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Modification of cardiopulmonary hemodynamics and vasoactive mediators by extracorporeal membrane oxygenation in newborn lambs
Modification of Cardiopulmonary Hemodynamics and Vasoactive Mediators by Extracorporeal Membrane Oxygenation in Newborn Lambs By Charles J.H. Stolar and Peter W. Dillon N e w York, N e w York 9 W e asked if prolonged venoarterial extracorporeal membrane oxygenation (ECMO) causes alterations in cardiopulmonary hemodynamics that might be reflected in arbiters of vascular tone: thromboxane, prostac~[~n, norepinephrine, and epinephrine. Newborn lambs undergoing ECMO demonstrated significant augmentation of systemic and pulmonary arterial blood pressure that was temporally related to rises in all vasoactive mediators measured. Although the prostanoids returned to baseline within 30 minutes, the catecholamines remained elevated significantly throughout bypass. Long-term bypass, however, was not associated with sustained systemic hypertension. Pulmonary hypertension was achieved only after 6 hours of bypass. These acute and chronic changes may exacerbate a pathophysiological state for which ECMO is indicated. 9 1990 by W.B. Saunders Company. INDEX WORDS: Extracorporeal membrane oxygenation (ECMO); cardiopulmonary bypass.
membrane oxygenation E XTRACORPOREAL (ECMO) has been used successfully for the management of a variety of neonatal respiratory illnesses all of which share pulmonary hypertension and some degree of lung injury as a final common pathway) Although the short-term effects of cardiopulmonary bypass have been studied in adult animal models, 2'3 little is known about the effects of ECMO on normal newborn cardiopulmonary physiology. The circulatory hemodynamics of a newborn animal are in much more flux because of the resistance changes occurring in the pulmonary capillary bed at time of birth--the transitional circulation. Consequently little is known of the effects of ECMO on the normal newborn animal. While ECMO with its attendant "lung rest" may seem benign, the therapy itself may create hemodynamic aberrations in the pulmonary and systemic circulation that could in turn be superimposed on a pathophysiological state for which ECMO might be indicated. We asked what effects prolonged ECMO might have on cardiopulmonary hemodynamics in early newborn animals and if these ECMO induced hemodynamic changes might be at least temporally related to modification of some of the family of vasoactive substances that contribute to net pulmonary vasomotor tone. Alterations in prostaglandin and catecholamine levels have been linked to hemodynamic changes induced by extracorporeal circulation)'4 Specifically thromboxane A:, a potent vasoconstrictor, and Journal of Pediatric Surgery, Vo125, No 1 (January), 1990: pp 33-37
prostacycline PGI2, a counter balancing vasodilator, have both been implicated in the altered physiology.4 Similarly augmentations of epinephrine and norepinephrine generation have been implicated: If cardiopulmonary tone reflects the net effects of a variety of vasoactive compounds, then the effects of ECMO might also be reflected in modifications of a vasoactive profile. Second, if healthy newborn animals were studied then some of the effects of ECMO could be observed independent of pathological derangements. MATERIALS AND METHODS Five newborn lambs, less than 24 hours old underwent left thoracotomy after intubation and anesthesia with halothane/ oxygen/pentobarbital. Polyvinyl pressure/sampling catheters (I.2 mm OD) were placed in the right atrium, left atrium, pulmonary artery, and femoral artery/vein. All catheters were brought out through subcutaneous tunnels, fixed in position, and flushed with heparinized saline. A nylon mesh harness secured the externalized catheters. The lamb was recovered with the ewe and adequacy of recovery confirmed by feeding observation, body temperature, respiratory rate, and blood gas analysis. After 48 to 72 hours recovery time the lambs were placed on venoarterial ECMO following light sedation with pentobarbital (5 to 10 mg/kg); local anesthesia with I% xylocaine was used for the cervical extrathoracic cannulation. The venous drainage cannula was passed into the right atrium via the right internal jugular vein and the arterial perfusion cannula passed to the aortic arch/ ascending aorta by the common carotid artery. The ECMO circuit was configured as described by Bartlett et al 6 and used 0.8 m 2 silicone membrane lung (Sci-Med Life Systems lnc, Minneapolis, MN): The priming solution consisted of Ringers's lactate/25% albumin displaced by whole maternal sheep blood, ACD preserved. Sodium heparin 500 U and calcium gluconate 300 meq were added to each unit of whole blood. The blood filled circuit was corrected for pO2 (75 to 100 torr) and the CO2 (35 to 45 torr) by sweep gas adjustments while pH was balanced by sodium bicarbonate administration (7.35 to 7.45). After attachment to the ECMO circuit the animals were intubated and assisted with positive pressure ventilation (FiO2 0.3 to 0.4, PIP 25 em H20, PEEP 5 cm H20, 1MV 20). Prone positioning in a supporting sling was maintained by minimal sedation (pentobarbital 5 to 10 mg/kg). After a period of at least 30
