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Lung damage following lymph fistula preparation in sheep
JOURNAL OF SURGICAL RESEARCH
42, 1% 197 ( 1987)
Lung Damage following Lymph Fistula Preparation LENNARTSMITH,
SVEN-ERIK
ANDREASSON,
TOM SALDEEN,*
in Sheep’
AND
Bo
RISBERG
Department of Surgery I, University of Giiteborg, Sweden and *Department of Forensic Medicine, University of Uppsala, Sweden Submitted for publication September 4, 1985 The effect of preparative trauma on lung microvascular permeability during lung lymph fistula prep aration in sheep was investigated. Two groups of anesthetized sheep were used. In group I (controls: n = 3) the lymph fistula was prepared 3 to 4 days before the experiment. In group II (experimental group: n = 13): left atrial and lymph catheters were introduced on the day of the experiment. All animals had catheters in the aorta and pulmonary artery. The stable prostanoid metabolites tromboxane B2 (TxBr) and 6-keto-PGFla (6-keto) were measured in lymph. In group 1 systemic (P,) and pulmonary pressures(Pm) remained constant. Leukocytes and platelets were unchanged as was pulmonary lymph flow (Qr) and the lymph to plasma total protein concentration ratio (L/p). In group II P, and Pp. remained constant. All sheep developed a temporary leukopenia and trombocytopenia. TxBr and 6-keto were low and stable during anesthesia in group I but were elevated immediately following surgery in group II but values normalized during a 4-hr observation period. Qr during the first half hour was 1.9 f 0.3 ml/30 min and increased over 5 hr to 3.1 + 0.7 ml/30 min corresponding to 60 + 15% over baseline (P < 0.0 1). L/P did not change. These changescould be due either to changing permeability or surfacearea. These effectsof preparative trauma indicated that the model was not in a steady state. This observation should be kept in mind when evaluating studies using the lung lymph fistula in acute experiments. Q 1987 Academic Press,Inc.
complement activation and the trapping of platelets and leukocytes in the lung [9]. InMany different experimental models have creasedlevels of 6-keto-PGF 1CYand tromboxbeen used to study the mechanisms behind ane B2 were found 2 hr postoperatively after the development of the acute respiratory dislung lymph fistula preparation in sheep, intress syndrome. Staub [2 1] presented the lung dicative of local lung injury [7]. lymph fistula preparation in sheepas a means In a previous study Minnear et al. [ 141 of studying pulmonary capillary exchange. compared acutely prepared and chronically This approach has the advantage that animals instrumented unanesthetized sheep and conwith a chronic lung lymph fistula can be studcluded that lung endothelial permeability was ied in the conscious state. This chronic model not increased following acute preparation. is, however, cumbersome to use and many Townsley et al. [23] on the other hand found authors have changed to an acute preparation a decreasein the sieving properties of the capin the anesthetized sheep [3, 4, 6, 141. The illary membrane for proteins after preparation operative procedure involves bilateral thoraindicating increased permeability. cotomies with deflection of the lungs during The effect of anesthesia and the operative the preparation. It has been stated that this procedure per se on lung fluid dynamics must model is stable for at least 8 hr [6]. Previous be defined when performing acute experistudies have demonstrated that trauma caused ments. In the present study we examined such effectson lung fluid exchange. INTRODUCTION
’ Supported by grants from the Swedish Medical Research Council (Project 0660), The Iaerdal Foundation, Gijteborg Medical Society,the Medical Faculty, University of Goteborg, and the SwedishNational Association against Heart and Chest Diseases. 0022-4804187 $1SO Copyright 0 1987 by Academic F&s?.,Inc. AU rights of rqmxh3ion in my form reserved.
METHODS
Animals. Sixteen fasted sheep with an averageweight of 3 1.4 kg (range 24-35 kg) were
192
SMITH
ET AL.: LUNG
used in the present experiment. The animals were divided into two groups. Control group (n = 3). For the operative procedure the animals were anesthetized with Ketamine (500 mg im) and Thiopenthal sodium (20 mg/kg body weight, iv) intubated and ventilated with air in an Engstriim respirator. Anesthesia was maintained during surgery by a continuous infusion of Ketamine. Following the lymph fistula preparation the animals were allowed to recuperate for 3 to 4 days before the experiment. On the day of the experiment after confirming a steady flow of clear lymph the sheep were anesthetized and ventilated in the prone position. A carotid artery was catheterized and a thermistor tipped double lumen flow directed catheter was positioned in the pulmonary artery. This group was used to study the influence of anesthesia alone and the animals were followed for 5 hr. Experimental group (n = 13). The sheep were anesthetizedwith thiopenthal sodium (20 mg/kg body weight). Muscle relaxation was achieved with pancuroniumbromide (0.1 mg/ kg body weight). Ventilation with a mixture of oxygen and nitrous oxide (30/70%) using a tidal volume of 10 ml/kg body weight at a frequency of 15/min with continuous positive endexpiratory pressure (PEEP) of 5 cm water to prevent atelectasiswas used. Following surgery for introduction of the lymph catheter the right sided thoracotomies were closed. A catheter for pressure registration was placed in the left atrium via a left-sided thoracotomy. A catheter was then placed in a carotid artery and a thermistor tipped double lumen flow directed catheter was positioned in the pulmonary artery. In 5 sheepa 5 Fr-catheter with attached thermistor was placed in the aorta via a carotid artery for measurement of extravascular lung water (EVTV). After the operative procedure, which lasted about 2 hr, the animals were placed in the prone position and observed for 5 hr. Preparation of lung lymph jstula. Lung lymph was collected using the technique described by Staub et al. [21]. Through a rightsided thoracotomy in the ninth intercostal spacethe caudal mediastinal lymph node was
