests that the processes controlling urea permeability and serotonin uptake are not tightly linked or that serotonin uptake is relatively insensitive to such changes. The extravascular volume accessible to the 3HOH was quite close to the gravimetrically determined extravascular volume in this preparation, and it tracked the increase with perfusion time. This suggests that this measurement may have utility in in vivo studies in which whole lung gravimetric measurements are impractical. For in vivo perfusion the use of a nonblood perfusate has a number of potential advantages. The ability to manipulate substrate composition and the absence of plasma proteins are two useful features. The latter is advantageous because many chemotherapeutic agents bind to plasma proteins. The elimination of these proteins
from the perfusate would allow for much smaller vascular concentrations to be used, with the result that effective pulmonary-systemic crossover after reconnection of the pulmonary circuit would be virtually eliminated. In the present study we could detect no disadvantages to the particular salt solution used. There was no attempt to find an optimal perfusate. However, the lobes appeared to tolerate both the particular solution used and the hyperthermia as well as the blood-perfused lungs. The tendency was for a smaller weight gain than with the blood-perfused lungs. The salt solution did contain approximately 1% autologous blood, which we suspect is an important addition to this particular perfusate, based on previous observations. The observations made in this study cannot be taken as evidence that hyperthermia up to about 44 0 C had no deleterious influence, It is not practical to evaluate all the variables that might be important. We chose a few that have been used as measures oflung injury in the past. The changes in these measures associated with lung injury are well documented. However, their sensitivity to those aspects of the injury that determine the long-term outcome is not known. The important test in the future will be to determine whether the hyperthermic exposure alters recovery oflung function invivo. The present studies suggest that such studies are feasible and that acuteinjury during the perfusion period is not likely to be the limiting factor in such studies.
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Tolerance of lung to hyperthermia
April 1991
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