From the College of Physicians and Surgeons, Columbia University, and The Division of l~ediatric Surgery, The Babies Hospital, Columbia-Presbyterian Medical Center, New York, NY. Supported by The Charles Edison Fund and The Anya Fund. Presented at the 20th Annual Meeting of the American Pediatric Surgical Association, Baltimore, Maryland, May 28-31, 1989: Address reprint requests to Charles J.H. Stolar, MD, Room 203 N, The Babies Hospital, 3959 Broadway, New York, N Y 10032. ~) 1990 by W.B. Saunders Company. 0022-3468/90/2501-0006503.00/0 33
34
STOLAR AND DILLON
minutes with a stable blood pressure and heart rate, extracorporeal flow rates were initiated at 15 cc/kg/min and increased to 100 cc/kg/min over 5 minutes. The 100 cc/kg/min flow was maintained for 21 hours, then changed to 30 cc/kg/min for 2 hours. Bypass was terminated after 23 hours, and the animals were observed for an additional hour. The animals were continuously heparinized with 30 U/kg/min sodium heparin, lntravascular volume/urine output was maintained by 120 cc/kg/24 hours Ringer's lactate solution. Blood volume removed for sampling was replaced with whole sheep blood as needed. Hemodynamic parameters were continuously monitored by pressure transducers (Bentley-Trantec, Ardsley, NY) and viewed with a multichannel analog recorder (Sensor Medics, Anaheim, CA). Blood gas samples were drawn from pulmonary/femoral arteries and left atrium and were analyzed for pOz, pCO2, and pH by blood gas analyzer (Instrumentation Laboratories, #713, Boston, MA), calibrated to commercial standards.
150
*p<0.01
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125 S-rv
E E .o
100
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75 50
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30min lh 4h Bypass
8h 10h 20h 2~4h I
TIME
V~soactive Mediator Analysis Whole blood samples were obtained from the dorsal aorta via femoral artery at preselected times for serum assay of 6-keto prostacyclin Fla, thromboxane B2, epinephrine, and norepinephrine. Initial baseline samples were drawn 30 minutes after reading stable hemodynamics following cannulation and intubation but before ECMO initiation. Additional samples were obtained at ECMO initiation and at 30 minutes, 90 minutes, 4 hours, 8 hours, 21 hours, 23 hours, and 1 hour after bypass termination. Samples for prostacyclin determination were collected and refrigerated in 3-cc plastic syringes transferred to refrigerated polypropylene tubes containing 0.1 mol/L indomethacin and centrifuged at 3,000 rpm for 15 minutes. Serum was decanted to refrigerated polypropylene tubes and stored at -80~ until assayed. Prostacyclin levels were inferred from direct radioimmunoassay (New England Nuclear, Boston, MA) of the stable metabolite 6-keto prostaglandin Fla. The assay was based on an iodinated analog of 6-keto-prostaglandin F1, as tracer and rabit anti-6-keto-prostaglandin FI as antiserum. Thromboxane A= was inferred from radioimmunoassay of its stable metabolite thromboxane B2 by identical methodology with a tritiated tracer (New England Nuclear). Samples (2 cc) for catecholamine levels were collected in plastic syringes from the femoral artery and transferred to commercially prepared CAT-tubes (Upjohn Diagnostics, Kalamazoo, MI) containing heparin and glutathione. Refrigerated samples were centrifuged at 3,000 rpm and the serum stored at -80~ The assay was accomplished by an enzymatic method whereby catechoI-O-methyltransferase is used to catalyze transfer of the 3H-methyl group from S adenosyl-tmethionine-(3H-methyl) to norepinephrine and epinephrine. All assays were performed in duplicate. Results are presented as mean • SEM. Statistical analysis was performed by paired Student's t test. Significance was attributed to values less than .05.
RESULTS
Fig 1. R e s p o n s e of m e a n S A P to prolonged E C M O ; m e a n • S E M , n = 5.
line values. The effects of E C M O on mean pulmonary artery pressure (PAP) are shown in (Fig 2). E C M O causes a rise from a baseline of 23 _+ 2 mm Hg to 48 + 7 mm Hg by 2 minutes of bypass (P < .01). With full bypass flow of 100 c c / k g / m i n reached at 5 minutes, mean PAP remained elevated at 47 _+ 7 mm Hg. PAP returned to baseline values by 30 minutes of bypass and then gradually decreased during continued bypass t o a nadir of 16 _+ 3 m m H g a t 6 hours (P < .01). With the termination of bypass, mean PAP returned to p r e - E C M O baseline values.