LYMPH
FISTULA
193
resected below the inferior pulmonary ligament. All visible contributory vesselsfrom the diaphragm and chest wall to the lymph node were sectioned. Through a second thoracotomy in the fifth intercostal space the efferent duct from the lymph node was cannulated with a heparin-glutaraldehyde treated silastic catheter (o.d. 1.19 mm). With this preparation lung lymph was collected [2 11. Measurements. Pulmonary arterial (Ppa) and aortic pressures(Pm)were recorded continuously in all animals. Left atrial pressure (P,J was measured in 12 sheep in the experimental group. In some of these the measurements were discontinuous due to clotting of the catheters. The reference level for pressures was the apex of the shoulder. Cardiac output (Q) was measured by the thermodilution technique in 5 sheepusing the aorta catheter. In these animals EVTV was measured using a thermal dye technique [ 1, lo] and an Edwards lung water computer 93 10 (Edwards Laboratories, Santa Ana, Calif.). In the other animals Qt was measured by a thermodilution technique using the thermistor-tipped pulmonary artery catheter. Measurements were made in duplicate. Lymph was continuously collected and flow (Qr) was measured at 15-min intervals. Samples for total protein concentration in plasma and lymph were taken at 30-min intervals. Leukocyte and platelet counts and hematocrit (hct) were analyzed using standard methods. The partial pressuresof oxygen (PO*), carbon dioxide (X0,), and pH were measured using an automized blood gas analyzer. Biochemical analysis. The total protein concentrations were analyzed in lymph and plasma using the Biuret-method and the lymph to plasma ratio (L/P) was calculated. Tromboxane B2 (TxB2) and 6-ketoPGF1 (Y (6-keto), the stable metabolites of tromboxane A2 (TxAJ and prostacyclin, were analyzed in lymph in all control animals and in 5 experimental animals. The metabolites were measured using a RIA technique [20]. Statistics. Data were expressed as mean f SEM. Significance calculations were made using Wilcoxon Rank test and the Rank sum
194
JOURNAL OF SURGICAL RESEARCH: VOL. 42, NO. 2, FEBRUARY 1987
test between the groups. P < 0.05 was considered to be statistically significant. RESULTS
PsammHg
100
\r,T~\?\~
120 1
0 In the control group the hemodynamic parameters P, , Pw , and Qt remained stable durPpa mm Hg ing the experimental period. QL was 1.2 + 0.1 25 ml/30 min during the first hour and the :-T-X-, -T-T changesduring the experiment were less than 15 10%.L/P for total protein was after 1 hr 0.82 0I + 0.04 and remained stable for 3 hr but dePh mmHg creased the last hour to 0.72 f 0.04 (Fig. 1). *i T The leukocyte and platelet counts were 5.8 T T/ 4 ?-?,T/-* f 1.4 and 248 f 58 X lo9 X I-‘, respectively, . before anesthesia and remained stable during 0 the whole experiment. TxBz and 6-keto were 0 1 2 3 4 5 h post op. low and stable and did not change during the FIG. 2. Mean arterial (P,) and mean pulmonary arterial experiment. POZ, pCO2, and hct remained stable and within normal limits during the ex- (P& pressure in 13 and mean left atrial pressure (&,) in 12sheepin the experimental group (2 + SEM). Preparative periment.
!
I
trauma did not alter these parameters.
In the experimental group P,, Ppa,and PI, remained stable for 5 hr after the operation (Fig. 2). Qt (n = 10) was 88 + 14 ml min-’ X kg-’ body weight after 1 hr and there were no changesduring the 4 hr observation period. In 5 sheepEVTV was 10.7 + 1.4 ml kg-’ body weight initially and 9.6 f 2 ml kg-’ body PpamnHg weight just before termination of the experi20 ment. 10 During the first half hour QL was 1.9 + 0.3 OJ QL ml.30 mir-’ ml/30 min and increased successively to 3.1 1.5 f 0.7 ml/30 min (P < 0.01) after 5 hr which 1.0 corresponded to 60 + 15% over base line 0.5 (Fig. 3). -0 IIn 10 sheepthe L/P for total protein could LIP be measured from the start to the end of exn=2 periment. The ratio was 0.77 f 0.04 during 0.7 the first hour and 0.85 + 0.04 during the last hour of experiment. The changes were not 0 statistically significant. 0 1 2 3 4 5 h after Leukocytes and platelets were counted preanaesthesia operatively, early (60-90 min) and late (270FlG. I. Mean arterial pressure (Pm), mean pulmonary 300 min after operation) postoperatively. The arterial pressure (P,,& lymph tlow (a), and lymph to leukocytes decreasedfrom 5.3 + 0.4 X IO9 1-l plasma ratio for total protein (L/P) in three control sheep (X + SEM). These parameters remained stable during the before the operation to 2.4 + 0.5 X lo9 1-l in experiment. the early (P < 0.02) and 5.1 +_0.9 X lo9 1-l psa?m
Hg
1‘--L-+-IT
0.9 iy”-
195
SMITH ET AL.: LUNG LYMPH FISTULA
(P < 0.02) and stayed at a low value in the late phase 220 f 26 X lo9 I-’ (Fig. 4). Hct did not change after the operation. PO2 and pC02 were in the normal range. The levels of TxBz and 6-keto were high in the immediate postoperative period and then decreasedsuccessively.TxB2 was alter 4 hr still significantly higher than in the control group whereas 6-keto was significantly higher in the early postoperative phase but attained the same level as the controls after 4 hr (Fig. 5).
UP Or Ad--0.7
1
oJ
DISCUSSION 0
1
2
3
4
5 h post op.
FIG. 3. Lymph flow (Qr) in 13 sheep and lymph to plasma ratio for total protein (L/P) in 10 sheep in the experimental group (.V+ SEM). QL was significantly increasedcompared to the first 30 min of lymph sampling from 120 min until the end of the experiment. L/P was not significantly altered.
in the late postoperative period. The platelet count decreasedfrom 446 + 56 X lo9 1-l preoperatively to 236 f 42 X lo9 1-l in the early
In the present experiments we studied the effectson transvascular lung fluid exchange in sheep following the lung lymph fistula preparation. The effect of anesthesia per se was studied in anesthetized sheep with a chronic TxB2 in lymph pglO.1 ml
Leukocytes 109.1-’
6 0
4
240
min. post op.
6-keto in lymph pglO.1 ml
.
2
500
0 In
400
0
control
300
Platelets 109.1-1
experimental
200
‘j fl i;l r;l Pre op.
60-90
270-300 mh. post op
FIG. 4. Leukocytes in 9 and platelets in 7 sheep in the experimental group (2 + SEM). Leukocytes were temporarily depressedat 60-90 min while platelets were significantly reduced during the experiment compared to baseline values. * = significantly different from preoperative values.
100 0~
h
240
min. post op
FIG. 5. Thromboxane Bz (TxB2) and 6-keto PGF~Lv(6keto) in lymph in 3 sheep in the control and 5 sheep in the experimental group after induction of anesthesiaand postoperatively (2 * SEM). Preparative trauma signiticantly elevated levels of TxBz in lymph compared to the control animals during the whole experiment. 6-keto levels were only increased immediately following surgery. * = significantly different from control group.
196
JOURNAL OF SURGICAL RESEARCH: VOL. 42, NO. 2, FEBRUARY 1987
lung fistula. These animals had stable hemodynamic and lymph parameters during anesthesia for 5 hr. Acutely prepared sheep had a progressive increase in QL with unchanged L/P and a decreasein leukocytes and platelets. In the postoperative period the sheep showed evidence of activation of the eicosanoid system with high levels of the stable metabolites of tromboxane A2 and prostacyclin in lung lymph. The depth of anesthesiacould have affected the results. In our experiments anesthesiawas adequate to keep vascular pressures and cardiac output at normal and stable levels in both groups but during the operation it might have been insufficient. In previous studies where stable lymph flow was found following preparation [6, 131 the sheep were more heavily anesthetized. Although the animals in one of these studies were fully anesthetized and only had an unilateral thoracotomy severalanimals were rejected becauseof unstable lymph flow [6]. Increased lymph flow by two to three times over base line is generally considered to be a sign of increased microvascular permeability if the L/P for total protein is stable or increased.Even with this considerable increase in flow it is difficult to differ increased permeability from an increased surface area. To be able to differ between increased permeability and surface area a high stable lymph flow with a filtration independent L/P ratio is necessary.At these high flows the convective flux dominates over diffusive and it is possible to calculate the filtration properties of the microvascular membrane [ 161.With this protein wash-out technique Townsley et al. [23] demonstrated recently increased pulmonary microvascular permeability following the acute lung lymph fistula preparation in sheep. That study unfortunately lacked a proper control group as their data were compared to those of unanesthetized sheepfrom another study [ 171. The ways by which trauma may affect the lungs is a matter of speculation. Surgical operations and trauma are known to activate the complement cascade[9, 181.Activated com-
plement and leukocytes have been shown to damage endothelial cells in vitro [ 191. Activation of complement with cobra venom factor or by artificial membranes was followed by leukopenia, pulmonary sequestration especially of polymorphonuclear neutrophils, and damageor destruction of endothelial cells [22, 241. Similar changes were evident from lymph flow data in sheepindicating increased permeability [ 1I]. Complement activation was followed by increased plasma levels of TxB2 and 6-keto PGFla! [5,24]. Thesechangesafter complement activation were attenuated in leukocyte depleted animals [22]. Leukopenia has also been found to reduce the changesafter microembolization or sepsis[6, 81.Depletion of complement prior to any blood contact with artificial membranes did not give leukopenia or trombocytopenia and did not stimulate tromboxane A2 [24]. Activation of the prostaglandin system after surgery or sepsis as manifested by increased levels of stable metabolites of tromboxane A2 and prostacyclin in lymph has been demonstrated [7]. Cyclooxygenase blockade did not affect the increasedpermeability after sepsisbut blocked the pressure increase in the pulmonary artery [ 151.In our experiment the levels of TxB2 and 6-keto PGFl (Ywere increased in lymph following acute operation. Manipulation of the lung causing sequestration of leukocytes and platelets with endothelial damage can induce local eicosanoid production [ 121. TxA2 is a known vasoconstrictor and can mediate the increase in Ppaseen after sepsis.Prostacyclin is a dilatator but there seemsto be a balance between constrictor and dilatator responses after operation as pressures remained stable. In spite of this lymph flow increased. Data from our experiment could not differentiate between increased permeability or increased surface area but the changes in leukocytes, platelets and prostaglandin levels were supportive of a permeability change. Further support for this assumption was found in the study by Townsley et al. [23]. The model using an acute lung lymph fistula preparation in sheep can thus not be consid-