Prostanoid Analysis Before the initiation of bypass, plasma arterial thromboxane Bz was 358 +_ 158 p g / m L (zero time) (Fig 3). At the 30-minute sample there was an increase to 1,182 _+ 350 p g / m L (P < .01). Measurements at 90 minutes showed an elevated level of 462 _+ 65 p g / m L that was not statistically different from prebypass
A
601 50"
o~ E E o
40" 30"
Hemodynamics The effects of extracorporeal membrane oxygenation bypass on newborn lamb systemic arterial hemodynamics are shown in Fig 1. The initiation of venoarterial bypass is marked by an increase in mean systemic arterial pressure (SAP) from baseline of 95 _+ 7 mm Hg to a peak of 127 _+ 5 mm Hg (P < .01) by 3 minutes bypass. SAP then returns to baseline by 15 minutes of bypass. For the remainder of the perfusion mean SAP remains stable and unchanged from base-
*p<0.01 **p<0.05
P
20"
..... i
Ix 10" 0
/
0
I
/
./
30rain ih
Byp ,
4h
./,
8h 10h 20h 214h
]
TIME Fig 2. R e s p o n s e of m e a n PAP to prolonged E C M O ; m e a n + S E M . n = 5.
ECMO & VASOACTIVE MEDIATORS IN LAMBS
35
1500'
*p<0.05
*p<0.01
3000 1250"
_~ 1000' E
--~2000 750'
J
v
500"
1000"
250" U
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"
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'
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12 Bypass
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24 I
TIME (hours)
Fig 3. T h r o m b o x a n a A 2 expressed as tromboxane B2 generation during prolonged ECMO; mean _+ SEM. n = 5.
Fig 5. Norepinephrine generation during prolonged E C M O ; mean -+ SEM, n = 5.
values. There was no significant change in Thromboxane B2 levels as bypass was weaned and terminated. At the initiation of bypass (zero time) plasma arterial 6-keto-prostaglandin Fla, concentration was 320 +_ 191 p g / m L (Fig 4). With the initiation of bypass there was an increase to 778 _+ 385 p g / m L at the 30-minute time point (P < .02). By 90 minutes the plasma concentration was not statistically different from baseline. With the decrease in bypass support at the end of the experiment there was no significant change noted in 6-keto-prostaglandin Fla level.
2,303 _+ 999 n g / m L was reached (P < .05). One hour after discontinuing bypass plasma norepinephrine levels had fallen to half that level or 890 _+ 50 ng/mL. Serum arterial epinephrine levels followed a similar but less dramatic course (Fig 6). Zero time epinephrine levels were 79 _+ 25 n g / m L before the initiation of bypass. There was a progressive rise during bypass to a maximum level of 632 _+ 320 n g / m L (P < .05) at the end of the perfusion. One hour after the termination of ECMO serum epinephrine levels had fallen to 230 _+ 78 ng/mL.
Catecholamine Analysis
DISCUSSION
Immediately before bypass initiation, plasma arterial norepinephrine concentration was 324 +_ 29 ng/ mL (Fig 5). Although not statistically significant at each time point, plasma norepinephrine levels increased throughout bypass. Just before terminating ECMO at 23 hours the maximum concentration of
These studies document cardiopulmonary and vasoactive mediator alterations induced by extracorporeal membrane oxygenation in the newborn lamb and illustrate the inherently nonphysiologicai nature of the therapy. Self limited augmentations in both thromboxane B2 and prostacyclin generation appear to be at 1500
1250
p<0 05
*p<0.05
1250
1000'
1000 --~ 750
E
750
r
v
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500 25O
250 ,
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I
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Bypass TIME (hours)
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,
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Fig 4. Prostacyclin expressed as 6-keto-prostaglandin FI= generation during prolonged ECMO; mean + S E a , n = 5.
0
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8
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12 Bypass
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generation during prolonged E C M O ,
36
STOLAR AND DILLON
least temporally related to initial episodes of pulmonary and systemic arterial hypertension when commencing bypass. Despite maintaining extracorporeal circulatory support of at least 50% of the anticipated cardiac output, PAP did not immediately fall with bypass. Maximum pulmonary artery hypotension was reached only after several hours. One of the proposed benefits of venoarterial ECMO is the hemodynamic consequence of pre-load and after-load reduction of the right ventricle in an hypoxic setting with pulmonary arterial hypertension. It appears that this benefit to the right ventricle accrues only with time, not acutely. Wonders et al 7 and Zapol et al 2 have previously demonstrated transient alterations in cardiopulmonary hemodynamics and prostaglandin metabolism as a result of ECMO in adult sheep during short perfusions. These results were confirmed by Peterson et al 3 who concluded that activation of the vasoactive prostaglandins was a direct function of extracorporeai perfusion. More recent clinical investigations in the pediatric population undergoing open heart surgery in cardiopulmonary bypass also demonstrated similar significant changes in prostacyclin and thromboxane generation. 4 In these studies the extracorporeal circuit itself has not been shown to be the site of prostaglandin metabolism. Proposed mechanisms for the generation and release of thromboxane B2 and 6-keto-prostaglandin Fla are speculative. Prostaglandin release has been documented as a result of pulmonary sequestration of platelets, leukocytes, and microemboli. 8'9 Cardiopulmonary bypass has also been implicated in complement activation with subsequent effect on prostaglandin metabolism. 1~ Tissue ischemia, hypotension, and hypoxia are known to cause thromboxane generation in particular. 9 However, at no time during our perfusion was hypotension, hypoxia, or acidosis encountered. We therefore believe that the release of the prostaglandins and activation of the arachidonic cascade was stimulated by initiation of cardiopulmonary bypass rather than tissue ischemia or poor perfusion. The catecholamine response to partial bypass shows a progressive rise in both epinephrine and norepineph-
rine levels as perfusion time increases. Only with the conclusion of bypass do epinephrine and norepinephrine levels decrease. Similar responses in catecholamines have been documented in adults with cardiopulmonary bypass. 5 The exact roles of these catecholamines in the control of cardiopulmonary hemodynamics of the newborn lamb while on bypass is unclear since pulse and blood pressure remained stable throughout the entire perfusion. The rising catecholamines may reflect a physiological reaction to stress induced by cardiopuimonary bypass that cannot be delineated by the simple parameters of pulse and blood pressure alone. Areas such as temperature regulation, pain, caloric intake, and blood volume were controlled as uniformly as possible. This study reports that in healthy newborn lambs initiation of E C M O is associated with hemodynamic and vasoactive mediator changes that are at least temporally related. Specifically, E C M O perfusion in a newborn lamb model can be viewed as having two phases. The early or acute phase is limited to the first 4 hours of bypass and is characterized by significant but self-limited augmentation of both SAP and PAP. The blood pressure changes are concomitant with augmentation of thromboxane B2 and.6-keto-prostaglandin Fla generation. The acute phase of ECMO is replaced by a chronic phase, which is characterized by return of SAP to baseline for the duration of bypass. The PAP falls below baseline perhaps reflecting preload reduction by ECMO. If a goal of ECMO is to relieve pulmonary hypertension, it can be accomplished in this model only after several hours. A persistent stress to the animal continues throughout the perfusion and is marked by elevated catecholamine levels despite the absence of systemic hypertension. The elevated catecholamine levels reflect the unphysiological nature of extracorporeal circulation. E C M O in a healthy newborn lamb creates a constellation of acute and chronic changes in hemodynamics and vasoactive mediators that are self limited. Although the salutary effects of ECMO are well documented, in a pathophysiological state for which ECMO is indicated, these E C M O induced changes may be superimposed on or exacerbate that state.
REFERENCES
1. Toomasian JM, Snedecor SM, Cornell RG, et al: National experiencewith extracorporealmembraneoxygenationfor newborn respiratoryfailere. Data'from 715 cases. Trans Am Soc Artif Intern Organs 34:140-147, 1988 2. Zapol WM, Peterson MB, WondersTR, et al: Plasma thromboxaneand prostacyclinmediatorsin sheepduring partial cardiopulmonary bypass. Trans Am Soc Artif Intern Organs 26:556-559, 1980 3. PetersonMB, HuttemeierPC, Zapol WM, etal: Thromboxane
mediates acute pulmonary hypertension in sheep extracorporea~ perfusion. Am J Physiol234:H471-H479, 1982 4. Greeley WJ, Bushman GA, Kong DL, et al: Effectsof cardiopulmonary bypass on eicosanoid metabolism during pediatric cardiovascular surgery. J Thorac CardiovascSurg 95:842-849, 1988 5. Replogle R, Levy M, Dewall RA, et al: Catecholamine and serotonin responseto cardiopulmonarybypass. J Thorac Cardiovasc Surg 44:638-648, 1962 6. Bartlett RH, Gazzaniga AB, Jefferies MR, et at: Extracorpo-
ECMO & VASOACTIVE MEDIATORS IN LAMBS
real membrane oxygenation (ECMO) cardiopulmonary support in infancy. Trans Am Soc Artif Intern Organs 25:386-389, 1979 7. Wonders TR, S0uthmayd JR, Schuette AH, et al: Reduced plasma thromboxane elevation during partial cardiopulmonary bypass in thrombocytopenic sheep. Trans Am Soc Artif Intern Organs 27:276-279, 1981 8. Hamburg M, Sevnsson J, Samuelson B: Thromboxanes: A new group of biochemically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci USA 72:2994-2998, 1975