SMITH ET AL.: LUNG LYMPH FISTULA
ered to be a stable preparation and this should be taken into consideration when performing experiments using this model. REFERENCES I. Andreasson, S., Bylock, A., Smith, L., and Risberg, B. Extravascular lung water measurement in septic sheep.J. Surg. Res. 40: 95-104, 1985. 2. Barie, P. S., Tahamont, M. V., and Malik, A. B. Prevention of increasedpulmonary, vascular permeability after pancreatitis by granulocyte depletion in sheep. Amer. Rev. Respir. Dis. 126: 904, 1982. 3. Binder, A. S., Nakahava, K., Dhkuda, K., Kageler, W., and Staub, N. C. Effect of heparin or fibrinogen depletion on lung fluid balance in sheepafter emboli. J. Appl. Physiol. 47: 2 13, 1979. 4. Binder, A. S., Kageler, W., Perel, A., Flick, M. R., and Staub, N. C. Effect of platelet depletion on lung vascular permeability after microemboli in sheep. J. Appl. Physiol. 48: 414, 1980. 5. Cooper, J. D., McDonald, J. W., Ah, M., Menkes, E., Masterson, J., and Klement, P. Prostaglandin production associated with the pulmonary vascular response to complement activation. Surgery 88: 215, 1980. 6. Flick, M. R., Perel, A., and Staub, N. C. Leukocytes are required for increased lung microvascular permeability after microembolization in sheep.Circ. Res. 48: 344, 1981. 7. Flynn, J. T., Gee, M. H., and Demling, R. H. Effects of anesthesiaand surgeryon prostanoid concentrations in plasma and pulmonary lymph. Prostugkmdins, Leukotrienes and Medicine 10: 2 13, 1983. 8. Heflin, A. C., and Brigham, K. L. Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia. J. Clin. Invest. 68: 1253, 1981. 9. Heideman, M. Complement activation by injury. Academic dissertation, University of Goteborg, Sweden, 1978. 10. Lewis, F. R., and Elings, V. J. Microprocessor determination of lung water using thermal-greendye double indicator dilution. Surg. Forum 24: 182, 1978. 11. Malik, A. B., Johnson, A., and Blumenstock, F. A. Interaction between formed elements and the pulmonary endothelium. Gen.Phurmacol. 14: 197, 1982. 12. McDonald, J. W., Ali, M., Morgan, E., Townsend,
197
E. R., and Cooper, J. D. Tromboxane synthesis by sourcesother than platelets in association with complement-induced pulmonary leukostasis and pulmonary hypertension in sheep. Circ. Res. 52: 1, 1983. 13. Minnear, F. L., Barie, P. S., and Malik, A. B. Lung fluid and protein exchange in the acute sheep preparation. J. Appl. Physiol. 50: 1358, 1981. 14. Ohkuda, K., Nakahara, K., Weidner, W. J., Binder, A., and Staub, N. C. Lung fluid exchangeafter uneven pulmonary artery obstruction in sheep. Circ. Res. 43: 152, 1978. 15. Ogletree, M. L., and Brigham, K. L. Effects of cyklooxygenase inhibitors on pulmonary vascular responsesto endotoxin in unanesthetized sheep. Prostaglandins, Leukotrienes and Medicine 8: 489, 1982. 16. Parker,J. C., Parker, R. E., Granger, D. N., and Taylor, A. E. Vascular permeability and transvascular fluid and protein transport in the dog lung. Circ. Res. 48: 549, 1981. 17. Parker, R. E., Roselli, R. J., Harris, T. R., and Brigham, K. L. Effectsof gradedincreasein pulmonary vascular pressureson lung fluid balance in unanesthetized sheep. Circ. Res. 49: 1164, 1981. 18. Pedersen,J. H., Sorensen,H., and Kehlet, H. Complement activation during surgical procedures. Surg. Gynecol. Obstet. 146: 66, 1978. 19. Sachs,T., Moldow, C. F., Craddock, P. R., Bowers, T. K., and Jacob, H. S. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. J. Clin. Invest. 61: 1161, 1978. 20. Saldeen, P., Esquivel, C. O., Bjiirck, C. G., Bergqvist, D., and Saldeen,T. Thromboxane production in umbilical vein grafts. Thromb. Res. 33: 259, 1984. 21. Staub, N. C., Bland, R. D., Brigham, K. L., Demling, R. H., Erdman, A. J., and Woolverton, W. C. Preparation of chronic lung lymph fistulasin sheep.J. Surg. Res. 19: 315, 1975. 22. Till, G. O., Johnson, K. J., Kunkel, R., and Ward, P. A. Intravascular activation of complement and acute lung injury. J. C/in. Invest. 69: 1126, 1982. 23. Townsley, M. J., McClure, D. E., and Weidner, W. J. Assessmentof pulmonary microvascular permeability in acutely prepared sheep.J. Appl, Physiol. 56: 857, 1984. 24. Wonders, T., Huttemeier, P., Berry, D., Schnette, A., Kong, D., Watkins, W. D., and Zapol, W. M. Complement depletion prevents pulmonary hypertension and leukopenia in sheep extracorporeal membrane oxygenation. Trans. Amer. Artif Intern. Org. 29: 210, 1983.