37
9. Smith JB, Ingerman C, Kocsis BH: Formation of an intermediate in prostaglandin biosynthesis and its association with the platelet release reaction. J Clin Invest 53:1468-1472, 1974 10. Kirklin JK, Wisstaby S, Blackstone EH, et al: Compliment in the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 86:845-857, 1983 11. Howard R J, Cran C, Franzini DA, et al: Effects of cardiopulmonary bypass on pulmonary leukostasis and compliment activation. Arch Surg 123:1496-1501, 1988
membrane oxygenation E XTRACORPOREAL (ECMO) has been used successfully for the management of a variety of neonatal respiratory illnesses all of which share pulmonary hypertension and some degree of lung injury as a final common pathway) Although the short-term effects of cardiopulmonary bypass have been studied in adult animal models, 2'3 little is known about the effects of ECMO on normal newborn cardiopulmonary physiology. The circulatory hemodynamics of a newborn animal are in much more flux because of the resistance changes occurring in the pulmonary capillary bed at time of birth--the transitional circulation. Consequently little is known of the effects of ECMO on the normal newborn animal. While ECMO with its attendant "lung rest" may seem benign, the therapy itself may create hemodynamic aberrations in the pulmonary and systemic circulation that could in turn be superimposed on a pathophysiological state for which ECMO might be indicated. We asked what effects prolonged ECMO might have on cardiopulmonary hemodynamics in early newborn animals and if these ECMO induced hemodynamic changes might be at least temporally related to modification of some of the family of vasoactive substances that contribute to net pulmonary vasomotor tone. Alterations in prostaglandin and catecholamine levels have been linked to hemodynamic changes induced by extracorporeal circulation)'4 Specifically thromboxane A:, a potent vasoconstrictor, and Journal of Pediatric Surgery, Vo125, No 1 (January), 1990: pp 33-37
prostacycline PGI2, a counter balancing vasodilator, have both been implicated in the altered physiology.4 Similarly augmentations of epinephrine and norepinephrine generation have been implicated: If cardiopulmonary tone reflects the net effects of a variety of vasoactive compounds, then the effects of ECMO might also be reflected in modifications of a vasoactive profile. Second, if healthy newborn animals were studied then some of the effects of ECMO could be observed independent of pathological derangements. MATERIALS AND METHODS Five newborn lambs, less than 24 hours old underwent left thoracotomy after intubation and anesthesia with halothane/ oxygen/pentobarbital. Polyvinyl pressure/sampling catheters (I.2 mm OD) were placed in the right atrium, left atrium, pulmonary artery, and femoral artery/vein. All catheters were brought out through subcutaneous tunnels, fixed in position, and flushed with heparinized saline. A nylon mesh harness secured the externalized catheters. The lamb was recovered with the ewe and adequacy of recovery confirmed by feeding observation, body temperature, respiratory rate, and blood gas analysis. After 48 to 72 hours recovery time the lambs were placed on venoarterial ECMO following light sedation with pentobarbital (5 to 10 mg/kg); local anesthesia with I% xylocaine was used for the cervical extrathoracic cannulation. The venous drainage cannula was passed into the right atrium via the right internal jugular vein and the arterial perfusion cannula passed to the aortic arch/ ascending aorta by the common carotid artery. The ECMO circuit was configured as described by Bartlett et al 6 and used 0.8 m 2 silicone membrane lung (Sci-Med Life Systems lnc, Minneapolis, MN): The priming solution consisted of Ringers's lactate/25% albumin displaced by whole maternal sheep blood, ACD preserved. Sodium heparin 500 U and calcium gluconate 300 meq were added to each unit of whole blood. The blood filled circuit was corrected for pO2 (75 to 100 torr) and the CO2 (35 to 45 torr) by sweep gas adjustments while pH was balanced by sodium bicarbonate administration (7.35 to 7.45). After attachment to the ECMO circuit the animals were intubated and assisted with positive pressure ventilation (FiO2 0.3 to 0.4, PIP 25 em H20, PEEP 5 cm H20, 1MV 20). Prone positioning in a supporting sling was maintained by minimal sedation (pentobarbital 5 to 10 mg/kg). After a period of at least 30
From the College of Physicians and Surgeons, Columbia University, and The Division of l~ediatric Surgery, The Babies Hospital, Columbia-Presbyterian Medical Center, New York, NY. Supported by The Charles Edison Fund and The Anya Fund. Presented at the 20th Annual Meeting of the American Pediatric Surgical Association, Baltimore, Maryland, May 28-31, 1989: Address reprint requests to Charles J.H. Stolar, MD, Room 203 N, The Babies Hospital, 3959 Broadway, New York, N Y 10032. ~) 1990 by W.B. Saunders Company. 0022-3468/90/2501-0006503.00/0 33
34
STOLAR AND DILLON
minutes with a stable blood pressure and heart rate, extracorporeal flow rates were initiated at 15 cc/kg/min and increased to 100 cc/kg/min over 5 minutes. The 100 cc/kg/min flow was maintained for 21 hours, then changed to 30 cc/kg/min for 2 hours. Bypass was terminated after 23 hours, and the animals were observed for an additional hour. The animals were continuously heparinized with 30 U/kg/min sodium heparin, lntravascular volume/urine output was maintained by 120 cc/kg/24 hours Ringer's lactate solution. Blood volume removed for sampling was replaced with whole sheep blood as needed. Hemodynamic parameters were continuously monitored by pressure transducers (Bentley-Trantec, Ardsley, NY) and viewed with a multichannel analog recorder (Sensor Medics, Anaheim, CA). Blood gas samples were drawn from pulmonary/femoral arteries and left atrium and were analyzed for pOz, pCO2, and pH by blood gas analyzer (Instrumentation Laboratories, #713, Boston, MA), calibrated to commercial standards.
150
*p<0.01
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125 S-rv
E E .o
100
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.
.
75 50
Ix
25 0
0 [
30min lh 4h Bypass
8h 10h 20h 2~4h I
TIME
V~soactive Mediator Analysis Whole blood samples were obtained from the dorsal aorta via femoral artery at preselected times for serum assay of 6-keto prostacyclin Fla, thromboxane B2, epinephrine, and norepinephrine. Initial baseline samples were drawn 30 minutes after reading stable hemodynamics following cannulation and intubation but before ECMO initiation. Additional samples were obtained at ECMO initiation and at 30 minutes, 90 minutes, 4 hours, 8 hours, 21 hours, 23 hours, and 1 hour after bypass termination. Samples for prostacyclin determination were collected and refrigerated in 3-cc plastic syringes transferred to refrigerated polypropylene tubes containing 0.1 mol/L indomethacin and centrifuged at 3,000 rpm for 15 minutes. Serum was decanted to refrigerated polypropylene tubes and stored at -80~ until assayed. Prostacyclin levels were inferred from direct radioimmunoassay (New England Nuclear, Boston, MA) of the stable metabolite 6-keto prostaglandin Fla. The assay was based on an iodinated analog of 6-keto-prostaglandin F1, as tracer and rabit anti-6-keto-prostaglandin FI as antiserum. Thromboxane A= was inferred from radioimmunoassay of its stable metabolite thromboxane B2 by identical methodology with a tritiated tracer (New England Nuclear). Samples (2 cc) for catecholamine levels were collected in plastic syringes from the femoral artery and transferred to commercially prepared CAT-tubes (Upjohn Diagnostics, Kalamazoo, MI) containing heparin and glutathione. Refrigerated samples were centrifuged at 3,000 rpm and the serum stored at -80~ The assay was accomplished by an enzymatic method whereby catechoI-O-methyltransferase is used to catalyze transfer of the 3H-methyl group from S adenosyl-tmethionine-(3H-methyl) to norepinephrine and epinephrine. All assays were performed in duplicate. Results are presented as mean • SEM. Statistical analysis was performed by paired Student's t test. Significance was attributed to values less than .05.
RESULTS
Fig 1. R e s p o n s e of m e a n S A P to prolonged E C M O ; m e a n • S E M , n = 5.
line values. The effects of E C M O on mean pulmonary artery pressure (PAP) are shown in (Fig 2). E C M O causes a rise from a baseline of 23 _+ 2 mm Hg to 48 + 7 mm Hg by 2 minutes of bypass (P < .01). With full bypass flow of 100 c c / k g / m i n reached at 5 minutes, mean PAP remained elevated at 47 _+ 7 mm Hg. PAP returned to baseline values by 30 minutes of bypass and then gradually decreased during continued bypass t o a nadir of 16 _+ 3 m m H g a t 6 hours (P < .01). With the termination of bypass, mean PAP returned to p r e - E C M O baseline values.
Prostanoid Analysis Before the initiation of bypass, plasma arterial thromboxane Bz was 358 +_ 158 p g / m L (zero time) (Fig 3). At the 30-minute sample there was an increase to 1,182 _+ 350 p g / m L (P < .01). Measurements at 90 minutes showed an elevated level of 462 _+ 65 p g / m L that was not statistically different from prebypass
A
601 50"
o~ E E o
40" 30"
Hemodynamics The effects of extracorporeal membrane oxygenation bypass on newborn lamb systemic arterial hemodynamics are shown in Fig 1. The initiation of venoarterial bypass is marked by an increase in mean systemic arterial pressure (SAP) from baseline of 95 _+ 7 mm Hg to a peak of 127 _+ 5 mm Hg (P < .01) by 3 minutes bypass. SAP then returns to baseline by 15 minutes of bypass. For the remainder of the perfusion mean SAP remains stable and unchanged from base-
*p<0.01 **p<0.05
P
20"
..... i
Ix 10" 0
/
0
I
/
./
30rain ih
Byp ,
4h
./,
8h 10h 20h 214h
]
TIME Fig 2. R e s p o n s e of m e a n PAP to prolonged E C M O ; m e a n + S E M . n = 5.