42, 1% 197 ( 1987)
Lung Damage following Lymph Fistula Preparation LENNARTSMITH,
SVEN-ERIK
ANDREASSON,
TOM SALDEEN,*
in Sheep’
AND
Bo
RISBERG
Department of Surgery I, University of Giiteborg, Sweden and *Department of Forensic Medicine, University of Uppsala, Sweden Submitted for publication September 4, 1985 The effect of preparative trauma on lung microvascular permeability during lung lymph fistula prep aration in sheep was investigated. Two groups of anesthetized sheep were used. In group I (controls: n = 3) the lymph fistula was prepared 3 to 4 days before the experiment. In group II (experimental group: n = 13): left atrial and lymph catheters were introduced on the day of the experiment. All animals had catheters in the aorta and pulmonary artery. The stable prostanoid metabolites tromboxane B2 (TxBr) and 6-keto-PGFla (6-keto) were measured in lymph. In group 1 systemic (P,) and pulmonary pressures(Pm) remained constant. Leukocytes and platelets were unchanged as was pulmonary lymph flow (Qr) and the lymph to plasma total protein concentration ratio (L/p). In group II P, and Pp. remained constant. All sheep developed a temporary leukopenia and trombocytopenia. TxBr and 6-keto were low and stable during anesthesia in group I but were elevated immediately following surgery in group II but values normalized during a 4-hr observation period. Qr during the first half hour was 1.9 f 0.3 ml/30 min and increased over 5 hr to 3.1 + 0.7 ml/30 min corresponding to 60 + 15% over baseline (P < 0.0 1). L/P did not change. These changescould be due either to changing permeability or surfacearea. These effectsof preparative trauma indicated that the model was not in a steady state. This observation should be kept in mind when evaluating studies using the lung lymph fistula in acute experiments. Q 1987 Academic Press,Inc.
complement activation and the trapping of platelets and leukocytes in the lung [9]. InMany different experimental models have creasedlevels of 6-keto-PGF 1CYand tromboxbeen used to study the mechanisms behind ane B2 were found 2 hr postoperatively after the development of the acute respiratory dislung lymph fistula preparation in sheep, intress syndrome. Staub [2 1] presented the lung dicative of local lung injury [7]. lymph fistula preparation in sheepas a means In a previous study Minnear et al. [ 141 of studying pulmonary capillary exchange. compared acutely prepared and chronically This approach has the advantage that animals instrumented unanesthetized sheep and conwith a chronic lung lymph fistula can be studcluded that lung endothelial permeability was ied in the conscious state. This chronic model not increased following acute preparation. is, however, cumbersome to use and many Townsley et al. [23] on the other hand found authors have changed to an acute preparation a decreasein the sieving properties of the capin the anesthetized sheep [3, 4, 6, 141. The illary membrane for proteins after preparation operative procedure involves bilateral thoraindicating increased permeability. cotomies with deflection of the lungs during The effect of anesthesia and the operative the preparation. It has been stated that this procedure per se on lung fluid dynamics must model is stable for at least 8 hr [6]. Previous be defined when performing acute experistudies have demonstrated that trauma caused ments. In the present study we examined such effectson lung fluid exchange. INTRODUCTION
’ Supported by grants from the Swedish Medical Research Council (Project 0660), The Iaerdal Foundation, Gijteborg Medical Society,the Medical Faculty, University of Goteborg, and the SwedishNational Association against Heart and Chest Diseases. 0022-4804187 $1SO Copyright 0 1987 by Academic F&s?.,Inc. AU rights of rqmxh3ion in my form reserved.
METHODS
Animals. Sixteen fasted sheep with an averageweight of 3 1.4 kg (range 24-35 kg) were
192
SMITH
ET AL.: LUNG
used in the present experiment. The animals were divided into two groups. Control group (n = 3). For the operative procedure the animals were anesthetized with Ketamine (500 mg im) and Thiopenthal sodium (20 mg/kg body weight, iv) intubated and ventilated with air in an Engstriim respirator. Anesthesia was maintained during surgery by a continuous infusion of Ketamine. Following the lymph fistula preparation the animals were allowed to recuperate for 3 to 4 days before the experiment. On the day of the experiment after confirming a steady flow of clear lymph the sheep were anesthetized and ventilated in the prone position. A carotid artery was catheterized and a thermistor tipped double lumen flow directed catheter was positioned in the pulmonary artery. This group was used to study the influence of anesthesia alone and the animals were followed for 5 hr. Experimental group (n = 13). The sheep were anesthetizedwith thiopenthal sodium (20 mg/kg body weight). Muscle relaxation was achieved with pancuroniumbromide (0.1 mg/ kg body weight). Ventilation with a mixture of oxygen and nitrous oxide (30/70%) using a tidal volume of 10 ml/kg body weight at a frequency of 15/min with continuous positive endexpiratory pressure (PEEP) of 5 cm water to prevent atelectasiswas used. Following surgery for introduction of the lymph catheter the right sided thoracotomies were closed. A catheter for pressure registration was placed in the left atrium via a left-sided thoracotomy. A catheter was then placed in a carotid artery and a thermistor tipped double lumen flow directed catheter was positioned in the pulmonary artery. In 5 sheepa 5 Fr-catheter with attached thermistor was placed in the aorta via a carotid artery for measurement of extravascular lung water (EVTV). After the operative procedure, which lasted about 2 hr, the animals were placed in the prone position and observed for 5 hr. Preparation of lung lymph jstula. Lung lymph was collected using the technique described by Staub et al. [21]. Through a rightsided thoracotomy in the ninth intercostal spacethe caudal mediastinal lymph node was
LYMPH
FISTULA
193
resected below the inferior pulmonary ligament. All visible contributory vesselsfrom the diaphragm and chest wall to the lymph node were sectioned. Through a second thoracotomy in the fifth intercostal space the efferent duct from the lymph node was cannulated with a heparin-glutaraldehyde treated silastic catheter (o.d. 1.19 mm). With this preparation lung lymph was collected [2 11. Measurements. Pulmonary arterial (Ppa) and aortic pressures(Pm)were recorded continuously in all animals. Left atrial pressure (P,J was measured in 12 sheep in the experimental group. In some of these the measurements were discontinuous due to clotting of the catheters. The reference level for pressures was the apex of the shoulder. Cardiac output (Q) was measured by the thermodilution technique in 5 sheepusing the aorta catheter. In these animals EVTV was measured using a thermal dye technique [ 1, lo] and an Edwards lung water computer 93 10 (Edwards Laboratories, Santa Ana, Calif.). In the other animals Qt was measured by a thermodilution technique using the thermistor-tipped pulmonary artery catheter. Measurements were made in duplicate. Lymph was continuously collected and flow (Qr) was measured at 15-min intervals. Samples for total protein concentration in plasma and lymph were taken at 30-min intervals. Leukocyte and platelet counts and hematocrit (hct) were analyzed using standard methods. The partial pressuresof oxygen (PO*), carbon dioxide (X0,), and pH were measured using an automized blood gas analyzer. Biochemical analysis. The total protein concentrations were analyzed in lymph and plasma using the Biuret-method and the lymph to plasma ratio (L/P) was calculated. Tromboxane B2 (TxB2) and 6-ketoPGF1 (Y (6-keto), the stable metabolites of tromboxane A2 (TxAJ and prostacyclin, were analyzed in lymph in all control animals and in 5 experimental animals. The metabolites were measured using a RIA technique [20]. Statistics. Data were expressed as mean f SEM. Significance calculations were made using Wilcoxon Rank test and the Rank sum
194
JOURNAL OF SURGICAL RESEARCH: VOL. 42, NO. 2, FEBRUARY 1987
test between the groups. P < 0.05 was considered to be statistically significant. RESULTS
PsammHg
100
\r,T~\?\~
120 1
0 In the control group the hemodynamic parameters P, , Pw , and Qt remained stable durPpa mm Hg ing the experimental period. QL was 1.2 + 0.1 25 ml/30 min during the first hour and the :-T-X-, -T-T changesduring the experiment were less than 15 10%.L/P for total protein was after 1 hr 0.82 0I + 0.04 and remained stable for 3 hr but dePh mmHg creased the last hour to 0.72 f 0.04 (Fig. 1). *i T The leukocyte and platelet counts were 5.8 T T/ 4 ?-?,T/-* f 1.4 and 248 f 58 X lo9 X I-‘, respectively, . before anesthesia and remained stable during 0 the whole experiment. TxBz and 6-keto were 0 1 2 3 4 5 h post op. low and stable and did not change during the FIG. 2. Mean arterial (P,) and mean pulmonary arterial experiment. POZ, pCO2, and hct remained stable and within normal limits during the ex- (P& pressure in 13 and mean left atrial pressure (&,) in 12sheepin the experimental group (2 + SEM). Preparative periment.
!
I
trauma did not alter these parameters.
In the experimental group P,, Ppa,and PI, remained stable for 5 hr after the operation (Fig. 2). Qt (n = 10) was 88 + 14 ml min-’ X kg-’ body weight after 1 hr and there were no changesduring the 4 hr observation period. In 5 sheepEVTV was 10.7 + 1.4 ml kg-’ body weight initially and 9.6 f 2 ml kg-’ body PpamnHg weight just before termination of the experi20 ment. 10 During the first half hour QL was 1.9 + 0.3 OJ QL ml.30 mir-’ ml/30 min and increased successively to 3.1 1.5 f 0.7 ml/30 min (P < 0.01) after 5 hr which 1.0 corresponded to 60 + 15% over base line 0.5 (Fig. 3). -0 IIn 10 sheepthe L/P for total protein could LIP be measured from the start to the end of exn=2 periment. The ratio was 0.77 f 0.04 during 0.7 the first hour and 0.85 + 0.04 during the last hour of experiment. The changes were not 0 statistically significant. 0 1 2 3 4 5 h after Leukocytes and platelets were counted preanaesthesia operatively, early (60-90 min) and late (270FlG. I. Mean arterial pressure (Pm), mean pulmonary 300 min after operation) postoperatively. The arterial pressure (P,,& lymph tlow (a), and lymph to leukocytes decreasedfrom 5.3 + 0.4 X IO9 1-l plasma ratio for total protein (L/P) in three control sheep (X + SEM). These parameters remained stable during the before the operation to 2.4 + 0.5 X lo9 1-l in experiment. the early (P < 0.02) and 5.1 +_0.9 X lo9 1-l psa?m
Hg
1‘--L-+-IT
0.9 iy”-
195
SMITH ET AL.: LUNG LYMPH FISTULA
(P < 0.02) and stayed at a low value in the late phase 220 f 26 X lo9 I-’ (Fig. 4). Hct did not change after the operation. PO2 and pC02 were in the normal range. The levels of TxBz and 6-keto were high in the immediate postoperative period and then decreasedsuccessively.TxB2 was alter 4 hr still significantly higher than in the control group whereas 6-keto was significantly higher in the early postoperative phase but attained the same level as the controls after 4 hr (Fig. 5).