ECMO & VASOACTIVE MEDIATORS IN LAMBS
35
1500'
*p<0.05
*p<0.01
3000 1250"
_~ 1000' E
--~2000 750'
J
v
500"
1000"
250" U
'
"
0 I
'
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"
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12 Bypass TIME (hours)
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9
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0 I
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12 Bypass
16
20
24 I
TIME (hours)
Fig 3. T h r o m b o x a n a A 2 expressed as tromboxane B2 generation during prolonged ECMO; mean _+ SEM. n = 5.
Fig 5. Norepinephrine generation during prolonged E C M O ; mean -+ SEM, n = 5.
values. There was no significant change in Thromboxane B2 levels as bypass was weaned and terminated. At the initiation of bypass (zero time) plasma arterial 6-keto-prostaglandin Fla, concentration was 320 +_ 191 p g / m L (Fig 4). With the initiation of bypass there was an increase to 778 _+ 385 p g / m L at the 30-minute time point (P < .02). By 90 minutes the plasma concentration was not statistically different from baseline. With the decrease in bypass support at the end of the experiment there was no significant change noted in 6-keto-prostaglandin Fla level.
2,303 _+ 999 n g / m L was reached (P < .05). One hour after discontinuing bypass plasma norepinephrine levels had fallen to half that level or 890 _+ 50 ng/mL. Serum arterial epinephrine levels followed a similar but less dramatic course (Fig 6). Zero time epinephrine levels were 79 _+ 25 n g / m L before the initiation of bypass. There was a progressive rise during bypass to a maximum level of 632 _+ 320 n g / m L (P < .05) at the end of the perfusion. One hour after the termination of ECMO serum epinephrine levels had fallen to 230 _+ 78 ng/mL.
Catecholamine Analysis
DISCUSSION
Immediately before bypass initiation, plasma arterial norepinephrine concentration was 324 +_ 29 ng/ mL (Fig 5). Although not statistically significant at each time point, plasma norepinephrine levels increased throughout bypass. Just before terminating ECMO at 23 hours the maximum concentration of
These studies document cardiopulmonary and vasoactive mediator alterations induced by extracorporeal membrane oxygenation in the newborn lamb and illustrate the inherently nonphysiologicai nature of the therapy. Self limited augmentations in both thromboxane B2 and prostacyclin generation appear to be at 1500
1250
p<0 05
*p<0.05
1250
1000'
1000 --~ 750
E
750
r
v
500
500 25O
250 ,
0
I
.
,
4
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,
8
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,
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Bypass TIME (hours)
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,
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,
24
I
Fig 4. Prostacyclin expressed as 6-keto-prostaglandin FI= generation during prolonged ECMO; mean + S E a , n = 5.
0
,
0
,
4
I
.
,
8
.
,
.
12 Bypass
,
16
.
,
,
20
24 I
TIME (hours) Fig 6. Epinephrine m e a n -+ S E a , n = 5.
generation during prolonged E C M O ,
36
STOLAR AND DILLON
least temporally related to initial episodes of pulmonary and systemic arterial hypertension when commencing bypass. Despite maintaining extracorporeal circulatory support of at least 50% of the anticipated cardiac output, PAP did not immediately fall with bypass. Maximum pulmonary artery hypotension was reached only after several hours. One of the proposed benefits of venoarterial ECMO is the hemodynamic consequence of pre-load and after-load reduction of the right ventricle in an hypoxic setting with pulmonary arterial hypertension. It appears that this benefit to the right ventricle accrues only with time, not acutely. Wonders et al 7 and Zapol et al 2 have previously demonstrated transient alterations in cardiopulmonary hemodynamics and prostaglandin metabolism as a result of ECMO in adult sheep during short perfusions. These results were confirmed by Peterson et al 3 who concluded that activation of the vasoactive prostaglandins was a direct function of extracorporeai perfusion. More recent clinical investigations in the pediatric population undergoing open heart surgery in cardiopulmonary bypass also demonstrated similar significant changes in prostacyclin and thromboxane generation. 4 In these studies the extracorporeal circuit itself has not been shown to be the site of prostaglandin metabolism. Proposed mechanisms for the generation and release of thromboxane B2 and 6-keto-prostaglandin Fla are speculative. Prostaglandin release has been documented as a result of pulmonary sequestration of platelets, leukocytes, and microemboli. 8'9 Cardiopulmonary bypass has also been implicated in complement activation with subsequent effect on prostaglandin metabolism. 1~ Tissue ischemia, hypotension, and hypoxia are known to cause thromboxane generation in particular. 9 However, at no time during our perfusion was hypotension, hypoxia, or acidosis encountered. We therefore believe that the release of the prostaglandins and activation of the arachidonic cascade was stimulated by initiation of cardiopulmonary bypass rather than tissue ischemia or poor perfusion. The catecholamine response to partial bypass shows a progressive rise in both epinephrine and norepineph-