UP Or Ad--0.7
1
oJ
DISCUSSION 0
1
2
3
4
5 h post op.
FIG. 3. Lymph flow (Qr) in 13 sheep and lymph to plasma ratio for total protein (L/P) in 10 sheep in the experimental group (.V+ SEM). QL was significantly increasedcompared to the first 30 min of lymph sampling from 120 min until the end of the experiment. L/P was not significantly altered.
in the late postoperative period. The platelet count decreasedfrom 446 + 56 X lo9 1-l preoperatively to 236 f 42 X lo9 1-l in the early
In the present experiments we studied the effectson transvascular lung fluid exchange in sheep following the lung lymph fistula preparation. The effect of anesthesia per se was studied in anesthetized sheep with a chronic TxB2 in lymph pglO.1 ml
Leukocytes 109.1-’
6 0
4
240
min. post op.
6-keto in lymph pglO.1 ml
.
2
500
0 In
400
0
control
300
Platelets 109.1-1
experimental
200
‘j fl i;l r;l Pre op.
60-90
270-300 mh. post op
FIG. 4. Leukocytes in 9 and platelets in 7 sheep in the experimental group (2 + SEM). Leukocytes were temporarily depressedat 60-90 min while platelets were significantly reduced during the experiment compared to baseline values. * = significantly different from preoperative values.
100 0~
h
240
min. post op
FIG. 5. Thromboxane Bz (TxB2) and 6-keto PGF~Lv(6keto) in lymph in 3 sheep in the control and 5 sheep in the experimental group after induction of anesthesiaand postoperatively (2 * SEM). Preparative trauma signiticantly elevated levels of TxBz in lymph compared to the control animals during the whole experiment. 6-keto levels were only increased immediately following surgery. * = significantly different from control group.
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JOURNAL OF SURGICAL RESEARCH: VOL. 42, NO. 2, FEBRUARY 1987
lung fistula. These animals had stable hemodynamic and lymph parameters during anesthesia for 5 hr. Acutely prepared sheep had a progressive increase in QL with unchanged L/P and a decreasein leukocytes and platelets. In the postoperative period the sheep showed evidence of activation of the eicosanoid system with high levels of the stable metabolites of tromboxane A2 and prostacyclin in lung lymph. The depth of anesthesiacould have affected the results. In our experiments anesthesiawas adequate to keep vascular pressures and cardiac output at normal and stable levels in both groups but during the operation it might have been insufficient. In previous studies where stable lymph flow was found following preparation [6, 131 the sheep were more heavily anesthetized. Although the animals in one of these studies were fully anesthetized and only had an unilateral thoracotomy severalanimals were rejected becauseof unstable lymph flow [6]. Increased lymph flow by two to three times over base line is generally considered to be a sign of increased microvascular permeability if the L/P for total protein is stable or increased.Even with this considerable increase in flow it is difficult to differ increased permeability from an increased surface area. To be able to differ between increased permeability and surface area a high stable lymph flow with a filtration independent L/P ratio is necessary.At these high flows the convective flux dominates over diffusive and it is possible to calculate the filtration properties of the microvascular membrane [ 161.With this protein wash-out technique Townsley et al. [23] demonstrated recently increased pulmonary microvascular permeability following the acute lung lymph fistula preparation in sheep. That study unfortunately lacked a proper control group as their data were compared to those of unanesthetized sheepfrom another study [ 171. The ways by which trauma may affect the lungs is a matter of speculation. Surgical operations and trauma are known to activate the complement cascade[9, 181.Activated com-
plement and leukocytes have been shown to damage endothelial cells in vitro [ 191. Activation of complement with cobra venom factor or by artificial membranes was followed by leukopenia, pulmonary sequestration especially of polymorphonuclear neutrophils, and damageor destruction of endothelial cells [22, 241. Similar changes were evident from lymph flow data in sheepindicating increased permeability [ 1I]. Complement activation was followed by increased plasma levels of TxB2 and 6-keto PGFla! [5,24]. Thesechangesafter complement activation were attenuated in leukocyte depleted animals [22]. Leukopenia has also been found to reduce the changesafter microembolization or sepsis[6, 81.Depletion of complement prior to any blood contact with artificial membranes did not give leukopenia or trombocytopenia and did not stimulate tromboxane A2 [24]. Activation of the prostaglandin system after surgery or sepsis as manifested by increased levels of stable metabolites of tromboxane A2 and prostacyclin in lymph has been demonstrated [7]. Cyclooxygenase blockade did not affect the increasedpermeability after sepsisbut blocked the pressure increase in the pulmonary artery [ 151.In our experiment the levels of TxB2 and 6-keto PGFl (Ywere increased in lymph following acute operation. Manipulation of the lung causing sequestration of leukocytes and platelets with endothelial damage can induce local eicosanoid production [ 121. TxA2 is a known vasoconstrictor and can mediate the increase in Ppaseen after sepsis.Prostacyclin is a dilatator but there seemsto be a balance between constrictor and dilatator responses after operation as pressures remained stable. In spite of this lymph flow increased. Data from our experiment could not differentiate between increased permeability or increased surface area but the changes in leukocytes, platelets and prostaglandin levels were supportive of a permeability change. Further support for this assumption was found in the study by Townsley et al. [23]. The model using an acute lung lymph fistula preparation in sheep can thus not be consid-
SMITH ET AL.: LUNG LYMPH FISTULA
ered to be a stable preparation and this should be taken into consideration when performing experiments using this model. REFERENCES I. Andreasson, S., Bylock, A., Smith, L., and Risberg, B. Extravascular lung water measurement in septic sheep.J. Surg. Res. 40: 95-104, 1985. 2. Barie, P. S., Tahamont, M. V., and Malik, A. B. Prevention of increasedpulmonary, vascular permeability after pancreatitis by granulocyte depletion in sheep. Amer. Rev. Respir. Dis. 126: 904, 1982. 3. Binder, A. S., Nakahava, K., Dhkuda, K., Kageler, W., and Staub, N. C. Effect of heparin or fibrinogen depletion on lung fluid balance in sheepafter emboli. J. Appl. Physiol. 47: 2 13, 1979. 4. Binder, A. S., Kageler, W., Perel, A., Flick, M. R., and Staub, N. C. Effect of platelet depletion on lung vascular permeability after microemboli in sheep. J. Appl. Physiol. 48: 414, 1980. 5. Cooper, J. D., McDonald, J. W., Ah, M., Menkes, E., Masterson, J., and Klement, P. Prostaglandin production associated with the pulmonary vascular response to complement activation. Surgery 88: 215, 1980. 6. Flick, M. R., Perel, A., and Staub, N. C. Leukocytes are required for increased lung microvascular permeability after microembolization in sheep.Circ. Res. 48: 344, 1981. 7. Flynn, J. T., Gee, M. H., and Demling, R. H. Effects of anesthesiaand surgeryon prostanoid concentrations in plasma and pulmonary lymph. Prostugkmdins, Leukotrienes and Medicine 10: 2 13, 1983. 8. Heflin, A. C., and Brigham, K. L. Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia. J. Clin. Invest. 68: 1253, 1981. 9. Heideman, M. Complement activation by injury. Academic dissertation, University of Goteborg, Sweden, 1978. 10. Lewis, F. R., and Elings, V. J. Microprocessor determination of lung water using thermal-greendye double indicator dilution. Surg. Forum 24: 182, 1978. 11. Malik, A. B., Johnson, A., and Blumenstock, F. A. Interaction between formed elements and the pulmonary endothelium. Gen.Phurmacol. 14: 197, 1982. 12. McDonald, J. W., Ali, M., Morgan, E., Townsend,
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E. R., and Cooper, J. D. Tromboxane synthesis by sourcesother than platelets in association with complement-induced pulmonary leukostasis and pulmonary hypertension in sheep. Circ. Res. 52: 1, 1983. 13. Minnear, F. L., Barie, P. S., and Malik, A. B. Lung fluid and protein exchange in the acute sheep preparation. J. Appl. Physiol. 50: 1358, 1981. 14. Ohkuda, K., Nakahara, K., Weidner, W. J., Binder, A., and Staub, N. C. Lung fluid exchangeafter uneven pulmonary artery obstruction in sheep. Circ. Res. 43: 152, 1978. 15. Ogletree, M. L., and Brigham, K. L. Effects of cyklooxygenase inhibitors on pulmonary vascular responsesto endotoxin in unanesthetized sheep. Prostaglandins, Leukotrienes and Medicine 8: 489, 1982. 16. Parker,J. C., Parker, R. E., Granger, D. N., and Taylor, A. E. Vascular permeability and transvascular fluid and protein transport in the dog lung. Circ. Res. 48: 549, 1981. 17. Parker, R. E., Roselli, R. J., Harris, T. R., and Brigham, K. L. Effectsof gradedincreasein pulmonary vascular pressureson lung fluid balance in unanesthetized sheep. Circ. Res. 49: 1164, 1981. 18. Pedersen,J. H., Sorensen,H., and Kehlet, H. Complement activation during surgical procedures. Surg. Gynecol. Obstet. 146: 66, 1978. 19. Sachs,T., Moldow, C. F., Craddock, P. R., Bowers, T. K., and Jacob, H. S. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. J. Clin. Invest. 61: 1161, 1978. 20. Saldeen, P., Esquivel, C. O., Bjiirck, C. G., Bergqvist, D., and Saldeen,T. Thromboxane production in umbilical vein grafts. Thromb. Res. 33: 259, 1984. 21. Staub, N. C., Bland, R. D., Brigham, K. L., Demling, R. H., Erdman, A. J., and Woolverton, W. C. Preparation of chronic lung lymph fistulasin sheep.J. Surg. Res. 19: 315, 1975. 22. Till, G. O., Johnson, K. J., Kunkel, R., and Ward, P. A. Intravascular activation of complement and acute lung injury. J. C/in. Invest. 69: 1126, 1982. 23. Townsley, M. J., McClure, D. E., and Weidner, W. J. Assessmentof pulmonary microvascular permeability in acutely prepared sheep.J. Appl, Physiol. 56: 857, 1984. 24. Wonders, T., Huttemeier, P., Berry, D., Schnette, A., Kong, D., Watkins, W. D., and Zapol, W. M. Complement depletion prevents pulmonary hypertension and leukopenia in sheep extracorporeal membrane oxygenation. Trans. Amer. Artif Intern. Org. 29: 210, 1983.