rine levels as perfusion time increases. Only with the conclusion of bypass do epinephrine and norepinephrine levels decrease. Similar responses in catecholamines have been documented in adults with cardiopulmonary bypass. 5 The exact roles of these catecholamines in the control of cardiopulmonary hemodynamics of the newborn lamb while on bypass is unclear since pulse and blood pressure remained stable throughout the entire perfusion. The rising catecholamines may reflect a physiological reaction to stress induced by cardiopuimonary bypass that cannot be delineated by the simple parameters of pulse and blood pressure alone. Areas such as temperature regulation, pain, caloric intake, and blood volume were controlled as uniformly as possible. This study reports that in healthy newborn lambs initiation of E C M O is associated with hemodynamic and vasoactive mediator changes that are at least temporally related. Specifically, E C M O perfusion in a newborn lamb model can be viewed as having two phases. The early or acute phase is limited to the first 4 hours of bypass and is characterized by significant but self-limited augmentation of both SAP and PAP. The blood pressure changes are concomitant with augmentation of thromboxane B2 and.6-keto-prostaglandin Fla generation. The acute phase of ECMO is replaced by a chronic phase, which is characterized by return of SAP to baseline for the duration of bypass. The PAP falls below baseline perhaps reflecting preload reduction by ECMO. If a goal of ECMO is to relieve pulmonary hypertension, it can be accomplished in this model only after several hours. A persistent stress to the animal continues throughout the perfusion and is marked by elevated catecholamine levels despite the absence of systemic hypertension. The elevated catecholamine levels reflect the unphysiological nature of extracorporeal circulation. E C M O in a healthy newborn lamb creates a constellation of acute and chronic changes in hemodynamics and vasoactive mediators that are self limited. Although the salutary effects of ECMO are well documented, in a pathophysiological state for which ECMO is indicated, these E C M O induced changes may be superimposed on or exacerbate that state.
REFERENCES
1. Toomasian JM, Snedecor SM, Cornell RG, et al: National experiencewith extracorporealmembraneoxygenationfor newborn respiratoryfailere. Data'from 715 cases. Trans Am Soc Artif Intern Organs 34:140-147, 1988 2. Zapol WM, Peterson MB, WondersTR, et al: Plasma thromboxaneand prostacyclinmediatorsin sheepduring partial cardiopulmonary bypass. Trans Am Soc Artif Intern Organs 26:556-559, 1980 3. PetersonMB, HuttemeierPC, Zapol WM, etal: Thromboxane
mediates acute pulmonary hypertension in sheep extracorporea~ perfusion. Am J Physiol234:H471-H479, 1982 4. Greeley WJ, Bushman GA, Kong DL, et al: Effectsof cardiopulmonary bypass on eicosanoid metabolism during pediatric cardiovascular surgery. J Thorac CardiovascSurg 95:842-849, 1988 5. Replogle R, Levy M, Dewall RA, et al: Catecholamine and serotonin responseto cardiopulmonarybypass. J Thorac Cardiovasc Surg 44:638-648, 1962 6. Bartlett RH, Gazzaniga AB, Jefferies MR, et at: Extracorpo-
ECMO & VASOACTIVE MEDIATORS IN LAMBS
real membrane oxygenation (ECMO) cardiopulmonary support in infancy. Trans Am Soc Artif Intern Organs 25:386-389, 1979 7. Wonders TR, S0uthmayd JR, Schuette AH, et al: Reduced plasma thromboxane elevation during partial cardiopulmonary bypass in thrombocytopenic sheep. Trans Am Soc Artif Intern Organs 27:276-279, 1981 8. Hamburg M, Sevnsson J, Samuelson B: Thromboxanes: A new group of biochemically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci USA 72:2994-2998, 1975
37
9. Smith JB, Ingerman C, Kocsis BH: Formation of an intermediate in prostaglandin biosynthesis and its association with the platelet release reaction. J Clin Invest 53:1468-1472, 1974 10. Kirklin JK, Wisstaby S, Blackstone EH, et al: Compliment in the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 86:845-857, 1983 11. Howard R J, Cran C, Franzini DA, et al: Effects of cardiopulmonary bypass on pulmonary leukostasis and compliment activation. Arch Surg 123:1496-1501, 1988